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UBC Theses and Dissertations

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. Gibson B . S c , U n i v e r s i t y of 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 Laboratory of N e u r o l g g i c a l Research Department of P s y c h i a t r y We accept t h i s t h e s i s as conforming to the required- sj^afldard THE/ UNIVERSITY OF BRITISH COLUMBIA May 3rd, 1968 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e Head o f my D e p a r t m e n t o r b y h its r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f Vt H LA T/<V The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a n a d a D a t e M/9-V 6. ABSTRACT I n t e r e s t i n b r a i n catecholamines has grown conside r a b l y i n the l a s t few years i n view of t h e i r p o s s i b l e r o l e as neuro-t r a n s m i t t e r s . This i n v e s t i g a t i o n deals p r i m a r i l y w i t h the enzyme t y r o s i n e hydroxylase which i s thought to be the r a t e l i m i t i n g step i n catecholamine s y n t h e s i s . Using t y r o s i n e hydroxylase measurements and c a t e c h o l -amine d e p l e t i o n techniques,attempts were made to determine the s i t e of increased synthesis of catecholamines i n animals exposed to c o l d . B r a i n , heart and spleen do not appear to be the organs i n v o l v e d i n t h i s change. Adrenals may be of s i g n i f i c a n c e but the r e s u l t s were suggestive r a t h e r than c o n c l u s i v e . Tyrosine hydroxylase d i s t r i b u t i o n i n b r a i n was determined i n various regions of r a t , r a b b i t and cat b r a i n , and a c t i v i t y was shown to be highest i n the caudate, s e p t a l area, nucleus accombens, and a n t e r i o r p e r f o r a t i n g substance, w i t h much lower a c t i v i t i e s i n other regions such as hippocampus, amygdala, hypothalamus, thalamus, c o r t e x , cerebellum and b r a i n stem. Using 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 of normal t y r o s i n e hydroxylase a c t i v i t y i n areas of 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 techniques, studies were c a r r i e d out to determine noradrenergic and dopanergic 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 midbrain were traced i n the midbrain and diencephalon to the caudate and s e p t a l area, and the r e l a t i v e p o s i t i o n s of each group of f i b e r s determined along t h e i r course. i i i INDEX Page INTRODUCTION 1 1. CATECHOLAMINES 1 a) Chemistry 1 b) Metabolism 1 c) L o c a t i o n .. .. .. .. .. .. .. 1 d) Function 4 2. TYROSINE HYDROXYLASE 6 a) As a c o n t r o l of CA synthesis 6 b) C h a r a c t e r i s t i c s .. 7 c) L o c a t i o n 8 i ) S u b c e l l u l a r l o c a t i o n .. 8 i i ) Anatomical l o c a t i o n .. .. .. .. 8 d) I n h i b i t o r s 8 3. DOPAMINERGIC AND NORADRENERGIC PATHWAYS IN BRAIN.. 9 4. POSSIBLE ROLE OF CATECHOLAMINES .. . . . . .. 10 a) Emotion and behavior 10 b) Memory and l e a r n i n g .. .. .. .. .. .. 13 c) Sleep 13 d) Temperature r e g u l a t i o n 13 e) Basal g a n g l i a and Parkinsonism 13 5. AIMS OF THESIS 14 METHODS AND MATERIALS 16 a) Tyrosine hydroxylase a n a l y s i s .. 16 i v i ) Procedure 16 i i ) R a t i o n a l f o r procedure 16 b) Tyrosine a n a l y s i s 22 i ) Procedure .. .. .. .. . . . . . . 22 i i ) R a t i o n a l f o r procedure 22 c) C a l c u l a t i o n of V 24 max d) Noradrenaline and Dopamine determinations .. 25 i ) I s o l a t i o n 25 i i ) NA a n a l y s i s 25 i i i ) DA a n a l y s i s 26 i v ) R a t i o n a l f o r catecholamine determination. 26 e) Separation of noradrenaline and dopamine and some of t h e i r metabolites 28 i ) Ion-exchange chromatography 28 i i ) T h i n-layer chromatography 30 f ) 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 determination i n ur i n e .. 31 i i ) Tyrosine Hydroxylase i n c o l d a c c l i m a t i z e d r a t s 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 31 g) E f f e c t of a l t e r e d catecholamine l e v e l s on Tyrosine Hydroxylase 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 of catecholamine pathways i n cat b r a i n 32 RESULTS 35 1. SEPARATION OF NA, DA AND SOME OF THEIR METABOLITES 35 a) Ion-exchange chromatography 35 b) Thin-layer chromatography 35 2. CATECHOLAMINE ACTIVITY IN RATS EXPOSED TO COLD .. 35 a) Urine 35 b) Tissue 39 c) Tyrosine hydroxylase a c t i v i t y . . 39 d) Turnover r a t e s 39 3. EFFECTS OF ALTERING CATECHOLAMINE LEVELS ON IN VITRO TYROSINE HYDROXYLASE ACTIVITY 43 4. DISTRIBUTION STUDIES OF TYROSINE HYDROXYLASE IN RAT, RABBIT AND CAT BRAIN 43 5. EFFECT ' OF LESIONS ON TYROSINE HYDROXYLASE LEVELS IN VARIOUS REGIONS OF CAT BRAIN 49 a) Lesions of diencephalon 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 .. 54 c) Habenula 56 d) Substantia n i g r a 60 e) M i d l i n e midbrain 64 f ) Raphe 68 V DISCUSSION .. 71 1. TYROSINE HYDROXYLASE ACTIVITY IN VITRO 71 2. SEPARATION OF NA, DA AND SOME OF THEIR METABOLITES .. 72 3. CATECHOLAMINE ACTIVITY IN RATS EXPOSED TO COLD .. 73 4. TYROSINE HYDROXYLASE ACTIVITY IN BRAIN .. .. 76 a) D i s t r i b u t i o n 76 b) As a c o n t r o l of CA synthesis 77 5. TYROSINE HYDROXYLASE CONTAINING FIBERS IN 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 diencephalon 82 i ) Mid-diencephalon . . 82 i i ) p o s t e r i o r diencephalon .. .. .. .. 83 c) F i b e r s i n the midbrain 84 i ) to the caudate 84 i i ) to the s e p t a l area 86 d) Summary and Conclusions ... .. .. .. .. 87 SUMMARY AND CONCLUSIONS. 90 REFERENCES . . 92 APPENDIX .. .. Papers - Some C h a r a c t e r i s t i c s of B r a i n Tyrosine Hydroxyase D i s t r i b u t i o n of Tyrosine Hydroxylase A c t i v i t y i n A d u l t and Developing B r a i n v i INDEX TO TABLES Table Page 1 Summary f o r d i s t r i b u t i o n data f o r major areas of b r a i n 5 2 E f f e c t of DMPH^ on t y r o s i n e hydroxylase a c t i v i t y i n c a t , r a b b i t and r a t b r a i n 20 3. E f f e c t of phosphate a d d i t i o n on s i z e of blank and Dopa recovery from alumina column 21 4. values f o r NA, DA and some of the precursors and metabolites on polyamide 38 5 Catecholamine content of 24 hour u r i n e samples of r a t s exposed to c o l d .. 40 6 Catecholamine l e v e l s i n t i s s u e s of r a t s exposed to c o l d 41 7 Tyrosine hydroxylase a c t i v i t y i n r a t s exposed to c o l d 42 8 E f f e c t s of c e r t a i n drugs on catecholamine l e v e l s and t y r o s i n e hydroxylase a c t i v i t y i n r a t b r a i n .. 44 9 D i s t r i b u t i o n of t y r o s i n e hydroxylase i n a d u l t r a t , r a b b i t and cat b r a i n 45 10 Tissue weights of d i f f e r e n t b r a i n areas .. .. 46 11 Tyrosine concentrations i n v a r i o u s areas of r a t , r a b b i t and cat b r a i n . . . 50 12 Tyrosine hydroxylase a c t i v i t y i n s u b d i v i s i o n s of s e p t a l area of cat b r a i n 51 13 E f f e c t s of l e s i o n s i n f l o o r ofdiencephalon on t y r o s i n e hydroxylase and catecholamines i n r o s t r a l areas . . . . . . . . . . . . .. . . . . 52 14 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 Tyrosine hydroxylase and catecholamines i n r o s t r a l areas .. 58 15 E f f e c t s of attempted habenular l e s i o n s on t y r o s i n e hydroxylase a c t i v i t y i n caudate, s e p t a l area, amygdala and hippocampus .. .. .. .. .. .. 59 v i i 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 hydroxylase a c t i v i t y and catecholamines i n caudate and septum .. .. .. .. .. .. . . 62 17 E f f e c t of b i l a t e r a l midbrain l e s i o n s on t y r o s i n e hydroxylase a c t i v i t y i n caudate and septum .. .. 65 18 R e l a t i v e t y r o s i n e hydroxylase a c t i v i t y and noradrenaline turnover r a t e s i n r a t b r a i n .. 79 v i i i INDEX TO FIGURES Figure Page 1 Metabolism of Catecholamines 2 2 Summary of noradrenergic and dopaminergic path-way i n b r a i n .. . . . . . . . . . . 11 3 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 hydroxylase a c t i v i t y .. 18 4 E f f e c t of pH of i n c u b a t i o n mixture on the a c t i v i t y of t y r o s i n e hydroxylase i n b r a i n .. .. .. .. 18 5 A c t i v i t y of t y r o s i n e hydroxylase w i t h respect to time of i n c u b a t i o n .. ... .. .. 19 6 Recovery of catechols from alumina at various pH's of the sample as i t i s placed i n the column . . . 19 7 Standard curve f o r t y r o s i n e determination .. .. 23 8 Recovery of NA and DA from alumina w i t h respect to pH of sample as i t i s placed i n the column .. .. 23 9 K determination f o r r a b b i t and cat b r a i n .. .. 27 m 10 K determination f o r r a t b r a i n 27 m 11 Standard curve f o r NA and DA determinations .. 29 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 the method of d i s s e c t i o n .. .. .. .. .. .. .. 33 13 Median s a g i t t a l s e c t i o n of cat 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 area 34 14 Separation by ion-exchange chromatography of NA, NM and DA- 1 4C . . . 36 14 15 Separation of NA, DA, MT and C-NA by i o n -exchange chromatography .. .. .. . . . . . . 37 16 (a) D e p l e t i o n of NA w i t h -methyl-m-tyrosine from organs of normal r a t s and r a t s exposed to c o l d .. 47 16 (b) D e p l e t i o n of NA with<A-methy1-p-tyrosine from various organs of normal r a t s and r a t s exposed to c o l d .. .. .. .. .. .. .. .. .. 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 to 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 of the diencephalon and midbrain 53 18 L e s i o n i n the f l o o r of the mid-diencephalon .. 55 19 M e d i a l l y placed l e s i o n i n the p o s t e r i o r diencephalon at the l e v e l of the mammillary bodies 57 20 Lesi o n i n the p o s t e r i o r diencephalon 57 21 Le s i o n made i n an attempt to destroy the habenular r e g i o n but w i t h damage v e n t r a l to i t 61 22 L e s i o n i n the habenular area that extended i n t o the v e n t r a l diencephalon .. .. .. .. .. .. .. 63 23 L e s i o n of the habenula only 63 24 Dors a l c e r v e a u - i s o l e l e s i o n .. .. .. .. .. 66 25 V e n t r a l c e r v e a u - i s o l e l e s i o n 66 26 Le s i o n of the m i d l i n e of the midbrain w i t h only s l i g h t l a t e r a l extension r . .. .. .. 67 27 L e s i o n of the m i d l i n e midbrain w i t h a large l a t e r a l extension . . .. . . . . .. . . 67 28 Lesi o n of the m i d l i n e midbrain w i t h l a t e r a l extension of the l e s i o n more on the r i g h t .. .. 69 29 Le s i o n of the m i d l i n e midbrain i n the d o r s a l raphe 70 X INDEX OF DIAGRAMS Diagram Page 1 S u b c e l l u l a r l o c a t i o n of no r a d r e n a l i n i n the nerve endings of the sympathetic nervous system 3 2 Summary of t y r o s i n e hydroxylase c o n t a i n i n g f i b e r s of the caudate and s e p t a l area 88 ACKNOWLEDGEMENTS I am g r a t e f u l l y indebted to Drs. P a t r i c k and Edie McGeer, Dr. Juhn Wada and Dr. C. Loeser 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 advice 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 to Mr. Ron Tsujikawa and Mrs. G. Turbis along w i t h other members of the Kinsmen Laboratory of N e u r o l o g i c a l Resear f o r t h e i r h e l p f u l t e c h n i c a l a s s i s t a n c e . 1 . INTRODUCTION Recently i t has become evident that the catecholamines (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 . Therefore, a study of these compounds should a i d i n e l u c i d a t i n g some of the biochemical mechanisms of 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 of the CA i s a dihydroxy aromatic r i n g ( c a t e c h o l moiety) w i t h a two carbon s i d e chain c o n t a i n i n g an amine group. V a r i a t i o n s w i t h i n the CA a r i s e from s u b s t i t u t i o n s on the ' J 3 carbon and/or the amine group. The important members of the CA f a m i l y , noradrenaline (NA) , dopamine (DA) and adrenaline, are shown i n F i g . 1 . b) Metabolism The steps i n the i n v i v o synthesis and catabolism of the CA have been worked out. The sequence and necessary enzymes are presented i n F i g . 1 . I t i s worth n o t i n g that 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 being precursors i n a d r e n a l i n e s y n t h e s i s . The CAs are i n a c t i v a t e d i n v i v o not only by O-methylation and o x i d a t i o n as shown i n F i g . 1 , but by re-uptake i n t o t i s s u e and by d i f f u s i o n away from the s i t e of a c t i v i t y . The CA and metabolites are e v e n t u a l l y excreted v i a the kidney and can be detected i n the u r i n e . c) L o c a t i o n The CAs.- are found i n various organs of the body: e.g. b r a i n and adrenals 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 innervated t i s s u e . I n the adrenals NA and adrenaline are contained i n v e s i c l e s w i t h i n chromaffin c e l l s , and are r e l e a s e d on s t i m u l a t i o n of the splanchnic nerve. DA i s a l s o present but probably only as a precursor ( 1 ) . In the r e s t of the p e r i p h e r y , NA i s contained i n the nerve endings, both i n v e s i c l e s , that are depleted on nerve s t i m u l a t i o n , and i n the cytoplasm (see Diag. 1 ) . 2. 0CH o IH COOH COOH Horaovanillic a c i d 3,4-dihydroxyphenyl-a c e t i c a c i d C H 3 ° HO -/^"X.CHOHCH^NH^ Normetanephrine JHOHCH^NH, Noradrenaline (NA) N-met l y l t r a n s f e r a s e V OH HOHCOOH COMT CHOHCH NHCH-2 3 Adrenaline 3,4-dihydroxymandelic CHOHCOOH a c i d 5j3-methoxy-4-hydroxymandelic •^r a c i d CH 30 / H O _ ^ ~ y , CH0HCH2NHCH3 Metanephrine COMT catecho1-0-methyltransferase MAO monamine oxidase F i g . 1: Metabolism of Catecholamine 3. TYROSINE TYROSINE HYDROXYLASE DOPA DOPAMINE GRANULE NA RESERVE POOL • \IN VESICLE DEAMINATED MAO. MOBILE POOL OF TABOLITES NA IN CYTOPLASM RELEASE REUPTA: f ^COMT i DIFFUSION — — — — EFFECTOR CELL Diag. 1: S u b c e l l u l a r l o c a t i o n of noradrenaline i n the nerve endings of the sympathetic nervous system. 4. NA and DA are present i n b r a i n at approximately 1% of the c o n c e n t r a t i o n found i n adrenals. Here DA appears to have some r o l e i n a d d i t i o n to that as a precursor f o r NA s y n t h e s i s . As shown i n Table 1. both chemical (2,3,4,5) and h i s t o -chemical (6,7) analyses i n d i c a t e an uneven d i s t r i b u t i o n of NA and DA i n the b r a i n . DA i s concentrated i n the s t r i a t u m , NA i n the hypothalamus, but both are found i n s i g n i f i c a n t concentrations throughout the l i m b i c system. I t has a l s o been found by r a d i o -a c t i v e t r a c e r work that NA and DA i n j e c t e d i n t o the b r a i n are taken 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 areas (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. For example, i f r a d i o -a c t i v e NA 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 a f t e r one hour i n the hypothalamus i s 10 times that of the c o r t e x . A l s o i n d i c a t e d i n Table 1 are the d i f f e r e n c e s i n r a t e of i n v i v o s y n t h e s i s of CA i n the various regions of b r a i n ( 9 ) . By i n j e c t -i o n of the r a d i o a c t i v e precursor t y r o s i n e i t has been demonstrated th a t the type of CA synthesis v a r i e s throughout the b r a i n ; the major product 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 , NA i n the b r a i n has been detected i n presynaptic v e s i c l e s of nerve endings (11,12). Histochemical s t u d i e s a l s o i n d i c a t e that DA i s l o c a t e d p r i m a r i l y i n nerve endings (13). d) F u n c t i o n The f u n c t i o n s of CA i n the periphery are f a i r l y w e l l e s t a b l i s h e d . When an animal i s placed under s t r e s s there i s an increased neuronal a c t i v i t y i n the sympathetic nervous system, r e s u l t i n g i n NA and adrenaline r e l e a s e from the adrenals and a discharge of NA from sympathetic neurons. NA causes vaso-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 of 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 i n c r e a s e i n heart r a t e and f o r c e of c o n t r a c t i o n . Adrenaline has 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 heart 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 muscle, d i l a t i o n of b r o n c h i , m o b i l i z a t i o n of 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 * + 0 Area Biochemical Histochemical Turnover NA DA D e s c r i p t i o n of Monoamine NA DA Terminals Caudate Septum Hypothalamus Amygdala Hippocampus Thalamus Mi d b r a i n Pons-Medulla Oblongata Cerebellum Cortex V/gm 0.3 - 0.6 0.7 - 1.5 0.7 0.2 - 0.3 0.1 - 0.2 0.2 - 0.4 0.3 - 0.5 0.1 - 0.4 0.1 - 0.2 0.1 - 0.3 Y/gm 3.1 - 7.5 1.6 1.8 - 3.6 0.2 0.1 - 0.2 0.5 0.2 0.1 - 0.3 0.03- 0.1 0.1 - 0.3 i n t e n s i t y and type strong d i f f u s e fluorescence of DA low to medium of NA; dotted strong i n t e n s i t y of DA low to very s trong of NA ( i n d i f f e r e n t n u c l e i ) medium of NA low to high of NA predominantly low of NA -two very high n u c l e i wide v a r i a t i o n depending on n u c l e i wide v a r i a t i o n low - mostly NA mug/gm/hr 900 - 2400 234 33 90 42 36 summary of data from references 2,3,4 and 5 + from reference 7 0 from reference 9 6. These p h y s i o l o g i c a l changes enable the animal to meet many l i f e t h r e a t e n i n g 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 the periphery NA, but not ad r e n a l i n e , acts as a neurotransmitter i n p o s t g a n g l i o n i c neurons of the sympathetic nervous system. Although the evidence i s not as c o n c l u s i v e , i t i s a l s o thought that NA and DA are neurotransmitters i n the CNS (14,15). The i n d i c a t i o n s f o r t h i s r o l e are: 1) NA and DA are present i n b r a i n i n the appropriate concentrations (16), 2) enzymes f o r t h e i r formation and d e s t r u c t i o n are present (17,2,18,19), 3) agents that a c t as agonists and antagonists a t noradrenergic synapses i n the periphery a l s o a f f e c t the c e n t r a l nervous system (15), 4) r e l e a s e of GA i s observed on s t i m u l a t i o n of c e r t a i n nerve t r a c t s (20,21) 5) the CAsrare l o c a t e d i n nerve endings (11,12,13)and 6) there i s some i n d i c a t i o n that a p p l i c a t i o n of CA to synapses a f f e c t post s y n a p t i c p o t e n t i a l s (22)'. 2. Tyrosine Hydroxylase a) As a c o n t r o l of CA synthesis The enzymes a c t i v e i n the b i o s y n t h e s i s of CA, th a t i s t y r o s i n e hydroxylase, dopa decarboxylase, dopamine oxidase and N-methyl t r a n s f e r a s e have been s t u d i e d a great d e a l i n adrenal medulla (23,24,25",26) . In b r a i n , only t y r o s i n e hydroxylase (27) and dopa decarboxylase (2) have been worked on extensively,' (N-methyl t r a n s f e r a s e i s not present to any e x t e n t ) . From these s t u d i e s t y r o s i n e hydroxylase appears to be the r a t e l i m i t i n g enzyme, as would be expected s i n c e i t i s the f i r s t enzyme i n the b i o s y n t h e t i c r o u t e . The other enzymes i n the sequence have a c t i v i t i e s i n the order of 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 of 4-100 mjumoles/gm/hr. The K m f o r 7. o v e r a l l c o n v e r s i o n o f t y r o s i n e t o CAs i s 1 x 10 , t h e appr o x i m a t e f o r t y r o s i n e h y d r o x y l a s e ( 2 8 ) . I n h i b i t o r s o f t y r o s i n e 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 CA s y n t h e s i s ; t h e r e d u c t i o n i n NA i s p r o p o r t i o n a l t o t h e degree 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 i o n ( 2 9 ) . 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) prop o s e d t h a t t h e c o n v e r s i o n 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 s t e p i n CA s y n t h e s i s , because t h e amount o f enzyme p r e s e n t i n t h e t i s s u e i s l i m i t i n g . . A c c o r d i n g t o t h e i n v i v o t u r n o v e r s t u d i e s o f G l o w i n s k i (30,31,32) CA c o n c e n t r a t i o n s do n o t n e c e s s a r i l y r e f l e c t CA t u r n -o v e r . G l o w i n s k i f o u n d , f o r example, t h a t t h e c e r e b e l l u m , a l t h o u g h i t i s low i n CA, has one o f t h e h i g h e s t t u r n o v e r r a t e s . Such i n v i v o s t u d i e s o f CA t u r n o v e r i n v o l v e l o n g . a n d cumbersome p r o c e d u r e s and g i v e i n c o n s i s t e n t r e s u l t s . T h e r e f o r e measurement o f t y r o s i n e h y d r o x y l a s e i n v i t r o a c t i v i t y may be more h e l p f u l i n o b t a i n i n g an 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) C h a r a c t e r i s t i c s A l t h o u g h t h e p r e s e n c e 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 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 ( 1 0 ) , i t had n o t been p o s s i b l e t o d e t e c t i n v i t r o a c t i v i t y . Low e n z y m a t i c a c t i v i t y 14 and h i g h t y r o s i n e c o n c e n t r a t i o n s i n t i s s u e meant t h a t _ : . C -t y r o s i n e o f h i g h 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 . U d e n f r i e n d (33,34) was t h e f i r s t t o show an e n z y m a t i c c o n v e r s i o n o f L - t y r o s i n e t o Dopa. U s i n g p u r i f i e d b e e f a d r e n a l , t h e c o n d i t i o n s 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 a c e t a t e b u f f e r , pH 6.0y 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 ^ 2-amino-4-hydroxy-6,7-d i m e t h y l t e t r a h y d r o p t e r i d i n e (DMPH^) i n m e r c a p t o e t h a n o l . Work by U d e n f r i e n d and Kaufman (35) i n d i c a t e t h a t maximum a c t i v i t y o f t y r o s i n e h y d r o x y l a s e o c c u r s i n t h e p r e s e n c e o f p t e r i d i n e c o f a c t o r w h i c h a p p a r e n t l y i n c r e a s e s 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 i n v o l v e s t h e r e d u c t i o n o f enzyme by DMPH^ e n a b l i n g t h e enzyme t o a e r o b i c a l l y o x i d i z e t y r o s i n e t o Dopa. 8. Work i n t h i s l a b o r a t o r y has c o n f i r m e d t h e c h a r a c t e r i s t i c s o f t y r o s i n e h y d r o x y l a s e i n cr u d e b e e f a d r e n a l homogenate. B u t i n c r u d e 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 r e s u l t s were o b t a i n e d ( 2 7 , 3 6 ) . Maximum i n v i t r o a c t i v i t y was f o u n d t o be w i t h BO, b u f f e r pH 6.2, i n a i r ; DMPH, does n o t enhance a c t i v i t y . - 5 - 5 The 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 f o r t h e m -5 above t i s s u e s as compared t o 2 x 10 f o r a d r e n a l . c) L o c a t i o n i ) S u b c e l l u l a r l o c a l i z a t i o n U s i n g s u c r o s e g r a d i e n t t e c h n i q u e s , 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 has been l o c a t e d i n t h e n e r v e e n d i n g s ( 3 7 ) . However t h e d i s t r i b u t i o n w i t h i n t h e synaptosome.has n o t been p r e c i s e l y e s t a b l i s h e d . O r i g i n a l l y 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 most 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 a f t e r 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 ( 3 8 ) . I n s p l a n c h n i c n e r v e i t has been d e m o n s t r a t e d t h a t t y r o s i n e h y d r o x y l a s e and dopa d e c a r b o x y l a s e a r e i n t h e c y t o p l a s m s ( 3 9 , 4 0 ) . Dopamine B o x i d a s e i s a p a r t i c l e bound enzyme p o s s i b l y p a r t o f , o r w i t h i n , -la-t h e 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 groups o f w o r k e r s (41,37) have 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. i i ) A n a t o m i c a l l o c a t i o n 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 most a c t i v e i n a d r e n a l s and 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 ) . 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 v a r i o u s b r a i n a r e a s has n o t been t h o r o u g h l y i n v e s t i g a t e d . d) I n h i b i t o r s 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 have been f o u n d , c a t e c h o l s and a r o m a t i c amino a c i d s (41,42,43,44). A c c o r d i n g t o k i n e t i c s t u d i e s o f U d e n f r i e n d , Dopa and 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^ and n o n - c o m p e t i t i v e i n h i b i t o r s 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 t y r o s i n e f o r s i t e s on t h e enzyme. The CAs th e m s e l v e s c a n i n h i b i t t y r o s i n e ^ ! : hydroxylase 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 potent catechol-type i n h i b i t o r s a r e ^ - m e t h y l Dopa, epinine and methylaminoacetocatechol. Examples of potent amino a c i d i n h i b i t o r s areo 1 -methyl-p-tyrosine, 3-iodo-tyrosine and h a l o -tryptophans. These compounds have been u s e f u l f o r studying t u r n -over r a t e s of CAs 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 CA l e v e l s (45,46). 3. Dopanergic and Noradrenergic Pathways i n B r a i n For many years anatomists have used nerve degeneration r e s u l t i n g from l e s i o n s to study neuronal pathways i n the c e n t r a l nervous system and periphery. Biochemical changes can a l s o be observed i n degenerated axons. C u t t i n g of the p o s t - g a n g l i o n i c neuron i n the sympathetic nerve r e s u l t s i n decreases i n NA content of innervated organs (47,48). These p r i n c i p l e s can be a p p l i e d to the c e n t r a l nervous system: u s i n g s t e r e o t a x i c apparatus, 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 placed i n b r a i n , and subsequent measurement of changes i n CA concentrations. can be used to tr a c e dopanergic and noradrenergic pathways The most extensive mapping of such paths has been done by a group of Swedish (6,7,49,50,51) workers using h i s t o c h e m i c a l techniques on r a t s . They describe DA-containing neurons o r i g i n a t -i n g i n the midbrain n u c l e i , i . e . zona compacta of 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 formation, and c r a n i a l 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 bodies send axons c a u d a l l y to the s t r i a t u m , tuberculum o l f a c t o r i u m and nucleus accombens on the i p s i l a t e r a l s i d e . The c e l l bodies of NA neurons are i n the pons and medulla oblongata. Most of the noradrenergic f i b r e s descend to the s p i n a l cord. 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 of the r e t i c u l a r formation send axons r o s t r a l l y through the tegmentum of the midbrain and medial f o r e b r a i n bundle (MFB) to the hypothalamus, p r e - o p t i c area, s e p t a l area, amygdala, hippocampus and c i n g u l a t e gyrus. The r e s u l t s of these experiments are summarized i n -.Fig. 2. 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 w o r k e r s 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 . P o i r i e r and Sourkes (53,54) u s i n g c a t s and monkeys, 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 l e s i o n s 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 b o d i e s i n 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 e o l i s p i g m e n t o s i s . 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 w i t h MAO i n h i b i t o r s . G o l d s t e i n (54,55,56) a l s o w o r k i n g on t h e n i g r o s t r i a t a l t r a c t , d e m o n s t r a t e d t h a t l e s i o n s o f the v e n t r o m e d i a l tegmentum o f t h e m i d b r a i n caused d e c r e a s e s i n DA c o n c e n t r a t i o n s , 14 d e c r e a s e i n s y n t h e s i s o f DA f r o m C - t y r o s i n e and d e c r e a s e s i n u p t a k e o f r a d i o a c t i v e DA i n the i p s i l a t e r a l c a u d a t e and putamen. Moore and H e l l e r (57,58,59) have r e p o r t e d d e c r e a s e s i n NA w i t h l e s i o n s o f t h e MFB w i t h i n the l a t e r a l h ypothalamus; a r e a s showing d e c r e a s e i n c l u d e t h e septum, s t r i a t u m , amygdala and 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 d e m o n s t r a t i n g t h e s e C A - c o n t a i n i n g t r a c t s ( 6 0 , 6 1 , 6 2 ) . 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 i n d i c a t e e f f e r e n t s t o t h e r e d n u c l e u s , s u p e r i o r c o l l i c u l i , thalamus and g l o b u s p a l l i d u s . Nauta (63) c l a i m s t h e r e a r e a s c e n d i n g f i b r e s 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 th e i n t e r n a l c a p s u l e . He (64) and o t h e r s (65,66) a l s o d e s c r i b e f i b e r s i n t h e MFB t o t h e amygdala, t h a l a m u s , s e p t a l n u c l e i , d i a g o n a l band n u c l e i , hippocampus and hypothalamus. 4. P o s s i b l e R o l e o f C a t e c h o l a m i n e s i n B r a i n F u n c t i o n S i n c e t h e CAs a r e p r o b a b l y n e u r o t r a n s m i t t e r s i n t h e c e n t r a l n e r v o u s system; i t w o u l d be assumed t h e y i n f l u e n c e s p e c i f i c f u n c t i o n s i n t h e b r a i n . Some work has been done i n t r y i n g t o r e l a t e CA and b r a i n f u n c t i o n , a) E m o t i o n and B e h a v i o r Papez (67) was t h e f i r s t t o p u t t h e n e b u l o u s term e m o t i o n < i n t o s o m e t h i n g " 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 o f h y p o t h a l a m u s , t h a l a m u s , c i n g u l a t e g y r u s and h i p p o -campus. T h i s c i r c u i t i s now t h o u g h t t o i n v o l v e most o f t h e l i m b i c s y s t e m ( 1 4 ) . The hippocampus and amygdala appear t o be t h e c e n t r a l 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 t r a n s d u c e d i n t o a p r e c i s e s o m a t i c 11.' P A T H W A Y S D O P A M I N E i N O R A D R E N A L I N E I I F i g . 2: Summary of noradrenergic and dopaminergic t r a c t s i n b r a i n LF = l i m b i c f o r e b r a i n MFB = medial f o r e b r a i n bundle ST = s t r i a t u m ME = mesencephalon TH = thalamus MO = medulla oblongata HY =» hypothalamus From reference 15 12. emotional responses which i s expressed v i a the other components of the c i r c u i t . The s e p t a l area may f u n c t i o n as a type of 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 to breakdown. The r e s u l t of such a d i s r u p t i o n could be mental i l l n e s s . As already i n d i c a t e d , these areas are r e l a t i v e l y h i g h i n CAs. I t has been hypothesized that they may p l a y some r o l e as neurotransmitters 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 theory i t has been found that CA agonists and antagonists can be used i n the treatment of mental i l l n e s s . I n general, drugs that antagonize CA a c t i v i t y are mood depressants and those that a c t as agonists are mood ele v a t o r s (15). The hypothesis (15,68) i s that mood changes are caused by a c t i v a t i o n or depression of synapses i n the l i m b i c lobe. Since the method of mood development i s not 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 other systems, the exact mechanism of a c t i o n of these drugs i n chaig.ng mood i s obscure. Numerous animal experiments have been done i n an attempt to r e l a t e emotion, behavior and CA. Examples of these are: 1) s t i m u l a t i o n of the amygdala i n cats produces sham rage along w i t h a decrease i n NA i n the b r a i n (69), 2) brainstem t r a n s e c t i o n s that evoke defense r e a c t i o n s cause decrease i n NA i n b r a i n (70), 3) s e l f s t i m u l a t i o n of pleasure centres of the hypo-thalamus i s i n h i b i t e d by decreasing NA co n c e n t r a t i o n and enhanced by the NA a g o n i s t , metanephrine, suggesting that NA may be the neurotransmitter i n t h i s pleasure system (46), and 4) animals i n groups ( s o c i a l s t r e s s ) have more CA and se r o t o n i n i n the b r a i n , tend to be l e s s aggressive and responsive to s t i m u l i than do i s o l a t e d animals (71,72,73). The suggestion i s that i n the grouped animals w i t h excess t r a n s m i t t e r s the receptors are d e s e n s i t i z e d and therefore l e s s responsive. This could be a s o r t of emotional s a f e t y mechanism. Other s i m i l a r s t u d i e s (74,75,45) have been done but 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, behavior and emotion have been obtained. 13. b) Memory and Learning As w e l l as being p a r t of the "emotion c i r c u i t " the hippocampus i s a l s o important i n memory (77). Whether the CAs:, present there or i n other regions of the b r a i n are s i g n i f i c a n t i n memory i s not known. Wada and McGeer (78) showed that an i n c r e a s e 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 reported that decreases i n c e r e b r a l CA and s e r o t o n i n concentrations enhanced l e a r n i n g . With respect to the already learned response, i t has been reported (80,81,82) t h a t antagonists of NA a c t i v i t y blocked conditioned behavior response. c) Sleep .Sleep i s a p o o r l y understood phenomenon i n which CA may p l a y an important r o l e . .During a sleep period animals a l t e r n a t e between l i g h t sleep and p a r a d o x i c a l sleep. Jouvet (83,84) has proposed t h a t NA from the n u c l e i locus caeruleus i n the tegmentum of the pons acts as mediator 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 s l e e p . Narcolepsy, a p a t h o l o g i c a l c o n d i t i o n which causes a sudden onset 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 formation can cause an a r o u s a l response (86) . These l a t t e r observations would i n d i c a t e t h e i r may be a r e l a t i o n s h i p between CA and s l e e p . d) Temperature Re 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 that the hypothalamus i s prominently concerned i n body temperature c o n t r o l . Feldbury and Meyer (87) and others (88) have p o s t u l a t e d that temperature r e g u l a t i o n takes place by a balance of the hyper-thermic e f f e c t s of s e r o t o n i n and of 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) that the CA, whose e x c r e t i o n i s increased when animals are placed i n the c o l d , are important i n adaption to c o l d . 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 only or whether there i s a l s o a CNS component. e) Basal Ganglia and Parkinsonism Through s t u d i e s of DA metabolism and dopanergic pathways the 14. f u n c t i o n and mechanism of a c t i o n of the b a s a l g a n g l i a are being e l u c i d a t e d . I n p a t i e n t s s u f f e r i n g from Parkinsonism, a disease apparently caused by malfunction of 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 DA and i t s metabolites are decreased (90). A f t e r death 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 decrease i n DA content (91). Agents such as r e s e r p i n e that (among other a c t i o n s ) deplete DA from the b a s a l g a n g l i a cause Parkinsonism - l i k e symptoms (92), 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 reported to a l l e v i a t e the symptoms of Parkinsonism i n some cases (93). Lesions of 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 (94). From there and other f i n d i n g s i t has been suggested that DA acts as an i n h i b i t o r y neurotransmitter i n the b a s a l g a n g l i a and that Parkinsonism may i n p a r t be a r e s u l t of inadequate i n h i b i t i o n i n the extra-pyramidal motor system as a r e s u l t of l a c k of neurotransmitter DA (95,96). In a l l the aspects of b r a i n f u n c t i o n mentioned there are h i n t s of r e l a t i o n s h i p s between CA and b r a i n mechanisms, but no d e f i n i t e working hypothesis can be e s t a b l i s h e d . Much more work must be done on the CA i n b r a i n . 5. Aims of Thesis The purpose of t h i s i n v e s t i g a t i o n i s to study s e v e r a l aspects 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. There w i l l be three main c o n s i d e r a t i o n s : 1) The e f f e c t s on t y r o s i n e hydroxylase a c t i v i t y i n b r a i n of a l t e r i n g CA l e v e l s i n the body w i l l be determined. These a l t e r a t i o n s are brought about by p l a c i n g the animal i n the cold) 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 that deplete CA stores and MAO i n h i b i t o r s that i n c r e a s e CA c o n c e n t r a t i o n s ) . 2) An extensive study w i l l be made of t y r o s i n e hydroxylase a c t i v i t y i n various regions of b r a i n i n s e v e r a l animals. 3) On the b a s i s of, these 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 that t y r o s i n e hydroxylase i s contained i n nerve endings, the t h i r d aim of t h i s study w i l be the i n v e s t i g a t i o n of the anatomy of the noradrenergic and dopanergic pathways i n b r a i n . Le s i o n techniques coupled 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 Tissue was homogenized i n 4 - 9 volumes of sucrose. The in c u b a t i o n mixture c o n s i s t e d of 0.1 ml 0.28 M PO^ b u f f e r pH 6.2, 0.1'ml of homogenate, and 0.1 ml of a s o l u t i o n of uniform 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. ac t . 360-375 mc/mmoles) -3 which 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 of 0.40 M PO. -4 4 4 b u f f e r and 0.03 ml of 2 x 10 DMPH^ i n 0.02M 2-mercaptoethanol were added i n s t e a d of the 0.1 ml of 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 out i n a i r at 37°C f o r 30 minutes and stopped w i t h the a d d i t i o n of 2 ml of a 1:1 mixture of 0.2M HAc and 0.4M HC10, 4 co n t a i n i n g 0.1 ug/ml of Dopa, NA and DA. Blanks were run at the same time using t i s s u e that had been heated at 80 - 90°C f o r 10 - 15 minutes. D u p l i c a t e s were run f o r each sample. In most cases the a c i d i f i e d incubates were f r o z e n before i s o l a t i o n of the ca t e c h o l s . I s o l a t i o n of the r a d i o a c t i v e catechols formed was on aluminum oxide. The i n c u b a t i o n mixture was thawed, c e n t r i f u g e d and the supernatant poured i n t o a 20 ml beaker c o n t a i n i n g 1 ml of 0.2M EDTA. The p r e c i p i t a t e was washed w i t h 3 ml of 0.32M PO^, r e c e n t r i f u g e d and the supernatant pooled w i t h the other. Each sample was taken to pH 8.8 - 9.2 w i t h NaOH and 300 mg of alumina was added to the beaker. The mixture was s t i r r e d f o r 4 - 5 minutes and then washed i n t o a small glass column (4:mm diameter) stoppered w i t h glass wool. Gentle s u c t i o n was used to draw the l i q u i d through the column. The column was f u r t h e r washed w i t h 2 l o t s of water (ca 10 ml each). The catechols were el u t e d from the column w i t h 2 ml of 0.5N HAc i n t o a small v i a l . Ten ml of Bray'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 spectrophotometer. The whole i s o l a t i o n procedure took approximately 10 minutes per sample. i i ) R a t i o n a l f o r Procedure Conditions 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 out (27) and diagrams presented i n t h i s t h e s i s are co n f i r m a t i o n 17. of these. The volumes of sucrose were chosen to give 10 - 20 mg of t i s s u e per i n c u b a t i o n . The amount used i n any given i n c u b a t i o n depended on the amount of t i s s u e a v a i l a b l e and the a c t i v i t y of the t i s s u e . As shown i n F i g . 3 the a c t i v i t y i s l i n e a r w i t h mg of t i s s u e used i n t h i s range. Using sucrose of m o l a r i t y 0.2 - 0.3 i t has been shown (27) t h a t 0.28 gives the best conversion. As shown i n F i g . 4 the maximum pH f o r the 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 (27) PO, b u f f e r 14 gave maximal r e s u l t s f o r b r a i n t i s s u e . The L - t y r o s i n e - C from the manufacturer was d i l u t e d so that each i n c u b a t i o n mixture con-tained 1/12^uc (150,000 cpm). For whole r a t b r a i n t h i s r e s u l t e d i n approximately 2000 cpm of catechols formed w i t h a blank of 150 - 200 cpm. Therefore, t h i s amount appeared to be a good compromise between reasonable conversion, low blank and cost. NSD-1034 i s a potent Dopa decarboxylase i n h i b i t o r (97) , and blocks the r e a c t i o n i s a t the Dopa stage. I f NA and DA were formed they would f u r t h e r be metabolized by 0-methyl t r a n s f e r a s e and monoamine oxidase. This would make i s o l a t i o n more d i f f i c u l t . A d d i t i o n of c o l d catechols before i s o l a t i o n improves the recovery. Incubation i s l i n e a r w i t h time up to 45mihutes ( F i g . 5 ) . T h i r t y minutes was chosen f o r con-venience . I t has been reported that DMPH^ i s necessary f o r maximum t y r o s i n e hydroxylase a c t i v i t y i n p u r i f i e d beef adrenal (23). Incubations c a r r i e d out w i t h and without DMPH^ are ...presented i n Table 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 hydroxylase a c t i v i t y of b r a i n homogenates w i t h and without 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 adre n a l s , i n d i c a t i n g that the DMPH^ had not been decomposed. Because of these r e s u l t s the co f a c t o r was not used r o u t i n e l y i n the b r a i n t y r o s i n e hydroxylase homogenates. Oxygen i s necessary f o r the r e a c t i o n but 20% saturates the enzyme (23). The incubations are therefore c a r r i e d out i n a i r . Alumina has proven to be one of the most s a t i s f a c t o r y and si m p l e s t methods f o r i s o l a t i o n catechols (98). Uptake on the colunn i s 18. I , ; i 8 • I i 0 5 10 15 20 MG. T I S S U E / I N C U B A T I O N F i g . 3: 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 hydroxylase a c t i v i t y F i g . 4: E f f e c t of pH of i n c u b a t i o n mixture on the a c t i v i t y of t y r o s i n e hydroxylase 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 Tyrosine hydroxylase w i t h respect to ; time of i n c u b a t i o n — „ — 50 60 70-_ L U > 65-o o LU _ 60* "S-5- 9.0 9^ 5 10© PH : F i g . 6: Recovery of catechols from' alumina at various pH's of the J sample as i t i s placed on the column 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 of A c t i v i t y Without 11 Areas of Rabbit B r a i n 18 Areas of Cat B r a i n 36 Rat B r a i n s Cat Adrenal Medulla 109% - 17 92% - 21 104% 1030% TABLE 3 EFFECT OF PHOSPHATE ADDITION ON SIZE OF BLANK AND DOPA RECOVERY FROM ALUMINA COLUMN cpm 3 5 257 249 4000 4210 Ml of Phosphate 0 1 Blank (150,000 cpm of 1343 606 1 4 ^ .Tyrosine- C) Dopa (6200 cpm) 4020 4136 22. s p e c i f i c f o r the c a t e c h o l moiety a t a l k a l i n e pH's. EDTA i s added to prevent o x i d a t i o n by heavy metals. 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 at a l k a l i n e pH. The pH f o r maximum recovery i s 9.0 as shown i n F i g . 6. PO^ i s added because i t was found to prevent 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 which can give i n a very l a r g e blank as shown i n Table 3. The phosphate does not i n t e r f e r e w i t h the Dopa recovery which i s 65 - 70%. The samples c o n s i s t e n t l y counted at 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 spectrophotometer. b) Tyrosine A n a l y s i s For the c a l c u l a t i o n of V f o r t y r o s i n e hydroxylase i t max i s necessary to know the endogenous t y r o s i n e l e v e l s . Incubations are run below s a t u r a t i o n and endogenous l e v e l s are h i g h enough to a f f e c t the value. i ) Procedure Tyrosine was determined by a m o d i f i c a t i o n of the procedure of Waalkes and Udenfriend (99). 0.2 ml of the sucrose homogenate were added to 0.2 ml of 30% TCA and 0.6 ml of H 20 ( i f s u f f i c i e n t 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 give more supernatant to work w i t h ) . A f t e r c e n t r i g u g a t i o n the supernatant was poured o f f and u s u a l l y f r o z e n before a n a l y s i s . The analysate mixture c o n s i s t e d of 0.6 ml supernatant, 0.6 ml of 0.1% n i t r o s o - 2 -naphthol i n 95%, ethanol and 0.6 ml of a mixture of 24.5 ml of 1:5 n i t r i c a c i d and 0.5 ml of 2.5% NaNO^- A f t e r thorough mixing the mixture was placed i n a water bath at 55°C f o r 30 minutes. The unreacted l - n i t r o s o - 2 - n a p h t h o l was e x t r a c t e d w i t h 2.5 ml of ethylene d i c h l o r i d e . The aqueous l a y e r was placed i n a small c l e a r t e s t tube and the fluorescence determined i n a spectrophotoflUorometer at 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 blank and tY/ml standard s o l u t i o n were analyzed at the same time. | i i ) R a t i o n a l f o r A n a l y s i s : Tyrosine has been shown to conjugate w i t h l - n i t r o s o - 2 -naphthol 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. Fluorescence 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 normally used ( F i g . 7 ) . Samples u s u a l l y gave readings of 0 0.5 T!o™ 1.5 C O N C E N T R A T I O N 8ml.) F i g . 7: Standard curve f o r t y r o s i n e determination, fluorescence versus co n c e n t r a t i o n _!o~ — i 70-8.5 9.0 9.5 m O 10.5 PH F i g . 10: Recovery of NA and DA from alumina w i t h respect to pH of sample as i t i s placed i n • the column . ' 24. 0.06 - 0.15 f l u o r e s c e n t u n i t s and the blank was approximately 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 ^ m a x f° r t y r o s i n e hydroxylase i t was necessary to determined the K value. K determinations were c a r r i e d out m m using whole r a t b r a i n , cat and r a b b i t and cat and r a b b i t caudate. Incubations were c a r r i e d out as p r e v i o u s l y described except that i n c r e a s i n g amounts of c o l d t y r o s i n e were added i n the phosphate b u f f e r . P l o t s were done according to the standard i / V vs I/S method and by the S/V vs S method as recommended by Dowd and Riggs (108). F i g . 8 i s a r e p r e s e n t a t i v e p l o t f o r t y r o s i n e hydroxylase i n r a t b r a i n . t Many determinations i n t h i s l a b o r a t o r y showed the average K m f o r r a t b r a i n to be 0.45 x 10 ^ (27). For cat and r a b b i t b r a i n the K f o r t y r o s i n e hydroxylase i s of the order of 1 x 10 Sample p l o t s are shown i n F i g . 9. These were the values used i n c a l c u l a t i o n of V max The V i n mu moles of Dopa formed/gm of t i s s u e / h r max r ° usin g the Michaelis-Menten formula: V = v (1 + |P) max K - i n moles/l was determined experimentally f o r each animal as discussed above. c-.. - t o t a l c o n c e n t r a t i o n of t y r o s i n e i n moles/1 of incubate. This in 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 of Dopa formed/gm/hr. This i s deter-mined from the p r o p o r t i o n of r a d i o a c t i v e t y r o s i n e converted to Dopa w i t h c o r r e c t i o n s f o r the non-l a b e l l e d t y r o s i n e present. Since the i n c u b a t i o n volume, amount and a c t i v i t y of the r a d i o a c t i v e t y r o s i n e used, column recovery, counting e f f i c i e n c y and time of i n c u b a t i o n were a l l kept constant, a formula could be 25. derived r e l a t i n g V . o n l y , to the amount of t i s s u e used, the ° max J t y r o s i n e c o n c e n t r a t i o n i n that t i s s u e , the K f o r the r e a c t i o n and J m the measured cpm. v = ( 5 4 - 3 y 4 6 ) + T max I B K = Km '  . To"6 -4 1.3 x 10 x cpm S = mg of 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 d) Noradrenaline and Dopamine Determinations i ) I s o l a t i o n Tissue was homogenized i n a minimum volume of a 1:1 mixture of 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 sucrose f o r enzyme incubations the volume was noted and 0.2 ml of concentrated HC10. was added. The 4 samples were cooled f o r 10 minutes,. ..'centrifuged f o r 10 minutes, and the supernatant was poured i n t o a t e s t tube c o n t a i n i n g 0.5 ml of EDTA. The p r e c i p i t a t e was taken up i n 2 ml of HCIO^: HAc mixture and the procedure repeated. At 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 alumina, as p r e v i o u s l y described^except the mixture was taken to pH 9.0 - 9.5 before — ab s o r p t i o n on to the column. The eluant of 0.5N HAc was c o l l e c t e d i n a graduated t e s t tube. 0.5 ml of 1.0M Ac b u f f e r pH 6.0 was added and the pH was adjusted to 6.0 w i t h NaOH. Water was added to make the f i n a l volume 3.0 ml. Standards of c o l d and r a d i o a c t i v e catecholamines were run at the same time to determine percent recovery. i i ) NA A n a l y s i s For each sample 3 small tubes were used: 1. sample 2. i n t e r n a l standard 3. blank To each tube 0.5 ml of sample was added. Tubes 1 and 3 were buf f e r e d w i t h 0.5 ml of 0.5M NaAc pH 6.4, and tube 2 w i t h 26. 0.5 ml of b u f f e r c o n t a i n i n g 0.1 ug of NA. To a l l tubes 0.5 ml of 0.01N i o d i n e was added followed i n 4 minutes by 0.25 ml of 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 ml of a mixture of 7 ml of 5N NaOH and 0.5 ml of 0.57o a s c o r b i c a c i d , tube 3 w i t h 0.35 ml NaOH. The tubes were l e f t standing i n l i g h t f o r 90 minutes. Before readings were taken, i n the spec t r o -photofluorometer, 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 added to the blank. 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 peak 505 mu. i i i ) DA a n a l y s i s A l l reagents must be at room temperature. Two l a r g e t e s t tubes were used f o r each sample. 1. sample 2. i n t e r n a l standard Two eluant blanks were run at the same time f o r each group of sample. To each tube 0.5 ml of sample was added to tube 1. 0.5 ml of 1.0M A C b u f f e r pH 6.0 was added, and to tube 2 0.5 ml of b u f f e r c o n t a i n i n g 0.1 p.g!;DA. Tubes were then t r e a t e d w i t h 0.25 ml of 0.01N i o d i n e f o l l o w e d i n 10 minutes by 0.25 ml of 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 of anhydrous Na^S^O^. Three minutes l a t e r 0.5 ml of 5N HCl was added to each. The tubes were l e f t to stand i n l i g h t f o r 12 -.24 hours and then read i n the spec t r o -photofluorometer 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. i v ) R a t i o n a l f o r Catecholamine Determination DA and NA were taken up on alumina as p r e v i o u s l y described f o r Dopa. However as shown i n F i g . 10, the recovery was found to be maximum between pH 9.0 - 9.5. The recovery was more v a r i a b l e (60 - 807c) and c o l d standards were th e r e f o r e run w i t h each group. For very 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 catecholamine standards were used to determine the per cent recovery 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 of catecholamines 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 (100). The equation f o r the noradrenaline r e a c t i o n i s : m 0 T.O 2 . 0 3.0 4.0 5.0 S * 1 0 s F i g . 9: K determination f o r r a b b i t (x) and cat (.) b r a i n m F i g . 8: K determination f o r r a t b r a i n m 28. Because the f l u o r e s c e n t compound i s unstable i n a l k a l i , a s c o r b i c a c i d i s added. Dopamine undergoes a s i m i l a r r e a c t i o n . The pH f o r DA a n a l y s i s i s lowered w i t h HCI to decrease the wave-length of a c t i v a t i o n and fluorescence (to d i s t i n g u i s h i t from NA) and to i n t e n s i f y the fluorescence (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 that there i s a l i n e a r r e l a t i o n s h i p between co 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 . This method i s very u s e f u l because very small q u a n t i t i e s ^ 0 . 1 Vcan e a s i l y be detected and measured a c c u r a t e l y . e) Separation of Noradrenaline, Dopamine and Some of Their M e t a b o l i t e s Experiments were c a r r i e d out to develop methods of s e p a r a t i n g noradrenaline, dopamine and t h e i r m e t a b o l i t e s . 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 (102,103,104), 2. 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 c a r r i e d out on Dowex 50 x 8, 200 - 400 mesh, which was kept under F i g . 11: Standard curve f o r NA and DA determinations. Fluorescence verus c o n c e n t r a t i o n 30. 2N HCI p r i o r to use. .A small glass column, 4 mm i n diameter was plugged w i t h glass wool. The Dowex i n 2N HCI was added so that the f i n a l height of r e s i n was 5 cm. The r e s i n was washed w i t h d i s t i l l e d water u n t i l the washings were n e u t r a l (with pH paper) and then 10 - 15 mis of 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 approximately 2 ml 0.1N HCI f o r a p p l i c a t i o n to the column. U s u a l l y 25 V of each were used plus various amounts of r a d i o a c t i v e -14 C DA and NA. The r e s i n was washed w i t h 10 mis of water and 10 mis of 0.1N HCI. E l u t i o n was c a r r i e d out w i t h 0.4N HCI a t a r a t e of 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 of catecholamine fluorescence determined by measuring each f r a c t i o n i n the spectrophotofluorometer at a c t i v a t i o n peak of 285 mu and emission of 330 mu. A h a l f ml a l i q u o t of each sample was added to 10 mis of Bray'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 -s c i n t i l l a t i o n spectrophotometer. i i ) Thin Layer Chromatography Precoated sheets of polyamide were used as the s t a t i o n a r y phase. The mobile phase was a mixture of i s o b u t a n o l , a c e t i c a c i d and cyclohexane (80:7:10). The tank was saturate d w i t h the so l v e n t f o r 15 - 20 minutes p r i o r to use. The chromatograms ran f o r 3 -44 hours (8 - 10 cm above the o r i g i n ) . One - f i v e jag of NA, DA, normetanephrine, methyoxytyramine, Dopa, t y r o s i n e , 3,4-dihydroxy-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 spotted. Those c o n t a i n i n g the cat e c h o l moiety (NA, DA, Dopa and dihydroxy-ph e n y l a c e t i c a c i d ) were detected using a s o l u t i o n of ethylenediamine (1:1 w i t h water) and examination under UV l i g h t . The remaining compounds were observed using a p - n i t r o - a n i l i n e reagent made by combining the f o l l o w i n g three 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 are: 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 2 i n 100 ml H^O, and, 3. 10% KC0 2 s o l u t i o n . A l l compounds gave purple spots. f ) Catecholamine A c t i v i t y i n Rats i n the Cold i ) Catecholamine determination i n u r i n e Rats were placed i n i n d i v i d u a l metabolic cages i n the c o l d room (3°C) and at room temperature. Both sets of r a t s were exposed to a c y c l e of 12 hours l i g h t and 12 hours darkness. Twenty four hour u r i n e samples were c o l l e c t e d and analyzed f o r NA and DA as p r e v i o u s l y d e s c ribed. Before i s o l a t i o n on the alumina the urine s are taken to pH 4, heated i n a . b o i l i n g water bath f o r 10 minutes to hydrolyze catecholamines conjugates. i i ) Tyrosine hydroxylase a c t i v i t y i n c o l d a c c l i m a t i z e d r a t s Experimental and c o n t r o l r a t s were housed as described i n the previous s e c t i o n f o r periods of 4 hours to s e v e r a l weeks. The animals 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 , heart and spleen removed. The b r a i n and adrenals were analyzed f o r t y r o s i n e hydroxylase a c t i v i t y and f o r catecholamines. The hearts and spleens were analyzed only f o r catecholamines. 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 described were i n j e c t e d w i t h the t y r o s i n e hydroxylase 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 animals (2 experimental and 2 c o n t r o l s a t each time period) 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 , adrenals, heart and spleen were analyzed f o r NA and DA. The l o g of the c o n c e n t r a t i o n of amine was p l o t t e d a g a i n s t time to determine turnover r a t e s . g) E f f e c t of A l t e r e d Catecholamine Levels on Tyrosine Hydroxylase 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 tranylcypramine, and the catecholamine depletors r e s e r p i n e and guan-e t h i d i n e . The doses were p a r g y l i n e 75 mg/kg fo l l o w e d by 5 mg/kg 32. 12 hours l a t e r ; tranylcypramine 30 mg/kg and 5 mg/kg; r e s e r p i n e 25 mg/kg and 15 mg/kg and guanethidine 5 mg/kg both times.. The animals 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 of the f i r s t dose. The b r a i n s were analyzed f o r t y r o s i n e hydroxylase 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 Rats, r a b b i t s and cats were used i n t h i s study. Rats were s a c r i f i c e d by a blow to the head, r a b b i t s by c e r v i c a l d i s -l o c a t i o n and cats by n i t r o g e n a s p h y x i a t i o n . The bra 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 area i n some of the cats 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 analyzed f o r t y r o s i n e hydroxylase a c t i v i t y and the V f o r each area determined. J J J max i ) Study of Catecholamine Pathways i n Cat 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 placed i n the midbrain and diencephalon of cats using s t e r e o t a x i c techniques. The s t e r e o t a x i c a t l a s e s of Snider (106) and Jasper (107) were used i n placement of the l e s i o n s as to 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 p o s i t i o n i n g . The s i z e of the l e s i o n s could 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 of needle placements. Seventy two hours a f t e r the operation the cats 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 the areas described under d i s t r i b u t i o n s t u d i e s . Tyrosine hydroxylase a n a l y s i s was done on a l l areas. Catecholamine determinations were done on caudate and s e p t a l area. Areas from each side of the b r a i n were analyzed s e p a r a t e l y even i n the case of 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 values i n the case of b i l a t e r a l max l e s i o n s and w i t h the unlesioned s i d e i n the case of u n i l a t e r a l . The l e s i o n e d area was removed and placed i n formaldehyde and given to the Pathology and/or Anatomy departments. There i t was placed i n a p a r a f f i n b l o c k , cut and s t a i n e d by the Luxol Fast Blue method. The sections^-.." examined by a neuroanatomist who reported on the p o s i t i o n and extent of the l e s i o n . 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 the method of d i s s e c t i o n Landmarks - A - corpus callosum, B - f o r n i x , C - o p t i c chiasm, D - mammillary bodies and E - a n t e r i o r commissure Areas - 1 - s p i n a l cord, 2 - pons and medulla oblongata, 3 - midbrain 4 - cerebellum, 5 - hypothalamus, 6 - s e p t a l area, 7 - thalamus 8 - c o r t e x 34. I I I I I I I- I I I I I -I I I i I I I I I I I .1 I + 30 mm •2-2 0 mm +1 0mm M E D I A N S A G G I T A L S E C T I O N T R A N S V E R S E S E C T I O N F i g . 13: Median s a g i t t a l and transverse s e c t i o n of cat 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 area Landmarks - A - corpus callosum, B - f o r n i x , C - a n t e r i o r commissure, D - o p t i c chiasm Areas - 1 - a n t e r i o r hypothalamus 4 2 - p r e o p t i c area 5 a n t e r i o r s e p t a l area r a n t e r i o r perf. substance 3 - s e p t a l n u c l e i 6 - a r e a 0 f nucleus accombens 35. RESULTS i 1. Separation of NA, DA and Some of Their M e t a b o l i t e s a) Ion exchange chromatography Since 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 flu o r e s c e n c e , the behavior of the compounds on the Dowex column was determined by running the amines 14 14 one a t a time and by using NA- C and DA- C. When e l u t i o n from the column was c a r r i e d out w i t h 0.4N HCI, only NA waseluted 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 present, s e p a r a t i o n of NA and DA could be c a r r i e d out more r a p i d l y by using 2N HCI as an eluant a f t e r the NA had been removed from the column w i t h 0.4N HCI. DA could then be c o l l e c t e d ^ immediately a f t e r e l u t i o n w i t h 2N HCI beganjin tubes 1 - 9 and MT could be e l u f e d i n tubes 11 - 20. These r e s u l t s are summarized by t y p i c a l e l u t i o n curves i n F i g . 14 and 15. I t was a l s o found that t y r o s i n e and Dopa could be separated from the amines by washing the column w i t h 10 ml of phosphate b u f f e r pH 5.0. The ac i d metabolites of DA and NA, ho m o v a n i l l i c a c i d and 3-methoxy74-hydroxymandelic a c i d , are probably not absorbed, or are only very weakly absorbed on t h i s column. When no MAO i n h i b i t o r was added to an in c u b a t i o n mixture of b r a i n homogenate c o n t a i n i n g 14 DA- C, over 507o of the r a d i o a c t i v i t y appeared i n the washings when t h i s mixture was a p p l i e d to the column. With an MAO i n h i b i t o r £-< Vk was contained i n the washings. Other i n v e s t i g a t o r s (102) have reported that these a c i d metabolites are not adsorbed i n t h i s type of column. b) l Thin Layer chromatography R^ values obtained from t h i n l a y e r chromatography of DA, /YVs NA and some of t h e i r metabolites are presented i n Table 4. 2. Catecholamine A c t i v i t y i n Rats Exposed to Cold a) Urine 1.6- n 36. 1.4-1.2- N E 1.0-. L U o z LU o in LU _ o 0.8-0.6-0.4-0.2-0 10 f) •u © 20 30 0.4 N H C I -T U B E N U M B E R F i g . 14: Separation by ion-exchange chromatography of NA, NM and DA- C - fluorescence at 258 mu/330 nyu — counts per minute f o r C-DA 1.6-Jl ru 37, 1.4c 1.2-l . G c U J O z UJ U tf) UJ o Z3 0.8= 0.6* 0.4= 0.2-4-D A M T v 10 •0.4 N HCi -20 30 40 -2N HCI 50 F i g . 15: 14, T U B E N U M B E R Separation of NA, DA and MT and NA-"C by ion-exchange chromatography fluorescence at 285 mu/330.mu — Counts per minute f o r -C-NA ' TABLE 4 •R VALUES^FOR NA, DA AND SOME OF THEIR PRECURSORS 1AND METABOLITES ON POLYAMIDE Compound (as obtained i n (as reported 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 a c i d 0 .18 0 .27 3-Methoxy-4-hydroxymandelic a c i d 0 .19 0 .35 39. As i n d i c a t e d i n Table 5 there was a l a r g e increase ( 2 - 3 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 . There was a s l i g h t increase i n DA e x c r e t i o n . The increase occurred i n the f i r s t 24 hours i n the c o l d and the concentrations of DA and NA stayed a t the same l e v e l f o r up to two weeks. The values given are the averages 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 of b r a i n between normal r a t s and those exposed to c o l d . There was an increase (407o) of NA i n adrenal but no change i n DA. There may be an increase i n spleen NA and a decrease i n heart NA but the r e s u l t s on these t i s s u e s are not 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 large standard d e v i a t i o n . The r e s u l t s are presented i n Table 6. Spleen and heart DA concentrations are not presented because they were very v a r i a b l e . c) Tyrosine hydroxylase a c t i v i t y There was no s i g n i f i c a n t change i n t y r o s i n e hydroxylase 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 adrenals of c o l d exposed animals as shown i n Table 7. However, since the s i z e of the adrenals increased i n r a t s exposed to c o l d , there was an increase of 707o i n t y r o s i n e hydroxylase a c t i v i t y per adrenal which was s i g n i f i c a n t (p = 0.01). d) Turnover r a t e s In another attempt to assess the e f f e c t of c o l d exposure on catecholamine metabolism the r a t e of d e p l e t i o n of noradrenaline was measured a f t e r treatment w i t h the t y r o s i n e hydroxylase i n h i b i t o r s , alpha-methyl-p-tyrosine and alpha-methyl-mrtyrosine. At 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 are not reported, and NA l e v e l s i n heart w i t h v\-methyl-m-tyrosine. In b r a i n , heart and spleen the curves f o r noradrenaline l e v e l s a gainst time are very 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 that the apparent noradrenaline turnover 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 at .01 Le v e l DA 100 - 3% (II)" 1" 112 - 47 (10) +127, NA 100 -107, (14) 286 - 2.57, (13) +1867, * Expressed as 7> of c o n t r o l •j- No. of animal i n brackets TABLE 6 CATECHOLAMINE LEVELS IN TISSUES OF RATS EXPOSED TO COLD* Con t r o l Cold % S i g n i f i c a n t Change at .01 L e v e l B r a i n NA 100 + 137o ( 8)t 106 + 187o ( 9) -DA 100 + 357c ( 5) 122 + 87o ( 6) -Adrenal NA 100 + 47o ( 6) 140 + 77o ( 6) +407o DA 100 + 367, ( 5) 93 + 507o (15) -Spleen NA 100 + 277o ( 5) • 185 + 5570 ( 6) -Heart NA 100 + 367o ( 5) 67 + 317o ( 6) -Expressed as 7, of c o n t r o l No. of animals i n brackets TABLE 7 TYROSINE HYDROXYLASE ACTIVITY IN RATS EXPOSED TO COLD* Co n t r o l Cold B r a i n 100 - 15% (10) + 92 - 17% (12) Adrenals (per gm) ( 100 - 20% ( 7) 140 - 25% ( 7) (per ad) 100 - 14% 170 - 10% * Expressed as %, of c o n t r o l + No. of animals i n brackets ' adrenal 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 at about 4 hours suggesting that the noradrenaline may have more than one turnover r a t e , or may be recovery as appears to ^be the case i n h e a r t , spleen and adrenals. ( F i g . 16 (a) and (b) ) Data f o r the adrenals are u n s a t i s f a c t o r y i n that the r a t e of d e p l e t i o n of noradrenaline 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 alpha-methyl-p-tyrosine, p o s s i b l y due to a p l u r a l i t y of a c t i o n s . In the c o l d adapted animals 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 both 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 turnover r a t e than i n e i t h e r group of c o n t r o l s . Further e x p l o r a t i o n of these phenomena was not done because of an i n s u f f i c i e n t supply of the i n h i b i t o r s and the pressure of other programs. 3. E f f e c t s of A l t e r i n g Catecholamine Levels on In V i t r o Tyrosine  Hydroxylase A c t i v i t y As i n d i c a t e d i n Table 8, a number of drugs which s i g n i f i c a n a l t e r e d catecholamine 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 hydroxylase a c t i v i t y of these brains as measured i n v i t r o . 4. D i s t r i b u t i o n Studies of Tyrosine Hydroxylase 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 hydroxylase i n various regions of c a t , r a b b i t and r a t b r a i n are presented i n Table 9. A c t i v i t y r e l a t i v e to the cerebellum i s a l s o shown. The caudate i s by f a r the most a c t i v e area being 5 x greater than the next two most a c t i v e areas, the septum and a n t e r i o r p e r f o r a t i n g substance. The caudate i s almost 100 times more a c t i v e i n t y r o s i n e hydroxylase than the cerebellum which i s the l e a s t a c t i v e area. The r e l a t i v e values as compared to the cerebellum, are q u i t e c o n s i s t e n t i n the three species s t u d i e d . To show that there was a consistency i n d i s s e c t i o n , the average weight * standard d e v i a t i o n i s shown i n Table 10. Tyrosine values were a l s o quite c o n s i s t e n t f o r each area TABLE 8 EFFECTS (OF CERTAIN DRUGS ON CATECHOLAMINE LEVELS AND TYROSINE HYDROXYLASE ACTIVITY IN RAT BRAIN* NA DA Tyrosine Hydroxylase C o n t r o l ( 4 ) T 100 - 10% 100 - 17% 100 - 8% P a r g y l i n e (4) 180% 147% 99% T r a n y l -cypramine (2) 155% 152% 94% Reserpine (4) 14% 40% 95% Guanethidine (2) 110% 89% 106% t Expressed as % of c o n t r o l No. of animals i n brackets TABLE 9 DISTRIBUTION OF TYROSINE HYDROXYLASE IN ADULT RAT, RABBIT AND CAT BRAIN * Area V A c t i v i t y R e l a t i v e to Cerebellum max J r a t r a b b i t 4 c a t r a t r a b b i t cat Caudate 22.3 5.5 (6) 70.9 + 9.2 (5) 98.5 9 (11) , 56 101 0 70 Septal." area,;, 15.0 + 2.4 (6) 20.3 + 1.6 (4) 19.0 + 5 (12) 1.1 29 13.5 A n t e r i o r P e r f o r a t i n g Substance - - 28.6 11 ( 4) - 20 Amygdala 4.2 + 0.5 (3) - 4.2 + 1 ( I D 3.1 - 3.0 Hypothalamus \ 4- 3.7 + 1.0 (5) 3.9 + 0.7(11) \ 5.3 2.8 Thalamus > 5.0 i 1.6 (-6) 0.6 + 0.1 (5) 2.7 + 0.2( 9) > 3.7 0.9 1.9 Midbr a i n 3.6 + 1.0 (6) 2.2 + 0.5 (5) 3.1 + 0.2( 5) J %J 3.1 2.2 Pons-Medulla 2.9 + 0.3 (6) 1.7 + 0.2 (5) 1.8 + 0.5(13) 2.2 2.4 1.3 Cortex-Whole 3.1 + 0.8 (6) 2.2 + 1.0 (5) 2.5 + 0.6( 9) 2.3 3.1 1.8 - V i s u a l - - 2.5 + 1.5( 9) - - 1.8 -Auditory - - 2.1 + 1.8( 9) - - 1.5 - A s s o c i a t i o n - - 2.1 + 1.6( 9) - - 1.5 -Cingulate Gyrus - - 2.2 + 1.9( 9) - - 1.6 Hippocampus 1.8 + 0.4 (6) 2.1 + 0.6 (5) 1.6 + 0.4(10) 1.3 3.0 1.1 S p i n a l Cord 1.2 + 0.7 (5) 2.2 + 0.9 (4) - 1.0 3.1 - u Cerebellum 1.3 + 0.4 (6) 0.7 + 0.2 (4) 1.4 + 0.2( 4) 1.0 1.0 1.0 * No. of animals i n parenthesis •f In mumoles DOPA/hr/gm of t i s s u e TABLE 10 TISSUE WEIGHTS OF DIFFERENT BRAIN AREAS Area Rat Rabbit Cat Caudate 33 + 6 77 + 7 416 + 58 Septum 48 + 23 70 + 7 124 + 26 Pons-Medulla 214 + 30 :801 + 118 1408 + 163 Midbr a i n 118 + 15 536 + 98 1074 + 154 Hypothalamus I 115 + 21 110 + 30 Thalamus > 224 67 400 + 23 1278 + 246 * Hippocampus 96 + 26 507 + 74 582 + 110 Amygdala 76 + 14 - 400 + 62 Cerebellum 256 + 9 1144 + 128 3438 + 201 A n t e r i o r P e r f . _1_ Substance 35: A T 9 i n mg. 47:. Z o < I-Z ui O z o o o BRAIN I I I T T Legend: C o n t r o l Cold I range of values f o r 2 or 3 animals h a l f l i f e \ = 0.8 T 2 "I 6 8~ T I M E ( H O U R S ) SPLEEN ADRENAL F i g . l 6 & " . D e p l e t i o n of NA w i t h (A -methy 1-m-tyrosine organs of normal r a t s and r a t s exposed to co 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 various 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 Table 11. There were s l i g h t d i f f e r e n c e s between areas, but i n general the values ranged from 15 - 30 V/gm of wet t i s s u e . The t y r o s i n e hydroxylase a c t i v i t i e s of the various s u b d i v i s i o n s of the s e p t a l area f o r cat are presented i n Table 12, along w i t h the average weights and t y r o s i n e values. 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 area are r e l a t i v e l y i n a c t i v e . Most of the t y r o s i n e hydroxylase a c t i v i t y of the s e p t a l area, as we defined i t , i s contained 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 the r e g i o n of the nucleus . accombens. This r e g i o n may be part of the tuberculum olfactorum or nucleus of the diagonal band of Broca. The r e g i o n of the nucleus accombens has about h a l f the a c t i v i t y of the caudate and i s therefore the second most a c t i v e area 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 of Lesions on the Tyrosine Hydroxylase Levels i n Various Regions of Cat B r a i n S i x main types of bra i n s l e s i o n s were done: 1. f l o o r of mid diencephalon, 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. s u b s t a n t i a n i g r a , 5. m i d l i n e midbrain and 6$ raphe The l o c a t i o n s of these l e s i o n s are shown i n F i g . 17. a) Lesions of diencephalon f l o o r Nine animals were le s i o n e d at t h i s l e v e l of the b r a i n , 3 animals w i t h medial l e s i o n s and 6 w i t h l a t e r a l ones. The r e s u l t s are presented i n Table 13. The medial l e s i o n s had l i t t l e e f f e c t on t y r o s i n e hydroxylase a c t i v i t y i n the caudate; only one of the 3 cats showed a drop i n a c t i v i t y to 63% of 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 hydroxylase a c t i v i t y decreased TABLE 11 TYROSINE CONCENTRATIONS IN VARIOUS AREAS OF RAT, RABBIT AND CAT BRAIN* Area Rat Rabbit Cat Pons-Medulla 17.3 + 3.2 24.1 + 10.1 13.4 + 2.8 Caudate 28.8 + 10.2 27.8 + 5.2 24.6 + 6.5 Septum 16.5 + 6.3 24.9 + 3.5 17.6 + 6.0 Hippocampus 22.7 + 7.1 28.3 + 4.0 13.2 + 3.2 Thalamus 1 JL. 25.3 + 6.2 14.5 + 5.7 Hypo thalamus > 20.8 T 5.4 23.5 + 5.1 19.8 + 5.5 Amygdala 23.9 + 5.1 - 16.1 + 3.5 Cortex 15.7 + 4.5 31.2 + 4.8 14.8 + 5.1 Mid b r a i n 15.6 + 4.2 25.8 + 4.2 14.3 + 7.0 Cerebellum 17.3 + 5.5 22.9 + 8.5 18.9 + 4.6 S p i n a l Cord 22.7 3.5 22.7 + 3.7 -A n t e r i o r P e r f o r a t i n g Substance - - 23.3 + 3.1 * ugm/gm - standard d e v i a t i o n 51. TABLE 12 TYROSINE HYDROXYLASE ACTIVITY IN SUBDIVISIONS OF SEPTAL AREA OF CAT BRAIN Region V Tyrosine Tissue Wt. max J mumole/DOPA/ I % mg g/hr A n t e r i o r Hypo-thalamus 5.9 + 5.2 26.5 + 5.1 25 + 6 P r e o p t i c 8.0 + 3.8 21.2 + 5.7 18 + 5 A n t e r i o r Septum 32.6 + 13.9 17.4 + 3.5 37 + 10 Region - Nucleus Accombens 61.0 + 18.5 25.5 + 7.5 28 + 2 C l a s s i c a l S e p t a l N u c l e i 3.8 + 0.9 17.1 + 1.7 39 + 18 TABLE 13 EFFECTS OF LESIONS IN FLOOR OF DIENCEPHALON ON TYROSINE HYDROXYLASE AND CATECHOLAMINES IN ROSTRAL AREAS'' Area M e d i a l + (3) L a t e r a l (6) Tyrosine DA NA Tyrosine DA NA Hydroxy- Hydroxy-l a s e l a s e Caudate 110 — - 63, 34, 118, 41, 87, 105, 63 74 89 63, 59 -, -, 104 105 77 15, 75 65, 41 110, 114 Septum 64 60, 65, -14.5 - - 125, 33, 56.5 - 21, 103 * a c t i v i t y expressed as % of non-lesioned + no. of animals i n brackets ' medial caudate F i g . 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 of the l e s i o n s of the diencephalon and midbrain. Landmarks - A - corpus callosum, B - f o r n i x , C - o p t i c chiasm D - i n t e r p e d u n c u l a r nucleus, E - s u p e r i o r c o l l i c u l i E - i n f e r i o r c o l l i c u l i <2 Lesioned areas - 1. f l o o r of mid-diencephalon, 2. p o s t e r i o r diencephalon 4- habenula 5. m i d l i n e midbrain and raphe 6. cerveau i s o l e 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. L a t e r a l l y placed 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 both caudate (15 - 65% of normal) and s e p t a l area (21 - 65%,) i n 4 of the animals. The other two l a t e r a l l e s i o n s showed decreases i n caudate but none i n the s e p t a l area. 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 other areas of b r a i n , measured: i . e . amygdala, hippo-campus, thalamus, hypothalamus, cortex and pons and medulla oblongata. Caudate catecholamine l e v e l s were determined i n 6 of these animals; s e p t a l area l e v e l s could not be measured because of i n s u f f i c i e n t t i s s u e . There were no s i g n i f i c a n t changes i n caudate noradrenaline concentrations. In one animal where there was no s i g n i f i c a n t change i n caudate t y r o s i n e hydroxylase a c t i v i t y , there was l i k e w i s e no change i n dopamine l e v e l . Four of the 5 cats which showed decreases i n enzyme a c t i v i t y i n the caudate also showed decreases i n dopamine content, but these decreases were not as large as those of the enzyme (15% as compared to 45%,). No h i s t o l o g i c a l s tudies were done on the l a t e r a l l y placed l e s i o n s . One medial l e s i o n , i n which the s e p t a l t y r o s i n e hydroxylase a c t i v i t y was 56.5%, of normal, was 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 diencephalon that d i d not extend f a r i n t o the diencephalon. This l e s i o n i s shown i n F i g . 18. b) P o s t e r i o r diencephalon - 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 ge c e n t r a l l y placed one (4 cats) and i i ) small m e d i a l l y placed ones (7 c a t s ) . With the l a r g e r l e s i o n s there were s u b s t a n t i a l decreases i n t y r o s i n e hydroxylase a c t i v i t y , to as low as 11% of normal, i n both caudate and s e p t a l areas; the smaller l e s i o n s r e s u l t e d i n lower drops, to about 50% of normal. With both types of l e s i o n the caudate and s e p t a l area decreased by approximately the same amount. F i g . 18: Lesion i n the f l o o r of the mid-diencephalon 56. These r e s u l t s are presented ihaTable 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 (to approximately 30% of normal) w i t h the caudate of both of the cats i n the large l e s i o n s on which amine analyses were done. The only other areas of b r a i n i n which there were changes i n t y r o s i n e hydroxylase a c t i v i t y were the amygdala and hippocampus. Of the 11 cats 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. The r e s u l t s f o r the hippocampus were however, very i n c o n s t a n t . Although 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 seventh. And i n 2 other cases the hippocampus on the non-lesioned side showed no a c t i v i t y at 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 large l e s i o n s . Three of the small medial l e s i o n s , which had caused decreases i n t y r o s i n e hydroxylase a c t i v i t y i n both caudate and s e p t a l area, were confirmed h i s t o l o g i c a l l y . Examples of 2 of these are shown i n F i g . 19 and 20. 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 diencephalon at the l e v e l of the mammillary bodies. F i g . 20 shows a very d i s c r e t e l e s i o n at 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 placed. The entrance of the e l e c t r o d e s can a l s o be seen i n t h i s p i c t u r e , c) Habenular. Eleven animals were l e s i o n e d i n an attempt to destroy the habenulafregion, which would be at 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 . Four of these habenular 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 examination ( h i s t o l o g i c a l ) i t was found that not a l l the l e s i o n s were i n the habenular area. The biochemical r e s u l t s are presented i n Table 15 w i t h values f o r r i g h t and l e f t sides of the b r a i n w i t h some b i l a t e r a l l e s i o n s . i ) Three of the 4 cats w i t h u n i l a t e r a l l e s i o n s had approximately 50%, decreases i n t y r o s i n e hydroxylase i n the s e p t a l F i g . 19: M e d i a l l y placed l e s i o n i n p o s t e r i o r diencephalon at the l e v e l of the mammillary bodies F i g . 2 0 : Lesion i n the p o s t e r i o r diencephalon (only h a l f of 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 animals showing changes presented) Area Large (4) Small (7) (Cen t r a l ) (Medial) Tyrosine DA NA Tyrosine DA NA Hydroxy- Hydroxy-l a s e l a s e Caudate \23 31 28 34, 36, 45 27 33 59, 56, 49 62 -Septum 11 - - 41, 57, - -17 - - 43, 55, 75 - - 44, 65 Hippocampus B l (NL) - - 220, 67 B l (NL) - - 13, 58 B l - - B l , 50 Amygdala 49 - - 60, 58, 54 — — 56 ~ — * A c t i v i t y expressed as ' 'A of c o n t r o l + no. of animal i n brackets B l No a c t i v i t y ( i e . equal to blank) NL non-lesioned s i d e 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 Septum Hippocampus Amygdala 109,108,114,85 46,85,49,49 64,75,100,39 42,115,132,47 11°, 36,66,70 20°,27,71,60°,61 17°,48,34,B1°,35, 210,B1,B1°,9°,24 19,218°,150,185,236, 141 * expressed as % of c o n t r o l + no. of animals i n brackets B l no a c t i v i t y ( i e . equal to blank) 0 designates from the same animal r i g h t and l e f t s ide r e s p e c t i v e l y 60. area, and amygdala but no changes i n other areas. I t was found however, that i n a l l of these cats 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 to i t . In 2 of the animals that had changes i n a c t i v i t y i n the s e p t a l area there were a l s o evidences of damage on the m i d l i n e of the diencephalon below the p o s t e r i o r commissure. An example of t h i s type of l e s i o n i s seen i n F i g . 21. i i ) Only 3 of the 7 b i l a t e r a l habenular l e s i o n s resulted', i n decreases i n t y r o s i n e hydroxylase a c t i v i t y i n the caud-ate and s e p t a l area. In one animal, showing a very large drop i n a c t i v i t y to 10 - 307„ of normal, the l e s i o n was found on h i s t o l o g i c a l examination.jto i n v o l v e not only the habenular r e g i o n but to 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 almost to the f l o o r of the diencephalon (see F i g . 22). Three of the other b i l a t e r a l l e s i o n s that 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 to be i n the habenula area, only 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 hydroxylase 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 habenula l e s i o n s . In the same animal that had had la r g e decreases i n caudate and s e p t a l area the a c t i v i t y i n the amygdala was 197o of normal; i n 4 of the animals there was increased amygdala a c t i v i t y (141 - 2367, of normal). The hippocampus a c t i v i t y was l e s s than normal i n 6 out of 7 animals, d) Substantia n i g r a Only 2 animals were le s i o n e d i n the s u b s t a n t i a n i g r a since so much had p r e v i o u s l y been done i n t h i s area by other workers (51 - 56). As i n d i c a t e d i n Table 16, the caudate i n both cases showed the expected l a r g e drops, to as l i t t l e as 2% of normal, i n t y r o s i n e hydroxylase a c t i v i t y i n the caudate, as w e l l as smaller decreases i n DA.(.2% c f i : 3 K ) . The NA l e v e l was a l s o below normal i i n one of the c a t s . 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 hydroxylase. H i s t o l o g i c a l l y , the l e s i o n s were F i g . 21: Lesion made i n an attempt to destroy 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 DA NA Hydroxylase.'. Caudate 2, 5 31, 39 51, 50 Septum 20, 9.5 25, - 60,102 expressed as % of c o n t r o l F i g . 23 : Lesion of the habenular only 64. confirmed to be i n the s u b s t a n t i a n i g r a , extending medial from i t to the m i d l i n e . e) M i d l i n e midbrain The 10 m i d l i n e midbrain l e s i o n s were b i l a t e r a l . There were two types of l e s i o n s : i ) c e r v e a u - i s o l e - l e s i o n s placed at 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 formation on both sides of the m i d l i n e (5 animals), i i ) l e s i o n s extending r o s t r a l to t h i s cerveau i s o l e under the superior c o l l i c u l i and confined 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 formation (5 animals). (a) The c e r v e a u - i s o l e l e s i o n s had l i t t l e e f f e c t on t y r o s i n e hydroxylase a c t i v i t y i n e i t h e r caudate or s e p t a l area, as shown i n Table 17. Four of these l e s i o n s were confirmed h i s t o l o g i c a l l y . A d o r s a l l y l o c a t e d l e s i o n was described as d e s t r u c t i o n of the m i d l i n e teg-mentum of the midbrain, extending 2 mm on e i t h e r side at the l e v e l of the caudal 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 medial encroachment on the s u b s t a n t i a n i g r a , but sparing the interpeduncular nucleus ( 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 destroyed the interpeduncular nucleus ( F i g . 25). (b) The l e s i o n s j u s t r o s t r a l to t h i s c e r v e a u - i s o l e produced very i n t e r e s t i n g changes i n tyrosine hydroxylase a c t i v i t y i n caudate and s e p t a l area. A l e s i o n on the m i d l i n e at the s u p e r i o r c o l l i c u l i l e v e l , w i t h l i t t l e • 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 animal, a t the same l e v e l , but extending more l a t e r a l l y (see F i g . 27) produced drops i n both caudate and s e p t a l area to about 50% of normal. The importance of l a t e r a l extent of the l e s i o n was evident i n one animal where h i s t o l o g i c a l examination showed that TABLE 17 EFFECT OF BILATERAL MIDBRAIN LESIONS ON TYROSINE HYDROXYLASE ACTIVITY IN CAUDATE AND SEPTUM* Area Cerveau M i d l i n e (5) Raphe (4) I s o l e (5) Caudate 60 71 95 56 70 Septum 108 103 97 70 75 * expressed as % of c o n t r o l + no. of animals i n brackets B l a c t i v i t y equal to blank 0 designates from same animal - r i g h t and l e f t s i d e r e s p e c t i v e l y F i g . 27: Lesion of the m i d l i n e midbrain w i t h a large l a t e r a l extension 68. the m i d l i n e midbrain tegmentum was destroyed more l a t e r a l l y i n the r i g h t side than on the l e f t ( F i g . 28). In t h i s animal 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 sides being 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 other two animals of t h i s group had s i m i l a r . r e s u l t s , i n that 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 hydroxylase a c t i v i t y on the r i g h t and l e f t could be c o r r e l a t e d w i t h the l a t e r a l extent of the l e s i o n as determined h i s t o l o g i c a l l y . The caudate and s e p t a l area were not always comparable, i n one animal the caudate t y r o s i n e hydroxylase a c t i v i t y was normal on the r i g h t side w h i l e the s e p t a l a c t i v i t y was 20%, of normal. In a l l these m i d l i n e midbrain 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 midbrain i n c l u d i n g the interpeduncular nucleus, the s u b s t a n t i a n i g r a and other p e r i p h e r a l n u c l e i were l a r g e l y spared, f ) Raphe The 4 raphe l e s i o n s were a l l confirmed 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 not extend as v e n t r a l l y as those 5b but other-wise 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 midbrain, such as the d o r s a l raphe, medial 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 caudal i n f e r i o r c o l l i c u l i and caudal superior c o l l i c u l i (see F i g . 29). As shown i n Table 17 t y r o s i n e hydroxylase a c t i v i t y i n the caudate and s e p t a l area could 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 on the l a t e r a l extent of thfe l e s i o n . 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 could be a f f e c t e d or j u s t s e p t a l area but never j u s t caudate a c t i v i t y . 69. F i g . 28: Lesion of the m i d l i n e midbrain w i t h l a t e r a l extension of the l e s i o n more on the r i g h t F i g . 29: L e s i o n of the m i d l i n e midbrain i n the d o r s a l raphe DISCUSSION 1. Tyrosine Hydroxylase A c t i v i t y In V i t r o The method, described 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 to be a r a p i d and p r e c i s e one; r a p i d i n that up to 80 incubations could be done i n 2 days and p r e c i s e i n that d u p l i c a t e incubations were w i t h i n s e v e r a l per cent of each other. Accuracy a l s o , i s guggested by the f a c t that c o n s i s t e n t r e s u l t s are obtained f o r each area of the b r a i n (Table 9 ). Another method f o r measurement has been described i n the l i t e r a t u r e 3 (108,110) using 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 of the aromatic r i n g i s r e l e a s e d i n t o the water during the r e a c t i o n and can be r e a d i l y measured. This method has been 14 reported to give s i m i l a r r e s u l t s to the C-tyrosine method. The values f o r t y r o s i n e hydroxylase a c t i v i t y were i n the range of 1 - 100 njumoles of Dopa formed/gm/hr f o r b r a i n . This i s of the same order of magnitude as reported by others (27,28) who obtained values i n the range of 4 - 100 mjimoles/gm/hr. The a c t i v i t y i n adrenal i s reported (28) to be 15 times that of b r a i n ; but i n other p e r i p h e r a l t i s s u e such as heart and spleen i t i s d i f f i c u l t to detect any a c t i v i t y i n v i t r o . Sympathetic endings make up only a small p r o p o r t i o n of such t i s s u e which may account f o r the r e l a t i v e i n a c t i v i t y . In v i t r o s tudies show d i f f e r e n c e s i n t y r o s i n e hydroxylase i n b r a i n andadrenal. The c o n d i t i o n s f o r maximum a c t i v i t y of crude adrenal homogenate (28) or of p a r t i a l l y p u r i f i e d adrenal enzyme (23) are acetate b u f f e r pH 6.0 i n the presence of DMPH^ . The a c t i v i t y drops o f f a f t e r 20 minutes. We have found that b r a i n t y r o s i n e hydroxylase i s most a c t i v e i n phosphate b u f f e r pH 6.2, without 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 at l e a s t 30 - 45 minutes. The enzymes may be d i f f e r e n t i n b r a i n and adrenal but 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 could a l s o be explained by d i f f e r e n c e s i n the i o n i c environment of the t i s s u e and the f a c t that there may be s u f f i c i e n t DMPH^ i n crude b r a i n homogenate already. In support of t h i s suggestion, Ikeda and L e v i t t (111) has reported that 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 hydroxylase. A l s o i t was noted during these i n v e s t i g a t i o n s of K values that at very high concentrations of t y r o s i n e the curve 1/v vs 1/s was no longer l i n e a r without DMPH, but remained l i n e a r w i t h c o f a c t o r . This 4 may i n d i c a t e DMPH^ was becoming l i m i t i n g at high t y r o s i n e c o n c e n t r a t i o n s . The K values obtained f o r t y r o s i n e hydroxylase appear to be species and organ dependant. In b r a i n the values as reported i n t h i s work ranged from 0.5 x 10 to 1 x 10 ^  f o r the various species ( c a t , r a b b i t and r a t ) and ^ i t has been reported that f o r crude beef adrenal the K i s i n -5 m the order of 2 x 10 (28). Udenfriend has reported the K ^ -5 -5 m may range from 2 x 10 to 0.1 x 10 (23). 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 species and organs. Tyrosine hydroxylase has a l s o been reported to be capable of converting 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 adrenal t i s s u e (112). 2. Separation of NA, DA and Some of Their M e t a b o l i t e s a) Ion 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 separation of NA, DA and MT; NA, NM and MT, w i t h incomplete separation of NM and DA. I t i s a l s o a u s e f u l technique f o r the removal of the precursors of CA s y n t h e s i s , t y r o s i n e and Dopa, and of t h e i r a c i d metabolites from s o l u t i o n s o f DA and NA. Although d i f f e r e n t s i z e s of columns of Dowex and d i f f e r e n t e l u t i o n schedules have been used by other workers (102-104) the types of separation achieved are s i m i l a r . This chromatographic technique has been used to separate the precursor DA from the product NA i n the a n a l y s i s of dopamine p-oxidase (113,114), and f o r the study of i n v i v o metabolism of CA (30,31). Since t y r o s i n e hydroxylase may not be the only c e n t r a l l i n g f a c t o r i n CA synthesis i t i s u s e f u l to study other enzymes i n v o l v e d i n CA synthesis 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 product 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) Thin layer chromatography In the o r i g i n a l reference (105) t h i s method of separation of CAs and t h e i r metabolites was claimed to be 100 times more s e n s i t i v e than paper chromatography. With use of the cfetector ethylene diamine they were able to detect4. 0.05 Tf^of CA. In our hands the method was a good means of separating DA, NA, MT and NM f o r as i n d i c a t e d i n Table 4 there was at l e a s t 0.1 u n i t between each amine. The precursors t y r o s i n e and Dopa and the a c i d metabolites of DA and NA had much lower R^ values and could be separated out from the amines that ran ahead of them. The R^ values obtained 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 reported (Table 4 ) . The s e p a r a t i o n of the amines obtained using t h i n l a y e r chromatography i s comparable w i t h that of paper chromatography (115), and has the advantage of being more r a p i d , r e q u i r e s smaller 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 . I t could be a p p l i e d to studies of i n v i v o CA metabolism i n place of the paper chromatographic techniques o f t e n used. Another t h i n l a y e r chromatographic method using c e l l u l o s e i n s t e a d of poly-amide has a l s o given s i m i l a r r e s u l t s (116). 3. Catecholamine A c t i v i t y i n Rats Exposed to Cold The large increase 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 other r e p o r t s (89,117). Leduc (89) claims a 5 f o l d increase i n NA and a 40% increase i n DA e x c r e t i o n i n c o l d a c c l i m a t i z e d animals. However there 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'. Leduc (21) has reported no change i n NA of adrenals and a decrease i n c o n c e n t r a t i o n i n heart and spleen; others have shown (117) a 40 - 507, increase i n NA i n adrenals and no change i n b r a i n NA content. The r e s u l t s presented i n Table 6 (407, increase i n NA i n adrenals 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 extensive work w i t h c o l d a c c l i m a t i z e d r a t s . From h i s studies 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 agents, he concludes that NA r e l e a s e d from sympathetic endings i s the f i r s t , and adrenaline r e l e a s e d from the adrenals i s the second meansy; of p r o v i d i n g non-.shivering.r thermogenesis i n animals placed i n the c o l d . Both , NA and adrenaline have been shown to increase glycogen breakdown 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 increased metabolism could perhaps account f o r the r e q u i r e d increase i n heat production i n c o l d a c c l i m a t i z e d animals (118). Muscle and brown f a t , which i s r i c h l y innervated w i t h sympathetic endings, have been proposed as s i t e s of t h i s increased heat formation (119). From the r e s u l t s obtained 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 could be drawn concerning t y r o s i n e hydroxylase a c t i v i t y i n r a t s exposed to c o l d . The increase i n t y r o s i n e hydroxylase a c t i v i t y per adrenal would i n d i c a t e an increase i n NA and adrenaline synthesis i n that gland. The turnover s t u d i e s w i t h NA, however, could 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 values f o r NA turnover were not obtained. There are s e v e r a l p o s s i b l e reasons f o r the d i s c r e p e n c i e s between the values obtained 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*- -methyl-p-tyrosine used was i n s u f f i c i e n t to lower NA l e v e l s i n h e a r t ) . -Methyl-m-tyrosine i s a l s o a Dopa decarboxy-l a s e i n h i b i t o r (120) w h i l e C(-methy-p-tyrosine i s not and t h i s d i f f e r e n c e i n a c t i o n may have been a f a c t o r . The 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 metabolite could i n t e r f e r e w i t h storage 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 . In a d r e n a l , heart and spleen there were a l s o i n d i c a t i o n s that the t i s s u e was r e c o v e r i n g from the i n h i b i t o r s a f t e r 2 - 4 hours. Other i n v e s t i g a t o r s (122, 123) have reported much longer h a l f - l i v e s , i'in the order of 300 hours, f o r adrenals our work i s not comparable to t h e i r s . Glowinski (124) has shown that two t h i r d s of the CAs i n u r i n e are from the periphery so i t i s probable that a large part 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 that some may come from the adrenal. I t does not appear to come from heart or spleen, but^rown f a t and other s y m p a t h e t i c a l l y innervated t i s s u e s , not considered i n t h i s i n v e s t i g a t i o n , may be an important source. Even though no changes i n t y r o s i n e hydroxylase a c t i v i t y (Table 7) or i n turnover r a t e of NA ( F i g . 16 and 17) were obtained 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 excluded as important i n c o l d adaption, probably i n a r e g u l a t o r y c a p a c i t y . I t has been suggested (87) that NA i n the hypothalamus and lower b r a i n stem i s important i n c o n t r o l of body temperature. Since i t i s thought to have a hypothermic e f f e c t i t might be expected that the hypothalamic NA might be decreased or i t s a c t i o n be i n h i b i t e d during c o l d adaption. The hypothermic a c t i o n of NA Is supposedly confined to very l i m i t e d b r a i n areas and measurements of CA c o n c e n t r a t i o n and t y r o s i n e hydroxylase a c t i v i t y i n whole b r a i n may not r e f l e c t changes i n these s p e c i f i c areas. From the d i s t r i b u t i o n s tudies /; A (Table 9) i t can be seen that the hypothalamus and pons-medulla are only l / 2 0 t h and l/100th as a c t i v e as the caudate, and changes 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 a c t i v i t y of t y r o s i n e hydroxylase i n whole b r a i n . A l s o i n v i t r o assays of t y r o s i n e hydroxylase may only measure maximum ca p a c i t y of the t i s s u e to produce CA, since a l l the f a c t o r s a f f e c t i n g CA synthesis are not known (125). An a l t e r e d i n v i v o a c t i v i t y of enzyme might not be detected by i n v i t r o measurement. G o l d s t e i n (126) has reported a s l i g h t increase i n turnover 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 aspect of the turnover s t u d i e s of NA i n b r a i n i s the p o s s i b i l i t y that there are 2 turnover r a t e s f o r t h i s CA. The break i n the curves ( F i g . 5) may represent recovery from the i n h i b i t o r , but could a l s o be a t t r i b u t e d to a second turnover r a t e s ; Glowinski (22) has reported two turnover r a t e s f o r NA of r a t b r a i n ( t x = 3 hrs and 17 h r s ) . Since the t j 's obtained are of the same order of magnitude (eg. i n one case 3.8 and 18 hrs) i t i s p o s s i b l e that t h i s phenomena i s being observed. The two turnover r a t e s would i n d i c a t e two stores of NA; one store being l a b i l e ( tx = 3 hrs) and probably i n the cytoplasm as unbound NA; and the second store 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 noadrenergic nerve endings, and r e l e a s e d more g r a d u a l l y during neuronal a c t i v i t y . 4. Tyrosine Hydroxylase A c t i v i t y i n B r a i n a) D i s t r i b u t i o n L i k e the CA t y r o s i n e hydroxylase has a very 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 high c o n c e n t r a t i o n i n the caudate s e p t a l area and nucleus accombens; i n moderate con c e n t r a t i o n i n the hypothalamus and midbrain and i n low co n c e n t r a t i o n i n the pons, medulla oblongata, thalamus, hippocampus, cerebellum and cortex. NA i s high i n conce n t r a t i o n i n the hypothalamus, moderate i n co n c e n t r a t i o n i n the s t r i a t u m and medulla oblongata and low i n conc e n t r a t i o n i n the cortex and cerebellum. Tyrosine hydroxylase has a high a c t i v i t y t h e r e f o r e and appears to be more c l o s e l y r e l a t e d to 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 of the s e p t a l area ( a n t e r i o r s e p t a l area) may correspond t o parts of the tuberculum olfactorum which i s high i n DA and/or the nucleus of the diagonal band of Broca. The s e p t a l area, other than the c l a s s i c a l s e p t a l nuclei, which has l i t t l e t y r o s i n e hydroxylase a c t i v i t y , i s a poorly defined r e g i o n . Therefore 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 hydroxylase a c t i v i t y there to a s p e c i f i c s t r u c t u r e . The t y r o s i n e hydroxylase d i s t r i b u t i o n i s i n agreement w i t h other i n v e s t i g a t i o n of CA synthesis i n various regions of b r a i n . The i n v i v o synthesis 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 synthesis i n caudate from p r e l i m i n a r y r e p o r t s (31) appears to be much higher. I n j e c t i o n of the r a d i o a c t i v e precursor 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 being more a c t i v e w i t h DA as the major product (10). R a d i o a c t i v e DA i s converted to NA i n the amygdala and hypothalamus but not i n the s t r i a t u m , i n d i c a t i n g a d i f f e r e n t type of CA synthesis i n various regions of b r a i n as w e l l as d i f f e r e n t degree (31). 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 synthesis (127). I t had p r e v i o u s l y been demonstrated that t y r o s i n e hydroxylase a c t i v i t y i s contained i n nerve endings (37). To s u b s t a n t i a t e t h i s , i t has now been shown that t y r o s i n e hydroxylase i s most a c t i v e i n areas c o n t a i n i n g a large number of nerve endings (eg. caudate and septum) as compared to the area c o n t a i n i n g c e l l bodies (eg. pons, medulla and midbrain). A l s o there i s a l o s s of t y r o s i n e hydroxylase a c t i v i t y when axons l e a d i n g to these areas are cut (see l e s i o n s t u d i e s ) . I t has been suggested (127) that t y r o s i n e hydroxylase or a precursor i s transported from the c e l l bodies to the nerve endings by axon flow. The 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 the increases found i n t y r o s i n e hydroxylase a c t i v i t y i n the areas of nerve endings ( i . e . caudate,' s e p t a l area, hippocampus and amygdala), during development of the b r a i n (128). The enzyme a c t i v i t y may be an i n d i c a t i o n of the formation of nerve endings or of u s e f u l dopaminergic and noradrenergic synapses. I t i s perhaps of i n t e r e s t that mentally retarded i n d i v i d u a l s do not respond to amphetamine, a drug that i s thought to a f f e c t the CNS by i t s a c t i o n on CA c o n t a i n i n g synapses, b) As a c o n t r o l of CA synthesis 78. Although Udenfriend (28) has proposed that t y r o s i n e hydroxylase i n the r a t e l i m i t i n g step i n the synthesis of CA, i t can not be assumed that i t i s the only c o n t r o l l i n g f a c t o r i n t h e i r formation. Some potent 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 hydroxylase 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 reported (129,130) to increase 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 t y r o s i n e hydroxylase a c t i v i t y i n v i t r o (42). DA neurons recover more r a p i d l y from ff^ methyl-p-tyrosine i n h i b i t i o n than do NA terminals ( 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 reducing NA l e v e l s than <\-methyl-p-tyrosine (131). Therefore there may be another c o n t r o l of NA synthesis at the dopa-mine B-oxidase stage. In assessing the importance of t y r o s i n e hydroxylase 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 values f o r the i n v i t r o enzyme a c t i v i t y of various areas of b r a i n s w i t h the i n v i v o synthesis as obtained by Glowinski (9,30,31,32). As shown i n Table 18, there i s good agreement between the r e l a t i v e i n v i v o synthesis r a t e s , and the i n v i t r o t y r o s i n e hydroxylase a c t i v -i t i e s v Tyrosine hydroxylase i s probably 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 rious regions of b r a i n than CA concentrations which do not n e c e s s a r i l y represent turnover, the cerebellum, f o r example, has a very low l e v e l of CA but has a f a i r l y r a p i d turnover. However, when absolute values of CA i n v i v o synthesis and t y r o s i n e hydroxylase a c t i v i t y are considered the comparison i s not so s t r a i g h t forward. Using G l o w i n s k i 1 s f i g u r e s that DA concentration 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 hours, the i n v i v o synthesis;:would be 937 - 3750 mpg/gm/hr; f o r hypothalamus the formation of NA i s 234 mug/gm/hr. According to t y r o s i n e hydroxylase a c t i v i t y f i g u r e s about 15,000 miag/gm/hr of DA could 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. Therefore 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 hydroxylase i s 3 to 16 times greater than that r e q u i r e d f o r i n v i v o s y n t h e s i s . Udenfriend's (27) suggestion that 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 R e l a t i v e Tyrosine Hydroxylase 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 R e l a t i v e NA Turnover based on reference 31 80. i s the r a t e l i m i t i n g step 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 decarboxylase or w i t h dopamine B-oxidase. But i f there i s an excessoof enzyme over that r e q u i r e d there must be some other f a c t o r s or f a c t o r c o n t r o l l i n g i t s a c t i v i t y . One e x c e l l e n t p o s s i b i l i t y i s feedback i n h i b i t i o n by the CAs. From work done i n the periphery S t j a r n e (132,133) proposed that the NA content of sympathetic nerve stays r e l a t i v e l y constant through v a r y i n g degrees of a c t i v i t y because of the increased synthesis r e s u l t i n g from the removal of the i n h i b i t o r NA. This i n h i b i t i o n may be at the t y r o s i n e hydroxylase or dopamine B-oxidase l e v e l . NA and DA i n h i b i t b r a i n t y r o s i n e hydroxylase by about 40% and 70% -4 r e s p e c t i v e l y at 10 M (42). Normal CA concentrations i n nerve endings have been estimated to be i n the order of 8000 ug/gm -2 (134) i . e . about 10 M but they are d i l u t e d out during the homogenizing process. 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 a c t i v i t y a r e : ^ 1) a r e l a t i v e l a c k of c o f a c t o r or precursor that may 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) non-ideal 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 other than the CA. I t has been reported that s l i c e s of s t r i a t u m show lower a c t i v i t y than homogenates; whereas some other areas had greater a c t i v i t y i n s l i c e s (127). This may i n d i c a t e that 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 a c t i v i t y . 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 drugs, changed t y r o s i n e hydroxylase a c t i v i t y as measured i n v i t r o . The r e s u l t s were negative but t h i s may be due to the d i l u t i o n f a c t o r mentioned above. The f a c t that the hypothalamus i s high i n NA content but _ l a t i v e l y low i n t y r o s i n e hydroxylase suggests that the enzyme i n the hypothalamus i s working c l o s e r to i t s maximum c a p a c i t y than i n the caudate. This may be r e l a t e d to feedback i n h i b i t i o n since 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 there i s a 81. higher c o n c e n t r a t i o n of 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 but f o r a true i n d i c a t i o n of CA s y n t h e s i s , other p o s s i b l e f a c t o r s must a l s o be considered. Since the CA and t y r o s i n e hydroxylase 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 that they have a s p e c i f i c f u n c t i o n . The CAs have been connected w i t h emotion, behavior and l e a r n i n g but i t f i s d i f f i c u l t to lo c a t e these anatomically. Perhaps however, there i s some r e l a t i o n s h i p between the high c o n c e n t r a t i o n of DA, large c a p a c i t y to synthesis CA and high turnover 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 other hand, the hypothalamus w i t h i t s high NA conc e n t r a t i o n but lower c a p a c i t y to form CA and smaller turnover r a t e s , appears to bed p r i m a r i l y concerned w i t h autonomic f u n c t i o n . 5. Tyrosine Hydroxylase Containing F i b e r s i n Cat B r a i n a) 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 the r e s u l t s (Table 13 - 17) the caudate and s e p t a l area were the primary regions a f f e c t e d by the l e s i o n s , i n midbrain s t r u c t u r e s of the ca t . Decreases i n a c t i v i t y i n these areas could be determined w i t h confidence since 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 small ( 10 - 20%). Most of the d i s c u s s i o n w i l l be concerned w i t h t r a c t s to these areas In many other areas i t was d i f f i c u l t to determine changes since the counts f o r the c o n t r o l s were very low and there was a large v a r i a t i o n around the nornu . However where there 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 other workers i n t h i s f i e l d leave the animals 2 - 1 0 weeks before s a c r i f i c i n g (49,59). I t was found i n t h i s study that up to 80%, decreases could be obtainedjin t y r o s i n e hydroxylase a c t i v i t y w i t h i n 72 hours of 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 or 3 weeks. So f o r / t h e m a j o r i t y of animals there 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 . The short i n t e r v a l however, made i t d i f f i c u l t to observe chromolysis or extensive retrograde degeneration d i - f f - i c u i t ^ so no r e s u l t s as to p o s s i b l e l o c a t i o n of c e l l bodies could be presented. I n c o n s i s t e n t r e s u l t s obtained w i t h CA measurements may a l s o be accounted f o r the short i n t e r v a l between l e s i o n i n g and s a c r i f i c i n g . Moore and H e l l e r (57) have shown the amines do not begin to 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 that there i s a gradual 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 degeneration i s complete a f t e r three days. The i n d i c a t i o n then i s that the nerve degenerates r a p i d l y and that t y r o s i n e hydroxylase i s l o s t as r a p i d l y as the nerve i s destroyed but t h a t CAs are s t i l l present and undergo a more gradual d e s t r u c t i o n . Moore and H e l l e r argue that the slowness of the CA decrease as compared to v i s i b l e neuronal degeneration i n d i c a t i n g a t r a n s y n a p t i c e f f e c t . The Swedish workers (49) maintain, however, that the f i b e r s are d i r e c t but are so small that the degeneration cannot be observed. The f i n d i n g s i n t h i s study would be i n agreement w i t h the l a t t e r theory, b) F i b e r s i n the diencephalon 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 conclusions can be drawn: 1. CA c o n t a i n i n g f i b e r s going to the caudate and s e p t a l area are present at the l e v e l of the mid t h i r d of the diencephalon. 2. f i b e r s going to the caudate are l a t e r a l to those going to the s e p t a l area. 3. f i b e r s to the caudate may be going p r i m a r i l y to the medial p a r t . 4. at t h i s l e v e l at l e a s t 85% of the f i b e r s to the s e p t a l area can be account f o r . Therefore i n t h i s l a t e r a l hypothalamic r e g i o n of the mid-diencephalon there i s some d i s t i n c t i o n between caudate f i b e r s ( l a t e r a l ) and s e p t a l area (medial), but the separation i s incomplete. Moore and H e l l e r (58) a l s o obtained decreases i n NA and dopa decarboxylase 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 area ( l a t e r a l hypothalamic, complete t r a n s e c t i o n of the MFB), as w e l l as decreases i n the c i n g u l a t e gyrus, hypothalamic 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 hydroxylase were obtained f o r these l a t t e r areas. The r e s u l t s of t h i s i n v e s t i g a t i o n i n the diencephalon 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 (49,50). With l e s i o n s of the MFB at the l e v e l s of the a n t e r i o r commissure, they obtained decreases i n DA i n the tuberculum olfactorum and nucleus accombens, as w e l l as NA changes i n the hypothalamus, p r e o p t i c area, s e p t a l area and c i n g u l a t e gyrus, but the s t r i a t u m showed no change. They consider that the f i b e r s to the caudate leave the MFB i n the p o s t e r i o r diencephalon to enter the i n t e r n a l capsule and terminate p r i m a r i l y i n the l a t e r a l and ventromedial caudate, but they do not account f o r f i b e r s to the dorsomedial caudate. Our r e s u l t s would i n d i c a t e that the f i b e r s to the caudate go to a t l e a s t the mid-diencephalon and that these f i b e r s may be going p r i m a r i l y to the medial caudate. This would be i n agreement w i t h P o i r i e r and Sourkes (53) who describe DA f i b e r s to the s t r i a t u m of cats and monkeys going more r o s t r a l l y to those described 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 hypothalamic r e g i o n have been shown to go to the caudate, diagonal band of Broca, hypothalamus, s e p t a l n u c l e i , hippocampus, amygdala and thalamus (64,65,66). In 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 behavior, i t was found that l e s i o n s of the MFB caused increased esdape l a t e n c y time during l e a r n i n g (135,136). i i ) P o s t e r i o r diencephalon Lesions i n the caudal p o r t i o n of the diencephalon at the beginning of the MFB d i d not separate f i b e r s going to the caudate from those to the s e p t a l area. Even w i t h very 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 placed 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 hydroxylase i n both areas (Table 12). AT t h i s l e v e l i n r a t s the Swedish group (62) were able to determine NA f i b e r s to the diencephalon were most m e d i a l l y l o c a t e d , that DA to nucleus accombens and tuberculum olfactorum were s l i g h t l y 84. l a t e r a l to the NA f i b e r s and that these DA f i b e r s to the caudate were most l a t e r a l l y l o c a t e d . We could not confirm these f i n d i n g s . AT l e a s t 807o of. the f i b e r s to the caudate and s e p t a l area must pass through the F i e l d s of F o r e l r e g i o n ; the m a j o r i t y of caudate f i b e r s presumably have not yet l e f t the MFB to enter the i n t e r n a l capsule as they are thought to do (49). True habenular l e s i o n s appeared to have no e f f e c t on caudate and s e p t a l area t y r o s i n e hydroxylase. C l a s s i c a l neuro-anafomical methods have shown t'some r e c i p t o c a l connections between the habenularand s e p t a l n u c l e i (but these n u c l e i are 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 hydroxylase ) (Table 12). Those l e s i o n s which r e s u l t e d i n a change were not true habenular l e s i o n s but probably extended more v e n t r a l l y i n t o the F i e l d s of F o r e l . I t i s of i n t e r e s t that i n 3 of the 4 cats there were 507, decreases i n s e p t a l area t y r o s i n e hydroxylase 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 described p r e v i o u s l y , so f i b e r s going to the s e p t a l area might be more d o r s a l than those to the caudate. In both these p o s t e r i o r diencephalon l e s i o n s , ( F i e l d s of F o r e l and habenula) there 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 hydroxylase. R e c i p r o c a l connections between these areas and the r e t i c u l a r formation have been described (66,65). Some of these may be crossed, as a p o s s i b l e e x p l a n a t i o n f o r the decreases i n a c t i v i t y obtained on the les i o n e d s i d e . 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 described changes i n CA concentrations i n these areas w i t h MFB l e s i o n s . c) Fiberstinrthe.-midbrain' The midbrain area would appear to be the c r i t i c a l r e g i o n f o r the o r i g i n of 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 that 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 of 80 - 907. of the f i b e r s to the caudate. Lesions caudal to the 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 hydroxylase of the caudate, thus e s t a b l i s h i n g the caudal extent of origin of f i b e r to the nucleus. Some mi d l i n e l e s i o n s of the midbrain that d i d not a f f e c t the s u b s t a n t i a n i g r a could decrease caudate t y r o s i n e hydroxy-l a s e 20 - 30% of normal, the f i b e r s apparently course m e d i a l l y from the 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 Hstochemical 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 the s u b s t a n t i a n i g r a through the crus c e r e b r i i n t o the caudal 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 capsule to the caudate and putamen. Theyfound a d i r e c t r e l a t i o n s h i p between the extent 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 of DA te r m i n a l s . P o i r i e r and Sourkes (51,52) confirmed t h i s , general t r a c t i n the monkeys and c a t s . They give the o r i g i n of the t r a c t as the s u b s t a n t i a n i g r a and parabrachiole pigmentosis and place 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 that described by the Swedes and suggest that 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 of the midbrain and showed decreases i n t y r o s i n e hydroxylase a c t i v i t y , i n v i v o DA synthesis and DA uptake on the i p s i l a t e r a l s i d e . I t i s however, d i f f i c u l t to 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 t r a c t s a n a t o m i c a l l y . Nauta (63) has described connections from the midbrain to caudate and putamen. Other r e p o r t s (60,61) i n d i c a t e that l e s i o n s of the su b s t a n t i a n i g r a cause degeneration only i n the red nucleus, superior c o l l i c u l i and globus p a l l i d u s . The great i r t e r e s t 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 connection w i t h Parkinsonism; and experiment have been done w i t h animals i n an attempt to r e l a t e Parkinsonism, NA and n i g r o s t r i a t a l l e s i o n s . In r a t s , l e s i o n s alone^did not produce . P a r k i n s o n - l i k e symptoms but when re 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 the si d e c o n t r a l a t e r a l to the l e s i o n . It^was pos t u l a t e d that there i s a balance between c h o l i n e r g i c 86. and dopanergic f i b e r s to the s t r i a t u m . This i s r e f l e c t e d at the s p i n a l l e v e l i n the c o n t r o l of the output of and f i b e r s . When one system (eg. dopanergic) i s upset 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 . P o i r i e r (137) was able 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 of the MAO i n h i b i t o r harmaline i n t e n s i f i e d the motor abnormality but t h i s could not 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 exact 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 ganglion and Parkinsonism i s not yet e s t a b l i s h e d . i i ) To the s e p t a l area Less work has been done on the o r i g i n and course of the f i b e r s to the s e p t a l area. The Swedish workers (6) have post u l a t e d that two groups of CA c e l l s ( p r i m a r i l y DAD i n the midbrain are the o r i g i n of f i b e r s to the nucleus accombens and tuberculum olfactorum. One group (designated as A 10) i s along the mi d l i n e j u s t d o r s a l to the interpeduncular nucleus extending over most of i t s length; the second group (A8) i s j u s t l a t e r a l to t h i s at approximately the same l e v e l . These workers c l a i m that NA f i b e r s i n the s e p t a l area o r i g i n a t e i n the pons and medullary r e g i o n . Since c e r v e a u - i s o l e 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 area, we are 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 midbrain r o s t r a l to the i n f e r i o r c o l l i c u l i . Up to 1007o of the s e p t a l f i b e r s could be accounted f o r i n t h i s r e g i o n , depending on the l a t e r a l extent of the i e s i o n . The f i b e r s and/or the c e l l bodies to the s e p t a l area must therefore be a few mm from the m i d l i n e but medial to the f i b e r s to the caudate (from the su 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 to a f f e c t the s e p t a l f i b e r s without touching the caudate ones. Whether the o r i g i n of 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 the 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 or 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 not known. The r e s u l t s obtained could be c o n s i s t e n t w i t h e i t h e r or both exp l a n a t i o n s . 87 d) Summary Diag. 3 i s a summary of the p o s s i b l e o r i g i n s and course 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 hydroxylase to 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 , turn m e d i a l l y as they go through the midbrain and p o s t e r i o r diencephalon, where most of them can 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 course through the i n t e r n a l capsule to the caudate, thoughlisomeawould appear to course more a n t e r i o r l y to the midp diencephalon before e n t e r i n g the caudate, mos.t-probablrytto the medial aspects. The f i b e r s to the s e p t a l area could o r i g i n a t e i n those c e l l s , designated as A 10 and A 8 by the Swedish workers, course 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 midbrain s l i g h t l y medial to the n i g r o s t r i a t a l f i b e r s . In the p o s t e r i o r diencephalon they may be i n t e r m i n g l e d or d o r s a l to the f i b e r s to the caudate. However, as the two group of f i b e r s to s e p t a l area and caudate course through the diencephalon they begin to separate i n t o medial and l a t e r a l p o s i t i o n s respectively. Whether these f i b e r s j u s t described are DA or NA c o n t a i n i n g i s not known. Probably these to the caudate are DA c o n t a i n i n g . I f r a t s are comparable to c a t s , i t would appear from the Swedish work that the f i b e r s we are 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 midbrain and going to the tuberculum olfactorum and nucleus accombens. I t i s d i f f i c u l t 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 techniques, since the only means i s d i f f e r e n t recovery times a f t e r , d e p l e t i o n w i t h «K^methyl-p-tyrosine. Therefore i t cannot be st a t e d at t h i s time whether a l l c e l l s of the midbrain are 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 that there were no changes i n t y r o s i n e hydroxylase 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 ) although many other 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 l e s i o n . S i m i l a r l e s i o n s s tudies w i t h the enzyme dopamine B-oxidase could 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 . Diag 2: Summary of t y r o s i n e hydroxylase c o n t a i n i n g f i b r e s to the caudate and s e p t a l area medulla oblongata midbrain supe r i o r c o l l i c u l i i n f e r i o r c o l l i c u l i A 8, A 10, A 9 - r e f e r to groups "of CA c e l l s , (as designated by r e c t a n g l e s ) i n midbrain as described i n reference 49, that are 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. SA = s e p t a l area MO CA = caudate MB DE = diencephalon SC ic"= i n t e r n a l capsule IC MFB = medial 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 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 courses of DA and NA c o n t a i n i n g f i b e r s without determing exact d o r s a l , v e n t r a l and l a t e r a l medial p o s i t i o n s , i n order to p i n poi n t exact n u c l e i of o r i g i n and courses of 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 are r e q u i r e d . 90. SUMMARY AND CONCLUSIONS The r e s u l t s of 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 conclusions that are drawn from these can be summarized as f o l l o w s : 1) Ion exchange chromatography on Dowex 50 x 8 resin/' and ~~ t h i n l a y er chromatography on polyamide have proven to be success-f u l i n the separation of NA and DA; t h e i r precursors t y r o s i n e and Dopa; and some of t h e i r m e t abolites. P o s s i b l e a p p l i c a t i o n s of these techniques i n enzyme a n a l y s i s and i n v i v o metabolic s t u d i e s of CA have been discussed. 2) I n c o n c l u s i v e r e s u l t s were obtained i n determining the e f f e c t of exposure to c o l d on CA metabolism i n r a t s . Although an i ncrease i n CA s e c r e t i o n i n urines of animals i n the c o l d was observed, the exact s i t e at which t h i s increase i n synthesis takes place was not determined. There was some i n d i c a t i o n that the adrenal gland may be one organ of increased CA synthesis during c o l d adaption. 3) The d i s t r i b u t i o n of t y r o s i n e hydroxylase i n b r a i n was determined and found to p a r a l l e l CA concentrations and i n v i v o determinations of CA s y n t h e s i s , w i t h highest a c t i v i t y i n the caudate, nucleus accombens, s e p t a l area 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 of CA metabolism i n various regions of the b r a i n , but i t may not be the only c o n t r o l . 4) F i b e r s c o n t a i n i n g t y r o s i n e hydroxylase were t e n t a t i v e l y traced i n the cat from the midbrain ( t h e i r apparent s i t e of o r i g i n ) through the diencephalon to the caudate and s e p t a l area by means of l e s i o n experimentation. The experiments f i b e r s to the s e p t a l area appear to course m e d i a l l y to those of the caudate i n the midbrain and mid-diencephalon, but i n the p o s t e r i o r diencephalon the two groups of f i b e r s are i n close p r o x i m i t y . There were a l s o i n d i c a t i o n s of t y r o s i n e hydroxylase c o n t a i n i n g f i b e r s to the amygdala and hippocampus i n the p o s t e r i o r diencephalon. 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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 cate-cholinergic 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 distribu-tions 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 to-3,4-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 bio-chemical 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 OF B I O C H E M I S T R Y . V O L . 45, 1967 Tissue TABLE I weights of various brain areas* Species Areaf Rat Rabbit Cat Caudate Septal area Pons-medulla Midbrain Hypothalamus Thalamus Hippocampus Amygdala Cerebellum 3 3 ± 6 48 ± 2 3 2 1 4 ± 3 0 118±15 J 2 2 4 ± 6 7 9 6 ± 2 6 7 6 ± 1 4 258 ± 9 7 7 ± 7 7 0 ± 7 801±118 536±98 115±21 400±23 507 ± 7 4 1114±128 4 1 6 ± 5 8 124 ± 2 6 1408 ± 1 6 3 1074±154 1 1 0 ± 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. M a t e r i a l s and Methods R a t s were k i l l e d b y a sharp b l o w to the head, rabbi t s b y cerv ica l d i s loca t ion , a n d adu l t cats b y n i t rogen a sphyx ia t i on . T h e brains were r a p i d l y removed , a n d dissected in to the areas ind ica ted i n T a b l e I and F i g . 1. T h e tissue was h o m o -genized i n 4 -19 vo lumes of ice-cold 0.25 M sucrose, the larger vo lumes of sucrose be ing used w i t h the more ac t ive 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 dissect ion procedure is ind ica ted b y the d a t a on weights g iven i n T a b l e I . K i t t e n s of k n o w n ages f rom 2 to 47 days were k i l l e d b y decap i t a t ion . S i x l i t te rs i n a l l were used: four l i t ters of four k i t t ens each, a n d two of three. S ib l ings were a lways k i l l e d a t w e e k l y in te rva ls (see T a b l e V for deta i l s ) . T h e b r a in was d i v i d e d as shown i n F i g . 1 except tha t the h y p o t h a l a m u s was pooled F I G . 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. M c G E E R 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 1945 TABLE II Endogenous tyrosine levels (Average ± standard deviation in /xg/g of tissue) Area Adult rat Adult rabbit Adult cat Kitten* Caudate 2 8 . 8 ± 1 0 . 2 2 7 . 8 ± 5 . 2 2 4 . 6 ± 6 . 5 2 2 . 6 ± 5 . 7 Septal area 1 6 . 5 ± 6 . 3 2 4 . 9 ± 3 . 5 1 7 . 6 ± 6 . 0 1 9 . 4 ± 4 . 7 Pons-medulla 1 7 . 3 ± 3 . 2 2 4 . 1 ± 1 0 . 1 1 3 . 4 ± 2 . 8 1 9 . 4 ± 3 . 8 Midbrain 1 5 . 6 ± 4 . 2 2 5 . 8 ± 4 . 2 1 4 . 3 ± 7 . 0 1 9 . 1 ± 4 . 7 Hippocampus 2 2 . 7 ± 7 . 1 28.3-1-4.0 1 3 . 2 ± 3 . 2 [ l S . 4 ± 3 . 6 Amygdala 2 3 . 9 ± 5 . 1 — 1 6 . 1 ± 3 . 5 Hypothalamus Thalamus J 2 0 . 8 ± 5 . 4 2 3 . 5 ± 5 . 1 2 5 . 3 ± 6 . 2 1 9 . 8 ± 5 . 5 1 4 . 5 ± 5 . 7 J 2 0 . 1 ± 4 . 2 Cortex 1 5 . 7 ± 4 . 5 3 1 . 2 ± 4 . 8 1 4 . 8 ± 5 . 1 2 0 . 7 ± 4 . 5 Cerebellum 1 7 . 3 ± 5 . 5 2 2 . 9 ± 8 . 5 1 8 . 9 ± 4 . 6 2 3 . 4 ± 4 . 2 Spinal cord 2 2 . 7 ± 3 . 5 2 2 . 7 ± 3 . 7 — — *No consistent change with age was evident. w i t h the tha lamus a n d the a m y g d a l a was pooled w i t h the h ippocampus . E a c h b r a i n area was homogenized i n 5.6 vo lumes of sucrose. I n those cases where p inea l a c t i v i t y was measured, the p inea l was dissected ou t a n d i m m e d i a t e l y homogenized i n 0.3 m l of 0.25 M sucrose. M e a s u r e m e n t of tyros ine hyd roxy la se was done i n a l l cases b y the p r e v i o u s l y repor ted m e t h o d (6) i n w h i c h an a l iquo t of b r a in homogenate is incuba ted w i t h rad ioac t ive tyros ine i n the presence of a D O P A decarboxylase i nh ib i to r , and the r ad ioac t ive catecholamines formed are isolated a n d counted . 2 - A m i n o - 4 -hyd roxy -6 ,7 -d ime thy l t e t r ahyd rop t e r i d ine ( D M P H 4 ) i n c o m b i n a t i o n w i t h mer-cap toe thanol was no t used rou t ine ly since this adrenal cofactor sys tem has been repeatedly shown to have no signif icant effect on ra t b r a i n tyros ine hyd roxy la se a c t i v i t y (6). S i m i l a r l y , i n this s tudy , i t was found to have no signif icant effect on convers ions i n ca t or r abb i t b r a in homogenates. W i t h 11 areas of r a b b i t b ra in , for example , convers ion w i t h cofactor was 109 ± 1 7 % of tha t w i t h o u t cofactor, whereas i n 18 different b r a i n areas from several a d u l t cats, the convers ion was 92 ± 2 1 % of tha t w i t h o u t . S i m i l a r co m p a r a t i v e studies w i t h each area in each of four s ib l ing k i t t ens s tud ied at 5-27 days of age again ind ica ted tha t the 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 no signif icant enhancement of convers ion . T h e same D M P H 4 w h i c h h a d l i t t l e or no effect on the convers ion i n var ious ca t b r a in areas increased the convers ion i n cat adrena l homogenate more than 10-fold. A l l incuba t ions were r u n at tyros ine concent ra t ions be low sa tu ra t ion of the enzyme. T h e ca l cu l a t i on of Vm&K requi red , therefore, knowledge of the endo-genous tissue tyros ine level a n d of the M i c h a e l i s constant for the reac t ion . Endogenous tyros ine levels (Tab le I I ) were de te rmined b y a modi f i ca t ion of the me thod of W a a l k e s and Udenf r i end (6, 7). Repea t ed de t e rmina t ion of M i c h a e l i s constants for r a b b i t b r a i n , for whole ca t b r a i n , and for ca t caudate y i e lded an average figure of 1 X 10~ 5 for Km i n each case. S i m i l a r results i n a l l cases were ob ta ined w i t h a n d w i t h o u t added D M P H 4 p lus mercap toe thano l . Represen ta t ive plots used i n the de te rmina t ion 1946 C A N A D I A N J O U R N A L OF 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 DMPH 4 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. of the Michaelis constant are given in F ig . 2. The previously determined Michaelis constant for whole rat brain homogenate (4.5 X 10~6) was used in calculation of the rat data (6). The measured velocity, Vmeas, 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: V = Vm SE ~t~ Sn where 5 E = concentration of endogenous tyrosine and 5 R = concentration of radioactive tyrosine. The Vm in each case could be calculated from V by the equation: Km F m a x = V 1 + 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. For this purpose the volume was measured, and then the homogenate was made 0.2 N in perchloric acid. The further pro-cedure for isolation and determination of the catecholamines was as reported previously for brain tissue homogenates (9). M c G E E R 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 1947 TABLE III Distribution of tyrosine hydroxylase activity in adult rat, rabbit, and cat brain* in nmoles DOPA/h/g of tissue Activity relative to cerebellum Area Rat Rabbit Cat Rat Rabbit Cat Caudate 72.3±5.5 (6)* 70.9±9.2 (5) 98.5±9 (11) 56 101 70 Septal area 15.0±2.4 (6) 20.3±1.6 (4) 19.0±5 (12) 11 .5 29 13 .5 Amygdala 4.2±0.5 (3) — 4.2±1 (11) 3 .2 — 3 .0 Hypothalamus Thalamus | 5.Oil.6 (6) 3.7±1.0 0.6±0.1 (5) (5) 3.9±0.7 2.7±0.2 (11) (9) {3 .8 5.3 0.9 2. 1. .8 .9 Midbrain 3.6±1.0 (6) 2.2±0.5 (5) 3.1±0.2 (5) 2. .8 3.1 2 .2 Pons-medulla 2.9±0.3 (6) 1.7±0.2 (5) 1.8±0.5 (13) 2. .2 2.4 1. .3 Cortex 3.1±0.8 (6) 2.2±1.0 (5) 2.5±0.6 (9) 2. .4 3.1 1 .8 Hippocampus 1.8±0.4 (4) 2.1±0.6 (5) 1.6±0.4 (10) 1. 4 3.0 1 .1 Spinal cord 1.2±0.7 (5) 2.2±0.9 (4) — 0. .9 3.1 Cerebellum 1.3±0.4 (6) 0.7±0.2 (4) 1.4±0.2 (4) 1. 0 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 mid-brain, 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 3H-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 agree-ment, the absolute conversions indicated by the tyrosine hydroxylase data are 1948 C A N A D I A N J O U R N A L OF B I O C H E M I S T R Y . V O L . 45, 1967 TABLE IV Relative tyrosine hydroxylase activities and noradrenaline turnover rates in rat brain areas Relative tyrosine hydroxylase Relative NA turnover* Hypothalamus >3.8f 5.6 Medulla 2.2 2.1 Cortex 2.4 0.9 Hippocampus 1.4 0.8 Cerebellum 1.0 1.0 *Based on data of Iversen and Glowinski (5). tGiven as > 3.8 because the hypothalamic activity in rats was un-doubtedly 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 m a x under in vitro con-ditions where feedback inhibition by product amine would be negligible, and where saturation of the enzyme with tyrosine could be assumed. This dis-crepancy, 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, pons-medulla, 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 c G E E R 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 1949 i — i — i i i — i — i — i — i 1 i • • • i r — i — i • i — n — • — i 1 i 1 1 1 1 1 ' i 2 3 4 5 6 8 10 15 2 0 3 0 50 2 3 4 5 6 8 1 0 15 2 0 3 0 5 0 8 0 AGE IN DAYS AGE IN 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 OF 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* Hypothalamus Litter Age in days Cerebellum Midbrain -thalamus designation 2 0.8±0.3 3.3±1.3 3.0±0.7 A,B,C,Dt 5 1.0 2.3 4.3 E 8 1.7±0.3 3.3±1.0 3.3±0.6 A,B,C 12 1.1 3.3 3.1 E 15 1.9±0.3 3.9±1.4 3.3±0.9 A,B,C 19 1.1 3.9 2.6 E 22 1.1±0.2 3.0±0.6 2.5±0.8 A,B,C 26 1.3 3.6 2.7 E 33 1.5 4.1 3.2 F 40 0.7 2.3 2.7 F 47 0.9 3.8 3.3 F Average of above 1.3±0.4 3.4±0.9 3.0±0.7 Adult level 1.4±0.2 3.1±0.2 *Pmaxin nmoles of DOPA formed/h/g of tissue. fLittermates of this kitten were lost. TABLE VI Tissue : weights of selected brain areas in neonatal cats Age in days Caudate Cortex Midbrain Pons-medulla 2 101 ±21 3070±330 272±56 242 ±48 5 88 3620 300 270 8 149 ±62 5760±220 316±62 372±89 12 156 6090 370 432 15 203 ±44 781S±348 408±17 409 ±126 19 455* 8410 500 443 22 260 ±20 10160±330 482 ±64 504±39 25 328 10600 530 658 33 281 12600 592 720 40 307 12060 665 803 47 367 13874 672 875 *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). 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 1951 s I O J r- 1 1 1 1—I—i—I I 1 1 1 1 1 — 2 3 4 5 6 7 8 9 10 2 0 3 0 40 50 60 A G E I N D A Y S 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 hydroxy-lase 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 s References 1. S. UDENFRIEND. Pharmacol. Rev. 18, 43 (1966). 2. T . NAGATSU, et al. J . Biol. Chem. 239, 2910 (1964). 3. P. L. MCGEER, S. P. BAGCHI, and E . G . MCGEER. Life Sci. 4, 1839 (1965). 4. N. A. HILLARP, et al. Pharmacol. Rev. 18, 727 (1966). 5. L. L. IVERSEN and J . GLOWINSKI. J . Neurochem. 13, 671 (1966). 6. E . G . MCGEER, S. GIBSON, and P. L. MCGEER. Can. J . Biochem. 45, 1557 (1966). 7. J . P. WAALKES and S. UDENFRIEND. J . Lab. Clin. Med. 50, 73 (1957). 8. J . E . DOWD and D . S. RIGGS. J . Biol. Chem. 240, 863 (1965). 9. J . A. WADA and E . G . MCGEER. 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). SOME C H A R A C T E R I S T I C S 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 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 hydroxyl-ase 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-dimethyltetrahydro-pteridine ( 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 Km for tyrosine in beef adrenal homogenates was 4 X 10-5 M, and in rat , brain homogenates was 0.45 X 10-6 M. Conversion in beef adrenal homogenates was maximum in 0.6 M sodium acetate buffer, pH 6.0, and in rat brain homog-enates was maximum in 0.28 M phosphate buffer, pH 6.2. I n t r o d u c t i o n Much 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). Kinet ic studies have been done chiefly with the enzyme from adrenal medulla (2-7). The enzyme from brain seems to differ in some respects from the enzyme from the adrenal medulla (2-4). This paper reports some further kinetic data on brain tyrosine hydroxylase and gives the basis for the assay method used in this laboratory. M a t e r i a l s 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). The buffer was often made 3 X 10~3 or 10 - 4 M in 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine ( D M P H 4 ) and 6 X 10" 2 M in 2-mercaptoethanol. Typical ly the radioactive tyrosine solution con-tained 140,000-280,000 c.p.m. (1/6-1/12 mCi) of L-tyrosine- 1 4C (uniformly labelled; specific activity 330 mCi/mmole) and was made 3 X 1 0 - 3 M in iV-methyl-A^-3-hydroxyphenylhydrazine (NSD-1034), a D O P A decarboxylase inhibitor. Blanks were run by incubating with tyrosine- 1 4C 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 Canadian Journal of Biochemistry. Volume 45 (1967) 1557 1558 CANADIAN JOURNAL OF BIOCHEMISTRY. VOL. 45, 1967 D O P A , dopamine , and noradrenal ine (0.2 jug of each). T h e mix tures were frozen u n t i l w o r k - u p . T h e D M P H 4 used was obta ined f rom C a l b i o c h e m , and the N S D - 1 0 3 4 f rom D r . D . J . D r a i n of S m i t h a n d N e p h o w Research . If D I V I P H 4 was to be employed , an aqueous so lu t ion 10~ 2 or 10~ 3 M i n D M P H 4 and 0.2 M i n 2-mercaptoethanol was prepared jus t p r io r to the exper i -ment . Such a so lu t ion retained a c t i v i t y for several hours if kep t on ice i n the dark . F o r incubat ions , a 0 .3-ml a l iquo t of this so lu t ion was mixed w i t h 0.7 m l of ei ther 0.4 M phosphate buffer, p H 6.2, or 0.85 M acetate buffer, p H 6.0; 0.1 m l of the mix tu re was used i m m e d i a t e l y i n each incuba t ion . Since the tyrosine hydroxy lase i n c u b a t i o n is run a t substrate concentra t ions be low sa tura t ion , knowledge of endogenous tyrosine levels is necessary for ca lcu la t ion of Vm&x. A sma l l a l i quo t of the homogenate was mixed w i t h an equal vo lume of 3 0 % t r ich lorace t ic ac id a n d three vo lumes of water , a n d the supernate was used for analysis of endogenous tyros ine b y the method of W a a l k e s a n d Udenf r i end (8) modified o n l y i n the use of 0.6 m l each of supernate, l -n i t roso-2-naphthol and d i lu te n i t r i c ac id , a n d 2.5 m l of e thylene d ich lo r ide . Isola t ion and measurement of the rad ioac t ive catechols was on an a l u m i n a c o l u m n . T h e incuba t ion mix tu re was thawed a n d centrifuged brief ly. T h e clear supernate was poured in to a 20-ml beaker con ta in ing 1.25 m l of 0.2 M ethylenediaminete t race t ic a c i d ( E D T A ) . T h e contents of the incuba t ion tube were rinsed in to the centrifuge tube w i t h 1.5 m l of 0.35 M K H 2 P O 4 fol lowed b y a l i t t le water . T h e m i x t u r e was centr ifuged again and the clear supernate combined w i t h the first supernate plus E D T A . T h e so lu t ion was then taken to p H 8.S-9.2 w i t h d i lu te s o d i u m hydrox ide , a p p r o x i m a t e l y 350 m g of ac id -washed a l u m i n a was added i m m e d i a t e l y , and the mix tu re was s t i r red for 4 -5 m i n on a magnet ic st irrer . I t was then poured in to a glass c o l u m n plugged w i t h cellulose fiber. A s l ight v a c u u m was used to promote r a p i d passage of the so lu t ion th rough the c o l u m n . T h e a l u m i n a was washed f rom the beaker onto the c o l u m n w i t h app rox ima te ly 20 m l of water . T h i s and a second 20 m l wash were passed th rough the c o l u m n under a s l ight v a c u u m . T h e co lumn was then removed from the v a c u u m flask a n d eluted 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 eluate was col lected i n a sc in t i l l a t ion v i a l and was counted in a l i qu id - sc in t i l l a t i on spectrophotometer after a d d i t i o n of 10 m l of B r a y ' s mix tu re (9). T h e whole process f rom the t ime of m a k i n g the incuba t ion mix tu re a lka l ine to beginning of the ac id e lu t ion occupied about 10 m i n bu t never longer than 15 m i n . T h e incuba t ion mixtures were occas iona l ly d i lu ted further (to about 15-20 ml) 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 closely comparable so long as the amounts of phosphate a n d E D T A were increased accord ing ly . T h e p H used to pu t the catechols upon the a l u m i n a c o l u m n was chosen after experiments w i t h this pa r t i cu la r lo t of a l u m i n a and w i t h sma l l amounts of rad ioac t ive D O P A added to mock- incuba t i on mixtures . U n d e r the condi t ions specified, the recovery of rad ioac t ive D O P A was consis tent ly 6 5 - 7 0 % . I n the ca lcu la t ions , a recovery of 6 5 % was assumed. M C G E E R E T A L . : BRAIN T Y R O S I N E 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* 1.5 ml of . 3 ml of No phosphate 0.35 M KH2PO* 0.35 M KH 2P0 4 Freshly prepared tyrosine solution (140,000 c.p.m.; 0.05 ug) 370 192-217 206 Tyrosine frozen 50 days in solution (140,000 cp.m.; 0.05 Mg) 1,651-2,358 247 . 259 DOPA (2,150 c.p.m.; 0.11 jug) 1,425 1,460 1,410 DOPA (6,450 c.p.m.; 0.33 ug) 4,302 4,209 4,298 *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 DOPA 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. DOPA-"C. Results The blanks were typically 190-250 c.p.m. when 140,000 c.p.m. of tyrosine-14C 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 Vma.x(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 adre-nal homogenate using 2.5, 10, 15, and 20 mg of tissue gave calculated Vmax(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. 1560 CANADIAN "JOURNAL OF BIOCHEMISTRY. VOL. 45, 1967 TABLE II Relative tyrosine hydroxylase activities of rat brain, cat brain, and beef adrenal homogenates in various buffers* Relative activity, % Buffer Rat Cat Beef 0.28 M K3P04, pH 6.2f 100 100 100 0.28 M Na3P04, pH 6.2 101 — 98 0.6 M KAc, pH 6.2 48 59 127 0.6 M NaAc, pH 6.2 64.5 74 124 0.6 M NaAc, pH 6.0 41 55 132 0.6 M Tris, pH 6.2 61 51 — *Data cited were obtained with 10-3 M D M P H 4 and 0.02 M 2-mercaptoethanol; com-parable relative results were obtained in each series without these additives. fChosen as reference conditions. 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 hydroxy-lase (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 60% 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 10~4 M final concentration (Zn 2 +, Fe 2 +, Mg 2 +, Ca 2 +, Cu 2 +, Co 2 +, or Fe 3 +) 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 insigni-ficantly in the presence of DMPH 4 (2). Addition of 10~4 M Fe 2 + ion to a beef 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. M C G E E R E T A L . : BRAIN T Y R O S I N E H Y D R O X Y L A S E 1501 TABLE III Relative tyrosine hydroxylase activities of a rat brain homogenate using different molarities of phosphate and sucrose Phosphate buffer, pH 6.2 S ucrose 0.2 M 0.25 M 0.28 M 0.30 M 0.2 M 0.24 M 0.28 M 0.32 M 0.35 M 22% 41% 24% 37% 20% 44% 48% 100%* 96% 85% 17% 54% 89% 104% 93% 39% 50% 72% 78% 64% *Chosen as reference conditions. adrenal incuba t ion in the presence of 1 0 - 4 M D M P H 4 gave an a c t i v i t y 1 2 7 % of that i n its absence. JVTg 2 + , C a 2 + , and F e 3 + had no effect whatsoever. F l u s h i n g the incuba t ion tubes w i t h oxygen d i d not s igni f icant ly increase observed conversions, bu t flushing w i t h ni t rogen caused ve ry large decreases. T h e most s ignif icant difference in tyrosine hydroxy lase a c t i v i t y between rat b ra in and beef adrenal homogenates is the dependence of the la t ter on D M P H 4 and 2-mercaptoethanol . These cofactors i n c o m b i n a t i o n d i d not s igni f icant ly affect a c t i v i t y in crude rat b ra in homogenates, a l though they increased adrenal homogenate a c t i v i t y 15-fold. T h e da ta in T a b l e I V i l lus t ra te not on l y this effect, bu t also the i n h i b i t o r y effect of 2-mercaptoethanol b y itself. T h i s i n h i b i t i o n is not surpr i s ing i n v iew of the i n h i b i t o r y effect found for related compounds such as e thanol and L-cysteine (10). D M P H 4 b y itself increased the a c t i v i t y of adrenal homogenates roughly threefold, bu t had no effect on b ra in homogenates. In c o m p i l i n g the da ta shown in a l l tables, a separate b l a n k was run for each test cond i t ion . T h e b lanks were general ly unaffected b y var ia t ions in p H , mo la r i t y , or buffer type bu t tended to be 5 0 - 6 0 % higher i n the presence of D M P H 4 than in its absence. TABLE IV Relative tyrosine hydroxylase activities in the presence of various additives Relative activity, % Additive Beef adrenal Rat brain (final concentration) (acetate buffer) (phosphate buffer) None* 100 100 10"3 M DMPH, plus 0.02 M 2-mercaptoethanol (SH) 1520 92 10^3 M DMPH 4 plus 0.02 M SH, preincubated for 10 min — 75 10"4 ikTDMPH4 plus 0.02 M SH 1495 96 lO"3 M DMPH 4 280 98 0.02 M SH 89 79 10-3 M TPNH — 97 10~3 M folic acid — 98 •Chosen as reference condition. 1562 CANADIAN JOURNAL OF BIOCHEMISTRY. VOL. 45, 1967 TABLE V Km (X10~5) for tyrosine determined with and without added DMPH 4 (0.001 M) plus 2-mercaptoethanol (0.02 M) With cofactor Without cofactor No. of runs Rat brain 0.45±0.08 0.47±0.05 7 Beef adrenal 4.Oil.2 nonlinear 4 I n another approach to the cofactor p rob lem, 36 i n d i v i d u a l ra t b ra in homogenates were run w i t h and w i thou t the c o m b i n a t i o n of D M P H 4 plus 2-mercaptoethanol under the usual condi t ions of r e la t ive ly low tyrosine concent ra t ion . T h e convers ion in the presence of D M P H 4 was 104 ± 9 % of tha t i n its absence. I t appears possible tha t the effectiveness of exogenous D M P H 4 m igh t be related to the length of t ime between sacrifice of the a n i m a l a n d incuba t ion . T h e b ra in studies were done w i t h an imals sacrificed i n the l abora to ry a n d the usual t ime between sacrifice a n d assay was general ly less than 2 h . T h e beef adrenal was obta ined from the s laughter house a n d kept on ice d u r i n g its t ranspor t to the labora tory . T h e r e were general ly about 4 -5 h between dissec-t ion and assay. T r i a l of 12 ra t b r a in homogenates a l lowed to s tand in ice for 5 h between the i n i t i a l assay and reassay, however , ind ica ted no change i n a c t i v i t y d u r i n g such a per iod of s tand ing , a n d no s ignif icant effect of exogenous D M P H 4 plus 2-mercaptoethanol at e i ther t ime. T h e conversions w i t h D M P H 4 i n this series were 96 ± 8 % of the conversions wi thou t . Different K m values were ob ta ined for rat b ra in a n d beef adrenal homogenates (Tab le V ) , i n conf i rmat ion of previous reports (3, 4) . T h e va lue of 4.0 ( ± 1 . 2 ) X 10~ 5 M for beef adrenal homogenates is somewhat lower , bu t i n reasonable agreement w i t h the va lue of 1 X 10~ 4 M for purif ied adrenal tyros ine hydroxy lase reported b y Ikeda et al. (5). A nonl inear reciprocal p lo t of convers ion versus tyrosine concent ra t ion was obta ined for adrenal homog-enates i n the absence of added cofactor, m a k i n g i t impossible to ob t a in a K m va lue for such condi t ions . A t higher tyrosine concentrat ions , convers ion r a p i d l y fell off. F o r crude rat b ra in homogenates, however , iden t ica l reciprocal plots were obta ined w i t h and w i t h o u t added cofactor. T h e K m va lue ob ta ined , 0.45 ( ± 0 . 0 8 ) X 1 0 - 5 M , was s ignif icant ly lower than that for adrenal homogenates. Discussion A l t h o u g h b ra in tyros ine hydroxy lase appears to be different f rom adrena l tyrosine hydroxy lase i n being par t ic le -bound, insensi t ive to cofactors, respon-sive to s l igh t ly different condi t ions of p H a n d buffer, and possessing a lower M i c h a e l i s constant for tyros ine , f i rm conclusions regarding differences canno t be d r a w n from crude enzyme preparat ions. A t t e m p t s to solubi l ize b ra in tyros ine hydroxy lase as a p repara to ry step to MCGEER ET AL.: BRAIN TYROSINE HYDROXYLASE 1563 pur i fy ing i t have led , i n our hands , to loss of a c t i v i t y . T h e enzyme sediments w i t h the crude m i t o c h o n d r i a l f rac t ion, a n d upon further separa t ion is shown to be concentrated in the nerve-ending par t ic les (11). D i s r u p t i o n of the nerve-ending par t ic les b y osmot ic shock a n d other means has a lways led to disap-pearance of tyrosine hydroxy lase a c t i v i t y . T h e da t a presented here on b ra in tyros ine hydroxy lase m a y a p p l y d i r ec t l y to the enzyme. B u t i t is equa l ly possible tha t they a p p l y to the enzyme plus a par t ic le to w h i c h the enzyme is a t tached or w i t h i n w h i c h i t is con ta ined . S u c h a par t ic le cou ld con ta in essential cofactors w h i c h w o u l d no t be d i lu ted b y the homogeniza t ion process. T h i s w o u l d exp la in the insens i t iv i ty of b r a in tyros ine hydroxy lase to added co-factors. T h e par t ic le cou ld also l i m i t the a v a i l a b i l i t y of tyrosine to the enzyme, and thus account for the lower K m for tyros ine i n brain compared w i t h tha t in adrenal . A l t h o u g h b r a in tyrosine hydroxy lase is h igh ly local ized to nerve endings, h is tochemical studies (12) fo l lowing lesions to ca techolamine axons have shown accumula t ion of catecholamines p r o x i m a l to the lesion and disappearance of them a t the nerve endings. T h i s result is consistent w i t h the concept of con-t inuous t ranspor t of tyros ine hydroxy lase from the cel l b o d y to the nerve t e rmina l , bu t the nature of the t ranspor t poses problems. If the enzyme were free a t the t ranspor t stage, some soluble tyrosine hydroxy lase should be found fol lowing homogeniza t ion . C l e a r l y , the true nature of b r a in tyros ine hydroxy lase mus t awa i t methods wh ich w i l l lead to its pur i f ica t ion and w h i c h w i l l exp la in its associat ion w i t h b ra in part icles. Acknowledgments T h i s w o r k was suppor ted b y the M e d i c a l Research C o u n c i l of C a n a d a ( M A - 1 4 2 1 ) , and 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. 239, 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. 50, 733 (1957). 9. G . A. B R A Y. 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. BAGCHI, 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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