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Study of several aspects of the enzyme tyrosine hydroxylase Gibson, Sheila M. 1968

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A STUDY OF SEVERAL ASPECTS OF THE ENZYME TYROSINE HYDROXYLASE  by  S h e i l a M. G i b s o n B . S c , U n i v e r s i t y o f B r i t i s h Columbia,  1966  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF  Master of Science i n the Kinsmen L a b o r a t o r y o f N e u r o l g g i c a l  Research  Department o f P s y c h i a t r y  We a c c e p t t h i s t h e s i s as conforming t o the required- sj^afldard  THE/ UNIVERSITY OF BRITISH COLUMBIA May 3 r d , 1968  In  presenting  for  an  that  advanced  shall  I further  thesis  thesis  degree  the Library  Study.  or by  publication  without  my  Department  at  M/9-V  i t freely  may  be  h its r e p r e s e n t a t i v e s .  written  of  thesis  Vt  H LA  6.  T/<V Columbia  the  granted  by  requirements  Columbia,  t h e Head  shall  and  copying  It i s understood  gain  I agree  for reference  f o r extensive  for financial  permission.  of  of British  available  permission  purposes  of this  fulfilment  the U n i v e r s i t y  that  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada Date  in partial  make  agree  for scholarly  Department  or  this  of  of  this  my  that  n o t be  copying  allowed  ABSTRACT  I n t e r e s t i n b r a i n c a t e c h o l a m i n e s has grown c o n s i d e r a b l y i n the l a s t few y e a r s i n view of t h e i r p o s s i b l e r o l e as neurotransmitters. T h i s i n v e s t i g a t i o n d e a l s p r i m a r i l y w i t h the enzyme t y r o s i n e h y d r o x y l a s e w h i c h i s thought t o be the r a t e step i n catecholamine  limiting  synthesis.  U s i n g t y r o s i n e h y d r o x y l a s e measurements and c a t e c h o l amine d e p l e t i o n  t e c h n i q u e s , a t t e m p t s were made t o determine the  s i t e o f i n c r e a s e d s y n t h e s i s o f c a t e c h o l a m i n e s i n animals to c o l d .  exposed  B r a i n , h e a r t and s p l e e n do n o t appear t o be the  organs i n v o l v e d i n t h i s change.  A d r e n a l s may be o f s i g n i f i c a n c e  but t h e r e s u l t s were s u g g e s t i v e r a t h e r than c o n c l u s i v e . T y r o s i n e h y d r o x y l a s e d i s t r i b u t i o n i n b r a i n was determined i n v a r i o u s r e g i o n s o f r a t , r a b b i t and c a t b r a i n , and a c t i v i t y was shown t o be h i g h e s t i n the caudate, a r e a , n u c l e u s accombens, and a n t e r i o r p e r f o r a t i n g  septal  substance,  w i t h much lower a c t i v i t i e s i n o t h e r r e g i o n s such as hippocampus, amygdala, hypothalamus,  thalamus, c o r t e x , c e r e b e l l u m and b r a i n  stem. U s i n g these d i s t r i b u t i o n s t u d i e s as i n d i c a t i o n s o f normal  t y r o s i n e h y d r o x y l a s e a c t i v i t y i n areas o f r a t b r a i n , and  e l e c t r o l y t i c l e s i o n t e c h n i q u e s , s t u d i e s were c a r r i e d o u t to determine n o r a d r e n e r g i c and d o p a n e r g i c pathways i n b r a i n . Catecholamine  f i b e r s from t h e i r o r i g i n i n the m i d b r a i n were  t r a c e d i n the m i d b r a i n and d i e n c e p h a l o n t o the caudate and s e p t a l a r e a , and the r e l a t i v e p o s i t i o n s of each group o f f i b e r s determined a l o n g t h e i r c o u r s e .  iii  INDEX Page  1.  2.  INTRODUCTION  1  CATECHOLAMINES  1  a)  Chemistry  1  b)  Metabolism  1  c)  Location  d)  Function  ..  ..  ..  ..  ..  ..  ..  4  TYROSINE HYDROXYLASE  6  a)  As a c o n t r o l of CA s y n t h e s i s  b)  Characteristics  c)  Location i)  6 ..  Subcellular  l o c a t i o n ..  8 ..  ..  ..  Inhibitors  8 8  3.  DOPAMINERGIC AND NORADRENERGIC PATHWAYS IN BRAIN..  4.  POSSIBLE ROLE OF CATECHOLAMINES  5.  7 8  i i ) A n a t o m i c a l l o c a t i o n .. d)  1  ..  ....  ..  9  10  a)  Emotion and b e h a v i o r  10  b)  Memory and l e a r n i n g  c)  Sleep  13  d)  Temperature r e g u l a t i o n  13  e)  Basal ganglia  13  ..  ..  ..  ..  ..  ..  and P a r k i n s o n i s m  13  AIMS OF THESIS  14  METHODS AND MATERIALS  16  a)  Tyrosine hydroxylase analysis  ..  16  iv  b)  i)  Procedure  16  ii)  R a t i o n a l f o r procedure  16  Tyrosine analysis  22  i)  Procedure  ..  ..  ..  ii)  R a t i o n a l f o r procedure  ..  . .  . .  c)  Calculation of V max  d)  N o r a d r e n a l i n e and Dopamine d e t e r m i n a t i o n s  e)  f)  . .  22 24 ..  Isolation  25  ii)  NA a n a l y s i s  25  i i i ) DA a n a l y s i s  26  iv)  26  R a t i o n a l f o r catecholamine determination.  S e p a r a t i o n o f n o r a d r e n a l i n e and dopamine and some o f t h e i r m e t a b o l i t e s  28  i)  Ion-exchange  28  ii)  T h i n - l a y e r chromatography  chromatography  30  Catecholamine a c t i v i t y i n r a t s i n the c o l d ..  31  i)  Catecholamine d e t e r m i n a t i o n i n u r i n e  31  ii)  Tyrosine Hydroxylase i n c o l d a c c l i m a t i z e d  ..  31  i i i ) Turnover r a t e s i n c o l d a c c l i m a t i z e d r a t s  1.  2.  25  i)  rats  g)  22  31  E f f e c t o f a l t e r e d c a t e c h o l a m i n e l e v e l s on T y r o s i n e H y d r o x y l a s e a c t i v i t y i n r a t b r a i n ..  31  h)  D i s t r i b u t i o n of Tyrosine Hydroxylase i n b r a i n  32  i)  Study o f c a t e c h o l a m i n e pathways i n c a t b r a i n  32  RESULTS  35  SEPARATION OF NA, DA AND SOME OF THEIR METABOLITES  35  a)  Ion-exchange  35  b)  T h i n - l a y e r chromatography  chromatography  CATECHOLAMINE ACTIVITY IN RATS EXPOSED TO COLD a)  Urine  35 ..  35 35  3.  b)  Tissue  c)  Tyrosine hydroxylase a c t i v i t y  d)  Turnover r a t e s  39 . .  39  EFFECTS OF ALTERING CATECHOLAMINE LEVELS ON IN VITRO TYROSINE HYDROXYLASE ACTIVITY  4.  43  DISTRIBUTION STUDIES OF TYROSINE HYDROXYLASE IN RAT, RABBIT AND CAT BRAIN  5.  39  43  EFFECT ' OF LESIONS ON TYROSINE HYDROXYLASE LEVELS IN VARIOUS REGIONS OF CAT BRAIN  49  a)  L e s i o n s of d i e n c e p h a l o n f l o o r  49  b)  P o s t e r i o r diencephalon - F i e l d s of F o r e l  c)  Habenula  56  d)  Substantia nigra  60  e)  Midline midbrain  64  f)  Raphe  68  ..  54  V  DISCUSSION  ..  71  1.  TYROSINE HYDROXYLASE ACTIVITY IN VITRO  2.  SEPARATION OF NA, DA AND SOME OF THEIR METABOLITES  71  ..  72  3.  CATECHOLAMINE ACTIVITY I N RATS EXPOSED TO COLD  ..  73  4.  TYROSINE HYDROXYLASE ACTIVITY I N BRAIN  ..  76  5.  ..  a)  Distribution  76  b)  As a c o n t r o l o f CA s y n t h e s i s  77  TYROSINE HYDROXYLASE CONTAINING FIBERS I N CAT BRAIN  81  a)  General c o n s i d e r a t i o n s  81  b)  F i b e r s i n the d i e n c e p h a l o n  82  c)  d)  i)  Mid-diencephalon  . .  ii)  p o s t e r i o r diencephalon  ..  ..  ..  ..  82 83  F i b e r s i n the m i d b r a i n  84  i)  t o t h e caudate  84  ii)  to the s e p t a l a r e a  86  Summary and C o n c l u s i o n s  ... ..  ..  ..  ..  SUMMARY AND CONCLUSIONS.  REFERENCES  APPENDIX Papers -  90  . .  ..  87  92  ..  Some C h a r a c t e r i s t i c s o f B r a i n T y r o s i n e Hydroxyase D i s t r i b u t i o n of Tyrosine Hydroxylase i n A d u l t and D e v e l o p i n g B r a i n  Activity  vi  INDEX TO TABLES  Table 1  Page Summary f o r d i s t r i b u t i o n d a t a f o r major areas of b r a i n  2  5  E f f e c t of DMPH^ on t y r o s i n e h y d r o x y l a s e  activity  i n c a t , r a b b i t and r a t b r a i n 3.  20  E f f e c t of phosphate a d d i t i o n on s i z e of b l a n k and Dopa r e c o v e r y from alumina column  4.  v a l u e s f o r NA,  21  DA and some of the p r e c u r s o r s  and m e t a b o l i t e s on polyamide 5  Catecholamine  c o n t e n t of 24 hour u r i n e  of r a t s exposed to c o l d 6  Catecholamine  38 samples  ..  40  l e v e l s i n t i s s u e s of r a t s exposed to  cold 7  41  T y r o s i n e h y d r o x y l a s e a c t i v i t y i n r a t s exposed to cold  8  42  E f f e c t s of c e r t a i n drugs on c a t e c h o l a m i n e  levels  and t y r o s i n e h y d r o x y l a s e a c t i v i t y i n r a t b r a i n 9  ..  D i s t r i b u t i o n of t y r o s i n e h y d r o x y l a s e i n a d u l t r a t , r a b b i t and c a t b r a i n  45  10  T i s s u e w e i g h t s of d i f f e r e n t b r a i n areas  11  T y r o s i n e c o n c e n t r a t i o n s i n v a r i o u s areas of r a t , r a b b i t and c a t b r a i n  12  ..  ..  ..  .  Tyrosine hydroxylase a c t i v i t y i n subdivisions  of 51  E f f e c t s of l e s i o n s i n f l o o r o f d i e n c e p h a l o n on t y r o s i n e h y d r o x y l a s e and c a t e c h o l a m i n e s areas  14  15  46  50  s e p t a l a r e a of c a t b r a i n 13  44  ..  ..  ..  ..  ..  ..  in rostral ..  ..  ..  52  E f f e c t s of l e s i o n s i n F i e l d s of F o r e l on T y r o s i n e h y d r o x y l a s e and c a t e c h o l a m i n e s  i n r o s t r a l areas  E f f e c t s of attempted h a b e n u l a r  l e s i o n s on  ..  58  tyrosine  hydroxylase a c t i v i t y i n caudate, s e p t a l area, amygdala and hippocampus ..  ..  ..  ..  ..  ..  59  vii  16  E f f e c t s of s u b s t a n t i a n i g r a l e s i o n s on t y r o s i n e h y d r o x y l a s e a c t i v i t y and c a t e c h o l a m i n e s i n caudate and septum  17  ..  ..  ..  ..  ..  ..  62  ..  65  ..  79  E f f e c t of b i l a t e r a l m i d b r a i n l e s i o n s on t y r o s i n e h y d r o x y l a s e a c t i v i t y i n caudate and septum ..  18  ..  R e l a t i v e t y r o s i n e h y d r o x y l a s e a c t i v i t y and noradrenaline turnover rates i n r a t b r a i n  viii  INDEX TO FIGURES  Figure  Page  1  M e t a b o l i s m of Catecholamines  2  Summary of n o r a d r e n e r g i c and way  3  i n brain  2 dopaminergic p a t h -  ..  ..  ..  ..  ..  ..  11  ..  18  R e l a t i o n s h i p between amount of b r a i n t i s s u e used i n incubation  4  and  tyrosine hydroxylase a c t i v i t y  E f f e c t of pH of i n c u b a t i o n m i x t u r e on the of t y r o s i n e h y d r o x y l a s e i n b r a i n  5  ..  activity  ..  ..  ..  A c t i v i t y of t y r o s i n e h y d r o x y l a s e w i t h r e s p e c t time of i n c u b a t i o n  6  ..  Recovery of c a t e c h o l s  ...  ..  ..  19  i n the column  7  Standard curve f o r t y r o s i n e determination  8  R e c o v e r y o f NA and  K  pH's ...  ..  f o r r a b b i t and  determination  for r a t brain  cat brain  23  to  i n the column ..  determination  19 ..  DA from a l u m i n a w i t h r e s p e c t  pH of sample as i t i s p l a c e d 9  to  from a l u m i n a a t v a r i o u s  of the sample as i t i s p l a c e d  18  ..  ..  23  ..  27  m 10  K  27  m 11  S t a n d a r d curve f o r NA  and DA d e t e r m i n a t i o n s  12  Median s a g i t t a l s e c t i o n of b r a i n to i l l u s t r a t e method of d i s s e c t i o n ..  13  ..  ..  ..  ..  ..  29  the  ..  ..  33  Median s a g i t t a l s e c t i o n of c a t b r a i n to i l l u s t r a t e the s u b d i v i s i o n s of the s e p t a l a r e a  14  Separation  34  by ion-exchange chromatography of  NM and D A - C . . 14  NA,  .  36 14  15  Separation  of NA,  DA,  MT  exchange chromatography 16  (a)  Depletion  of NA w i t h  and ..  C-NA ..  by i o n ..  ..  ..  (b)  Depletion various cold  37  ..  47  - m e t h y l - m - t y r o s i n e from  organs of normal r a t s and r a t s exposed to c o l d 16  ..  of NA with<A-methy1-p-tyrosine from  organs of normal r a t s and r a t s exposed to ..  ..  ..  ..  ..  ..  ..  ..  ..  48  -''ix  17  Median s a g i t t a l s e c t i o n of the b r a i n t o i l l u s t r a t e the l o c a t i o n of l e s i o n s o f the d i e n c e p h a l o n and midbrain  53  18  L e s i o n i n the f l o o r of the m i d - d i e n c e p h a l o n  19  M e d i a l l y p l a c e d l e s i o n i n the p o s t e r i o r diencephalon  ..  55  a t the l e v e l of the mammillary b o d i e s  20  L e s i o n i n the p o s t e r i o r d i e n c e p h a l o n  21  L e s i o n made i n an attempt t o d e s t r o y the habenular  57 57  r e g i o n b u t w i t h damage v e n t r a l t o i t 22  L e s i o n i n the habenular v e n t r a l diencephalon  ..  61  a r e a t h a t extended i n t o the ..  ..  ..  ..  ..  ..  23  L e s i o n of the habenula o n l y  24  Dorsal cerveau-isole lesion  25  Ventral cerveau-isole lesion  26  L e s i o n o f the m i d l i n e o f the m i d b r a i n w i t h o n l y s l i g h t l a t e r a l extension  27  63 ..  ..  ..  ..  66 66  r.  ..  ..  . .  L e s i o n of the m i d l i n e m i d b r a i n  ..  . .  ..  with  . .  67  ..  . .  67  lateral  e x t e n s i o n of the l e s i o n more on the r i g h t 29  ..  L e s i o n o f the m i d l i n e m i d b r a i n w i t h a l a r g e l a t e r a l extension  28  63  ..  ..  69  L e s i o n of the m i d l i n e m i d b r a i n i n the d o r s a l raphe  70  X  INDEX OF DIAGRAMS  Diagram 1  Page Subcellular l o c a t i o n of noradrenalin  i n the  n e r v e endings o f t h e s y m p a t h e t i c nervous system 2  3  Summary o f t y r o s i n e h y d r o x y l a s e c o n t a i n i n g f i b e r s o f the caudate and s e p t a l a r e a  88  ACKNOWLEDGEMENTS I am g r a t e f u l l y i n d e b t e d t o Drs. P a t r i c k and E d i e McGeer, Dr. Juhn Wada and Dr. C. L o e s e r f o r c o n s t r u c t i v e and s t i m u l a t i n g a d v i c e f o r t h i s t h e s i s , and I am e s p e c i a l l y g r a t e f u l t o Mr. Ron T s u j i k a w a and Mrs. G. T u r b i s a l o n g w i t h o t h e r members of the Kinsmen L a b o r a t o r y o f N e u r o l o g i c a l Resear for their helpful technical assistance.  1.  INTRODUCTION  R e c e n t l y i t has become e v i d e n t t h a t the (CA) have a s i g n i f i c a n t r o l e i n b r a i n f u n c t i o n .  catecholamines Therefore,  s t u d y o f t h e s e compounds s h o u l d a i d i n e l u c i d a t i n g some o f  a the  b i o c h e m i c a l mechanisms o f the c e n t r a l nervous system (CNS).  1.  Catecholamines a)  Chemistry The b a s i c s t r u c t u r e o f the CA i s a d i h y d r o x y  aromatic  r i n g ( c a t e c h o l m o i e t y ) w i t h a two c a r b o n s i d e c h a i n c o n t a i n i n g an amine group. on  the'J3  V a r i a t i o n s w i t h i n the CA a r i s e f r o m s u b s t i t u t i o n s  c a r b o n and/or the amine group.  the CA f a m i l y , n o r a d r e n a l i n e  The  i m p o r t a n t members o f  (NA) , dopamine (DA)  and a d r e n a l i n e ,  a r e shown i n F i g . 1 . b)  Metabolism The  s t e p s i n the i n v i v o s y n t h e s i s and c a t a b o l i s m o f  CA have been worked o u t . presented  in Fig. 1 .  The  sequence and n e c e s s a r y  the  enzymes a r e  I t i s w o r t h n o t i n g t h a t NA and DA have  s i g n i f i c a n t p h y s i o l o g i c a l a c t i v i t y as w e l l as b e i n g p r e c u r s o r s i n adrenaline synthesis.  The CAs a r e i n a c t i v a t e d i n v i v o n o t o n l y by  O - m e t h y l a t i o n and o x i d a t i o n as shown i n F i g . 1 , but by r e - u p t a k e i n t o t i s s u e and by d i f f u s i o n away f r o m the s i t e o f a c t i v i t y . CA and m e t a b o l i t e s a r e e v e n t u a l l y e x c r e t e d v i a the k i d n e y and  The can  be d e t e c t e d i n the u r i n e . c)  Location The  CAs.- a r e found i n v a r i o u s organs of the body:  e.g.  b r a i n and a d r e n a l s as w e l l as i n a l l s y m p a t h e t i c a l l y i n n e r v a t e d tissue. I n the a d r e n a l s NA and a d r e n a l i n e a r e c o n t a i n e d i n v e s i c l e s w i t h i n c h r o m a f f i n c e l l s , and a r e r e l e a s e d on s t i m u l a t i o n o f the s p l a n c h n i c n e r v e . a precursor  (1).  DA i s a l s o p r e s e n t but p r o b a b l y o n l y as  I n the r e s t o f the p e r i p h e r y , NA i s c o n t a i n e d  i n the n e r v e e n d i n g s , b o t h i n v e s i c l e s , t h a t a r e d e p l e t e d on n e r v e s t i m u l a t i o n , and i n the c y t o p l a s m  (see D i a g . 1 ) .  2.  0CH  o  IH COOH Horaovanillic acid  COOH 3,4-dihydroxyphenylacetic acid C H  3°  HO -/^"X.CHOHCH^NH^ Normetanephrine JHOHCH^NH,  Noradrenaline (NA)  N-met l y l transferase V  OH  HOHCOOH COMT 3,4-dihydroxymandelic CHOHCOOH acid 5j3-methoxy-4hydroxymandelic •^r acid CH 0 / 3  CHOHCH NHCH2 3 Adrenaline  H O _ ^ ~ y , CH0HCH NHCH 2  Metanephrine  COMT  catecho1-0-methyltransferase  MAO  monamine o x i d a s e  F i g . 1: M e t a b o l i s m o f Catecholamine  3  3.  TYROSINE  TYROSINE HYDROXYLASE  GRANULE  DOPA  NA  DOPAMINE  RESERVE POOL • \IN VESICLE  DEAMINATED TABOLITES  f  MAO. MOBILE POOL OF NA IN CYTOPLASM  RELEASE  ^COMT —  EFFECTOR  D i a g . 1:  —  —  REUPTA:  i DIFFUSION  —  CELL  Subcellular l o c a t i o n of noradrenaline i n the nerve endings o f the s y m p a t h e t i c nervous system.  4.  NA  and DA a r e p r e s e n t i n b r a i n a t a p p r o x i m a t e l y 1%  the c o n c e n t r a t i o n  found i n a d r e n a l s .  of  Here DA appears to have some  r o l e i n a d d i t i o n to t h a t as a p r e c u r s o r  f o r NA  synthesis.  As shown i n T a b l e 1. b o t h c h e m i c a l (2,3,4,5) and h i s t o c h e m i c a l (6,7)  a n a l y s e s i n d i c a t e an uneven d i s t r i b u t i o n o f NA  DA i n the b r a i n .  DA i s c o n c e n t r a t e d i n the s t r i a t u m , NA  and  i n the  hypothalamus, but b o t h are found i n s i g n i f i c a n t c o n c e n t r a t i o n s t h r o u g h o u t the l i m b i c system. a c t i v e t r a c e r work t h a t NA  I t has  a l s o been found by r a d i o -  and DA i n j e c t e d i n t o the b r a i n  are  t a k e n up p r e f e r e n t i a l l y i n t o c e r t a i n a r e a s (8) w i t h a d i s t r i b u t i o n s i m i l a r to t h a t of the endogenous amine. a c t i v e NA  For example, i f r a d i o -  i s i n j e c t e d i n t o r a t b r a i n , the c o n c e n t r a t i o n  after  hour i n the hypothalamus i s 10 times t h a t o f the c o r t e x .  one  Also  i n d i c a t e d i n T a b l e 1 a r e the d i f f e r e n c e s i n r a t e o f i n v i v o s y n t h e s i s of CA i n the v a r i o u s r e g i o n s i o n o f the r a d i o a c t i v e p r e c u r s o r  of b r a i n ( 9 ) .  By  inject-  t y r o s i n e i t has been demonstrated  t h a t the type of CA s y n t h e s i s v a r i e s throughout the b r a i n ; the major p r o d u c t of CA s y n t h e s i s  i s DA i n the s t r i a t u m and NA  i n the hypo-  thalamus (10) . As i n the p e r i p h e r y , i n presynaptic  NA  i n the b r a i n has been  v e s i c l e s of n e r v e endings (11,12).  s t u d i e s a l s o i n d i c a t e t h a t DA  detected  Histochemical  i s l o c a t e d p r i m a r i l y i n n e r v e endings  (13). d)  Function The  f u n c t i o n s of CA i n the p e r i p h e r y  established.  are f a i r l y w e l l  When an a n i m a l i s p l a c e d under s t r e s s t h e r e i s an  i n c r e a s e d n e u r o n a l a c t i v i t y i n the s y m p a t h e t i c nervous system, r e s u l t i n g i n NA  and  adrenaline  r e l e a s e f r o m the a d r e n a l s and  d i s c h a r g e o f NA  from s y m p a t h e t i c neurons.  NA  a  causes v a s o -  c o n s t r i c t i o n i n the g a s t r o i n t e s t i n a l t r a c t , s k i n and  kidney;  i n h i b i t i o n o f i n t e s t i n a l smooth muscle c o n t r a c t i o n , p u p i l d i l a t i o n and Adrenaline h e a r t and  i n c r e a s e i n h e a r t r a t e and f o r c e of  has  contraction.  s i m i l a r e f f e c t s on the p u p i l s , smooth muscle  and  i n a d d i t i o n , causes v a s o d i l a t i o n i n m u s c l e , d i l a t i o n o f  b r o n c h i , m o b i l i z a t i o n o f f a t t y a c i d s and  increased  glucose metabolism.  TABLE 1 SUMMARY OF DISTRIBUTION DATA FOR MAJOR AREAS OF BRAIN  Area  Biochemical NA  DA  Histochemical D e s c r i p t i o n o f Monoamine Terminals  V/gm  Y/gm  i n t e n s i t y and type  Caudate  0.3 - 0.6  Septum  0.7 - 1.5  Hypothalamus  *  0.7  3.1 - 7.5 1.6 1.8 - 3.6  +  900 - 2400  strong d i f f u s e fluorescence of DA low to medium o f NA; d o t t e d s t r o n g i n t e n s i t y o f DA 234  low t o v e r y s t r o n g o f NA (in different nuclei)  Hippocampus  0.1 - 0.2  Thalamus  0.2 - 0.4  0.5  p r e d o m i n a n t l y low o f NA two v e r y h i g h n u c l e i  Midbrain  0.3 - 0.5  0.2  wide v a r i a t i o n depending on nuclei  Pons-Medulla Oblongata  0.1 - 0.4  0.1 - 0.3  wide v a r i a t i o n  90  Cerebellum  0.1 - 0.2  0.03- 0.1  low - m o s t l y NA  42  Cortex  0.1 - 0.3  0.1 - 0.3  summary o f d a t a from r e f e r e n c e s 2,3,4 and 5  DA  mug/gm/hr  0.2 - 0.3  0.1 - 0.2  0  NA  Amygdala  0.2  Turnover  medium o f NA low t o h i g h o f NA  33  36  +  from r e f e r e n c e 7  0  from r e f e r e n c e 9  6.  These p h y s i o l o g i c a l changes e n a b l e t h e a n i m a l t o meet many l i f e threatening s i t u a t i o n s w i t h a " f i g h t or f l i g h t " response. Throughout t h e p e r i p h e r y NA, b u t n o t a d r e n a l i n e , a c t s as a neurotransmitter  i n p o s t g a n g l i o n i c neurons o f t h e s y m p a t h e t i c  nervous system. A l t h o u g h t h e e v i d e n c e i s n o t as c o n c l u s i v e , i t i s a l s o thought t h a t NA and DA a r e n e u r o t r a n s m i t t e r s The  i n t h e CNS  (14,15).  indications f o r this r o l eare: 1)  NA and DA a r e p r e s e n t  i n b r a i n i n the appropriate  concentrations (16), 2)  enzymes f o r t h e i r f o r m a t i o n and d e s t r u c t i o n a r e  present 3)  (17,2,18,19),  agents t h a t a c t as a g o n i s t s and a n t a g o n i s t s a t  noradrenergic  synapses i n t h e p e r i p h e r y a l s o a f f e c t t h e  c e n t r a l nervous system ( 1 5 ) , 4)  r e l e a s e o f GA i s o b s e r v e d on s t i m u l a t i o n o f c e r t a i n  n e r v e t r a c t s (20,21) 5)  t h e C A s r a r e l o c a t e d i n n e r v e endings (11,12,13)and  6)  t h e r e i s some i n d i c a t i o n t h a t a p p l i c a t i o n o f CA  to synapses a f f e c t p o s t s y n a p t i c p o t e n t i a l s (22)'.  2.  Tyrosine a)  Hydroxylase  As a c o n t r o l o f CA s y n t h e s i s The  enzymes a c t i v e i n t h e b i o s y n t h e s i s o f CA, t h a t i s  tyrosine hydroxylase,  dopa d e c a r b o x y l a s e ,  dopamine  o x i d a s e and  N-methyl t r a n s f e r a s e have been s t u d i e d a g r e a t d e a l i n a d r e n a l m e d u l l a (23,24,25",26) . and dopa d e c a r b o x y l a s e  I n b r a i n , only t y r o s i n e hydroxylase  (27)  (2) have been worked on e x t e n s i v e l y , '  (N-methyl t r a n s f e r a s e i s n o t p r e s e n t studies t y r o s i n e hydroxylase  t o any e x t e n t ) .  appears t o be t h e r a t e  From t h e s e limiting  enzyme, as would be e x p e c t e d s i n c e i t i s t h e f i r s t enzyme i n t h e biosynthetic route.  The o t h e r enzymes i n t h e sequence have  a c t i v i t i e s i n t h e o r d e r o f 10,000 imomoles/gm/hr whereas t y r o s i n e hydroxylase  has an a c t i v i t y o f 4-100 mjumoles/gm/hr.  The K  m  for  7.  o v e r a l l conversion for  o f t y r o s i n e t o CAs i s 1 x 10  tyrosine hydroxylase  (28).  , the approximate  Inhibitors of tyrosine  h y d r o x y l a s e a r e more e f f e c t i v e t h a n i n h i b i t o r s o f dopamine ^ o x i d a s e and dopa d e c a r b o x y l a s e i n r e d u c i n g reduction  i n NA i s p r o p o r t i o n a l  hydroxylase i n h i b i t i o n ( 2 9 ) .  CA s y n t h e s i s ;  the  to the degree o f t y r o s i n e  From t h e s e f i n d i n g s U d e n f r i e n d (28)  proposed that  the conversion  o f t y r o s i n e t o Dopa i s t h e r a t e  l i m i t i n g step  i n CA s y n t h e s i s ,  b e c a u s e t h e amount o f enzyme  present i n the tissue i s l i m i t i n g . .According to the i n vivo ( 3 0 , 3 1 , 3 2 ) CA c o n c e n t r a t i o n s over.  Glowinski  turnover studies  studies  o f CA t u r n o v e r i n v o l v e inconsistent results.  tyrosine hydroxylase i n v i t r o  obtaining  turn-  found, f o r example, t h a t t h e c e r e b e l l u m ,  p r o c e d u r e s and g i v e of  Glowinski  do n o t n e c e s s a r i l y r e f l e c t CA  a l t h o u g h i t i s l o w i n CA, h a s o n e o f t h e h i g h e s t Such i n v i v o  of  turnover  rates.  l o n g . a n d cumbersome T h e r e f o r e measurement  a c t i v i t y may b e more h e l p f u l i n  a n a c c u r a t e p i c t u r e o f CA t u r n o v e r i n v i v o ,  b)  Characteristics Although the presence of tyrosine hydroxylase i n b r a i n  had  been e s t a b l i s h e d by i n v i v o work b e f o r e  possible  to detect  and  tyrosine concentrations  high  tyrosine of high  i n vitro  activity.  (10),  i t had n o t been  Low e n z y m a t i c  activity 14  i n t i s s u e meant t h a t  _ : . C -  i s o t o p i c e n r i c h m e n t was r e q u i r e d .  Udenfriend  ( 3 3 , 3 4 ) was t h e f i r s t  t o show a n e n z y m a t i c c o n v e r s i o n  L-tyrosine  Using p u r i f i e d beef adrenal,  t o Dopa.  f o u n d f o r maximum i n v i t r o  a c t i v i t y were acetate  i n a i r and i n t h e p r e s e n c e o f t h e c o f a c t o r ^ dimethyltetrahydropteridine  of  the conditions  b u f f e r , pH  6.0y  2-amino-4-hydroxy-6,7-  (DMPH^) i n m e r c a p t o e t h a n o l .  Work b y  U d e n f r i e n d a n d K a u f m a n ( 3 5 ) i n d i c a t e t h a t maximum a c t i v i t y o f tyrosine hydroxylase occurs i n the presence of p t e r i d i n e which apparently  increases  t h e a f f i n i t y o f t h e enzyme f o r t y r o s i n e .  The p r o p o s e d mechanism o f h y d r o x y l a t i o n enzyme b y DMPH^ e n a b l i n g to Dopa.  cofactor  involves  the reduction  t h e enzyme t o a e r o b i c a l l y o x i d i z e  of  tyrosine  8.  Work i n t h i s of  l a b o r a t o r y has confirmed  t y r o s i n e h y d r o x y l a s e i n crude beef  the characteristics  a d r e n a l homogenate.  But i n  crude b e e f , r a t , r a b b i t and c a t b r a i n homogenates, d i f f e r e n t were o b t a i n e d  (27,36).  Maximum i n v i t r o  a c t i v i t y was f o u n d  w i t h BO, b u f f e r p H 6.2, i n a i r ;  DMPH, d o e s n o t e n h a n c e 5 5 T h e K v a l u e i n b r a i n r a n g e d f r o m 0.5 x 10 t o 1 x 10 -5 a b o v e t i s s u e s a s c o m p a r e d t o 2 x 10 f o r adrenal.  t o be  activity. f o r the  m  c)  results  Location i)  Subcellular  localization  Using sucrose gradient techniques, tyrosine a c t i v i t y has been l o c a t e d i n t h e nerve distribution within established.  endings  (37).  t h e synaptosome.has n o t been  Originally  hydroxylase However t h e  precisely  t y r o s i n e h y d r o x y l a s e was t h o u g h t  t o be  a s o l u b l e enzyme i n a d r e n a l , s i n c e m o s t o f t h e a c t i v i t y was f o u n d to be i n t h e s u p e r n a t a n t  after  c e n t r i f u g a t i o n a t 105,000 g ( 3 3 ) .  More r e c e n t l y i t has been r e p o r t e d t o b e o p a r t i c l e bound  (38).  splanchnic nerve  hydroxylase  and  i t has been demonstrated  dopa d e c a r b o x y l a s e  that tyrosine  a r e i n the cytoplasms(39,40).  In  Dopamine  B o x i d a s e i s a p a r t i c l e b o u n d enzyme p o s s i b l y p a r t o f , o r w i t h i n , -la-  the v e s i c l e s  s t o r i n g NA.  I n b r a i n two s e p a r a t e g r o u p s o f w o r k e r s  ( 4 1 , 3 7 ) h a v e shown t h a t t y r o s i n e h y d r o x y l a s e i s p a r t i c l e  bound,  b u t i t s e x a c t l o c a t i o n i s n o t known. ii)  Anatomical  location  C o n s i d e r i n g g r o s s anatomy, t y r o s i n e h y d r o x y l a s e i s m o s t a c t i v e i n a d r e n a l s a n d b r a i n w i t h some i n d i c a t i o n t h a t i t i s p r e s e n t i n h e a r t and s p l e e n ( 3 4 ) .  Tyrosine hydroxylase  i n v a r i o u s b r a i n areas has n o t been t h o r o u g h l y d)  distribution  investigated.  Inhibitors Two m a i n c l a s s e s o f t y r o s i n e h y d r o x y l a s e i n h i b i t o r s  been found, According  c a t e c h o l s and a r o m a t i c amino a c i d s  to kinetic  have  (41,42,43,44).  s t u d i e s o f U d e n f r i e n d , Dopa a n d t h e c a t e c h o l s  a r e c o m p e t i t i v e i n h i b i t o r s o f DMPH^ a n d n o n - c o m p e t i t i v e  inhibitors  o f t y r o s i n e , w h i l e t h e amino a c i d s a r e c o m p e t i t i v e w i t h  tyrosine  for  s i t e s o n t h e enzyme.  T h e CAs t h e m s e l v e s  can i n h i b i t  tyrosine^!:  h y d r o x y l a s e and p o s s i b l y a c t as feedback r e g u l a t o r s of s y n t h e s i s . Other p o t e n t c a t e c h o l - t y p e i n h i b i t o r s a r e ^ - m e t h y l Dopa, e p i n i n e and m e t h y l a m i n o a c e t o c a t e c h o l .  Examples o f p o t e n t amino a c i d  i n h i b i t o r s a r e o - m e t h y l - p - t y r o s i n e , 3 - i o d o - t y r o s i n e and h a l o 1  tryptophans.  These compounds have been u s e f u l f o r s t u d y i n g t u r n -  over r a t e s o f CAs CA l e v e l s  3.  i n v i v o (32) and b e h a v i o r a l e f f e c t s of a l t e r e d  (45,46).  Dopanergic  and N o r a d r e n e r g i c Pathways i n B r a i n  F o r many y e a r s a n a t o m i s t s have used nerve d e g e n e r a t i o n r e s u l t i n g from l e s i o n s to s t u d y n e u r o n a l pathways i n the c e n t r a l nervous  system and p e r i p h e r y .  o b s e r v e d i n degenerated  B i o c h e m i c a l changes can a l s o be  axons.  C u t t i n g o f the p o s t - g a n g l i o n i c  neuron i n the s y m p a t h e t i c n e r v e r e s u l t s i n d e c r e a s e s i n NA of  i n n e r v a t e d organs  (47,48).  to  the c e n t r a l nervous  These p r i n c i p l e s can be  content  applied  system: u s i n g s t e r e o t a x i c a p p a r a t u s , p r e c i s e  e l e c t r o l y t i c l e s i o n s can be p l a c e d i n b r a i n , and  subsequent  measurement o f changes i n CA c o n c e n t r a t i o n s . can be used t o t r a c e d o p a n e r g i c and n o r a d r e n e r g i c pathways The most e x t e n s i v e mapping o f such paths has been done by a group o f Swedish  (6,7,49,50,51) workers u s i n g h i s t o c h e m i c a l  t e c h n i q u e s on r a t s .  They d e s c r i b e D A - c o n t a i n i n g neurons o r i g i n a t -  ing  i n the m i d b r a i n n u c l e i , i . e . zona compacta o f the s u b s t a n t i a  n i g r a , v e n t r o l a t e r a l p o r t i o n of the r e t i c u l a r f o r m a t i o n , and h a l f of nucleus i n t e r p e d u n c u l a r i s .  These c e l l b o d i e s send axons  c a u d a l l y t o the s t r i a t u m , t u b e r c u l u m o l f a c t o r i u m and accombens on the i p s i l a t e r a l s i d e .  t o the s p i n a l c o r d .  nucleus  The c e l l b o d i e s of NA neurons  a r e i n the pons and m e d u l l a o b l o n g a t a . f i b r e s descend  cranial  Most of the n o r a d r e n e r g i c  However, f i b r e s from c e l l s i n  the v e n t r o l a t e r a l p a r t o f the r e t i c u l a r f o r m a t i o n send axons r o s t r a l l y t h r o u g h the tegmentum o f the m i d b r a i n and m e d i a l f o r e b r a i n bundle  (MFB)  t o the hypothalamus,  pre-optic area, septal area,  amygdala, hippocampus and c i n g u l a t e g y r u s . e x p e r i m e n t s are summarized i n -.Fig. 2.  The r e s u l t s of these  10.  U s i n g b i o c h e m i c a l t e c h n i q u e s o t h e r workers have c o n f i r m e d some o f t h e s e r e s u l t s .  Poirier  and S o u r k e s  (53,54)  using  a n d m o n k e y s , showed d e c r e a s e s i n DA o f t h e s t r i a t u m w i t h and c h r o m a t o l y s i s o f n e r v e c e l l parabracheolis pigmentosis. w i t h MAO  inhibitors.  nigrostriatal  (54,55,56)  a l s o w o r k i n g on  within  concentrations,  C - t y r o s i n e and d e c r e a s e s i n  i n the i p s i l a t e r a l  (57,58,59)  l e s i o n s o f t h e MFB  from  the  t h a t l e s i o n s of the v e n t r o m e d i a l  t e g m e n t u m o f t h e m i d b r a i n c a u s e d d e c r e a s e s i n DA 14  Moore and H e l l e r  and  These d e c r e a s e s c o u l d n o t be r e v e r s e d  t r a c t , demonstrated  u p t a k e o f r a d i o a c t i v e DA  lesions  bodies i n the s u b s t a n t i a n i g r a  Goldstein  d e c r e a s e i n s y n t h e s i s o f DA  cats  c a u d a t e and  putamen.  h a v e r e p o r t e d d e c r e a s e s i n NA  the l a t e r a l hypothalamus;  d e c r e a s e i n c l u d e the septum, s t r i a t u m , amygdala  areas  and  with showing  hippocampus.  . A n a t o m i c a l s t u d i e s have been g e n e r a l l y u n s u c c e s s f u l i n demonstrating these CA-containing tracts the  substantia nigra indicate efferents  colliculi,  thalamus  ascending fibres  the  internal capsule. i n t h e MFB  He  (63) c l a i m s t h e r e  (64) and o t h e r s (65,66)  to the amygdala,  also  describe  thalamus, s e p t a l n u c l e i ,  diagonal  hypothalamus.  P o s s i b l e Role of Catecholamines i n B r a i n F u n c t i o n S i n c e t h e CAs  c e n t r a l nervous  r e l a t e CA a n d b r a i n a)  Some w o r k h a s b e e n d o n e i n t r y i n g  the f i r s t  term emotion  t h a l a m u s , c i n g u l a t e g y r u s and  T h i s c i r c u i t i s now (14).  to put the nebulous  <  " c o n c r e t e " by d e s c r i b i n g an " e m o t i o n a l c i r c u i t , "  c o n s i s t i n g of hypothalamus,  system  to  Behavior  P a p e z ( 6 7 ) was  campus.  specific  function,  E m o t i o n and  something  are probably n e u r o t r a n s m i t t e r s i n the  s y s t e m ; i t w o u l d be a s s u m e d t h e y i n f l u e n c e  f u n c t i o n s i n the b r a i n .  into  Nauta  f r o m t h e m i d b r a i n t o c a u d a t e and putamen v i a  band n u c l e i , hippocampus and 4.  Lesions of  to the r e d n u c l e u s , s u p e r i o r  and g l o b u s p a l l i d u s .  are  fibers  (60,61,62).  The  hippo-  thought t o i n v o l v e most of the  hippocampus and amygdala  appear  t o be t h e  t u r n o v e r p o i n t s where a s t i m u l u s i s transduced i n t o a p r e c i s e  limbic central somatic  11.'  PATHWAYS DOPAMINE  i  NORADRENALINE  I I F i g . 2:  Summary o f n o r a d r e n e r g i c and d o p a m i n e r g i c tracts i n brain LF = l i m b i c f o r e b r a i n  MFB = m e d i a l f o r e b r a i n b u n d l e  ST = s t r i a t u m  ME = mesencephalon  TH = thalamus  MO = m e d u l l a o b l o n g a t a  HY =» hypothalamus From r e f e r e n c e 15  12.  e m o t i o n a l r e s p o n s e s w h i c h i s e x p r e s s e d v i a the o t h e r components o f the c i r c u i t .  The s e p t a l a r e a may f u n c t i o n as a type o f a c t i v a t i n g  system f o r the hippocampus.  I t has f u r t h e r been suggested  that  the hippocampus-amygdala complex i s s u s c e p t i b l e t o breakdown. r e s u l t o f such a d i s r u p t i o n c o u l d be m e n t a l i l l n e s s . i n d i c a t e d , t h e s e areas a r e r e l a t i v e l y h i g h i n CAs. hypothesized  The  As a l r e a d y I t has been  t h a t they may p l a y some r o l e as n e u r o t r a n s m i t t e r s i n  the "emotion c i r c u i t . "  To s u b s t a n t i a t e t h i s t h e o r y i t has been  found t h a t CA a g o n i s t s and a n t a g o n i s t s of mental i l l n e s s .  c a n be used i n t h e t r e a t m e n t  I n g e n e r a l , drugs t h a t a n t a g o n i z e CA a c t i v i t y  a r e mood d e p r e s s a n t s and those t h a t a c t as a g o n i s t s a r e mood elevators  (15).  The h y p o t h e s i s  caused by a c t i v a t i o n o r d e p r e s s i o n Since  (15,68) i s t h a t mood changes a r e o f synapses i n t h e l i m b i c l o b e .  t h e method o f mood development i s n o t known, and these agents  can have s e v e r a l e f f e c t s on CA and p o s s i b l y o t h e r  systems, the e x a c t  mechanism o f a c t i o n o f t h e s e drugs i n chaig.ng mood i s o b s c u r e . Numerous a n i m a l experiments have been done i n an a t t e m p t to r e l a t e emotion, b e h a v i o r and CA. 1)  Examples o f these a r e :  s t i m u l a t i o n o f t h e amygdala i n c a t s produces sham  r a g e a l o n g w i t h a d e c r e a s e i n NA i n t h e b r a i n 2)  b r a i n s t e m t r a n s e c t i o n s t h a t evoke d e f e n s e r e a c t i o n s  cause d e c r e a s e i n NA i n b r a i n 3)  (69),  (70),  s e l f s t i m u l a t i o n of pleasure  c e n t r e s o f t h e hypo-  thalamus i s i n h i b i t e d by d e c r e a s i n g  NA c o n c e n t r a t i o n and  enhanced by the NA a g o n i s t , metanephrine, s u g g e s t i n g NA may be t h e n e u r o t r a n s m i t t e r  i n t h i s pleasure  that  system  ( 4 6 ) , and 4)  a n i m a l s i n groups ( s o c i a l s t r e s s ) have more CA and  s e r o t o n i n i n t h e b r a i n , tend t o be l e s s a g g r e s s i v e and responsive The  suggestion  the r e c e p t o r s  t o s t i m u l i than do i s o l a t e d a n i m a l s (71,72,73).  i s t h a t i n t h e grouped a n i m a l s w i t h excess t r a n s m i t t e r s a r e d e s e n s i t i z e d and t h e r e f o r e l e s s r e s p o n s i v e .  c o u l d be a s o r t o f e m o t i o n a l s a f e t y mechanism. studies  Other s i m i l a r  (74,75,45) have been done b u t no c o n c l u s i v e r e s u l t s  r e l a t i n g CA, b e h a v i o r and emotion have been  obtained.  This  13.  b)  Memory and  Learning  As w e l l as b e i n g p a r t of the "emotion c i r c u i t " hippocampus i s a l s o i m p o r t a n t present  i n memory ( 7 7 ) .  the  Whether the  CAs:,  t h e r e or i n o t h e r r e g i o n s of the b r a i n a r e s i g n i f i c a n t i n  memory i s n o t known.  Wada and McGeer (78) showed t h a t an  increase  i n DA and NA enhanced l e a r n i n g w h i l e low l e v e l s i n h i b i t e d l e a r n i n g . Others (79) have r e p o r t e d serotonin concentrations  t h a t d e c r e a s e s i n c e r e b r a l CA enhanced l e a r n i n g .  W i t h r e s p e c t to the  a l r e a d y l e a r n e d r e s p o n s e , i t has been r e p o r t e d a n t a g o n i s t s of NA a c t i v i t y b l o c k e d c)  and  (80,81,82) t h a t  conditioned behavior  response.  Sleep .Sleep i s a p o o r l y u n d e r s t o o d phenomenon i n w h i c h CA  p l a y an i m p o r t a n t  role.  may  .During a s l e e p p e r i o d a n i m a l s a l t e r n a t e  between l i g h t s l e e p and p a r a d o x i c a l s l e e p .  J o u v e t (83,84) has  proposed t h a t NA from the n u c l e i l o c u s c a e r u l e u s  i n the tegmentum  of the pons a c t s as m e d i a t o r i n the i n i t i a t i o n of p a r a d o x i c a l Narcolepsy,  sleep.  a p a t h o l o g i c a l c o n d i t i o n w h i c h causes a sudden o n s e t  of s l e e p , can be c o n t r o l l e d by the NA a g o n i s t , amphetamine (85)j. and NA a p p l i e d to the r e t i c u l a r f o r m a t i o n can cause an r e s p o n s e (86) . may  These l a t t e r o b s e r v a t i o n s would i n d i c a t e t h e i r  be a r e l a t i o n s h i p between CA and d)  arousal  sleep.  Temperature R e g u l a t i o n By l e s i o n and s t i m u l a t i o n s t u d i e s i t has been e s t a b l i s h e d  t h a t the hypothalamus i s p r o m i n e n t l y control.  concerned i n body temperature  F e l d b u r y and Meyer (87) and o t h e r s  (88) have p o s t u l a t e d  t h a t temperature r e g u l a t i o n takes p l a c e by a b a l a n c e of the h y p e r thermic  e f f e c t s of s e r o t o n i n and o f NA.  I t has a l s o been f a i r l y  w e l l e s t a b l i s h e d by Leduc (89) t h a t the CA, whose e x c r e t i o n i s i n c r e a s e d when a n i m a l s are p l a c e d i n the c o l d , a r e i m p o r t a n t adaption  to c o l d .  in  I t has not been determined i f t h i s CA i n f l u e n c e  on c o l d a c c l i m i t i z a t i o n i s p e r i p h e r a l o n l y or whether t h e r e i s a l s o a CNS  component. e)  B a s a l G a n g l i a and  Parkinsonism  Through s t u d i e s of DA m e t a b o l i s m and d o p a n e r g i c pathways the  14.  f u n c t i o n and mechanism o f a c t i o n of the b a s a l g a n g l i a a r e elucidated.  I n p a t i e n t s s u f f e r i n g from Parkinsonism,  a  being disease  a p p a r e n t l y caused by m a l f u n c t i o n o f the b a s a l g a n g l i a and some of i t s connections  (e.g. s u b s t a n t i a n i g r a ) , u r i n a r y s e c r e t i o n s of  and i t s m e t a b o l i t e s  are decreased (90).  A f t e r d e a t h a n a l y s i s of  the b a s a l g a n g l i a of these p a t i e n t s show a d e c r e a s e i n DA (91).  DA  content  Agents such as r e s e r p i n e t h a t (among o t h e r a c t i o n s )  DA f r o m the b a s a l g a n g l i a cause P a r k i n s o n i s m  deplete  - l i k e symptoms ( 9 2 ) ,  and a d m i n i s t r a t i o n of l a r g e doses of Dopa have been r e p o r t e d  to  a l l e v i a t e the symptoms of P a r k i n s o n i s m  Lesions  i n some cases ( 9 3 ) .  o f the n i g r o s t r i a t a l t r a c t i n monkeys i n some cases can produce tremor and r i g i d i t y ( 9 4 ) .  From t h e r e and o t h e r  f i n d i n g s i t has  been suggested t h a t DA a c t s as an i n h i b i t o r y n e u r o t r a n s m i t t e r the b a s a l g a n g l i a and  that Parkinsonism  may  i n a d e q u a t e i n h i b i t i o n i n the e x t r a - p y r a m i d a l r e s u l t of l a c k of n e u r o t r a n s m i t t e r I n a l l the a s p e c t s  DA  i n p a r t be a r e s u l t of motor system as a  (95,96).  of b r a i n f u n c t i o n mentioned t h e r e  h i n t s o f r e l a t i o n s h i p s between CA and b r a i n mechanisms, but d e f i n i t e working hypothesis  in  can be e s t a b l i s h e d .  are  no  Much more work  must be done on the CA i n b r a i n .  5.  Aims o f The  aspects  Thesis purpose of t h i s i n v e s t i g a t i o n i s to s t u d y  several  of the s i g n i f i c a n t enzyme i n CA s y n t h e s i s , t y r o s i n e  hydroxylase. 1)  There w i l l be t h r e e main c o n s i d e r a t i o n s : The  e f f e c t s on t y r o s i n e h y d r o x y l a s e  activity i n brain  o f a l t e r i n g CA l e v e l s i n the body w i l l be d e t e r m i n e d . These a l t e r a t i o n s a r e b r o u g h t about by p l a c i n g the a n i m a l i n the c o l d ) and by a r t i f i c i a l l y chang.ng the l e v e l s w i t h drugs (substances  t h a t d e p l e t e CA s t o r e s and  i n h i b i t o r s t h a t i n c r e a s e CA 2)  MAO  concentrations).  An e x t e n s i v e s t u d y w i l l be made of t y r o s i n e  a c t i v i t y i n v a r i o u s r e g i o n s of b r a i n i n s e v e r a l 3)  On  hydroxylase animals.  the b a s i s of, t h e s e normal d i s t r i b u t i o n s t u d i e s , and  15.  the f a c t t h a t t y r o s i n e h y d r o x y l a s e i s c o n t a i n e d i n nerve e n d i n g s , the t h i r d aim o f t h i s s t u d y w i l be the i n v e s t i g a t i o n o f the anatomy o f the n o r a d r e n e r g i c and d o p a n e r g i c pathways i n b r a i n . Le s i o n t e c h n i q u e s c o u p l e d w i t h enzyme measurements w i l l be used.  16.  METHODS AND MATERIALS a)  Tyrosine Hydroxylase A n a l y s i s i)  Procedure  T i s s u e was homogenized i n 4 - 9 volumes o f s u c r o s e .  The  i n c u b a t i o n m i x t u r e c o n s i s t e d o f 0.1 ml 0.28 M PO^ b u f f e r pH 6.2, 0.1'ml o f homogenate, and 0.1 ml o f a s o l u t i o n o f u n i f o r m l y 14 l a b e l l e d L - t y r o s i n e - C (150,000 cpm, sp. a c t . 360-375 mc/mmoles) -3 w h i c h was 3 x 10 M i n N-methyl-N-3-hydroxyphenylhydrazine (NSD-1034) i n d i s t i l l e d water. When DMPH. was used, 0.07 ml o f 0.40 M PO. -4 b u f f e r and 0.03 ml o f 2 x 10 DMPH^ i n 0.02M 2-mercaptoethanol 4  4  were added i n s t e a d of t h e 0.1 ml o f 0.28M b u f f e r .  The i n c u b a t i o n  was  c a r r i e d o u t i n a i r a t 37°C f o r 30 minutes and stopped w i t h  the  a d d i t i o n o f 2 ml o f a 1:1 m i x t u r e o f 0.2M HAc and 0.4M HC10, 4  c o n t a i n i n g 0.1 ug/ml of Dopa, NA and DA.  B l a n k s were r u n a t the  same time u s i n g t i s s u e t h a t had been heated a t 80 - 90°C f o r 10 - 15 minutes.  D u p l i c a t e s were r u n f o r each sample.  I n most cases the  a c i d i f i e d i n c u b a t e s were f r o z e n b e f o r e i s o l a t i o n o f the c a t e c h o l s . I s o l a t i o n o f the r a d i o a c t i v e c a t e c h o l s formed was on aluminum oxide.  The i n c u b a t i o n m i x t u r e was thawed, c e n t r i f u g e d and the  s u p e r n a t a n t poured i n t o a 20 ml beaker c o n t a i n i n g 1 ml o f 0.2M EDTA. The p r e c i p i t a t e was washed w i t h 3 ml o f 0.32M PO^, r e c e n t r i f u g e d and t h e s u p e r n a t a n t p o o l e d w i t h the o t h e r .  Each sample was t a k e n  to pH 8.8 - 9.2 w i t h NaOH and 300 mg o f alumina was added t o the beaker.  The m i x t u r e was s t i r r e d f o r 4 - 5  minutes and then washed  i n t o a s m a l l g l a s s column (4:mm d i a m e t e r ) s t o p p e r e d w i t h g l a s s w o o l . G e n t l e s u c t i o n was used t o draw the l i q u i d through the column. column was f u r t h e r washed w i t h 2 l o t s of water  The  ( c a 10 ml e a c h ) .  The c a t e c h o l s were e l u t e d from the column w i t h 2 ml o f 0.5N HAc i n t o a small v i a l .  Ten ml o f B r a y ' s s o l u t i o n were added and the v i a l s  were counted i n a l i q u i d - s c i n t i l l a t i o n s p e c t r o p h o t o m e t e r .  The  whole i s o l a t i o n procedure took a p p r o x i m a t e l y 10 minutes per sample. ii)  R a t i o n a l f o r Procedure  C o n d i t i o n s f o r i n c u b a t i o n and i s o l a t i o n had p r e v i o u s l y been worked o u t (27) and diagrams p r e s e n t e d i n t h i s t h e s i s a r e c o n f i r m a t i o n  17.  of these. The volumes o f s u c r o s e were chosen to g i v e 10 - 20 mg o f t i s s u e per i n c u b a t i o n .  The amount used i n any g i v e n i n c u b a t i o n depended  on t h e amount o f t i s s u e a v a i l a b l e and t h e a c t i v i t y o f t h e t i s s u e . As shown i n F i g . 3 t h e a c t i v i t y i s l i n e a r w i t h mg o f t i s s u e used i n t h i s range.  U s i n g s u c r o s e o f m o l a r i t y 0.2 - 0.3 i t has been shown  (27) t h a t 0.28 g i v e s the b e s t c o n v e r s i o n .  As shown i n F i g . 4 t h e  maximum pH f o r t h e r e a c t i o n i s 6.0 - 6.4.  Therefore the intermediate  pH 6.2 was chosen.  As p r e v i o u s l y demonstrated  gave maximal r e s u l t s f o r b r a i n t i s s u e .  (27) PO, b u f f e r 14  The L - t y r o s i n e -  C from  the m a n u f a c t u r e r was d i l u t e d so t h a t each i n c u b a t i o n m i x t u r e cont a i n e d 1/12^uc (150,000 cpm).  F o r whole r a t b r a i n t h i s r e s u l t e d  i n a p p r o x i m a t e l y 2000 cpm o f c a t e c h o l s formed w i t h a b l a n k o f 150 - 200 cpm.  T h e r e f o r e , t h i s amount appeared  t o be a good  compromise between r e a s o n a b l e c o n v e r s i o n , low b l a n k and c o s t . 1034 i s a p o t e n t Dopa d e c a r b o x y l a s e i n h i b i t o r r e a c t i o n i s a t t h e Dopa s t a g e .  NSD-  (97) , and b l o c k s t h e  I f NA and DA were formed they would  f u r t h e r be m e t a b o l i z e d b y 0-methyl t r a n s f e r a s e and monoamine o x i d a s e . T h i s would make i s o l a t i o n more d i f f i c u l t . b e f o r e i s o l a t i o n improves  the recovery.  time up t o 45mihutes ( F i g . 5 ) .  Addition of cold catechols Incubation i s linear with  T h i r t y minutes was chosen f o r con-  venience . I t has been r e p o r t e d t h a t DMPH^ i s n e c e s s a r y f o r maximum t y r o s i n e h y d r o x y l a s e a c t i v i t y i n p u r i f i e d beef a d r e n a l ( 2 3 ) . c a r r i e d o u t w i t h and w i t h o u t  Incubations  DMPH^ a r e ...presented i n T a b l e 2.  There was no s i g n i f i c a n t d i f f e r e n c e i n t y r o s i n e h y d r o x y l a s e a c t i v i t y o f b r a i n homogenates w i t h and w i t h o u t c o f a c t o r .  There was a 10 f o l d  i n c r e a s e i n a c t i v i t y w i t h c a t a d r e n a l s , i n d i c a t i n g t h a t t h e DMPH^ had n o t been decomposed.  Because o f t h e s e r e s u l t s t h e c o f a c t o r was  n o t used r o u t i n e l y i n t h e b r a i n t y r o s i n e h y d r o x y l a s e homogenates. Oxygen i s n e c e s s a r y f o r t h e r e a c t i o n b u t 20% s a t u r a t e s the enzyme (23).  The i n c u b a t i o n s a r e t h e r e f o r e c a r r i e d o u t i n a i r . Alumina has proven t o be one o f the most s a t i s f a c t o r y and s i m p l e s t  methods f o r i s o l a t i o n c a t e c h o l s ( 9 8 ) .  Uptake on t h e colunn i s  18.  I  ,  ;  5  0  3:  8  10 MG.  Fig.  i  •  I  15  i  20  TISSUE/INCUBATION  R e l a t i o n s h i p between amount of b r a i n t i s s u e used i n i n c u b a t i o n and t y r o s i n e h y d r o x y l a s e  F i g . 4:  activity  E f f e c t o f pH of i n c u b a t i o n m i x t u r e on the a c t i v i t y of t y r o s i n e h y d r o x y l a s e i n b r a i n  < LU  _  ©J  ~3Q  —  40  T I M E (MIN.) '• F i g . 5:  A c t i v i t y of T y r o s i n e h y d r o x y l a s e w i t h r e s p e c t to  ;  time of i n c u b a t i o n  70-  _  LU  >  o o  65-  LU _  60*  "S-5: F i g . 6:  9.0  9^5  10©  PH R e c o v e r y of c a t e c h o l s from' alumina a t v a r i o u s pH's o f the  J  sample as i t i s p l a c e d on the column  „  —  50  60  TABLE 2.  EFFECT OF DMPH. ON TYROSINE HYDROXYLASE ACTIVITY 4 IN CAT, RABBIT AND RAT BRAIN  A c t i v i t y w i t h DMPH^ as Percent o f A c t i v i t y Without  11 Areas o f R a b b i t B r a i n 18 Areas o f C a t B r a i n 36 R a t B r a i n s Cat A d r e n a l M e d u l l a  109% - 17 92% - 21 104% 1030%  TABLE 3  EFFECT OF PHOSPHATE ADDITION ON SIZE OF BLANK AND DOPA RECOVERY FROM ALUMINA COLUMN  cpm M l o f Phosphate B l a n k (150,000 cpm o f 14^ . T y r o s i n e - C) Dopa (6200 cpm)  0  1  3  5  1343  606  257  249  4136  4000  4210  4020  22.  s p e c i f i c f o r the c a t e c h o l m o i e t y a t a l k a l i n e pH's. added t o p r e v e n t o x i d a t i o n by heavy m e t a l s .  EDTA i s  Catechols are  p a r t i c u l a r l y s u s c e p t i b l e to d e s t r u c t i o n a t a l k a l i n e pH. f o r maximum r e c o v e r y i s 9.0 because i t was  as shown i n F i g . 6.  PO^  The  pH  i s added  found to p r e v e n t the uptake of i m p u r i t i e s i n the  o r i g i n a l t y r o s i n e s o l u t i o n w h i c h can g i v e i n a v e r y l a r g e b l a n k as shown i n T a b l e 3.  The phosphate does n o t i n t e r f e r e w i t h the Dopa  r e c o v e r y w h i c h i s 65 - 70%.  The samples c o n s i s t e n t l y counted a t  75% e f f i c i e n c y i n the l i q u i d - s c i n t i l l a t i o n b)  spectrophotometer.  Tyrosine Analysis For the c a l c u l a t i o n o f V  f o r tyrosine hydroxylase i t max  i s n e c e s s a r y t o know the endogenous t y r o s i n e l e v e l s .  Incubations  a r e r u n below s a t u r a t i o n and endogenous l e v e l s a r e h i g h enough t o a f f e c t the v a l u e . i)  Procedure  T y r o s i n e was  determined by a m o d i f i c a t i o n o f the  of Waalkes and U d e n f r i e n d ( 9 9 ) .  0.2 ml o f the s u c r o s e homogenate  were added to 0.2 ml of 30% TCA and 0.6 ml o f H 0  ( i f sufficient  2  t i s s u e was  a v a i l a b l e , the q u a n t i t i e s were doubled to g i v e more  s u p e r n a t a n t to work w i t h ) . was  procedure  A f t e r c e n t r i g u g a t i o n the s u p e r n a t a n t  poured o f f and u s u a l l y f r o z e n b e f o r e a n a l y s i s .  The a n a l y s a t e  m i x t u r e c o n s i s t e d of 0.6 ml s u p e r n a t a n t , 0.6 ml o f 0.1%  nitroso-2-  n a p h t h o l i n 95%, e t h a n o l and 0.6 ml o f a m i x t u r e o f 24.5 ml o f n i t r i c a c i d and 0.5 ml of 2.5% NaNO^m i x t u r e was  A f t e r thorough m i x i n g the  p l a c e d i n a water b a t h a t 55°C f o r 30 m i n u t e s .  u n r e a c t e d l - n i t r o s o - 2 - n a p h t h o l was dichloride.  The aqueous l a y e r was  1:5  The  e x t r a c t e d w i t h 2.5 ml o f e t h y l e n e placed i n a small clear  test  tube and the f l u o r e s c e n c e determined i n a s p e c t r o p h o t o f l U o r o m e t e r a t 406 wp. a c t i v a t i o n and 570 m/as f l u o r e s c e n c e .  A sucrose-TCA  b l a n k and t Y / m l s t a n d a r d s o l u t i o n were a n a l y z e d a t the same t i m e . | ii)  Rational for Analysis:  T y r o s i n e has been shown to c o n j u g a t e w i t h l - n i t r o s o - 2 n a p h t h o l under the c o n d i t i o n s i n d i c a t e d (99) to form a f l u o r e s c e n t compound.  F l u o r e s c e n c e i s p r o p o r t i o n a l to c o n c e n t r a t i o n over the  range n o r m a l l y used ( F i g . 7 ) .  Samples u s u a l l y gave r e a d i n g s o f  0  T!o™  1.5  _!o~  CONCENTRATION  8ml.)  0.5  F i g . 7:  —i  Standard c u r v e f o r t y r o s i n e determination, fluorescence versus concentration  70-  8.5  9.0  mO  9.5 PH  F i g . 10: Recovery of NA and DA  from  alumina w i t h r e s p e c t to pH of sample as i t i s p l a c e d i n the column  • . '  10.5  24.  0.06 - 0.15 f l u o r e s c e n t u n i t s and the b l a n k was a p p r o x i m a t e l y 0.02. c)  C a l c u l a t i o n of V max To c a l c u l a t e ^ f ° t y r o s i n e h y d r o x y l a s e i t was n e c e s s a r y to d e t e r m i n e d the K v a l u e . K d e t e r m i n a t i o n s were c a r r i e d o u t m m r  m a x  u s i n g whole r a t b r a i n , c a t and r a b b i t and c a t and r a b b i t caudate. I n c u b a t i o n s were c a r r i e d o u t as p r e v i o u s l y d e s c r i b e d  except that  i n c r e a s i n g amounts o f c o l d t y r o s i n e were added i n the phosphate buffer.  P l o t s were done a c c o r d i n g  to the s t a n d a r d i / V vs I/S  method and by t h e S/V vs S method as recommended by Dowd and R i g g s (108).  Fig. 8 i s a representative  rat brain.t average K  p l o t f o r tyrosine hydroxylase i n  Many d e t e r m i n a t i o n s i n t h i s l a b o r a t o r y showed t h e f o r r a t b r a i n to be 0.45 x 10 ^ ( 2 7 ) .  m  r a b b i t b r a i n the K 1 x 10  F o r c a t and  f o r t y r o s i n e hydroxylase i s of the order of  Sample p l o t s a r e shown i n F i g . 9.  These were the  v a l u e s used i n c a l c u l a t i o n o f V max The V i n mu moles o f Dopa formed/gm o f t i s s u e / h r max ° using the Michaelis-Menten formula: r  V  =  v (1 +  |P)  max K  - i n m o l e s / l was d e t e r m i n e d e x p e r i m e n t a l l y f o r each a n i m a l as d i s c u s s e d  c-.. - t o t a l c o n c e n t r a t i o n incubate.  above.  o f t y r o s i n e i n moles/1 o f  T h i s i n c l u d e s r a d i o a c t i v e and endo-  genous t y r o s i n e .  v  - mnmoles o f Dopa formed/gm/hr. mined from t h e p r o p o r t i o n  This i s deter-  of r a d i o a c t i v e  tyrosine  c o n v e r t e d to Dopa w i t h c o r r e c t i o n s f o r the nonl a b e l l e d tyrosine present.  S i n c e t h e i n c u b a t i o n volume, amount and a c t i v i t y o f t h e r a d i o a c t i v e t y r o s i n e used, column r e c o v e r y , c o u n t i n g e f f i c i e n c y and time o f i n c u b a t i o n were a l l k e p t c o n s t a n t , a f o r m u l a c o u l d  be  25.  derived r e l a t i n g V . o n l y , to the amount o f t i s s u e used, the ° max t y r o s i n e c o n c e n t r a t i o n i n t h a t t i s s u e , the K f o r the r e a c t i o n and m the measured cpm. J  J  v  max K = m K  .  d)  =  (  5 4  - y  I  3  4 6  -4  )+ T  1.3 x 10  B  x  cpm  ''  To"  6  S  =  mg o f t i s s u e / i n c u b a t i o n  T  -  endogenous t y r o s i n e c o n c e n t r a t i o n i n j i g / g m  N o r a d r e n a l i n e and Dopamine D e t e r m i n a t i o n s i)  Isolation  T i s s u e was  homogenized i n a minimum volume of a  1:1  m i x t u r e o f 0.4N  HC10. and 0.2N HAc. I f the t i s s u e had been 4 p r e v i o u s l y homogenized i n s u c r o s e f o r enzyme i n c u b a t i o n s the volume was noted and 0.2 ml o f c o n c e n t r a t e d HC10. was added. 4  The  samples were c o o l e d f o r 10 minutes,. ..'centrifuged f o r 10 m i n u t e s , and the s u p e r n a t a n t was of EDTA.  poured i n t o a t e s t tube c o n t a i n i n g 0.5  The p r e c i p i t a t e was  ml  t a k e n up i n 2 ml o f HCIO^: HAc  m i x t u r e and the procedure r e p e a t e d .  A t t h i s p o i n t the samples  were u s u a l l y f r o z e n u n t i l i s o l a t i o n on a l u m i n a , as p r e v i o u s l y d e s c r i b e d ^ e x c e p t the m i x t u r e was a b s o r p t i o n on to the column. i n a graduated t e s t tube. added and the pH was  taken to pH 9.0  - 9.5  The e l u a n t o f 0.5N 0.5 ml o f 1.0M  HAc was  Water was  Standards of c o l d and  c a t e c h o l a m i n e s were r u n a t the same time t o determine  was added  radioactive  percent  recovery. ii)  NA A n a l y s i s  F o r each sample 3 s m a l l tubes were used: 1.  sample  2.  i n t e r n a l standard  3.  blank  To each tube 0.5 ml o f sample was were b u f f e r e d w i t h 0.5 ml of 0.5M  added.  NaAc pH 6.4,  —  collected  Ac b u f f e r pH 6.0  a d j u s t e d t o 6.0 w i t h NaOH.  to make the f i n a l volume 3.0 m l .  before  Tubes 1 and 3  and tube 2 w i t h  26.  0.5 ml o f b u f f e r c o n t a i n i n g 0.1 ug o f NA. 0.01N  To a l l tubes 0.5 ml o f  i o d i n e was added f o l l o w e d i n 4 minutes by 0.25 ml o f 0.05N  sodium t h i o s u l p h a t e s o l u t i o n .  Tubes 1 and 2 were t r e a t e d w i t h  0.5 m l o f a m i x t u r e o f 7 ml o f 5N NaOH and 0.5 ml of a c i d , tube 3 w i t h 0.35 ml NaOH. l i g h t f o r 90 m i n u t e s .  0.57o  ascorbic  The tubes were l e f t s t a n d i n g i n  B e f o r e r e a d i n g s were t a k e n , i n the s p e c t r o -  p h o t o f l u o r o m e t e r , 0.15 ml of 0.5% a s c o r b i c a c i d s o l u t i o n was to  the b l a n k .  peak 505  added  The a c t i v a t i o n peak was 395 mp and the f l u o r e s c e n t  mu. i i i ) DA a n a l y s i s A l l r e a g e n t s must be a t room t e m p e r a t u r e .  Two  large  test  tubes were used f o r each sample. 1.  sample  2.  i n t e r n a l standard  Two of  sample.  e l u a n t b l a n k s were r u n a t the same time f o r each group To each tube 0.5 ml o f sample was added t o tube 1.  0.5 ml o f 1.0M  A C b u f f e r pH 6.0 was added, and to tube 2 0.5 ml o f  b u f f e r c o n t a i n i n g 0.1 p.g!;DA. of  0.01N  Tubes were then t r e a t e d w i t h 0.25  ml  i o d i n e f o l l o w e d i n 10 minutes by 0.25 ml o f a 4.5N NaOH  s o l u t i o n c o n t a i n i n g 25 mg/ml o f anhydrous Na^S^O^. l a t e r 0.5 ml o f 5N HCl was added t o each.  Three minutes  The tubes were l e f t  to s t a n d i n l i g h t f o r 12 -.24 hours and then r e a d i n the s p e c t r o p h o t o f l u o r o m e t e r a t a c t i v a t i o n peak 330 mju and f l u o r e s c e n t peak 380. iv)  R a t i o n a l f o r Catecholamine D e t e r m i n a t i o n  DA and NA were t a k e n up on a l u m i n a as p r e v i o u s l y d e s c r i b e d for  Dopa.  However as shown i n F i g .  be maximum between pH 9.0 (60 For  807c)  - 9.5.  10, the r e c o v e r y was found to  The r e c o v e r y was more v a r i a b l e  and c o l d s t a n d a r d s were t h e r e f o r e r u n w i t h each group.  v e r y p r e c i s e work i n t e r n a l r a d i o a c t i v e c a t e c h o l a m i n e s t a n d a r d s  were used t o d e t e r m i n e the per c e n t r e c o v e r y f o r each sample. The f l u o r i m e t r i c a n a l y s i s i s based on the o x i d a t i o n o f c a t e c h o l a m i n e s and t h e i r re-arrangement i n a l k a l i n e to form f l u o r e s c e n t compounds ( 1 0 0 ) . reaction i s :  The e q u a t i o n f o r the n o r a d r e n a l i n e  m  0 F i g . 9:  F i g . 8:  2.0  T.O K  K  S  m  m  *10  3.0  4.0  5.0  s  d e t e r m i n a t i o n f o r r a b b i t (x) and c a t (.)  determination for r a t brain  brain  28.  Because the f l u o r e s c e n t compound i s u n s t a b l e i n a l k a l i , a s c o r b i c a c i d i s added. The  Dopamine undergoes a s i m i l a r r e a c t i o n .  pH f o r DA a n a l y s i s i s lowered w i t h HCI  l e n g t h o f a c t i v a t i o n and f l u o r e s c e n c e and  to d e c r e a s e the wave-  ( t o d i s t i n g u i s h i t from  NA)  to i n t e n s i f y the f l u o r e s c e n c e ( 6 ) . The  optimum c o n d i t i o n f o r these r e a c t i o n s had p r e v i o u s l y  been worked out i n t h i s l a b o r a t o r y (101).  From F i g . 11 i t can  be seen t h a t t h e r e i s a l i n e a r r e l a t i o n s h i p between c o n c e n t r a t i o n of catecholamine  and f l u o r e s c e n c e .  because v e r y s m a l l q u a n t i t i e s ^ 0 . 1  T h i s method i s v e r y u s e f u l V c a n e a s i l y be d e t e c t e d  and  measured a c c u r a t e l y . e)  S e p a r a t i o n o f N o r a d r e n a l i n e , Dopamine and Some of T h e i r Metabolites Experiments were c a r r i e d out to d e v e l o p methods of  s e p a r a t i n g n o r a d r e n a l i n e , dopamine and  their metabolites.  Two  methods were t r i e d : 1.  i o n exchange chromatography as o u t l i n e d i n s e v e r a l  references 2.  (102,103,104),  t h i n l a y e r chromatography as suggested by Randeruth  (105). i)  i o n exchange ."chromatography - s e p a r a t i o n was  out on Dowex 50 x 8, 200  - 400 mesh, w h i c h was  carried  k e p t under  Fig.  11:  S t a n d a r d c u r v e f o r NA and DA d e t e r m i n a t i o n s . Fluorescence verus c o n c e n t r a t i o n  30.  2N HCI p r i o r t o use.  .A s m a l l g l a s s column, 4 mm  i n diameter was plugged w i t h g l a s s w o o l .  The Dowex  i n 2N HCI was added so t h a t t h e f i n a l h e i g h t o f r e s i n was 5 cm. until  The r e s i n was washed w i t h d i s t i l l e d water  the washings were n e u t r a l ( w i t h pH paper) and  then 10 - 15 mis o f 1M NaAc pH 6.0 were passed through. The amines (NA, DA and t h e i r O-methyl d e r i v a t i v e s ) were d i s s o l v e d i n a p p r o x i m a t e l y 2 ml 0.1N HCI f o r a p p l i c a t i o n t o the column. U s u a l l y 25 V of each were used p l u s v a r i o u s amounts o f r a d i o a c t i v e 14 C of  DA and NA.  The r e s i n was washed w i t h 10 mis o f water and 10 mis  0.1N HCI. E l u t i o n was c a r r i e d o u t w i t h 0.4N HCI a t a r a t e o f 0.2 -  0.3 mls/min.  One ml f r a c t i o n s were c o l l e c t e d and peaks o f  c a t e c h o l a m i n e f l u o r e s c e n c e d e t e r m i n e d by measuring each f r a c t i o n i n the  s p e c t r o p h o t o f l u o r o m e t e r a t a c t i v a t i o n peak o f 285 mu and  e m i s s i o n o f 330 mu.  A h a l f ml a l i q u o t o f each sample was added to  10 mis o f B r a y ' s s o l u t i o n i n a v i a l and counted i n the l i q u i d scintillation ii)  spectrophotometer. T h i n Layer Chromatography  P r e c o a t e d s h e e t s o f polyamide were used as t h e s t a t i o n a r y phase.  The m o b i l e phase was a m i x t u r e o f i s o b u t a n o l , a c e t i c a c i d  and c y c l o h e x a n e (80:7:10). for  The tank was s a t u r a t e d w i t h t h e s o l v e n t  15 - 20 minutes p r i o r t o use.  hours (8 - 10 cm above the o r i g i n ) . n o r m e t a n e p h r i n e , methyoxytyramine,  The chromatograms r a n f o r 3 -44 One - f i v e jag o f NA, DA, Dopa, t y r o s i n e , 3 , 4 - d i h y d r o x y -  p h e n y l a c e t i c a c i d and 3-methyl-4-hydroxymandelic  a c i d were s p o t t e d .  Those c o n t a i n i n g t h e c a t e c h o l m o i e t y (NA, DA, Dopa and d i h y d r o x y p h e n y l a c e t i c a c i d ) were d e t e c t e d u s i n g a s o l u t i o n o f e t h y l e n e d i a m i n e (1:1 w i t h w a t e r ) and e x a m i n a t i o n under UV l i g h t .  The r e m a i n i n g  compounds were observed u s i n g a p - n i t r o - a n i l i n e r e a g e n t made by combining t h e f o l l o w i n g t h r e e s o l u t i o n s , i n a 1:1:2 r a t i o j u s t p r i o r to use.  These a r e : 1.  0.1 g p - n i t r o - a n i l i n e i n 2 ml cone. HCI made up to  31.  100 m i s , 2.  0.2 g NaN0  3.  10% K C 0  2  i n 100 ml H^O,  2  and,  solution.  A l l compounds gave p u r p l e s p o t s . f)  Catecholamine i)  A c t i v i t y i n Rats i n the C o l d  Catecholamine  determination i n urine  Rats were p l a c e d i n i n d i v i d u a l m e t a b o l i c cages i n the c o l d B o t h s e t s o f r a t s were exposed  room (3°C) and a t room temperature.  t o a c y c l e o f 12 hours l i g h t and 12 hours d a r k n e s s .  Twenty f o u r  hour u r i n e samples were c o l l e c t e d and a n a l y z e d f o r NA and DA as previously described.  B e f o r e i s o l a t i o n on the alumina  the u r i n e s  a r e taken t o pH 4, heated i n a . b o i l i n g water b a t h f o r 10 minutes to h y d r o l y z e c a t e c h o l a m i n e s ii)  conjugates.  Tyrosine hydroxylase a c t i v i t y i n cold a c c l i m a t i z e d rats  E x p e r i m e n t a l and c o n t r o l r a t s were housed as d e s c r i b e d i n the p r e v i o u s s e c t i o n f o r p e r i o d s o f 4 hours t o s e v e r a l weeks.  The  a n i m a l s were s a c r i f i c e d by a blow to the head, and the b r a i n s , a d r e n a l s , h e a r t and s p l e e n removed.  The b r a i n and a d r e n a l s were  a n a l y z e d f o r t y r o s i n e h y d r o x y l a s e a c t i v i t y and f o r c a t e c h o l a m i n e s . The h e a r t s and s p l e e n s were a n a l y z e d o n l y f o r c a t e c h o l a m i n e s . i i i ) Turnover s t u d i e s f o r c o l d a e c l i m i t i z e d r a t s Rats housed as p r e v i o u s l y d e s c r i b e d were i n j e c t e d w i t h the t y r o s i n e h y d r o x y l a s e inhibitorsCT' - m e t h y l - p - t y r o s i n e j l 0 0 mg/kg^ and cZ-methyl-m-tyrosine^100 mg/kg.  The a n i m a l s  (2 e x p e r i m e n t a l  and 2 c o n t r o l s a t each time p e r i o d ) were s a c r i f i c e d a t 2,4, and 6 hours a f t e r i n j e c t i o n .  The b r a i n s , a d r e n a l s , h e a r t and s p l e e n were  a n a l y z e d f o r NA and DA.  The l o g o f the c o n c e n t r a t i o n o f amine was  p l o t t e d a g a i n s t time to determine g)  turnover r a t e s .  E f f e c t o f A l t e r e d Catecholamine Hydroxylase  L e v e l s on T y r o s i n e  A c t i v i t y i n Rat B r a i n  Rats were i n j e c t e d w i t h the MAO i n h i b i t o r s p a r g y l i n e and t r a n y l c y p r a m i n e , and the c a t e c h o l a m i n e ethidine.  d e p l e t o r s r e s e r p i n e and guan-  The doses were p a r g y l i n e 75 mg/kg f o l l o w e d by 5 mg/kg  32.  12 hours l a t e r ; t r a n y l c y p r a m i n e 30 mg/kg and 5 mg/kg; r e s e r p i n e 25 mg/kg and 15 mg/kg and g u a n e t h i d i n e 5 mg/kg b o t h times..  The  a n i m a l s were s a c r i f i c e d 24 hours a f t e r a d m i n i s t r a t i o n o f the f i r s t dose.  The b r a i n s were a n a l y z e d f o r t y r o s i n e h y d r o x y l a s e and  catecholamines. h)  D i s t r i b u t i o n of Tyrosine Hydroxylase i n B r a i n R a t s , r a b b i t s and c a t s were used i n t h i s s t u d y .  Rats  were s a c r i f i c e d by a blow t o the head, r a b b i t s by c e r v i c a l l o c a t i o n and c a t s by n i t r o g e n a s p h y x i a t i o n .  dis-  The b r a i n s were  q u i c k l y removed and d i s s e c t e d i n t o the areas as i n d i c a t e d i n Table ? and F i g . 12.  The s e p t a l a r e a i n some o f the c a t s was f u r t h e r  d i v i d e d as i n d i c a t e d i n F i g . 13.  The t i s s u e was a n a l y z e d f o r  t y r o s i n e h y d r o x y l a s e a c t i v i t y and the V max J  J  i)  J  Study o f Catecholamine  f o r each a r e a  determined.  Pathways i n C a t B r a i n  D i s c r e t e e l e c t r o l y t i c l e s i o n s were p l a c e d i n the m i d b r a i n and d i e n c e p h a l o n o f c a t s u s i n g s t e r e o t a x i c t e c h n i q u e s .  The  s t e r e o t a x i c a t l a s e s o f S n i d e r (106) and J a s p e r (107) were used i n placement o f t h e l e s i o n s as t o a n t e r i o r h o r i z o n t a l and v e r t i c a l positioning.  The s i z e o f the l e s i o n s c o u l d be v a r i e d by time and  e l e c t r i c c u r r e n t i n t e n s i t y and by the number o f n e e d l e Seventy  placements.  two hours a f t e r the o p e r a t i o n t h e c a t s were s a c r i f i c e d .  The  b r a i n was removed and d i s s e c t e d i n t o t h e areas d e s c r i b e d under distribution studies. a l l areas. septal area.  T y r o s i n e h y d r o x y l a s e a n a l y s i s was done on  Catecholamine  d e t e r m i n a t i o n s were done on caudate and  Areas from each s i d e o f the b r a i n were a n a l y z e d  s e p a r a t e l y even i n the case o f b i l a t e r a l l e s i o n s .  The c a l c u l a t e d  V 's were compared w i t h c o n t r o l v a l u e s i n the case o f b i l a t e r a l max l e s i o n s and w i t h t h e u n l e s i o n e d s i d e i n the case o f u n i l a t e r a l . The l e s i o n e d a r e a was removed and p l a c e d i n formaldehyde to the P a t h o l o g y and/or Anatomy departments.  and g i v e n  There i t was p l a c e d  i n a p a r a f f i n b l o c k , c u t and s t a i n e d by the L u x o l F a s t B l u e method. The sections^-.." examined by a n e u r o a n a t o m i s t p o s i t i o n and e x t e n t o f the l e s i o n .  who r e p o r t e d on the  Median s a g i t t a l s e c t i o n of b r a i n t o i l l u s t r a t e  the  method of d i s s e c t i o n Landmarks - A - corpus c a l l o s u m , Areas -  B - fornix,  C - optic  chiasm,  D - mammillary b o d i e s and E - a n t e r i o r commissure 1 - s p i n a l c o r d , 2 - pons and m e d u l l a o b l o n g a t a , 3 - m i d b r a i n 4 - c e r e b e l l u m , 5 - hypothalamus, 6 - s e p t a l a r e a , 7 - thalamus 8 - cortex  34.  I  + 30  I  I  I  I  I  I-  mm  MEDIAN  I  I  I  I  I -I I I  •2-2 0 mm  SAGGITAL F i g . 13:  i  I I I  I  +1  I  I I .1  0mm  I  TRANSVERSE  SECTION  SECTION  Median s a g i t t a l and t r a n s v e r s e s e c t i o n o f c a t b r a i n to i l l u s t r a t e the  subdivisions  of the s e p t a l a r e a Landmarks - A - corpus c a l l o s u m , B - f o r n i x , C - a n t e r i o r commissure, D - o p t i c Areas  - 1 - a n t e r i o r hypothalamus 2 - preoptic  area  3 - septal nuclei  chiasm  4  anterior septal area 5 anterior perf. substance 6 f nucleus accombens r  a  r  e  a  0  35. RESULTS i  1.  S e p a r a t i o n o f NA, DA and Some o f T h e i r M e t a b o l i t e s a)  I o n exchange chromatography S i n c e a l l the amines of i n t e r e s t (NA, DA, normetanephrine  (NM)  and methoxytyramine (MT))  have the same f l u o r e s c e n t peak,  285 mu a c t i v a t i o n and 330 imu f l u o r e s c e n c e , the b e h a v i o r  o f the  compounds on the Dowex column was determined by r u n n i n g 14 14  the amines  one a t a time and by u s i n g NA-  C and DA-  the column was c a r r i e d o u t w i t h 0.4N HCI,  C.  When e l u t i o n from  o n l y NA w a s e l u t e d i n  f r a c t i o n s 4 - 15, NM i n 20 - 35 and DA i n 25 - 4 5 .  I f no NM was  p r e s e n t , s e p a r a t i o n o f NA and DA c o u l d be c a r r i e d o u t more r a p i d l y by u s i n g 2N HCI as an e l u a n t a f t e r t h e NA had been removed from the column w i t h 0.4N HCI. immediately  DA c o u l d then be c o l l e c t e d ^  a f t e r e l u t i o n w i t h 2N HCI b e g a n j i n tubes 1 - 9 and MT  c o u l d be e l u f e d i n tubes 11 - 20. by t y p i c a l e l u t i o n curves  These r e s u l t s a r e summarized  i n F i g . 14 and 15.  found t h a t t y r o s i n e and Dopa c o u l d be s e p a r a t e d  I t was a l s o from t h e amines  by washing the column w i t h 10 ml o f phosphate b u f f e r pH 5.0.  The  a c i d m e t a b o l i t e s o f DA and NA, h o m o v a n i l l i c a c i d and 3-methoxy74h y d r o x y m a n d e l i c a c i d , a r e p r o b a b l y n o t absorbed, o r a r e o n l y v e r y w e a k l y absorbed on t h i s column. When no MAO i n h i b i t o r was added t o an i n c u b a t i o n m i x t u r e o f b r a i n homogenate c o n t a i n i n g 14 DA-  C, over 507o o f the r a d i o a c t i v i t y appeared i n the washings when  t h i s m i x t u r e was a p p l i e d to the column. was  contained  i n the w a s h i n g s .  W i t h an MAO i n h i b i t o r £-< Vk  Other i n v e s t i g a t o r s  (102) have  r e p o r t e d t h a t these a c i d m e t a b o l i t e s a r e n o t adsorbed i n t h i s  type  of column. b) l  T h i n Layer chromatography R^ v a l u e s o b t a i n e d from t h i n l a y e r chromatography o f DA, /YVs  NA and some o f t h e i r m e t a b o l i t e s a r e p r e s e n t e d 2.  i n Table 4.  Catecholamine A c t i v i t y i n R a t s Exposed t o C o l d a)  Urine  1.6-  n  36.  1.4-  1.2-  NE  1.0f)  .LU  o z LU o  in  •u  0.8-  LU  _  ©  o 0.6-  0.4-  0.2-  0  10  20  30 0.4 N H C I -  TUBE F i g . 14:  NUMBER  S e p a r a t i o n by ion-exchange chromatography of NA, NM and DA- C - f l u o r e s c e n c e a t 258 mu/330 nyu — counts per minute f o r C-DA  1.6-  ru  Jl  37,  1.4  c  1.2-  l.G  c  UJ  O  z UJ U  DA  0.8=  MT  tf) UJ  o  Z3  0.6* 4-  0.4=  0.2-  10 •0.4  20  40  30  N HCi-  -2N TUBE  F i g . 15:  v  50  HCI  NUMBER  14, S e p a r a t i o n o f NA, DA and MT and NA-"C by ion-exchange chromatography f l u o r e s c e n c e a t 285 mu/330.mu — Counts per minute f o r  -C-NA  '  TABLE 4 •R  VALUES^FOR NA, DA AND SOME  OF THEIR PRECURSORS AND METABOLITES ON POLYAMIDE 1  Compound (as o b t a i n e d i n (as r e p o r t e d t h i s work) i n 105) Noradrenaline  0 .29  0 .42  Dopamine  0 .39  0 .51  Normetanephrine  0 .69  0 .69  Me thoxytytamine  0 .75  0 .75  Dopa  0 .16  Tyrosine  0 .15  3,4-Dihydroxyphenylacetic  acid  3-Methoxy-4-hydroxymandelic a c i d  0 .18  0 .27  0 .19  0 .35  39. As i n d i c a t e d i n T a b l e 5 t h e r e was (2-3 was  a large increase  f o l d ) i n NA e x c r e t i o n by c o l d a c c l i m a t i z e d r a t s .  a s l i g h t i n c r e a s e i n DA e x c r e t i o n .  There  The i n c r e a s e o c c u r r e d i n  the f i r s t 24 hours i n the c o l d and the c o n c e n t r a t i o n s of DA NA s t a y e d a t the same l e v e l f o r up to two weeks. g i v e n a r e the averages  The  and  values  f o r u r i n e samples from r a t s i n the c o l d f o r  from one day to two weeks. b)  Tissue There was  no s i g n i f i c a n t d i f f e r e n c e i n NA or DA  contents  o f b r a i n between normal r a t s and those exposed to c o l d . was  There  an i n c r e a s e (407 ) of NA i n a d r e n a l but no change i n DA. o  There may  be an i n c r e a s e i n s p l e e n NA and a decrease i n h e a r t NA  but the r e s u l t s on these t i s s u e s a r e n o t s i g n i f i c a n t a t the p = 0.5  l e v e l because of the l a r g e s t a n d a r d d e v i a t i o n .  r e s u l t s are p r e s e n t e d i n Table 6.  S p l e e n and h e a r t  The  DA  c o n c e n t r a t i o n s are not p r e s e n t e d because they were v e r y v a r i a b l e . c)  Tyrosine hydroxylase There was  activity  no s i g n i f i c a n t change i n t y r o s i n e h y d r o x y l a s e  a c t i v i t y per gram of t i s s u e i n e i t h e r b r a i n or a d r e n a l s of c o l d exposed animals as shown i n Table 7.  However, s i n c e the s i z e of  the a d r e n a l s i n c r e a s e d i n r a t s exposed to c o l d , t h e r e was  an  i n c r e a s e of 707 i n t y r o s i n e h y d r o x y l a s e a c t i v i t y per a d r e n a l w h i c h o  was  significant d)  Turnover  (p = 0.01). rates  I n another attempt on c a t e c h o l a m i n e metabolism  to assess the e f f e c t of c o l d exposure the r a t e of d e p l e t i o n of n o r a d r e n a l i n e  was measured a f t e r treatment w i t h the t y r o s i n e h y d r o x y l a s e i n h i b i t o r s , a l p h a - m e t h y l - p - t y r o s i n e and  alpha-methyl-mrtyrosine.  A t the dosage used (100 mg/kg), dopamine l e v e l s i n a l l t i s s u e were not a f f e c t e d and so a r e not r e p o r t e d , and NA l e v e l s i n h e a r t w i t h v\-methyl-m-tyrosine.  I n b r a i n , h e a r t and s p l e e n the curves  f o r n o r a d r e n a l i n e l e v e l s a g a i n s t time a r e v e r y s i m i l a r f o r c o l d a c c l i m i t i z e d and f o r normal r a t s , i n d i c a t i n g t h a t the  apparent  n o r a d r e n a l i n e t u r n o v e r r a t e s i n these t i s s u e s are not markedly  TABLE 5  CATECHOLAMINE CONTENT OF 24 HOUR URINE SAMPLES OF RATS EXPOSED TO COLD*  Change a t .01 Level  DA  100 - 3% (II)" " 112 - 47 (10)  NA  100 -107, (14) 286 - 2.57, (13)  1  *  E x p r e s s e d as 7> o f c o n t r o l  •j-  No. o f a n i m a l i n b r a c k e t s  +127, +1867,  TABLE 6  CATECHOLAMINE LEVELS IN TISSUES OF RATS EXPOSED TO COLD*  Control  Brain Adrenal  NA  100  DA  100  Heart  137o  8)t  106  +  % Significant Change a t .01 Level  187o ( 9)  -  ( 6)  +407o  o  (15)  -  0  ( 6)  NA NA  100 + 367o ( 5)  NA  67 + 317o ( 6)  E x p r e s s e d as 7, o f c o n t r o l No. o f a n i m a l s i n b r a c k e t s  -  ( 6)  + 357c ( 5) 122 + 87o ( 100 + 47o ( 6) 140 + 77o 100 + 367, ( 5) 93 + 507 100 + 277o ( 5) •185 + 557  DA Spleen  +  Cold  -  TABLE 7  TYROSINE HYDROXYLASE ACTIVITY IN RATS EXPOSED TO COLD*  Control  Brain Adrenals  Cold  100 - 15% (10)  +  (per gm) 100 - 20% ( 7) (  (per ad)  100 - 14%  *  Expressed  as %, o f c o n t r o l  +  No. o f a n i m a l s i n b r a c k e t s  '  adrenal  92 - 17% (12) 140 - 2 5 % ( 7) 170 - 10%  43.  a f f e c t e d by the c o l d exposure.  The  curves f o r b r a i n show a break  a t about 4 hours s u g g e s t i n g t h a t the n o r a d r e n a l i n e may than one  t u r n o v e r r a t e , or may  have more  be r e c o v e r y as appears to ^be  case i n h e a r t , s p l e e n and a d r e n a l s .  ( F i g . 16 (a) and  the  (b) )  Data f o r the a d r e n a l s are u n s a t i s f a c t o r y i n t h a t the r a t e of d e p l e t i o n o f n o r a d r e n a l i n e i n normal animals was more r a p i d a f t e r alpha-methyl-m-tyrosine  than a f t e r a l p h a - m e t h y l - p - t y r o s i n e ,  p o s s i b l y due to a p l u r a l i t y o f a c t i o n s .  I n the c o l d  adapted  a n i m a l s the r a t e s of d e p l e t i o n were approximately the same a f t e r b o t h i n h i b i t o r s , a n d i n d i c a t e d a f a s t e r t u r n o v e r r a t e than i n e i t h e r group of c o n t r o l s . phenomena was  F u r t h e r e x p l o r a t i o n of  these  not done because of an i n s u f f i c i e n t s u p p l y of the  i n h i b i t o r s and the p r e s s u r e of other programs.  3.  E f f e c t s of A l t e r i n g Catecholamine Hydroxylase  L e v e l s on I n V i t r o T y r o s i n e  Activity  As i n d i c a t e d i n Table 8, a number of drugs w h i c h a l t e r e d catecholamine  significan  l e v e l s i n the b r a i n had no e f f e c t on t y r o s i n e  h y d r o x y l a s e a c t i v i t y of these b r a i n s as measured i n v i t r o .  4.  D i s t r i b u t i o n S t u d i e s of T y r o s i n e H y d r o x y l a s e  i n Rat, Rabbit  and Cat B r a i n In v i t r o  a c t i v i t y of t y r o s i n e h y d r o x y l a s e i n v a r i o u s  r e g i o n s of c a t , r a b b i t and r a t b r a i n are p r e s e n t e d i n Table A c t i v i t y r e l a t i v e to the c e r e b e l l u m i s a l s o shown.  The  9.  caudate  i s by f a r the most a c t i v e area b e i n g 5 x g r e a t e r than the n e x t two most a c t i v e a r e a s , the septum and a n t e r i o r p e r f o r a t i n g The  caudate  substance.  i s almost 100 times more a c t i v e i n t y r o s i n e h y d r o x y l a s e  than the c e r e b e l l u m w h i c h i s the l e a s t a c t i v e a r e a .  The  r e l a t i v e v a l u e s as compared to the c e r e b e l l u m , are q u i t e c o n s i s t e n t i n the t h r e e s p e c i e s s t u d i e d . To show t h a t t h e r e was  a consistency i n dissection,  average w e i g h t * s t a n d a r d d e v i a t i o n i s shown i n T a b l e  the  10.  T y r o s i n e v a l u e s were a l s o q u i t e c o n s i s t e n t f o r each a r e a  TABLE 8  EFFECTS (OF CERTAIN DRUGS ON CATECHOLAMINE LEVELS AND TYROSINE HYDROXYLASE ACTIVITY IN RAT BRAIN*  NA  DA  100 - 10%  100 - 17%  Tyrosine Hydroxylase 100 -  Control  (4)  Pargyline  (4)  180%  147%  99%  cypramine  (2)  155%  152%  94%  Reserpine  (4)  14%  40%  95%  G u a n e t h i d i n e (2)  110%  89%  106%  T  Tranyl-  E x p r e s s e d as % o f c o n t r o l  t  No. o f a n i m a l s i n b r a c k e t s  8%  TABLE 9 DISTRIBUTION OF TYROSINE HYDROXYLASE IN ADULT RAT, RABBIT AND CAT BRAIN *  Area  V max  + 2.4 15.0  Anterior Perforating Substance Amygdala Hypothalamus Thalamus Midbrain Pons-Medulla Cortex-Whole  \  4.2  4-  + 1.0 (6) 2.9 + 0.3 (6) 3.1 + 0.8 (6)  2.2  + 0.4 (6) + 0.7 (5) 1.2 1.3 + 0.4 (6) 1.8  No. o f a n i m a l s i n p a r e n t h e s i s I n mumoles DOPA/hr/gm o f t i s s u e  + 1.0 (5)  -  -  - C i n g u l a t e Gyrus  Cerebellum  + 1.0 (5) 0.6 + 0.1 (5) 2.2 + 0.5 (5) 1.7 + 0.2 (5) 3.7  1.6 (-6)  3.6  -Association  S p i n a l Cord  -  + 0.5 (3)  > 5.0 i  -Auditory  Hippocampus  (6)  cat 9 (11)  + 1.6 (4) 19.0 + 5 (12) 20.3  -  -Visual  * •f  rabbit 5.5 (6) 70.9 + 9.2 (5) 98.5  22.3  Septal." area,;,  J  4  rat Caudate  A c t i v i t y R e l a t i v e to C e r e b e l l u m  + 0.6 (5) + 0.9 (4) 2.2 0.7 + 0.2 (4) 2.1  28.6 4.2  ,  56  +  1 (ID  + 0.7(11) + 0.2( 9) 2.7 3.1 + 0.2( 5) 1.8 + 0.5(13)  rabbit 101  cat 0  70  1.1  29  13.5 20  3.1  -  11 ( 4)  \  3.0 5.3  2.8  0.9  1.9  3.1  2.2  2.4  1.3  2.3  3.1  1.8  -  -  1.8  -  1.5  -  1.6  1.3  3.0  1.1  -  1.0  3.1  -  + 0.2( 4)  1.0  1.0  1.0  3.9  + 0.6( 9) 2.5 + 1.5( 9) 2.1 + 1.8( 9) 2.5  + 1.6( 9) 2.2 + 1.9( 9) 1.6 + 0.4(10) 2.1  1.4  rat  > 3.7  J %J 2.2  1.5  u  TABLE 10  TISSUE WEIGHTS OF DIFFERENT BRAIN AREAS  Area  Rat  Caudate  33  Septum  +  Midbrain Hypothalamus  Hippocampus Amygdala Cerebellum Anterior Perf. Substance i n mg.  *  7  124  + 118 536 + 98 115 + 21 + 23 400  1408  + 23 214 + 30 118 + 15  70  > 224  Thalamus  416  77  I  67  + 26 76 + 14 256 + 9 96  +  Cat 7  6  48  Pons-Medulla  Rabbit  +  :801  507  +  74  1144  + 128  + +  58 26  + 163 1074 + 154 110 + 30 + 246 1278 + 110 400 + 62 3438 + 201 582  _1_  AT  35:  9  47:.  Legend: Control Cold range o f v a l u e s f o r 2 or 3 animals  I  half  life  Z  o < IZ ui O  BRAIN  z o o o  \  = 0.8  I  I  T  I  T TIME  T  "I  6  8~  ( H O U R S )  SPLEEN Fig. l 6 & " .  2  D e p l e t i o n of NA w i t h  ADRENAL (A -methy 1-m-tyrosine  organs o f normal r a t s and r a t s exposed to c o l d  48.  T I M E ( H O U R S ) F i g . 16b; D e p l e t i o n of NA w i t h C<-methy1-p-tyrosine from v a r i o u s organs of normal r a t s and r a t s exposed to the c o l d ( f o r legend see F i g . 15)  49.  as i n d i c a t e d i n T a b l e 11.  There were s l i g h t d i f f e r e n c e s between  a r e a s , b u t i n g e n e r a l the v a l u e s ranged from 15 - 30 V/gm  o f wet  tissue. The  t y r o s i n e h y d r o x y l a s e a c t i v i t i e s of the v a r i o u s  s u b d i v i s i o n s o f the s e p t a l a r e a f o r c a t a r e p r e s e n t e d i n T a b l e 12, a l o n g w i t h the average w e i g h t s and t y r o s i n e v a l u e s .  The  c l a s s i c a l s e p t a l n u c l e i and p r e o p t i c a r e a a r e r e l a t i v e l y  inactive.  Most o f the t y r o s i n e h y d r o x y l a s e a c t i v i t y o f the s e p t a l a r e a , as we d e f i n e d i t , i s c o n t a i n e d i n the a n t e r i o r p o r t i o n j u s t v e n t r a l to the s e p t a l nuclei o v e r l y i n g t h e r e g i o n o f the n u c l e u s . accombens. T h i s r e g i o n may be p a r t o f the tuberculum o l f a c t o r u m or nucleus of  the d i a g o n a l band o f B r o c a .  The r e g i o n o f the n u c l e u s  accombens has about h a l f the a c t i v i t y o f the caudate and i s t h e r e f o r e the second most a c t i v e a r e a we found i n the b r a i n .  The  a n t e r i o r hypothalamus has about the same a c t i v i t y as does the whole hypothalamus.  5. .  ;  E f f e c t o f L e s i o n s on the T y r o s i n e H y d r o x y l a s e L e v e l s i n V a r i o u s Regions o f C a t B r a i n S i x main types o f b r a i n s l e s i o n s were done: 1.  f l o o r o f mid d i e n c e p h a l o n ,  2.  p o s t e r i o r diencephalon ( F i e l d s of F o r e l ) ,  3.  habenular,  4.  substantia nigra,  5.  m i d l i n e m i d b r a i n and  6$  raphe  The l o c a t i o n s o f these l e s i o n s a r e shown i n F i g . 17. a)  Lesions of diencephalon  floor  Nine animals were l e s i o n e d a t t h i s l e v e l o f the b r a i n , 3 a n i m a l s w i t h m e d i a l l e s i o n s and 6 w i t h l a t e r a l ones. a r e p r e s e n t e d i n Table 13.  The r e s u l t s  The m e d i a l l e s i o n s had l i t t l e  effect  on t y r o s i n e h y d r o x y l a s e a c t i v i t y i n the caudate; o n l y one o f the 3 c a t s showed a drop i n a c t i v i t y t o 63% o f normal.  However f o r  these same l e s i o n s the s e p t a l t y r o s i n e h y d r o x y l a s e a c t i v i t y  decreased  TABLE 11 TYROSINE CONCENTRATIONS IN VARIOUS AREAS OF RAT, RABBIT AND CAT BRAIN*  Area  Rat  Pons-Medulla  17.3  Caudate  28.8  Septum Hippocampus  1>  Thalamus Hypo thalamus  +  Rabbit  +  Cat  3.2  24.1  + 10.2 16.5 + 6.3 22.7 + 7.1  27.8  +  5.2  24.9  +  3.5  JL.  20.8  T  +  5.4  10.1  + 4.0 25.3 + 6.2 + 5.1 23.5 28.3  -  Amygdala  23.9  Cortex  4.5  Cerebellum  + 15.6 + 17.3 +  5.5  + 4.8 25.8 + 4.2 22.9 + 8.5  S p i n a l Cord  22.7  3.5  22.7  15.7  Midbrain  Anterior Perforating *  ugm/gm  Substance  - standard d e v i a t i o n  -  5.1 4.2  31.2  +  -  13.4  +  2.8  + 6.5 17.6 + 6.0 13.2 + 3.2  24.6  + 5.7 + 19.8 5.5 16.1 + 3.5 14.5  + 5.1 14.3 + 7.0 18.9 + 4.6 14.8  -  3.7 23.3  + 3.1  51.  TABLE 12  TYROSINE HYDROXYLASE ACTIVITY IN SUBDIVISIONS OF SEPTAL AREA OF CAT BRAIN  Region  V max  Tyrosine  mumole/DOPA/ g/hr A n t e r i o r Hypothalamus  + 5.2  A n t e r i o r Septum R e g i o n - Nucleus Accombens Septal  I%  26.5  + 3.8 32.6 + 13.9 61.0 + 18.5  21.2  + 0.9  17.1  8.0  Preoptic  Classical Nuclei  5.9  3.8  T i s s u e Wt.  J  + 5.1  + 5.7 17.4 + 3.5 25.5 + 7.5 + 1.7  mg  25  + 6  + 5 37 + 10 28 + 2 18  39  + 18  TABLE 13 EFFECTS OF LESIONS IN FLOOR OF DIENCEPHALON ON TYROSINE HYDROXYLASE AND CATECHOLAMINES IN ROSTRAL AREAS''  Area  Caudate  Septum  Medial  +  (3)  L a t e r a l (6)  Tyrosine Hydroxylase  DA  NA  Tyrosine Hydroxylase  110  —  -  63,  34, 118, 4 1 , 8 7 , 105,  63  74  89  63,  59  104  105  77  15,  75  60,  65,  64 14.5  -  56.5  -  -  DA  -,  NA  -,  65, 41 110, 114 -  125, 33, 2 1 , 103  *  a c t i v i t y e x p r e s s e d as % o f n o n - l e s i o n e d  +  no. o f animals i n b r a c k e t s  '  m e d i a l caudate  Fig.  17:  Median s a g i t t a l s e c t i o n of the b r a i n to i l l u s t r a t e the l o c a t i o n s o f the l e s i o n s of the d i e n c e p h a l o n and m i d b r a i n .  Landmarks - A - corpus c a l l o s u m , B - f o r n i x , C - o p t i c chiasm D - interpeduncular nucleus, E - superior c o l l i c u l i E - inferior colliculi L e s i o n e d a r e a s - 1. f l o o r of m i d - d i e n c e p h a l o n , 2. p o s t e r i o r d i e n c e p h a l o n 4- h a b e n u l a 5. m i d l i n e m i d b r a i n and raphe 6. c e r v e a u i s o l e  <2  54.  i n a l l c a t s , to as l i t t l e as 15% of normal i n one case.  Laterally  p l a c e d l e s i o n s caused s u b s t a n t i a l decreases i n a c t i v i t y of the enzyme i n b o t h caudate  (15 - 65% of normal) and s e p t a l a r e a (21 - 65%,)  i n 4 of the a n i m a l s .  The o t h e r two l a t e r a l l e s i o n s showed d e c r e a s e s  i n caudate but none i n the s e p t a l a r e a . There were no s i g n i f i c a n t or c o n s i s t e n t changes i n t y r o sine hydroxylase a c t i v i t y ,  w i t h these d i e n c e p h a l i c l e s i o n s i n  any of the o t h e r areas of b r a i n , measured: i . e . amygdala, h i p p o campus, thalamus, hypothalamus,  c o r t e x and pons and  medulla  oblongata. Caudate c a t e c h o l a m i n e l e v e l s were determined  i n 6 of  these a n i m a l s ; s e p t a l area l e v e l s c o u l d n o t be measured because of insufficient tissue.  There were no s i g n i f i c a n t changes i n  caudate n o r a d r e n a l i n e c o n c e n t r a t i o n s . was  no s i g n i f i c a n t change i n caudate  t h e r e was  I n one a n i m a l where t h e r e  tyrosine hydroxylase a c t i v i t y ,  l i k e w i s e no change i n dopamine l e v e l .  Four of the 5  c a t s w h i c h showed d e c r e a s e s i n enzyme a c t i v i t y i n the caudate  also  showed decreases i n dopamine c o n t e n t , but these d e c r e a s e s were n o t as l a r g e as those of the enzyme (15% as compared to 45%,). No h i s t o l o g i c a l s t u d i e s were done on the l a t e r a l l y p l a c e d lesions.  One m e d i a l l e s i o n , i n w h i c h the s e p t a l  h y d r o x y l a s e a c t i v i t y was  56.5%, o f n o r m a l , was  tyrosine  confirmed h i s t o l o g i c a l l y  as a l e s i o n of the m i d l i n e f l o o r of the d i e n c e p h a l o n t h a t d i d n o t extend f a r i n t o the d i e n c e p h a l o n . b)  T h i s l e s i o n i s shown i n F i g . 18.  P o s t e r i o r d i e n c e p h a l o n - F i e l d s of F o r e l The  11 l e s i o n s done i n t h i s area can be d i v i d e d i n t o  two  groups: i)  l a r g e c e n t r a l l y p l a c e d one  (4 c a t s ) and  ii)  s m a l l m e d i a l l y p l a c e d ones (7 c a t s ) .  W i t h the l a r g e r l e s i o n s t h e r e were s u b s t a n t i a l d e c r e a s e s i n t y r o s i n e h y d r o x y l a s e a c t i v i t y , to as low as 11% of n o r m a l , i n both caudate and s e p t a l a r e a s ; the s m a l l e r l e s i o n s r e s u l t e d i n lower d r o p s , to about 50% of normal.  W i t h b o t h types of l e s i o n the  caudate and s e p t a l a r e a decreased by a p p r o x i m a t e l y the same amount.  F i g . 18:  L e s i o n i n the f l o o r of the m i d - d i e n c e p h a l o n  56.  These r e s u l t s are p r e s e n t e d i h a T a b l e 14. There were s i g n i f i c a n t decreases  i n NA and  DA  c o n c e n t r a t i o n ( t o a p p r o x i m a t e l y 30% of normal) w i t h the  caudate  of b o t h of the c a t s i n the l a r g e l e s i o n s on w h i c h amine a n a l y s e s were done. The o n l y o t h e r areas of b r a i n i n w h i c h t h e r e were changes i n t y r o s i n e h y d r o x y l a s e a c t i v i t y were the amygdala and hippocampus.  Of the 11 c a t s l e s i o n e d , 5 had drops i n a c t i v i t y  of about 50% i n the amygdala; and 9 animals had changes i n a c t i v i t y i n the hippocampus. however, v e r y i n c o n s t a n t .  The r e s u l t s f o r the hippocampus were A l t h o u g h the hippo.campal '. a c t i v i t y  was  l e s s than normal on the l e s i o n e d s i d e i n 6 c a t s , i t was more than normal i n a s e v e n t h .  And i n 2 o t h e r cases the hippocampus on  the n o n - l e s i o n e d s i d e showed no a c t i v i t y a t a l l . Therewere no h i s t o l o g i c a l s t u d i e s f o r the l a r g e lesions. decreases  Three of the s m a l l m e d i a l l e s i o n s , w h i c h had  caused  i n t y r o s i n e h y d r o x y l a s e a c t i v i t y i n b o t h caudate  s e p t a l a r e a , were c o n f i r m e d h i s t o l o g i c a l l y . these are shown i n F i g . 19 and 20.  and  Examples of 2 of  F i g . 19 shows a l e s i o n c l o s e  to the m i d l i n e i n the v e n t r a l d i e n c e p h a l o n a t the l e v e l of the mammillary b o d i e s .  F i g . 20 shows a v e r y d i s c r e t e l e s i o n a t the  same l e v e l but s l i g h t l y more l a t e r a l l y p l a c e d . the e l e c t r o d e s can a l s o be seen i n t h i s c)  The e n t r a n c e of  picture,  Habenular. E l e v e n a n i m a l s were l e s i o n e d i n an attempt  to d e s t r o y  the h a b e n u l a f r e g i o n , w h i c h would be a t the same l e v e l as the F i e l d s of F o r e l , but more d o r s a l l y l o c a t e d . habenular  Four of these  l e s i o n s were u n i l a t e r a l (a) and b i l a t e r a l -7 , ( b ) .  On e x a m i n a t i o n  ( h i s t o l o g i c a l ) i t was  l e s i o n s were i n the habenular  area.  found t h a t n o t a l l the The b i o c h e m i c a l r e s u l t s a r e  p r e s e n t e d i n Table 15 w i t h v a l u e s f o r r i g h t and l e f t s i d e s of the b r a i n w i t h some b i l a t e r a l i)  lesions.  Three of the 4 c a t s w i t h u n i l a t e r a l l e s i o n s  a p p r o x i m a t e l y 50%, decreases  had  i n t y r o s i n e h y d r o x y l a s e i n the s e p t a l  F i g . 19:  M e d i a l l y p l a c e d l e s i o n i n p o s t e r i o r d i e n c e p h a l o n a t the l e v e l of the mammillary b o d i e s  Fig. 20:  L e s i o n i n the p o s t e r i o r d i e n c e p h a l o n ( o n l y h a l f o f the b r a i n )  TABLE 14 EFFECTS OF LESIONS IN FIELDS OF FOREL ON TYROSINE HYDROXYLASE AND CATECHOLAMINES IN ROSTRAL AREAS*' (Only those a n i m a l s showing changes p r e s e n t e d )  Area  Caudate  L a r g e (4) (Central)  S m a l l (7) (Medial)  Tyrosine Hydroxylase  DA  NA  Tyrosine Hydroxylase  \23  31  28  34, 36,  45  27  33  59, 56,  49 Septum  11 17 75  Hippocampus  B l (NL) B l (NL) Bl  Amygdala  49 54  *  —  A c t i v i t y e x p r e s s e d as ''A o f c o n t r o l  Bl  No a c t i v i t y ( i e . e q u a l to b l a n k )  NL  non-lesioned side  NA  62  -  4 1 , 57,  -  -  56  ~  —  +  no. o f a n i m a l i n b r a c k e t s  -  220, 67  -  13, 58  -  B l , 50  —  DA  43, 55, 44, 65  60, 58,  TABLE 15  EFFECTS OF ATTEMPTED HABENULAR LESIONS ON TYROSINE HYDROXYLASE ACTIVITY IN CAUDATE, SEPTUM, AMYGDALA AND HIPPOCAMPUS'  Area  U n i l a t e r a l (4)  B i l a t e r a l (7)  Caudate  109,108,114,85  11°, 36,66,70  Septum  46,85,49,49  20°,27,71,60°,61  Hippocampus  64,75,100,39  17°,48,34,B1°,35, 21 ,B1,B1°,9°,24 0  Amygdala  42,115,132,47  19,218°,150,185,236, 141  *  e x p r e s s e d as % o f c o n t r o l  +  no. o f animals i n b r a c k e t s  Bl  no a c t i v i t y ( i e . e q u a l to b l a n k )  0  d e s i g n a t e s from the same a n i m a l r i g h t and l e f t respectively  side  60.  a r e a , and amygdala b u t no changes i n o t h e r a r e a s .  I t was found  however, t h a t i n a l l o f these c a t s the habenular r e g i o n had been spared w i t h damage p r i m a r i l y d o r s o l a t e r a l t o i t .  I n 2 o f the  a n i m a l s t h a t had changes i n a c t i v i t y i n the s e p t a l a r e a t h e r e were a l s o e v i d e n c e s o f damage on the m i d l i n e o f the d i e n c e p h a l o n below the p o s t e r i o r commissure.  An example o f t h i s  type of l e s i o n i s  seen i n F i g . 21. ii)  Only 3 of the 7 b i l a t e r a l habenular  lesions  r e s u l t e d ' , i n decreases i n t y r o s i n e h y d r o x y l a s e a c t i v i t y i n the caudate  and s e p t a l a r e a .  I n one a n i m a l , showing a v e r y l a r g e drop  i n a c t i v i t y t o 10 - 307„ o f n o r m a l , the l e s i o n was found on histological  examination.jto i n v o l v e n o t o n l y the habenular r e g i o n  but t o extend l a t e r a l l y and v e n t r a l l y from the v e n t r i c l e to t h e f l o o r  o f the d i e n c e p h a l o n (see F i g . 2 2 ) .  almost  Three o f the  o t h e r b i l a t e r a l l e s i o n s t h a t had no change i n enzyme a c t i v i t y were confirmed h i s t o l o g i c a l l y  t o be i n the habenula a r e a , o n l y as  shown i n F i g . 23. There were a l s o changes i n t y r o s i n e h y d r o x y l a s e a c t i v i t y iril.amygda l a and hippocampus w i t h the b i l a t e r a l lesions.  habenula  I n the same a n i m a l t h a t had had l a r g e d e c r e a s e s i n  caudate and s e p t a l a r e a the a c t i v i t y i n the amygdala was 197o o f normal; i n 4 o f the a n i m a l s t h e r e was i n c r e a s e d amygdala (141 - 2367, o f n o r m a l ) .  activity  The hippocampus a c t i v i t y was l e s s  than  normal i n 6 o u t o f 7 a n i m a l s , d)  Substantia nigra Only 2 a n i m a l s were l e s i o n e d i n the s u b s t a n t i a n i g r a  s i n c e so much had p r e v i o u s l y been done i n t h i s a r e a by o t h e r workers  (51 - 56).  As i n d i c a t e d i n Table 16, the caudate i n b o t h  cases showed the expected l a r g e d r o p s , t o as l i t t l e as 2% o f n o r m a l , i n t y r o s i n e h y d r o x y l a s e a c t i v i t y i n the caudate, as w e l l as s m a l l e r d e c r e a s e s i n DA.(.2% c f i : 3 K ) . i i n one o f the c a t s .  The NA l e v e l was a l s o below normal  There were a l s o s u b s t a n t i a l decreases i n  s.eptal t y r o s i n e h y d r o x y l a s e .  Histologically,  the l e s i o n s were  F i g . 21:  L e s i o n made i n an attempt to d e s t r o y the habenular r e g i o n but w i t h damage v e n t r a l to i t .  TABLE 16  EFFECTS OF SUBSTANTIA NIGRA LESIONS ON TYROSINE HYDROXYLASE ACTIVITY AND CATECHOLAMINES IN CAUDATE AND SEPTUM"  Area  Tyrosine Hydroxylase.'.  Caudate  2, 5  Septum  20, 9.5  e x p r e s s e d as % o f c o n t r o l  DA  NA  3 1 , 39  5 1 , 50  25,  60,102  -  F i g . 23 :  L e s i o n of the h a b e n u l a r o n l y  64.  confirmed  to be i n the s u b s t a n t i a n i g r a , e x t e n d i n g m e d i a l  from  i t to the m i d l i n e . e)  Midline The  midbrain  10 m i d l i n e m i d b r a i n  were two types of i)  l e s i o n s were b i l a t e r a l .  There  lesions:  c e r v e a u - i s o l e - l e s i o n s p l a c e d a t a l e v e l between  the i n f e r i o r and s u p e r i o r c o l l i c u l i and i n c l u d i n g most of the r e t i c u l a r f o r m a t i o n on b o t h s i d e s of the m i d l i n e (5 a n i m a l s ) , ii)  l e s i o n s e x t e n d i n g r o s t r a l to t h i s c e r v e a u i s o l e under  the s u p e r i o r c o l l i c u l i and c o n f i n e d to the m i d l i n e s t r u c t u r e s of the r e t i c u l a r f o r m a t i o n (a)  The  c e r v e a u - i s o l e l e s i o n s had  tyrosine hydroxylase  l e s i o n was  l i t t l e effect  on  a c t i v i t y i n e i t h e r caudate or  s e p t a l a r e a , as shown i n T a b l e 17. were c o n f i r m e d  (5 a n i m a l s ) .  histologically.  Four of these l e s i o n s  A d o r s a l l y located  d e s c r i b e d as d e s t r u c t i o n of the m i d l i n e t e g -  mentum o f the m i d b r a i n , e x t e n d i n g  2 mm  on e i t h e r s i d e  a t the l e v e l of the c a u d a l i n f e r i o r c o l l i c u l i , w i t h perhaps a s l i g h t m e d i a l encroachment on the s u b s t a n t i a n i g r a , but s p a r i n g the i n t e r p e d u n c u l a r n u c l e u s  ( F i g . 24).  More v e n t r a l l y l o c a t e d l e s i o n s of t h i s type a l s o the i n t e r p e d u n c u l a r nucleus (b)  The  ( F i g . 25).  l e s i o n s j u s t r o s t r a l to t h i s  cerveau-isole  produced v e r y i n t e r e s t i n g changes i n t y r o s i n e a c t i v i t y i n caudate and  destroyed  s e p t a l area.  m i d l i n e a t the s u p e r i o r c o l l i c u l i  hydroxylase  A l e s i o n on  l e v e l , with  the  little  • l a t e r a l e x t e n s i o n , as shown i n F i g . 26, produced no change i n caudate or s e p t a l area a c t i v i t y (see Table 17). However a l e s i o n i n another a n i m a l , a t the same l e v e l ,  but  e x t e n d i n g more l a t e r a l l y (see F i g . 27) produced drops i n b o t h caudate and s e p t a l area to about 50% of normal. The  importance of l a t e r a l e x t e n t of the l e s i o n was  i n one a n i m a l where h i s t o l o g i c a l e x a m i n a t i o n  evident  showed t h a t  TABLE 17  EFFECT OF BILATERAL MIDBRAIN LESIONS ON TYROSINE HYDROXYLASE ACTIVITY IN CAUDATE AND SEPTUM*  Area  Caudate  Cerveau I s o l e (5)  Midline  (5)  Raphe (4)  60 71 95 56 70  Septum  108 103 97 70 75  *  e x p r e s s e d as % o f c o n t r o l  +  no. o f a n i m a l s i n b r a c k e t s  Bl  a c t i v i t y equal to blank  0  d e s i g n a t e s from same a n i m a l - r i g h t and l e f t respectively  side  F i g . 27:  L e s i o n of the m i d l i n e m i d b r a i n w i t h a l a r g e l a t e r a l extension  68.  the m i d l i n e m i d b r a i n tegmentum was  d e s t r o y e d more  l a t e r a l l y i n the r i g h t s i d e than on the l e f t In  ( F i g . 28).  t h i s a n i m a l the decrease i n enzyme a c t i v i t y were on  the l e f t and r i g h t s i d e s b e i n g 307, and 18% of  normal  r e s p e c t i v e l y f o r caudate and 55% and 25%, f o r s e p t a l . a r e a The o t h e r two animals of t h i s group had  similar  . r e s u l t s , i n t h a t d i f f e r e n c e s i n the decreases i n t y r o s i n e h y d r o x y l a s e a c t i v i t y on the r i g h t and  left  c o u l d be c o r r e l a t e d w i t h the l a t e r a l e x t e n t of the l e s i o n as determined h i s t o l o g i c a l l y . s e p t a l a r e a were n o t always comparable, the caudate  The caudate i n one  t y r o s i n e h y d r o x y l a s e a c t i v i t y was  animal normal  on the r i g h t s i d e w h i l e the s e p t a l a c t i v i t y was of  and  20%,  normal. I n a l l these m i d l i n e m i d b r a i n l e s i o n s the damage  extended from the aquaduct (and i n some cases above i t ) to  the f l o o r of the m i d b r a i n i n c l u d i n g the i n t e r p e d u n c u l a r  n u c l e u s , the s u b s t a n t i a n i g r a and o t h e r p e r i p h e r a l n u c l e i were l a r g e l y s p a r e d , f)  Raphe The 4 raphe l e s i o n s were a l l c o n f i r m e d h i s t o l o g i c a l l y .  These l e s i o n s d i d n o t extend as v e n t r a l l y as those 5b but o t h e r w i s e they were h i s t o l o g i c a l l y s i m i l a r , i . e . d e s t r u c t i o n of the m i d l i n e s t r u c t u r e s of the m i d b r a i n , such as the d o r s a l r a p h e , m e d i a l l o n g i t u d i n a l f a s c i c u l u s and occulomotor  complex, between the  c a u d a l i n f e r i o r c o l l i c u l i and c a u d a l s u p e r i o r c o l l i c u l i (see Fig.  29). As shown i n Table 17 t y r o s i n e h y d r o x y l a s e a c t i v i t y i n the  caudate and s e p t a l a r e a c o u l d s e l e c t i v e l y be l e f t unchanged or decreased  to p r a c t i c a l l y n i l depending  thfe l e s i o n .  on the l a t e r a l e x t e n t o f  As w i t h the l e s i o n s of 5b type both caudate  and  s e p t a l a c t i v i t y c o u l d be a f f e c t e d or j u s t s e p t a l a r e a but never j u s t caudate  activity.  69.  Fig.  28:  L e s i o n o f the m i d l i n e m i d b r a i n w i t h l a t e r a l of the l e s i o n more on the r i g h t  extension  F i g . 29:  L e s i o n of the m i d l i n e m i d b r a i n  i n the d o r s a l raphe  DISCUSSION  1.  Tyrosine Hydroxylase A c t i v i t y I n V i t r o The method, d e s c r i b e d i n t h i s i n v e s t i g a t i o n f o r  determination of t y r o s i n e hydroxylase  a c t i v i t y i n v i t r o has  proven t o be a r a p i d and p r e c i s e one; r a p i d i n t h a t up t o 80 i n c u b a t i o n s c o u l d be done i n 2 days and p r e c i s e i n t h a t d u p l i c a t e i n c u b a t i o n s were w i t h i n s e v e r a l per c e n t o f each o t h e r . A c c u r a c y a l s o , i s guggested by the f a c t t h a t c o n s i s t e n t r e s u l t s are o b t a i n e d f o r each area o f the b r a i n (Table 9 ) .  Another  method f o r measurement has been d e s c r i b e d i n t h e l i t e r a t u r e 3 (108,110) u s i n g t r i t i a t e d t y r o s i n e .  The  H on the meta-  p o s i t i o n o f the a r o m a t i c r i n g i s r e l e a s e d i n t o the water d u r i n g the r e a c t i o n and can be r e a d i l y measured. 14 r e p o r t e d t o g i v e s i m i l a r r e s u l t s t o the  T h i s method has been C - t y r o s i n e method.  The v a l u e s f o r t y r o s i n e h y d r o x y l a s e  a c t i v i t y were i n the  range o f 1 - 100 njumoles o f Dopa formed/gm/hr f o r b r a i n . i s o f t h e same o r d e r o f magnitude as r e p o r t e d by o t h e r s  This (27,28)  who o b t a i n e d v a l u e s i n t h e range o f 4 - 100 mjimoles/gm/hr. a c t i v i t y i n adrenal i s reported  (28) t o be 15 times  The  that of  b r a i n ; b u t i n o t h e r p e r i p h e r a l t i s s u e such as h e a r t and s p l e e n i t i s d i f f i c u l t t o d e t e c t any a c t i v i t y i n v i t r o .  Sympathetic  endings make up o n l y a s m a l l p r o p o r t i o n o f such t i s s u e w h i c h may account f o r the r e l a t i v e  inactivity.  I n v i t r o s t u d i e s show d i f f e r e n c e s i n t y r o s i n e i n b r a i n andadrenal.  hydroxylase  The c o n d i t i o n s f o r maximum a c t i v i t y o f  crude a d r e n a l homogenate (28) o r of p a r t i a l l y p u r i f i e d  adrenal  enzyme (23) a r e a c e t a t e b u f f e r pH 6.0 i n the presence o f DMPH^ . The a c t i v i t y drops o f f a f t e r 20 m i n u t e s . b r a i n t y r o s i n e hydroxylase  We have found t h a t  i s most a c t i v e i n phosphate b u f f e r  pH 6.2, w i t h o u t DMPH^ and w i t h a c t i v i t y s t a y i n g l i n e a r f o r a t l e a s t 30 - 45 m i n u t e s .  The enzymes may be d i f f e r e n t i n b r a i n and  a d r e n a l b u t the d i f f e r e n c e s i n i n v i t r o a c t i v i t y c o u l d a l s o be e x p l a i n e d by d i f f e r e n c e s i n the i o n i c environment o f t h e t i s s u e  and t h e f a c t t h a t t h e r e may be s u f f i c i e n t DMPH^ i n crude b r a i n homogenate a l r e a d y .  I n support o f t h i s s u g g e s t i o n , Ikeda and  L e v i t t (111) has r e p o r t e d t h a t DMPH^ enhances a c t i v i t y i n p u r i f i e d dog caudate t y r o s i n e h y d r o x y l a s e . d u r i n g these i n v e s t i g a t i o n s o f K  A l s o i t was noted  values that a t very high  c o n c e n t r a t i o n s o f t y r o s i n e the curve 1/v vs 1/s was no l o n g e r l i n e a r w i t h o u t DMPH, b u t remained l i n e a r w i t h c o f a c t o r . 4 may  This  i n d i c a t e DMPH^ was becoming l i m i t i n g a t h i g h t y r o s i n e  concentrations. The K  values obtained for tyrosine hydroxylase  appear t o be s p e c i e s and organ dependant.  I n b r a i n the  v a l u e s as r e p o r t e d i n t h i s work ranged from 0.5 x 10  to  1 x 10 ^ f o r the v a r i o u s s p e c i e s ( c a t , r a b b i t and r a t ) and  ^  i t has been r e p o r t e d t h a t f o r crude beef a d r e n a l the K i s i n -5 the o r d e r o f 2 x 10 ( 2 8 ) . U d e n f r i e n d has r e p o r t e d the K ^ -5 -5 m  m  may  range from 2 x 10  t o 0.1 x 10  ( 2 3 ) . He may have been  "  r e f e r r i n g to d i f f e r e n c e s i n s p e c i e s and organs. T y r o s i n e h y d r o x y l a s e has a l s o been r e p o r t e d t o be capable of c o n v e r t i n g phenylalanine to t y r o s i n e i n v i t r o i n b r a i n and a d r e n a l t i s s u e (112). 2. S e p a r a t i o n o f NA, DA and Some o f T h e i r M e t a b o l i t e s a)  I o n exchange chromatography From the r e s u l t s  (as shown i n F i g . 15 and 14) t h i s i o n  exchange chromatographic method i s s u i t a b l e f o r the s e p a r a t i o n of NA, DA and MT; NA, NM and MT, w i t h i n c o m p l e t e NM and DA.  separation of  I t i s a l s o a u s e f u l technique f o r t h e removal o f the  p r e c u r s o r s o f CA s y n t h e s i s , t y r o s i n e and Dopa, and o f t h e i r m e t a b o l i t e s from s o l u t i o n s o f DA and NA.  Although  acid  different  s i z e s o f columns o f Dowex and d i f f e r e n t e l u t i o n schedules  have  been used by o t h e r workers (102-104) the types o f s e p a r a t i o n achieved are s i m i l a r . T h i s chromatographic technique has been used t o s e p a r a t e the p r e c u r s o r DA from t h e p r o d u c t NA i n the a n a l y s i s o f dopamine  p-oxidase (113,114), CA  (30,31).  and f o r the study of i n v i v o metabolism o f  S i n c e t y r o s i n e h y d r o x y l a s e may  n o t be the o n l y  c e n t r a l l i n g f a c t o r i n CA s y n t h e s i s i t i s u s e f u l to s t u d y o t h e r enzymes i n v o l v e d i n CA s y n t h e s i s and to i n v e s t i g a t e the i n v i v o metabolism.  I t would f o r example, be of i n t e r e s t to  determine  whether NA or DA i s the major CA p r o d u c t i n the s e p t a l area which i s high i n t y r o s i n e hydroxylase, b)  T h i n l a y e r chromatography I n the o r i g i n a l r e f e r e n c e (105) t h i s method of s e p a r a t i o n  of CAs and  t h e i r m e t a b o l i t e s was  c l a i m e d to be 100 times more  s e n s i t i v e than paper chromatography. e t h y l e n e diamine  they were a b l e to detect4. 0.05 Tf^of CA.  I n our hands the method was DA,  NA, MT  W i t h use of the cfetector a good means of s e p a r a t i n g  and NM f o r as i n d i c a t e d i n T a b l e 4 t h e r e was  l e a s t 0.1  u n i t between each amine.  at  The p r e c u r s o r s t y r o s i n e  and Dopa and the a c i d m e t a b o l i t e s of DA and NA had much lower  R^  v a l u e s and c o u l d be s e p a r a t e d out from the amines t h a t r a n ahead of them.  The R^ v a l u e s o b t a i n e d i n t h i s i n v e s t i g a t i o n are  s l i g h t l y lower than those r e p o r t e d (Table 4 ) . The s e p a r a t i o n of the amines o b t a i n e d u s i n g t h i n l a y e r chromatography i s comparable w i t h t h a t of paper chromatography ( 1 1 5 ) , and has  the advantage of b e i n g more r a p i d , r e q u i r e s s m a l l e r  q u a n t i t i e s and no p r e l i m i n a r y a c e t y l a t i o n i s r e q u i r e d .  It  c o u l d be a p p l i e d to s t u d i e s of i n v i v o CA m e t a b o l i s m i n p l a c e of the paper chromatographic l a y e r chromatographic  techniques o f t e n used.  Catecholamine The  thin  method u s i n g c e l l u l o s e i n s t e a d of p o l y -  amide has a l s o g i v e n s i m i l a r r e s u l t s  3.  Another  (116).  A c t i v i t y i n Rats Exposed to C o l d  l a r g e i n c r e a s e i n DA e x c r e t i o n i n r a t s exposed to c o l d  are i n agreement w i t h o t h e r r e p o r t s (89,117).  Leduc (89) c l a i m s  a 5 f o l d i n c r e a s e i n NA and a 40% i n c r e a s e i n DA e x c r e t i o n i n c o l d acclimatized  animals.  However t h e r e are c o n f l i c t i n g r e p o r t s on the changes i n  CA c o n c e n t r a t i o n i n t i s s u e s of r a t s exposed to cold'. (21) has r e p o r t e d no change i n NA of a d r e n a l s and  Leduc  a decrease i n  c o n c e n t r a t i o n i n h e a r t and s p l e e n ; o t h e r s have shown (117)  a  40 - 507, i n c r e a s e i n NA i n a d r e n a l s and no change i n b r a i n NA content.  The r e s u l t s p r e s e n t e d  i n Table 6 (407, i n c r e a s e i n NA  i n a d r e n a l s and no change i n b r a i n ) would be i n agreement w i t h the l a t t e r r e p o r t . Leduc (89) has done the most e x t e n s i v e work w i t h c o l d acclimatized rats.  From h i s s t u d i e s u s i n g  adrenalectomized  r a t s and g a n g l i o n i c b l o c k i n g a g e n t s , he concludes r e l e a s e d f r o m sympathetic  that  endings i s the f i r s t , and  r e l e a s e d from the a d r e n a l s  NA  adrenaline  i s the second meansy; of p r o v i d i n g  . s h i v e r i n g . r thermogenesis i n animals  p l a c e d i n the c o l d .  NA and a d r e n a l i n e have been shown to i n c r e a s e g l y c o g e n and cause f a t t y a c i d m o b i l i z a t i o n ; the r e s u l t i n g  non-  Both ,  breakdown  increased  m e t a b o l i s m c o u l d perhaps account f o r the r e q u i r e d i n c r e a s e i n h e a t p r o d u c t i o n i n c o l d a c c l i m a t i z e d animals  (118).  and brown f a t , w h i c h i s r i c h l y i n n e r v a t e d w i t h endings,  Muscle  sympathetic  have been proposed as s i t e s of t h i s i n c r e a s e d heat  formation  (119). From the r e s u l t s o b t a i n e d i n t h i s i n v e s t i g a t i o n no  d e f i n i t e c o n c l u s i o n c o u l d be drawn c o n c e r n i n g a c t i v i t y i n r a t s exposed to c o l d . hydroxylase NA  The  hydroxylase  increase i n tyrosine  a c t i v i t y per a d r e n a l would i n d i c a t e an i n c r e a s e i n  and a d r e n a l i n e s y n t h e s i s i n t h a t g l a n d .  w i t h NA,  tyrosine  The  turnover  studies  however, c o u l d not s u b s t a n t i a t e t h i s i n d i c a t i o n because  c o n s i s t e n t c o n t r o l v a l u e s f o r NA  t u r n o v e r were not  obtained.  There a r e s e v e r a l p o s s i b l e r e a s o n s f o r the d i s c r e p e n c i e s between the v a l u e s o b t a i n e d w i t h the two i n h i b i t o r s .  The doses may  have  been i n s u f f i c i e n t to produce complete i n h i b i t i o n of the enzyme (the dose of a*- - m e t h y l - p - t y r o s i n e NA  levels i n heart).  lase i n h i b i t o r  used was  -Methyl-m-tyrosine  i s a l s o a Dopa decarboxy-  (120) w h i l e C(-methy-p-tyrosine  d i f f e r e n c e i n a c t i o n may  i n s u f f i c i e n t to lower  have been a f a c t o r .  i s n o t and The  this  formation  of  <^-methyl NA from the i n h i b i t o r s i s a l s o a p o s s i b i l i t y  (121).  This  m e t a b o l i t e c o u l d i n t e r f e r e w i t h s t o r a g e s i t e s of the CA as w e l l as i n t e r f e r e w i t h the a n a l y s i s .  I n a d r e n a l , h e a r t and  t h e r e were a l s o i n d i c a t i o n s t h a t the t i s s u e was the i n h i b i t o r s a f t e r 2 - 4 h o u r s .  spleen  r e c o v e r i n g from  Other i n v e s t i g a t o r s (122,  have r e p o r t e d much l o n g e r h a l f - l i v e s , i ' i n the order of 300 f o r a d r e n a l s our work i s n o t comparable to t h e i r s . (124) has  shown t h a t two  t h i r d s of the CAs  p e r i p h e r y so i t i s p r o b a b l e  123)  hours,  Glowinski  i n u r i n e a r e from the  t h a t a l a r g e p a r t of the excess  CAs  i n u r i n e of c o l d a c c l i m a t i z e d r a t s i s from p e r i p h e r a l t i s s u e . Our r e s u l t s i n d i c a t e t h a t some may  come from the a d r e n a l .  It  does not appear to come from h e a r t or s p l e e n , but^rown f a t and o t h e r s y m p a t h e t i c a l l y i n n e r v a t e d t i s s u e s , not c o n s i d e r e d t h i s i n v e s t i g a t i o n , may  be an i m p o r t a n t  in  source.  Even though no changes i n t y r o s i n e h y d r o x y l a s e (Table 7) or i n t u r n o v e r r a t e of NA  ( F i g . 16 and  activity  17) were o b t a i n e d  w i t h b r a i n t i s s u e from r a t s exposed to c o l d , the b r a i n cannot be e x c l u d e d  as i m p o r t a n t  regulatory capacity. hypothalamus and temperature.  i n c o l d adaption, probably  in a  I t has been suggested (87) t h a t NA i n the  lower b r a i n stem i s i m p o r t a n t  i n c o n t r o l of body  S i n c e i t i s thought to have a hypothermic  e f f e c t i t might be expected t h a t the h y p o t h a l a m i c NA might be d e c r e a s e d or i t s a c t i o n be i n h i b i t e d d u r i n g c o l d a d a p t i o n . The hypothermic a c t i o n of NA I s s u p p o s e d l y c o n f i n e d to v e r y l i m i t e d b r a i n a r e a s and measurements of CA c o n c e n t r a t i o n tyrosine hydroxylase  a c t i v i t y i n whole b r a i n may  changes i n these s p e c i f i c a r e a s . /; A  not  reflect  From the d i s t r i b u t i o n s t u d i e s  (Table 9) i t can be seen t h a t the hypothalamus and a r e o n l y l / 2 0 t h and  pons-medulla  l / 1 0 0 t h as a c t i v e as the c a u d a t e , and  i n a c t i v i t y i n these areas would have l i t t l e e f f e c t on of t y r o s i n e h y d r o x y l a s e tyrosine hydroxylase t i s s u e to produce CA, are not known (125).  i n whole b r a i n .  may  and  changes  activity  A l s o i n v i t r o assays of  o n l y measure maximum c a p a c i t y of  s i n c e a l l the f a c t o r s a f f e c t i n g CA  the synthesis  An a l t e r e d i n v i v o a c t i v i t y of enzyme  m i g h t n o t be d e t e c t e d by i n v i t r o measurement.  G o l d s t e i n (126)  has r e p o r t e d a s l i g h t i n c r e a s e i n t u r n o v e r of NA i n b r a i n of r a t s i n the c o l d . An i n t e r e s t i n g a s p e c t o f the t u r n o v e r s t u d i e s o f NA i n b r a i n i s the p o s s i b i l i t y t h a t t h e r e a r e 2 t u r n o v e r r a t e s f o r t h i s CA.  The break i n the curves  ( F i g . 5) may r e p r e s e n t r e c o v e r y from  the i n h i b i t o r , b u t c o u l d a l s o be a t t r i b u t e d t o a second t u r n o v e r r a t e s ; G l o w i n s k i (22) has r e p o r t e d two t u r n o v e r r a t e s f o r NA of rat brain ( t  x  = 3 h r s and 17 h r s ) .  S i n c e the t j 's o b t a i n e d a r e  of the same o r d e r o f magnitude (eg. i n one case 3.8 and 18 h r s ) i t i s p o s s i b l e t h a t t h i s phenomena i s b e i n g observed.  The two  t u r n o v e r r a t e s would i n d i c a t e two s t o r e s of NA; one s t o r e b e i n g labile  ( t  x  = 3 h r s ) and p r o b a b l y i n t h e c y t o p l a s m as unbound NA;  and the second s t o r e more t i g h t l y bound, p o s s i b l y i n the v e s i c l e s i n n o a d r e n e r g i c nerve e n d i n g s , and r e l e a s e d more g r a d u a l l y d u r i n g neuronal  4.  activity.  Tyrosine Hydroxylase A c t i v i t y i n B r a i n a)  Distribution L i k e the CA t y r o s i n e h y d r o x y l a s e has a v e r y d e f i n i t e  d i s t r i b u t i o n i n b r a i n (compare Table 1 and Table 9 ) .  DA i s  i n h i g h c o n c e n t r a t i o n i n the caudate s e p t a l a r e a and n u c l e u s accombens; i n moderate c o n c e n t r a t i o n i n the hypothalamus and m i d b r a i n and i n low c o n c e n t r a t i o n i n the pons, medulla thalamus,  hippocampus, c e r e b e l l u m and c o r t e x .  oblongata,  NA i s h i g h i n  c o n c e n t r a t i o n i n the hypothalamus, moderate i n c o n c e n t r a t i o n i n the s t r i a t u m and medulla o b l o n g a t a and low i n c o n c e n t r a t i o n i n the c o r t e x and c e r e b e l l u m .  T y r o s i n e h y d r o x y l a s e has a h i g h a c t i v i t y  t h e r e f o r e and appears t o be more c l o s e l y r e l a t e d t o d i s t r i b u t i o n of DA r a t h e r than NA.  The most a c t i v e r e g i o n o f the s e p t a l area  ( a n t e r i o r s e p t a l a r e a ) may c o r r e s p o n d t o p a r t s of the tuberculum o l f a c t o r u m w h i c h i s h i g h i n DA and/or the n u c l e u s of the d i a g o n a l band o f B r o c a .  The s e p t a l a r e a , o t h e r than t h e c l a s s i c a l s e p t a l  n u c l e i , w h i c h has l i t t l e  tyrosine hydroxylase a c t i v i t y , i s a poorly  defined region.  T h e r e f o r e i t i s d i f f i c u l t to r e l a t e the  high  t y r o s i n e h y d r o x y l a s e a c t i v i t y t h e r e to a s p e c i f i c s t r u c t u r e . The w i t h other The  t y r o s i n e h y d r o x y l a s e d i s t r i b u t i o n i s i n agreement  i n v e s t i g a t i o n of CA s y n t h e s i s i n v a r i o u s r e g i o n s of b r a i n .  i n v i v o s y n t h e s i s of NA  i n b r a i n v a r i e s from 234 mug/g/hr i n  hypothalamus to 33 mpg/g/hr i n c o r t e x , and r a t e of DA caudate from p r e l i m i n a r y r e p o r t s  synthesis  in  (31) appears to be much h i g h e r .  I n j e c t i o n of the r a d i o a c t i v e p r e c u r s o r  t y r o s i n e , a l s o shows  d i f f e r e n t r a t e s of s y n t h e s i s ; the s t r i a t u m b e i n g more a c t i v e w i t h DA as the major p r o d u c t (10). NA  Radioactive  DA  i s c o n v e r t e d to  i n the amygdala and hypothalamus but n o t i n the  striatum,  i n d i c a t i n g a d i f f e r e n t type of CA s y n t h e s i s i n v a r i o u s r e g i o n s b r a i n as w e l l as d i f f e r e n t degree ( 3 1 ) .  Work w i t h b r a i n s l i c e s  and homogenates showed s i m i l a r d i s t r i b u t i o n of CA s y n t h e s i s I t had  of  (127).  p r e v i o u s l y been demonstrated t h a t t y r o s i n e  hydroxylase a c t i v i t y i s contained s u b s t a n t i a t e t h i s , i t has  now  i n nerve endings (37).  To  been shown t h a t t y r o s i n e h y d r o x y l a s e  i s most a c t i v e i n areas c o n t a i n i n g a l a r g e number of nerve endings (eg. caudate and  septum) as compared to the a r e a  c e l l b o d i e s (eg. pons, m e d u l l a and m i d b r a i n ) .  containing  A l s o there i s a l o s s  of t y r o s i n e h y d r o x y l a s e a c t i v i t y when axons l e a d i n g to these areas are c u t (see l e s i o n s t u d i e s ) .  I t has  t y r o s i n e h y d r o x y l a s e or a p r e c u r s o r  been suggested (127)  i s transported  that  f r o m the  cell  b o d i e s to the nerve endings by axon f l o w . The increases  s i g n i f i c a n c e of t h i s l o c a t i o n i s emphasized by  found i n t y r o s i n e h y d r o x y l a s e a c t i v i t y i n the areas of  n e r v e endings ( i . e . caudate,' s e p t a l a r e a , hippocampus and d u r i n g development of the b r a i n The  enzyme a c t i v i t y may  amygdala),  (128). be an i n d i c a t i o n of the  of nerve endings or of u s e f u l dopaminergic and synapses.  the  formation  noradrenergic  I t i s perhaps of i n t e r e s t t h a t m e n t a l l y  retarded  i n d i v i d u a l s do not respond to amphetamine, a drug t h a t i s thought to a f f e c t the CNS b)  by i t s a c t i o n on CA c o n t a i n i n g  As a c o n t r o l of CA  synthesis  synapses,  78.  Although Udenfriend  (28) has proposed  that tyrosine  h y d r o x y l a s e i n the r a t e l i m i t i n g s t e p i n the s y n t h e s i s of  CA,  i t can not be assumed t h a t i t i s the o n l y c o n t r o l l i n g f a c t o r i n their formation.  Some p o t e n t i n v i v o and i n v i t r o i n h i b i t o r s of  t y r o s i n e h y d r o x y l a s e do not cause a p r o p o r t i o n a l decrease i n CA content of t i s s u e (43).  Chlorpromazine  i s r e p o r t e d (129,130) to  i n c r e a s e t y r o s i n e h y d r o x y l a t i o n i n v i v o , but has no e f f e c t on tyrosine hydroxylase a c t i v i t y i n v i t r o  (42).  DA neurons  more r a p i d l y from ff^ m e t h y l - p - t y r o s i n e i n h i b i t i o n than do  recover NA  t e r m i n a l s ( 7 ) , and d i s u l f u r a m (dopamine p-oxidase i n h i b i t o r ) i s more e f f e c t i v e i n r e d u c i n g NA l e v e l s than <\-methyl-p-tyrosine T h e r e f o r e t h e r e may mine B-oxidase  (131).  be another c o n t r o l of NA s y n t h e s i s a t the dopa-  stage.  I n a s s e s s i n g the importance  of t y r o s i n e h y d r o x y l a s e  c a p a c i t y i n the c o n t r o l of CA s y n t h e s i s , i t i s of i n t e r e s t to compare v a l u e s f o r the i n v i t r o enzyme a c t i v i t y of v a r i o u s areas of b r a i n s w i t h the i n v i v o s y n t h e s i s as o b t a i n e d by G l o w i n s k i  (9,30,31,32).  As shown i n Table 18, t h e r e i s good agreement between the r e l a t i v e i n v i v o s y n t h e s i s r a t e s , and itiesv  the i n v i t r o t y r o s i n e h y d r o x y l a s e  activ-  T y r o s i n e h y d r o x y l a s e i s p r o b a b l y a b e t t e r i n d i c a t i o n of  CA a c t i v i t y i n v a r i o u s r e g i o n s of b r a i n than CA c o n c e n t r a t i o n s which do not n e c e s s a r i l y r e p r e s e n t t u r n o v e r , the c e r e b e l l u m , f o r example, has a v e r y low l e v e l of CA but has a f a i r l y r a p i d t u r n o v e r . However, when a b s o l u t e v a l u e s of CA i n v i v o  s y n t h e s i s and  t y r o s i n e h y d r o x y l a s e a c t i v i t y are c o n s i d e r e d the comparison so s t r a i g h t f o r w a r d .  i s not  U s i n g G l o w i n s k i s f i g u r e s t h a t DA c o n c e n t r a t i o n 1  i n the s t r i a t u m i s 7.5 ^ig/gm, w i t h a h a l f - l i f e of 1-4 h o u r s , i n v i v o synthesis;:would be 937  the  - 3750 mpg/gm/hr; f o r hypothalamus  the f o r m a t i o n of NA i s 234 mug/gm/hr.  A c c o r d i n g to t y r o s i n e  h y d r o x y l a s e a c t i v i t y f i g u r e s about 15,000 miag/gm/hr of DA c o u l d be produced i n the s t r i a t u m and about 750 mug/gm/hr of NA i n the hypothalamus.  T h e r e f o r e the i n v i t r o a c t i v i t y of t y r o s i n e  h y d r o x y l a s e i s 3 to 16 times g r e a t e r than t h a t r e q u i r e d f o r i n v i v o synthesis.  Udenfriend's  (27) s u g g e s t i o n t h a t the h y d r o x y l a t i o n  TABLE 18 RELATIVE TYROSINE HYDROXYLASE ACTIVITIES AND NORADRENALINE TURNOVER RATES IN RAT BRAIN  Area  Relative Tyrosine Hydroxylase  Relative NA Turnover  Hypothalamus  3.7  5.6  Medulla  2.2  2.1  Cortex  2.3.  0.9  Hippocampus  1.3  0.8  Cerebellum  1.0  1.0  based on r e f e r e n c e 31  80.  i s the r a t e l i m i t i n g s t e p i s based on the much lower K  f o r the  h y d r o x y l a t i o n as compared w i t h the d e c a r b o x y l a s e or w i t h dopamine B - o x i d a s e .  But i f t h e r e i s an e x c e s s o o f enzyme over  t h a t r e q u i r e d t h e r e must be some o t h e r f a c t o r s or f a c t o r controlling i t s activity. i n h i b i t i o n by the CAs.  One  e x c e l l e n t p o s s i b i l i t y i s feedback  From work done i n the p e r i p h e r y  S t j a r n e (132,133) proposed  t h a t the NA c o n t e n t of  nerve s t a y s r e l a t i v e l y c o n s t a n t  sympathetic  through v a r y i n g degrees of  a c t i v i t y because of the i n c r e a s e d s y n t h e s i s r e s u l t i n g from removal o f the i n h i b i t o r NA.  T h i s i n h i b i t i o n may  t y r o s i n e h y d r o x y l a s e or dopamine B-oxidase  level.  the  be a t the NA and  DA  i n h i b i t b r a i n t y r o s i n e h y d r o x y l a s e by about 40% and 70% -4 r e s p e c t i v e l y a t 10 M ( 4 2 ) . Normal CA c o n c e n t r a t i o n s i n nerve endings have been e s t i m a t e d to be i n the o r d e r of 8000 ug/gm -2 (134) i . e . about 10  M but they are d i l u t e d out d u r i n g the  homogenizing p r o c e s s .  Other p o s s i b l e l i m i t i n g f a c t o r s i n v i v o  activity are:^ 1) may  a r e l a t i v e l a c k of c o f a c t o r or p r e c u r s o r t h a t become more r e a d i l y a v a i l a b l e a f t e r homogenation;  2)  n o n - i d e a l c o n d i t i o n i n v i v o (eg. pH),  3)  some endogenous i n h i b i t o r o t h e r than the  I t has been r e p o r t e d t h a t s l i c e s of s t r i a t u m show lower  CA. activity  than homogenates; whereas some o t h e r areas had g r e a t e r a c t i v i t y in slices  (127).  T h i s may  i n d i c a t e t h a t the s t a t e of the t i s s u e  i s important i n t y r o s i n e hydroxylase  activity.  An attempt was made i n t h i s i n v e s t i g a t i o n , to  determine  i f a l t e r i n g CA l e v e l s , by d r u g s , changed t y r o s i n e h y d r o x y l a s e a c t i v i t y as measured i n v i t r o . t h i s may  be due  The r e s u l t s were n e g a t i v e but  to the d i l u t i o n f a c t o r mentioned above.  The f a c t t h a t the hypothalamus i s h i g h i n NA c o n t e n t but _ l a t i v e l y low i n t y r o s i n e h y d r o x y l a s e suggests t h a t the enzyme i n the hypothalamus i s w o r k i n g c l o s e r to i t s maximum c a p a c i t y than i n the caudate.  T h i s may  be r e l a t e d to feedback i n h i b i t i o n s i n c e  DA i s a b e t t e r enzyme i n h i b i t o r i n v i t r o than NA and t h e r e i s a  81. h i g h e r c o n c e n t r a t i o n o f DA i n the caudate than i n the hypothalamus. Tyrosine hydroxylase i s therefore a b e t t e r i n d i c a t i o n of CA a c t i v i t y than CA l e v e l s b u t f o r a t r u e i n d i c a t i o n o f CA s y n t h e s i s , o t h e r p o s s i b l e f a c t o r s must a l s o be c o n s i d e r e d . S i n c e the CA and t y r o s i n e h y d r o x y l a s e have a s p e c i f i c d i s t r i b u t i o n i n b r a i n , i t i s assumed t h a t they have a s p e c i f i c function.  The CAs have been connected w i t h emotion, b e h a v i o r and  l e a r n i n g b u t i t f i s d i f f i c u l t t o l o c a t e these a n a t o m i c a l l y .  Perhaps  however, t h e r e i s some r e l a t i o n s h i p between t h e h i g h c o n c e n t r a t i o n of DA, l a r g e c a p a c i t y t o s y n t h e s i s CA and h i g h t u r n o v e r r a t e i n the caudate and i t s p o s s i b l e r o l e i n motor f u n c t i o n .  On the o t h e r  hand, the hypothalamus w i t h i t s h i g h NA c o n c e n t r a t i o n b u t lower c a p a c i t y t o form CA and s m a l l e r t u r n o v e r r a t e s , appears t o bed p r i m a r i l y concerned  5.  w i t h autonomic f u n c t i o n .  Tyrosine Hydroxylase a)  C o n t a i n i n g F i b e r s i n Cat B r a i n  General c o n s i d e r a t i o n As i n d i c a t e d i n t h e r e s u l t s (Table 13 - 17) the caudate  and s e p t a l area were the p r i m a r y r e g i o n s a f f e c t e d by the l e s i o n s , i n m i d b r a i n s t r u c t u r e s o f the c a t . these areas c o u l d be determined  Decreases i n a c t i v i t y i n  w i t h c o n f i d e n c e s i n c e the v a r i a t i o n  around the mean f o r normal animals i s r e l a t i v e l y s m a l l ( 10 - 2 0 % ) . Most of the d i s c u s s i o n w i l l be concerned I n many o t h e r areas i t was d i f f i c u l t  w i t h t r a c t s t o these  t o determine  areas  changes s i n c e the  counts f o r the c o n t r o l s were v e r y low and t h e r e was a l a r g e v a r i a t i o n around the nornu .  However where t h e r e were l a r g e and  c o n s i s t e n t changes such as found i n the amygdala and hippocampus, which are probably  meaningful.  Most o t h e r workers i n t h i s f i e l d l e a v e the animals weeks b e f o r e s a c r i f i c i n g (49,59). t h a t up t o 80%, decreases  I t was found i n t h i s  2-10  study  c o u l d be o b t a i n e d j i n t y r o s i n e h y d r o x y l a s e  a c t i v i t y w i t h i n 72 hours o f l e s i o n i n g and the r e s u l t s were the same as i n animals w i t h s i m i l a r l e s i o n s l e f t f o r 6 days o r 3 weeks. So f o r / t h e m a j o r i t y o f animals  t h e r e was 72 hours between the time  82.  of  l e s i o n i n g and s a c r i f i c i n g .  made i t d i f f i c u l t  The s h o r t i n t e r v a l however,  t o observe c h r o m o l y s i s or e x t e n s i v e r e t r o g r a d e  d e g e n e r a t i o n d i - f f - i c u i t ^ so no r e s u l t s as t o p o s s i b l e l o c a t i o n o f c e l l b o d i e s c o u l d be p r e s e n t e d .  Inconsistent r e s u l t s obtained  w i t h CA measurements may a l s o be accounted between l e s i o n i n g and s a c r i f i c i n g .  f o r the s h o r t i n t e r v a l  Moore and H e l l e r (57)  have shown the amines do n o t b e g i n t o decrease u n t i l the t h i r d day a f t e r l e s i o n i n g and t h a t t h e r e i s a g r a d u a l r e d u c t i o n u n t i l the 12th day.  They a l s o claimthate. W a l l e r i a n d e g e n e r a t i o n i s complete  a f t e r t h r e e days.  The i n d i c a t i o n then i s t h a t the nerve  degenerates  r a p i d l y and t h a t t y r o s i n e h y d r o x y l a s e i s l o s t as r a p i d l y as the nerve i s d e s t r o y e d b u t t h a t CAs a r e s t i l l p r e s e n t and undergo a more gradual destruction.  Moore and H e l l e r argue t h a t the slowness of  the CA decrease as compared to v i s i b l e n e u r o n a l indicating a transynaptic effect.  degeneration  The Swedish workers (49)  m a i n t a i n , however, t h a t the f i b e r s a r e d i r e c t b u t a r e so s m a l l t h a t the d e g e n e r a t i o n cannot be o b s e r v e d .  The f i n d i n g s i n t h i s  s t u d y would be i n agreement w i t h the l a t t e r b)  theory,  F i b e r s i n the d i e n c e p h a l o n i)  Mid-diencephalon  From the r e s u l t s i n Table 13, the f o l l o w i n g c o n c l u s i o n s can be drawn: 1.  CA c o n t a i n i n g f i b e r s g o i n g to the caudate and s e p t a l  a r e a a r e p r e s e n t a t the l e v e l o f the mid t h i r d o f the d i e n c e p h a l o n . 2.  f i b e r s g o i n g t o the caudate  a r e l a t e r a l to those  g o i n g to the s e p t a l a r e a . 3.  f i b e r s t o the caudate may be g o i n g p r i m a r i l y to the  medial p a r t . 4.  a t t h i s l e v e l a t l e a s t 8 5 % of the f i b e r s t o the  s e p t a l a r e a can be account f o r . T h e r e f o r e i n t h i s l a t e r a l h y p o t h a l a m i c r e g i o n o f the midd i e n c e p h a l o n t h e r e i s some d i s t i n c t i o n between caudate f i b e r s  (lateral)  and s e p t a l area ( m e d i a l ) , b u t the s e p a r a t i o n i s i n c o m p l e t e . Moore and H e l l e r (58) a l s o o b t a i n e d decreases i n NA and dopa d e c a r b o x y l a s e i n the caudate and s e p t a l areas w i t h l e s i o n s i n  83.  the same a r e a ( l a t e r a l h y p o t h a l a m i c , complete MFB),  t r a n s e c t i o n of the  as w e l l as decreases i n the c i n g u l a t e g y r u s , h y p o t h a l a m i c  r e g i o n , hippocampus and amygdala.  No c o n s i s t e n t changes i n  t y r o s i n e h y d r o x y l a s e were o b t a i n e d f o r these l a t t e r a r e a s . The r e s u l t s o f t h i s i n v e s t i g a t i o n i n the d i e n c e p h a l o n c o r r e l a t e d w i t h the f i n d i n g s of Swedish workers l e s i o n s of the MFB  (49,50).  With  a t the l e v e l s of the a n t e r i o r commissure,  they o b t a i n e d decreases i n DA i n the tuberculum o l f a c t o r u m and n u c l e u s accombens, as w e l l as NA changes i n the  hypothalamus,  p r e o p t i c a r e a , s e p t a l a r e a and c i n g u l a t e g y r u s , but the s t r i a t u m showed no change. l e a v e the MFB  They c o n s i d e r t h a t the f i b e r s to the  caudate  i n the p o s t e r i o r d i e n c e p h a l o n to e n t e r the i n t e r n a l  c a p s u l e and t e r m i n a t e p r i m a r i l y i n the l a t e r a l and v e n t r o m e d i a l c a u d a t e , but they do n o t account f o r f i b e r s to the d o r s o m e d i a l caudate.  Our r e s u l t s would i n d i c a t e t h a t the f i b e r s t o the  go to a t l e a s t the m i d - d i e n c e p h a l o n  and t h a t these f i b e r s may  g o i n g p r i m a r i l y to the m e d i a l caudate. w i t h P o i r i e r and Sourkes  (53) who  caudate be  T h i s would be i n agreement  d e s c r i b e DA f i b e r s to the s t r i a t u m  of c a t s and monkeys g o i n g more r o s t r a l l y to those d e s c r i b e d by the Swedes.  Using c l a s s i c a l neuroanatomical techniques f i b e r s i n  the M F B , l a t e r a l h y p o t h a l a m i c r e g i o n have been shown to go to the c a u d a t e , d i a g o n a l band of B r o c a , hypothalamus, hippocampus, amygdala and thalamus  septal nuclei,  (64,65,66).  I n attempts to c o r r e l a t e these types of l e s i o n s w i t h b e h a v i o r , i t was  found t h a t l e s i o n s of the MFB  esdape l a t e n c y time d u r i n g l e a r n i n g ii)  Posterior  caused i n c r e a s e d  (135,136).  diencephalon  L e s i o n s i n the c a u d a l p o r t i o n of the d i e n c e p h a l o n a t the b e g i n n i n g of the MFB  d i d not s e p a r a t e f i b e r s g o i n g to the caudate  from those to the s e p t a l a r e a .  Even w i t h v e r y p r e c i s e l e s i o n s  as shown i n F i g . 20 m e d i a l l y or l a t e r a l l y p l a c e d l e s i o n s  produced  s i m i l a r decreases i n t y r o s i n e h y d r o x y l a s e i n b o t h areas (Table 12). AT t h i s l e v e l i n r a t s the Swedish  group (62) were a b l e to determine  NA f i b e r s to the d i e n c e p h a l o n were most m e d i a l l y l o c a t e d , t h a t DA to n u c l e u s accombens and tuberculum o l f a c t o r u m were s l i g h t l y  84.  l a t e r a l t o the NA f i b e r s and t h a t these DA f i b e r s t o the caudate were most l a t e r a l l y l o c a t e d .  We c o u l d n o t c o n f i r m these f i n d i n g s .  AT l e a s t 807o of. the f i b e r s t o the caudate  and s e p t a l  a r e a must pass through the F i e l d s o f F o r e l r e g i o n ; the m a j o r i t y o f caudate f i b e r s presumably have n o t y e t l e f t the MFB t o e n t e r t h e i n t e r n a l c a p s u l e as they a r e thought t o do (49). True habenular caudate  l e s i o n s appeared  t o have no e f f e c t on  and s e p t a l a r e a t y r o s i n e h y d r o x y l a s e .  Classical  n e u r o - a n a f o m i c a l methods have shown t'some r e c i p t o c a l between t h e habenularand  connections  s e p t a l n u c l e i (but these n u c l e i a r e  r e l a t i v e l y i n a c t i v e i n t y r o s i n e h y d r o x y l a s e ) ( T a b l e 12).  Those  l e s i o n s w h i c h r e s u l t e d i n a change were n o t t r u e h a b e n u l a r  lesions  but p r o b a b l y extended more v e n t r a l l y i n t o the F i e l d s o f F o r e l . I t i s o f i n t e r e s t t h a t i n 3 o f t h e 4 c a t s t h e r e were 507, d e c r e a s e s i n s e p t a l a r e a t y r o s i n e h y d r o x y l a s e w i t h no change i n the caudate. These l e s i o n s were more d o r s a l than those d e s c r i b e d p r e v i o u s l y , so f i b e r s g o i n g t o t h e s e p t a l a r e a might be more d o r s a l than those t o the  caudate. In b o t h these p o s t e r i o r d i e n c e p h a l o n l e s i o n s , ( F i e l d s o f  F o r e l and habenula) t h e r e appeared  to be s i g n i f i c a n t and  c o n s i s t e n t changes i n amygdala and hippocampus t y r o s i n e h y d r o x y l a s e . R e c i p r o c a l c o n n e c t i o n s between these areas and the r e t i c u l a r f o r m a t i o n have been d e s c r i b e d (66,65).  Some o f these may be c r o s s e d , as a  p o s s i b l e e x p l a n a t i o n f o r t h e decreases lesioned side.  i n a c t i v i t y o b t a i n e d on the  As p r e v i o u s l y mentioned Moore and H e l l e r and the  Swedish groups have d e s c r i b e d changes i n CA c o n c e n t r a t i o n s i n these areas w i t h MFB l e s i o n s . c)  Fiberstinrthe.-midbrain' The m i d b r a i n a r e a would appear t o be t h e c r i t i c a l r e g i o n  for  the o r i g i n o f CA c o n t a i n i n g f i b e r s E n d i n g i n the caudate and  s e p t a l area. i)  To the Caudate  From these r e s u l t s i t would appear t h a t the s u b s t a n t i a n i g r a i s the o r i g i n o f 80 - 907. o f the f i b e r s t o the caudate. L e s i o n s c a u d a l t o t h e s u b s t a n t i a n i g r a eg. c e r v e a u - i s o l e , produced  85. no s i g n i f i c a n t changes i n t y r o s i n e h y d r o x y l a s e  of t h e c a u d a t e ,  thus e s t a b l i s h i n g t h e c a u d a l e x t e n t o f origin o f f i b e r t o t h e nucleus.  Some m i d l i n e l e s i o n s of the m i d b r a i n  that did not  a f f e c t the s u b s t a n t i a n i g r a c o u l d d e c r e a s e caudate t y r o s i n e hydroxyl a s e 20 - 3 0 % o f n o r m a l , t h e f i b e r s a p p a r e n t l y  course m e d i a l l y  f r o m t h e s u b s t a n t i a n i g r a as they a l s o go r o s t r a l l y . Using  t h e i r H s t o c h e m i c a l techniques  on r a t s the  Swedish groups (51) demontrated t h i s n i g r o - s t r i a t a l DA t r a c t from t h e s u b s t a n t i a n i g r a t h r o u g h t h e c r u s c e r e b r i i n t o the c a u d a l MFB and then t u r n i n g l a t e r a l through the i n t e r n a l to t h e caudate and putamen.  capsule  Theyfound a d i r e c t r e l a t i o n s h i p  between t h e e x t e n t of s u b s t a n t i a n i g r a d e s t r u c t i o n and l o s s o f DA t e r m i n a l s . general  P o i r i e r and Sourkes (51,52) c o n f i r m e d  t r a c t i n t h e monkeys and c a t s .  this,  They g i v e t h e o r i g i n o f  the t r a c t as t h e s u b s t a n t i a n i g r a and p a r a b r a c h i o l e  pigmentosis  and p l a c e i t s p o s i t i o n more d o r s o l a t e r a l l y than t h a t d e s c r i b e d by the Swedes and s u g g e s t t h a t i t may extend more r o s t r a l l y i n t o the diencephalon.  G o l d s t e i n (71,72,73) made l e s i o n s i n the v e n t r o l a t e r a l  tegmentum o f the m i d b r a i n hydroxylase  and showed d e c r e a s e s i n t y r o s i n e  a c t i v i t y , i n v i v o DA s y n t h e s i s and DA uptake on the  i p s i l a t e r a l side. I t i s however, d i f f i c u l t t o s u b s t a n t i a t e these n i g r o s t r i a t a l tracts anatomically. connections  from the m i d b r a i n  Nauta (63) has d e s c r i b e d t o caudate and putamen.  Other  r e p o r t s (60,61) i n d i c a t e t h a t l e s i o n s o f the s u b s t a n t i a n i g r a cause degeneration  only i n the r e d nucleus,  s u p e r i o r c o l l i c u l i and globus  pallidus. The  great irterest i n t h i s n i g r o - s t r i a t a l t r a c t o r i g i n a t e s  from i t s p o s s i b l e c o n n e c t i o n w i t h P a r k i n s o n i s m ;  and experiment have  been done w i t h animals i n an attempt to r e l a t e P a r k i n s o n i s m , nigrostriatal lesions.  NA and  I n r a t s , l e s i o n s a l o n e ^ d i d n o t produce  P a r k i n s o n - l i k e symptoms b u t when r e s e r p i n e was a l s o  .  administered  there was r i g i d i t y and tremor on t h e s i d e c o n t r a l a t e r a l to t h e lesion.  It^was p o s t u l a t e d t h a t there i s a b a l a n c e between c h o l i n e r g i c  86.  and d o p a n e r g i c f i b e r s t o the s t r i a t u m .  T h i s i s r e f l e c t e d a t the  s p i n a l l e v e l i n t h e c o n t r o l o f the o u t p u t o f  and  fibers.  When one system (eg. d o p a n e r g i c ) i s u p s e t there i s h y p e r e x c i t a b i l i t y of the o\ system and r e d u c t i o n i n the V .  Poirier  (137) was a b l e  to produce P a r k i n s o n - l i k e symptoms i n monkeys w i t h l e s i o n s i n the n i g r o s t r i a t a l system.  A d m i n i s t r a t i o n o f t h e MAO i n h i b i t o r  harmaline  i n t e n s i f i e d the motor a b n o r m a l i t y b u t t h i s c o u l d n o t be r e l a t e d to any a f f e c t on the n i g r o s t r i a t a l t r a c t .  Therefore  the e x a c t  r e l a t i o n s h i p between DA, s e r o t o n i n , b a s a l g a n g l i o n and P a r k i n s o n i s m i s not y e t established. ii)  To the s e p t a l a r e a  L e s s work has been done on the o r i g i n and course f i b e r s t o the s e p t a l a r e a . t h a t two groups o f CA c e l l s  The Swedish workers (6) have p o s t u l a t e d ( p r i m a r i l y DAD i n t h e m i d b r a i n  o r i g i n o f f i b e r s t o the n u c l e u s One  o f the  a r e the  accombens and tuberculum o l f a c t o r u m .  group ( d e s i g n a t e d as A 10) i s a l o n g t h e m i d l i n e j u s t d o r s a l t o  the i n t e r p e d u n c u l a r n u c l e u s  e x t e n d i n g over most o f i t s l e n g t h ; the  second group (A8) i s j u s t l a t e r a l t o t h i s a t a p p r o x i m a t e l y level.  the same  These workers c l a i m t h a t NA f i b e r s i n the s e p t a l a r e a  o r i g i n a t e i n t h e pons and m e d u l l a r y  region.  Since  cerveau-isole  l e s i o n s (Table 17) produced l i t t l e change i n t y r o s i n e  hydroxylase  a c t i v i t y i n the s e p t a l a r e a , we a r e most l i k e l y d e a l i n g w i t h f i b e r s o r i g i n a t i n g i n the m i d b r a i n r o s t r a l t o the i n f e r i o r Up  colliculi.  t o 1007o o f the s e p t a l f i b e r s c o u l d be accounted f o r i n t h i s r e g i o n ,  depending on t h e l a t e r a l e x t e n t o f t h e i e s i o n . the c e l l b o d i e s  The f i b e r s and/or  t o t h e s e p t a l a r e a must t h e r e f o r e be a few mm from  the m i d l i n e b u t m e d i a l  to t h e f i b e r s t o t h e caudate (from the  s u b s t a n t i a n i g r a ) s i n c e i t was p o s s i b l e t o a f f e c t the s e p t a l f i b e r s without  t o u c h i n g t h e caudate ones.  Whether t h e o r i g i n o f  the s e p t a l f i b e r s i n the group A 10 c e l l s w i t h t h e f i b e r s  coursing  l a t e r a l l y and r o s t r a l l y o r whether the o r i g i n i n the A 8 c e l l s w i t h the f i b e r s t r a v e l l i n g m e d i a l l y and r o s t r a l l y i s n o t known. The  r e s u l t s o b t a i n e d c o u l d be c o n s i s t e n t w i t h e i t h e r or  both explanations.  87  d)  Summary D i a g . 3 i s a summary of the p o s s i b l e o r i g i n s and c o u r s e  of f i b e r s c o n t a i n i n g t y r o s i n e h y d r o x y l a s e t o the caudate and s e p t a l area.  The diagram i n d i c a t e s the f i b e r s to the caudate  probably  o r i g i n a t e l a r g e l y i n the s u b s t a n t i a n i g r a , t u r n m e d i a l l y as they go through the m i d b r a i n and p o s t e r i o r d i e n c e p h a l o n , where most of them c a n s t i l l be accounted f o r .  The m a j o r i t y of f i b e r s  then probably c o u r s e through t h e i n t e r n a l c a p s u l e t o the caudate, thoughlisomeawould  appear to c o u r s e more a n t e r i o r l y to the midp  d i e n c e p h a l o n b e f o r e e n t e r i n g the c a u d a t e , mos.t-probablrytto the medial aspects.  The f i b e r s to the s e p t a l a r e a c o u l d o r i g i n a t e  i n those c e l l s , d e s i g n a t e d as A 10 and A 8 by the Swedish  workers,  c o u r s e m e d i a l l y or l a t e r a l l y r e s p e c t i v e l y as they go through the m i d b r a i n s l i g h t l y m e d i a l to the n i g r o s t r i a t a l f i b e r s .  I n the  p o s t e r i o r d i e n c e p h a l o n they may be i n t e r m i n g l e d o r d o r s a l t o the f i b e r s to the caudate.  However, as the two group of f i b e r s t o  s e p t a l a r e a and caudate c o u r s e through the d i e n c e p h a l o n they b e g i n t o s e p a r a t e i n t o m e d i a l and l a t e r a l p o s i t i o n s r e s p e c t i v e l y . Whether these f i b e r s j u s t d e s c r i b e d a r e DA o r NA c o n t a i n i n g i s n o t known. DA c o n t a i n i n g .  P r o b a b l y these to the caudate a r e  I f r a t s a r e comparable to c a t s , i t would appear  from the Swedish work t h a t the f i b e r s we a r e d e a l i n g w i t h are p p r i m a r i l y dopanminergic,  o r i g i n a t i n g i n the m i d b r a i n and g o i n g  to the tuberculum o l f a c t o r u m and n u c l e u s accombens.  It is difficult  to d i s t i n g u i s h NA and DA c o n t a i n i n g f i b e r s by h i s t o c h e m i c a l t e c h n i q u e s , s i n c e the o n l y means i s d i f f e r e n t r e c o v e r y times d e p l e t i o n w i t h «K^methyl-p-tyrosine.  after,  T h e r e f o r e i t cannot be  s t a t e d a t t h i s time whether a l l c e l l s of the m i d b r a i n a r e p r i m a r i l y DA c o n t a i n i n g .  I t i s perhaps  of i n t e r e s t though t h a t  t h e r e were no changes i n t y r o s i n e h y d r o x y l a s e a c t i v i t y i n the hypothalamus ( p r i m a r i l y NA c o n t a i n i n g f i b e r s ) a l t h o u g h many o t h e r r e p o r t s i n d i c a t e a decrease i n NA w i t h s i m i l a r MFB  lesion.  S i m i l a r l e s i o n s s t u d i e s w i t h the enzyme dopamine B-oxidase c o u l d d i s t i n g u i s h the NA and DA c o n t a i n i n g f i b e r s .  D i a g 2:  Summary of t y r o s i n e h y d r o x y l a s e c o n t a i n i n g f i b r e s t o the caudate and s e p t a l a r e a SA = s e p t a l a r e a  MO  medulla oblongata  CA = caudate  MB  midbrain  DE = d i e n c e p h a l o n  SC  superior  colliculi  ic"= i n t e r n a l  IC  inferior  colliculi  capsule MFB = m e d i a l f o r e b r a i n bundle FF = F i e l d s of F o r e l C l = Cerveau i s o l e  A 8, A 10, A 9 - r e f e r to groups "of CA c e l l s , (as d e s i g n a t e d by rectangles) i n midbrain as d e s c r i b e d i n r e f e r e n c e 49, t h a t a r e p o s s i b l e o r i g i n s of the f i b e r s i n the diagram.  89.  This i n v e s t i g a t i o n d e a l t p r i m a r i l y w i t h determining approximate o r i g i n s and c o u r s e s of DA and NA c o n t a i n i n g  fibers  w i t h o u t d e t e r m i n g e x a c t d o r s a l , v e n t r a l and l a t e r a l m e d i a l  positions,  i n o r d e r t o p i n p o i n t e x a c t n u c l e i o f o r i g i n and c o u r s e s o f f i b e r s , more d i s c r e t e l e s i o n s on a l a r g e r number of animals a r e required.  90.  SUMMARY AND CONCLUSIONS  The r e s u l t s o f t h i s i n v e s t i g a t i o n and p o s s i b l e c o n c l u s i o n s t h a t a r e drawn from these can be summarized as f o l l o w s : 1)  I o n exchange chromatography on Dowex 50 x 8 r e s i n / ' and  ~~  t h i n l a y e r chromatography on polyamide have p r o v e n t o be s u c c e s s f u l i n the s e p a r a t i o n o f NA and DA; t h e i r p r e c u r s o r s Dopa; and some o f t h e i r m e t a b o l i t e s . these t e c h n i q u e s  t y r o s i n e and  P o s s i b l e a p p l i c a t i o n s of  i n enzyme a n a l y s i s and i n v i v o m e t a b o l i c  studies  of CA have been d i s c u s s e d . 2)  I n c o n c l u s i v e r e s u l t s were o b t a i n e d i n d e t e r m i n i n g  the e f f e c t o f exposure t o c o l d on CA m e t a b o l i s m i n r a t s . an i n c r e a s e i n CA s e c r e t i o n i n u r i n e s o f animals  Although  i n the c o l d was  o b s e r v e d , the e x a c t s i t e a t w h i c h t h i s i n c r e a s e i n s y n t h e s i s p l a c e was n o t d e t e r m i n e d .  takes  There was some i n d i c a t i o n t h a t the  a d r e n a l gland may be one organ o f i n c r e a s e d CA s y n t h e s i s d u r i n g cold  adaption. 3)  The d i s t r i b u t i o n o f t y r o s i n e h y d r o x y l a s e  i n b r a i n was  determined and found to p a r a l l e l CA c o n c e n t r a t i o n s and i n v i v o determinations nucleus  o f CA s y n t h e s i s , w i t h h i g h e s t a c t i v i t y i n the c a u d a t e ,  accombens, s e p t a l a r e a and a n t e r i o r p e r f o r a t i n g  substance.  From these d i s t r i b u t i o n s s t u d i e s i t would appear t y r o s i n e hydroxylase  i s a good i n d i c a t i o n o f CA m e t a b o l i s m i n v a r i o u s  regions  of the b r a i n , b u t i t may n o t be the o n l y c o n t r o l . 4)  Fibers containing t y r o s i n e hydroxylase  t r a c e d i n the c a t from the m i d b r a i n through the d i e n c e p h a l o n  were t e n t a t i v e l y  ( t h e i r a p p a r e n t s i t e of o r i g i n )  t o the caudate and s e p t a l a r e a by means  of l e s i o n e x p e r i m e n t a t i o n .  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A s s o c , J .  94, 53,  DISTRIBUTION OF TYROSINE HYDROXYLASE ACTIVITY IN ADULT AND DEVELOPING BRAIN E . G . M C G E E R , S. GIBSON, J . A . WADA, AND P. L . M C G E E R Kinsmen Laboratory of Neurological Research, Faculty of Medicine, The University of British Columbia, Vancouver, B.C.  Received August 25, 1967 The distribution of tyrosine hydroxylase activity in various areas of brain was studied in mature rats, rabbits, cats, and in kittens of various ages. Distribution was closely similar in all species. Areas known to have high concentrations of catecholinergic nerve endings, such as the caudate, septal area and pineal, showed very high adult levels and sharp increases during the neonatal period. Areas such • as the pons-medulla and midbrain, known to contain predominantly catecholinergic cell bodies, showed, on the other hand, relatively low adult levels and little or no change in the neonatal period. Developmental data correlate with known neonatal changes in endogenous catecholamine levels and adult distributions correlate with known turnover rates. The results are in conformity with previous findings that tyrosine hydroxylase is concentrated largely in nerve endings and suggest that measurement of this enzymic activity may provide a more convenient and more sensitive index of catecholinergic nerve activity under various conditions than is provided by measurement of the amines themselves.  Tyrosine hydroxylase is the enzyme responsible for conversion of tyrosine to3,4-dihydroxyphenylalanine (Dopa) (1). This initial step in the biosynthesis of catecholamines is the slow step, and is therefore considered to be rate-limiting for the overall conversion in vivo (2). The enzyme has greatest activity in such tissues as the adrenal medulla and brain, which have high concentrations of catecholamines. In brain, tyrosine hydroxylase has been shown to be particle-bound, and highly localized in nerve endings (3). This biochemical information ties in closely with histochemical studies showing that noradrenaline and dopamine are also highly localized in nerve endings. Lower concentrations of these amines are found in cell bodies. The concentration of catecholamines in various brain areas, as revealed by histochemical studies, is more or less in accord with biochemical studies showing the distribution of dopamine and noradrenaline (4). However, the turnover rate of radioactive catecholamines is not equal in all brain areas and does not parallel the concentration. For example, the cerebellum has an unusually high turnover rate and yet has a very low concentration of catecholamines (5). Tyrosine hydroxylase activity should reflect a combination of turnover rate and density of catecholinergic nerve endings in any given region of brain. Consequently, measurement of tyrosine hydroxylase may provide a more sensitive index of catecholinergic nerve activity under various conditions than measurement of the amines themselves. This paper reports the distribution of tyrosine hydroxylase in various areas of the developing and mature brain. The rat, rabbit, and cat were used for the adult study, and kittens for the developmental study. Canadian Journal of Biochemistry. Volume 45 (1967)  1943  1944  C A N A D I A N J O U R N A L O F B I O C H E M I S T R Y . V O L . 45, 1967  TABLE I Tissue weights of various brain areas* Species Areaf Caudate Septal area Pons-medulla Midbrain Hypothalamus Thalamus Hippocampus Amygdala Cerebellum  Rat  Rabbit  33±6 48 ± 2 3 214±30 118±15  77±7 70±7 801±118 536±98 115±21 400±23 507 ± 7 4  J224±67 96±26 76±14 258 ± 9  1114±128  Cat 416±58 124 ± 2 6 1408 ± 1 6 3 1074±154 110± 30 1278 ± 2 4 6 582 ± 1 1 0 400 ± 6 2 3438 ± 2 0 1  * Average ± standard deviation in mg. fData on weights of cortex and spinal cord are not given since only a portion of these areas was used. For subcortical areas, weights are for tissue pooled from both brain halves.  Materials and Methods R a t s were k i l l e d b y a s h a r p b l o w to the h e a d , r a b b i t s b y c e r v i c a l d i s l o c a t i o n , a n d a d u l t cats b y n i t r o g e n a s p h y x i a t i o n . T h e b r a i n s were r a p i d l y r e m o v e d , a n d dissected i n t o the areas i n d i c a t e d i n T a b l e I a n d F i g . 1. T h e tissue w a s h o m o g e n i z e d i n 4 - 1 9 v o l u m e s of i c e - c o l d 0.25 M sucrose, the larger v o l u m e s of sucrose b e i n g used w i t h the m o r e a c t i v e b r a i n regions. T h e r e p r o d u c i b i l i t y of the d i s s e c t i o n p r o c e d u r e is i n d i c a t e d b y the d a t a o n w e i g h t s g i v e n i n T a b l e I . K i t t e n s of k n o w n ages f r o m 2 to 47 d a y s were k i l l e d b y d e c a p i t a t i o n . S i x l i t t e r s i n a l l were u s e d : f o u r l i t t e r s of f o u r k i t t e n s each, a n d t w o of three. S i b l i n g s were a l w a y s k i l l e d a t w e e k l y i n t e r v a l s (see T a b l e V for d e t a i l s ) . T h e b r a i n w a s d i v i d e d as s h o w n i n F i g . 1 e x c e p t t h a t the h y p o t h a l a m u s w a s p o o l e d  FIG. 1. Sagittal section showing some of areas dissected (numbers) and some landmarks used in dissection (letters) 1 = spinal cord, 2 = pons-medulla, 3 = midbrain, 4 = cerebellum, 5 = hypothalamus, 6 = septal area, 7 = thalamus, 8 = cortex, A = corpus callosum, B = fornix, C = optic chiasma, D = mammillary body, E = anterior commissure.  McGEER E T AL.: TYROSINE HYDROXYLASE ACTIVITY  1945  T A B L E II Endogenous tyrosine levels (Average ± standard deviation in /xg/g of tissue) Area Caudate Septal area Pons-medulla Midbrain Hippocampus Amygdala Hypothalamus Thalamus Cortex Cerebellum Spinal cord  Adult rat  Adult rabbit  Adult cat  Kitten*  28.8±10.2 16.5±6.3 17.3±3.2 15.6±4.2 22.7±7.1 23.9±5.1  27.8±5.2 24.9±3.5 24.1±10.1 25.8±4.2 28.3-1-4.0  24.6±6.5 17.6±6.0 13.4±2.8 14.3±7.0 13.2±3.2 16.1±3.5 19.8±5.5 14.5±5.7 14.8±5.1 18.9±4.6  22.6±5.7 19.4±4.7 19.4±3.8 19.1±4.7  —  23.5±5.1 25.3±6.2 31.2±4.8 22.9±8.5 22.7±3.7  J20.8±5.4 15.7±4.5 17.3±5.5 22.7±3.5  [lS.4±3.6 J20.1±4.2 20.7±4.5 23.4±4.2  —  —  *No consistent change with age was evident.  w i t h the thalamus a n d the a m y g d a l a was pooled w i t h the hippocampus. E a c h b r a i n area w a s h o m o g e n i z e d i n 5.6 v o l u m e s of sucrose. I n those cases w h e r e p i n e a l a c t i v i t y w a s m e a s u r e d , t h e p i n e a l w a s dissected o u t a n d i m m e d i a t e l y h o m o g e n i z e d i n 0.3 m l of 0.25 M sucrose. M e a s u r e m e n t of t y r o s i n e h y d r o x y l a s e w a s d o n e i n a l l cases b y t h e p r e v i o u s l y r e p o r t e d m e t h o d (6) i n w h i c h a n a l i q u o t of b r a i n h o m o g e n a t e is i n c u b a t e d w i t h r a d i o a c t i v e t y r o s i n e i n t h e presence of a D O P A d e c a r b o x y l a s e i n h i b i t o r , a n d the r a d i o a c t i v e c a t e c h o l a m i n e s f o r m e d a r e i s o l a t e d a n d c o u n t e d . 2 - A m i n o - 4 hydroxy-6,7-dimethyltetrahydropteridine ( D M P H ) i n combination with merc a p t o e t h a n o l w a s n o t u s e d r o u t i n e l y since t h i s a d r e n a l c o f a c t o r s y s t e m h a s been r e p e a t e d l y s h o w n t o h a v e n o s i g n i f i c a n t effect o n r a t b r a i n t y r o s i n e h y d r o x y l a s e a c t i v i t y (6). S i m i l a r l y , i n t h i s s t u d y , i t w a s f o u n d t o h a v e n o s i g n i f i c a n t effect o n c o n v e r s i o n s i n c a t o r r a b b i t b r a i n h o m o g e n a t e s . W i t h 11 areas of r a b b i t b r a i n , for e x a m p l e , c o n v e r s i o n w i t h c o f a c t o r w a s 109 ± 1 7 % of t h a t w i t h o u t cofactor, whereas i n 18 different b r a i n areas from s e v e r a l a d u l t cats, t h e c o n v e r s i o n w a s 9 2 ± 2 1 % of t h a t w i t h o u t . S i m i l a r c o m p a r a t i v e s t u d i e s w i t h each area i n each of four s i b l i n g k i t t e n s s t u d i e d a t 5 - 2 7 d a y s of age a g a i n i n d i c a t e d t h a t t h e D M P F U - m e r c a p t o e t h a n o l c o m b i n a t i o n caused n o s i g n i f i c a n t e n h a n c e m e n t of c o n v e r s i o n . T h e same D M P H w h i c h h a d l i t t l e o r n o effect o n t h e c o n v e r s i o n i n v a r i o u s c a t b r a i n areas increased t h e c o n v e r s i o n i n c a t a d r e n a l h o m o g e n a t e m o r e t h a n 10-fold. 4  4  A l l i n c u b a t i o n s w e r e r u n a t t y r o s i n e c o n c e n t r a t i o n s b e l o w s a t u r a t i o n of t h e e n z y m e . T h e c a l c u l a t i o n of V r e q u i r e d , therefore, k n o w l e d g e of t h e e n d o genous tissue t y r o s i n e l e v e l a n d of the M i c h a e l i s c o n s t a n t for t h e r e a c t i o n . E n d o g e n o u s t y r o s i n e l e v e l s ( T a b l e I I ) were d e t e r m i n e d b y a m o d i f i c a t i o n o f the m e t h o d of W a a l k e s a n d U d e n f r i e n d (6, 7 ) . m&K  R e p e a t e d d e t e r m i n a t i o n of M i c h a e l i s c o n s t a n t s for r a b b i t b r a i n , f o r w h o l e c a t b r a i n , a n d f o r c a t c a u d a t e y i e l d e d a n average figure of 1 X 10~ f o r K i n e a c h case. S i m i l a r results i n a l l cases were o b t a i n e d w i t h a n d w i t h o u t a d d e d D M P H p l u s m e r c a p t o e t h a n o l . R e p r e s e n t a t i v e p l o t s used i n t h e d e t e r m i n a t i o n 5  m  4  1946  C A N A D I A N J O U R N A L O F B I O C H E M I S T R Y . V O L . 45.  1967  S xio F I G . 2. Sample curves for the determination of the Michaelis constant for rabbit and cat brain. Plots drawn as recommended by Dowd and Riggs (8). Solid symbols with D M P H plus mercaptoethanol; empty symbols without. Rabbit data ( A — and A — ) obtained on two different tissue samples; cat data ( 9 — and O — ) obtained in parallel analyses on aliquots of the same brain homogenate. 4  of the Michaelis constant are given in F i g . 2. T h e previously determined Michaelis constant for whole rat brain homogenate (4.5 X 10~ ) was used in calculation of the rat data (6). The measured velocity, V , could be calculated from the amount of radioactive tyrosine converted to radioactive catecholamines. A n overall velocity, V, was related to the measured velocity by the equation: 6  meas  V =  V  SE  ~t~ Sn  m  where 5 = concentration of endogenous tyrosine and 5 = concentration of radioactive tyrosine. The V in each case could be calculated from V by the equation: E  R  m  F  max  =V 1 +  K  m  In the developmental study, the tissue homogenate remaining after removal of aliquots for enzyme and tyrosine assay was used in many cases for analysis of endogenous catecholamine levels. F o r this purpose the volume was measured, and then the homogenate was made 0.2 N in perchloric acid. T h e further procedure for isolation and determination of the catecholamines was as reported previously for brain tissue homogenates (9).  McGEER ET AL.  1947  TYROSINE H Y D R O X Y L A S E ACTIVITY  T A B L E III Distribution of tyrosine hydroxylase activity in adult rat, rabbit, and cat brain* Activity relative to cerebellum  in nmoles DOPA/h/g of tissue Area  Rat  Caudate 72.3±5.5 (6)* Septal area 15.0±2.4 (6) Amygdala 4.2±0.5 (3) Hypothalamus | 5.Oil.6 (6) Thalamus Midbrain 3.6±1.0 (6) Pons-medulla 2.9±0.3 (6) Cortex 3.1±0.8 (6) Hippocampus 1.8±0.4 (4) Spinal cord 1.2±0.7 (5) Cerebellum 1.3±0.4 (6)  Rabbit  Cat  70.9±9.2 (5) 20.3±1.6 (4)  98.5±9 (11) 19.0±5 (12) 4.2±1 (11) 3.9±0.7 (11) 2.7±0.2 (9) 3.1±0.2 (5) 1.8±0.5 (13) 2.5±0.6 (9) 1.6±0.4 (10)  —  3.7±1.0 (5) 0.6±0.1 (5) 2.2±0.5 (5) 1.7±0.2 (5) 2.2±1.0 (5) 2.1±0.6 (5) 2.2±0.9 (4) 0.7±0.2 (4)  —  1.4±0.2 (4)  Rat  Rabbit  Cat  56 11 .5 3 .2 3 .8 2..8 2..2 2..4 1.4 0..9 1.0  101 29  70 13 .5 3 .0 2..8 1..9 2 .2 1..3 1 .8 1 .1  {  —  5.3 0.9 3.1 2.4 3.1 3.0 3.1 1.0  1..0  *Number of animals given in parentheses.  Results and Discussion The data obtained on the distribution of tyrosine hydroxylase activity in various areas of adult rat, rabbit, and cat brain are summarized in Table III. It is evident that the distribution follows a similar pattern in all three species, and that tyrosine hydroxylase activity is highly concentrated in the caudate and septal area, regions with extremely high endogenous dopamine levels and very rich in catecholinergic nerve endings (10). The hypothalamus and midbrain, areas rich in noradrenaline but relatively low in dopamine, contain much less tyrosine hydroxylase than does the striatum. Since tyrosine hydroxylase activity may be expected to correlate with total catecholamine turnover rather than endogenous levels, it is interesting to compare the relative activities found with such estimates of relative turnover as are available. Iversen and Glowinski (5) have estimated noradrenaline turnover rates in five areas of rat brain from data on disappearance rates for injected H-noradrenaline and from endogenous noradrenaline levels. Their estimates, expressed as relative to estimated cerebellar turnover, are given in Table IV along with the relative tyrosine hydroxylase activities found for these areas. The agreement, except for the cortex, is remarkably close. Striatal tyrosine hydroxylase levels might be expected to reflect dopamine rather than noradrenaline turnover. Iversen and Glowinski (5), using two different methods, arrive at a half-life for striatal dopamine of 1.5-4 h, and Glowinski and Iversen (11) report endogenous striatal dopamine levels in the rat of 7.5 yug/g. These data would correspond to a turnover rate of 6-16 nmoles of dopamine per gram per hour, or approximately 23-62 times the turnover rate estimated by these authors for noradrenaline in the cerebellum. This ratio again appears to be of the same order of magnitude as the ratio of striatal tyrosine hydroxylase activity to that of cerebellum. It must be pointed out that, although the relative data are in general agreement, the absolute conversions indicated by the tyrosine hydroxylase data are 3  1948  C A N A D I A N J O U R N A L OF BIOCHEMISTRY. VOL.  45, 1967  T A B L E IV Relative tyrosine hydroxylase activities and noradrenaline turnover rates in rat brain areas Relative tyrosine hydroxylase Hypothalamus Medulla Cortex Hippocampus Cerebellum  >3.8f 2.2 2.4 1.4 1.0  Relative NA turnover* 5.6 2.1 0.9 0.8 1.0  *Based on data of Iversen and Glowinski (5). tGiven as > 3.8 because the hypothalamic activity in rats was undoubtedly diluted by the thalamus which has a lower order of activity (see Table III).  five- to sixfold greater than those indicated by the turnover rates. The tyrosine hydroxylase figures given, of course, are for F under in vitro conditions where feedback inhibition by product amine would be negligible, and where saturation of the enzyme with tyrosine could be assumed. This discrepancy, however, accords with other indications that it is not enzyme capacity but rather enzyme utilization that is the rate-limiting factor of in vivo catecholamine synthesis. Data obtained in the developmental study generally confirmed the results on distribution. However, there were some significant changes with postnatal age in certain areas which accord with previous data of Pscheidt and Himwich (12). They reported that many regions of cat brain show a progressive increase in serotonin and noradrenaline levels with increasing postnatal age and that each of these regions has a distinctive pattern of accumulation of amines. The data for tyrosine hydroxylase for six areas (caudate, pineal, septal, ponsmedulla, cortex, and amygdala-hippocampus) similarly indicated a change in activity with postnatal development. These data are plotted in Fig. 3A. Figure 3B shows corresponding data for endogenous noradrenaline levels. Those data of Pscheidt and Himwich (12) for noradrenaline levels are included in Fig. 3B where the brain area studied seemed comparable. It is evident that there is good agreement between our data for noradrenaline and those of Pscheidt and Himwich, considering the probable differences in dissection. It is also evident that in those areas where sizeable changes in noradrenaline levels occur, similar changes in tyrosine hydroxylase activity occur and often precede the changes in amine level. Thus, the drop-off in noradrenaline levels in the pons-medulla at 30-90 days noted by Pscheidt and Himwich correlates with a fall in tyrosine hydroxylase activity between the 22nd and the 40th day after birth. Similarly the sharp rise in caudate noradrenaline found by Pscheidt and Himwich at about the 80th day is preceded by a sharp increase in tyrosine hydroxylase. A large relative increase in enzyme activity occurs in the cortex between the 8th and 15th days which corresponds to the time when Pscheidt and Himwich noted the beginning of a steady rise in noradrenaline. m a x  McGEER  i—i—i  2  3  4  ET AL.: TYROSINE HYDROXYLASE  ii—i—i—i—i  5 6  AGE  8 10 15  IN  1  i • • • i  20 30  DAYS  50  2  r—i—i 3  1949  ACTIVITY  •i — n — • — i  1  4 5 6 8 1 0 15 2 0 3 0  AGE  IN  i 1 ' i 1 1 1  50  1  8 0  DAYS  F I G . 3. Tyrosine hydroxylase activity and endogenous noradrenaline levels in some areas of developing cat brain. Log-log scale. Each symbol represents one tissue sample in the case of tyrosine hydroxylase activity and an average value for noradrenaline levels, c = caudate, s = septal area, p = pons-medulla, o = cortex, a = amygdala-hippocampus, e = pineal. Tyrosine hydroxylase activity in terms of Vma* in nmoles/g tissue/h except for pineal where Fnun is in pmoles/pineal/h. Noradrenaline levels in M g / g of tissue. Solid lines showing changes in noradrenaline levels represent data of Pscheidt and Himwich (12); symbols and dotted lines show our data. Solid line without any symbols is cortical data of Pscheidt and Himwich.  Data for tyrosine hydroxylase levels for the cerebellum, midbrain, and hypothalamus-thalamus are given in Table V; these data are not plotted since they do not indicate any significant postnatal change. All the brain areas studied increased in weight as the kittens aged from 2 to 47 days. The cerebellum showed the largest (ninefold) increase and the septal area the smallest (twofold). There was no correlation between changes in weight and in tyrosine hydroxylase activity per unit weight. This is illustrated in Table VI which indicates the weights at various ages of the cortex, caudate, midbrain, and pons-medulla.  1950  C A N A D I A N J O U R N A L O F B I O C H E M I S T R Y . V O L . 45, 1967  TABLE V Tyrosine hydroxylase activity in cerebellum, midbrain, and hypothalamus-thalamus in neonatal cats* Litter Hypothalamus -thalamus designation  Age in days Cerebellum  Midbrain  2 5 8 12 15 19 22 26 33 40 47 Average of above Adult level  0.8±0.3 1.0 1.7±0.3 1.1 1.9±0.3 1.1 1.1±0.2 1.3 1.5 0.7 0.9  3.3±1.3 2.3 3.3±1.0 3.3 3.9±1.4 3.9 3.0±0.6 3.6 4.1 2.3 3.8  3.0±0.7 4.3 3.3±0.6 3.1 3.3±0.9 2.6 2.5±0.8 2.7 3.2 2.7 3.3  1.3±0.4 1.4±0.2  3.4±0.9 3.1±0.2  3.0±0.7  A,B,C,Dt E A,B,C E A,B,C E A,B,C E F F F  *Pmaxin nmoles of DOPA formed/h/g of tissue. fLittermates of this kitten were lost.  T A B L E VI Tissue: weights of selected brain areas in neonatal cats Age in days  Caudate  Cortex  Midbrain  Pons-medulla  2 5 8 12 15 19 22 25 33 40 47  101 ±21 88 149 ±62 156 203 ±44 455* 260 ±20 328 281 307 367  3070±330 3620 5760±220 6090 781S±348 8410 10160±330 10600 12600 12060 13874  272±56 300 316±62 370 408±17 500 482 ±64 530 592 665 672  242 ±48 270 372±89 432 409 ±126 443 504±39 658 720 803 875  *Data on this sample are not included in Figs. 3 or 4 since the weight indicates a major variation in dissection.  In all the kittens studied, as well as in the adult cats, the caudate was by far the most active area in terms of tyrosine hydroxylation per gram of tissue. When the weight of tissue is considered, however, it is evident that, in the older cats, the cortex, despite its lower unit activity, makes a greater contribution to total tyrosine hydroxylase activity, and presumably also to catecholamine synthesis, than does any other brain area including the caudate. The caudate contribution is 30-45% of the total tyrosine hydroxylase activity of brain, with the higher figures tending to occur in the younger kittens. The percentage contribution of the cortex rises from a figure of less than 30% in the 2-day-old kittens to more than 40% in the older kittens. The time at which the cortical contribution becomes greater than the caudate is about 12 days (Fig. 4), which is coincident with the first signs of maturation of the EEG in kittens (12).  1951  McGHER E T A L . : T Y R O S I N E H Y D R O X Y L A S E A C T I V I T Y  s  IO  J  r-  2  1  1  1  3  4  5 AGE  1—I—i—I  I  6 7 8 9 10 IN  1  1  20  30  1  1  1—  4 0 50 6 0  DAYS  F I G . 4. Percentage contribution of caudate and cortex to total brain tyrosine hydroxylase activity in kittens. Semilog scale. Circles and dashed line for cortex; triangles and solid line for caudate.  The pons-medulla and midbrain, where catecholinergic cell bodies are almost entirely concentrated (10, 13), showed no appreciable rise in tyrosine hydroxylase activity during postnatal development. The caudate, the limbic structures, and the cortex, on the other hand, which contain the terminal nerve endings of these cell bodies, showed sharp increases in tyrosine hydroxylase activity. The pineal gland, which contains sympathetic nerve endings but not cell bodies (14), also showed a sharp increase with age. Tyrosine hydroxylase is known to be concentrated in nerve ending particles (3). Presumably the postnatal increases in tyrosine hydroxylase activity reflect growth and development of catecholinergic nerve endings, and the marked differences in activity per gram of tissue found in the various adult brain areas reflect the wide variation in concentration of such nerve endings. Both types of data are in general accord with such data as have so far been accumulated through histochemical studies (4, 10, 13). Acknowledgments This work was supported by Medical Research Council of Canada grants MA-1421, MA-2504, and 100-3W-2; Federal-Provincial public health grant 609-7-108; National Science Foundation grant GB-S27; and National Institutes of Health Grant NB-2812.  1952  C A N A D I A N J O U R N A L O F B I O C H E M I S T R Y . V O L . 45, 1967  References 1. 2. 3. 4. 5. 6. 7. 8. 9.  S. UDENFRIEND. Pharmacol. Rev. 18, 43 (1966). T . NAGATSU, et al. J . Biol. Chem. 239, 2910 (1964). P. L. M C G E E R , S. P. BAGCHI, and E . G . M C G E E R . Life Sci. 4, 1839 (1965). N. A. HILLARP, et al. Pharmacol. Rev. 18, 727 (1966). L. L. IVERSEN and J . GLOWINSKI. J . Neurochem. 13, 671 (1966). E . G . M C G E E R , S. GIBSON, and P. L. M C G E E R . Can. J . Biochem. 45, 1557 (1966). J . P . W A A L K E S and S. UDENFRIEND. J . Lab. Clin. Med. 50, 73 (1957). J . E . DOWD and D . S. RIGGS. J . Biol. Chem. 240, 863 (1965). J . A. WADA and E . G . M C G E E R . Arch. Neurol. 14, 129 (1966).  10. A. DAHLSTROM, et al.  Acta Physiol. Scand. Suppl. 64, 247 (1965).  11. - J . GLOWINSKI and L. L. IVERSEN. J . Neurochem. 13, 655 (1966). 12. G . R. PSCHEIDT and H . E . HIMWICH. Brain Res. 1, 363 (1966).  13. A. DAHLSTROM and K . FUXE. Acta Physiol. Scand. Suppl. 62, 232 (1964). 14. A. J . KAPPERS. Z. Zellforsch. Mikroskop. Anat. Abt. Histochem. 52, 163 (1961).  s  SOME CHARACTERISTICS  OF BRAIN  TYROSINE  HYDROXYLASE E. G. MCGEER, S. GIBSON, AND P . L . MCGEER Kinsmen Laboratory of Neurological Research, Faculty of Medicine, The University of British Columbia, Vancouver, British Columbia  Received April 20, 1967 Tyrosine hydroxylase from brain homogenates differed from tyrosine hydroxylase from adrenal homogenates in being particle-bound, insensitive to cofactors, possessing a lower Michaelis constant for tyrosine, and being responsive to slightly different optimum conditions of pH and buffer. The combination of 0.02 M mercaptoethanol and 0.1-1.0 mM 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine ( D M P H 4 ) increased tyrosine hydroxylase activity in beef adrenal homogenates 15-fold, but was without effect on activity in rat brain homogenates. The K for tyrosine in beef adrenal homogenates was 4 X 10 M, and in rat brain homogenates was 0.45 X 10 M. Conversion in beef adrenal homogenates was maximum in 0.6 M sodium acetate buffer, pH 6.0, and in rat brain homogenates was maximum in 0.28 M phosphate buffer, pH 6.2. -5  m  -6  ,  Introduction M u c h interest has been aroused in tyrosine hydroxylase, the enzyme which converts tyrosine to 3,4-dihydroxyphenylalanine ( D O P A ) , because it appears to be the rate-limiting enzymatic step in catecholamine synthesis (1). Kinetic studies have been done chiefly with the enzyme from adrenal medulla (2-7). T h e enzyme from brain seems to differ in some respects from the enzyme from the adrenal medulla (2-4). T h i s paper reports some further kinetic data on brain tyrosine hydroxylase and gives the basis for the assay method used in this laboratory. Materials and Methods Tissue was rapidly removed from animals sacrificed by a sharp blow to the neck. It was weighed and homogenized in 4-9 volumes of ice-cold 0.25 M sucrose. A 0.1-ml aliquot was incubated in an air atmosphere with 0.1 ml of a radioactive tyrosine solution and 0.1 ml of buffer. The buffer was either 0.28 M phosphate, p H 6.2 (preferred for brain), or 0.6 M acetate, p H 6.0 (preferred for adrenal). T h e buffer was often made 3 X 10~ or 1 0 M in 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine ( D M P H ) and 6 X 10" M in 2-mercaptoethanol. T y p i c a l l y the radioactive tyrosine solution contained 140,000-280,000 c.p.m. (1/6-1/12 mCi) of L-tyrosine- C (uniformly labelled; specific activity 330 mCi/mmole) and was made 3 X 1 0 M in iV-methyl-A^-3-hydroxyphenylhydrazine (NSD-1034), a D O P A decarboxylase inhibitor. Blanks were run by incubating with tyrosine- C tissue homogenates, initially heated to 80-90 °C for 12-15 min and then cooled in ice. Incubations at 37 °C were generally 30 min for brain homogenates and 10 min for adrenal homogenates. The reactions were stopped by the addition of 2 ml of a 1:1 mixture of 0.4 N perchloric acid and 0.2 N acetic acid, containing cold carrier 3  -4  2  4  14  -3  14  Canadian Journal of Biochemistry. Volume 45 (1967)  1557  CANADIAN JOURNAL OF BIOCHEMISTRY. VOL. 45, 1967  1558  D O P A , dopamine, and noradrenaline frozen u n t i l w o r k - u p .  (0.2 jug of each). T h e m i x t u r e s  were  T h e D M P H used was o b t a i n e d f r o m C a l b i o c h e m , a n d the N S D - 1 0 3 4 f r o m D r . D . J . D r a i n of S m i t h a n d N e p h o w R e s e a r c h . 4  If D I V I P H 4 was to be e m p l o y e d , a n a q u e o u s s o l u t i o n 10~ or 10~ M i n D M P H 4 a n d 0.2 M i n 2 - m e r c a p t o e t h a n o l was prepared j u s t p r i o r to the e x p e r i m e n t . S u c h a s o l u t i o n r e t a i n e d a c t i v i t y for several hours if k e p t o n ice i n the d a r k . F o r i n c u b a t i o n s , a 0 . 3 - m l a l i q u o t of this s o l u t i o n was m i x e d w i t h 0.7 m l of either 0.4 M p h o s p h a t e buffer, p H 6.2, or 0.85 M a c e t a t e buffer, p H 6 . 0 ; 0.1 m l of the m i x t u r e was used i m m e d i a t e l y i n each i n c u b a t i o n . 2  3  S i n c e the t y r o s i n e h y d r o x y l a s e i n c u b a t i o n is r u n a t s u b s t r a t e c o n c e n t r a t i o n s b e l o w s a t u r a t i o n , k n o w l e d g e of endogenous t y r o s i n e levels is necessary for c a l c u l a t i o n of V . A s m a l l a l i q u o t of the h o m o g e n a t e was m i x e d w i t h a n e q u a l v o l u m e of 3 0 % t r i c h l o r a c e t i c a c i d a n d three v o l u m e s of w a t e r , a n d the s u p e r n a t e was used for a n a l y s i s of endogenous t y r o s i n e b y the m e t h o d of W a a l k e s a n d U d e n f r i e n d (8) modified o n l y i n the use of 0.6 m l each of s u p e r n a t e , l - n i t r o s o - 2 - n a p h t h o l a n d d i l u t e n i t r i c a c i d , a n d 2.5 m l of e t h y l e n e d i c h l o r i d e . I s o l a t i o n a n d m e a s u r e m e n t of the r a d i o a c t i v e catechols was on a n a l u m i n a c o l u m n . T h e i n c u b a t i o n m i x t u r e was t h a w e d a n d centrifuged b r i e f l y . T h e clear s u p e r n a t e was p o u r e d i n t o a 2 0 - m l beaker c o n t a i n i n g 1.25 m l of 0.2 M e t h y l e n e d i a m i n e t e t r a c e t i c a c i d ( E D T A ) . T h e c o n t e n t s of the i n c u b a t i o n t u b e were rinsed i n t o the centrifuge t u b e w i t h 1.5 m l of 0.35 M K H 2 P O 4 f o l l o w e d b y a l i t t l e w a t e r . T h e m i x t u r e was centrifuged a g a i n a n d the clear s u p e r n a t e c o m b i n e d w i t h the first s u p e r n a t e p l u s E D T A . T h e s o l u t i o n was t h e n t a k e n to p H 8.S-9.2 w i t h d i l u t e s o d i u m h y d r o x i d e , a p p r o x i m a t e l y 350 m g of a c i d w a s h e d a l u m i n a was a d d e d i m m e d i a t e l y , a n d the m i x t u r e was s t i r r e d for 4 - 5 m i n on a m a g n e t i c stirrer. I t was t h e n p o u r e d i n t o a glass c o l u m n p l u g g e d w i t h cellulose fiber. A s l i g h t v a c u u m was used to p r o m o t e r a p i d passage of the s o l u t i o n t h r o u g h the c o l u m n . T h e a l u m i n a was w a s h e d f r o m the b e a k e r o n t o the c o l u m n w i t h a p p r o x i m a t e l y 20 m l of w a t e r . T h i s a n d a second 20 m l w a s h were passed t h r o u g h the c o l u m n u n d e r a s l i g h t v a c u u m . T h e c o l u m n was then r e m o v e d f r o m the v a c u u m flask a n d e l u t e d i m m e d i a t e l y w i t h 2 m l of 0.5 N acetic a c i d ; the e l u a t e was c o l l e c t e d i n a s c i n t i l l a t i o n v i a l a n d was c o u n t e d i n a l i q u i d - s c i n t i l l a t i o n s p e c t r o p h o t o m e t e r after a d d i t i o n of 10 m l of B r a y ' s m i x t u r e (9). T h e w h o l e process f r o m the t i m e of m a k i n g the i n c u b a t i o n m i x t u r e a l k a l i n e to b e g i n n i n g of the a c i d e l u t i o n o c c u p i e d a b o u t 10 m i n b u t n e v e r longer t h a n 15 m i n . T h e i n c u b a t i o n m i x t u r e s were o c c a s i o n a l l y d i l u t e d further (to a b o u t 1 5 - 2 0 m l ) before m a k i n g alkaline~and s t i r r i n g w i t h a l u m i n a ; the results were c l o s e l y c o m p a r a b l e so l o n g as the a m o u n t s of p h o s p h a t e a n d E D T A were increased a c c o r d i n g l y . m&x  T h e p H used to p u t the catechols u p o n the a l u m i n a c o l u m n was chosen after e x p e r i m e n t s w i t h this p a r t i c u l a r l o t of a l u m i n a a n d w i t h s m a l l a m o u n t s of r a d i o a c t i v e D O P A a d d e d to m o c k - i n c u b a t i o n m i x t u r e s . U n d e r the c o n d i t i o n s specified, the r e c o v e r y of r a d i o a c t i v e D O P A was c o n s i s t e n t l y 6 5 - 7 0 % . I n the c a l c u l a t i o n s , a r e c o v e r y of 6 5 % was a s s u m e d .  M C G E E R E T A L . : BRAIN TYROSINE H Y D R O X Y L A S E  1559  TABLE I Effect of phosphate on alumina-column absorption of radioactive products c.p.m. recovered from column with 6-8 ml of solution*  Freshly prepared tyrosine solution (140,000 c.p.m.; 0.05 ug) Tyrosine frozen 50 days in solution (140,000 cp.m.; 0.05 g) DOPA (2,150 c.p.m.; 0.11 jug) DOPA (6,450 c.p.m.; 0.33 ug) M  No phosphate  1.5 ml of 0.35 M KH PO*  370  192-217  206  247 . 1,460 4,209  259 1,410 4,298  1,651-2,358 1,425 4,302  2  . 3 ml of 0.35 M K H P 0 2  4  *The solution in each case was a mock-incubation mixture and contained 1 ml 0.4 N perchloric acid, 1 ml 0.2 N acetic acid, 1.25 ml 0.2 M E D T A , 0.1 ml 0.25 M sucrose, 0.1 ml 0.28 M phosphate, 0.2 fig each of cold tyrosine, DOPA, dopamine, noradrenaline, and water to make up to volume. The cold D O P A was decreased to 0.1 /tg in the solution with 2,150 c.p.m. DOPA-KC, and omitted from the solution with 6,450 c.p.m. D O P A - " C .  Results  The blanks were typically 190-250 c.p.m. when 140,000 c.p.m. of tyrosine- C were used, and 350-425 when 280,000 c.p.m. were used. Tests varied from about 600 to 30,000 depending on the tissue used. Heated tissue from various brain areas or from the adrenal always gave the same blank. Incubations of test and blank were always run in triplicate for the first sixty or so experiments. Agreement was invariably within a few percent, so that only duplicates were usually run in the later experiments. An important modification to the normal procedure for isolating catechols was the use of phosphate in the mixture to be put on the column. Radioactive tyrosine which has been in solution for some time contains as much as 1-2% of impurity which may be absorbed on the alumina column. Phosphate appears to prevent or minimize the absorption of these impurities without affecting the absorption of DOPA (Table I). Purification df the radioactive tyrosine each time before use involves tedious procedures. In practice, it proved more satisfactory to use phosphate in the absorption mixture and to prepare dilutions of the radioactive tyrosine in small quantities which were rapidly used. The volume of sucrose used for homogenation was chosen in order to yield 5-20 mg of tissue per incubation; generally 15-20 mg were used. With all but the most active brain regions, 5 mg tended to give counts which were lower than desirable for accuracy; above 20 mg of tissue, the conversion tended to fall off. A typical experiment with a rat brain homogenate using 5, 10, 15, and 20 mg of tissue gave calculated V . (s) of, respectively, 22.6, 25.6, 25.4, and 24.9 nmoles per hour per gram of tissue. A typical experiment with a beef adrenal homogenate using 2.5, 10, 15, and 20 mg of tissue gave calculated V (s) of, respectively, 1201, 1090, 1180, and 1120 nmoles per hour per gram of tissue. Repeated tests with rat brain homogenate indicated that the reaction was linear at least up to 40 min. Thirty minutes was chosen as the usual incubation time. A few experiments with beef adrenal homogenate suggested that the period of linearity was shorter than with brain homogenate and a 10-min time of incubation seemed preferable. 14  ma x  max  1560  CANADIAN "JOURNAL OF BIOCHEMISTRY. VOL. 45, 1967 T A B L E II Relative tyrosine hydroxylase activities of rat brain, cat brain, and beef adrenal homogenates in various buffers* Relative activity, % Buffer 0.28 M K P0 , pH 6.2f 0.28 M Na P0 , pH 6.2 0.6 M KAc, pH 6.2 0.6 M NaAc, pH 6.2 0.6 M NaAc, pH 6.0 0.6 M Tris, pH 6.2 3  4  3  4  Rat  Cat  Beef  100 101 48 64.5 41 61  100 — 59 74 55 51  100 98 127 124 132 —  *Data cited were obtained with 10 M D M P H 4 and 0.02 M 2-mercaptoethanol; comparable relative results were obtained in each series without these additives. fChosen as reference conditions. -3  The choice of phosphate buffer for brain homogenates and of acetate buffer for adrenal homogenates was based on comparative experiments illustrated in Table II. Acetate buffer has been used by others for adrenal tyrosine hydroxylase (2, 3, 5) and there appears to be little pH change during the incubation. Beef caudate resembled rat and cat brain rather than beef adrenal in that the activity in 0.6 M acetate buffer, pH 6.0, was 6 0 % of that in 0.28 M phosphate buffer,.pH 6.2. Figure 1 shows the effect of buffer pH on tyrosine hydroxylase activity in adrenal (Fig. la) and rat brain (Fig. 16) homogenates, and Table III indicates the effect of some changes in molarity of sucrose and phosphate on the activity of a rat brain homogenate. Addition of a variety of ions at 10~ M final concentration (Zn , Fe , Mg , Ca , Cu , Co , or Fe ) failed to have any significant effect on enzyme activity in rat brain homogenates either in the presence or absence of D M P H 4 . Ferrous ion has been reported to stimulate the activity in beef adrenal to a small extent itself, markedly in the presence of tetrahydrofolate and insignificantly in the presence of D M P H (2). Addition of 10~ M F e ion to a beef 4  2+  2+  2+  2+  2+  2+  3+  4  2+  4  pH of Buffer  pH of Buffer  FIG. 1. Effect of buffer pH on tyrosine hydroxylase activities in beef adrenal (a) and rat brain (b) homogenates. Graphs are based on three series for rat brain and two for adrenal homogenates. In each series, activity at each given pH was expressed as a percentage of that found at pH 6.0. Range of data is indicated by lines.  1501  M C G E E R E T A L . : BRAIN TYROSINE H Y D R O X Y L A S E  T A B L E III Relative tyrosine hydroxylase activities of a rat brain homogenate using different molarities of phosphate and sucrose Phosphate buffer, pH 6.2  Sucrose 0.2  0.2 M 0.24 M 0.28 M 0.32 M 0.35 M  M  0.25  22% 41% 24% 37% 20%  M  44% 48% 100%* 96% 85%  0.28 M  0.30 M  17% 54% 89% 104% 93%  39% 50% 72% 78% 64%  *Chosen as reference conditions.  a d r e n a l i n c u b a t i o n i n the presence of 1 0 M D M P H g a v e a n a c t i v i t y 1 2 7 % of t h a t i n its absence. JVTg , C a , a n d F e h a d n o effect w h a t s o e v e r . F l u s h i n g the i n c u b a t i o n tubes w i t h o x y g e n d i d n o t s i g n i f i c a n t l y increase o b s e r v e d c o n v e r s i o n s , b u t flushing w i t h n i t r o g e n caused v e r y large decreases. T h e m o s t s i g n i f i c a n t difference i n t y r o s i n e h y d r o x y l a s e a c t i v i t y between rat b r a i n a n d beef a d r e n a l homogenates is the dependence of the l a t t e r o n D M P H a n d 2 - m e r c a p t o e t h a n o l . T h e s e cofactors i n c o m b i n a t i o n d i d n o t s i g n i f i c a n t l y affect a c t i v i t y i n c r u d e rat b r a i n homogenates, a l t h o u g h t h e y increased a d r e n a l h o m o g e n a t e a c t i v i t y 15-fold. T h e d a t a i n T a b l e I V i l l u s t r a t e n o t o n l y this effect, b u t also the i n h i b i t o r y effect of 2 - m e r c a p t o e t h a n o l b y itself. T h i s i n h i b i t i o n is n o t s u r p r i s i n g i n v i e w of the i n h i b i t o r y effect f o u n d for related c o m p o u n d s s u c h as e t h a n o l a n d L-cysteine (10). D M P H b y itself increased the a c t i v i t y of a d r e n a l homogenates r o u g h l y threefold, b u t h a d n o effect o n b r a i n homogenates. - 4  4  2+  2 +  3 +  4  4  In c o m p i l i n g the d a t a s h o w n i n a l l tables, a separate b l a n k was r u n for each test c o n d i t i o n . T h e b l a n k s were g e n e r a l l y unaffected b y v a r i a t i o n s i n p H , m o l a r i t y , o r buffer t y p e b u t t e n d e d to be 5 0 - 6 0 % h i g h e r i n the presence of D M P H t h a n i n its absence. 4  T A B L E IV Relative tyrosine hydroxylase activities in the presence of various additives Relative activity, % Additive (final concentration) None* 10" M DMPH, plus 0.02 M 2-mercaptoethanol (SH) 10^ M D M P H plus 0.02 M SH, preincubated for 10 min 10" ikTDMPH plus 0.02 M SH lO" M D M P H 0.02 M SH 10 M TPNH 10~ M folic acid  Beef adrenal (acetate buffer)  Rat brain (phosphate buffer)  100  100  1520  92  3  3  4  4  4  3  4  —  1495 280 89  -3  —  3  —  •Chosen as reference condition.  75 96 98 79 97 98  1562  CANADIAN JOURNAL OF BIOCHEMISTRY. VOL. 45, 1967 K  TABLE V (X10~ ) for tyrosine determined with and without added D M P H (0.001 M) plus 2-mercaptoethanol (0.02 M) 5  m  Rat brain Beef adrenal  4  With cofactor  Without cofactor  No. of runs  0.45±0.08 4.Oil.2  0.47±0.05 nonlinear  7 4  I n a n o t h e r a p p r o a c h to the cofactor p r o b l e m , 36 i n d i v i d u a l r a t b r a i n homogenates were r u n w i t h a n d w i t h o u t the c o m b i n a t i o n of D M P H plus 2 - m e r c a p t o e t h a n o l u n d e r the u s u a l c o n d i t i o n s of r e l a t i v e l y l o w t y r o s i n e c o n c e n t r a t i o n . T h e c o n v e r s i o n i n the presence of D M P H was 104 ± 9 % of t h a t i n its absence. I t a p p e a r s possible t h a t the effectiveness of exogenous D M P H m i g h t be related to the l e n g t h of t i m e between sacrifice of the a n i m a l a n d i n c u b a t i o n . T h e b r a i n studies were done w i t h a n i m a l s sacrificed i n the l a b o r a t o r y a n d the u s u a l t i m e between sacrifice a n d assay was g e n e r a l l y less t h a n 2 h . T h e beef a d r e n a l was o b t a i n e d f r o m the s l a u g h t e r house a n d k e p t o n ice d u r i n g its t r a n s p o r t to the l a b o r a t o r y . T h e r e were g e n e r a l l y a b o u t 4 - 5 h between dissect i o n a n d assay. T r i a l of 12 r a t b r a i n homogenates a l l o w e d to s t a n d i n ice for 5 h between the i n i t i a l assay a n d reassay, h o w e v e r , i n d i c a t e d n o change i n a c t i v i t y d u r i n g s u c h a p e r i o d of s t a n d i n g , a n d n o s i g n i f i c a n t effect of exogenous D M P H p l u s 2 - m e r c a p t o e t h a n o l at e i t h e r t i m e . T h e c o n v e r s i o n s w i t h D M P H i n t h i s series were 96 ± 8 % of the c o n v e r s i o n s w i t h o u t . D i f f e r e n t K v a l u e s were o b t a i n e d for r a t b r a i n a n d beef a d r e n a l homogenates ( T a b l e V ) , i n c o n f i r m a t i o n of p r e v i o u s reports (3, 4 ) . T h e v a l u e of 4.0 ( ± 1 . 2 ) X 10~ M for beef a d r e n a l homogenates is s o m e w h a t l o w e r , b u t i n reasonable a g r e e m e n t w i t h the v a l u e of 1 X 10~ M for purified a d r e n a l t y r o s i n e h y d r o x y l a s e r e p o r t e d b y I k e d a et al. (5). A n o n l i n e a r r e c i p r o c a l p l o t of c o n v e r s i o n versus t y r o s i n e c o n c e n t r a t i o n was o b t a i n e d for a d r e n a l h o m o g enates i n the absence of a d d e d cofactor, m a k i n g i t i m p o s s i b l e to o b t a i n a K v a l u e for s u c h c o n d i t i o n s . A t h i g h e r t y r o s i n e c o n c e n t r a t i o n s , c o n v e r s i o n r a p i d l y fell off. 4  4  4  4  4  m  5  4  m  F o r c r u d e r a t b r a i n homogenates, h o w e v e r , i d e n t i c a l r e c i p r o c a l p l o t s were o b t a i n e d w i t h a n d w i t h o u t a d d e d cofactor. T h e K v a l u e o b t a i n e d , 0.45 ( ± 0 . 0 8 ) X 1 0 M , was s i g n i f i c a n t l y l o w e r t h a n t h a t for a d r e n a l homogenates. m  - 5  Discussion A l t h o u g h b r a i n t y r o s i n e h y d r o x y l a s e a p p e a r s to be different f r o m t y r o s i n e h y d r o x y l a s e i n b e i n g p a r t i c l e - b o u n d , i n s e n s i t i v e to cofactors, sive to s l i g h t l y different c o n d i t i o n s of p H a n d buffer, a n d possessing M i c h a e l i s c o n s t a n t for t y r o s i n e , firm c o n c l u s i o n s r e g a r d i n g differences be d r a w n from c r u d e e n z y m e p r e p a r a t i o n s . A t t e m p t s to s o l u b i l i z e b r a i n t y r o s i n e h y d r o x y l a s e as a p r e p a r a t o r y  adrenal respona lower cannot step to  MCGEER ET AL.: BRAIN TYROSINE HYDROXYLASE  1563  p u r i f y i n g i t h a v e l e d , i n o u r h a n d s , t o loss of a c t i v i t y . T h e e n z y m e s e d i m e n t s w i t h the c r u d e m i t o c h o n d r i a l f r a c t i o n , a n d u p o n f u r t h e r s e p a r a t i o n is s h o w n to be c o n c e n t r a t e d i n t h e n e r v e - e n d i n g p a r t i c l e s (11). D i s r u p t i o n of the n e r v e e n d i n g p a r t i c l e s b y o s m o t i c s h o c k a n d o t h e r m e a n s has a l w a y s l e d t o d i s a p p e a r a n c e of t y r o s i n e h y d r o x y l a s e a c t i v i t y . T h e d a t a presented here o n b r a i n t y r o s i n e h y d r o x y l a s e m a y a p p l y d i r e c t l y to t h e e n z y m e . B u t i t is e q u a l l y possible t h a t t h e y a p p l y t o t h e e n z y m e p l u s a p a r t i c l e t o w h i c h t h e e n z y m e is a t t a c h e d o r w i t h i n w h i c h i t is c o n t a i n e d . S u c h a p a r t i c l e c o u l d c o n t a i n essential cofactors w h i c h w o u l d n o t be d i l u t e d b y the h o m o g e n i z a t i o n process. T h i s w o u l d e x p l a i n t h e i n s e n s i t i v i t y of b r a i n t y r o s i n e h y d r o x y l a s e to a d d e d c o factors. T h e p a r t i c l e c o u l d also l i m i t the a v a i l a b i l i t y of t y r o s i n e t o the e n z y m e , a n d thus a c c o u n t for the l o w e r K for t y r o s i n e i n b r a i n c o m p a r e d w i t h t h a t in a d r e n a l . m  A l t h o u g h b r a i n t y r o s i n e h y d r o x y l a s e is h i g h l y l o c a l i z e d t o n e r v e e n d i n g s , h i s t o c h e m i c a l studies (12) f o l l o w i n g lesions to c a t e c h o l a m i n e a x o n s h a v e s h o w n a c c u m u l a t i o n of c a t e c h o l a m i n e s p r o x i m a l to the lesion a n d d i s a p p e a r a n c e of t h e m a t the nerve endings. T h i s result is c o n s i s t e n t w i t h the c o n c e p t of c o n t i n u o u s t r a n s p o r t of t y r o s i n e h y d r o x y l a s e f r o m the cell b o d y to the n e r v e t e r m i n a l , b u t the n a t u r e of the t r a n s p o r t poses p r o b l e m s . If the e n z y m e were free a t the t r a n s p o r t stage, some s o l u b l e t y r o s i n e h y d r o x y l a s e s h o u l d be f o u n d following homogenization. C l e a r l y , the true n a t u r e of b r a i n t y r o s i n e h y d r o x y l a s e m u s t a w a i t m e t h o d s w h i c h w i l l lead t o its p u r i f i c a t i o n a n d w h i c h w i l l e x p l a i n its a s s o c i a t i o n w i t h b r a i n particles.  Acknowledgments T h i s w o r k w a s s u p p o r t e d b y the M e d i c a l R e s e a r c h C o u n c i l of C a n a d a ( M A - 1 4 2 1 ) , a n d a F e d e r a l - P r o v i n c i a l P u b l i c H e a l t h G r a n t (609-7-108).  References 1. S. U D E N F R I E N D . Pharmacol. Rev. 18, 43 (1966). 2. T. N A G A T S U , M . L E V I T T , and S. U D E N F R I E N D . J. Biol. Chem. 2 3 9 , 2910 (1964). 3. T. N A G A T S U , M . L E V I T T , and S. U D E N F R I E N D . Biochem. Biophys. Res. Commun. 14, 543 (1964). 4. S. P. B A G C H I and P . ' L . M C G E E R . Life Sci. 3, 1195 (1964). 5. M . I K E D A , L . A. F A H I E N , and S. U D E N F R I E N D . J. Biol. Chem. 241, 4452 (1966). 6. A. R. B R E N N E M A N and S. K A U F M A N . Biochem. Biophys. Res. Commun. 17, 177 (1964). 7. L . E L L E N B O G E N , R. J . T A Y L O R , JR., and G . B. B R U N D A G E . Biochem. Biophys. Res. Commun. 19, 708 (1965). 8. T. P. W A A L K E S and S. U D E N F R I E N D . J . Lab. Clin. Med. 5 0 , 733 (1957). 9. G . A. BRAY. Anal. Biochem. 1, 279 (1960). 10. E . G . M C G E E R and P. L. M C G E E R . Can. J . Biochem. 45, 115 (1967). 11. P. L. M C G E E R , S. P. B A G C H I , and E . G . M C G E E R . Life Sci. 4, 1859 (1965). 12. A. D A H L S T R O M and K. F U X E . Acta Physiol. Scand. Suppl. 64, 247 (1965).  

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