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An investigation into the possible relationship of adenosine triphosphate to sensory synaptic transmitter… Muirhead, Christopher Robert 1962

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AN INVESTIGATION INTO THE POSSIBLE RELATIONSHIP OP ADENOSINE TRIPHOSPHATE TO SENSORY SYNAPTIC TRANSMITTER SUBSTANCES  by  C h r i s t o p h e r Robert Muirhead B.A., U n i v e r s i t y of B r i t i s h Columbia, 1955  A T h e s i s Submitted i n P a r t i a l The Requirements  F u l f i l m e n t of  f o r the Degree of  Master of Science i n the Department of Physiology  We accept t h i s t h e s i s as conforming t o the  r e q u i r e d standard  THE UNIVERSITY OP BRITISH COLUMBIA A p r i l , 1962  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study.  I further agree that permission  for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be alloiired without my written permission.  Department of  Physiology  The University of British Columbia, Vancouver 8, Canada. Date  26th A p r i l . 1962.  ABSTRACT  An  attempt has been made t o determine the r e l a t i o n -  s h i p , i f any, between adenosine t r i p h o s p h a t e substance r e s p o n s i b l e of v a r i o u s  and the t r a n s m i t t e r  f o r antidromic v a s o d i l a t a t i o n .  Extracts  areas of t h e c e n t r a l nervous system have been made  by d i a l y z i n g b o i l e d , ground b r a i n t i s s u e against These e x t r a c t s were analyzed f o r ; l a b i l e  d i s t i l l e d water.  phosphate content by  the method o f Berenblum and Chain ( 6 6 ) , adenosine  triphosphate  content by paper chromatography and by t h e l u c i f e r i n - l u c i f e r a s e enzyme method o f S t r e h l e r and T o t t e r The the  (69)..  content of v a s o d i l a t o r a c t i v i t y  of e x t r a c t s  same areas was determined by the method o f Holton  e x t r a c t s t o be t e s t e d were i n j e c t e d i n t o the f a c i a l r a b b i t and allowed t o flow Into the  ear.  a r t e r y of a  The changes produced In t h e e a r were d e t e c t e d  passing  through t h e ear.  decrease i n the amount o f l i g h t v a s o c o n s t r i c t i o n as an  ( 3 5 ) . The  the a u r i c u l a r a r t e r y and through  of a p h o t o e l e c t r i c c e l l which measured d i f f e r e n c e s of l i g h t  from  by means  i n the amount  V a s o d i l a t a t i o n appeared as a  transmitted  through the ear and  increase.  I t was thought that  i f t h e r e was a r e l a t i o n s h i p between  adenozine triphosphate' and the t r a n s m i t t e r m a t e r i a l the areas containing  t h e most ATP should a l s o c o n t a i n t h e most v a s o d i l a t o r  activity.  A comparison o f t h e l o c a t i o n and c o n c e n t r a t i o n  two  substances r e v e a l e d The  1.  no such c o r r e l a t i o n .  two most important  questions t o be answered were:  i s ATP the substance r e s p o n s i b l e  t i o n ? and 2.  o f the  f o r antidromic v a s o d i l a t a -  i f so, i s i t a l s o a sensory s y n a p t i c  transmitter  substance?  I t was  concluded that ATP  substance r e s p o n s i b l e  was  f o r antidromic v a s o d i l a t a t i o n .  accepts Dale's h y p o t h e s i s (48) t h a t  a neurone may  same t r a n s m i t t e r substance at a l l branches i t would a l s o seem t o r u l e out ATP mitter  u n l i k e l y t o be  I f one  employ the  of the axon, then  as a sensory s y n a p t i c  agent.  Hugh McLennan,  the  Ph.D.  trans-  ACKNOWLEDGMENT  I wish t o acknowledge w i t h g r a t i t u d e t h e guidance and encouragement g i v e n me throughout  the course of these  i n v e s t i g a t i o n s by Dr. H, McLennan. I would l i k e  t o thank Dr. D.H. Copp f o r h i s k i n d  a s s i s t a n c e d u r i n g my course of s t u d i e s .  My thanks  a r e due  a l s o t o Mr. R. Walker f o r h i 3 h e l p with t h e r a b b i t e a r prepara t i o n s and t o Mr. K. Henze f o r making the i l l u s t r a t i o n s .  TABLE OP CONTENTS Page I. II.  INTRODUCTION  1  MATERIALS AND METHODS 1.  18  The D i s t r i b u t i o n of " V a s o d i l a t o r " A c t i v i t y i n the C e n t r a l Nervous System .  18  Method f o r P r e p a r i n g D i a l y s a t e s  18  of B r a i n  Method f o r P r e p a r i n g the Rabbit E a r ' , 2.  . .  Method f o r Measuring the Amount of Vasodilatation D i s t r i b u t i o n of Adenosine Triphosphate In the C e n t r a l Nervous System  19 21 22  Methods f o r Determining ATP i n C e n t r a l Nervous System 3.  Chromatography  4.  Chemical P r o p e r t i e s  25 of the V a s o d i l a t o r  Substance and ATP  27  Acid-Base S t a b i l i t y  27  D i a l y s i s of the V a s o d i l a t o r Substance and ATP Treatment o f D i a l y s a t e s w i t h Exchange  28  Resins III.  22  .  RESULTS 1. 2. 3.  4. 5.  The D i s t r i b u t i o n of V a s o d i l a t o r A c t i v i t y i n the C e n t r a l Nervous System D i s t r i b u t i o n of Adenosine Triphosphate i n the C e n t r a l Nervous System A Comparison of V a s o d i l a t o r A c t i v i t y w i t h Adenosine Triphosphate Content of the C e n t r a l Nervous System  28 30 30 32 34  A Comparison of the Chemical P r o p e r t i e s of the V a s o d i l a t o r A c t i v i t y and ATP . .  35  Chromatography of A c t i v e E x t r a c t s f o r V a s o d i l a t o r M a t e r i a l and ATP  40  Page 6.  Recordings  of V a s o d i l a t a t i o n Produced  i n the Rabbit IV.  DISCUSSION  BIBLIOGRAPHY  Ear Preparation  42 44 52  LIST OP TABLES Table 1.  2.  3. 4.  5.  6.  Page D i s t r i b u t i o n of V a s o d i l a t o r A c t i v i t y i n E x t r a c t s from V a r i o u s Areas of the B r a i n of the Cow  31  Adenosine Triphosphate Content of Areas of Cow B r a i n Expressed i n ug./gm. Fresh Brain  33  Comparison o f the Amounts of ATP V a s o d i l a t o r Substance  36  and  The E f f e c t o f Various Ion Exchange Resins on A c t i v e D i a l y s a t e s and S o l u t i o n s of ATP  38  V a s o d i l a t o r A c t i v i t y , Not Due t o A c e t y l c h o l i n e or Histamine, of E x t r a c t s From D i f f e r e n t Regions of the Horse B r a i n  47  D i s t r i b u t i o n of N e u r o l o g i c a l A c t i v e Compounds i n Areas of C e n t r a l Nervous System . .  48  . . . . .  - 1 -  I.  INTRODUCTION: A f o r m o f v a s c u l a r r e f l e x phenomenon, w h i c h h a s s i n c e  b e e n named " a n t i d r o m i c Loveh  v a s o d i l a t a t i o n " was f i r s t  described  by  ( l ) , who showed t h a t when t h e c e n t r a l e n d o f t h e g r e a t  a u r i c u l a r n e r v e i n t h e r a b b i t ' s e a r was s t i m u l a t e d , s t r i c t i o n 'occurred  In other  organs a l t h o u g h  t i o n was i n i t i a t e d  i n the ear i t s e l f .  This  vasocon-  a marked v a s o d i l a t a same phenomenon was  also described  by G o l t z  certain  fibres  of the s c i a t i c  nerve would induce a p e r i p h e r a l  vasodilatation  i n a localized  area  (3)  confirmed  this  ( 2 ) , who o b s e r v e d t h a t s t i m u l a t i o n o f  of the skin.  I n 1876 S t r i e k e r  f i n d i n g , and f u r t h e r r e p o r t e d t h a t  i t could  s i m i l a r l y be b r o u g h t a b o u t b y s t i m u l a t i o n o f t h e d i s t a l of a s e c t i o n e d direct law  p o s t e r i o r r o o t , a f i n d i n g w h i c h seemed t o be i n  o p p o s i t i o n t o the Bell-Magendie law.  states that  portion  posterior roots  t h e r e f o r e do n o t conduct  contain  'The B e l l - M a g e n d i e  sensory f i b r e s  o n l y , and  impulses towards t h e p e r i p h e r y .  Because  of t h e apparent c o n t r a d i c t i o n t o t h e Bell-Magendie law, S t r i e k e r ' s w o r k was n o t g e n e r a l l y a c c e p t e d , u n t i l and  extended the s t u d i e s .  B a y l i s s (4-8) confirmed  B a y l i s s showed t h a t t h e f i b r e s  c e r n e d seemed i n d i s t i n g u i s h a b l e f r o m o r d i n a r y s e n s o r y and  fibres,  he s u g g e s t e d t h a t t h e r e m i g h t b e a p e r i p h e r a l n e r v e  around the a r t e r i o l e s fibres.  con-  network  common t o b o t h s e n s o r y a n d v a s o d i l a t o r  He named t h e phenomenon " a n t i d r o m i c v a s o d i l a t a t i o n "  because t h e impulses passed i n a d i r e c t i o n opposite was u s u a l l y t h e c a s e . theory with with cocaine  Bruce  (9) was a b l e  t o that  which  t o confirm B a y l i s s '  some e x p e r i m e n t s i n w h i c h he p a r a l y z e d  sensory  fibres  a n d f o u n d t h a t no v a s o d i l a t a t i o n was p r o d u c e d on  Figure 1.  Diagram of Axon R e f l e x  - 2 applying  o i l of mustard t o the  sensory f i b r e s .  He  degenerate no oil  i t d i d not  a l s o found that  He  that  branches of a neurone and give  bhe  allowed  When the  therefore  s k i n was  d i r e c t i o n over the  could  also studied  pass t o a  arterioles  antidromic  innervated.  vasodilata-  of p o s t e r i o r r o o t s  cat caused f l u s h i n g i n the  s k i n of the  foot.  become quite on one  side.  pale He  and  clamped o f f he  then s t i m u l a t e d  the  various  vasodilator  stimulation  was  in  limb on the s t i m u l a t e d  the  In experiments i n allowed the f e e t  lumbar p o s t e r i o r  and  toes of the  s i d e , which he  was  a r e s u l t of c a p i l l a r y d i l a t a t i o n , while there was  the  opposite s i d e .  pads  concluded a pallor  the  experiment, d i l a t a t i o n of the  a r t e r i e s might take b l o o d from  the  capillaries.  occur, he  t o have been due  t h i s d i d not  conditions  on  of  As  reasoned t h a t under the  to  roots  observed a combination of f l u s h i n g of the  He  an  stimu-  demonstrated t h a t  abdominal aorta  to  sensory nerve concerned contained  pass i n any  (10,11,12)  area  a p p l i c a t i o n of mustard  r i s e t o a d i l a t a t i o n of the  Langley  which the  the  the v a s o d i l a t a t i o n r e q u i r e d  f i b r e s t o the a r t e r i o l e s .  l a t e d nerve impulses could  t i o n and  when the nerve was  concluded t h a t  i n t a c t nerve supply and  branch and  by  prevent inflammation from  inflammatory r e a c t i o n to the  occurred.  vasodilator  skin supplied  However, s e c t i o n i n g the nerve trunk t o the  without a n a e s t h e t i z i n g occurring.  area of the  considered the  to a decrease of c a p i l l a r y tone which allowed  them t o be s l i g h t l y d i s t e n d e d by the venous p r e s s u r e . ested that  the v a s o d i l a t a t i o n might be the  s p e c i a l connection of a f f e r e n t that  the  flushing  stimulated  afferent  He  sugg-  r e s u l t e i t h e r of a  f i b r e s with the  capillaries,  or  f i b r e s set f r e e m e t a b o l i t e s which  -  secondarily latter  gave r i s e  theory  t o the  and  Bayliss  also noticed.  the  an  "axon r e f l e x " .  to  occur  i n the  the in  i s not  arterioles, a state  blood, root  the  fibres.  there  must  capillaries  found  that  they  can  and be  chemical  be  long  that  and  man the  of blood  r e s t i n g state the closed  to  stimulation  local  reactions  s t i m u l i were due  that  that  any  capillaries  shown  (15). c a l i b r e of coming  cap-  from  capillaries  are  the  passage  of  of  posterior  of  skin  vessels  t o axon r e f l e x e s , calibre  capillaries  and of  capillaries  are  set  lends  f r e e by  logically  be  credance  the  substance r e l e a s e d  would  which  a v a s o d i l a t o r mechanism must  of the  metabolites as  been  m a i n l y s i t u a t e d In the  conclusion  between  involved  since  some mechanism f o r r e g u l a t i n g the  capillaries,  site  volume  delay  pathway  show t h a t  opened by  immediate a r e a  the  to  the  ( 2 t o 8 seconds)  the  (14)  therefore  w h i c h w o u l d be  c a l i b r e of the  afferent  to  regulate  released  at  suggestion  as  of i t s a c t i o n . Gaskell  possible  tion  by  skin  cells.  (18),  nature  i n 1916,  of the  postulating that  transmission  first  mediator  S p e c i a l a t t e n t i o n has since  since  substance  of the  offered to  causing  i n 1921  heart,  on (19)  from  humoral i n which  several pieces  strengthen  the  to  vasodila-  were r e l e a s e d  been f o c u s e d  Loewi's experiments  have been  made t h e  acid metabolites  demonstrated humoral c o n t r o l evidence  rabbit  i n the  concluded  theory  i n the  the  of the  the  the  (13),  a function  Krogh's  i n the  to Langley's fibres  Langley c a l l e d  able  He  relatively  dilatation  (16,17) was  and  that  themselves. be  of the  of c o n t r a c t i o n  but  of the  Langley favoured  A n t i d r o m i c v a s o d i l a t a t i o n has  t o m e c h a n i c a l and that  start  frog  Krogh illaries  dilatation.  c h i e f l y because  stimulation had  3 -  theory  he  of that  -  humoral t r a n s m i s s i o n e l i c i t s tioned the of  above t h e most  l o n g d e l a y which  4  -  antidromic vasodilatation.  obvious evidence  As men-  f o r humoral c o n t r o l  o c c u r r e d between s t i m u l a t i o n  and  the  was onset  dilatation. Before  any  particular  possible  humoral substance  criteria  must be (a)  (c)  can be  c o n s i d e r e d as a  for antidromic vasodilatation  certain  met:  can t h e s u b s t a n c e the  (b)  compound  be  found  i n the nerves  or i n  endings,  are the nerve  fibres  thesizing  substance,  the  i s t h e r e any  enzyme  concerned  capable  capable  of syn-  of d e s t r o y i n g the  sub-  stance, (d)  can the any  presence  special  of the s u b s t a n c e  a r e a , s u c h as n e r v e  be  localized  endings  to  i n the  capillaries, (e)  i f a specialized tion  of the substance  to the nerve Lewis lation  and  a r e a can be  and  one  Marvin  dilatation  skin was  and  (20,2l)  and went  the  prolonged, which  dilator  substance"  stimulation  identical of the  on t o do some e x p e r i m e n t s the d i l a t a t i o n circulation  f l u s h produced  they  effect  applica-  n o t e d t h e d e l a y between  They o c c l u d e d the  found t h a t  an  can  involved?  seemed u n e q u i v o c a l l y t o show t h a t a humoral agent.  produce  obtained through  fibres  found,  interpreted  by  was  stimu-  which  produced  t o an a r e a  by  of  antidromic stimulation  t o mean t h a t  the  "vaso-  continued t o act d u r i n g c i r c u l a t o r y  arrest  - 5and  was only removed when the c i r c u l a t i o n was r e s t o r e d ,  as the  subsidence of the f l u s h seemed t o depend on i r r i g a t i o n of the tissues.  T  h e y a l s o pointed  out that while the d e c l i n e  of vaso-  d i l a t a t i o n was d e l a y e d by c i r c u l a t o r y a r r e s t , the time of onset of the v a s o d i l a t a t i o n was not a l t e r e d ; they found t h a t the peripheral vessels relaxed stimulated  a few seconds a f t e r t h e nerve had been  even though the c i r c u l a t i o n was stopped.  o f f e r e d more evidence f o r humoral t r a n s m i s s i o n which r e l a t e d the l e n g t h flush.  i n experiments  of s t i m u l a t i o n w i t h t h e d u r a t i o n  Lewis and Marvin claimed t h i s i n d i c a t e d t h a t  s t i m u l a t i o n a v a s o d i l a t o r substance was r e l e a s e d spaces and that to r i s e .  They a l s o  of the  during  i n t o the t i s s u e  i f s t i m u l a t i o n continued, the c o n c e n t r a t i o n  With longer  tended  o c c l u s i o n of the c i r c u l a t i o n the r e a c t i o n  was prolonged, but only up t o a point; a b a l a n c e was  eventually  a t t a i n e d between l i b e r a t i o n and removal of the substance so 'that a longer  r e a c t i o n was not induced by lengthening  the s t i m u l a t i o n .  Kibjakow (22) presented evidence which a l s o ened the humoral t r a n s m i s s i o n dilator activity  strength-  t h e o r y when he observed a vaso-  i n the r a b b i t ear p r e p a r a t i o n  present i n b l o o d  c o l l e c t e d from a r e g i o n o f a n t i d r o m i c v a s o d i l a t a t i o n In the 'cat. It has been found that when s k i n i s i n j u r e d by s c r a t c h i n g with a needle, by a p p l i c a t i o n of burning heat, b y f r e e z i n g or by other p h y s i c a l means, the v a s c u l a r The  vessels  r e a c t i o n Is t h r e e f o l d .  d i l a t e l o c a l l y , the s k i n b l i s t e r s , and the surround-  i n g s k i n shows a b r i g h t r e d f l a r e .  The f i r s t  two r e a c t i o n s are  independent of a nerve supply, and the t h i r d r e a c t i o n i s known to be r e f l e x i n o r i g i n .  Lewis (23) has shown that the surrounding  - 6 r e d f l a r e w h i c h c a n he p r o d u c e d  by p u n c t u r e  n o r m a l s k i n d e p e n d s on an i n t a c t  observations  of Dale  a general loss  In view of these f i n d i n g s  i n the c a p i l l a r i e s ,  mine-like  L e w i s and G r a n t  s u b s t a n c e was  released  in injured  and  i n t h e nerves which  t i o n but  little  He  f o r a n t i d r o m i c vaso-  skin,  a  stimulation.  claimed that  and T a l a a t  m i n e was  antihistamine d i d not a f f e c t tion  fibres  directly  by  concluded posterior  released  concluded that  s u p p l i e d by the n e r v e s  E v i d e n c e has  histarather  They a l s o have found  p r o d u c e d by  intra-arterial  effect  by  that  but injec-  w h i c h t h e y c l a i m e d t o be e v i d e n c e t h a t  compounds were p r o d u c i n g t h e i r  on h i s t a m i n e  from  after antidromic stimulation  themselves.  the d i l a t a t i o n  from  stimulation  compounds r e d u c e d a n t i d r o m i c v a s o d i l a t a t i o n  of a c e t y l c h o l i n e ,  antihistamine  Kwiatkowski  substance.  (28) have a l s o  s u p p l y i n g the a r e a , but  r e l e a s e d from the t i s s u e s  than from the nerve  hista-  this strengthened  T h e y f o u n d t h a t h i s t a m i n e was  the s k i n but not f r o m the muscles of the nerve f i b r e s  a  histamine-like  t h a t h i s t a m i n e causes the v a s o d i l a t a t i o n produced root  while  a l s o found that  the t h e o r y that h i s t a m i n e i s the v a s o d i l a t o r Ibrahim, S t e l l a ,  suggest-  of sensory nerves  i n the cat l i b e r a t e d  s u b s t a n c e i n t o v e n o u s b l o o d , and  to  produce a n t i d r o m i c v a s o d i l a t a -  i n motor f i b r e s .  of cut p o s t e r i o r r o o t s  parts  the  related  (26) were a b l e t o show t h a t  (27) f o u n d h i s t a m i n e i n t h e d i s t a l the s k i n  as w e l l as  h i s t a m i n e was  ed as t h e s u b s t a n c e l i k e l y t o be r e s p o n s i b l e dilatation.  was  s u p p l y t o t h e r e g i o n had "been  ( 2 4 , 2 5 ) t h a t h i s t a m i n e s h o c k was  o f tone  into  nerve supply; the f l a r e  f o u n d t o d i s a p p e a r when t h e n e r v e allowed to degenerate.  of h i s t a m i n e  the  acting  itself. a l s o been o f f e r e d by Unger  (29,30,31) to  - 7support the theory dilatation. released  that histamine  He h a s shown t h a t  release  causes a n t i d r o m i c  a h i s t a m i n e - l i k e substance i s  f r o m p e r i p h e r a l ends o f a f f e r e n t f i b r e s d u r i n g  activity.  vaso-  antidromic  He c a l l e d t h e s u b s t a n c e h i s t a m i n e - l i k e b e c a u s e a l -  though i t has n o t been d e f i n i t e l y  identified  ties  As a b i o l o g i c a l t e s t he has  s u g g e s t i t t o be h i s t a m i n e .  shown t h a t gastric  the substance l i b e r a t e d  segments o f i s o l a t e d  tion was  proper-  of augmenting  only by comparably  Von E u l e r and A s t r o m  (32),  p e r i p h e r a l nerve from c a t t l e ,  t h a t under c e r t a i n c o n d i t i o n s leased  was c a p a b l e  s e c r e t i o n , i n a manner d u p l i c a t e d  s m a l l doses of h i s t a m i n e .  i t s chemical  studying  have  found  a h i s t a m i n e - l i k e s u b s t a n c e was r e -  f r o m one e n d o f t h e segment f o l l o w i n g e l e c t r i c a l  of the other, which they claimed  t o indicate that  the t r a n s m i t t e r substance involved i n antidromic  stimulahistamine  vasodilata-  tion. On bhe o t h e r upon t h e l i k e l i h o o d stance  i n t h e body.  Lefebvre (35) block  hand, c o n s i d e r a b l e  of histamine Unlike  doubt has been  a c t i n g as a v a s o d i l a t o r s u b -  I b r a h i m e t a l . ^28),  h a v e a l l b e e n u n a b l e t o show t h a t  antihistamine  substance.  Further,  Chauchard  actually inhibits  roots  Parrot  compounds  transmission  (37) f o u n d t h a t  caused t h e r e l e a s e  were t h e t r a n s m i t t e r  (36) h a s b e e n a b l e  w h i c h w o u l d n o t be e x p e c t e d i f h i s t a m i n e question.  and P e r r y  v a s o d i l a t a t i o n produced by nerve s t i m u l a t i o n ,  w h i c h would be e x p e c t e d t o happen i f h i s t a m i n e  histamine  P a r r o t and  ( 3 3 ) ; P r u m i n , % a i , a n d Wang ( 3 4 ) a n d H o l t o n  antidromic  cast  t o show t h a t  i n sensory neurones w e r e t h e compound i n  s t i m u l a t i o n o f lumbar p o s t e r i o r  o f a compound whose p r o p e r t i e s  s i m i l a r t o those of a d e r i v a t i v e of adrenaline.  were  He  also  showed t h a t  released  by t h e i n j u r e d  peripheral dilator  endings  origin.  the  histamine  present  evidence  acetylcholine  of the hind  presence  in  that  for  voluntary  this  during  t o support antidromic  compound  the theory vasodilatation  of experiments  roots,  he was a b l e  on t h e i s o l a t e d f r o g ' s  acetylcholine  dilatation  it  of a c e t y l -  on t h e p e r -  antidromic  t o demonstrate the  o f a s u b s t a n c e w h i c h h a d t h e same p h a r m a c o l o g i c a l  antidromic  ally  suggested that  In a s e r i e s  m u s c l e and t h e b l o o d  eserine,  obscure.  f i b r e s t o perfused  of a cat c o l l e c t e d during  However, o t h e r cate  significance  of antidromic v a s o d i l a t a t i o n (42).  i s released  limb  (38) have n o t s u p p o r t e d  system remains  has b e e n o f f e r e d  of posterior  acetylcholine  leech  a vaso-  h i s t a m i n e may n o t a c t as  demonstrated the r e l e a s e  o f motor  (43,44,45,46).  stimulation  as  and F e l d b e r g  a l s o be t h e m e d i a t o r  fusate  t o stimulate the  t h e o r y and the f u n c t i o n a l  (39,40,41) and i t has b e e n  Wybauw  that  i n t h e nervous  on s t i m u l a t i o n  Experimental  by  h i s t a m i n e was  f i b r e s and e l i c i t  investigations  transmitter  Dale  that  a n d t h i s was a b l e  He s u g g e s t e d  More r e c e n t  of h i s t a m i n e  might  cells,  of the skin,  a t t h e end of t h e a x o n r e f l e x b u t a s t h e s t i m u l u s a t  its  choline  Irritation  of the v a s o d i l a t o r  axon r e f l e x .  a mediator  muscle  after  8 -  pressure  of a chloralosed c a t .  i s u n l i k e l y t o be t h e c h e m i c a l Holton  (35) f o u n d  was n o t i n h i b i t e d by a t r o p i n e although the release  on s t i m u l a t i o n  of transected  seems u n l i k e l y t h a t  the e s e r i n i z e d  o b s e r v a t i o n s have b e e n made w h i c h  vasodilatation.  so t h a t  heart,  this  that  substance  indi-  mediator t h e vaso-  n o r was i t e n h a n c e d by  of acetylcholine  posterior roots  antidromic vasodilatation (47).  effects  itself  could  peripher-  i s not d e n i e d be  responsible  Dale r e l e a s e the and  (48)  reasoned that a sensory neurone would  same t r a n s m i t t e d  substance p e r i p h e r a l l y and c e n t r a l l y ,  i f t h i s were true i t would be expected that the  r o o t s but  not  substance.  the  a n t e r i o r r o o t s would c o n t a i n the  Hellauer  likely  posterior transmitter  and Umrath (49,50) have found p o s t e r i o r root  e x t r a c t s t o c o n t a i n a h i g h l y a c t i v e v a s o d i l a t o r m a t e r i a l which was  absent i n a n t e r i o r r o o t e x t r a c t s .  They a l s o found t h a t  v a s o d i l a t o r a c t i v i t y of the e x t r a c t s was w i t h f r e s h b r a i n t i s s u e and by the  t o the mixture.  that the v a s o d i l a t o r substance was and  enzymic breakdown, the  that  i d e n t i c a l t o the  strychnine  substance.  would be expected that s t r y c h n i n e  should  e r a l ends of the case  This has  I f t h i s were so, i t  potentiate  antidromic  a c t i o n of e s e r i n e  not  one  in cholin-  at the  been found t o be  periphthe  (35). In order  has  enzyme being  i f the enzyme i s present  neurone.  central  i n h i b i t e d i t s normal  of the c r i t e r i a f o r a t r a n s m i t t e r  v a s o d i l a t a t i o n , by analogy with the  inhibited  They suggested  presence of a d e s t r o y i n g  e r g i c nerve t r a n s m i s s i o n ,  incubation  that t h i s d e s t r u c t i o n was  a d d i t i o n of s t r y c h n i n e  sensory t r a n s m i t t e r  reduced by  the  to c l a r i f y  the problem f u r t h e r Holton  (35)  attempted t o measure the e f f e c t s of s p e c i f i c antagonists  synergists  of a c e t y l c h o l i n e and histamine,  of s t r y c h n i n e  on antidromic  c h o l i n e i t s e l f was  fcund  t a t i o n of l e s s than a minute's d u r a t i o n , l a t i o n produced a d i l a t a t i o n l a s t i n g t i o n of 1Q>^  as w e l l as the e f f e c t  vasodilatation.  measured and was  and  The  e f f e c t of a c e t y l -  to produce a v a s o d i l a while antidromic  stimu-  from 3 t o 4 minutes.  Injec-  histamine produced a v a s o c o n s t r i c t i o n , while  smaller  do3es tended t o give a m i l d d i l a t a t i o n .  Atropine,  which i n h i b i t s  - 10 most p e r i p h e r a l a c t i o n s  of a c e t y l c h o l i n e , was found t o a b o l i s h  the e f f e c t s of i n j e c t e d a c e t y l c h o l i n e but not those of antidromic nerve s t i m u l a t i o n . diminish tiate  E s e r i n e , an a n t i c h o l i n e s t e r a s e , was found t o  the e f f e c t s of antidromic  s t i m u l a t i o n r a t h e r than poten-  them, while the a c t i o n of i n j e c t e d a c e t y l c h o l i n e was en-  hanced.  Mepyramine, an a n t i h i s t a m i n e ,  was found t o a b o l i s h the  e f f e c t s of i n j e c t e d histamine completely but not the e f f e c t s of stimulation. From these f i n d i n g s Holt on suggested that  antidromic  v a s o d i l a t a t i o n i s brought about by the l i b e r a t i o n of a chemical t r a n s m i t t e r but t h e long l a t e n c y and d u r a t i o n as w e l l as t h e pharmacological evidence, make i t u n l i k e l y t h a t the t r a n s m i t t e r i s e i t h e r a c e t y l c h o l i n e or histamine.  Two other  u n l i k e l y that histamine i s the t r a n s m i t t e r ,  f a c t s make i t  (a) histamine does  not disappear from sensory nerves of t h e r a b b i t ' s ear on degenera t i o n and (b) histamine appears t o i n h i b i t sensory neurones  (36).  response t o antidromic  As s t r y c h n i n e  did  d i d not p o t e n t i a t e the  s t i m u l a t i o n i t seems u n l i k e l y that the  t r a n s m i t t e r l i b e r a t e d i s destroyed enzyme.  transmission i n  by a s t r y c h n i n e - s e n s i t i v e  Holton suggested, however, that t h i s l a s t  observation  not exclude the p o s s i b i l i t y t h a t t h e t r a n s m i t t e r  i s i d e n t i c a l w i t h the c e n t r a l s y n a p t i c t r a n s m i t t e r , enzyme r e s p o n s i b l e  substance and that the  f o r i t s d e s t r u c t i o n may be present  i n the  s p i n a l cord but not i n t h e p e r i p h e r a l nerve endings. In another paper Holton  (51) found that b o i l e d s a l i n e  e x t r a c t s of b o t h p o s t e r i o r and a n t e r i o r r o o t s contained  a sub-  stance which causes v a s o d i l a t a t i o n very s i m i l a r t o antidromic v a s o d i l a t a t i o n when i n j e c t e d a r t e r i a l l y  into the rabbit's ear.  -  A vasodilator  effect  acetone d r i e d  powders  difference  i n the  from the  two  was  stable  heat  the  at  pH7  kallikrein  (54),  and  their  and  as  (56)  the  not on  show an  tried  perfused,  increase  of the  chemical  compounds  in  be  acetone  dialysable  (55).  The  roots  in saline.  their  which  might  i s not  anterior  boil-  properties  P is soluble  and  lost  Fresh  a c t i v i t y when mashed  fresh anterior  roots  lost  their  powders.  various  vasodilator  compounds  produce a d i l a t a t i o n r e s e m b l i n g She  chemical  found that  (ATP)  satisfied  s h a r e d by (AMP)  adenosine or  isolated rabbit's  in absorption  this above.  diphosphate  H o l t on  In was  of u l t r a - v i o l e t l i g h t  c h a r a c t e r i s t i c of  a u r i c u l a r nerve.  that  adenosine. ear  purine  She  to  injection  specifications given  a d e n o s i n e monophosphate the  d e s t r o y e d by  necrosin  stimulation.  the  was  to b o i l i n g acid  were shown t o be  2 5 5 - 2 6 5 ^ range,  stimulation  (53),  to r e t a i n  substance  which t h e r e f o r e  when i n c u b a t e d  could  fulfilled  properties  to  has  active  The  no  extracts  The  various  of a d e n o s i n e t r i p h o s p h a t e  and  experiments  out  acetone-dried  from a n t i d r o m i c  (ADP), but  able  labile  saline while  d e t e r m i n e w h e t h e r any  These  alkali.  is resistant  as r a p i d l y  criterion  but  of  a p p e a r e d t o be  s u b s t a n c e when  Thus s u b s t a n c e  were f o u n d  incubated with  solutions  by  a d i l a t a t i o n and  i s heat  H o l t on  of  there  active  dialysable,  enzymically  roots  resulting  and  powders o f b o t h p o s t e r i o r  activity  activity  obtained from e x t r a c t s  substance r u l e  bradykinin  posterior  and  involved.  (52),  acetone d r i e d  of the  a l t h o u g h not  cause  transmitter  be  o f r o o t s were c o m p a r e d .  of t h i s v a s o d i l a t o r upon I n j e c t i o n  also  -  of both r o o t s  content  sets  ing mineral acid  could  11  compounds,  suggested these  in  on results  indicated lation  that  of t h e  ATP  of v a s o d i l a t o r has  areas.  She  found  not  v a r i e d from b r a i n  it  was  extract  nucleus unlikely  although the  to b r a i n ,  showed no  of  these  activity  p a t t e r n of  However, she  chemical  I t was  admits  i n any  substance  maintained  i n v e r s e or d i r e c t  of i n c u b a t e d e x t r a c t s system  content  nervous  contents  and  from  one  other  that the  of  than  results  between t h e  different  the d i s t r i b u t i o n  that  s i n c e the  d i s c r i m i n a t e between s u b s t a n c e s  histamine.  nervous  central  a definite  cuneatus.  clear relationship  central  of the  the  w i t h h i g h e s t c o n c e n t r a t i o n s i n the  to a single  c o u l d not  activity  to determine  a l l the v a s o d i l a t o r a c t i v i t y  due  stimu-  absolute vasodilator  t h e r e was  nucleus  that  and  r e l e a s e d on  compared t h e s e w i t h t h e ATP  that  and  c o u l d be  acetylcholine  the  made an e f f o r t  w i t h i n each b r a i n ,  methods u s e d  dilator  ha3  m a t e r i a l i n v a r i o u s areas  but  caudate  compound was  fibres.  (57)  system,  activity  -  or a s i m i l a r  sensory  Holton  12  vaso-  regions  of  cholinergic  neurones. Holton  thought  dilatation to injection to the dilates the  site the  of action large  of e x t r a c t s  (58).  vessels,  substance result through over.  dilated  the  as t h e the  rapidly  only the  injected  capillaries.  away by  capillaries  would expect  vaso-  as w e l l as  which opening  should g i v e a quick response  swept  dilator  I f the  of the  c o u l d g i v e some i n f o r m a t i o n  capillaries  the b l o o d .  w o u l d have t o  b e f o r e the  response  a d e l a y while the  as  I f the  a prolonged response  substance  whole c a p i l l a r y b e d  A l s o , one  of response  F o r example, a s u b s t a n c e  a r t e r i o - v e n o u s anastomoses,  t h e m a t e r i a l w o u l d be  the  that the type  should  flow  would  substance  be reaches  l a r g e r v e s s e l s were a l r e a d y d i l a t e d  and  - 13 the a r t e r i o - v e n o u s  anastomoses were open before the  response should be  short  lived.  Holton's experiments demonstrated that were the vided  s t i m u l a t i o n was  she  short enough.  give  or not.  S p i n a l root  large  closed  venous anastomoses were open the response was  over  the  that the  at some d i s t a n c e  c a p i l l a r i e s may  from the  t r a c t s acted  p r i m a r i l y on the c a p i l l a r i e s  antidromic stimulation.  The  liberated  i n the  and  substance.  same way  prolonged nature of the  exas does  vasodilata-  quickly  destroyed  blood. Holton has  done v a r i o u s  b i o c h e m i c a l analyses i n an  e f f o r t t o I d e n t i f y more c l o s e l y the vasodilatation  (59).  Extracts  substance causing  absorption  occurred  a c t i v i t y was  a c t i v i t y was  ultraviolet  i n p o s i t i o n s corresponding to ATP  These spots were e l u t e d and  antidromic  of acetone powders of p o s t e r i o r  a n t e r i o r r o o t s were chromatograpbed, intense  of the  be  s p i n a l root  t i o n i n d i c a t e s that the d i l a t o r substance i s not i n the  arterio-  larger vessels  be more s e n s i t i v e t o the  Furthermore, the v a s o d i l a t o r substance of the  i n pre-  quickly.  Holt on concluded that the v a s o d i l a t o r substance may c a p i l l a r y bed  to  arterio-venous  anastomoses, i f the l a r g e r v e s s e l s were d i l a t e d and  i n the  trans-  vessels,  e x t r a c t s , however, were found  w i t h c o n s t r i c t e d v e s s e l s and  pro-  r a b b i t ' s ear v e s s e l s were  a prolonged response as w e l l as a degree of l a t e n c y  parations  and  capillaries  In t e s t i n g p o s s i b l e  found that adenosine, which d i l a t e s the  produced a quick response whether the dilated  the  only v e s s e l s t o respond t o a n t i d r o m i c s t i m u l a t i o n ,  the  mitters  i n j e c t i o n the  and  ADP.  assayed f o r v a s o d i l a t o r a c t i v i t y ,  recovered from the ATP  recovered from the ADP  p o r t i o n and  4 $ of  p o r t i o n w i t h respect  to  16$ the  the  -  original the  extract.  A series  a c t i v e substance  was  enzymic d e t e r m i n a t i o n s Ultraviolet  -  of d e t e r m i n a t i o n s  compared a g a i n s t  of l a b i l e  a b s o r p t i o n was  of the  adenine n u c l e o t i d e  cluded  that the  three  14  phosphate  present  a b l e t o say t h a t t h e  e x t r a c t s was  due  t o a mixture  rabbit's the  ear  a f t e r the ated;  as  the  was  injected duces  amount  account amount  ed  o f ATP  from the  (62) ine  f o r the  lation crepancy  She  o f ATP  and  ADP  and  extracts.  Holton  agreed  as  estimation con-  closely  vasodilator activity  firefly  observed  i n the  small  amounts  l u m i n e s c e n c e method,  and  t h a t ATP  from the of the  not  c o u l d not  present  to give  she  amount  necessary  o f 10^  injected  to e l i c i t In  in recovery  amount to  order,  This  between  ATP re-  be intro-  p r o b l e m , how  t h e maximum amount  l/lOO  of  to  the  t o d u p l i c a t e the v a s o d i l a t a -  stimulation.  of c h o l i n e r g i c nerves. order  of the  amount  the s o u r c e  w h i c h had  only  degener-  t o the  However, t h e  of ATP  into  nerve  i t had  a comparable d i l a t a t i o n .  o f s t i m u l a t i o n and after  sensory  related  which  stimulated  released  after  be  nerve.  large discrepancy  perfusate  was  concluded  most p u z z l i n g a s p e c t s  be  from the  s t i m u l a t i o n of the  liberated  w h i c h must  o f 10^  of the  extracts.  on p e r f u s a t e s  have b e e n a b l e t o r e c o v e r injected  i n the  b e e n s e c t i o n e d but  o n l y 0.16$  of the  effects  f o r ATP,  erythrocytes  arterially  one  the  antidromic  intracellular  covered  tion  on  n e r v e had  of hemolyzed t o be  used  (60,61).  perfusate  as w e l l  products.  has  Is h i g h l y s p e c i f i c  content  determinations  enough t o be  Holton  pure ATP,  a l s o done t o o b t a i n a r o u g h  separate  o f f u r t h e r breakdown  were done i n w h i c h  Brown, D a l e  of the  a response light  of ATP  amount  of i n j e c t e d  and  to  research  ATP  Peldberg  of a c e t y l c h o l -  comparable  of t h i s  recover-  stimua  seems t o o  dislarge  to  i n d i c a t e that  i t i s the  15  -  substance r e s p o n s i b l e  f o r the  vaso-  dilatation. Holton crepancy.  In  stimulation  (61)  perfusion  had  o f the  method  cannot out  ignore  the  closely  mitter  the  vessels,  Since  that  neurones.  and  there  enzymic  ATP  i s present  Thus ATP as  the  t h e y must  i s no  of t h e  out  the  c e n t r a l nervous  n e u r o n e s are there (the  which are  a three  of  substance.  and  that  system.  I t has  for a  enzyme  neurone  i s absent.  and  acetylase power.  first while  It  and the  over des-  compound apparthroughnot a l l that  acetylase  content  Sensory  pathway, t h e  synthesizing  shown t h a t  a c e t y l c h o l i n e , and  acetylcholine)  not  including  such a  choline  so  trans-  T h i s becomes more  been  is  i t is  d i s t r i b u t i o n of a c e t y l c h o l i n e  d e f i c i e n t i n choline  acetylcholine  cells  synthesizing  enzyme w h i c h s y n t h e s i z e s  the b r a i n by  nerve  through-  be  of s y n t h e s i z i n g  In w h i c h the  In m e t a b o l i s m  have some c o n t r o l  capable  the  also  n e u r o n e s must  i s a tendency f o r neurones w i t h h i g h  with c e l l s  high  capable  the  one  in a l l cells,  a transmitter considers  reaching  convenienb  criteria  would  when one  cells  of the n e u r o n e  I t seems u n l i k e l y , t h e r e f o r e ,  ent  of  mentioned,  t r o y i n g ATP. a l s o be  layer before  in a l l  i s present  dis-  collected after  plays  metabolism  f o r the  breakdown.  discrepancy  w o u l d f i t some  Secondly,  metabolism,  that  against  w i t h the  ATP  substance.  ATP  l e a s t one  w e l l known r o l e ATP  associated  explanation  adenosine triphosphatase  a d d i t i o n to the  body.  surprising  their  blood  an  experiments the  o f p r o t e c t i n g ATP In  suggested  t o pass t h r o u g h at  which would c o n t a i n lumen  has  to  alternate  impulses third  reach  of  s e c o n d has  seems r e a s o n a b l e  to  a  -  believe  that  activities  the  synapses  mediator i n the  stated  above, ATP  of t r a n s m i s s i o n  in contrast,  i t is the  suitable blocking out  agents  histamine  f o r which b l o c k i n g  which  indicates that  a large  (63).  Just  are  the  McLennan ATP  so  Basing  i s not  have  the  out  great  nerves to degenerate.  responsible tions the  then the  should  case.  into the  of the  this  the  be  denervated  were  r e a c t i o n t o ATP  When e x t r a c t s area  responses to v e n t r a l  their  a u r i c u l a r nerves  potentiated.  the  i n the  However, t h i s  of p o s t e r i o r the  as  is  because  its effects  substance. the  and  and  area  of r a b b i t s  not  were  solutions  allowed  prepara-  found  very  cut  substance  denervated was  substance.  and  transmitter  It i s  formerly  McLennan  spinal roots  extract  and  transmitter  v a s o d i l a t a t i o n was  roots  still  p r o d u c e d some e v i d e n c e  phenomenon, P l o r e y  I f ATP  distri-  known.  transmitter  them becomes s e n s i t i z e d t o  t h e i r work on  segments the  by  exist,  e f f e c t s of a c e t y l c h o l i n e  known t h a t when s e n s o r y f i b r e s have d e g e n e r a t e d supplied  of  such a  i n question  known f o r ATP  (64)  prob-  cells.  substance  agents are  non-  is  number  b e e n shown t o  i n a l l body  are  ruled  and  acetylcholine  does c a u s e v a s o d i l a t a t i o n , t h e r e  c a n n o t be  Plorey  across  not  transmitter  as  that  system  has  i s present  A l t h o u g h ATP  no  a l t e r a t i o n o f c h o l i n e r g i c and  c e n t r a l nervous  o f ATP,  that  an  elements which suggests  bution  doubt  -  a l t e r a t i o n o f n e u r o n e s w i t h d i f f e r e n t enzyme  corresponds to  cholinergic ably  this  16  to  be  injected  marked o f ATP  while were  decreased. The is  responsible  assumes g r e a t  question  which n a t u r a l l y a r i s e s  for antidromic  vasodilatation?  i m p o r t a n c e when one  considers  i s what This  Dale's  substance question  (48)  remark  - 17 that  since  the b i p o l a r a f f e r e n t neurones i n v o l v e d  i n cutaneous  f l a r e do not synapse u n t i l they are w i t h i n  the c e n t r a l nervous  system, the v a s o d i l a t o r  i n the p e r i p h e r y dur-  substance r e l e a s e d  ing a n t i d r o m i c v a s o d i l a t a t i o n may a l s o be the t r a n s m i t t e r c e n t r a l synapse of the a f f e r e n t  neurones, which would  at the  further  suggest that sensory neurones only and not motor neurones, would contain  the humoral agent.  Peldberg  same idea when he says that  (65) a l s o expresses the  d i f f e r e n t branches of the same axon  are u n l i k e l y t o have d i f f e r e n t t r a n s m i t t e r  substances.  In t h i s  case any Information made a v a i l a b l e on the substance  responsible  for antidromic v a s o d i l a t a t i o n  a valuable  insight  i n t o the f u n c t i o n  could  a l s o perhaps give  of the c e n t r a l nervous system  It has been proposed that s i b l e f o r antidromic v a s o d i l a t a t i o n produced t o support t h i s s u g g e s t i o n .  itself.  A T P i s the substance respon(56) and evidence has been However, by the same token,  evidence has a l s o come t o l i g h t which appears t o c o n t r a d i c t  this  p r o p o s a l (61,64), consequently the problem remains unsolved. S p e c i f i c a l l y i t was i n an e f f o r t t o r e s o l v e contradictions  these apparent  and to determine j u s t what r o l e ATP had, i f any,  in antidromic v a s o d i l a t a t i o n that the following undertaken.  research  was  - 18 II.  MATERIALS AND 1.  The  -  METHODS:  D i s t r i b u t i o n of " v a s o d i l a t o r "  a c t i v i t y In  the  C e n t r a l Nervous System. (a)  Method f o r P r e p a r i n g D l a l y s a t e s  Beef b r a i n t i s s u e was  of  Brain  obtained from f r e s h l y k i l l e d  c a t t l e , known weights of t i s s u e were taken from the cerebral  areas  of  c o r t e x , caudate nucleaus, thalamus, s u b - c o r t i c a l white  matter, r e t i c u l a r formation, f l o o r of the cerebellar  fourth v e n t r i c l e ,  cortex, o p t i c chiasma, l e n t i f o r m nucleus,  cuneatus et g r a c i l i s ,  superior  corpora quadrigemina,  corpora quadrigemina, d o r s a l r o o t s , columns of s p i n a l cord, and As  nuclei  ventral roots,  deep c e r e b e l l a r  q u i c k l y as p o s s i b l e  the  inferior  dorsal  structures.  f r e s h t i s s u e was  boiled  f o r f i v e minutes i n a volume of d i s t i l l e d water e q u a l t o times the  weight of the  order t o d e s t r o y any The  boiled material  fresh tissue.  T h i s was  necessary i n  i n a c t i v a t i n g enzymes present i n the was  then homogenized by g r i n d i n g  involved.  The  t u b i n g and  d i a l y z e d against  The  homogenate was  stored  the  redissolved  in physiological  the  5°C,  volume of the f r e s h t i s s u e . u n t i l used.  Certain  s a l i n e which These e x t r a c t s  extracts  were t r e a t e d  remove the m a j o r i t y of potassium present, and adding one-tenth the out  dialysis  d i a l y s a t e s were evaporated t o dryness i n a f l a s h  at -5°C.  filtering  volume  d i s t i l l e d water twenty times  homogenate f o r f o r t y - e i g h t hours at  evaporator and one-third  placed i n cellophane  tissue.  i n a mortar  w i t h f i n e sand or i n a Waring blendor, depending on the  volume of the  two  the  extract  resultant  volume of 11.6 p r e c i p i t a t e , and  t h i s was  N.  was were to  done by  perchloric  neutralizing  acid, to  - 19 pH7 by adding 1 N. sodium hydroxide. stored  These s o l u t i o n s were  at -5°C. a l s o . (b) The  Method f o r P r e p a r i n g the Rabbit E a r vasodilator  a c t i v i t y of t h e e x t r a c t s was  mined by i n j e c t i n g 0.125 c c . of the m a t e r i a l the  deter-  t o be t e s t e d  into  r a b b i t ' s e a r and measuring the amount of v a s o d i l a t a t i o n  produced.in the e a r . The r a b b i t was f i r s t nembutal (50 mg./ml.).  anaesthetized with  A t r a c h e a l cannula was i n s e r t e d  t o ensure adequate v e n t i l a t i o n , a cannula was then i n t o the femoral v e i n o f one o f the hind t h e t i c could be g i v e n i f necessary.  inserted  l e g s so more anaes-  A t h i r d cannula  (a small  p o l y e t h y l e n e tube) was i n s e r t e d i n t o the f a c i a l a r t e r y , ing towards the h e a r t , ear t o be t e s t e d .  on the same side  first  point-  of the r a b b i t as the  A l l the major branches o f the c a r o t i d a r t e r y  i n c l u d i n g the i n t e r n a l c a r o t i d a r t e r y were t i e d e x c e p t i o n of the a u r i c u l a r a r t e r y . to the ear of the I n j e c t e d  extract.  This  o f f , w i t h the  allowed f r e e passage  On i n j e c t i o n the e x t r a c t  was f o r c e d t o flow backward.down the f a c i a l a r t e r y i n t o the external lar  a r t e r y by the b l o o d flow and thereby c a r r i e d t o the ear  itself. the  c a r o t i d a r t e r y where i t was then swept i n t o the a u r i c u -  To avoid  interference  from extraneous nerve  a u r i c u l a r nerve of the ear t o be t e s t e d was  Impulses  sectioned  b e f o r e the experiments began. The ( l mg./Kg.), (10 mg./Kg.),  r a b b i t was a l s o given an i n j e c t i o n of a t r o p i n e a blocking a blocking  agent f o r a c e t y l c h o l i n e ,  and b e n a d r y l  agent f o r h i s t a m i n e , b e f o r e experimen-  t a t i o n i n order t o r u l e out any e f f e c t s of a c e t y l c h o l i n e and histamine.  - 20  -  Key A. B. C. D. E. F. G. H. I. J.  Figure  2.  P o l y e t h y l e n e Cannula Auricular Artery F a c i a l A r t e r y Cannulated Superior T h y r o i d A r t e r y T i e d Off D i r e c t i o n of I n j e c t i o n Flow I n t e r n a l C a r o t i d T i e d Off D i r e c t i o n of Blood Flow Common C a r o t i d T r a c h e a l Cannula Trachea  Cannulation f o r Rabbit's Ear  Preparation  - 21 (c)  Method f o r Measuring the Amount of V a s o d i l a t a t i o n  V a s o d i l a t a t i o n i n the r a b b i t ' s e a r was rophotometrically; more t r a n s l u c e n t a green f i l t e r  the white r a b b i t ' s ear was  measured e l e c t  shaved and made A light  by s p r e a d i n g p a r a f f i n o i l over " i t .  over i t was  shone through the ear, which  taped t o a p l a s t i c box; beneath the ear was the box which allowed the l i g h t onto a p h o t o e l e c t r i c c e l l .  with  was  a c l e a r window i n  p a s s i n g through the ear t o  fall  Changes i n the i n t e n s i t y of l i g h t  passing through the e a r were d e t e c t e d as changes i n i n t e n s i t y of a current  produced by the p h o t o c e l l .  These  changes were  a m p l i f i e d and r e c o r d e d on a moving s t r i p of f i l m which  passed  In f r o n t of an o s c i l l o s c o p e s c r e e n ,  therefore,  A vasodilatation,  was  shown as a curve r i s i n g from a predetermined base l i n e  the  f i l m s t r i p and s i m i l a r l y ,  a f a l l below t h i s base  line  a v a s o c o n s t r i c t i o n was  (see F i g u r e 6.).  j e c t i o n of e x t r a c t , a photograph  on  shown as  Before each i n -  of the s t a b i l i z e d base  line  was taken t o enable any change i n base l i n e t o be measured.  Green F i l t e r  Rabbit Ear Clear  Window  Light-tight  Box  To A m p l i f i e r and Oscilloscope  F i g u r e 3.  System f o r Measuring V a s o d i l a t a t i o n  i n Rabbit's Ear  - 22 2.  Distribution  -  of Adenosine Triphosphate i n the  Central  Nervous System. (a)  Methods f o r Determining; ATP  in Central  Nervous  System (i)  Phosphomolybdic a c i d method (66).  od i s based on the  solubility  in isobutyl alcohol.  of r e d u c i b l e  the  a l c o h o l i c extract  stannous  l y s a t e s was  blue reduced form by  amount of i n o r g a n i c  determined f i r s t , then the  i n hot  1 N.  dialysates  of  measurements was  the  acid l a b i l e  assumed t o be  taken t o be  phate content was  water, 2.5  0.5  ml.  of the put  ml.  dialysate  between the  the  two  phosphate which  of 10 N.  of an 85%  the  to be  was  sulphuric  solution  p o r t i o n s of 5 ml.  stannous c h l o r i d e / ml. been d i l u t e d two  blue s o l u t i o n was  the  distilled  The  alcoholic  of a s o l u t i o n  solution  gm.  a c i d which  sulphuric  aqueous l a y e r d i s c a r d e d .  two  sulphuric  of 0.4  concentrated h y d r o c h l o r i c  poured i n t o a 10 ml.  and  shaken f o r  each of 1 N.  hundred times w i t h 1 N.  t h i r t y seconds, and  following  of ammonium molybdate,  This mixture was  then shaken with 15 ml.  the  a c i d , 2 ml.  aqueous l a y e r d i s c a r d e d .  washed w i t h two  determined f o r phos-  i n t o a s e p a r a t o r y f u n n e l and  of i s o b u t y l a l c o h o l .  minutes and  were hydro-  adenosine t r i p h o s p h a t e .  F i v e ml.  were added:  dia-  h y d r o c h l o r i d e a c i d f o r seven minutes and difference  a c i d and  shaking  phosphate present i n the  The  was  colour-  w i t h an a c i d i f i e d aqueous s o l u t i o n  phosphate again determined.  10 ml,  r e d u c t i o n of  chloride. The  lyzed  phosphomolybdic a c i d  I t i s e s s e n t i a l l y the  l e s s phosphomolybdic a c i d t o the  T h i s meth-  acid The  had  for  resulting  volumetric f l a s k ,  the  - 23 separately  funnel washed w i t h e t h y l a l c o h o l and  made up t o 10 ml. w i t h the washings. in a Klett  colorimeter  and  the  d e t e r m i n i n g ATP and  Totter  firefly  i t was  s o l u t i o n s were  luminescence enzyme method f o r  that  light  CNS.  Strehler  e m i s s i o n by  living  a product of a s e r i e s of chemical r e a c t i o n s  p o s s i b l e under c e r t a i n c o n d i t i o n s  to extract  luminous organs a heat s t a b l e substance, l u c i f e r i n , labile  read  prepared w i t h each experiment.  i n d i a l y s a t e s of areas of the  (68,69) have r e p o r t e d  organisms was that  The  solution  amount of phosphate determined  from a standard curve which was (ii)  The  the  and  and from  a heat  substance, l u c i f e r a s e , which when mixed t o g e t h e r would  emit l i g h t . of the  The  light  emitting  l u c i f e r i n molecule i n the  enzyme, l u c i f e r a s e . Cypridlna labile  luciferin  I t was  step depended upon the presence of oxygen and  shown t h a t a p a r t i a l l y  (from a s p e c i e s  r e a c t i o n and  i t was  from the breakdown of the  postulated  s i d e c h a i n was  that the  the  purified  of luminous f i s h )  phosphate groups which were removed d u r i n g  emitting  oxidation  the  contained light  energy  conserved as  derived  phosphate  energy bonds. Support f o r t h i s s u g g e s t i o n that groups are  concerned i n the  with extracts light ing  and  from f i r e f l y m a t e r i a l .  e x t r a c t s which had production  enon f o r the  I t was  obtained  possible to  restore  ceased t o luminesce by  add-  a d i v a l e n t i o n Mg+-r.  Implicit  the  phosphate  luminescent r e a c t i o n was  emission i n e x t r a c t s which had  ATP  labile  i n the  f i n d i n g that f i r e f l y  luminous organ  become dark would respond to added ATP  of l i g h t  was  assay of ATP  the  possibility  of u s i n g  i n l i v i n g m a t e r i a l under  by  t h i s phenom  various  - 24(a) -  j  Standard Curve of  3000  Luciferine - Luciferote Enzyme Method  n  ^jg / m l  Adenosine Triphosphate  conditions.  T h i s has  standard  highly refined  be  and  determined  i n g use , t o an  t o be  by H o l t o n .  and  enzyme m i x t u r e  seconds  M.  of d r i e d  other  liquid. was  the  For  placed  of the The  unknown light  firefly  i n a tube,  was  o f ATP  side facing  opposite  the  was  pH  7.4,  1.0  ml.  measured.  were r u n w i t h  e m i t t e d by  the  on  the  the mixture was 1.0  and  was  added ml.  to  of  this was  exactly  15  Standard  solutions  each set  of  dilu-  luminescent  reaction  was  scintillation  counter  from  A hole  thickness  i n , was  attached  r e p l a c e d by the  size  in a light-proof  a  t o the  clear  of the r e a c t i o n  of t h e window and  r e f l e c t e d back i n t o the  enclosed  added  of d i a l y s a t e  a mirror,  window on t h e  p h o t o m u l t i p l l e r t u b e s o t h a t most  e m i t t e d w o u l d be  ATP  dialysates.  into the  silvered  and  based  a stopwatch s t a r t e d ,  window o f p e r s p e x •§• i n c h t h i c k . drilled  can  l a n t e r n s were e x t r a c t e d  p h o s p h o r had b e e n removed and  was  ATP  T o t t e r w i t h seme m o d i f i c a t i o n s  m e a s u r e d by means o f a c o m m e r c i a l which the  output  each d e t e r m i n a t i o n  luminescence  o f known c o n c e n t r a t i o n s  both  compounds b y mak-  r e s e a r c h was  s r s e n a t e b u f f e r at  s o l u t i o n mixed,  later  using  20 mg./ml. of magnesium s u l p h a t e  supernatant  device  with  in this  S t r e h l e r and  mg.  o f 0.1  centrifuged  added, t h e  by  case  lanterns.  method u s e d  Fifty  5 ml.  tubes  the  light-measuring devices.  i n mixture  e x t r a c t of f i r e f l y  tions  -  of a l i n e a r r e l a t i o n between l i g h t  method d e v e l o p e d  the  been found  directly  The  with  24  of the  counter.  enclosure.  The  side  light whole  -  Light  25 -  Tight Box  Electronic Counter  P h o t o m n l t l n i 1ftr Tube C l e a r Window w i t h Hole Mirror  Figure 3.  5. ' S y s t e m f o r M e a s u r i n g  amounts  papers  Firefly  b a s i c t e c h n i q u e was t h e same i n a l l c a s e s ; v a r y -  o f ATP were a p p l i e d t o t h e p a p e r s  were d e v e l o p e d  the d i f f e r e n c e s b e i n g detecting  i n t h e s o l v e n t s used  papers  solvent n. for  full  5, 10, 20 and 30 lambda  size  sheets  run technique.  amounts  butanol,  The s e c o n d  picric  (0.005, 0.010,  o f ATP were s p o t t e d  u s i n g t h e two d i m e n s i o n a l  The f i r s t  dimension  p r o p a n o l , ammonium h y d r o x i d e , w a t e r 8 hours.  and t h e methods o f  o f Whatman #4 c h r o m a t o g r a p h y  were d e v e l o p e d  dimension  a c i d , water The p a p e r s  solvent  solvent  were t h e n s p r a y e d w i t h  ing  s o l u t i o n which c o n t a i n e d 5 m l . o f 60$ p e r c h l o r i c  for  15 m i n u t e s  solution,  and 60 m l . o f w a t e r . at 85°C.  system  was  tert:  20 m l . ) w h i c h was  f o r 5 hours.  acid,  descending  s y s t e m was  run  chloric  paper.  (60:30:10) a n d was r u n  (80 m l . , 4 gm.,  25 m l . o f 4 $ ammonium m o l y b d a t e  method,  chromatograms.  0.020, 0.030 ml.) o f a 1 mg./ml. s o l u t i o n on f o u r  and g e n e r a l l y t h e  b y t h e two d i m e n s i o n a l d e s c e n d i n g  t h e ATP i n t h e d e v e l o p e d (a)  The  Luminescence  Chromatography. The  ing  f o r R e a c t i o n Tubes  a  colourizacid,  10 m l . o f 1 N. h y d r o -  The p a p e r s  were t h e n  heated  - 26 (b)  25, 50, 75 and 100 lambda amounts of 1 mg./ml.  s o l u t i o n of ATP were s p o t t e d on sheets of Whatman #4 chromatogram paper as i n (a), the solvent the  systems  and techniques were  same as i n (a) except the second dimension s o l v e n t  was allowed t o run f o r 20 hours i n s t e a d of 5.  system  The c o l o u r i z i n g  s o l u t i o n used was the same as (a) except the sprayed papers were heated a f t e r b e i n g allowed t o d r y completely f i r s t . (c) of  20, 50, 75 and 100 lambdas of 1 mg./ml. s o l u t i o n  ATP were s p o t t e d on Whatman #4 papers, and the papers were  developed as above.  The c o l o u r i z i n g s o l u t i o n used t h i s  contained 1 gm. of ammonium molybdate of  time  In 8 ml. of water, 3 ml.  concentrated h y d r o c h l o r i c a c i d , 3 ml. o f 70$ p e r c h l o r i c  a c i d , and t h i s mixture then d i l u t e d t o 100 ml. w i t h acetone. The developed papers were dipped i n t h i s s o l u t i o n , allowed t o dry, out  and exposed t o u l t r a v i o l e t  l i g h t f o r 30 minutes t o b r i n g  the c o l o u r . (d)  equivalent  Four papers of Whatman #4 grade, each having the  of 100 mg. of ATP s p o t t e d on them, were r u n i n two  dimensions u s i n g t h e descending s o l v e n t run t e c h n i q u e .  The two  s o l v e n t systems were n-propanol, ammonium hydroxide, water (60:30:10) and i s o p r o p a n o l , s a t u r a t e d ammonium sulphate s o l u t i o n , and water the  (2:79:19); the f i r s t  second f o r 4-g- hours  (70) .  system was r u n f o r 18 hours,  The same c o l o u r i z i n g s o l u t i o n was  used as above. (e) of  a water  Four papers were s p o t t e d with 500 lambdas (0.5 ml.)  s o l u t i o n of acetone powders made from t h e caudate  nucleus, 50 mg. of ATP were a l s o s p o t t e d t o serve as a marker  - 27 compound; a second 50 mg. s o l v e n t system was  p o r t i o n was  first  run, and t h i s served as a marker compound  d u r i n g the second s o l v e n t run. first  a l s o put on a f t e r the  with u l t r a v i o l e t  The developed papers were  l i g h t and then dipped i n the  scanned  colourizing  s o l u t i o n used i n ( c ) . (f)  F i v e hundred  lambdas  (0.5 ml.)  of the  dialysate  from the thalamus  r e g i o n was  50 mg.  a l s o s p o t t e d as a marker compound.  of ATP was  spotted on each of 4 papers, The  and chrom-  atograms were developed and the spots c o i n c i d i n g w i t h the marker spots were e l u t e d w i t h d i s t i l l e d water and analyzed f o r a c i d l a b i l e phosphate and  as mentioned  above by the method of Berenblum  Chain. (g)  One hundred  lambda amounts (0.1 ml.)  of the d i a -  l y s a t e s of the caudate nucleus and the c e r e b r a l c o r t e x were s p o t t e d on 3 MM.  chromatogram paper, the papers were developed  by the descending technique i n n-propanol, ammonium hydroxide, water (60:30:10) f o r 16 hours.  The  chromatogram from o r i g i n t o solvent s e c t i o n s , each of these s e c t i o n s was  l e n g t h of the f r o n t was  developed  d i v i d e d i n t o ten  eluted with d i s t i l l e d  evaporated t o dryness, and r e d i s s o l v e d i n p h y s i o l o g i c a l  saline  of a volume approximately equal t o the o r i g i n a l d i a l y s a t e volume.  These samples,  4.  sample  r e p r e s e n t i n g the ten areas on the chrom-  atogram, were then t e s t e d on the r a b b i t above f o r v a s o d i l a t o r  water,  ear p r e p a r a t i o n d e s c r i b e d  activity,  Chemical p r o p e r t i e s of the v a s o d i l a t o r substance and (a)  Acid-base  One m i l l i l i t e r ate nucleus was  ATP,  stability.p o r t i o n of the d i a l y s a t e from the  caud-  b o i l e d with an equal volume of 2 N. h y d r o c h l o r i c  - 28 a c i d and n e u t r a l i z e d  on c o o l i n g , another p o r t i o n was b o i l e d  w i t h an equal amount of 1 N. sodium hydroxide and a l s o i z e d on c o o l i n g ,  a t h i r d p o r t i o n was b o i l e d f o r the same time  but no a c i d or base was added t o i t . followed area.  neutral-  with portions  The same procedure was  of the d i a l y s a t e from t h e c r e b r a l  These samples were then t e s t e d  tion for vasodilator a c t i v i t y .  cortex  on the r a b b i t ear prepara-  The same t h i n g was a l s o done  with a 1 mg./ml. s o l u t i o n of ATP. (b)  D i a l y s i s of the v a s o d i l a t o r  Experiments  substance and ATP  were done t o t r y and determine  how much  ATP was d i a l y s e d by the method used f o r d i a l y z i n g the areas o f brain f o r vasodilator  material.  Five m i l l i l i t e r s placed  of a 1 mg./ml. s o l u t i o n of ATP were  In a d i a l y s i s bag and d i a l y z e d against  i n t h e c o l d f o r 48 hours.  distilled  water  At the end o f t h i s time the d i a l y s a t e  and the contents of the bag were both a n a l y z e d f o r ATP content by the f i r e f l y luminescence (c)  Treatment  enzyme method mentioned  above.  of d i a l y s a t e s w i t h exchange r e s i n s  Two a c t i v e d i a l y s a t e s were t r e a t e d with i o n exchange r e s i n s i n an e f f o r t  t o determine  of the substance r e s p o n s i b l e  some b a s i c  chemical  properties  for vasodilatation.  Two m i l l i l i t e r p o r t i o n s  of the d i a l y s a t e s  caudate nucleus and the c e r e b r a l c o r t e x  from the  were t r e a t e d w i t h por-  t i o n s of Dowex-50 exchange r e s i n , separate p o r t i o n s were s i m i l a r l y t r e a t e d with Dowex-1 exchange r e s i n .  A f t e r f i l t e r i n g the  mixtures t o separate out the r e s i n s , the d i a l y s a t e s were then tested  on the r a b b i t  ear p r e p a r a t i o n  f o r vasodilator  activity.  - 29 Further. 1 m i l l i l i t e r p o r t i o n s of the same d i a l y s a t e s were t r e a t e d w i t h r e s i n IR-45, a weak anion exchanger, and IRC-50, a weak c a t i o n exchanger.  Before treatment both  were washed thoroughly; IR-45 i n 1 N. h y d r o c h l o r i c t a i n the c h l o r i d e form,  and then twice i n d i s t i l l e d  resins  a c i d t o obwater,  IRC-50 with 1 N. sodium hydroxide t o o b t s i n the hydroxide and then twice i n d i s t i l l e d  form,  water.  A f t e r shaking the d i a l y s a t e w i t h the r e s i n s , the liquid  was saved.  The remaining r e s i n IR-45, was e l u t e d  with  1 N. and 3 N. ammonium hydroxide, the e l u a t e was evaporated t o dryness and r e d i s s o l v e d  i n p h y s i o l o g i c a l s a l i n e and n e u t r a l i z e d .  S i m i l a r l y , IRC-50 was e l u t e d with 3 N. a c e t i c a c i d , t h i s e l u a t e being a l s o evaporated t o dryness and r e d i s s o l v e d s a l i n e and n e u t r a l i z e d . vasodilator  activity.  i n physiological  These samples were a l l a l s o t e s t e d f o r  - 30 III.  RESULTS: Holton (57) has t e s t e d various  areas of the c e n t r a l  nervous system f o r v a s o d i l a t o r a c t i v i t y and has r e p o r t e d a d e f i n i t e pattern  that  of d i s t r i b u t i o n e x i s t e d , the most a c t i v i t y  being found i n caudate nucleus and n u c l e a r  cuneatus.  The  f o l l o w i n g groups of experiments were undertaken i n an e f f o r t t o determine the d i s t r i b u t i o n o f the v a s o d i l a t o r a c t i v i t y and t o see i f the d i s t r i b u t i o n p a r a l l e l e d that Also,  i t was f e l t  that  claimed by H o l t o n .  i f ATP were the t r a n s m i t t e r  or r e l a t e d t o the substance i n q u e s t i o n ,  substance  the d i s t r i b u t i o n o f  ATP i n t h e c e n t r a l nervous system should be the same as, or c l o s e l y s i m i l a r t o , the d i s t r i b u t i o n of v a s o d i l a t o r therefore  t h e d i s t r i b u t i o n and c o n c e n t r a t i o n  activity,  of ATP i n the b r a i n  was a l s o determined. 1.  The d i s t r i b u t i o n of v a s o d i l a t o r a c t i v i t y i n the c e n t r a l nervous system Table I shows the r e l a t i v e amounts of the substance  responsible  f o r producing v a s o d i l a t a t i o n i n t h e r a b b i t ' s ear  found i n the areas of the c e n t r a l nervous system mentioned.  The  degree of v a s o d i l a t a t i o n produced by d i a l y s a t e s of the areas are graded from the s t r o n g e s t  e f f e c t t o t h e weakest by a number of  +'s; ++++being the s t r o n g e s t  e f f e c t and + being the weakest.  Where no v a s o d i l a t a t i o n was obtained an 0 i s i n d i c a t e d . Holton found caudate nucleus and nucleus cuneatus t o contain  the most v a s o d i l a t o r a c t i v i t y f o l l o w e d  by thalamus and  hypothalamus.  The r e s u l t s here agree g e n e r a l l y with those of  Holton i n t h a t  caudate nucleus contains  vasodilator material,  the g r e a t e s t  amount of  while the thalamus a l s o contains  much  - 31 TABLE I :  DISTRIBUTION OF VASODILATOR ACTIVITY IN EXTRACTS FROM VARIOUS AREAS OF THE BRAIN OF THE COW  Area of CNS. Tested Inferior  -  colliculus  IVth v e n t r i c l e f l o o r L e n t i c u l a r nucleus Thalamus  (mixed)  (cellular)  (cellular)  Caudate nucleus  (cellular)  R e t i c u l a r f o r m a t i o n (mixed) Cerebral cortex (precentral areas) Superior  colliculus  S u b c o r t i c a l white Hypothalamus  E x p t . 1 Expt. 2 Expt. 3 Expt.  matter  0 -++- + -n-  ++  0 +++  0  0  -  ++  -  •++-»-  +  -f-n-  -  0  0  +-  -t-+  0  -  0  0  -t--t-+-t-  -  0  -  Optic chiasma  0  -  Deep c e r e b e l l u m (mixed)  0  0  Dorsal roots  -  -  Ventral roots  -  -  +++  -f-+"t -i  -  N u c l e i c cuneatus et g r a c i l i o (cellular)  (white)  -  + ++  ++  P o s t e r i o r columns  -  -  A n t e r i o r r o o t s (white)  (white)  ++-++  -hi-  +++  (cellular)  Cerebellar cortex ( c e l l u l a r )  -  -n-t-  -  -  + "t-+ mt  - 32 activity. has  agreement ends.  Holton claims hypothalamus  much a c t i v i t y while these r e s u l t s i n d i c a t e i t has  ity;  these r e s u l t s a l s o show that  great the  There the  deal  same 2.  of a c t i v i t y but  c e r e b r a l cortex  Holton r e p o r t e d  little  no  activ-  contains a activity in  region. D i s t r i b u t i o n of adenosine t r i p h o s p h a t e i n the  central  nervous system An e f f o r t was  made t o d i s c o v e r  the d i s t r i b u t i o n of v a s o d i l a t o r of  ATP  i n the  a c o r r e l a t i o n between  a c t i v i t y and  c e n t r a l nervous system.  the d i s t r i b u t i o n  I f ATP  were i n some  way  connected w i t h a n t i d r o m i c v a s o d i l a t a t i o n , the d i s t r i b u t i o n of vasodilator  a c t i v i t y should be  The  method employed was  method which i s h i g h l y methods and  s i m i l a r to that  materials  the  The  ATP.  f i r e f l y luminescence enzyme  s p e c i f i c f o r ATP above.  of  and was  described  i n the  d e t e r m i n a t i o n s were c a r r i e d  out  on water d i a l y s a t e s as w e l l as acetone powders of areas  the  c e n t r a l nervous system In order t o determine whether  methods of i s o l a t i o n might be a f f e c t i n g the  of  the  amount of ATP  recov-  ered. Table II shows the  amounts of ATP  of f r e s h b r a i n t i s s u e i n water d i a l y s a t e s powders from the The ricle  water d i a l y s a t e  dilator  The  activity  which one  as w e l l as  in^g./gm. acetone  indicated.  contained the most ATP,  colliculus.  ATP  areas  expressed  of the  f l o o r of the  followed  f o u r t h vent-  by i n f e r i o r and  superior  caudate nucleus which contained the most vaso(see Table I) d i d not  would expect c o n s i s t e n t  contain  the  amounts of  with i t s v a s o d i l a t o r  - 33 TABLE 2:  ADENOSINE TRIPHOSPHATE CONTENT OP AREAS OP COW BRAIN EXPRESSED I N ^ g . / g m .  Area of B r a i n Tested  IVth v e n t r i c l e  floor  W  a  *  FRESH BRAIN e  r  X  Dialysate  Acetone 1  Cf  25.3  76.0  0  Powder (  0  0  Inferior  colliculus  20.7  62.0  45.0  18.0  Superior  colliculus  11.2  33.6  35.2  26.4  nucleus  4.1  12.2  26.7  20.0  cortex  3.7  11.0  24.6  24.6  Thalamus  3.3  10.0  80.0  60.0  S u b c o r t i c a l white matter  2.5  7.4  48.0  48.0  Lenticular  nucleus  2.0  6.0  39.5  29.6  Cerebellar  cortex  1.7  5.0  16.0  24.6  1.0  3.0  0  ' 0  0  0  0  ! 0  Caudate Cerebral  Reticular  formation  Hypothalamus  - 34 a c t i v i t y i f ATP were the t r a n s m i t t e r  substance.  The same can  be s a i d f o r c e r e b r a l cortex,thalamus, and c e r e b e l l a r cortex, the areas which a l s o contained The  l a r g e amounts of v a s o d i l a t o r  activity.  acetone powder of caudate nucleus a l s o d i d not  c o n t a i n t h e amounts of ATP c o n s i s t e n t with i t s v a s o d i l a t o r a c t i v i t y i f ATP were t h e t r a n s m i t t e r  substance concerned.  acetone powder of the thalamus contained  the g r e a t e s t  ATP but none o f the o t h e r areas which had v a s o d i l a t o r contained and  l a r g e amounts of ATP.  The  amount of activity  Caudate nucleus, c e r e b r a l  c e r e b e l l a r cortex a l l contained  cortex,  r e l a t i s e l y small amounts of  ATP by comparison w i t h the other areas t e s t e d . It i s i n t e r e s t i n g t o note that the amounts of ATP i n the  acetone powders were a l l c o n s i d e r a b l y  l a r g e r than the amounts  found i n t h e water d i a l y s a t e s and a l s o t h a t t h e order of decreasi n g amounts of ATP i s not the same i n both c a s e s .  This  seems t o  i n d i c a t e t h a t the d i a l y s i s performed was not s u f f i c i e n t t o extract  a l l the ATP present i n the b r a i n homogenate i n s i d e the  d i a l y s i s tubing.  However, the d i a l y s i s was s u f f i c i e n t  t r a c t the v a s o d i l a t o r a c t i v i t y p r e s e n t .  t o ex-  This would tend t o  i n d i c a t e that ATP and the v a s o d i l a t o r substance are two d i f f e r e n t compounds. 3t  A comparison o f v a s o d i l a t o r a c t i v i t y with triphosphate  adenosine  content of Ine c e n t r a l nervous system.  It was thought that the d i s t r i b u t i o n o f v a s o d i l a t o r a c t i v i t y could be best the  compared with t h e d i s t r i b u t i o n of ATP i n  c e n t r a l nervous system i n t a b u l a r form.  c e n t r a l nervous system are l i s t e d  The areas of the  i n order of d e c r e a s i n g  content  -  35  of the a p p r o p r i a t e substances Table 3 shows that  -  indicated. caudate n u c l e u s , c e r e b r a l c o r t e x ,  and c e r e b e l l a r c o r t e x were the areas where the g r e a t e s t of v a s o d i l a t o r  a c t i v i t y was  never found t o c o n t a i n is l i t t l e  c o n s i s t e n t l y found, but they were  the most ATP.  I t i s apparent  that t h e r e  c o r r e l a t i o n , d i r e c t or Inverse, between the ATP  of the water d i a l y s a t e s and acetone substance 4.  amount  powders and the v a s o d i l a t o r  content of areas i n the c e n t r a l nervous A comparison  of the chemical p r o p e r t i e s  d i l a t o r a c t i v i t y and It was  hoped that  nature of the v a s o d i l a t o r  content  system. of the  vaso-  ATP.  some i n d i c a t i o n of the  chemical  substance as compared t o ATP  would  a i d i n d e t e r m i n i n g i f any chemical r e l a t i o n s h i p e x i s t e d between the two was  compounds.  tested  The e f f e c t of s e v e r a l  i o n exchange r e s i n s  i n order t o see i f the compounds had molecules  were charged and I f so, whether they were anions or  which  cations.  The s t a b i l i t y of the v a s o d i l a t o r a c t i v i t y t o a c i d , a l k a l i , heat was  a l s o determined  these agents.  Another  and compared t o the s t a b i l i t y of ATP  simple experiment  was  The homogenates of b r a i n were d i a l y z e d against  agent.  d i s t i l l e d water  f o r t e s t i n g on the r a b b i t ear, so s o l u -  t i o n s of ATP were a l s o d i a l y z e d enough ATP  to  done which gave a  good i n d i c a t i o n of the m o l e c u l a r s i z e of the v a s o d i l a t o r  t o prepare the e x t r a c t s  and  i n the same manner t o see i f  passed through the membrane and would then i n d i c a t e  i t s molecule was  of a s i m i l a r s i z e .  In these experiments nucleus and c e r e b r a l where v a s o d i l a t o r  only water d i a l y s a t e s  of  caudate  c o r t e x were used as these were the two  a c t i v i t y was  consistently  found.  areas  - 36 TABLE 3t  COMPARISON  OP THE AMOUNTS OF ATP AND VASODILATOR SUBSTANCE  Water D i a l y s a t e s of Areas of CNS Cont a i n i n g ATP  Areas o f CNS Containing Vasodilator Substance  Acetone Powders of Areas of CNS Cont a i n i n g ATP  IVth v e n t r i c l e  caudate nucleus  thalamus  floor  inferior  colliculus  cerebral cortex  subcortical matter  superior  colliculus  cerebellar  inferior  caudate nucleus  thalamus  cerebral  lenticular  cortex  thalamus  cortex  colliculus  lenticular nucleus  superior  white  nucleus colliculus  r e t i c u l a r formation  caudate nucleus  subcortical matter  cerebral  subcortical matter  white  white  lenticular  nucleus  inferior  colliculus  cerebellar  cerebellar  cortex  superior  colliculus  IVth v e n t r i c l e floor  r e t i c u l a r formation  IVth v e n t r i c l e  hypothalamus  hypothalamus  floor  cortex cortex  r e t i c u l a r formation hypothalamus  - 37 (a)  Table 4 shows the e f f e c t  of s t r o n g and weak c a t i o n  and anion exchange r e s i n s on d i a l y s a t e s c o n t a i n i n g v a s o d i l a t o r a c t i v i t y and on s o l u t i o n s  of ATP.  The v a s o d i l a t o r a c t i v i t y  was  t e s t e d on the r a b b i t ear p r e p a r a t i o n as d e s c r i b e d above, the amount of d i l a t a t i o n i s shown by +'s, d i l a t a t i o n e f f e c t and  b e i n g the s t r o n g e s t  the weakest as r e c o r d e d on a f i l m  strip  moving past an o s c i l l o s c o p e s c r e e n . The amount of ATP present was measured by the amount of l i g h t  p a s s i n g through a known s o l u t i o n path at a wavelength  of 260 m^u .  The c o n c e n t r a t i o n of t h e ATP can be c a l c u l a t e d  the o p t i c a l d e n s i t y of t h e s o l u t i o n , the molecular coefficient  at pH 7.0,  from  extinction  and the measured path l e n g t h .  Thus, a  s o l u t i o n having an o p t i c a l d e n s i t y of 0.090, from the e q u a t i o n A = k c l ; where A coefficient, of l i g h t  a  o p t i c a l d e n s i t y , k = molecular  extinction  c = c o n c e n t r a t i o n i n m o l e s / l i t r e , and 1 = l e n g t h  path i n cms., w i l l have a c o n c e n t r a t i o n of 0.090. „  kl  k f o r ATP = 15.4 x 10° at pH 7.0 and 1 = 0 . 5 t i o n of ATP w i l l t h e r e f o r e be ° ^ ° ° 9  or expressed  in  Where  g./ml.  x  ^  cm., the concentraQ < 5  ^  or 0.011 x 1 0 "  3  m/l  6.85.  It can be seen from Table 4 t h a t ATP was  completely  taken out of s o l u t i o n by the two anion exchange r e s i n s Dowex-1 and Amberlite  IR-45.  The s t r o n g c a t i o n exchange r e s i n Dowex-50,  on the other hand, removed l e s s than h a l f the ATP p r e s e n t . weak c a t i o n exchange r e s i a , Amberlite one-tenth  of the t o t a l ATP p r e s e n t .  IRC-50 removed l e s s  The than  These data i n d i c a t e that  the ATP molecule  under n e u t r a l c o n d i t i o n s Is an anion, and t h e r e -  f o r e under these  c o n d i t i o n s i s s i m i l a r t o the v a s o d i l a t o r  substance.  - 38 TABLE 4:  THE EFFECT OF VARIOUS ION EXCHANGE RESINS ON ACTIVE DIALYSATES AND SOLUTIONS OF ATP  Sample  (Water D i a l y s a t e s )  caudate nucleus t r e a t e d wi t h Dowex-1 (anion exchanger)  Vasodilatation i n Rabbit E a r  0  caudate nucleus t r e a t e d w i t h Dowex-50 ( c a t i o n exchanger) c e r e b r a l c o r t e x t r e a t e d with Dowex-1 (anion exchanger) c e r e b r a l c o r t e x t r e a t e d w i t h Dowex-50 ( c a t i o n exchanger) caudate nucleus t r e a t e d w i t h IR-45 (weak anion exchanger) caudate nucleus t r e a t e d w i t h IRC-50 (weak c a t i o n exchanger)  0 +- +•+•  0  -t- +  c e r e b r a l c o r t e x t r e a t e d w i t h IR-45 (weak anion exchanger)  0  c e r e b r a l c o r t e x t r e a t e d w i t h IRC-50 (weak c a t i o n exchanger)  -tAmount o f ATP  10T/ml.) " " "  ATP s o l u t i o n t r e a t e d w i t h Dowex-1 (anion exchanger) ATP s o l u t i o n t r e a t e d w i t h Dowex-50 ( c a t i o n exchanger) ATP s o l u t i o n t r e a t e d with IR-45 (weak anion exchanger) A T P ' s o l u t i o n t r e a t e d w i t h IRC-50 (weak c a t i o n exchanger)  0 6.85 0 9.34  - 39 (b) tor material  A comparison of the s t a b i l i t i e s and ATP t o c o n d i t i o n s  of a l k a l i n e and a c i d i c  l y s i s as w e l l as heat was a l s o thought t o g i v e a possible  of the v a s o d i l a -  an Insight  r e l a t i o n s h i p between the two compounds.  experiments, as mentioned above, s u c c e s s i v e  into  In these  portions  d i a l y s a t e s from caudate nucleus and c e r e b r a l c o r t e x  hydro-  of water were heated  t o b o i l i n g wibh 2N.HC1 and IN.NaOH as w e l l a s b e i n g heated t o b o i l i n g w i t h no h y d r o l y z i n g a l s o done w i t h s o l u t i o n s remaining v a s o d i l a t o r solutions  agent present.  of ATP.  The same t h i n g was  The method of d e t e r m i n i n g the  a c t i v i t y was t o i n j e c t the n e u t r a l i z e d  i n t o the r a b b i t  ear p r e p a r a t i o n ,  measuring any vaso-  d i l a t a t i o n as mentioned b e f o r e . The  water d i a l y s a t e s which had been s u b j e c t e d  d i t i o n s of a l k a l i n e h y d r o l y s i s of v a s o d i l a t o r a c t i v i t y . to conditions  and t o heat alone showed no l o s s  The d i a l y s a t e s which had been  subjected  of a c i d i c h y d r o l y s i s , however, showed a complete  absence of any v a s o d i l a t o r  activity.  S i m i l a r l y , a s o l u t i o n of ATP when s u b j e c t e d hydrolysis  t o con-  to alkaline  or heat alone showed l i t t l e decrease i n v a s o d i l a t o r  a c t i v i t y , w h i l e a s o l u t i o n of ATP subjected showed a marked decrease of v a s o d i l a t o r (c)  to acidic  hydrolysis  activity.  To prepare the water d i a l y s a t e s homogenates of  t i s s u e from t h e areas of the b r a i n i n d i c a t e d were d i a l y z e d i n the  c o l d against  d i s t i l l e d water f o r 48 hours.  The r e s u l t i n g  d i a l y s a t e s were concentrated down t o a f i n a l volume which was one-third  t h e volume of the f r e s h t i s s u e . A s o l u t i o n o f ATP was a l s o t r e a t e d  i n the same manner  - 40 t o determine whether the ATP molecule would behave i n the same way  against  substance.  a concentration  gradient  as d i d t h e v a s o d i l a t o r  The amount of ATP which passed through the d i a l y s i s  membrane was measured by the f i r e f l y luminescence enzyme nique  (see m a t e r i a l s  &  tech-  methods).  The amounts of ATP which were found t o pass through the membrane were so small ATP was  as t o render i t most u n l i k e l y that  the v a s o d i l a t o r substance d i a l y z e d  t i s s u e homogenates. responsible  out of the b r a i n  I t would i n d i c a t e a l s o t h a t the substance  f o r the v a s o d i l a t a t i o n had a smaller  e t e r than ATP  m o l e c u l a r diam-  as i t passed through the d i a l y s i s membrane almost  completely a f t e r 48 hours. 5.  Chromatography o f a c t i v e e x t r a c t s material  and  ATP  An attempt was stance as w e l l as ATP dialysates. i a l and ATP eluted  I t was  made t o i s o l a t e the v a s o d i l a t o r  c h r o m a t o g r a p h i c a l l y from the a c t i v e  thought that  subwater  i f the a c t i v e v a s o d i l a t o r mater-  could be i s o l a t e d then these substances could  be  from the chromatograms and i n j e c t e d i n t o the r a b b i t ear  preparation  t o determine whether a n y • s i m i l a r i t y could  between the e l u t e d m a t e r i a l water  for vasodilator  and the v a s o d i l a t o r  be found  e f f e c t s of the  dialysates. Chromatograms of water d i a l y s a t e s were developed i n  n-propanol, ammonia, water  (60^30:10) f o r 18 hours and upon  being d r i e d were scanned under u l t r a v i o l e t areas  (which Is c h a r a c t e r i s t i c of ATP).  one border as a marker compound.  Several  f o r l i g h t absorbing  ATP was light  a l s o run along a b s o r b i n g areas  - 41 were found although none corresponded t o ATP.  Neither d i d  areas upon e l u t i o n produce v a s o d i l a t a t i o n i n the preparation. and  (2:79:19) f o r 4-| hours.  (NR^^SC^ s a t u r a t e d Ultraviolet light  were again t e s t e d u n s u c c e s s f u l l y  solution, absorbing areas  for vasodilator  activity.  S t r i p s of chromatogram paper were s p o t t e d water d i a l y s a t e s and tioned  above.  between the  ear  S i m i l a r l y , water d i a l y s a t e s were chromatographed  developed i n i s o p r o p a n o l ,  water  rabbit  any  developed i n the two  On d r y i n g the  o r i g i n and  the  solvent  s t r i p s were cut  solvent  f r o n t and  with active  systems men-  into ten  sections  each s e c t i o n  eluted with i s o t o n i c s a l i n e .  "Each e l u t i o n i n t u r n was  on the  for vasodilator  r a b b i t ear  preparation  was  tested  a c t i v i t y without  success. It was  hoped that  a c o r r e l a t i o n could be  between an area whose e l u a t e t i o n of the  caused v a s o d i l a t a t i o n and  same area t o s p e c i f i c c o l o u r i z i n g agents.  s t r i p s of paper were prepared at the various  established  same time and  a  reac-  Similar  treated  c o l o u r i z i n g agents s p e c i f i c f o r c e r t a i n species  with  of com-  pounds. N i n h y d r i n s o l u t i o n which y i e l d s coloured w i t h molecules possessing free-NHg groups was lysates yielded the  4-6  ninhydrin  used.  compounds Most d i a -  p o s i t i v e areas, probably i n d i c a t i n g  presence of amino acids which most c e r t a i n l y would be Molybdic a c i d spray was  used t o i n d i c a t e the  there.  presence  of organic phosphorous compounds, of which most d i a l y s a t e s gave four d i s t i n c t A Hg, the  areas. c o n t a i n i n g reagent was  used, which  Indicated  presence of t h i o u r a c i l , u r a c i l , u r i d i n e - 5 - P 0 , 3,4 4  dihydroxy-  - 42 p h e n y l a l a n i n e , and  -  L-adrenaline.  Most water d i a l y s a t e s  four areas of p o s i t i v e r e a c t i o n t o t h i s  reagent,  E h r l i c h ' s reagent, c o n t a i n i n g hyde, whose p o s i t i v e r e a c t i o n gives uracil,  p-dimethylaminobenzalde-  a yellow colour to dihydro-  thymine, p h e n y l a l a n i n e , t y r o s i n e ,  r i b o t i d e was  a l s o used.  l y s a t e s a l l y i e l d e d only  yielded  quanosine, and  quanine  The  chromatograms of a c t i v e water d i a -  one  area of p o s i t i v e r e a c t i o n t o t h i s  reagent. Sakaguchi reagent whose p o s i t ive,. r e a c t i o n y i e l d s orange colour with quanidine type compounds was water d i a l y s a t e s a l l had  only one  an  also t r i e d .  area which r e a c t e d  with  The  the  reagent. As no v a s o d i l a t o r the  eluates,  colouring 6.  found i n any  reacted  with the v a r i o u s  species'  the  spec-  reagent.  ear  preparation.  (a)  This t r a c i n g i s an example of the stimulating  There i s a lapse  stimulation  of  areas of  Recordings of v a s o d i l a t a t i o n produced i n the  produced by ally.  be  no c o r r e l a t i o n c o u l d be made w i t h the  chromatograms which had ific  a c t i v i t y could  and the  the great  rabbit  vasodilatation  a u r i c u l a r nerve a n t i d r o m i c -  of 5 seconds between the  onset of v a s o d i l a t a t i o n .  time of nerve  At the  end  of  55  seconds the v a s o d i l a t a t i o n i s s t i l l v e r y marked. (b)  This  curve i s a r e c o r d i n g  of the  caused by I n j e c t i o n of a s o l u t i o n of ATP there i s a s m a l l drop i n the  vasodilatation  ( l mg./ml.).  curve caused by the  c l e a r s o l u t i o n i n t o the blood v e s s e l s ,  entry  At  first  of  the  t h i s appears as a  - 42(a)  •  -  i 10  Figure 6A.  SEC.  This t r a c i n g i s an example of the v a s o d i l a t a t i o n produced i n the r a b b i t ear preparation by stimul a t i o n of the a u r i c u l a r nerve; the arrow i n d i c a t e s the point of s t i m u l a t i o n .  - 42(b) -  10  Figure  6B.  SEC.  This t r a c i n g i s an example o f v a s o d i l a t a t i o n produced i n the r a b b i t ear p r e p a r a t i o n byi n j e c t i o n i n t o the f a c i a l a r t e r y o f ATP s o l u t i o n ( l mg./ml.). The arrow i n d i c a t e s the p o i n t of i n j e c t i o n .  - 42(c) -  Figure  6C.  This t r a c i n g i s produced i n the t i o n of extract arrow i n d i c a t e s  an example of the v a s o d i l a t a t i o n r a b b i t ear p r e p a r a t i o n by i n j e c of the caudate n u c l e u s . The the p o i n t of i n j e c t i o n .  - 42(d) -  Figure  6D.  This t r a c i n g i s an example of the v a s o d i l a t a t i o n produced i n the r a b b i t ear p r e p a r a t i o n by i n j e c t i o n o f e x t r a c t o f p o s t e r i o r r o o t s o f the s p i n a l cord. The arrow i n d i c a t e s the p o i n t of i n j e c t i o n .  - 43 c o n s t r i c t i o n , the d i l a t a t i o n i s d e l a y e d by 5 seconds. •the d i l a t a t i o n produced by nerve s t i m u l a t i o n ,  Unlike  the d i l a t a t i o n  produced by ATP s t a r t s t o subside a f t e r 13 seconds. (c) extract  Shows the d i l a t a t i o n produced by i n j e c t i o n of the  of the caudate n u c l e u s .  Here t h e r e Is a s h o r t  t i o n a g a i n caused by the e n t r y of the c l e a r onset o f the d i l a t a t i o n i s delayed by 3-4 vasodilatation (d)  constric-  s o l u t i o n , a g a i n the  seconds.  The  peak  i s over a f t e r 13 seconds. This t r a c i n g  i l l u s t r a t e s the v a s o d i l a t a t i o n  duced b?/ i n j e c t i o n of an e x t r a c t  of posterior  roots.  pro-  The onset  of the d i l a t a t i o n i s delayed by 5 seconds and the d i l a t a t i o n i t s e l f s u b s i d e s a f t e r 8 seconds.  - 44 IV.  DISCUSSION: Dale  the  (48) and Feldberg  (65) have both put forward  same concept, that a neurone could be expected t o r e l e a s e  the same t r a n s m i t t e r agent at a l l synapses formed by the d i f f e r ent branches of the axon, and f u r t h e r , that a b i p o l a r neurone would possess the same t r a n s m i t t e r m a t e r i a l at the c e n t r a l end as w e l l as at the p e r i p h e r a l end of i t s processes.  I t would  t h e r e f o r e be expected t h a t any i n f o r m a t i o n  about the  obtained  t r a n s m i t t e r r e l e a s e d at the p e r i p h e r a l t e r m i n a l s  of a primary  sensory neurone c o u l d a l s o be a p p l i e d t o the t r a n s m i t t e r at the  c e n t r a l synapse. Evidence has come t o l i g h t r e c e n t l y which substan-  t i a t e s Dale's h y p o t h e s i s .  Among s p i n a l neurones a c e t y l c h o l i n e  i s known t o be the t r a n s m i t t e r substance e l a b o r a t e d  f o r the  propagation of a t r a i n of impulses from the motor neurones and l i b e r a t e d from t h e i r axonal endings t o cause t r a n s m i s s i o n neuromuscular j u n c t i o n s  l e a d i n g t o the a c t i v a t i o n o f the muscle.  I n h i b i t o r y Interneurones (Renshaw c e l l s ) , whose a c t i o n the by has  across  a c t i v i t y of the motor neurones, are present  inhibits  and are served  c o l l a t e r a l f i b r e s from the same motor neurones.  Acetylcholine  been found t o a c t i v a t e t h e muscle a t the neuromuscular  t i o n and i t w i l l a l s o a c t i v a t e the Renshaw c e l l s  (75);  junc-  this  i n d i c a t e s t h a t a c e t y l c h o l i n e i s a c t i n g as t h e t r a n s m i t t e r  sub-  stance a t more than one branch of the axon of the motor neurone. I t i s but a short  step t o p o s t u l a t e that the b i p o l a r sensory  neurone with i t s branches going t o the p e r i p h e r y i n g c e n t r a l l y i n the s p i n a l cord,  could  and a l s o synaps-  probably a l s o employ the  - 45 same t r a n s m i t t e r substance one  continuous  at e i t h e r end  of what i s e s s e n t i a l l y  process.  Sensory i n f o r m a t i o n reaches  the higher  centres of the  c e n t r a l nervous system by a t h r e e neurone pathway, the  first  synapse can e i t h e r be w i t h i n the segment at which the p o s t e r i o r root f i b r e enters the s p i n a l cord, or h i g h e r up i n the cuneatus et g r a c i l i s .  The  nuclei  second synapse i n the pathway is i n  the thalamus and the f i n a l synapse i n the a p p r o p r i a t e area  of  the c e r e b r a l c o r t e x . Evidence nervous system has way  are capable  i n c h o l i n e r g i c t r a n s m i s s i o n i n the  central  shown t h a t not a l l the neurones of the  path-  of s y n t h e s i z i n g a c e t y l c h o l i n e , the s y n t h e s i s of  which i s taken t o i n d i c a t e a t r a n s m i t t e r r o l e f o r a c e t y l c h o l i n e (63). for  A tendency appears t o e x i s t , In some cases at any r a t e ,  neurones of h i g h c h o l i n e a c e t y l a s e a c t i v i t y t o a l t e r n a t e i n  a chain with neurones.which' c o n t a i n no s y n t h e s i z i n g enzyme (71,72). Of the t h r e e neurones i n v o l v e d i n the primary sensory the f i r s t  and t h i r d are d e f i c i e n t  pathway,  i n c h o l i n e a c e t y l a s e while  second neurone has a h i g h a c t i v i t y of t h i s enzyme.  An a l t e r n a -  t i o n here seems t o e x i s t , of neurones having d i f f e r e n t a c t i v i t i e s which i s reasonable  the  to b e l i e v e corresponds  enzyme with  an  a l t e r n a t i o n of c h o l i n e r g i c and n o n - c h o l i n e r g i c elements of t r a n s mission. The  d i s t r i b u t i o n of v a s o d i l a t o r substance  was  determined i n the present  The  r e s u l t s agreed  Table  experiments  r o u g h l y with those  (see Table  i n beef b r a i n 1,  r e p o r t e d by Holton  results). (57),  5, In t h a t the area found t o c o n t a i n the most a c t i v i t y  was  - 46 the caudate n u c l e u s , w h i l e t h e thalamus  a l s o contained  of a c t i v i t y , as d i d the d o r s a l and v e n t r a l r o o t s . the agreement ends.  a degree  However, here  Holt on found the nucleus cuneatus t o have  much v a s o d i l a t o r m a t e r i a l while i n these experiments the n u c l e i cuneatus et g r a c i l i s was found t o possess almost no v a s o d i l a t o r material.  Holton a l s o r e p o r t s  little  a c t i v i t y i n the c e r e b r a l  c o r t e x whereas i n the above experiments the c e r e b r a l contained  cortex  almost as much v a s o d i l a t o r substance as the caudate  nucleus. At t h i s point bution  i t i s of i n t e r e s t t o compare the d i s t r i -  of v a s o d i l a t o r m a t e r i a l  i n areas of the c e n t r a l nervous  system w i t h the content of o t h e r n e u r o l o g i c a l l y a c t i v e substances.  Table 6 shows the d i s t r i b u t i o n of some compounds  t o be o f importance at c e n t r a l synapses.  likely  There does not appear  t o be any c o r r e l a t i o n between t h e d i s t r i b u t i o n of t h e v a r i o u s substances given  i n Table 6 and t h e d i s t r i b u t i o n of v a s o d i l a t o r  •material from the above experiments.  T h i s i s not s u r p r i s i n g i n  view of the f a c t t h a t the substances mentioned  have a l l been  r u l e d out as p o s s i b l e v a s o d i l a t o r agents e i t h e r because chemical or p h a r m a c o l o g i c a l p r o p e r t i e s . not  of t h e i r  Hence, h i s t a m i n e could  be the v a s o d i l a t o r m a t e r i a l because v a s o d i l a t a t i o n w i l l  occur i n the presence o f a n t i h i s t a m i n i c s  still  compounds; or substance  P cannot be the substance as i t w i l l not d i a l y z e through a semipermeable membrane w h i l e the a c t i v e m a t e r i a l w i l l d i a l y z e through. It  i s tempting t o p o s t u l a t e  stance r e s p o n s i b l e ponsible  that t h e t r a n s m i t t e r  sub-  f o r a n t i d r o m i c v a s o d i l a t a t i o n may a l s o be r e s -  f o r synaptic  transmission  a t the f i r s t  and t h i r d  neurones  - 47 TABLE 5:  VASODILATOR ACTIVITY, NOT DUE TO ACETYLCHOLINE OR  HISTAMINE, OF EXTRACTS FROM DIFFERENT REGIONS OF THE HORSE BRAIN"' Exp.  #1  #2  #3  #4  #5  100  100  100  100  caudate  nucleus  100  nucleus  cuneatus  300  nucleus  gracilis  5  fasiculus  gracilis  pyramidal  tracts  30 4  d o r s a l root optic  400  10  tract  1  trapezoid  2  fimbria  3  4  thalamus  5  8  cerebral cortex  1  4  2  4  i n t e r n a l capsule, post,  limb  anterior pituitary-  <2  hyp ot ha lamus  20  medial eminence of tuber cinereum standard a c e t o n e - d r i e d powder of d o r s a l r o o t s  20 constrictor  40  100  80  20*  20  100  " a f t e r treatment which i n a c t i v a t e d the p o s t e r i o r p i t u i t a r y p r i n c i p l e - e x t r a c t of caudate nucleus taken as the standard f o r each b r a i n , and the a c t i v i t y of the other samples was found by matching each with a d i l u t i o n of the standard. The a c t i v i t y i s t h e r e f o r e expressed as a percentage of t h a t i n the caudate nucleus. In a d d i t i o n , the caudate nucleus e x t r a c t was assayed a g a i n s t a s a t u r a t e d a c e t o n e - d r i e d p r e p a r a t i o n of d o r s a l r o o t s  1 P. H o l l i n , G.W. H a r r i s , J o u r n a l of P h y s i o l o g y , V o l ; 120, page 254, (1953).  TABLE 6:  DISTRIBUTION OF NEUROLOGICAL ACTIVE COMPOUNDS IN AREAS OF CENTRAL NERVOUS SYSTEM  Choline acetylase ^g./gm. powder  Noradrenaline  2  5 HT Histamine ^g./gm., ^«rg./gm.  •^g./gm.  CEF  %  %  -  ^20  0.01  0  4-11  o p t i c nerves  16  0  0.02  0  9  dorsal  33  0  0.3  deep r o o t s  Subst. Holt on's P vasodil. unit/gm. s u b s t .  40  10  100  6  1  100  27  30  -  -  100  1.6  1  100  I n t e r n a l capsule (posterior)  70  -  cerebellum  26  90  0.07  pyramids  42  -  0.06  0  -  -  3  15  573  11000  0.06  0  6-9  6  0  100  6  0  61  -  -  columns  v e n t r a l roots sympathetic g a n g l i a lateral body  geniculate  midbrain hypothalamus thalamus  2  -  -  0.01  ^0.1  -  -  325  2600  0.07  170  -  0.37  0.20  -  -  2000  1.03  0.28  12  823  3000  area postrema caudate nucleus  mm  437  13300  fO.24 med. 1-0.28 l a t . 1.04 0.10  -  0  -  100  -  -  0  70  20  -  460  ^0.2  46  0  -  -  f0.07 med, <0.4 lat. 0.24  lo  0  12.5  6  100  10.0  -  from C r o s s l a n d , J o u r n a l of Pharmacy & Pharmacology, V o l .12, page 10,  (1960/.  - 49 of the for  sensory pathway.  ATP  Indeed proposed such a r o l e  l a r g e l y on the grounds t h a t  d i l a t a t i o n and its  Holt on has  action.  discussed  i t g i v e s r i s e to a  capillary  no pharmacological agents are known which  She  has  block  a l s o produced some evidence, which has  above (see page 15,  I n t r o d u c t i o n ) , t h a t ATP  been  i s released  upon s t i m u l a t i o n of sensory f i b r e s . Measurements of the on v a r i o u s were the  amounts of ATP  areas of the b r a i n w i t h the  transmitter responsible  were c a r r i e d out  idea i n mind t h a t i f ATP  f o r antidromic  vasodilatation,  or r e l a t e d to i t , the areas c o n t a i n i n g the most v a s o d i l a t o r a c t i v i t y should Table 3 i n the l o c a t i o n and  a l s o c o n t a i n the most ATP.  As can be  seen from  r e s u l t s , no r e l a t i o n s h i p i s apparent between the  concentration  o f the two  substances.  In p o s t u l a t i n g the presence of the v a s o d i l a t o r " m a t e r i a l i n the sensory pathways i t Is n e c e s s a r y t o comment on the  possible  s i g n i f i c a n c e of d i s t r i b u t i o n i n the a r e a s . t e s t e d .  accepts  I f one  Dale's h y p o t h e s i s that a neurone w i l l u t i l i z e the  same t r a n s -  m i t t e r substance at a l l branches of the axon, and  there  evidence t o support t h i s , then one a c t i v e substance at the and  at the  would be  might w e l l expect t o f i n d  p e r i p h e r a l ends of the  c e n t r a l ends, i n which case the  present  i s some  i n the p o s t e r i o r r o o t s .  three neurone sensory pathways the f i r s t  the  sensory neurones  v a s o d i l a t o r substance In c o n s i d e r i n g  the  neurone synapses w i t h  the second neurone i n the thalamus, hence i t would seem reasonable the v a s o d i l a t o r m a t e r i a l be synapse of the  first  i n the thalamus a t the  sensory neurone.  Table l ) i n the r e s u l t s .  This has  central  been shown  I f the d i s t r i b u t i o n of the  (see  vasodilator  - 50 substance a l t e r n a t e d w i t h the  d i s t r i b u t i o n of a c e t y l c h o l i n e ,  then one would expect to f i n d v a s o d i l a t o r m a t e r i a l i n the and t h i r d neurone of the t h r e e  first  neurone pathway; i n which case  the c e r e b r a l c o r t e x should a l s o c o n t a i n v a s o d i l a t o r m a t e r i a l . T h i s has  been demonstrated, i n f a c t the c e r e b r a l c o r t e x  a l a r g e amount of v a s o d i l a t o r m a t e r i a l . s t i l l remains.  caudate nucleus was  est  amount.  The  a p p r e c i a b l e amounts o f most of the a c t i v e substances.  t o date to have motor f u n c t i o n s reason why  such an  in general,  only.  been shown  However, t h e r e  i s no  above evidence and from the f a c t  important r o l e  i t seems u n l i k e l y that i t can be  the substance r e s -  As ATP  i n c l u d i n g neurones, i t i s obvious that  some of the  criteria  f o r a t r a n s m i t t e r agent.  are; t h e r e must be  i s present  Since  Some of the  some system f o r s y n t h e s i z i n g  neurones must be able  in  i t would meet  substance, and t h e r e must a l s o be some mechanism f o r the substance.  that  as a source of energy f o r the body  f o r s e n s o r y nerve t r a n s m i s s i o n .  all cells,  criteria  f a c t that I t has  i n s e n s o r y pathways.  In view o f the  ponsible  caudate nucleus  the m a t e r i a l , i f i t i s an e x c i t a t o r y t r a n s m i t t e r ,  only be  has  great-  A 3 can be seen from Table 6. the caudate nucleus  i s d i f f i c u l t t o r e c o n c i l e w i t h the  ATP  material  c o n s i s t e n t l y found t o c o n t a i n the  presence of the v a s o d i l a t o r m a t e r i a l i n the  should  enigma  Of a l l the areas t e s t e d f o r v a s o d i l a t o r  the  contains  However, one  possesses  the  destroying  to control their  source of energy a c c u r a t e l y t h e y possess a system f o r s y n t h e s i z ing  ATP  and  a l s o a mechanism f o r d e s t r o y i n g  effect, f u l f i l l i n g  ATP  thereby, i r i  some o f the c r i t e r i a f o r a t r a n s m i t t e r  agent.  -  The  two  -  most important questions  were sought i n t h i s  r e s e a r c h were:  r e s p o n s i b l e f o r antidromic is  51  1.  f o r which answers  i s ATP  vasodilatation?  and  the  substance  2.  i f it is,  i t a l s o a sensory s y n a p t i c t r a n s m i t t e r substance?  from the above r e s e a r c h t h a t ATP  i s u n l i k e l y t o be  I t appears  the  substance  r e s p o n s i b l e f o r the v a s o d i l a t a t i o n , which would seem t o r u l e i t out as the  sensory s y n a p t i c t r a n s m i t t e r substance.  although t h i s has work, i f one  not been d e f i n i t e l y demonstrated i n the  accepts  present  Dale's p r o p o s a l , then the answer f o r both  questions  should be the same and  antidromic  v a s o d i l a t a t i o n should  transmitter  However,  substance.  the  substance r e s p o n s i b l e  for  a l s o be the c e n t r a l s y n a p t i c  BIBLIOGRAPHY 1.  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