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Studies on noradrenergic supersensitivity of the cyclic AMP response in rat cerebral cortex Kallstrom, Elizabeth 1979

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STUDIES ON NORADRENERGIC SUPERSENSITIVITY OF THE CYCLIC AMP RESPONSE IN RAT CEREBRAL CORTEX by Elizabeth M.Sc,  Kallstrom  The U n i v e r s i t y o f B r i t i s h Columbia, 1979  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in 'THE FACULTY ,OF GRADUATE STUDIES (Department  of Pathology)  We a c c e p t t h i s t h e s i s as conforming to t h e r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA October 1979  E l i z a b e t h K a l l s t r o m , 1979  In  presenting  an  advanced  the I  Library  further  for  his  of  this  written  shall  agree  at  it  purposes  for  6  It  financial  British  October 15. 1979  of  Columbia,  British for  gain  shall  Columbia  that  not  the  requirements  reference copying  by t h e Head  is understood  Pathology of  of  for extensive  permission.  University  fulfilment  available  may be g r a n t e d  2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  Date  freely  permission  representatives. thesis  in p a r t i a l  the University  make  that  Department o f The  thesis  degree  scholarly  by  this  of  I agree and this  study. thesis  o f my D e p a r t m e n t  copying  be a l l o w e d  or  th  o  publicatio  without  my  ABSTRACT  Intracerebral (DB)  injections  causes s e l e c t i v e  cortical  neurons  measurable period  of  of  depletion of  may t h e n d e v e l o p  by t h e  level  of  and t e n weeks was  At  days t h e r e  shift Kp,  The b a s e l i n e  cAMP s y s t e m a f t e r of  the  levels,  or V , of max  seven  curve  increase  of  the  second p o s t - n a t a l  cAMP and g r e a t e r  tested.  However,  d a y s was the  left,  The  c h o s e n as  a  supersensitive  a significant  significant,  of  the  shift  consisted  corresponding and m a x i m a l  week was  there  adenylate  cortical  slices.  These  ledge of  the  of  cyclase  characterized i n response  was no s i g n i f i c a n t  shift  of  permanent  increase  the  in  dose-  The  of a  effect  significant  to a l o w e r i n g  of  stimulation  system d u r i n g  by h i g h e r b a s e l i n e  t o a l l NA of  the  results  are  discussed  dose-response  cAMP as a component  of  of  curve.  and  posterior  our present  the p o s t - s y n a p t i c  levels  showing  i n both a n t e r i o r i n terms  the  concentrations  on t h e N A - s t i m u l a t e d cAMP r e s p o n s e ,  and m a x i m a l s t i m u l a t i o n l e v e l s  role  was  i n both baseline  cAMP a c c u m u l a t i o n  K i n d l i n g h a d no e f f e c t unchanged b a s a l  (NA) s t o r e s .  o f cAMP r e m a i n e d u n c h a n g e d .  the  bundle  cAMP.  The v e r y h i g h r e s p o n s i v e n e s s end o f  to  dorsal  l o n g - t e r m p e r i o d to measure  but not  values  of  t e n weeks p o s t - i n j e c t i o n  dose-response  and a s i g n i f i c a n t  development  the  t o NA and t h i s may be  Seven  the  s t i m u l a t i o n and a s l i g h t ,  the  supersensitivity  t a k e n as  maximal  curve.  c o r t i c a l noradrenaline  the  of  of  treatment.  to  effects  response  this  n e u r o t o x i n 6-OHDA i n t o  cAMP a c c u m u l a t i o n .  time from i n j e c t i o n  response,  the  receptor  knowcomplex.  - iii  -  TABLE OF CONTENTS  ABSTRACT  i i  TABLE OF CONTENTS LIST OF FIGURES LIST OF ABBREVIATIONS ACKNOWLEDGEMENT  i i i v v i i ix  I.  INTRODUCTION  1  1.  Catecholamine Systems  1  a) b) c)  1 3 3  Descending NA p a t h A s c e n d i n g NA p a t h NA paths o r i g i n a t i n g i n LC  2.  Some W e l l Known Drugs Which A f f e c t N o r a d r e n e r g i c and Dopaminic Systems  5  3.  cAMP and N e u r a l F u n c t i o n  8  4.  Supersensitivity  16  5.  cAMP i n D i s e a s e  24  a) b) c) d) e)  24 27 30 33 33  6.  7.  E t h a n o l , Morphine and cAMP Mental Disease Epilepsy Miscellaneous C l i n i c a l Conditions cAMP and C l i n i c a l C h e m i s t r y  Assay Methods f o r cAMP  33  a) b) c) d) e)  34 34 35 35 36  P h o s p h o r y l a s e A c t i v a t i o n Assay Enzymatic C y c l i n g Procedures P r o t e i n K i n a s e Assay P r o t e i n B i n d i n g Assays P r e l a b e l l i n g Techniques  Statement o f the Problem  37  - iv-  II.  EXPERIMENTAL PROCEDURE  39  1.  Materials  39  2.  Methods  39  a)  39  Treatment o f A n i m a l s i) ii) iii)  b)  K i n d l i n g Procedure 6-OHDA I n j e c t i o n s FLA-63 I n j e c t i o n s  39 40 41  Treatment o f B r a i n T i s s u e  41  i) ii) iii) iv) v) 3. III.  Tissue preparation I n c u b a t i o n Procedure and I s o l a t i o n o f cAMP D e t e r m i n a t i o n o f cAMP Measurement o f NA l e v e l s P r o t e i n Determination  A n a l y s i s o f Data RESULTS  41 42 43 45 48 48 50  1.  FLA-63  50  2.  Development o f Method and E a r l y F i n d i n g s  51  3.  K i n d l e d Rats  63  4.  Developmental E f f e c t on the cAMP System  68  5.  Supersensitivity  70  IV.  DISCUSSION  76  V.  REFERENCES  85  VI.  APPENDIX  94  1.  Table I  94  2.  Table I I  95  - v LIST OF FIGURES  1.  S a g i t t a l p r o j e c t i o n s o f NA pathways  2  2.  S a g i t t a l p r o j e c t i o n s o f DA pathways  4  3.  Schematic diagram o f s y n a p t i c c o n n e c t i o n s  6  4.  Proposed r o l e f o r cAMP and p r o t e i n p h o s p h o r y l a t i o n  11  5.  Schematic diagram o f n o r a d r e n e r g i c nerve ending  13  6.  Dose-response  curve o f 6 h r F L A - 6 3 - t r e a t e d animals  52  7.  Dose-response  curve o f 24 h r F L A - 6 3 - t r e a t e d animals  53  8.  Dose-response  curve o f animals s a c r i f i c e d by c e r v i c a l f r a c t u r e  54  and e t h e r 9.  Standard curve f o r the d e t e r m i n a t i o n o f cAMP c o n t e n t a t 1:200  57  dilution 10.  Standard curve f o r the d e t e r m i n a t i o n o f cAMP c o n t e n t a t 1:60  58  dilution 11.  Time c o u r s e o f the s t i m u l a t i o n o f cAMP f o r m a t i o n by NA  60  12.  The response o f d i f f e r e n t c o r t i c a l r e g i o n s t o NA  61  13.  The response o f d i f f e r e n t c o r t i c a l r e g i o n s to NA w i t h l e f t and  62  r i g h t hemispheres  combined  14.  R e l a t i v e r e c o v e r y o f NA from d i f f e r e n t alumina pH  15.  R e l a t i v e i n t e n s i t y of spectrophotofluorimeter readings  16.  S t i m u l a t i o n o f cAMP f o r m a t i o n by NA i n a n t e r i o r c o r t e x o f kindled  17.  65 66  animals  S t i m u l a t i o n o f cAMP f o r m a t i o n by NA i n p o s t e r i o r c o r t e x o f kindled  64  animals  67  - vi  -  Dose-response curve o f 15 day o l d animals Dose-response curve of v e h i c l e - i n j e c t e d  animals  Dose-response curve o f 7 day 6-OHDA-injected  animals  Dose-response curve of 10 week 6-OHDA-injected  animal  - viiLIST OF ABBREVIATIONS acetylcholine  Ach  —  Ct-MT  - alpha-methyl-p-tyrosine  A-P  - anterio-posterior  ATP  - adenosine t r i p h o s p h a t e  BSA  - b o v i n e serum albumin  CA  - catecholamine  cAMP  - c y c l i c 3 , 5 - a d e n o s i n e monophosphate  cGMP  - cyclic 35 -guanosine  CNS  - c e n t r a l nervous  CPZ  - chlorpromazine  DA  - dopamine  DB  - d o r s a l bundle  DPH  - diphenylhydantoin  DPG  - 3,4-dihydroxyphenyl glucolaldehyde  E C  50  1  1  1  monophosphate  system  - c o n c e n t r a t i o n o f drug p r o d u c i n g h a l f - m a x i m a l  EPSP  - fast excitatory post-synaptic potential  5-HT  - serotonin  GABA  - Y aminobutyric  [ H]  - tritiated  LC  - locus coeruleus  M  - molar  MFB  - medial forebrain  M-L  _  3  -  acid  compound  medio-lateral  bundle  response  - viii  -  NA  - noradrenaline  PDE  -  PK  - protein kinase  PKI  - p r o t e i n kinase  PPi  - i n o r g a n i c pyrophosphate  SCG  - superior c e r v i c a l ganglion  s-IPSP  - slow i n h i b i t o r y p o s t - s y n a p t i c p o t e n t i a l  6-OHDA  - 6-hydroxydopamine  SN  - substantia nigra  TB  - toothbar  TCA  - trichloroacetic  TFP  -  phosphodiesterase  trifluoperazine  inhibitor  acid  - i xACKNOWLEDGEMENTS I would l i k e to express my s i n c e r e g r a t i t u d e to Dr. E.G. McGeer, whose a d v i c e , encouragement and p a t i e n c e made t h e c o m p l e t i o n o f t h i s t h e s i s p o s s i b l e . I would l i k e to thank Dr. S.W. French f o r i n t r o d u c i n g me t o the t o p i c o f this thesis.  I am a l s o i n d e b t e d t o Dr. J . Nagy who p r o v i d e d many v a l u a b l e  s u g g e s t i o n s and i d e a s d u r i n g the experiments. F i n a n c i a l a s s i s t a n c e from t h e N a t i o n a l I n s t i t u t e o f H e a l t h ( g r a n t t o Dr. S.W. F r e n c h ) , U.B.C. Summer S t u d e n t s h i p and the Department o f P a t h o l o g y are g r a t e f u l l y acknowledged. F i n a l l y I would l i k e to thank Dr. R.H. Pearce and the Department o f Pathology f o r t h e i r understanding, a d m i n i s t r a t i v e f l e x i b i l i t y i n the c o m p l e t i o n o f t h i s  thesis.  and a s s i s t a n c e  - x -  "Problems worthy o f a t t a c k prove t h e i r worth by h i t t i n g back." P i e t Hein  - 1 -  I.  INTRODUCTION This  t h e s i s i s concerned with the p o s s i b l e r o l e of  adenylate c y c l a s e a c t i v i t y and  i n various  the development of t o l e r a n c e  Adenylate cyclase  noradrenaline-stimulated  pathological conditions  such as e p i l e p s y  or a d d i c t i o n to c e r t a i n drugs such as  ethanol.  i s the name f o r the enzyme or enzymes which c a t a l y z e the  reactions:  Mg2+ ATP Noradrenaline-stimulated which shows i n c r e a s e d In t h i s  >  cAMP + P P i  adenylate c y c l a s e r e f e r s to that form of the enyzme  activity  i n the presence of  noradrenaline.  i n t r o d u c t i o n the anatomy of c e n t r a l n o r a d r e n e r g i c  systems w i l l  f i r s t be d e s c r i b e d  and dopaminergic  b r i e f l y as w e l l as the i n t e r a c t i o n o f v a r i o u s  w e l l known drugs with these systems i n so f a r as such i n f o r m a t i o n  i s necessary  f o r the s e c t i o n s t h a t f o l l o w on the p o s s i b l e r o l e s o f cAMP i n n e u r a l  function,  s u p e r s e n s i t i v i t y and pathology. 1.  Catecholamine Systems The  anatomy of c e n t r a l n o r a d r e n e r g i c  demonstrated by u s i n g h i s t o c h e m i c a l  and dopaminergic systems was  f l u o r e s c e n t techniques and confirmed and  extended by l e s i o n and immunohistochemical s t u d i e s map  describes  initially  the f o l l o w i n g n o r a d r e n e r g i c  (1,2).  The U n g e r s t e d t (3)  (NA) neuronal systems i n the r a t  brain (Fig. 1): a)  Descending NA path: The  most caudal  cell  group  ( A l ) gives  rise  to one system of f i b e r s  cending i n the a n t e r i o r f u n i c u l u s and v e n t r a l p a r t of the l a t e r a l to terminate  desfuniculus  i n the v e n t r a l horn, and a second system descending i n the  d o r s a l p a r t of the l a t e r a l  f u n i c u l u s to terminate i n the d o r s a l horn.  F i g . 1. S a g i t t a l p r o j e c t i o n s o f a s c e n d i n g NA pathways i n the r a t . The descending pathways a r e not i n c l u d e d . The s t r i p e s i n d i c a t e the major nerve t e r m i n a l a r e a s . (From Ungerstedt 1971).  - 3 b)  A s c e n d i n g NA path ( " v e n t r a l b u n d l e " ) : C e l l b o d i e s i n the pons and m e d u l l a o b l o n g a t a to one of the major a s c e n d i n g NA pathways. b u n d l e t r a v e l through  ( A l , A2, A5, A7)  The  axons of t h i s v e n t r a l  the r e t i c u l a r f o r m a t i o n , c o n t i n u e through  and t e r m i n a t e i n the lower b r a i n stem, mesencephalon and  c)  the  MFB  diencephalon.  T h i s system i n n e r v a t e s the whole hypothalamus, a r c u a t e and n u c l e i , p r e o p t i c area and v e n t r a l p a r t of s t r i a  give r i s e  supraoptic  terminalis.  NA paths o r i g i n a t i n g i n LC: The  l o c u s c o e r u l e u s , A6, g i v e s r i s e to t h r e e d i f f e r e n t NA systems i n n e r -  v a t i n g almost a l l areas o f the b r a i n . i n n e r v a t e lower b r a i n stem n u c l e i .  One NA  t r a c t descends from A6  A second t r a c t t r a v e l s l a t e r a l l y to  e n t e r the c e r e b e l l u m and t e r m i n a t e s i n the c e r e b e l l a r c o r t e x . t r a c t , the " d o r s a l b u n d l e " , ascends i n the MFB the v e n t r a l bundle.  The  b o d i e s , hypothalamus and  to  A third  and septum t o g e t h e r w i t h  d o r s a l bundle g i v e s o f f branches to the g e n i c u l a t e the t h a l a m i c n u c l e i b e f o r e t e r m i n a t i n g i n the  c e r e b r a l c o r t e x and hippocampus.  F i b e r s i n n e r v a t i n g the hypothalamus are  p a r t l y c r o s s e d , w h i l e those t r a v e l l i n g to the c o r t e x are a l l u n c r o s s e d . L e s i o n s t u d i e s i n d i c a t e t h a t c e r t a i n b r a i n a r e a s , such as the m e d i a l p r e o p t i c n u c l e u s , v e n t r a l s t r i a t e r m i n a l i s , i n f e r i o r o l i v e , habenular p a r t s o f the thalamus,  and some  r e c e i v e few i f any f i b e r s from the l o c u s c o e r u l e u s  (4-6) but t h i s n u c l e u s i s c e r t a i n l y r e s p o n s i b l e f o r a l l , c o r t i c a l noradrenergic i n n e r v a t i o n (7,8). systems to the c o r t e x are  or almost a l l ,  o f the  I t i s worth re-emphasis t h a t the  NA  ipsilateral.  Dopaminergic systems are not the c h i e f concern of t h i s t h e s i s but  the  c h e m i s t r y and drug e f f e c t s are o f t e n c l o s e l y r e l a t e d and f r e q u e n t mention w i l l be made o f the b e s t known and most t h o r o u g h l y s t u d i e d dopaminergic  tract.  This  F i g . 2. S a g i t t a l terminal f i e l d s .  p r o j e c t i o n s o f dopamine pathways i n the r a t . (From U n g e r s t e d t 1971).  S t r i p e d areas i n d i c a t e dense  - 5 -  a r i s e s i n the pigmented c e l l s o f the s u b s t a n t i a n i g r a and ascends  rostrally  t h r o u g h t h e m e d i a l f o r e b r a i n bundle t o i n n e r v a t e t h e e n t i r e s t r i a t u m ( c a u d a t e and putamen) ( F i g . 2 ) . A s i m i l a r t r a c t a r i s e s i n the v e n t r a l tegmentum j u s t m e d i a l t o the s u b s t a n t i a n i g r a and i n n e r v a t e s v a r i o u s l i m b i c n u c l e i , p a r t i c u l a r l y the n u c l e u s accumbens. are not p e r t i n e n t t o t h i s  2.  The other dopaminergic systems i n b r a i n  thesis.  Some W e l l Known Drugs Which A f f e c t Dopaminergic and N o r a d r e n e r g i c Systems A schematic diagram o f a n o r a d r e n e r g i c nerve ending i s shown i n F i g . 3  w i t h an i n d i c a t i o n o f the s i t e s o f a c t i o n o f v a r i o u s drugs d e s c r i b e d  below.  6-Hydroxydopamine (6-OHDA) i s a s e l e c t i v e n e u r o t o x i n w h i c h , depending upon the method o f a d m i n i s t r a t i o n , can be used to d e s t r o y n o r a d r e n e r g i c and/or dopaminergic systems i n b r a i n .  Because o f i t s s e l e c t i v i t y , i t has been a v e r y  u s e f u l t o o l i n a wide v a r i e t y o f i n v e s t i g a t i o n s on the anatomy, b i o c h e m i s t r y , pharmacology  and f u n c t i o n a l i m p l i c a t i o n s o f t h e c a t e c h o l a m i n e r g i c systems ( 9 ) .  6-OHDA i s a s t r u c t u r a l a n a l o g o f the c a t e c h o l a m i n e s and i s accumulated  into  c a t e c h o l a m i n e neurons by the a c t i v e uptake p r o c e s s e s which r e c o v e r much o f t h e dopamine and n o r a d r e n a l i n e r e l e a s e d from such neurons (10,11).  The s p e c i f i c i t y  o f 6-OHDA p r o b a b l y depends upon the s p e c i f i c i t y o f n e u r o n a l uptake mechanisms - i . e . i t does not k i l l  c h o l i n e r g i c or s e r o t o n e r g i c neurons because i t i s not  accumulated by them. The m o l e c u l a r mechanism o f the n e u r o t o x i c a c t i o n o f 6-OHDA remains  specul-  a t i v e b u t p r o b a b l y depends upon i t s easy o x i d a t i o n which may l e a d t o o- and pquinones ( 1 2 ) , t o p e r o x i d e s o r , p o s s i b l y , t o the f o r m a t i o n o f a s u p e r o x i d e radical.  Any one o f s e v e r a l h i g h l y r e a c t i v e r a d i c a l s might be t h e n e u r o t o x i c  agent ( 1 3 ) .  - 6 -  6-OHDA oL-methyl-p tyrosine  FLA-63 Disulfiram PargylineBB  Reserpine Amphetamine  Noradrenaline  Cocaine Amphetamine Chlorpromazine  F i g . 3. Schematic diagram o f n o r a d r e n e r g i c nerve ending. White r e c t a n g l e s and w h i t e arrows show endogenous m e t a b o l i t e s and t h e i r l o c a t i o n s . Black r e c t a n g l e s and b l a c k arrows show the s i t e o f a c t i o n o f v a r i o u s n o r a d r e n e r g i c drugs. ( M o d i f i e d from McGeer, E c c l e s and McGeer, 1978).  - 1The  n e u r o t o x i c e f f e c t s o f 6-OHDA f o l l o w i n g i n t r a v e n t r i c u l a r i n j e c t i o n a r e  i n i t i a l l y seen i n nerve t e r m i n a l a r e a s , w i t h s t r u c t u r a l damage and l o s s o f uptake mechanisms appearing the 6-OHDA (14-18).  as e a r l y as two hours a f t e r t h e a d m i n i s t r a t i o n o f  The n e u r o n a l  p e r i k a r y a seem l e s s s u s c e p t i b l e t o the  n e u r o t o x i c a c t i o n s than the nerve endings,  b u t b o t h e l e c t r o n m i c r o s c o p i c and  b i o c h e m i c a l evidence i n d i c a t e t h a t the p e r i k a r y a i n the s u b s t a n t i a n i g r a and l o c u s c o e r u l e u s b e g i n t o degenerate w i t h i n 1 t o 2 days  (19-22).  Ot-Methyl-p- t y r o s i n e i s a s e l e c t i v e i n h i b i t o r o f t y r o s i n e h y d r o x y l a s e , the key enzyme i n the s y n t h e s i s o f b o t h dopamine and n o r a d r e n a l i n e .  Treatment o f  r a t s w i t h t h i s m a t e r i a l r e s u l t s i n l a r g e and s e l e c t i v e d e p l e t i o n s o f these amines from b o t h p e r i p h e r a l and c e n t r a l s t o r e s ( 2 3 ) . Reserpine  i s an agent which d e s t r o y s the c a p a c i t y o f v e s i c l e s to b i n d  dopamine, n o r a d r e n a l i n e  and c e r t a i n other n e u r o t r a n s m i t t e r s .  p r o t e c t i o n , the r e l e a s e d amines are m e t a b o l i z e d  rapidly.  causes p r o f o u n d d e p l e t i o n s i n the l e v e l s o f these lasts  Lacking v e s i c u l a r  Hence, r e s e r p i n e  transmitters.  The e f f e c t  f o r s e v e r a l days a f t e r a s i n g l e dose o f r e s e r p i n e because the v e s i c l e  b i n d i n g c a p a c i t y i s i r r e v e r s i b l y destroyed  and new v e s i c u l a r p r o t e i n must be  s y n t h e s i z e d and t r a n s p o r t e d from the p e r i k a r y a to the nerve endings b e f o r e e f f e c t i v e storage can be r e s t o r e d . C h l o r p r o m a z i n e and o t h e r p h e n o t h i a z i n e s  b l o c k n o r a d r e n a l i n e , dopamine and  s e r o t o n i n r e c e p t o r s , w i t h d i f f e r e n t members o f the s e r i e s showing some v a r i a b i l i t y i n t h e i r a c t i v i t y a t the v a r i o u s r e c e p t o r s . H a l o p e r i d o l and o t h e r butyrophenones a r e r e c e p t o r b l o c k e r s which tend t o be p a r t i c u l a t l y a c t i v e a t dopaminergic as opposed to n o r a d r e n e r g i c nergic receptors.  or s e r o t o -  - 8 Amphetamine i n h i b i t s t h e reuptake o f the c a t e c h o l a m i n e s , c a t e c h o l a m i n e r e l e a s e and i n h i b i t s MAO s l i g h t l y .  A l l three a c t i o n s tend to  p o t e n t i a t e the a c t i o n o f c a t e c h o l a m i n e s i n t h e s y n a p t i c The  t r i c y c l i c antidepressants  s t i m u l a t e s some  cleft.  are i n h i b i t o r s o f the r e u p t a k e o f n o r a d r e n -  a l i n e and s e r o t o n i n , w i t h some v a r i a t i o n i n t h e a c t i v i t y o f t h e v a r i o u s on the two systems.  drugs  S i n c e r e u p t a k e i s a major mechanism f o r removing the  amines from the s y n a p t i c c l e f t , the r e u p t a k e i n h i b i t o r s p o t e n t i a t e g r e a t l y t h e s y n a p t i c a c t i o n o f these amines. C o c a i n e i n h i b i t s the r e u p t a k e o f n o r a d r e n a l i n e  and dopamine i n t o  synaptic  nerve endings and thus p o t e n t i a t e s t h e i r a c t i o n s . P r o p r a n o l o l i s a 8-adrenergic receptor Isoproterenol i s a d i r e c t noradrenergic  blocker. agonist active at ^-adrenergic  receptors. Phenoxybenzamine i s an a - a d r e n e r g i c  receptor  blocker.  Apomorphine i s a d i r e c t dopaminergic a g o n i s t . 2+ FLA-63 i s a v e r y potent Cu  chelating disulfide.  T h i s drug i n h i b i t s  dopamine-8-hydroxylase a c t i v i t y and causes a v e r y r a p i d and complete d e p l e t i o n of c e n t r a l NA s t o r e s 3.  cAMP and N e u r a l  (24). Function  cAMP i s now b e l i e v e d to be an i n t r a c e l l u l a r r e g u l a t o r y agent f o r a l a r g e number o f c e l l u l a r p r o c e s s e s .  I t s o c c u r r e n c e has been demonstrated i n a l l  a n i m a l s p e c i e s and t i s s u e s i n v e s t i g a t e d ( 2 5 ) . synthesis i s adenylate  cyclase.  The c r i t i c a l enzyme f o r i t s  The h i g h e s t l e v e l s o f b o t h a d e n y l a t e  a c t i v i t y and cAMP i n most a d u l t animals are found i n the b r a i n ( 2 6 ) .  cyclase Therefore  - 9 a g r e a t d e a l o f a t t e n t i o n has been f o c u s s e d  on the problem o f how cAMP  f u n c t i o n s i n nervous t i s s u e . E a r l y support  f o r a r o l e f o r cAMP i n n e u r o t r a n s m i s s i o n  i n the CNS came from  the d i s c o v e r y t h a t e l e c t r i c a l s t i m u l a t i o n o f b r a i n s l i c e s r e s u l t e d i n i n c r e a s e d cAMP l e v e l s and from the demonstration  o f the s u b c e l l u l a r l o c a l i z a t i o n i n the  synaptosomal f r a c t i o n o f t h e enzymes i n v o l v e d i n t h e cAMP g e n e r a t i n g  system  (27). A r o l e f o r cAMP has been p o s t u l a t e d i n three aspects tioning:  mediation  of neurotransmitter  of neuronal  func-  a c t i o n on p o s t s y n a p t i c membranes,  r e g u l a t i o n o f n e u r o t r a n s m i t t e r b i o s y n t h e s i s ( 2 8 ) , and f u n c t i o n i n g o f m i c r o t u b u l e s ( 2 9 ) . The f i r s t o f these r o l e s i s t h e one f o r which t h e r e i s t h e most experimental  evidence and i t i s t h i s w i t h which t h i s t h e s i s w i l l be p r i m a r i l y  concerned. When n e u r o t r a n s m i t t e r s  are r e l e a s e d from p r e s y n a p t i c t e r m i n a l s , they a r e  thought t o induce a change i n p o s t s y n a p t i c membrane p o t e n t i a l by r e a c t i n g w i t h s p e c i f i c r e c e p t o r s on the p o s t s y n a p t i c membrane.  I n the case o f some t r a n s -  m i t t e r s and some type o f r e c e p t o r s , such as a c e t y l c h o l i n e a t t h e neuromuscular j u n c t i o n or w i t h GABA i n the c e n t r a l nervous system, the a c t i o n i s b e l i e v e d to i n v o l v e a r a p i d change i n i o n i c f l u x e s through t h e membrane.  In other  i n s t a n c e s , i t i s b e l i e v e d t h a t the p o s t s y n a p t i c a c t i o n o f the n e u r o t r a n s m i t t e r may be t h e i n d u c t i o n o f a s e r i e s o f c h e m i c a l  r e a c t i o n s w h i c h render t h e c e l l  membrane more or l e s s s e n s i t i v e to other i n p u t s .  Substances w i t h t h i s type o f  a c t i o n have been termed by some neuromodulators (30) and by o t h e r s  "metabo-  t r o p i c n e u r o t r a n s m i t t e r s " as opposed to the " i o n o t r o p i c n e u r o t r a n s m i t t e r s " e x e m p l i f i e d by t h e c l a s s i c a l case o f a c e t y l c h o l i n e a t t h e neuromuscular j u n c t i o n ( 3 1 ) . There i s c o n s i d e r a b l e evidence t h a t the c y c l i c n u c l e o t i d e s  - 10 (cAMP and cGMP), may p l a y key r o l e s i n the p o s t s y n a p t i c a c t i o n o f many o f these s o - c a l l e d neuromodulators or m e t a b o t r o p i c n e u r o t r a n s m i t t e r s . evidence i s a v a i l a b l e w i t h regard the c a t e c h o l a m i n e r g i c  The best  t o a key r o l e f o r cAMP i n the f u n c t i o n i n g o f  neurotransmitters,  dopamine and n o r a d r e n a l i n e ,  but there  i s a l s o e v i d e n c e t h a t cGMP i s i n v o l v e d i n t h e a c t i o n o f a c e t y l c h o l i n e a t m u s c a r i n i c , as opposed to n i c o t i n i c , The  initial,  receptors.  and perhaps s t i l l t h e b e s t , e v i d e n c e f o r a r e c e p t o r r o l e o f  cAMP comes from s t u d i e s o f the s u p e r i o r c e r v i c a l g a n g l i o n contains  c h o l i n e r g i c p r e g a n g l i o n i c f i b e r s and a dopaminergic  synapsing  f i b e r s are s t i m u l a t e d , a c e t y l c h o l i n e i s released at the  post-ganglionic n i c o t i n i c receptor not i n v o l v e any c y c l i c n u c l e o t i d e . muscarinic  interneuron  on the p o s t g a n g l i o n i c neuron ( F i g . 4 ) .  When p r e g a n g l i o n i c  receptors  c a u s i n g a f-EPSP; t h i s a c t i o n p r o b a b l y  does  These f i b e r s , however, a l s o synapse a t  on the p o s t g a n g l i o n i c neuron and the a c t i o n o f a c e t y l -  c h o l i n e a t these r e c e p t o r s cGMP.  (SCG), which  causes a s-EPSP which i s b e l i e v e d t o be mediated by  The p r e g a n g l i o n i c c h o l i n e r g i c f i b e r s a l s o synapse on dopaminergic  i n t e r n u n c i a l neurons; the r e l e a s e o f dopamine from these neurons onto t h e a adrenergic  postganglionic receptors  mediated by cAMP (32-34).  g e n e r a t e s a s-IPSP which i s thought to be  E v i d e n c e f o r such a mechanism comes from t h e f a c t  t h a t b o t h e l e c t r i c a l s t i m u l a t i o n o f the p r e g a n g l i o n i c f i b e r s and the a p p l i c a t i o n o f exogenous dopamine t o g a n g l i o n i c s l i c e s r e s u l t i n an i n c r e a s e d cAMP l e v e l i n the p o s t g a n g l i o n i c neuron (35-37).  A dopamine-sensitive  adenylate  c y c l a s e has been found i n t h e g a n g l i o n as w e l l as i n t h e caudate n u c l e u s and other areas o f the CNS which c o n t a i n e x t e n s i v e dopaminergic i n n e r v a t i o n ( 3 8 ) . Exogenous a p p l i c a t i o n o f cAMP t o the g a n g l i o n causes p o s t g a n g l i o n i c h y p e r p o l a r i z a t i o n (39).  The p o s t u l a t e d sequence o f steps i s i n d i c a t e d i n F i g . 5.  Muiconmc Cholinergic  ACh b l o c k e d by h e i o m t t h o n i u m PRE-  Nicotinic  GANGLIONIC  POSTGANGLIONIC  CHOLINERGIC FIBRES  blocked by otropme  C  e  NEURONE  R  - I - b l o c k e d by a - o d r e n t r g i c ' antagonists DOPAMINE  ^  °'C\ CAMP  POSTGANGLIONIC SYNAPTIC POTENTIALS  INTERNEURONE - | -  b l o c k e d by a t r o p i n e  ACh  T F i g . 4. Schematic diagram o f s y n a p t i c c o n n e c t i o n s i n the s u p e r i o r c e r v i c a l , g a n g l i o n and p o s t u l a t e d r o l e o f c y c l i c nucleotides i n the genesis o f postganglionic synaptic p o t e n t i a l s . (From Greengard 1976).  - 12 Dopamine s t i m u l a t e s a s p e c i f i c a d e n y l a t e of cAMP.  c y c l a s e t o produce i n c r e a s e d l e v e l s  The cAMP i n t u r n i s b e l i e v e d to s t i m u l a t e the p h o s p h o r y l a t i o n o f  s p e c i f i c s u b s t r a t e p r o t e i n s i n the membrane by cAMP-dependent p r o t e i n k i n a s e s (PK), such p h o s p h o r y l a t i o n  a f f e c t i n g membrane p e r m e a b i l i t y  characteristics.  Such cAMP-dependent p r o t e i n k i n a s e s have been found i n m i c r o s o m a l f r a c t i o n s o f bovine b r a i n and shown to be c a p a b l e o f c a t a l y z i n g the p h o s p h o r y l a t i o n o f endogenous membrane-bound s u b s t r a t e p r o t e i n s from s y n a p t i c membrane f r a c t i o n s (40,41).  The s p e c i f i c i t y o f cAMP a c t i o n i n d i f f e r e n t c e l l types c o u l d be  accounted f o r by the s p e c i f i c i t y o f v a r i o u s phosphokinases and the s u b s t r a t e proteins.  Two p r o t e i n s have a l r e a d y been found which seem t o be s p e c i f i c to  nervous t i s s u e and a r e p h o s p h o r y l a t e d kinases  w i t h i n 5 sec by endogenous phospho-  a f t e r cAMP a c t i v a t i o n . These p r o t e i n s are c o n c e n t r a t e d  i n the s y n a p t i c  membrane f r a c t i o n on s u b c e l l u l a r f r a c t i o n a t i o n and t h e i r c o n c e n t r a t i o n i n t i s sue  i n c r e a s e s i n the immediate p o s t n a t a l p e r i o d d u r i n g the time when s y n a p t i c  s t r u c t u r e s develop.  Moreover, t h e r e i s e v i d e n c e t h a t t h e r e i s a good c o r -  r e l a t i o n between the degree o f p h o s p h o r y l a t i o n the degree o f i o n i c f l u x o b t a i n e d  o f one o f these p r o t e i n s and  i n response t o t h e a c t i o n o f 8 - a g o n i s t s  (42,43). I n the c e n t r a l nervous system there i s evidence t h a t cAMP i s i n v o l v e d i n the p o s t s y n a p t i c a c t i o n s o f b o t h n o r a d r e n a l i n e  and dopamine and i t i s w i t h t h e  former catecholamine t h a t t h i s t h e s i s i s p r i m a r i l y concerned. s e c t i o n 1-1 ( F i g . 1 ) , the n o r a d r e n e r g i c  systems o f b r a i n have v e r y  s i t e s o f o r i g i n i n the b r a i n stem b u t r a m i f y v e r y b r o a d l y the b r a i n i n c l u d i n g the c e r e b e l l u m  As i n d i c a t e d i n  and c e r e b r a l c o r t e x .  to i n n e r v a t e most o f Most o f t h e a v a i l a b l e  evidence upon the p r o b a b l e importance o f cAMP i n the n o r a d r e n e r g i c comes from work on the c e r e b e l l u m  limited  receptor  and c e r e b r a l c o r t e x i n v i v o o r i n v i t r o .  Altered Microtubular Function Cyclic A  PRESYNAPTIC NEURON  Protein Kinase  PR0TEIN-P0 4  ion c o n d u c t a n c e ? eltctrogenic pump ?  to  I  POSTSYNAPTIC NEURON /  F i g . 5. of t h i s through synaptic  5'AMP ATP  Proposed r o l e s f o r cAMP and p r o t e i n p h o s p h o r y l a t i o n i n n e u r o n a l f u n c t i o n . A key element model i s t h a t t h e second messenger, dopamine, a f f e c t s t h e membrane p o t e n t i a l o f t h e c e l l the p r o t e i n k i n a s e system and t h e s u b s t r a t e p r o t e i n c o n t r o l s the p e r m e a b i l i t y o f the p o s t membrane. (From Greengard 1976).  - 14 I n v i v o i t has been shown t h a t the spontaneous d i s c h a r g e  of c e r e b e l l a r  P u r k i n j e c e l l s i s i n h i b i t e d by i o n t o p h o r e t i c a p p l i c a t i o n s o f n o r a d r e n a l i n e cAMP which cause a h y p e r p o l a r i z a t i o n o f the P u r k i n j e c e l l membrane. i n h i b i t o r y e f f e c t of noradrenaline  or  The  on P u r k i n j e c e l l s i s p o t e n t i a t e d by phospho-  d i e s t e r a s e i n h i b i t o r s ; such i n h i b i t i o n i s c o n s i s t e n t w i t h a m e d i a t i o n by cAMP since degradation  o f cAMP i s accomplished by a p h o s p h o d i e s t e r a s e ( F i g . 5) (44).  S i m i l a r l y , iontophoretic a p p l i c a t i o n s of noradrenaline  or cAMP t o p y r a m i d a l  t r a c t neurons o f the c e r e b r a l c o r t e x cause an i n h i b i t i o n o f t h e s e neurons, w h i l e a p p l i c a t i o n o f e i t h e r a c e t y l c h o l i n e or cGMP r e s u l t s i n p y r a m i d a l e x c i t a t i o n (45,46). I n i n v i t r o work, i t has been shown t h a t i n c u b a t i o n o f s l i c e s o f r a t c e r e b r a l c o r t e x w i t h 10  noradrenaline  causes a t w o - f o l d  i n c r e a s e i n the  cAMP l e v e l and t h a t t h i s e f f e c t can be enhanced by p r e t r e a t m e n t w i t h  reserpine.  -4 Maximal s t i m u l a t i o n o c c u r r e d i n c u b a t i o n (47). adrenaline (48),  a t 10  S i m i l a r increases  M noradrenaline  a f t e r a s i x minute  i n cAMP l e v e l s on i n c u b a t i o n w i t h n o r -  have a l s o been found i n c e r e b r a l c o r t i c a l s l i c e s from guinea p i g s  r a b b i t s (49,50), and humans (51).  The p r e f e r r e d time o f i n c u b a t i o n  v a r i e d from 2 to 6 min and the c o n c e n t r a t i o n s  - 5 - 4  a p p r o x i m a t e l y 10  t o 10  M.  .  .  .  I n some s p e c i e s , such as the guinea p i g ,  cAMP l e v e l s i n the s l i c e s a l s o i n c r e a s e d amines such as h i s t a m i n e  g i v i n g maximal response from  on i n c u b a t i o n w i t h other  and s e r o t o n i n (48);  as the human (51) and r a t (48),  i n s l i c e s from other  these other n e u r o t r a n s m i t t e r s  biogenic species  such  had no e f f e c t .  I t seems p r o b a b l e , i n any case, t h a t the e f f e c t s o f d i f f e r e n t t r a n s m i t t e r s are not mediated through e x a c t l y the same a d e n y l a t e c y c l a s e but e i t h e r through d i f f e r e n t forms o f the enzyme or through the same enzyme l i n k e d i n v a r y i n g f a s h i o n t o other components o f the membrane which c o n f e r r e c e p t o r  specificity.  cAMP f o r m a t i o n i n b r a i n s l i c e s can a l s o be s t i m u l a t e d by other m a t e r i a l s which are not now g e n e r a l l y accepted  as n e u r o t r a n s m i t t e r s .  Adenosine, f o r  example, which has been proposed as a n e u r o t r a n s m i t t e r but i s not y e t g e n e r a l l y accepted,  can a p p a r e n t l y a l s o s t i m u l a t e cAMP f o r m a t i o n by a c t i v a t i o n o f mem-  brane r e c e p t o r s ; i t can a l s o be i n c o r p o r a t e d i n t o the cAMP p r e c u r s o r p o o l D e p o l a r i z i n g agents,  (52).  such as p o t a s s i u m , v e r a t r i d i n e and ouabain, a l s o s t i m u l a t e  cAMP p r o d u c t i o n and i t has been suggested t h a t these may a c t through r e l e a s e of adenosine as an i n t e r m e d i a r y p r o c e s s .  D e p o l a r i z i n g agents cause an i n c r e a s e  i n i n t r a c e l l u l a r sodium c o n c e n t r a t i o n and t h i s e f f e c t can be b l o c k e d by membrane s t a b i l i z e r s such as cocaine which prevent  b o t h the i n c r e a s e i n sodium  p e r m e a b i l i t y and t h e r i s e i n cAMP l e v e l s ( 5 3 ) .  I n human b r a i n s l i c e s , t h e  effects of noradrenaline  and v e r a t r i d i n e on cAMP l e v e l s are r e p o r t e d t o be  a d d i t i v e (54). Sodium f l u o r i d e i s another agent which w i l l  s t i m u l a t e i n c r e a s e s i n cAMP  f o r m a t i o n i n b r a i n homogenates; i n t h i s case, however, adenosine has been r e p o r t e d to i n h i b i t the e f f e c t ( 5 5 ) . Increases  i n cAMP l e v e l s induced  by e i t h e r e l e c t r i c a l s t i m u l a t i o n o r  2+ d e p o l a r i z i n g agents are dependent upon the presence o f Ca . Low concen2+ . . . t r a t i o n s o f Ca c a n , by themselves, s t i m u l a t e cAMP a c c u m u l a t i o n , w h i l e h i g h c o n c e n t r a t i o n s have an i n h i b i t o r y e f f e c t ( 5 6 ) .  Excitation of cells results i n  2+ i n c r e a s e d i n t r a c e l l u l a r c o n c e n t r a t i o n s o f Ca  and these i n c r e a s e d concen-  t r a t i o n s r e s u l t i n a c t i v a t i o n o f the p h o s p h o d i e s t e r a s e a r a p i d c o n t r o l mechanism f o r cAMP l e v e l s  a c t i v i t y which p r o v i d e s  (57,58).  As might be expected f o r an enzyme b e l i e v e d t o be i n v o l v e d i n t i m a t e l y i n neuronal ation.  transmission, adenylate  c y c l a s e a c t i v i t y i n b r a i n changes w i t h matur-  I n r a t s , f o r example, the c e r e b r a l c o r t e x undergoes c o n s i d e r a b l e  post-  - 16 n a t a l development r e a c h i n g a d u l t morphology a t about 15 days o f age ( 5 9 ) . Basal adenylate cyclase a c t i v i t y i n t h i s  structure increases approximately  f o u r - f o l d from b i r t h t o about 21 days o f age w h i l e t h e maximum response  to the  2+ c a t e c h o l a m i n e s , t o Ca two weeks ( 6 0 ) .  and to sodium f l u o r i d e i s reached a t a p p r o x i m a t e l y  Receptor  s p e c i f i c i t y a l s o seems t o develop  c o r t i c a l a d e n y l a t e c y c l a s e o f newborn r a t s responds  postnatally;  to both dopamine and  s e r o t o n i n but t h i s r e s p o n s i v e n e s s decreases w i t h age. Responsiveness n o r a d r e n a l i n e i s markedly reflects  decreased  i n senescent  animals ( 6 1 ) ; t h i s  to probably  a l o s s o f d e n d r i t i c s p i n e s and t h e s y n a p t i c c o n t a c t s t h e r e o n .  o n l y p a r t o f the g e n e r a l p a t t e r n which  It is  i s b e g i n n i n g to emerge i n d i c a t i n g a  l o s s o f n e u r o n a l f u n c t i o n i n g i n normal a g i n g t h a t may account  f o r many o f t h e  decrements i n b e h a v i o r and changes i n p h a r m a c o l o g i c a l s e n s i t i v i t y which have been noted i n senescent animals and humans. 4.  Supersensitivity S u p e r s e n s i t i v i t y has been d e f i n e d as the phenomenon i n which  an a g o n i s t r e q u i r e d t o produce a g i v e n response measured by a s h i f t o f the dose-response  the amount o f  i s l e s s than normal.  It is  curve f o r t h a t a g o n i s t to the l e f t .  T h i s s h i f t may sometimes be accompanied by an i n c r e a s e i n t h e maximum response and/or a change i n the s l o p e o f the dose-response  curve.  the o p p o s i t e phenomenon w i t h t h e o p p o s i t e m a n i f e s t a t i o n s . D e n e r v a t i o n ' (62) has been m o d i f i e d by F l e m i n g Innervation': decreased  Subsensitivity i s Cannon's 'Law o f  (63) t o become the 'Law o f  "When f u n c t i o n a l nerve a c t i v i t y i s c h r o n i c a l l y i n c r e a s e d o r  (surgically,  p h y s i o l o g i c a l l y , p a t h o l o g i c a l l y or p h a r m a c o l o g i c a l l y ) ,  the s e n s i t i v i t y o f most d i s t a l e f f e c t o r s t o any p r o c e s s which i n i t i a t e s a  response i n the e f f e c t o r i s s l o w l y for  was  the a l t e r e d n e u r a l  a l t e r e d i n a d i r e c t i o n which w i l l  input".  The  procedures by which s u p e r s e n s i t i v i t y can be achieved  1)  s u r g i c a l d e n e r v a t i o n and  2)  pharmacological denervation,  3)  b l o c k a d e of r e c e p t o r or e f f e c t o r organ,  4)  p r e v e n t i o n of r e l e a s e  of  5)  a l t e r a t i o n of sensory  stimuli,  6)  s u r g i c a l a b l a t i o n of a f f e r e n t  The  f i r s t t i s s u e f o r which a mechanism of s u p e r s e n s i t i v i t y was  s k e l e t a l muscle (64).  an i n n e r v a t e d m u s c l e , but tivity  slowly  decentralization,  transmitter,  plate  S u p e r s e n s i t i v i t y was  (b) r e p r e s e n t a r e t u r n  and  due  i s s e n s i t i v e to a c e t y c h o l i n e  postulated  to:  p o t e n t i a l and will not  c a p a c i t a n c e , an i n c r e a s e  a s p e c i f i c i t y for acetylcholine  accept i n n e r v a t i o n  by  sensi-  due  to a spread  to a p r e - i n n e r v a t e d s t a t e of the muscle, and  S u p e r s e n s i t i v i t y i n s k e l e t a l muscle i s f u r t h e r c h a r a c t e r i z e d and  in  f i b e r responds  (a) be  to a l o s s of c o n t a c t between t r a n s m i t t e r  membrane r e s i s t a n c e  proposed  days a f t e r d e n e r v a t i o n , the a r e a of  of r e c e p t o r s , ( c ) to be  pathways.  Only the end several  include:  spreads outward u n t i l the e n t i r e s u r f a c e of the  to a c e t y l c h o l i n e .  compensate  transplanted  end  by an  i n the d u r a t i o n  (65).  organ. increased  of the  action  Denervated s k e l e t a l muscle  n e r v e , w h i l e i n n e r v a t e d muscle w i l l  (66). S u p e r s e n s i t i v i t y i n smooth muscle, u n l i k e t h a t i n s k e l e t a l muscle, i s not  s p e c i f i c for a single transmitter.  I t depends on a change i n p e r m e a b i l i t y  or  2+ c o n t r a c t i l e mechanisms and  a change i n Ca  binding  (67).  Exocrine glands  a l s o e x h i b i t s u p e r s e n s i t i v i t y , which tends to resemble t h a t i n smooth muscle rather  than t h a t  i n s k e l e t a l muscle.  S a l i v a r y g l a n d s , f o r example, e x h i b i t  - 18 non-specific increased  supersensitivity.  F u r t h e r m o r e , when t h e g l a n d i s exposed t o  amounts o f a c e t y l c h o l i n e by the a d m i n i s t r a t i o n  of a cholinesterase  i n h i b i t o r , such as p h y s o s t i g m i n e , t h e g l a n d becomes s u b s e n s i t i v e . S u p e r s e n s i t i v i t y to n o r a d r e n a l i n e been e x t e n s i v e l y  i n v o l v i n g changes i n the cAMP system has  s t u d i e d i n the p i n e a l g l a n d .  c i r c a d i a n i n f l u e n c e , so t h a t d u r i n g  The p i n e a l g l a n d i s under  the dark c y c l e n o r a d r e n e r g i c i n p u t  from  the SCG i n c r e a s e s .  NA a c t i v a t e s a cAMP system w h i c h , i n t u r n , a c t i v a t e s an  N-acetyltransferase  t h a t i s a r a t e - c o n t r o l l i n g enzyme i n the b i o s y n t h e s i s o f  melatonin.  L i g h t , denervation,  produce a s u p e r s e n s i t i v e input (68).  ganglionectomy and 6-OHDA i n j e c t i o n a l l  cAMP response to NA by d e p l e t i n g the n o r a d r e n e r g i c  S u p e r s e n s i t i v i t y o f the cAMP system i s accompanied by a s u p e r -  i n d u c t i o n o f the N - a c e t y l t r a n s f e r a s e  (69,70).  The enzyme i n d u c t i o n , but not  the cAMP response, i s dependent on p r o t e i n s y n t h e s i s . response can be a b o l i s h e d o f NA i t s e l f .  by i n j e c t i o n s o f the NA a g o n i s t ,  changes i n the p i n e a l g l a n d i s v e r y r a p i d .  denervation, generating binding  i s o p r o t e r e n o l , or  Repeated i n j e c t i o n s o f i s o p r o t e r e n o l or extended p e r i o d s  render the p i n e a l cAMP system s u b s e n s i t i v e .  proterenol  The s u p e r s e n s i t i v i t y  o f dark  The development o f s e n s i t i v i t y Subsensitivity, after iso-  a d m i n i s t r a t i o n , develops a f t e r 6 hours and s u p e r s e n s i t i v i t y , a f t e r w i t h i n 24 h o u r s .  The changes i n s e n s i t i v i t y o f the cAMP  system are p a r a l l e l e d by changes i n the number o f $ - a d r e n e r g i c  s i t e s (71,72).  Measurement o f t h e number and a f f i n i t y o f sodium-  independent, s a t u r a b l e b i n d i n g  s i t e s i s now w i d e l y  i n d e x o f the d e n s i t y and s e n s i t i v i t y o f r e c e p t o r s  accepted as a  biochemical  (73).  A l t h o u g h i t i s w e l l e s t a b l i s h e d t h a t d e n e r v a t i o n produces s u p e r s e n s i t i v i t y i n p e r i p h e r a l e f f e c t o r organs, i t i s not p o s s i b l e t o e x t r a p o l a t e mechanisms to the CNS.  peripheral  S h a r p l e s s (74) asks what k i n d o f f u n c t i o n a l changes  - 19 would be expected i n CNS  pathways i f neurons responded to d i s u s e .  s t r u c t u r e s are r e l a t i v e l y  simple to study, w h i l e CNS  Peripheral  s t r u c t u r e s are v e r y  c o m p l i c a t e d , w i t h e l a b o r a t e feed-back i n p u t s on each c e l l .  However, t h e r e  f u n c t i o n a l as w e l l as n e u r o c h e m i c a l o b s e r v a t i o n s  t h a t CNS  a l s o respond to d i s u s e .  The  suggesting  temperature-regulating  are  systems  c e n t e r o f the hypothalamus  s l o w l y develops a r e v e r s i b l e s u p e r s e n s i t i v i t y as a r e s u l t o f scopolamine administration  (75).  F r o n t a l lobectomy r e s u l t s i n an i n c r e a s e i n  convulsibility  and t h a l a m i c l e s i o n s r e s u l t i n h y p e r e x c i t a b i l i t y of the i p s i l a t e r a l c o r t e x (76).  Many b e h a v i o r a l r e p o r t s do not d e f i n e the t r a n s m i t t e r systems which are  i n v o l v e d i n the s u p e r s e n s i t i v i t y phenomenon.  There a r e , however, few  data  i n d i c a t i n g the development of s u p e r s e n s i t i v i t y to other t r a n s m i t t e r substances but the most e x t e n s i v e and p e r t i n a n t l i t e r a t u r e , however, i s concerned w i t h changes i n r e c e p t o r s e n s i t i v i t y to dopamine i n the s t r i a t u m and a d r e n a l i n e i n the c e r e b r a l c o r t e x .  There are c o n s i d e r a b l e b e h a v i o r a l ( 7 7 ) ,  p h y s i o l o g i c a l (78) and b i o c h e m i c a l  (79,80) data i n d i c a t i n g s u p e r s e n s i t i v i t y o f  s t r i a t a l dopaminergic r e c e p t o r s a f t e r d e n e r v a t i o n amine-depleting  to n o r -  or b l o c k i n g agents.  or c h r o n i c treatment  Most groups who  with  have s t u d i e d the problem  f i n d t h a t s u p e r s e n s i t i v i t y i s accompanied by an enhanced response of  adenylate  c y c l a s e i n the s t r i a t a l homogenates to exogenous dopamine (81-83) but there i s at l e a s t one group who adenylate  has  f a i l e d to f i n d any change i n d o p a m i n e - s t i m u l a t e d  c y c l a s e a c t i v i t y i n r a t s showing b e h a v i o r a l s u p e r s e n s i t i v i t y ( 8 4 ) .  S t u d i e s o f p o s s i b l e changes i n s e n s i t i v i t y i n n o r a d r e n e r g i c been more dependent upon c h e m i c a l  as opposed to b e h a v i o r a l measures because  t h e r e has been no c l e a r d e f i n i t i o n c o r t i c a l noradrenergic  systems.  response of c o r t i c a l a d e n y l a t e  r e c e p t o r s have  of the b e h a v i o r a l r o l e of the c e r e b r a l  S u p e r s e n s i t i v i t y , as measured by an enhanced c y c l a s e a c t i v i t y to n o r a d r e n a l i n e  i n ^n  vitro  - 20 i n c u b a t i o n s , has been found i n r a t s f o l l o w i n g d e n e r v a t i o n by a b l a t i o n o f t h e locus coeruleus reserpine (93).  (85), chemical  l e s i o n s w i t h 6-OHDA (86,87),  or treatment  The s u p e r s e n s i t i v e response i s s p e c i f i c f o r a and 8 a g o n i s t s ,  r e v e r s i b l e and c h a r a c t e r i z e d by a s h i f t to the l e f t o f the dose-response The  with  i n t e r p r e t a t i o n o f t h e data i n l e s i o n e d animals  curve.  can be complex a t c e r t a i n  times a f t e r the l e s i o n s because o f l e s i o n - i n d u c e d changes i n the p r e s y n a p t i c nerve t e r m i n a l s .  T h i s i s p a r t i c u l a r l y t r u e w i t h 6-OHDA l e s i o n s , because o f  the pronounced s e n s i t i v i t y o f the t e r m i n a l s to 6-OHDA (see s e c t i o n 1-2). Noradrenaline  i s g e n e r a l l y removed from t h e s y n a p t i c c l e f t l a r g e l y by t h e  h i g h a f f i n i t y (uM range) n e u r o n a l l e s i o n i n g i s the degeneration amine uptake mechanisms.  r e u p t a k e system.  An e a r l y e f f e c t o f 6-OHDA  o f t h e nerve t e r m i n a l s w i t h impairment o f t h e  One consequence i s t h a t exogenous  noradrenaline  added to i n c u b a t e d s l i c e s may remain i n c o n t a c t w i t h the p o s t s y n a p t i c l o n g e r than would be the case i n s l i c e s w i t h i n t a c t nerve t e r m i n a l s . r e s u l t i n an apparent " s u p e r s e n s i t i v i t y " . to t h a t o c c u r r i n g i f cocaine and  receptors T h i s may  This presynaptic e f f e c t i s s i m i l a r  (an uptake i n h i b i t o r ) i s added t o the i n c u b a t e s  develops 24 t o 72 hours a f t e r p r o d u c t i o n o f t h e l e s i o n i n 6-OHDA-treated  animals.  The t r u e s u p e r s e n s i t i v i t y which i s due to p o s t s y n a p t i c changes i s  not p o t e n t i a t e d by c o c a i n e and was found t o develop 72 t o 96 hours a f t e r 6-OHDA-treatment ( 8 9 ) . K a l i s k e r e_t a l . (89) d i d f u r t h e r s t u d i e s on the e a r l y and l a t e components o f t h e changes i n t h e cAMP system i n the c o r t e x f o l l o w i n g 6-OHDA-induced lesions.  C o n s i s t e n t w i t h the p o s t u l a t e t h a t the e a r l y component r e f l e c t s  f a i l u r e o f the p r e s y n a p t i c reuptake mechanisms, they found t h a t t h e e a r l y p o t e n t i a t i o n o f the s t i m u l a t i o n o f cAMP f o r m a t i o n was n o t apparent w i t h i s o p r o t e r e n o l , was l i m i t e d t o low NA c o n c e n t r a t i o n s  and f o l l o w e d t h e same t i m e  course  as the r e d u c t i o n o f h i g h a f f i n i t y  [ H]-NA a c c u m u l a t i o n . J  Isoproterenol  i s a NA a g o n i s t which i s not accumulated by the h i g h a f f i n i t y uptake system. H i g h c o n c e n t r a t i o n s o f NA (30 uM) accumulate by a low a f f i n i t y uptake system which i s not i n h i b i t e d by c o c a i n e and does not i n v o l v e s p e c i f i c , t r a n s p o r t systems.  presynaptic  The e f f e c t s o f b o t h i s o p r o t e r e n o l and NA on cAMP f o r m a t i o n  were p o t e n t i a t e d by 72 t o 96 h r s a f t e r t h e l e s i o n , i n d i c a t i n g t h e p o s t s y n a p t i c mechanism a t t h a t time.  I t was not c l e a r , however, from K a l i s k e r ' s d a t a ,  whether the p o s t s y n a p t i c e f f e c t i n v o l v e d a o r $ r e c e p t o r s , as b o t h p h e n t o l amine and p r o p r a n o l o l i n h i b i t e d the e f f e c t to the same degree. Recent r e p o r t s suggest t h a t 8 r e c e p t o r s a r e more l i k e l y than a t o be i n v o l v e d i n the changes i n r e s p o n s i v e n e s s 6-OHDA treatment.  o f the cAMP-generating system a f t e r  Membrane p r e p a r a t i o n s o f r a t c e r e b r a l c o r t e x were  incubated  3 3 w i t h the a and 8 r a d i o l i g a n d s , [ H]-WB-4101 and [ H ] - d i h y d r o a l p r e n o l o n e r e s p e c t i v e l y , a f t e r i n t r a v e n t r i c u l a r i n j e c t i o n s o f 6-OHDA. e x h i b i t e d a g r e a t e r degree o f 8 l i g a n d b i n d i n g ( 9 0 ) .  Treated  animals  The s l o p e s o f the  b i n d i n g curves were i d e n t i c a l f o r c o n t r o l and t r e a t e d a n i m a l s , p o i n t i n g t o an i n c r e a s e i n the number o f b i n d i n g s i t e s o f a s i n g l e h i g h - a f f i n i t y class.  The time course  w i t h the time course  receptor  of the increase i n receptor density c o r r e l a t e d w e l l  o f the i n c r e a s e i n N A - s t i m u l a t e d  cAMP l e v e l s .  Receptor  d e n s i t y s t a r t e d t o i n c r e a s e a f t e r 4 days and reached a maximum o f 150% a f t e r 16 days.  NA-stimulated  cAMP was 150% o f c o n t r o l l e v e l s a f t e r 4 days and  reached a maximum o f 200% a f t e r 11 days, a t which time the EC,.^ had a l s o reached a minimum ( 9 1 ) . percent  Both s t u d i e s (90,91) show a d i s c r e p a n c y between t h e  i n c r e a s e i n r e c e p t o r d e n s i t y (25 t o 50%) and the percent  the cAMP response (approx.  100%).  increase i n  The reason f o r t h i s remains u n c l e a r b u t i t  may w e l l depend upon the f a c t t h a t the r a d i o a c t i v e l i g a n d s used b i n d to  - 22 b o t h the p o s t s y n a p t i c r e c e p t o r and p r e s y n a p t i c a u t o r e c e p t o r s a s s o c i a t e d w i t h o n l y the p o s t s y n a p t i c r e c e p t o r s . this d i s t i n c t i o n i n c o r t i c a l noradrenergic  w h i l e cAMP i s  There i s l i t t l e e v i d e n c e f o r  systems b u t e x c e l l e n t e v i d e n c e i n  s t r i a t a l dopaminergic systems. The  a d m i n i s t r a t i o n o f 6-OHDA t o r a t s on the f i r s t day a f t e r b i r t h  prevents  the development o f p r e s y n a p t i c nerve t e r m i n a l s , b u t does not prevent t h e development o f 8 n o r a d r e n e r g i c  receptors.  The time course  o f the i n c r e a s e i n  8 r e c e p t o r s c o r r e l a t e d w e l l w i t h the time course o f t h e development o f t h e i s o p r o t e r e n o l - s t i m u l a t e d cAMP r e s p o n s e , b o t h r e a c h i n g a d u l t l e v e l s on day 16 i n b o t h c o n t r o l and 6-OHDA-treated a n i m a l s .  The 6-OHDA-treated animals  showed  a 45 to 75% h i g h e r r e c e p t o r d e n s i t y and a 40 t o 65% h i g h e r cAMP response (92) which i s another i n s t a n c e o f s u p e r s e n s i t i v i t y d e v e l o p i n g as a consequence o f disuse. S u b s e n s i t i v i t y of NA-stimulated  cAMP a c c u m u l a t i o n  i n c o r t i c a l s l i c e s has  a l s o been demonstrated i n mice or r a t s t r e a t e d w i t h an agent (d-amphetamine o r a t r i c y c l i c a n t i d e p r e s s a n t ) which would cause e x c e s s i v e  noradrenergic  s t i m u l a t i o n (93). I n s t u d i e s on p e r i p h e r a l organs, such as the c a t n i c t i t a t i n g membrane, T r e n d e l e n b u r g d e s c r i b e d what he c a l l e d two q u a l i t a t i v e l y d i f f e r e n t types o f s u p e r s e n s i t i v i t y (94).  One type was produced by d e c e n t r a l i z a t i o n ( p r e -  g a n g l i o n i c b l o c k a d e o f the neuronal  i n p u t ) and r e s u l t e d i n a moderate degree  of n o n - s p e c i f i c s u p e r s e n s i t i v i t y which developed r a t h e r s l o w l y over 7-14 days.  The other type was produced by d e n e r v a t i o n  e a r l y component resembled the e f f e c t o f cocaine 24-48 h o u r s . ization.  and had two components.  The  and developed r a p i d l y w i t h i n  The l a t e component was s i m i l a r t o t h e e f f e c t o f d e c e n t r a l -  A g a i n , as i n the case o f 6-OHDA-induced c e n t r a l l e s i o n s , t h e r e i s  - 23 considerable pharmacological  e v i d e n c e s u p p o r t i n g the v i e w t h a t the e a r l y  component i s a p r e s y n a p t i c e f f e c t r a t h e r than t r u e p o s t s y n a p t i c sensitivity.  Cocaine,  an uptake i n h i b i t o r , mimicked the e a r l y , but not  l a t e , component and o n l y the l a t t e r was agonist  not removed from i t s r e c e p t o r by r e u p t a k e mechanisms, a l s o i n v o l v e s only a slow-developing  Because of the s a t u r a b l e nature  the  r e v e a l e d by i s o p r o t e r e n o l as t h i s 8  i s not taken up i n t o the p r e s y n a p t i c membrane.  denervation  super-  Since a c e t y l c h o l i n e i s parasympathetic  p o s t s y n a p t i c component.  of amine uptake mechanisms, the amine  c o n c e n t r a t i o n at the r e c e p t o r i s not a l i n e a r f u n c t i o n o f the e x t e r n a l amine concentration.  This n o n - l i n e a r r e l a t i o n s h i p i s r e s p o n s i b l e f o r changes i n  s l o p e and n o n - p a r a l l e l s h i f t s o f the s l o p e s of dose-response c u r v e s .  Langer  found t h a t the s l o p e i n c r e a s e d w i t h a decrease i n the potency (an i n c r e a s e i n EC^Q)  o f d i f f e r e n t amines ( 9 5 ) .  T h i s e f f e c t was  only noticed i n innervated  or d e c e n t r a l i z e d organs where the amine-uptake, p r e s y n a p t i c systems were intact.  Cocaine treatment  or d e n e r v a t i o n  i t s dependence upon such uptake systems. s y n a p t i c changes i s o n l y one  abolished this e f f e c t , The  p o s s i b l e occurrence  indicating o f such p r e -  of the c o m p l i c a t i n g f a c t o r s t h a t must be  con-  s i d e r e d i n e v a l u a t i o n of data p u r p o r t i n g to i n d i c a t e sub- or s u p e r - s e n s i t i v i t y . Some of the problems encountered when a t t e m p t i n g  to f i n d the  exact  mechanism of s e n s i t i v i t y changes are the v a r i a t i o n s i n r e s u l t s o b t a i n e d d i f f e r e n t i n v e s t i g a t o r s . A l l s e n s i t i v i t y changes are m a n i f e s t e d the dose-response c u r v e s . r e s p o n s e , o t h e r s not.  K  m  Some authors  f o r adenylate  w i t h constant V  max  .  by s h i f t s i n  However, some s t u d i e s show an i n c r e a s e d maximum  but u s u a l l y t h i s does not occur. but constant K  by  a l s o r e p o r t a change i n b a s e l i n e v a l u e s ,  And  some r e p o r t s s t a t e a change i n V max c y c l a s e , w h i l e o t h e r s s t a t e a change i n the  F u r t h e r m o r e , the d i s c o n c e r t i n g r e p o r t s t h a t &  r  super-  - 24 s e n s i t i v i t y o f the N A - s t i m u l a t e d cAMP system never d e v e l o p s i n t h e g u i n e a p i g c e r e b r a l c o r t e x (96) or f o l l o w i n g 6-OHDA-induced l e s i o n s add t o t h e d i f f i c u l t y of e s t a b l i s h i n g a g e n e r a l model o f s e n s i t i v i t y changes i n the CNS.  .5 .  cAMP i n D i s e a s e M e n t a l d i s e a s e , e p i l e p s y and drug a d d i c t i o n are major u n s o l v e d problems  and  can be regarded as c h a l l e n g e s  probable that perturbations  f o r neuropathologists.  i n neurotransmission  I t seems v e r y  are fundamental and i t i s  t h e r e f o r e not s u r p r i s i n g t h a t e x t e n s i v e work has been done a t t e m p t i n g i d e n t i f y such p e r t u r b a t i o n s .  to  The l i t e r a t u r e on the p o s s i b l e r o l e o f cAMP i n  t h e s e c o n d i t i o n s i s reviewed h e r e .  I t must be remembered, however, t h a t  there  i s as much or more l i t e r a t u r e on the p o s s i b l e r o l e o f almost e v e r y known neurotransmitter  and r e l a t e d compound i n each o f these d i s e a s e s .  It is  becoming c l e a r t h a t the CNS i s a r a t h e r p l a s t i c s t r u c t u r e and t h a t any p e r t u r b a t i o n i n one n e u r o n a l system may cause compensating and secondary changes i n o t h e r s .  Hence i t has proven d i f f i c u l t to p i n p o i n t the e t i o l o g y ,  e s p e c i a l l y i n those c o n d i t i o n s w h i c h show no c o n s i s t e n t h i s t o p a t h o l o g y . more and b e t t e r r e s e a r c h A few r e f e r e n c e s  Only  data may g i v e the n e c e s s a r y c l u e s .  a r e a l s o c i t e d on the p o s s i b l e involvement o f cAMP i n  v a r i o u s other n e u r o p a t h o l o g i c a l  c o n d i t i o n s and the p o s s i b l e uses o f cAMP  assays i n CSF and u r i n e as d i a g n o s t i c a i d s . a)  E t h a n o l , Morphine and cAMP:  V o l i c i e r et_ al. (97) n o t i c e d a decrease  i n cAMP l e v e l s a f t e r acute a l c o h o l i n g e s t i o n .  T h i s decrease was m a i n l y  a t t r i b u t e d to a marked decrease i n c e r e b e l l a r cAMP.  Redos e_t a l . (98),  however, observed no change i n cAMP l e v e l s i n any o f the seven r e g i o n s o f b r a i n assayed a f t e r acute e t h a n o l , c h r o n i c e t h a n o l  or e t h a n o l  withdrawal.  - 25 -  There are a l s o some, not e n t i r e l y c o n s i s t e n t , o b s e r v a t i o n s on the e f f e c t s o f e t h a n o l on the cAMP g e n e r a t i n g (100)  system i n r a t b r a i n ( 9 9 ) .  I s r a e l et a l .  found t h a t c h r o n i c a l c o h o l a d m i n i s t r a t i o n i n c r e a s e d b a s a l  adenylate  c y c l a s e a c t i v i t y as measured i n b o t h c o r t i c a l s l i c e s and homogenates. a l c o h o l treatment  had no e f f e c t .  i n c r e a s e d the a d e n y l a t e the response to NA  Chronic a l c o h o l a d m i n i s t r a t i o n also  c y c l a s e response to NaF  in cortical slices.  of the c e r e b r a l c o r t e x was  The  i n homogenates but  phosphodiesterase  abolished  (PDE)  unchanged by c h r o n i c a l c o h o l treatment  a d d i t i o n o f e t h a n o l to s l i c e s i n v i t r o  activity  or by  (101).  French e t a l . (102,103) observed a s h i f t i n the s e n s i t i v i t y o f adenylate  Acute  c y c l a s e to NA d u r i n g c h r o n i c a l c o h o l treatment  A l c o h o l i n g e s t i o n f o r f o u r months, w i t h s a c r i f i c e  and  cortical  withdrawal.  two hours a f t e r the  last  d r i n k , r e s u l t e d i n a s h i f t to the r i g h t i n the dose-response curve compared to dextrose-fed receptor.  c o n t r o l s , i . e . an apparent s u b s e n s i t i v e response of the  T h i s c o u l d i n d i c a t e an i n c r e a s e i n the r e l e a s e and  during chronic ethanol  treatment  adrenergic  turnover  w i t h a compensatory change i n the  ( t r u e s u b s e n s i t i v i t y ) , or an apparent s u b s e n s i t i v i t y c o u l d be due  of  NA  receptor to decreased  r e t e n t i o n o f NA at the p o s t s y n a p t i c s i t e because of i n c r e a s e d m e t a b o l i s m or reuptake. On  the t h i r d day of w i t h d r a w a l  a f t e r f o u r months o f e t h a n o l i n g e s t i o n , the  dose-response curve s h i f t e d to the l e f t , i . e . the a d r e n e r g i c r e c e p t o r e x h i b i t e d an apparent s u p e r s e n s i t i v e response to NA s t i m u l a t i o n .  The  onset o f d e l i r i u m  tremens i n humans and f o o t - s h o c k h y p e r s e n s i t i v i t y i n r a t s b o t h occur on t h i r d day o f w i t h d r a w a l was  from c h r o n i c a l c o h o l .  shown to i n v o l v e 8 but not a - a d r e n e r g i c  The  the  adrenergic s u p e r s e n s i t i v i t y  r e c e p t o r s i n the c e r e b r a l c o r t e x ,  - 26 as p r o p r a n o l o l b u t not phenoxybenzamine b l o c k e d f o r maximal s t i m u l a t i o n ) (102,104).  the response to NA  (10  P r o p r a n o l o l a l s o d i m i n i s h e d the  4  M  clinical  symptoms of d e l i r i u m tremens. F r e n c h e t a l . (105) f u r t h e r e s t a b l i s h e d t h a t the s u p e r s e n s i t i v i t y response also occurred  during histamine  or a c e t y l c h o l i n e s t i m u l a t i o n .  and s e r o t o n i n s t i m u l a t i o n , but not d u r i n g GABA These f i n d i n g s l e d F r e n c h to p o s t u l a t e a non-  s p e c i f i c p o s t j u c t i o n a l s u p e r s e n s i t i v i t y phenomenon as the u n d e r l y i n g mechanism of a l c o h o l w i t h d r a w a l . Other i n v e s t i g a t o r s have examined the s e n s i t i v i t y of the dopamine r e c e p t o r s i n the s t r i a t u m and n u c l e u s  accumbens o f r a t s a f t e r c h r o n i c e t h a n o l  treatment.  Some b e h a v i o r a l evidence has been r e p o r t e d i n d i c a t i n g a s u p e r s e n s i t i v i t y (106, 107)  d u r i n g treatment  and a s u b s e n s i t i v i t y (77) d u r i n g w i t h d r a w a l ,  measurements o f d o p a m i n e - s e n s i t i v e  adenylate  but  c y c l a s e i n s t r i a t a l homogenates  from e t h a n o l - t r e a t e d r a t s have not supported  the h y p o t h e s i s  changes i n the p o s t s y n a p t i c r e c e p t o r (109).  Some evidence o f s u b s e n s i t i v i t y  i n mice d u r i n g w i t h d r a w a l of dopamine-sensitive  has, however, been found i n s i m i l a r in  adenylate  cyclase  adenylate  sensitivity  vitro  studies  (110).  There are a l s o a number o f r e p o r t s s u g g e s t i n g morphine may  of  t h a t some of the e f f e c t s o f  be r e l a t e d to changes i n a c t i v i t y o f a  catecholamine-sensitive  c y c l a s e a l t h o u g h , i n t h i s i n s t a n c e , the i n v e s t i g a t i o n s have been  concentrated  on the d o p a m i n e - s e n s i t i v e  adenylate  c y c l a s e i n the s t r i a t u m and  l i m b i c system r a t h e r than on the N A - s e n s i t i v e enzyme i n the c o r t e x . Rat  s t r i a t a l DA and  cAMP l e v e l s are markedly i n c r e a s e d d u r i n g morphine  dependence, and cAMP l e v e l s are markedly decreased d u r i n g w i t h d r a w a l 112).  (101,111,  Both acute and c h r o n i c a d m i n i s t r a t i o n of morphine are s a i d to i n c r e a s e  - 27 -  b a s a l a d e n y l a t e c y c l a s e a c t i v i t y but not t o change s i g n i f i c a n t l y t h e degree o f dopamine s t i m u l a t i o n (113).  D u r i n g w i t h d r a w a l , however, the s t i m u l a t o r y  e f f e c t s o f both dopamine and apomorphine on t h e enzyme seem t o be m a r k e d l y decreased  (101,111,113,114).  The a d d i t i o n o f morphine or r e l a t e d  enkephalins  to i n v i t r o assay systems has a l s o been r e p o r t e d t o i n h i b i t t h e s t i m u l a t i o n o f a d e n y l a t e c y c l a s e by dopamine (115,116) or n o r a d r e n a l i n e (117).  This  may c o m p l i c a t e i n t e r p r e t a t i o n o f r e s u l t s o b t a i n e d on d r u g - t r e a t e d  finding  animals,  however, i t i s s t i l l p o s s i b l e t h a t n a r c o t i c a n a l g e s i c s e x e r t t h e i r a c t i o n s through the r e g u l a t i o n o f a d e n y l a t e c y c l a s e a c t i v i t y i n c e n t r a l nervous system structures. b)  Mental Disease:  The search f o r b i o c h e m i c a l f a c t o r s i n the e t i o l o g y o f  the psychoses has been g o i n g on f o r many decades and has i n v o l v e d e x t e n s i v e i n v e s t i g a t i o n s o f the b l o o d , u r i n e and o t h e r t i s s u e s o f m e n t a l l y i l l i n an attempt  to d e f i n e some p a t h o g e n e t i c b i o c h e m i c a l a b n o r m a l i t y .  persons Each d i s -  c o v e r y o f a new c l a s s o f compounds important to b r a i n f u n c t i o n has i n i t i a t e d a new s e r i e s o f such i n v e s t i g a t i o n s .  Hormones, v i t a m i n s , t r a c e elements,  t r a n s m i t t e r s and c y c l i c n u c l e o t i d e s have a l l been e x t e n s i v e l y s t u d i e d .  neuroThe  work has produced many r e p o r t s o f a b n o r m a l i t i e s p e c u l i a r t o s c h i z o p h r e n i a o r a f f e c t i v e i l l n e s s b u t none have been c o n s i s t e n t l y c o n f i r m e d .  There are  s e r i o u s problems o f i n t e r p r e t a t i o n because o f the p o s s i b l e e f f e c t s o f d i e t , s t r e s s and s i m i l a r f a c t o r s o t h e r than the p s y c h o t i c i l l n e s s . The  advent o f the a n t i p s y c h o t i c drugs p r o v i d e d both a new s t i m u l u s and a  new approach to the search f o r b i o c h e m i c a l e t i o l o g i c a l f a c t o r s .  I t was argued  t h a t i d e n t i f i c a t i o n o f the b r a i n systems m o d i f i e d by t h e c l i n i c a l l y drugs would a l l o w the presumption  active  t h a t i t was p e r t u r b a t i o n s i n those systems  - 28 which l e d t o the p s y c h o t i c symptomatology.  T h i s assumption i s not n e c e s s a r i l y  v a l i d - a n t i c h o l i n e r g i c s were the treatment  o f c h o i c e f o r many y e a r s i n  P a r k i n s o n i s m , f o r example, b u t t h e r e i s now overwhelming evidence problem l i e s i n the dopaminergic  t h a t the  and not i n t h e c h o l i n e r g i c systems.  I t also  now seems p r o b a b l e t h a t v a r i o u s d i s t u r b a n c e s i n any one o f a number o f i n t e r connected  neuronal systems i n b r a i n c o u l d l e a d t o s i m i l a r symptomatology, so  t h a t s c h i z o p h r e n i a and a f f e c t i v e i l l n e s s may each comprise r a t h e r than an e t i o l o g i c a l e n t i t y .  a group o f d i s e a s e s  D e s p i t e these d i f f i c u l t i e s , some hypotheses  have been f o r m u l a t e d w i t h r e g a r d to the psychoses which are based on the g e n e r a l l y accepted modes o f a c t i o n o f a n t i p s y c h o t i c drugs and a r e o f i n t e r e s t here because they suggest  a key r o l e f o r the c a t e c h o l a m i n e - s t i m u l a t e d  adenylate c y c l a s e s . A p r e s e n t l y f a v o r e d h y p o t h e s i s on s c h i z o p h r e n i a i s t h a t i t i n v o l v e s hypera c t i v i t y o f a dopaminergic to  system o f b r a i n .  A key p i e c e o f e v i d e n c e l e a d i n g  t h i s h y p o t h e s i s was the e x c e l l e n t c o r r e l a t i o n found between the c l i n i c a l  potency  o f v a r i o u s p h e n o t h i a z i n e s and t h e i r a b i l i t y  to i n h i b i t the s t i m u l a t i o n  by dopamine o f a d e n y l a t e c y c l a s e a c t i v i t y i n s t r i a t a l homogenates (118). such c o r r e l a t i o n e x i s t e d between c l i n i c a l potency n e u r o t r a n s m i t t e r systems s t u d i e d .  No  and t h e e f f e c t s on o t h e r  U n f o r t u n a t e l y , a l t h o u g h t h e r e was a s i m i l a r  c o r r e l a t i o n w i t h i n the f a m i l y o f a n t i p s y c h o t i c butyrophenone d e r i v a t i v e s , t h e c o r r e l a t i o n d i d not h o l d when members o f the two groups o f drugs were compared. Thus, f o r example, h a l o p e r i d o l i s much more potent c l i n i c a l l y i n comparison w i t h chiorpromazine  than t h e i r r e l a t i v e e f f e c t s on  a d e n y l a t e c y c l a s e would suggest. groups o f drugs f a l l  dopamine-stimulated  I t has been found, however, t h a t the two  i n t o p l a c e i f b i n d i n g assays r a t h e r than  adenylate  c y c l a s e measurements a r e used as the b i o c h e m i c a l i n d e x o f dopamine r e c e p t o r s  - 29 (119) .  Whether t h i s i n d i c a t e s t h a t the important a n t i p s y c h o t i c a c t i o n i s a t  l e a s t p a r t i a l l y at a p r e s y n a p t i c dopamine r e c e p t o r which does not i n v o l v e cAMP or i n some n o n - s t r i a t a l l o c u s h a v i n g a dopamine-stimulated  adenylate c y c l a s e  w i t h p r o p e r t i e s which are somewhat d i f f e r e n t than those of the s t r i a t a l  enzyme  i s not c l e a r .  one  Whatever the outcome o f t h i s debate, a c t i v i t y w i t h i n any  s t r u c t u r a l c l a s s of r e c e p t o r b l o c k e r s does seem to c o r r e l a t e w i t h potency  clinical  and has been used as a s c r e e n i n g t e s t i n the development of new  drugs  (120) . An important  s i d e e f f e c t of c h r o n i c drug treatment  i n s c h i z o p h r e n i a i s the  development o f t a r d i v e d y s k i n e s i a , a movement d i s o r d e r b e l i e v e d to be due s u p e r s e n s i t i v i t y of the s t r i a t a l ponsiveness ically  of s t r i a t a l  dopaminergic  dopamine-sensitive  receptors.  The  enhanced r e s -  a d e n y l a t e c y c l a s e i n animals  chron-  t r e a t e d w i t h n e u r o l e p t i c s , presumably i n d i c a t i n g a s u p e r s e n s i t i v i t y  induced by the p r o l o n g e d dopamine r e c e p t o r b l o c k a d e , has been suggested model o f c l i n i c a l l y The  to  observed  as a  t a r d i v e d y s k i n e s i a (121).  t r i c y c l i c a n t i d e p r e s s a n t s w h i c h are agents o f c h o i c e f o r the  o f d e p r e s s i o n are a l l amine uptake b l o c k e r s .  treatment  Compounds which p r e f e r e n t i a l l y  i n h i b i t dopamine uptake are r e l a t i v e l y i n e f f e c t i v e c l i n i c a l l y ,  suggesting that  the dopamine systems are p r o b a b l y not p r i m a r i l y i n v o l v e d i n t h i s d i s o r d e r . Some p a t i e n t s w i t h d e p r e s s i o n respond b e t t e r to n o r a d r e n a l i n e uptake i n h i b i t o r s than to s e r o t o n i n uptake i n h i b i t o r s w h i l e the converse There i s now  evidence  i s true i n other  cases.  from s e v e r a l c e n t e r s t h a t some cases o f d e p r e s s i o n show  low l e v e l s of n o r a d r e n a l i n e m e t a b o l i t e s i n the pre-treatment  stage, w h i l e  o t h e r s show low l e v e l s o f the s e r o t o n i n m e t a b o l i t e , 5 - h y d r o x y i n d o l e a c e t i c acid.  R e p o r t e d l y , the pre-treatment  l e v e l s have p r e d i c t i v e v a l u e as to the  type of drug which w i l l be e f f e c t i v e i n a p a r t i c u l a r p a t i e n t (122).  This  has  - 30 -  l e d to the h y p o t h e s i s of d e p r e s s i o n , and  t h a t t h e r e a r e a t l e a s t two e t i o l o g i c a l l y d i s t i n c t forms  one i n which there  another i n which s e r o t o n e r g i c  i s hypofunctioning  o f noradrenergic  systems a r e d e f e c t i v e .  p l a y a key r o l e i n the p o s t s y n a p t i c  systems  S i n c e cAMP seems t o  a c t i o n o f these t r a n s m i t t e r s , i t would be  u s e f u l to have more data than are p r e s e n t l y a v a i l a b l e on p o s s i b l e  abnormalities  of t h i s substance i n these d i s o r d e r s . Urine  e x c r e t i o n o f b o t h NA and cAMP has been r e p o r t e d  to be e l e v a t e d  mania (123,124).  M a n i c - d e p r e s s i v e p a t i e n t s are o f t e n t r e a t e d w i t h L i  with considerable  success.  P a t i e n t s who improve on L i  show a decreased u r i n a r y e x c r e t i o n o f cAMP (125).  L i  +  +  therapy  +  during  salts  reportedly  reduces the r e l e a s e  o f NA and 5-HT from b r a i n s l i c e s and a n t a g o n i z e d b o t h NA- and  histamine-induced  a d e n y l a t e c y c l a s e a c t i v i t y i n b r a i n s l i c e s and NaF s t i m u l a t i o n i n homogenates (126).  These, however, a r e o n l y a few o f t h e many b i o c h e m i c a l  reported of L i  +  for L i  +  effects  and i t i s d i f f i c u l t to e x p l a i n the c h e m i c a l e f f e c t i v e n e s s  i n both mania and d e p r e s s i o n  data so f a r r e p o r t e d .  on the b a s i s o f any o f the b i o c h e m i c a l  Nor can u r i n a r y l e v e l s o f e i t h e r NA o r cAMP be t a k e n as  good i n d i c e s o f c o n t r o l n o r a d r e n e r g i c  activity.  Drugs which i n h i b i t or reduce PDE a c t i v i t y i n t h e CNS have a l s o been reported  to reduce a n x i e t y q u i t e e f f e c t i v e l y  (127). C a f f e i n e , t h e o p h y l l i n e and  theobromine b e l o n g t o t h i s group and t h e i r e f f e c t i s mimicked by a d m i n i s t r a t i o n of  di-butyryl-cAMP. c)  Epilepsy  E p i l e p s y i s a group o f n e u r o l o g i c a l d i s o r d e r s c h a r a c t e r i z e d by an abnormal s u s c e p t i b i l i t y o f n e u r o n a l aggregates t o g e n e r a t e and propagate a c t i o n p o t e n tials.  Factors  i n f l u e n c i n g the e l e c t r i c a l s t a t e and m e t a b o l i s m o f n e u r o n a l  systems are c e r t a i n t o c o n t r i b u t e to t h i s a b n o r m a l i t y .  I n order  to i n v e s t i g a t e  - 31  -  the m o r p h o l o g i c a l ,  biochemical,  before,  a f t e r the development of c o n v u l s i v e  d u r i n g and  p a t h o l o g i c a l and n e u r o p h y s i o l o g i c a l  t e c h n i q u e s to provoke s e i z u r e s have been developed. a u d i t o r y , p h o t o g e n i c , pharmacologic and a form of c h r o n i c e l e c t r i c a l  seizures,  changes  experimental  These t e c h n i q u e s  electrogenic stimulation.  include  Kindling is  s t i m u l a t i o n of the amygdala or o t h e r s e n s i t i v e  areas designed to produce i n animals a c h r o n i c s e i z u r e s u s c e p t i b i l i t y which c l o s e l y resembles the human c o n d i t i o n In attempting  (128).  to l o c a l i z e those areas of the b r a i n which a c t i v e l y  p a r t i c i p a t e i n the e x p r e s s i o n  of s e i z u r e s , i t has been observed t h a t r h y t h m i c ,  sharp waves develop c o n c u r r e n t w i t h c l i n i c a l m a n i f e s t a t i o n s convulsions  (129).  By l e s i o n i n g v a r i o u s  of amygdaloid  areas of the c e r e b r a l c o r t e x , i t has  a l s o been e s t a b l i s h e d t h a t o n l y those animals w i t h p r e f r o n t a l and  orbital  l e s i o n s r e q u i r e d more s t i m u l a t i o n s to develop k i n d l e d c o n v u l s i o n s . c i n g u l a t e and  p o s t e r i o r c o r t i c a l l e s i o n s were w i t h o u t e f f e c t .  Motor,  Stimulation  of  the f r o n t a l lobe of e p i l e p t i c p a t i e n t s a l s o evokes s e i z u r e s i d e n t i c a l to those s p o n t a n e o u s l y s u f f e r e d by the p a t i e n t s .  These o b s e r v a t i o n s  w o r t h w h i l e examining c o r t i c a l areas f o r b i o c h e m i c a l  suggest t h a t i t i s  changes r e l e v a n t  to  s e i z u r e phenomena. There i s an enormous l i t e r a t u r e on the p o s s i b l e r o l e of v a r i o u s t r a n s m i t t e r s i n c o n v u l s i v e b e h a v i o r and lesion-produced  i t seem c l e a r t h a t p h a r m a c o l o g i c a l  or  changes i n a number of t r a n s m i t t e r s can modify the development  or p o t e n t i a t i o n o f s e i z u r e s .  I n p a r t i c u l a r , a g r e a t body of e v i d e n c e suggests  t h a t c a t e c h o l a m i n e s e x e r t a suppressant e f f e c t on s e i z u r e a c t i v i t y . which were g e n e t i c a l l y prone to a u d i o g e n i c NA  neuro-  than d i d normal mice (128).  s e i z u r e s had  Mice  lower l e v e l s of b r a i n  C i r c a d i a n f l u c t u a t i o n s i n NA  l e v e l s i n the r a t  - 32 have a l s o been shown to be i n v e r s e l y r e l a t e d to s e i z u r e s e n s i t i v i t y Hippocampal and amygdalar NA l e v e l s were d e p l e t e d The  i n kindled cats  (129).  (130).  suppressant r o l e o f NA i n s e i z u r e development i s a l s o supported by  pharmacological  studies.  Drugs which d e p l e t e NA by i n h i b i t i n g s y n t h e s i s o r by  d e s t r o y i n g NA neurons, g e n e r a l l y tend to i n c r e a s e s e i z u r e s u s c e p t i b i l i t y and s e v e r i t y (131-136).  C o n v e r s e l y , i n t r a c e r e b r a l i n j e c t i o n s o f NA reduced  s u s c e p t i b i l i t y to a u d i o g e n i c s e i z u r e s (137).  Precursors  as w e l l as c h e m i c a l l y and e l e c t r i c a l l y  induced  o f NA and MAO i n h i b i t o r s had the same e f f e c t  (130,131). Some i n v e s t i g a t o r s have p o s t u l a t e d  s u p e r s e n s i t i v i t y as an u n d e r l y i n g  mechanism o f e p i l e p t i c s e i z u r e a c t i v i t y . served  Epileptogenic  f o c i have been ob-  to c o n s i s t o f aggregates o f p a r t i a l l y denervated neurons i n the c e r e b r a l  c o r t e x (138).  These neurons a l s o have v e r y u n s t a b l e  r e s t i n g membrane p o t e n -  t i a l s , a f e a t u r e o f t e n observed i n s u p e r s e n s i t i v e nerve c e l l s .  Undercut s l a b s  of c o r t e x v e r y q u i c k l y become e p i l e p t o g e n i c , i . e . , showed i n c r e a s e d to s u s t a i n paroxysmal a c t i v i t y  (epileptiform after  capacity  discharge).  S e v e r a l i n v e s t i g a t i o n s have a l s o i m p l i c a t e d cAMP i n t h e e x p r e s s i o n o f seizure a c t i v i t y .  A v a r i e t y o f agents capable o f i n c r e a s i n g cAMP l e v e l s i n  the r a t c o r t e x were found to be e p i l e p t o g e n i c Symptom-free e p i l e p t i c s have been r e p o r t e d  (139,140). t o have lower c o n c e n t r a t i o n s  cAMP i n t h e CSF than found i n p a t i e n t s who had undergone c o n v u l s i v e w i t h i n 0 to 3 days (141). CSF  of  attacks  I n more d e t a i l e d measurements o f cAMP l e v e l s i n the  o f e p i l e p t i c p a t i e n t s , i t was found t h a t the cAMP l e v e l was h i g h e s t  during  s e i z u r e , then d e c l i n e d g r a d u a l l y over 7 days a f t e r s e i z u r e and then remained at a normal l e v e l (142).  T h i s author suggested t h a t changes i n the cAMP  l e v e l s i n the CSF was a f a i r l y a c c u r a t e  measure o f changes i n n e u r o n a l / g l i a l  - 33 cAMP c o n c e n t r a t i o n s  and would not be a f f e c t e d by t o n i c and c l o n i c muscular  contractions. d)  Miscellaneous  clinical  conditions  A d m i n i s t r a t i o n of i s o p r e n a l i n e or adrenaline l e v e l s o f cAMP. asthma.  This  r e s u l t s i n elevated  response i s much lower i n p a t i e n t s w i t h  bronchial  A l l e r g i c c o n d i t i o n s may be a s s o c i a t e d w i t h a d e f e c t i v e B r e c e p t o r  response o f the cAMP system to a d r e n e r g i c  stimulation.  Low c o n c e n t r a t i o n s  Pb i n h i b i t s b o t h b a s a l and h o r m o n e - s t i m u l a t e d l e v e l s o f a d e n y l a t e activity.  Concentrations  needed to e l i c i t  cAMP and c l i n i c a l  i n vivo  cyclase  (143).  chemistry  cAMP assays may a l s o have some uses i n c l i n i c a l c h e m i s t r y .  Parathyroid  s t i m u l a t i o n o f kidney c o r t i c a l adenylate cyclase r e s u l t s i n increased e x c r e t i o n o f cAMP.  of  t h i s e f f e c t approach those which  are t o x i c and cause n e u r o l o g i c a l a b n o r m a l i t i e s e)  blood  urinary  Lack o f t h i s response i s d i a g n o s t i c f o r pseudo-  h y p o p a r a t h y r o i d i s m (144,145).  Vasopressin  stimulates kidney medullary  a d e n y l a t e c y c l a s e r e s u l t i n g i n an i n c r e a s e i n the l e v e l o f cAMP e x c r e t e d i n the u r i n e .  Lack o f t h i s response i s d i a g n o s t i c f o r r e n a l d i a b e t e s i n s i p i d u s .  Patients with cerebrovascular b o t h CSF and systemic  i n f a r c t i o n showed an e l e v a t e d  f l u i d compared t o c o n t r o l s (146).  l e v e l o f cAMP i n  Comatose p a t i e n t s  showed a lower CSF l e v e l o f cAMP; the more severe the coma the lower the cAMP level.  A change i n coma s t a t u s was accompanied by a change i n CSF cAMP l e v e l .  A l l plasma cAMP l e v e l s were normal (147,148).  6.  Assay Methods f o r cAMP Most i n v e s t i g a t i o n s o f a d e n y l a t e c y c l a s e a c t i v i t y depend upon assay o f the  amount o f cAMP formed d u r i n g a standard  incubation period.  The v a r i o u s  - 34 methods w h i c h have been r e p o r t e d f o r cAMP d e t e r m i n a t i o n a r e r e v i e w e d b r i e f l y h e r e ; c h o i c e o f method f o r t h i s study was based on s e n s i t i v i t y and ease o f performance. a)  Phosphorylase a c t i v a t i o n  H i s t o r i c a l l y , the f i r s t of  assay  assay procedure f o r cAMP was based on the a b i l i t y  cAMP to enhance the r a t e o f a c t i v a t i o n o f i n a c t i v e dog l i v e r p h o s p h o r y l a s e .  T h i s assay e v o l v e d from the experiments t h a t l e d t o the d i s c o v e r y o f cAMP (149,150). In  the f i r s t  s t a g e , i n a c t i v e l i v e r p h o s p h o r y l a s e i s c o n v e r t e d to the  a c t i v e enzyme; t h e r a t e o f c o n v e r s i o n i s dependent on t h e i n i t i a l cAMP concentration.  Dog l i v e r homogenate (11,000 x g s u p e r n a t a n t f r a c t i o n ) i s i n c u b a t e d  w i t h ATP, MgSO^, and c a f f e i n e t o g e t h e r w i t h a known amount o f cAMP o r an unknown sample.  I n the second  determined by measuring  s t a g e , the amount o f a c t i v e p h o s p h o r y l a s e i s  the c o n v e r s i o n o f g l u c o s e - l - P O ^ t o glycogen d u r i n g  i n c u b a t i o n at 37°C f o r 30 m i n u t e s .  I n the t h i r d s t a g e , g l y c o g e n i s d e t e r -  mined by i t s c o l o r i m e t r i c r e a c t i o n w i t h i o d i n e . The of  assay i s s e n s i t i v e to 0.1 pmole o f cAMP.  The most s e r i o u s d i s a d v a n t a g e  t h i s assay i s i t s v a r i a b i l i t y from day t o day; t h a t i s , the a b s o l u t e v a l u e s  for  cAMP v a r y a l t h o u g h the r e l a t i v e v a l u e s are c o n s t a n t .  are  t h e i n t e r f e r e n c e o f v a r i o u s o t h e r agents w i t h t h e assay and t h e n e c e s s i t y  to  Other  disadvantages  prepare the r e q u i r e d enzymes (151,152). b)  Enzymatic c y c l i n g  procedures  The  cAMP can be i s o l a t e d by t h i n - l a y e r chromatography  and c o n v e r t e d to 5'-  AMP w i t h p h o s p h o d i e s t e r a s e and then t o ATP w i t h myokinase and p y r u v a t e k i n a s e . The  enzymatic c y c l i n g system w i t h ATP as the c a t a l y t i c component, g e n e r a t e s  g l u c o s e - 6 - P 0 ^ , which can be measured f l u o r i m e t r i c a l l y w i t h  glucose-6-PO,  - 35 dehydrogenase (153).  A l t e r n a t i v e l y , ATP can be determined by i t s luminescent  r e a c t i o n w i t h f i r e f l y l u c i f e r i n and l u c i f e r a s e (154-156).  The assay  t i v e to 1 pmole cAMP and l i n e a r over t h r e e orders o f magnitude. disadvantage c)  i s sensi-  The main  i s the rigorous p u r i f i c a t i o n required.  P r o t e i n kinase  assay  Low c o n c e n t r a t i o n s o f cAMP w i l l a c t i v a t e cAMP-dependent p r o t e i n k i n a s e , w h i c h c a t a l y z e s t h e p h o s p h o r y l a t i o n o f p r o t e i n s u b s t r a t e s by ATP.  The  32 incubation mixture  i n c l u d e s [ y - P]-ATP, Mg a c e t a t e , cAMP s t a n d a r d or  unknown, h i s t o n e ( o r c a s e i n ) p r o t e i n , a c t i v a t e d p r o t e i n k i n a s e and Na a c e t a t e buffer.  The r e a c t i o n i s t e r m i n a t e d by the a d d i t i o n o f T C A - t u n g s t a t e - s u l f u r i c  a c i d , the p r e c i p i t a t e i s d i s s o l v e d i n NaOH and t h e r a d i o a c t i v i t y counted i n a s c i n t i l l a t i o n counter.  The e x t e n t o f h i s t o n e ( o r c a s e i n ) p h o s p h o r y l a t i o n i s  d i r e c t l y p r o p o r t i o n a l to t h e amount o f cAMP p r e s e n t The  i n the i n c u b a t i o n m i x t u r e .  s e n s i t i v i t y o f the assay i s 0.3 pmole cAMP (157,158). d)  Protein binding  assays  These depend upon the c o m p e t i t i o n between c o l d cAMP i n unknown or standard w i t h a known amount o f l a b e l l e d cAMP f o r s p e c i f i c b i n d i n g s i t e s on v a r i o u s proteins.  The amount o f l a b e l l e d cAMP which i s bound i s i n v e r s e l y r e l a t e d to  the amount o f c o l d cAMP. Radioimmunoassay:  Of the c o m p e t i t i v e p r o t e i n b i n d i n g s a t u r a t i o n a s s a y s ,  the radioimmunoassay i s the one g i v i n g the h i g h e s t s e n s i t i v i t y .  The method i s  based on the c o m p e t i t i o n o f cAMP w i t h an i s o t o p i c a l l y l a b e l l e d s u c c i n y l a t e d c y c l i c n u c l e o t i d e d e r i v a t i v e f o r b i n d i n g on a s p e c i f i c a n t i s e r u m . 2'-O-succinylated  cAMP i s conjugated  The  to a p r o t e i n as the f r e e c a r b o x y l end and  t h i s immunogenic c y c l i c n u c l e o t i d e d e r i v a t i v e i s i n j e c t e d i n t o r a b b i t s . A n t i s e r a produced c r o s s - r e a c t m i n i m a l l y w i t h other n u c l e o t i d e s (159,160).  The  a n t i s e r u m i s s a t u r a t e d w i t h an i o d i n a t e d ( "^ I o r " 'I) t y r o s i n e J  J  LJJ  m e t h y l e s t e r d e r i v a t i v e o f s u c c i n y l a t e d cAMP, which w i l l then compete w i t h t h e sample ( o r s t a n d a r d ) cAMP f o r b i n d i n g s i t e s on the a n t i b o d i e s . 125  .  .  Bound and f r e e  .  I d e r i v a t i v e can be s e p a r a t e d by ammonium s u l f a t e p r e c i p i t a t i o n and the p r e c i p i t a t e counted i n a gamma s p e c t r o m e t e r .  The r a d i o a c t i v i t y i s i n v e r s e l y  p r o p o r t i o n a l to the amount o f sample cAMP and the s e n s i t i v i t y i s 0.01 t o 2 pmole cAMP/tube.  The p r e p a r a t i o n o f t h e a n t i s e r a and t h e use o f s h o r t - l i v e d  i o d i n e i s o t o p e s poses t e c h n i c a l problems f o r r o u t i n e u s e . P r o t e i n k i n a s e b i n d i n g assay:  Because o f i t s s i m p l i c i t y and r e l a t i v e l y  h i g h s e n s i t i v i t y , t h e p r o t e i n k i n a s e b i n d i n g assay f o r cAMP i s a v e r y used method (161,162). The  widely  The r a t i o n a l e i s t h e same as f o r t h e radioimmunoassay.  b i n d i n g p r o t e i n i s most f r e q u e n t l y s k e l e t a l muscle p r o t e i n k i n a s e  of i t s h i g h b i n d i n g constant  f o r cAMP.  because  The t r i t i u m l a b e l l e d cAMP competes  w i t h u n l a b e l l e d sample cAMP f o r b i n d i n g s i t e s on the p r o t e i n k i n a s e  molecule.  I n a d d i t i o n , a b i n d i n g enhancement f a c t o r , p r o t e i n k i n a s e i n h i b i t o r p r o t e i n , i s added to i n c r e a s e the b i n d i n g o f the c y c l i c n u c l e o t i d e s . s e p a r a t e d from bound cAMP-protein complex by a d s o r p t i o n f i l t e r s or on c h a r c o a l  (163,164).  The p r o t e i n k i n a s e  on c e l l u l o s e e s t e r  i s so l a r g e a m o l e c u l e  t h a t i t i s r e t a i n e d on t h e s p e c i a l m i l l i p o r e f i l t e r s used. charcoal  Unbound cAMP i s  The f i l t e r s o r t h e  s u p e r n a t a n t s are counted i n a s c i n t i l l a t i o n counter.  The assay i s  s e n s i t i v e t o 0.05 t o 0.1 pmole o f cAMP. e)  Pre l a b e l l i n g  techniques  When mammalian t i s s u e s and c e l l s are incubated s o l u t i o n , t h i s purine  i n an adenine  containing  i s c o n v e r t e d i n t o 5'-AMP and f i n a l l y t o ATP. T h i s ATP  i s then a c t i v e l y c o n v e r t e d to cAMP.  By p u l s e - l a b e l l i n g c e l l s w i t h r a d i o a c t i v e  adenine, t h e r e l a t i v e amounts o f cAMP newly formed from ATP can be determined  by i s o l a t i n g the cAMP and measuring i t s r a d i o a c t i v i t y ( 1 6 5 ) .  I t i s thus  p o s s i b l e to measure changes i n the l e v e l s o f cAMP b u t not a b s o l u t e amounts o f the c y c l i c n u c l e o t i d e ( 1 6 6 ) .  The s e n s i t i v i t y o f t h i s method i s p r o p o r t i o n a l  to the s p e c i f i c a c t i v i t y o f the r a d i o a c t i v e adenine. 32  3  Homogenates can a l s o be i n c u b a t e d w i t h [ y - P]ATP and [ H]-cAMP and c o l d c a r r i e r cAMP.  The samples a r e a p p l i e d t o n e u t r a l hydrous alumina  oxide  columns, which s e l e c t i v e l y r e t a i n p o l y a n i o n i c n u c l e o t i d e s and P i .  The  e f f l u e n t , monovalent cAMP, can then be counted i n a t h r e e - c h a n n e l  Packard  counter. 7.  This method i s extremely  r a p i d and simple  (167).  STATEMENT OF THE PROBLEM The  o r i g i n a l aim o f t h i s t h e s i s was to i n v e s t i g a t e changes i n n o r a d r e n e r g i c  responsiveness  o f the r a t c e r e b r a l c o r t e x t a k i n g p l a c e d u r i n g the t h r e e days  f o l l o w i n g chronic a l c o h o l withdrawal.  The r e c e p t o r was r e p o r t e d to d i s p l a y  s u b s e n s i t i v i t y on day 0 but s u p e r s e n s i t i v i t y on day 3.  I f these  sensitivity  changes were due to f u n c t i o n a l d i s u s e , as a r e s u l t perhaps o f a decrease i n NA c o n t a c t w i t h the r e c e p t o r or a decrease i n NA r e l e a s e , i t s h o u l d be p o s s i b l e to mimic t h i s e f f e c t by b l o c k i n g NA s y n t h e s i s .  The FLA-63-induced  inhibition  o f dopamine-8-hydroxylase would h o p e f u l l y have produced t h e same f u n c t i o n a l d i s u s e and consequent s u p e r s e n s i t i v i t y o f the n o r a d r e n e r g i c  receptor.  Also,  the sequence o f such s e n s i t i v i t y changes c o u l d be compared w i t h t h e a l c o h o l withdrawal-induced  changes.  I n these experiments,  the same assay was t o be  used f o r cAMP as had been used i n the a l c o h o l w i t h d r a w a l  s t u d i e s (104).  These s e t s o f experiments were i n i t i a t e d but were not completed when the many problems w i t h the cAMP assay were r e c o g n i z e d .  The development o f a more  r e l i a b l e method i s o u t l i n e d i n the "RESULTS" s e c t i o n .  Some o f the major  - 38 d i f f i c u l t i e s with the original method are listed below: 1)  the 1 mm tissue slices were probably too thick for complete penetration of solutions  2)  the pH of the Krebs-Ringer buffer was 6.0 at the onset of incubation and gradually changed to pH 7.2 at the c r i t i c a l time of neurohormone addition  3)  the dilution of tissue with buffer was high so that the assay for cAMP was performed at a concentration where unreliable results might be expected  4)  the binding assay was done at a very high concentration of salt which interfered with the binding to protein kinase. FLA-63 injected animals had a very high mortality rate and the literature  contained very few reports on this drug at the time of experimentation.  A NA  depleting agent with better documented actions seemed preferable, so that any unusual results would not be attributed to d i f f i c u l t i e s with the drug. Therefore, 6-OHDA, a drug which consistently depletes central NA stores, was chosen to demonstrate supersensitivity.  By injecting 6-OHDA intracerebrally into the  dorsal bundle, the NA stores of the cortex are selectively depleted while DA remains unchanged (168).  The p o s s i b i l i t y that chronic supersensitivity of the  c o r t i c a l cAMP generating system might be induced i n this fashion had not been investigated when this work was undertaken. The very high sensitivity of the adenylate cyclase system during the end of the second post-natal week (60) could be the result of supersensitive noradrenergic receptors, due to neonatal "disuse" as a result of a lag i n the development of synapses compared to the development of 8 receptors. F i n a l l y , the p o s s i b i l i t y that supersensitivity might play a role i n convulsive behavior was examined by studying possible receptor sensitivity changes i n the kindled rat.  - 39 II. 1.  EXPERIMENTAL PROCEDURE Materials 3 . 3 U n i f o r m l y l a b e l l e d [ H]-cAMP (ammonium s a l t , 37.7 Ci/mmole) and [ H ] -  dopamine (12 Ci/mmole were o b t a i n e d from New England N u c l e a r .  Protein kinase  i n h i b i t o r , cAMP-dependent p r o t e i n k i n a s e , n o n - r a d i o a c t i v e cAMP, 1 - a r t e r e n o l - D b i t a r t r a t e and b o v i n e serum a l b u m i n ( c r y s t a l l i n e ) were a l l purchased from t h e Sigma Chemical Company. Sodium p e n t o b a r b i t a l was bought from Abbott L a b o r a t o r i e s , 6-hydroxydopamine hydro-bromide from R e g i s C h e m i c a l Company, L - a s c o r b i c a c i d from B r i t i s h Drug House and FLA-63 ( b i s - 4 - m e t h y l - l - h o m o p i p e r a z i n y l t h i o - c a r b o n y l d i s u l f i d e ) was a g i f t from AB A s t r a Lakemedel. Alumina ( a c i d washed, pH 3.0-5.0), Reagent grade, was purchased from t h e McArthur Chemical Company and D L - a r t e r e n o l h y d r o c h l o r i d e (B grade) from Calbiochem.  A l l o t h e r c h e m i c a l s , ACS grade, were o b t a i n e d from e i t h e r  M a l l i n c k r o d t or t h e F i s h e r Chemical Company.  Millipore filters  (cellulose  e s t e r , HA 0.45, 24 mm) were purchased from the M i l l i p o r e Company. A d u l t male W i s t a r a l b i n o r a t s were purchased from Woodlyn L a b o r a t o r i e s , male Long-Evans hooded r a t s from the Canadian B r e e d i n g Farms and 15-day o l d W i s t a r a l b i n o r a t s , male and female, were s u p p l i e d by t h e V i v a r i u m o f t h e U n i v e r s i t y o f B r i t i s h Columbia.  2.  Methods  a)  Treatment o f A n i m a l s i)  K i n d l i n g Procedure  K i n d l e d r a t s were o b t a i n e d from Dr. C o r c o r a n and the procedure used was the f o l l o w i n g .  Male Long-Evans hooded r a t s w e i g h i n g 300 g were a n a e s t h e t i z e d  - 40 -  with  sodium p e n t o b a r b i t a l (50 mg/kg, i . p . ) 30 min p r i o r t o t h e b i l a t e r a l  i m p l a n t a t i o n o f e l e c t r o d e s i n t o the m e d i a l s t e r o t a x i c c o o r d i n a t e s were: .8mm (from Bragma).  amygdaloid area, f o r which the  TB + 5mm + 5mm, D-V 8.5mm (from c o r t e x ) , A-P -  Electrical  s t i m u l a t i o n through i n s u l a t e d b i p o l a r  e l e c t r o d e s was p r o v i d e d by a c o n s t a n t - c u r r e n t intensity.  60~Hz s i n e wave source  The r e f e r e n c e e l e c t r o d e c o n s i s t e d o f a male plug connected to a  s u r g i c a l screw i n the f r o n t a l bone v i a u n i n s u l a t e d w i r e The day  r a t s were s t i m u l a t e d through the l e f t  f o r 10-12 days, u n t i l  (169).  amygdaloid e l e c t r o d e f o r 1 sec/  stage 4 o f k i n d l i n g was reached.  T h i s stage i s  c h a r a c t e r i z e d by the development o f g e n e r a l i z e d b i l a t e r a l clonus limbs, r e a r i n g o f the head and f a l l i n g . to was  have at l e a s t  10 f u l l  repeated w i t h  were f u r t h e r s t i m u l a t e d  s e i z u r e s , a f t e r which the e n t i r e k i n d l i n g  full  period  o f s e v e r a l months  s e i z u r e responses,  even i f the animals  had been allowed  a resting  (170).  6-OHDA I n j e c t i o n s  Male Wistar barbital  r a t s weighing 300-350g were a n a e s t h e t i z e d w i t h sodium pento-  (50 mg/kg, i . p . ) 30 min p r i o r  and K l i p p e l A t l a s c o o r d i n a t e s  to the s t e r e o t a x i c i n j e c t i o n s .  g r e a t e r weight o f animals  used i n t h i s  study,  so t h a t the f i n a l  TB - 4.2mm, A-P + 2.6mm, M-L + 1.1mm, D-V 3.7mm.  were placed  The  f o r the d o r s a l bundle (DB) n o r a d r e n e r g i c  t r a c t , which are based on 150g r a t s (171), had t o be a d j u s t e d  were:  procedure  s t i m u l a t i o n through the k i n d l i n g e l e c t r o d e s would always  elicit  Konig  The animals  o f the f o r e -  s t i m u l a t i o n through the r i g h t amygdaloid e l e c t r o d e . Any  subsequent e l e c t r i c a l  ii)  o f 160 uA  to a l l o w f o r t h e coordinates  A f t e r the animals  i n p o s i t i o n on the s t e r e o t a x i c apparatus (David Kopf S t e r e o t a x i c  Instruments),  two b u r r h o l e s were d r i l l e d  f o r the b i l a t e r a l  injection of 2  ul/10 min o f 2 ug/ul 6-OHDA, which was made up f r e s h l y before use i n a s o l u t i o n  - 41  of 0.3  mg  ascorbic  acid/ml of  -  isotonic saline.  u s i n g a 10 u l Hamilton s y r i n g e ,  34  T h i s v e r y slow i n j e c t i o n  gauge needle, ensured a g a i n s t  6-OHDA to proximal t r a c t s , i n p a r t i c u l a r the v e n t r a l tract  group of 8 r a t s was  week b e f o r e s a c r i f i c e and a control  solution  sacrifice.  left  assay.  The  T h i s was  iii)  FLA-63  vehicle,  and  was  left  injected  1 with  f o r 1 week b e f o r e  physical  damage to  the  assay r e s u l t s . require  tube-feeding,  ones (172).  Injections  dissolved  i n a few  to volume i n 0.9%  vehicle  drops of g l a c i a l a c e t i c a c i d  saline solution  (the volume was  as  C o n t r o l animals were i n j e c t e d w i t h an  (173)  close  and  to  1.5  equal volume of  solution.  Treatment of B r a i n i)  a second group of 8 f o r  done to determine whether p o s s i b l e  m l / r a t as p o s s i b l e ) .  Tissue  Tissue Preparation  Animals were s a c r i f i c e d by  c e r v i c a l d i s l o c a t i o n , the  b r a i n s removed  i n t o a beaker c o n t a i n i n g i c e - c o l d Krebs-Ringer b i c a r b o n a t e  (KRB-buffer), pH a bed  right  of  noradrenergic  A t h i r d group of 4 animals was  to i n t r a v e n t r i c u l a r l y i n j e c t e d  FLA-63 was  on  bundle (VB)  i n t r a c e r e b r a l l y 6-OHDA-injected animals d i d not  i n contrast  diluted  f o r 10 weeks and  c o n t a i n i n g only the  nerve t r a c t would a f f e c t the  put  diffusion  (172).  One  b)  rate,  and  matter.  7.4.  The  entire  tissue  of i c e i n a cold-room at 4°C. left The  sides  was  y i e l d was  c o r t e x were cut  dissected  out,  approximately 250  i n t o 0.3  mm  buffer  p r e p a r a t i o n procedure was The  cerebral  taking mg  per  cortices  care not adult  carried  out  from both  to i n c l u d e  rat.  and  The  any  slabs  white of  s l i c e s , u s i n g a M c l l w a i n t i s s u e chopper (Brinkman  - 42 Instrument cortical  Company), y i e l d i n g s e c t i o n s o f dimension  slices  5 x 1.5 x .3 mm.  from one animal were then suspended i n KRB-buffer, the  suspension v o r t e x e d and the s l i c e s d i s t r i b u t e d f l a s k s , each c o n t a i n i n g 3 ml KRB-buffer,  to s i x 25 ml Ehrlenmeyer  to a c h i e v e a random d i s t r i b u t i o n o f  the v a r i o u s areas o f the c o r t e x i n t o each o f the i n c u b a t i o n f l a s k s . KRB-buffer  The  The  was kept on i c e and aerated with 95% C>2-5% CC^ f o r one hour  b e f o r e and d u r i n g the subsequent i n c u b a t i o n ( 5 0 ) . The  composition  o f the KRB b u f f e r was as f o l l o w s :  b i c a r b o n a t e , 118 mM NaCl,  0  2  each day from 10 x c o n c e n t r a t e d stock  The  I n c u b a t i o n Procedure  I t was prepared  freshly  solutions.  and I s o l a t i o n of cAMP  i n c u b a t i o n f l a s k s were p l a c e d i n a 37°C shaking waterbath (70  c y c l e s / m i n ) , and gassed rubber  sodium  5mM KC1, 2.5 mM C a C l , 2 mM KHLPO., 2 mM 2 2 4  MgSO^, 7 H 0 and 0.02 mM EDTA and 12 mM g l u c o s e .  ii)  25 mM  stoppers.  f o r 30 min w i t h 95% 0^-5% CO2 through  T h i s marked the s t a r t o f the f i r s t  which was terminated  air-tight  preincubation period,  a f t e r 30 min by a s p i r a t i o n of the b u f f e r .  This pre-  i n c u b a t i o n p e r i o d was necessary because there i s an immediate post-mortem r i s e i n cAMP l e v e l s and time must be allowed by phosphodiesterase KRB-buffer, for  f o r the excess  cAMP to be m e t a b o l i z e d  to achieve constant b a s e l i n e l e v e l s  (174,175).  2.9 ml, was added to each o f the f l a s k s , which again were  Fresh gassed  15 min w i t h 95% 0 " 5 % C O ^ 2  At t h i s yield  time, NA was added i n 100 u l of 30 x c o n c e n t r a t e d s o l u t i o n s to  f i n a l c o n c e n t r a t i o n s o f 0 uM, 1 uM, 3 uM, 10 UM, 30 uM, and 100 uM.  Stock s o l u t i o n s o f 30 mM NA i n 0.004% (w/v) BSA were f r o z e n i n a l i q u o t s and were s e r i a l l y d i l u t e d  to 30 x c o n c e n t r a t i o n s b e f o r e the a d d i t i o n s to the  flasks.  of BSA has been found  The presence  empirically  to decrease the  - 43 -  o x i d a t i v e decomposition  of NA i n s o l u t i o n .  100 b l of BSA s o l u t i o n was added  to one f l a s k f o r each animal to a l l o w the e s t i m a t i o n o f the i n d i v i d u a l baseline.  F i v e minutes  after  these a d d i t i o n s , the r e a c t i o n was terminated by  c e n t r i f u g a t i o n o f the samples i n the c o l d a t 1000 x g f o r 30 sec, d e c a n t a t i o n of the b u f f e r and a d d i t i o n o f 1 ml o f 10% TCA.  The suspensions were  homogenized w i t h a T e f l o n p e s t l e and the p e s t l e was r i n s e d a f t e r homogenization  each  w i t h 0.4 ml 10% TCA.  The homogenates were c e n t r i f u g e d  at 12,000 x g f o r 15 min i n a S o r v a l l  c e n t r i f u g e and the supernatants decanted o f f i n t o 15 ml t e s t tubes. p e l l e t s were resuspended  The  i n an a d d i t i o n a l 0.4 ml 10% TCA, r e c e n t r i f u g e d and  the two s u p e r n a t a n t s , c o n t a i n i n g the cAMP, were combined to g i v e a t o t a l volume o f 1.8 ml.  The p e l l e t was d i s s o l v e d  i n 5 ml 2% (w/v) Na carbonate  c o n t a i n i n g 0.1 N NaOH i n a warm bath f o r 4 h r s and used protein.  The supernatant was e x t r a c t e d 6 times w i t h 2 volumes o f ether to  remove the TCA. test  f o r determination of  The remaining t r a c e s o f e t h e r were removed by p l a c i n g the  tubes i n a warm water bath under a l i g h t  stream of a i r .  To each  tube,  0.6 ml o f 200 mM Na a c e t a t e b u f f e r , pH 4.5, was added and the samples were s t o r e d at -20°C. Recovery  o f cAMP was determined  cAMP p r i o r  to homogenization.  procedures  and a l i q u o t s  taken at a l l t r a n s f e r  by adding a known amount o f r a d i o a c t i v e  The samples were taken through the i d e n t i c a l  taken f o r d e t e r m i n a t i o n o f r a d i o a c t i v i t y .  steps to minimize  c a r e , however, t r a n s f e r  l o s s e s o f the sample.  l o s s e s p r o b a b l y accounted  Care was  Despite t h i s  f o r most o f the e x p e r i m e n t a l  e r r o r i n the d e t e r m i n a t i o n o f cAMP. iii)  D e t e r m i n a t i o n o f cAMP  A modification  of the Gilman  b i n d i n g assay  measurement o f cAMP l e v e l s i n c o r t i c a l  tissue.  (161,162) was used  f o r the  - 44 -  a)  cAMP b i n d i n g  mixture  3 [ H]-cAMP ( s p . a c t . 37.7 C i / m m o l e ) was d i l u t e d buffer,  pH 4 . 5 , t o g i v e 70 p m o l e s p e r m l o r 5 x 10  w i t h 50 mM Na DPM p e r m l .  cAMP was d i s s o l v e d i n t h e same b u f f e r t o g i v e a s t o c k per  m l , w h i c h was l a t e r  per ml.  Protein kinase  concentration give a stock This  stock  reagents  diluted  o f 0.1 mg p e r m l .  o f 400 p m o l e s  ( P K I ) , was d i s s o l v e d i n w a t e r Protein kinase  Unlabelled  s o l u t i o n o f 40 n m o l e s  100 x t o g i v e a cAMP s t a n d a r d  inhibitor  acetate  to give a  ( P K ) was d i s s o l v e d i n w a t e r t o  s o l u t i o n o f 0.9 mg p e r m l , w h i c h was s t o r e d i n 600 u l a l i q u o t s .  s o l u t i o n was d i l u t e d  t o 8 7 . 1 mg p e r m l j u s t  before  use.A l l  w e r e s t o r e d a t -20°C, e x c e p t f o r t h e 50 mM Na a c e t a t e  b u f f e r , which  was r e f r i g e r a t e d . b)  cAMP b i n d i n g  assay  The  b i n d i n g a s s a y was c o n d u c t e d  on i c e i n a f i n a l v o l u m e o f 250 u l c o n -  t a i n i n g 50 u l P K I ( 5 u g ) , 50 u l [ H ] - c A M P ( 3 . 5 p m o l e s ) 5 t o 90 u l cAMP 3  s t a n d a r d o r 100 u l s a m p l e . T h e r e a c t i o n was i n i t i a t e d b y a d d i n g 50 u l P K ( 4 . 4 ug p r o t e i n ) . The c o n c e n t r a t i o n o f b i n d i n g p r o t e i n ( P K ) s h o u l d be s u f f i c i e n t 3 to  bind  l e s s t h a n 3 0 % o f t h e [ H ] - c A M P , t o e n s u r e t h a t PK i s s a t u r a t e d when  e q u i l i b r i u m i s reached a f t e r  1 h r o f i n c u b a t i o n a t 0°C.  A f t e r an a d d i t i o n a l  15 m i n , 1 m l o f c o l d 20 mM p o t a s s i u m p h o s p h a t e b u f f e r , pH 6.0 was a d d e d t o each assay tube. Millipore  Four min l a t e r  were p a s s e d  f i l t e r s ( c e l l u l o s e e s t e r , HA 0.45, 24 mm) m o u n t e d on a  manifold  (Millipore  acetate  buffer before  The  the assay mixtures  Company). being  The f i l t e r s w e r e r i n s e d w i t h  dried i n glass  counting  vials  r a d i o a c t i v i t y was d e t e r m i n e d i n a r e f r i g e r a t e d N u c l e a r  scintillation  counter,  300 s a m p l e r , u s i n g  c o n t a i n i n g 5 g PPO/1 t o l u e n e .  sampling  10 m l o f c o l d Na  a t 65°C f o r 1 h r . Chicago L i q u i d  10 m l o f s c i n t i l l a t i o n  The CPM r e c o r d e d  through  fluid  was c o r r e c t e d t o DPM f o r e a c h  •ll?  - 45 3 sample from a quench curve, w i t h the measured e f f i c i e n c y f o r [ H] v a r y i n g between 34-39%. The blank count, i . e . assay mixture without PK, was always below 100 DPM and was ignored s i n c e t e s t assays were always  over  10,000 DPM.  A standard curve was p l o t t e d on l o g - l o g paper, w i t h t o t a l pmoles cAMP (tritiated  + n o n - t r i t i a t e d ) on the a b s c i s s a e and DPM on the o r d i n a t e . The  s l o p e o f the s t r a i g h t  l i n e o b t a i n e d between 5 to 40 pmoles v a r i e d  inversely  w i t h the DPM r e c o r d e d , so that f o r i n c r e a s i n g amounts o f u n l a b e l l e d cAMP 3 competing  w i t h [ H]-cAMP f o r b i n d i n g s i t e s , t h e r e i s a r e d u c t i o n i n DPM.  The amount o f cAMP i n a sample can t h e r e f o r e e a s i l y be read from the graph, iv) The  Measurements o f NA L e v e l s procedure  f o l l o w e d f o r the e s t i m a t i o n o f NA was t h a t o f McGeer et^ a l .  (176,177). a)  E x t r a c t i o n o f NA  In order to e v a l u a t e the e f f e c t i v e n e s s o f the 6-OHDA l e s i o n s , NA l e v e l s were measured i n the hippocampi a t i o n s c o u l d not be performed cAMP e s t i m a t i o n s . one  o f c o n t r o l and l e s i o n e d animals.  NA determin-  on the c e r e b r a l c o r t i c e s as these were used f o r  An a l t e r n a t i v e  i s to use one group  o f animals  f o r adenylate c y c l a s e s t u d i e s , but the v a r i a b i l i t y  f o r NA and  found by a l l  i n v e s t i g a t o r s u s i n g 6-OHDA d i c t a t e d our d e c i s i o n to use the same animals f o r both a s s a y s . same group  Since i n n e r v a t i o n of both hippocampus and c o r t e x i s from the  o f NA f i b r e s  (see F i g . I ) the hippocampal  good index o f c o r t i c a l d e p l e t i o n . bilaterally  and weighed.  The hippocampi  NA l e v e l s should g i v e a  were d i s s e c t e d out  The y i e l d was u s u a l l y 100 mg.  The hippocampi  homogenized i n 1 ml 0.4 N p e r c h l o r i c c o n t a i n i n g 0.2 a c e t i c a c i d  were  (10 ml HCIO^  + 2.6 ml HAc + 437 ml ^ 0 ) , i n a T r i - R homogenizer w i t h 20 passes o f a Teflon pestle.  The homogenate was l e f t  s t a n d i n g i n i c e f o r 20 min and then  - 46 -  c e n t r i f u g e d at 1000 x g f o r 10 min. decanted  The  s u p e r n a t a n t , c o n t a i n i n g NA,  o f f i n t o a 5 ml t e s t tube, the p e l l e t resuspended  i n 0.5  was  ml  p e r c h l o r i c a c e t i c a c i d r e a g e n t , r e c e n t r i f u g e d , and the two s u p e r n a t a n t s were pooled and s t o r e d at -20°C u n t i l  assay.  NA d e t e r m i n a t i o n s were a l s o performed  on s u b c o r t i c a l t i s s u e from the  k i n d l e d r a t s , i n the hope of d e t e c t i n g a change i n NA l e v e l s as a r e s u l t of the k i n d l i n g p r o c e d u r e .  I n t h i s case, the c e r e b e l l u m was  d e c o r t i c a t e d b r a i n s and d i s c a r d e d .  The  removed from the  remaining s u b c o r t i c a l t i s s u e  d i v i d e d i n t o a n t e r i o r and p o s t e r i o r h a l v e s .  The t i s s u e was weighed (500-600  mg/half b r a i n ) , and homogenized i n 2 ml o f 0.4  N perchloric/acetic acid using  20 passes o f a T e f l o n p e s t l e i n a 10 ml T r i - R homogenizer. were s u b s e q u e n t l y t r e a t e d i n the same way  was  as those of the  These p r e p a r a t i o n s hippocampi.  3 Two ml o f 0.2 M EDTA and a known amount of [ H]-DA, f o r i n t e r n a l r e c o v e r y , was  added to the c l e a r s u p e r n a t a n t , which was  w i t h 5 N NaOH b e f o r e adding 300 mg  of acid-washed  a d j u s t e d to pH 8.5-9.0  alumina.  As each b a t c h of  alumina v a r i e s s l i g h t l y , the pH at which o p t i m a l r e c o v e r y was p r e v i o u s l y determined  o b t a i n e d was  to be between 8.5-9.0 ( F i g . 12) f o r the p a r t i c u l a r  alumina b a t c h used i n these e x p e r i m e n t s .  The alumina s l u r r y was  stirred for 4  min b e f o r e b e i n g i n t r o d u c e d i n t o a g l a s s column 10 cm l o n g , which had p r e v i o u s l y been plugged w i t h g l a s s wool. ml o f water under g e n t l e s u c t i o n . acetic acid without suction. b u f f e r , pH 6.0,  The column was washed t w i c e w i t h 3 to 5  The NA was  e l u t e d w i t h 0.6 ml o f 0.5  To t h i s e l u a n t was  N  added 0.6 ml of 1 M Na a c e t a t e  and 25 u l of 0.5 N NaOH, to g i v e a f i n a l pH o f 6.0, which  p r e v i o u s l y been shown to g i v e o p t i m a l f l u o r e s c e n c e i n t e n s i t i e s ( F i g . 15). b l a n k and three NA s t a n d a r d s (0.1 Ug, same procedure  to a l l o w comparison  0.2  ug, 0.4  Ug) were taken through  of the r e c o v e r y o f NA w i t h t h a t of the  had One the  - 47 -  r a d i o a c t i v e dopamine. for  A 0.1 ml a l i q u o t o f each pH-adjusted e l u a n t was t a k e n  r a d i o a c t i v i t y determination  i n 10 ml o f Bray's s c i n t i l l a t i o n m i x t u r e (180  g n a p h t h a l e n e , 12 g PPO, 0.45 g POPOP, 60 ml e t h y l e n e g l y c o l , 3 1 d i o x a n e ) . Extreme care was taken  throughout the e n t i r e procedure not to contaminate the  eluants with s c i n t i l l a t i o n  f l u i d , as t h e phosphor c o n t a i n e d i n these  i n t e r f e r e s g r e a t l y w i t h the s p e c t r o p h o t o f l u o r i m e t r i c i n t e n s i t y b)  mixtures  readings,  The NA Assay  A volume o f 0.4 ml 1 M Na a c e t a t e b u f f e r , pH 6.0, was added to 0.6 ml o f pH-adjusted e l u a n t .  Four min a f t e r t h e a d d i t i o n o f 0.5 ml o f an N i o d i n e  s o l u t i o n (0.254 g I  2  + 5 g K l i n 5 ml w a t e r , d i l u t e d to 222 m l ) , the samples  were t r e a t e d w i t h 0.25 ml o f 0.05 N sodium t h i o s u l f a t e f o l l o w e d by 0.5 ml o f a mixture for  o f 7 ml 5N NaOH and 3 ml 0.5% a s c o r b i c a c i d .  "Faded b l a n k s " were done  each sample i n w h i c h 0.35 ml o f 5 N NaOH was s u b s t i t u t e d f o r t h e NaOH/  ascorbic a c i d mixture. for  90 to 120 min.  to  the faded b l a n k s .  The t e s t tubes were l e f t under l i g h t i n an open r a c k  A f t e r t h i s p e r i o d , 0.15 ml o f 0.5% a s c o r b i c a c i d was added The f l u o r e s c e n c e was read  immediately  afterwards  i n an  Aminco-Bowman s p e c t r o p h o t o f l u o r i m e t e r (American Instrument Company), a t e x c i t a t i o n / e m i s s i o n wavelengths o f 402 nm/502 nm.  A l l assay steps were  c a r r i e d out a t room temperature. The  NA content  i n each sample was read o f f a l i n e a r s t a n d a r d  ug/ml-0.2 U g / m l , D L - a r t e r e n o l - H C l ) .  curve  (0.125  The NA standard was d i l u t e d t o 0.2 Ug/ml  from a s t o c k s o l u t i o n o f 10 ug/ml, which was s t o r e d a t -20°C i n 1 ml aliquots. The for  measured v a l u e s o f NA per 0.6 ml e l u a n t a l i q u o t (S^) were c o r r e c t e d  recovery  recovery  and f o r e l u a n t volume by u s i n g t h e i n d i v i d u a l r a d i o a c t i v e  (CPM/0.1 ml o f e l u a n t = C ) , t h e mean c o l d r e c o v e r y per 0.6 ml 1  - 48 a l i q u o t i n the standards c a r r i e d through the column procedure mean CPM/0.1 ml i n these same s t a n d a r d samples ( C ) . D  per sample was then c a l c u l a t e d as S, .C /C,R ,. 1  D  r  K  1 L  ( R ) and the £  The t o t a l i n i t i a l NA  The t o t a l r e c o v e r y o f  u n l a b e l l e d standards was 70 t o 80%; r a d i o a c t i v e r e c o v e r i e s were l i n e a r l y r e l a t e d to c o l d r e c o v e r i e s b u t a p p r e c i a b l y l e s s because o f t r i t i u m exchange 14 d u r i n g the procedure.  The use o f  C - l a b e l l e d catecholamine  permits  closer  r e a l c o r r e l a t i o n between hot and c o l d r e c o v e r i e s b u t was not used because a v a i l a b l e s p e c i f i c a c t i v i t i e s are so much lower than f o r the t r i t i a t e d 14 d e r i v a t i v e s t h a t the a d d i t i o n o f s i g n i f i c a n t  C CPM would add s u f f i c i e n t  m a t e r i a l t o i n f l u e n c e the f l u o r e s c e n t assay r e s u l t s .  The l a b e l l e d r e c o v e r i e s  were u s u a l l y i n the range o f 25 t o 30% and the u n l a b e l l e d ones 70 t o 80%. The data were expressed as ug NA/g wet b r a i n v)  tissue,  P r o t e i n Determination  A l l p r o t e i n d e t e r m i n a t i o n s were done by the method o f Lowry e t a l . ( 1 7 8 ) . The cAMP c o n t e n t had to be expressed as pmoles cAMP/mg p r o t e i n , as i t was never  f e a s i b l e to weigh the t i s s u e s l i c e s b e f o r e i n c u b a t i o n s .  However, i f one  assumes t h a t a d u l t r a t c e r e b r a l c o r t e x c o n t a i n s 10% p r o t e i n , a l l data can e a s i l y be c o n v e r t e d to pmoles cAMP/g wet weight b r a i n t i s s u e ( 1 7 9 ) . 3.  A n a l y s i s o f Data The v a r i a t i o n s o f NA l e v e l s were expressed as the means + S.E.M. ( s t a n d a r d  e r r o r o f the mean) and were a n a l y z e d u s i n g the student t - t e s t , w i t h d i f f e r e n c e s between groups c o n s i d e r e d to be s i g n i f i c a n t  i f p<.05.  In the s t a t i s t i c a l a n a l y s i s o f the dose-response c y c l a s e responses  curves, i . e . the adenylate  to d i f f e r e n t amounts o f n o r a d r e n a l i n e , q u a n t i t a t i v e a s s e s s -  ment can be accomplished by comparing the doses p r o d u c i n g e q u a l responses i n  - 49 -  c o n t r o l and e x p e r i m e n t a l p r e p a r a t i o n s . c e n t e r , o f a dose-response  curve  S i n c e the s t e e p e s t p a r t , i . e . the  i s the most a c c u r a t e p a r t , s t a t i s t i c a l  a n a l y s i s was done on the EC..Q v a l u e s , i . e . the dose o f a g o n i s t t h a t produces a half-maximal  response.  The EC^Q v a l u e s o f a g o n i s t s f o r most  appear to show a l o g normal d i s t r i b u t i o n .  tissues  Therefore, a l l s t a t i s t i c a l  compar-  i s o n s were done u s i n g l o g EC^Q data and the geometric means w i t h t h e i r 95% c o n f i d e n c e i n t e r v a l s have been p r e s e n t e d . from the EC^Q v a l u e s estimated Accurate  from  Geometric  the i n d i v i d u a l  means were c a l c u l a t e d curves.  e s t i m a t e s o f changes i n s e n s i t i v i t y may be o b t a i n e d by c a l c u l a t i n g  the r a t i o s o f geometric  means o f EC^Q's  or by the d e t e r m i n a t i o n o f the d i f -  f e r e n c e between the mean l o g s , i . e . the l o g s h i f t , mental groups.  T h i s i s probably  i n which the same animal  between c o n t r o l and e x p e r i -  the p r e f e r r e d method i n e x p e r i m e n t a l  serves as i t s own c o n t r o l .  In the p r e s e n t  a d e s i g n was not p o s s i b l e , as each animal c o u l d be used c o n t r o l or an e x p e r i m e n t a l animal. graphical estimations.  designs  study  such  o n l y once, e i t h e r  as a  In a d d i t i o n , EC^Q v a l u e s were always  Maximal s t i m u l a t i o n s , b a s e l i n e v a l u e s and p e r c e n t s o f  maximal s t i m u l a t i o n ( i . e . maximal s t i m u l a t i o n - b a s e l i n e v a l u e / b a s e l i n e v a l u e x 100) were a l s o analysed on the o r i g i n a l dose-response student's  t-test.  curve data by the  - 50 III. 1.  RESULTS FLA-63 I n an attempt to a c h i e v e n o r a d r e n e r g i c s u p e r s e n s i t i v i t y o f the c e r e b r a l  c o r t e x by p h a r m a c o l o g i c a l d e n e r v a t i o n , t h e drug FLA-63 was a d m i n i s t e r e d i n t r a p e r i t o n e a l l y at a dose of 25 mg per kg (180).  FLA-63 b i s ( 4 m e t h y l - 1 -  h o m o p i p e r a z i n y l - t h i o c a r b o n y l ) d i s u l f i d e i s a c h e l a t i n g agent and t h e r e f o r e . .  2+  i n h i b i t s the Cu  -dependent dopamine-8-hydroxylase  enzyme v e r y e f f e c t i v e l y .  The drug causes v e r y r a p i d and v e r y complete d e p l e t i o n o f c e n t r a l NA s t o r e s , w i t h o n l y m i n i m a l e f f e c t s on c e n t r a l DA s t o r e s (168,181).  A t a dose o f 25  mg/kg, FLA-63 caused a 65% r e d u c t i o n o f c o r t i c a l NA i n 4 hours w h i l e r a i s i n g DA l e v e l s by o n l y 10% (173,182).  FLA-63 u n f o r t u n a t e l y a l s o a f f e c t s  central  t h e r m o r e g u l a t o r y mechanisms (183) c a u s i n g s e v e r e hypothermia w i t h i n 2 h r s , so t h a t animals had to be kept a t 30°C (184). Most o f the animals d i e d w i t h i n 12 h r i f the e n v i r o n m e n t a l temperature was not r e g u l a t e d .  T h e i r body temperature f e l l 3°C 2 h r a f t e r i n j e c t i o n .  When  the room temperature was a d j u s t e d to 30°C, the r a t s s u r v i v e d and had normal body t e m p e r a t u r e s .  The v e h i c l e used t o d i s s o l v e the drug may have a l s o  c o n t r i b u t e d to the m o r t a l i t y r a t e .  A l t h o u g h g l a c i a l a c e t i c a c i d used to  d i s s o l v e the drug was d i l u t e d b e f o r e i n j e c t i o n , t h e pH was v e r y a c i d and c o u l d be expected to cause severe p e r i t o n e a l i n j u r y (185).  The c o n t r o l s , however,  r e c e i v e d the v e h i c l e and d i d not show such a h i g h m o r t a l i t y r a t e .  The assay  procedure employed i n t h i s e x p e r i - ment was the o r i g i n a l method d e s c r i b e d i n the f o l l o w i n g s e c t i o n ( s e c t i o n  III-2).  The animals were s a c r i f i c e d 6 h r s and 24 h r s p o s t - i n j e c t i o n . appears  There  to be a s u p e r s e n s i t i v e response a f t e r 6 h r s , as t h e dose response  c u r v e f o r the i n j e c t e d animals i s s h i f t e d to the l e f t o f t h a t f o r the  - 51 controls.  Both the b a s e l i n e and maximal response cAMP l e v e l s are lower i n the  e x p e r i m e n t a l animals ( F i g . 6 ) . The dose response curve f o r the 24 h r animals was s h i f t e d s l i g h t l y t o the r i g h t but the s h i f t was not s i g n i f i c a n t a t  p(0.05  (Fig.  7 ) .  The b a s e l i n e cAMP  response was d r a s t i c a l l y lowered from the 6 h r l e v e l s , but the maximal response was s l i g h t l y h i g h e r than both c o n t r o l and 6 h r maximal l e v e l s . However, the c o n t r o l v a l u e s appeared compared t o 6 h r r a t s .  to be d i f f e r e n t f o r 24 h r animals  The b a s e l i n e and cAMP l e v e l s were s i g n i f i c a n t l y  at 24 h r s than a t 6 h r s f o r c o n t r o l a n i m a l s . appeared  lower  The dose response curve a l s o  to s h i f t l e f t w a r d from 6 h r s t o 24 h r s .  I t s h o u l d perhaps be p o i n t e d  out t h a t c o n t r o l animals were a l s o k e p t a t 3 0 ° C room temperature and t h e i r body temperature a t 24 h r s was e l e v a t e d 1 . 5 ° C . 2.  Development o f method and e a r l y  findings  Due to the v a r i a b i l i t y o f the c o n t r o l a n i m a l s i n the F L A - 6 3  experiments,  and the l a c k o f r e p r o d u c i b i l i t y o f cAMP measurements i n mock e x p e r i m e n t s , i t was decided t h a t the e n t i r e e x p e r i m e n t a l procedure s h o u l d be i n v e s t i g a t e d . The o r i g i n a l method ( 1 0 4 )  employed e t h e r to s a c r i f i c e a n i m a l s .  Since ether i s  a v o l a t i l e a n a e s t h e t i c and t h e r e f o r e may a f f e c t the membrane o f CNS c e l l s , the method o f s a c r i f i c e seemed to be a good s t a r t i n g p o i n t . When the dose-response  curves f o r r a t s s a c r i f i c e d by e t h e r and f o r r a t s  s a c r i f i c e d by c e r v i c a l f r a c t u r e were compared, they were r a t h e r s i m i l a r , as shown i n F i g . 8.  The s l o p e s o f the curves were almost i d e n t i c a l , b u t the  b a s e l i n e cAMP response o f the c e r v i c a l f r a c t u r e animals was s i g n i f i c a n t l y h i g h e r than t h a t o f the e t h e r k i l l e d a n i m a l s .  The same was t r u e f o r the  maximal response (here 3 0 0 uM NA) and the EC^Q a l s o seemed t o be h i g h e r , b u t t h i s was not s i g n i f i c a n t .  A l t h o u g h t h e two curves a r e v e r y s i m i l a r , t h e d a t a  -  52 -  F i g . 6. Dose-response curve o f c e r e b r a l c o r t e x t o NA. C o r t i c a l s l i c e s from c o n t r o l ( O ) and 6 h r FLA-63 t r e a t e d (•) r a t s were exposed t o v a r i o u s c o n c e n t r a t i o n s o f NA f o r 5 min. T i s s u e cAMP content i n t h e absence o f NA (B) r e p r e s e n t s b a s e l i n e v a l u e s . Each p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 3 a n i m a l s . Semilog p l o t .  - 53 -  F i g . 7. Dose-response curve o f c e r e b r a l c o r t e x t o NA. C o r t i c a l s l i c e s from c o n t r o l ( O ) and 24 h r FLA-63 t r e a t e d ( • ) r a t s were exposed t o v a r i o u s c o n c e n t r a t i o n s o f NA f o r 5 min. T i s s u e cAMP c o n t e n t i n t h e absence o f NA (B) r e p r e s e n t s b a s e l i n e v a l u e s . Each p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 4 a n i m a l s . Semilog p l o t .  - 54 -  NA  concentration  (/uM)  F i g . 8. Dose-response curve o f c e r e b r a l c o r t e x t o NA. C o r t i c a l s l i c e s from animals s a c r i f i c e d by c e r v i c a l f r a c t u r e ( • ) and by e t h e r (O) were exposed t o v a r i o u s c o n c e n t r a t i o n s o f NA f o r 5 min. T i s u e cAMP content i n t h e absence o f NA (B) r e p r e s e n t s b a s e l i n e v a l u e s . Each p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 4 a n i m a l s . Semilog p l o t .  - 55 for  the e t h e r - k i l l e d r a t s were always more i n c o n s i s t e n t than those u s i n g  c e r v i c a l f r a c t u r e so t h a t the b e t t e r procedure was used t h e r e a f t e r . O r i g i n a l l y , r a t h e r s m a l l p i e c e s o f c o r t e x were removed w i t h a r a z o r b l a d e and  these p i e c e s were s l i c e d i n t o twelve 1 mm t h i c k s e c t i o n s .  tube then c o n t a i n e d  two such s e c t i o n s .  Each  incubation  I t i s v e r y u n l i k e l y t h a t such t h i c k  t i s s u e s l i c e s w i l l r e c e i v e u n i f o r m exposure to the b a t h i n g  s o l u t i o n and the  i o n s , O2 and neurohormones i n i t (186). I t proved p o s s i b l e to d i s s e c t out much l a r g e r p o r t i o n s o f t i s s u e by p e e l i n g c o r t i c a l slabs with a very f i n e s c a l p e l . 300 mg per r a t .  The y i e l d then became 250 t o  The new l a b o r a t o r y f o r t u n a t e l y had a M c l l w a i n  so the t i s s u e c o u l d be s e c t i o n e d  t i s s u e chopper,  one-way i n t o 0.3 mm t h i c k s l i c e s .  The  dimension o f the s l i c e s were 5 mm l o n g , 1.5 mm deep and 300 um t h i c k . use  o f t h i n n e r s l i c e s should  total This  f a c i l i t a t e r e s p i r a t i o n and ensure more u n i f o r m  exposure to i n c u b a t i o n s o l u t i o n s and more r a p i d uptake o f neurohormone from the medium. K r e b s - R i n g e r b i o c a r b o n a t e b u f f e r must be a e r a t e d p h y s i o l o g i c a l pH but a t atmospheric p r e s s u r e i s 95% 0  2  and 5% C 0  2  w i t h CO2 t o a c h i e v e  (760 mm Hg), the proper m i x t u r e  t o a c h i e v e pH 7.2 ( i d e a l l y i t should be 7.4).  I n the  o r i g i n a l method, d r y i c e (100% CC^) had been bubbled through the b u f f e r and t h i s r e s u l t e d i n a pH o f 6.0. i n c u b a t i o n f l a s k s and a e r a t e d increased  When t h i s pH 6.0 b u f f e r was t r a n s f e r r e d t o w i t h 95% 0^-5% CO^ m i x t u r e ,  to 7.2 over about 20 m i n u t e s .  the pH s t e a d i l y  D u r i n g the second i n c u b a t i o n  the neurohormone was added a t 14 minutes and i n c u b a t i o n c o n t i n u e d minutes.  The c r i t i c a l  was changing. C0„ b e f o r e  incubations  therefore occurred  for 6  a t a time when the pH  The K r e b s - R i n g e r b u f f e r was t h e r e f o r e a e r a t e d  as w e l l as d u r i n g the i n c u b a t i o n .  period,  w i t h 95% 0 2 5 % _  - 56 NA was  o r i g i n a l l y d i s s o l v e d i n BSA  b u f f e r , pH 7.2.  T h i s pH  a l k a l i n e pH and NA was  i s a b i t too b a s i c f o r NA,  The  t i s s u e was  pH  e t h e r to remove the TCA  by c e n t r i f u g a t i o n and and then l y o p h i l i z e d .  But h i g h c o n c e n t r a t i o n s  medium were s t i l l p r e s e n t  o r i g i n a l l y accomplished  The  the supernate e x t r a c t e d The  r e s i d u e was  the a d d i t i o n of TCA.  t e r m i n a t i o n procedure was  For  the same reason,  i n water r a t h e r than K r e b s - R i n g e r b i c a r b o n a t e  d i l u t i o n was and 1:60  another standard  pmoles i n F i g . 9 and 4.2  1:200  i n t e r f e r e n c e by i o n s i s  1:450.  The  shown to be  F i g u r e s 9 and  curve w i t h 100 u l a c e t a t e  The  10  buffer  amount of cAMP/100 u l of t i s s u e  pmoles i n F i g . 10.  i n F i g . 9 (a d i l u t i o n as low as 1:450  i n F i g . 10.  4.5.  curve w i t h a t i s s u e sample c o n t a i n i n g  u l b u f f e r added to each tube. 3.3  to  experiments.  each show a Gilman b i n d i n g assay standard  sample was  dissolved  originally  to be n e g l i g i b l e , and  I n the o r i g i n a l method the t i s s u e d i l u t i o n was  cAMP i n 100  of  The minute amounts of cAMP which might have  l e a k e d out i n t o the medium were c o n s i d e r e d  added to each tube and  therefore  PK was  r a i s e d from 4.0  A l t h o u g h b i n d i n g i s more e f f i c i e n t at the lower pH,  so by r a d i o a c t i v e r e c o v e r y  incubation  decantation  b u f f e r , which had  the pH of the NaAc b u f f e r was  m i n i m i z e d at the h i g h e r pH v a l u e .  with  these s a l t s would s u b s e q u e n t l y  changed to i n c l u d e a 30 sec low-speed c e n t r i f u g a t i o n step and  been the case, and  by  t a k e n up i n  of s a l t s from the o r i g i n a l  i n t h i s medium and  i n t e r f e r e w i t h cAMP b i n d i n g to PK.  the b u f f e r b e f o r e  The  to the i n c u b a t i o n medium c o n t a i n i n g the t i s s u e s l i c e s .  separated  acetate b u f f e r .  which w i l l decompose at  6.  of the neurohormone i n c u b a t i o n was  the a d d i t i o n of TCA  bicarbonate  s u b s e q u e n t l y d i s s o l v e d i n BSA-water i n s t e a d .  d i s t i l l e d water i n the l a b o r a t o r y was Termination  c o n t a i n i n g Krebs-Ringer  was  The  tissue  not  l i n e s are n o n - p a r a l l e l i n F i g . 9 and  attempted)  the amount of  -  57  -  cAMP concentration  (pM)  F i g . 9. Standard curve f o r the d e t e r m i n a t i o n o f cAMP c o n t e n t o f b r a i n s l i c e s . A t i s s u e sample c o n t a i n i n g 3.3 pm cAMP i n 100 u l b u f f e r was added (• ) t o t h e t y p i c a l s t a n d a r d curve ( O ) . T i s s u e d i l u t i o n 1:200. L o g - l o g p l o t .  - 58 -  cAMP  concentration (pM)  F i g . 10. Standard curve f o r the d e t e r m i n a t i o n o f cAMP c o n t e n t o f b r a i n s l i c e s . A t i s s u e sample c o n t a i n i n g 4.2 pm cAMP i n 100 u l b u f f e r was added (•) t o the t y p i c a l s t a n d a r d curve ( o ) . T i s s u e d i l u t i o n 1:60. L o g - l o g p l o t .  cAMP i n the t i s s u e c o u l d t h e r e f o r e not be a c c u r a t e l y determined u s i n g t h e s e conditions.  The l i n e s are p a r a l l e l i n F i g . 10 a l t h o u g h  the s l i g h t s h i f t i s w i t h i n e x p e r i m e n t a l  error.  not c o i n c i d e n t a l , b u t  (A S c a t c h a r d  p l o t gave a  = 0.5 x 10~ M). 9  Thus the e x p e r i m e n t a l  procedure f i n a l l y developed gave r e p r o d u c i b l e  i n agreement w i t h t h e l i t e r a t u r e (163,187). approximately The  The r e c o v e r y  values  o f cAMP was  73%.  time course o f the a c c u m u l a t i o n o f cAMP i n c o r t i c a l s l i c e s i s shown i n  F i g . 11. The use o f 100 uM NA f o r maximal s t i m u l a t i o n r e s u l t e d i n a v e r y r a p i d i n c r e a s e i n cAMP apparent a f t e r a 1 min i n c u b a t i o n and i n c r e a s i n g f u r t h e r a f t e r 5 min. The maximum was reached a f t e r 10 min but the d i f f e r e n c e between the v a l u e s  a t 5 and 10 min was not s i g n i f i c a n t .  d e c l i n e on l o n g e r i n c u b a t i o n p e r i o d s  The l e v e l s b e g i n t o  due to the h y d r o l y t i c a c t i o n o f PDE.  The  i n c u b a t i o n p e r i o d chosen was 6 min, so t h a t a PDE i n h i b i t o r such as aminop h y l l i n e or p a p a v e r i n e was not needed.  F i v e minute i n c u b a t i o n s  to w h i c h such  an i n h i b i t o r was added d i d not g i v e r e s u l t s s i g n i f i c a n t l y d i f f e r e n t from those i n d i c a t e d i n F i g . 11. The  c e r e b r a l c o r t i c a l s l i c e s were mixed and randomly d i s t r i b u t e d t o each  of the s i x i n c u b a t i o n v i a l s so t h a t any r e g i o n a l a n a t o m i c a l would not be d e t e c t e d .  To v e r i f y the a p p r o p r i a t e n e s s  d i f f e r e n c e i n cAMP  o f t h i s p r o c e d u r e , each  r e g i o n o f the c o r t e x was assayed s e p a r a t e l y as shown i n F i g . 12.  There was no  s i g n i f i c a n t d i f f e r e n c e between the c o r t i c a l lobes nor was there any d i f f e r e n c e between l e f t and r i g h t s i d e s f o r e i t h e r b a s e l i n e or m a x i m a l l y values.  stimulated  When the l e f t and r i g h t s i d e s were averaged, t h e r e was s t i l l no  s i g n i f i c a n t d i f f e r e n c e between p a r i e t a l , f r o n t a l or o c c i p i t o - t e m p o r a l This  cortex.  i s shown i n F i g . 13 and was t r u e f o r b o t h b a s e l i n e and s t i m u l a t e d (100 uM  NA) cAMP l e v e l s .  - 60 -  90  .E  -  80 -  0)  J _ I  01  i  5 T i m e  1  10  1  20  —i—  30  (min.)  F i g . 11. Time course o f the s t i m u l a t i o n o f cAMP f o r m a t i o n by NA i n c o r t i c a l s l i c e s o f a d u l t r a t s . B r a i n s l i c e s were i n c u b a t e d w i t h 100 uM NA f o r the time p e r i o d s i n d i c a t e d . Each p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 4 a n i m a l s .  - 61 -  140 h  C o r t i c a l  R e g i o n  F i g . 12. The response o f d i f f e r e n t c o r t i c a l r e g i o n s t o NA. The l e f t and r i g h t hemispheres were d i v i d e d i n t o F = f r o n t a l , P = p a r i e t a l and 0-T = o c c i p i t o temporal c o r t i c a l r e g i o n s . B r a i n s l i c e s were exposed t o 0 ^uM ( b a s e l i n e ) and 100 uM NA f o r 5 min. Each p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 6 a n i m a l s .  - 62 -  120 r  * o  100  yuM  NA  100  CL  co E < u  0) o E o o  80  60  Baseline  40 r  20 F  P C o r t i c a l  O-T R e g i o n  F i g . 13. The response o f d i f f e r e n t c o r t i c a l r e g i o n s t o NA. L e f t and r i g h t hemispheres were combined but d i v i d e d i n t o F = f r o n t a l , P = p a r i e t a l and 0-T = occipito-temporal c o r t i c a l regions. B r a i n s l i c e s were exposed t o 0 pK (basel i n e ) and 100 ^iM NA f o r 5 min. Each p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 6 a n i m a l s . F i g . 13 i s a r e - p l o t o f F i g . 12.  -  I n order to e s t a b l i s h o p t i m a l at which o p t i m a l e s t a b l i s h e d to be  63  conditions  r e c o v e r y was o b t a i n e d 8.5-9.0  (Fig.  -  14).  f o r the NA d e t e r m i n a t i o n s ,  w i t h the b a t c h o f alumina used was S i m i l a r l y , maximal s p e c t r o p h o t o -  f l u o r i m e t e r r e a d i n g s i n t h e NA assay were shown t o be o b t a i n e d eluant  was a d j u s t e d  the pH  i f the column  to pH 6 . 5 b e f o r e a l i q u o t s were taken f o r a n a l y s i s ( F i g .  15).  3.  Kindled  Rats  I n order to t e s t the h y p o t h e s i s t h a t permanent changes take p l a c e  during  the development o f s e i z u r e s u s c e p t i b i l i t y by k i n d l i n g , t h e NA s e n s i t i v e a d e n y l a t e c y c l a s e system was s t u d i e d kindled rats.  i n the e p i l e p t o g e n i c a l l y steady s t a t e o f  The c e r e b r a l c o r t e x was d i v i d e d i n t o a n t e r i o r and p o s t e r i o r  h a l v e s and i n c u b a t e d as d e s c r i b e d t i s s u e was s c a r c e  i n the "Methods" s e c t i o n .  Because c o r t i c a l  i n these e x p e r i m e n t s , o n l y t h r e e c o n c e n t r a t i o n s  ( b a s e l i n e ) , 10 UM and 100 UM, were t e s t e d . a p p r o x i m a t e l y 120 mg per c o r t i c a l  o f NA, 0  The average y i e l d was  half.  There was no s i g n i f i c a n t d i f f e r e n c e between k i n d l e d and c o n t r o l animals i n cAMP a c c u m u l a t i o n i n e i t h e r the a n t e r i o r or p o s t e r i o r c o r t i c a l s l i c e s a t any NA c o n c e n t r a t i o n .  Maximal s t i m u l a t i o n (100 uM NA) caused a 97% i n c r e a s e i n  cAMP i n the a n t e r i o r c o r t e x t o y i e l d 6 6 . 3 pmoles/mg p r o t e i n i n t h e k i n d l e d a n i m a l s and a 92% i n c r e a s e  to y i e l d 7 1 . 9 pmoles/mg p r o t e i n i n c o n t r o l s , as  shown i n F i g . 16 and T a b l e 1  (appendix).  I n the p o s t e r i o r s l i c e s ( F i g . 17) maximal NA s t i m u l a t i o n caused 121% increase  i n cAMP ( 6 7 . 3 pmoles/mg p r o t e i n ) i n k i n d l e d animals and 116% i n c r e a s e  ( 6 6 . 6 pmoles/mg p r o t e i n ) i n c o n t r o l s ( T a b l e 1,  appendix).  F i g . 14. R e l a t i v e r e c o v e r y o f NA from alumina batches at v a r i o u s pH's. Each p o i n t and v e r t i c a l bar r e p r e s e n t s the mean and S.E.M., r e s p e c t i v e l y , o f 4 determinations.  - 65 -  20  40  4-5 pH  5-0 of  5.5  6-0  6.5  7.0  7.5  Eluant  F i g . 15. R e l a t i v e i n t e n s i t y o f s p e c t r o p h o t o f l u o r i m e t e r r e a d i n g s as a f u n c t i o n o f t h e pH o f t h e Na A c e t a t e b u f f e r e l u a n t . Each p o i n t and v e r t i c a l b a r r e p r e s e n t s the mean and S.E.M., r e s p e c t i v e l y , o f 4 d e t e r m i n a t i o n s .  - 66 -  B  -fh  10 NA concentration  100 (yuM)  F i g . 16. S t i m u l a t i o n o f cAMP f o r m a t i o n by NA i n a n t e r i o r c o r t i c a l s l i c e s o f k i n d l e d (• ) and c o n t r o l ( o ) r a t s . T i s s u e cAMP c o n t e n t i n the absence o f NA (B) r e p r e s e n t s b a s e l i n e v a l u e s . Each p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 7 k i n d l e d and 4 c o n t r o l a n i m a l s . Semi-log plot.  - 67 -  B  -fh  10 NA  concentration  100 (yuM)  F i g . 17. S t i m u l a t i o n o f cAMP f o r m a t i o n by NA i n p o s t e r i o r c o r t i c a l s l i c e s o f k i n d l e d ( • ) and c o n t r o l ( o ) r a t s . T i s s u e cAMP c o n t e n t i n t h e absence o f NA (B) r e p r e s e n t s b a s e l i n e v a l u e s . Each p o i n t and v e r t i c a l b a r r e p r e s e n t s the mean and S.E.M., r e s p e c t i v e l y , o f 7 k i n d l e d and 4 c o n t r o l a n i m a l s . S e m i - l o g plot.  - 68 There were a l s o no s i g n i f i c a n t d i f f e r e n c e s between a n t e r i o r and p o s t e r i o r c o r t e x i n e i t h e r k i n d l e d or c o n t r o l animals a t any NA c o n c e n t r a t i o n I d e a l l y , a f u l l dose-response curve s h o u l d be c o n s t r u c t e d receptor  s e n s i t i v i t y changes a r e measured.  tested.  whenever  I t i s p o s s i b l e that there  isa  s h i f t o f the m i d p o i n t o f the curve i n s p i t e o f there b e i n g no d i f f e r e n c e i n b a s e l i n e , 10 uM NA and 100 uM N A - s t i m u l a t e d cAMP v a l u e s . Endogenous NA l e v e l s were measured i n the r e m a i n i n g s u b c o r t i c a l t i s s u e s . The average weight o f s u b c o r t i c a l " h a l f - b r a i n s " was 500-600 mg.  The NA l e v e l s  of a n t e r i o r b r a i n h a l v e s were not s i g n i f i c a n t l y d i f f e r e n t , as shown i n T a b l e 1 (appendix).  However, NA l e v e l s o f p o s t e r i o r b r a i n h a l v e s were  d i f f e r e n t at p<0.05 b u t not at p<0.025 (see T a b l e 1, a p p e n d i x ) .  significantly I t s h o u l d be  s t r e s s e d t h a t NA l e v e l s were measured on whole s u b c o r t i c a l t i s s u e s r a t h e r on s p e c i f i c r e g i o n s  than  o f the b r a i n thought to be i m p l i c a t e d i n s e i z u r e  s u s c e p t i b i l i t y mechanisms.  4.  Developmental E f f e c t on the cAMP System The r e s p o n s i v e n e s s o f the cAMP g e n e r a t i n g  system to NA was s t u d i e d i n  s l i c e s from whole c o r t e x i n 15 day o l d r a t s . at b i r t h but g r a d u a l l y  This  system i s i n s e n s i t i v e to NA  develops a r e s p o n s i v e n e s s which reaches a maximum a t 2  weeks p o s t n a t a l and t h e r e a f t e r decreases t o a d u l t l e v e l s (about 50% o f max) a t 25 days. As shown i n F i g . 18, the b a s e l i n e  l e v e l o f cAMP was s i g n i f i c a n t l y  higher  i n the young animals than i n the a d u l t c o n t r o l s , 50.7 pmoles/mg p r o t e i n i n t h e young and 31.0 pmoles/mg p r o t e i n i n the a d u l t s . tested e l i c i t e d a s i g n i f i c a n t l y higher compared to the a d u l t a n i m a l s .  Every concentration  o f NA  cAMP response i n the young r a t s as  T h i s was e s p e c i a l l y t r u e f o r the maximal  - 69 -  N A  concentration  (AJM)  F i g . 18. Dose-response curve o f c e r e b r a l c o r t e x t o NA. C o r t i c a l s l i c e s from 15 day o l d (O ) and a d u l t ( • ) r a t s were exposed t o v a r i o u s c o n c e n t r a t i o n s o f NA f o r 5 min. T i s s u e cAMP content i n t h e absence o f NA (B) r e p r e s e n t s b a s e l i n e v a l u e s . Each p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 5 young and 6 a d u l t a n i m a l s . Semi-log p l o t . * V a l u e s s i g n i f i c a n t l y d i f f e r e n t from a d u l t s (p<0.05).  - 70 response, which was 125.7 pmoles/mg p r o t e i n i n t h e young and 59.4 pmoles/mg p r o t e i n i n the a d u l t r a t s .  These v a l u e s correspond  t o an i n c r e a s e over  b a s e l i n e v a l u e s o f 164% i n the 15 day o l d animals and 125% i n the a d u l t s i n response  to 100 uM NA.  However, the l e f t w a r d h o r i z o n t a l s h i f t o f the EC,-Q v a l u e i n the young as compared to t h e o l d group was not s i g n i f i c a n t . very high responsiveness  I t t h e r e f o r e appears t h a t t h e  o f the cAMP system a t the end o f the second p o s t -  n a t a l week i s not due to s u p e r s e n s i t i v i t y o f t h e B r e c e p t o r a t t h i s stage o f development o f the b r a i n .  5.  Supersensitivity I n t r a v e n t r i c u l a r i n j e c t i o n o f 6-OHDA i n animals r e s u l t s i n a severe  t i o n o f b o t h NA and DA throughout  the b r a i n .  deple-  Numerous s t u d i e s have d e s c r i b e d  the b i o c h e m i c a l and h i s t o l o g i c a l e f f e c t s o f such i n j e c t i o n s , b u t a t the time o f the e x p e r i m e n t s ,  no one had performed an experiment  measuring  NA-stimulated  cAMP l e v e l s o f the c e r e b r a l c o r t e x a f t e r s p e c i f i c d e p l e t i o n o f NA u s i n g s e l e c t i v e a p p l i c a t i o n s o f 6-OHDA.  I n t r a c e r e b r a l i n j e c t i o n s o f 6-OHDA i n t o t h e  d o r s a l b u n d l e s p e c i f i c a l l y d e p l e t e c o r t i c a l NA s t o r e s and l e a v e almost a l l other c a t e c h o l a m i n e r g i c systems i n t a c t .  I t had been r e p o r t e d i n t h e  l i t e r a t u r e t h a t c o r t i c a l NA s t o r e s were a l r e a d y decreased D.B. l e s i o n s w i t h 6-OHDA and anterograde (188).  d e g e n e r a t i o n was complete i n 15 days  I n t r a v e n t r i c u l a r 6-OHDA i n j e c t i o n s r e s u l t e d i n a 50% decrease  s t o r e s a f t e r 7 days and an i n c r e a s e i n N A - s t i m u l a t e d days.  a t 5 days f o l l o w i n g  The i n c r e a s e d r e s p o n s i v e n e s s  4 weeks ( 1 8 9 ) .  cAMP a c c u m u l a t i o n  i n NA after 5  o f the cAMP system was s t i l l p r e s e n t  after  The one  present i n v e s t i g a t i o n consisted  o f two s e r i e s o f D.B. i n j e c t e d a n i m a l s  group o f 7 r a t s were assayed a f t e r 7 days and another group o f 7 r a t s were  s a c r i f i c e d a f t e r 10 weeks.  Only r a t s w i t h a r e d u c t i o n  l e v e l s o f a t l e a s t 75% were i n c l u d e d animals were i n j e c t e d w i t h served as u n t r e a t e d  i n the experiment.  i n hippocampal NA A t h i r d group o f 4  the v e h i c l e o n l y and a f o u r t h group o f 6 animals  controls.  F i g . 19 shows the dose-response curve f o r v e h i c l e (mock) i n j e c t e d and untreated c o n t r o l animals.  A l t h o u g h the v e h i c l e - i n j e c t e d animals seem t o  e x h i b i t a lower r e s p o n s i v e n e s s t o NA, t h i s i s n o t s t a t i s t i c a l l y s i g n i f i c a n t . When t h e NA l e v e l s were measured, 3 out o f 4 v e h i c l e i n j e c t e d r a t s were severely  depleted.  The reason f o r t h i s remains o b s c u r e , a l t h o u g h the  p o s s i b i l i t y e x i s t s t h a t t h i s group o f animals were s u b j e c t e d t o a s u r g i c a l i n j u r y or some i n t e r f e r e n c e temporary d e p l e t i o n of NA systems.  o f NA.  with axonal transport  non-specific  which l e d t o  There was no h i s t o l o g i c a l e v i d e n c e o f d e s t r u c t i o n  The evidence o f such n o n - s p e c i f i c  i n j u r y from v e h i c l e  i n j e c t i o n s c o m p l i c a t e i n t e r p r e t a t i o n s o f t h e r e s u l t s on 6-OHDA t r e a t e d a n i m a l s , a l t h o u g h these were c l e a r l y d i f f e r e n t from the v e h i c l e - i n j e c t e d  group  As shown i n F i g . 20 animals s a c r i f i c e d 7 days a f t e r t h e 6-OHDA i n j e c t i o n s showed a s i g n i f i c a n t l y h i g h e r maximal s t i m u l a t i o n v a l u e s o f cAMP remained unchanged.  than c o n t r o l s .  The b a s e l i n e  There appeared to be a s l i g h t  leftward  s h i f t i n the EC^Q o f the dose response c u r v e , i . e . a change i n K^, b u t t h i s s h i f t was not s t a t i s t i c a l l y s i g n i f i c a n t .  There a l s o appeared t o be a  c o n s i d e r a b l e change i n the s l o p e o f the c u r v e . The  e f f e c t on t h e cAMP system a f t e r 10 weeks p o s t - i n j e c t i o n i s shown i n  F i g . 21 and T a b l e 2 ( a p p e n d i x ) . baseline  There i s a s i g n i f i c a n t i n c r e a s e  i n the  l e v e l and maximum s t i m u l a t i o n , or V . There i s a l s o a max  -  72  -  100 -  c »  80 -  o  NA  concentration  (>uM)  F i g . 19. Dose-response o f c e r e b r a l c o r t e x t o NA. C o r t i c a l s l i c e s from v e h i c l e - i n j e c t e d ( O ) and c o n t r o l ( • ) animals were exposed t o v a r i o u s c o n c e n t r a t i o n s o f NA f o r 5 min. T i s s u e cAMP content i n t h e absence o f NA (B) r e p r e s e n t s b a s e l i n e v a l u e s . Each p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 3 m o d e - i n j e c t e d and 6 c o n t r o l a n i m a l s . Semi-log plot.  - 73 -  T  NA  concentration  Short-term  (/uM)  F i g . 2 0 . Dose-response curve o f c e r e b r a l c o r t e x t o NA. C o r t i c a l s l i c e s from 6-OHDA i n j e c t e d ( 7 days) ( O ) and c o n t r o l ( • ) r a t s were exposed t o v a r i o u s c o n c e n t r a t i o n s o f NA f o r 5 min. T i s s u e cAMP c o n t e n t i n t h e absence o f NA (B) r e p r e s e n t s b a s e l i n e v a l u e s . Each p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 7 s h o r t - t e r m i n j e c t e d and 6 c o n t r o l a n i m a l s . S e m i - l o g p l o t . * V a l u e s s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s .  -  74  -  120  c a>  100  k.  o  Q. CO  -term  80  a  < u  60  0) o  E a  40  20*  - —// B  ' 1  1  NA  1 3  1 10  concentration  i _  30  100  (>uM)  F i g . 21. Dose-response curve of c e r e b r a l c o r t e x to NA. C o r t i c a l s l i c e s from 6-OHDA i n j e c t e d (10 weeks) ( O ) and c o n t r o l ( • ) r a t s were exposed to v a r i o u s c o n c e n t r a t i o n s o f NA f o r 5 m i n . T i s s u e cAMP content i n the absence of NA (B) represents baseline values. Each p o i n t and v e r t i c a l bar r e p r e s e n t s the mean and S . E . M . , r e s p e c t i v e l y , o f 7 l o n g - t e r m i n j e c t e d and 6 c o n t r o l a n i m a l s . Semi-log p l o t . *Values s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s (p<0.05).  - 75 -  significant and of The  this  s h i f t to the l e f t  of the dose-response  i s p r o b a b l y the most r e l i a b l e evidence  curve, i . e . a lower  Kp,  o b t a i n e d f o r the m a n i f e s t a t i o n  s u p e r s e n s i t i v i t y of the n o r a d r e n e r g i c 6 r e c e p t o r i n the c e r e b r a l c o r t e x . s l o p e of the curve  i s a l s o s l i g h t l y changed.  - 76 DISCUSSION FLA-63 The causes o f the h i g h m o r t a l i t y among the i n j e c t e d a n i m a l s were a combina t i o n o f hypothermia and t o x i c i t y o f t h e a c e t i c a c i d v e h i c l e ( 1 7 3 ) .  Raising  the room temperature p a r t i a l l y s o l v e d the m o r t a l i t y problem and a d i f f e r e n t s o l v e n t f o r t h e drug might have f u r t h e r improved t h e experiment.  However, t h e  b i o c h e m i c a l methods employed i n a s s a y i n g cAMP a c c u m u l a t i o n were not r e l i a b l e as the dose response curve f o r c o n t r o l animals s h i f t e d and v a r i a b l e were o b t a i n e d w i t h s t a n d a r d s .  results  F u r t h e r m o r e , b o t h the b a s e l i n e and the maximal  response l e v e l s d i f f e r e n d markedly between t h e two s e t s o f c o n t r o l a n i m a l s . The  s l i g h t i n c r e a s e i n body temperature seemed u n l i k e l y to account f o r such  l a r g e changes i n the cAMP system.  R a t h e r , the e x p e r i m e n t a l procedure seemed  l i k e l y to be inadequate. The FLA-63 experiments were abandoned and the e n t i r e e x p e r i m e n t a l procedure r e - i n v e s t i g a t e d i n the hope o f d e v e l o p i n g a method which would y i e l d more consistent  results.  Development o f method The o r i g i n a l e x p e r i m e n t a l p r o c e d u r e , as p r e v i o u s l y d e s c r i b e d ( 1 0 4 ) , was i n c o n s i s t e n t i n the hands o f t h i s i n v e s t i g a t o r .  I t i s i m p o s s i b l e to make any  i n f e r e n c e s about e x p e r i m e n t a l treatments when the c o n t r o l treatments g i v e variable results.  The methodology  o f the e n t i r e cAMP f i e l d was undeveloped  when the work f o r t h i s t h e s i s was done.  Some p a r t i c u l a r l y s e r i o u s flaws i n  the method i n i t i a l l y used g r e a t l y exaggerated t h e i n h e r e n t v a r i a b i l i t y o f cAMP measurements. When w o r k i n g w i t h b r a i n s l i c e p r e p a r a t i o n s i t i s o f paramount importance to keep c o n d i t i o n s as p h y s i o l o g i c a l as p o s s i b l e .  T h e r e f o r e , t h e pH s h o u l d be  - 77 v e r y c l o s e t o 7.4 and the t i s s u e t h i n enough t o a l l o w u n i f o r m p e n e t r a t i o n o f substances added to the i n c u b a t i o n medium and adequate r e s p i r a t i o n throughout the e n t i r e t i s s u e s l a b . I t i s a l s o d e s i r a b l e to m i n i m i z e a l l i n t e r f e r e n c e s when w o r k i n g w i t h a s e n s i t i v e b i n d i n g assay such as t h e Gilman method and i t i s mandatory t o work at high  enough c o n c e n t r a t i o n s  o f cAMP t o a l l o w a c c u r a t e measurement.  Even a f t e r a l l apparent p r e c a u t i o n s  had been t a k e n , t h e v a r i a b i l i t y i n t h e  l e v e l s o f cAMP measured was d i s c o n c e r t i n g . literature.  This  i s g e n e r a l l y t r u e i n the  The reason f o r t h i s v a r i a b i l i t y has been a s c r i b e d  to i n d i v i d u a l  d i f f e r e n c e s i n the cAMP system ( 4 7 ) . Kindled The  Rats development o f c h r o n i c  s e i z u r e s u s c e p t i b i l i t y by the process known as  k i n d l i n g i s marked by p r o g r e s s i v e  stages o f s e i z u r e a c t i v i t y .  has  reached the f i n a l s t a g e , C - 5 , which i s c h a r a c t e r i z e d  and  generalized  always e l i c i t  has  taken p l a c e  s e i z u r e response even i f the animal has been r e s t i n g f o r  T h i s suggests t h a t a permanent change, p o s s i b l y  biochemical,  i n a t l e a s t some s t r u c t u r e s o f the b r a i n .  Evidence i s c i t e d i n the i n t r o d u c t i o n ( s e c t i o n I - 5 c ) noradrenaline  by l o s s o f b a l a n c e  c l o n i c j e r k i n g , any subsequent e l e c t r i c a l s t i m u l a t i o n w i l l  full  s e v e r a l months.  When t h e a n i m a l  exerts  which i n d i c a t e s  a suppressant e f f e c t on s e i z u r e a c t i v i t y .  This  that  evidence  suggests t h a t a t l e a s t c e r t a i n areas o f the k i n d l e d r a t b r a i n might have c h r o n i c a l l y depleted  NA s t o r e s .  And i f NA e x e r t s  i t s e f f e c t upon neurons v i a  the cAMP system i t was p l a u s i b l e t h a t a change i n cAMP a c c u m u l a t i o n , i n response to i n v i t r o NA s t i m u l a t i o n , would be observed i n the p r e s e n t gation.  investi-  I t was argued t h a t , i f the k i n d l e d b r a i n i s c h r o n i c a l l y d e p l e t e d o f  NA, then areas where t h i s occurs s h o u l d show a s u p e r s e n s i t i v e response t o NA  -  stimulation. (reviewed  78  -  This i s i n accord w i t h the s u g g e s t i o n s  i n s e c t i o n I-5c)  lying epileptic activity.  t h a t s u p e r s e n s i t i v i t y may  of s e v e r a l i n v e s t i g a t o r s be the mechanism under-  S i n c e Corcoran (132) showed t h a t the f r o n t a l  cortex  must be i n t a c t f o r the development o f k i n d l e d s e i z u r e s to o c c u r , the a n t e r i o r and  p o s t e r i o r h a l v e s o f the c o r t e x were i n v e s t i g a t e d s e p a r a t e l y . I t was  t h e r e f o r e d i s a p p o i n t i n g to f i n d no i n c r e a s e i n the cAMP response  and no d i f f e r e n c e i n endogenous NA  levels.  I t i s l i k e l y t h a t more d i s c r e t e  r e g i o n s o f the c o r t e x s h o u l d have been employed i n the cAMP assay.  Similarly,  NA  and  l e v e l s s h o u l d perhaps be measured e x c l u s i v e l y i n the hippocampal  amygdaloid r e g i o n s . The  present  g e n i c phase.  experiments were conducted d u r i n g the s t e a d y - s t a t e e p i l e p t o -  A more f r u i t f u l approach might be to i n v e s t i g a t e s e q u e n t i a l  changes i n b o t h NA kindling.  l e v e l s and cAMP response d u r i n g C-1  to C-5  stages  of  By s t u d y i n g these b i o c h e m i c a l parameters d u r i n g the development o f  s e i z u r e s u s c e p t i b i l i t y and  i n more a n a t o m i c a l  d e t a i l , more l i g h t might be shed  on the p o s s i b l e r o l e o f the NA-cAMP system i n t h i s p a r t i c u l a r  neuropathological  condition. The  development of r e s p o n s i v e n e s s  to NA o f the cAMP g e n e r a t i n g  p a r a l l e l s the i n c r e a s e i n endogenous NA  l e v e l s d u r i n g the f i r s t  l i f e and reaches a maximum at t h i s time (190).  Adenylate  system  two weeks o f  cyclase a c t i v i t y  and  r e c e p t o r d e n s i t y a l s o reach maximum l e v e l s at the end of the second p o s t - n a t a l week (191). the b r a i n . b l a s t has  I t i s a v e r y c r u c i a l time i n the development and m a t u r a t i o n The  c e r e b r a l c o r t e x appears h i s t o l o g i c a l l y mature and the neuro-  developed i n t o a mature neuron, d i s p l a y i n g a d u l t EEG  a c t i o n p o t e n t i a l s at the end of the second week. at t h i s time and  of  activity  and  Many enzyme systems develop  the f o r m a t i o n of synapses i s almost complete (192).  I t has  - 79 been suggested t h a t NA might i n f l u e n c e morphogenesis by a mechanism i n v o l v i n g the i n c r e a s e d s e n s i t i v i t y of the cAMP system (193). The  observed i n c r e a s e i n endogenous cAMP l e v e l s d u r i n g the f i r s t  few weeks  o f l i f e c o u l d be the r e s u l t of an i n c r e a s e i n the amount of a d e n y l a t e an i n c r e a s e i n the number of n o r a d r e n e r g i c  cyclase,  r e c e p t o r s , an i n c r e a s e i n the  s e n s i t i v i t y o f the r e c e p t o r s , an i n c r e a s e i n the c o n c e n t r a t i o n o f NA at r e c e p t o r or a slower  development of the PDE  activity.  The  PDE  the  a c t i v i t y i s not  f u l l y developed u n t i l the t h i r d p o s t - n a t a l week and t h e r e f o r e the g r e a t l y augmented cAMP l e v e l s observed at two weeks are p r o b a b l y  p a r t i a l l y due  to a  l e s s e f f i c i e n t breakdown of cAMP. Many of the other p o s s i b l e e x p l a n a t i o n s mentioned above were not t e s t e d i n the p r e s e n t  i n v e s t i g a t i o n but the r e s u l t s might i n d i c a t e t h a t the  cAMP l e v e l s were not due dose-response curve was  increased  to a change i n s e n s i t i v i t y o f the r e c e p t o r , as not s h i f t e d to the  the  left.  Responsiveness of the cAMP system develops at a s l i g h t l y d i f f e r e n t r a t e i n d i f f e r e n t r e g i o n s o f the c o r t e x .  P u r k i n j e c e l l s i n the c e r e b e l l u m may  i n a h y p e r s e n s i t i v e s t a t e d u r i n g the f i r s t n e o n a t a l weeks b e f o r e have become f u l l y i n n e r v a t e d ( 1 9 3 ) .  The  present  the  exist cells  experiment assayed cAMP  l e v e l s from whole c o r t e x and p o s s i b l y masked some r e g i o n a l d i f f e r e n c e s i n receptor  sensitivity.  When e s t i m a t i n g s e n s i t i v i t y changes of b i o l o g i c a l t a n t to use complete dose-response curves  r e c e p t o r s i t i s impor-  r a t h e r than s i n g l e doses o f d r u g s .  Q u a n t i t a t i v e measurements of s u p e r s e n s i t i v i t y s h o u l d always be based on magnitude o f h o r i z o n t a l s h i f t s of dose-response curves the curve  i s the most a c c u r a t e p a r t and  measured on t h i s  (194).  The  center  the h o r i z o n t a l s h i f t s s h o u l d  steep p o r t i o n of the curve.  the  E q u i e f f e c t i v e doses of  be an  of  - 80 a g o n i s t are n o r m a l l y d i s t r i b u t e d on a l o g r a t h e r than on an a r i t h m e t i c s c a l e and  t h e r e f o r e mean l o g s or g e o m e t r i c means o f e q u i e f f e c t i v e a g o n i s t  concentrations (195,196). presented  are the s t a t i s t i c a l l y v a l i d e s t i m a t e o f the mean s e n s i t i v i t i e s  A r i t h m e t i c means s h o u l d not be used. w i t h i t s 95% c o n f i d e n c e  Although  The g e o m e t r i c mean s h o u l d be  i n t e r v a l i n s t e a d o f a standard  e r r o r (197).  the c r i t e r i o n f o r s u p e r s e n s i t i v i t y i s l e f t w a r d s h i f t o f the dose-  response c u r v e , t h i s h o r i z o n t a l s h i f t i s sometimes accompanied by an i n c r e a s e i n b a s e l i n e v a l u e , an i n c r e a s e i n the maximum response and a change i n the s l o p e o f the curve.  A change i n the s l o p e i s due t o t h e s a t u r a b l e nature o f  the NA uptake mechanism, so t h a t the c o n c e n t r a t i o n o f NA a t the r e c e p t o r i s no l o n g e r a l i n e a r f u n c t i o n o f the e x t e r n a l NA c o n c e n t r a t i o n ( 9 5 ) . i n c r e a s e i n the maximum response i s an i n d i c a t i o n o f a more e f f i c i e n t  site  An coupling  of the r e c e p t o r t o i n t r a c e l l u l a r mechanisms and a s h i f t t o the l e f t i s a m a n i f e s t a t i o n o f i n c r e a s e d a f f i n i t y f o r the r e c e p t o r by the a g o n i s t  (92).  The p r e s e n t experiments on 6-OHDA-treated r a t s showed an i n c r e a s e i n t h e maximum response i n the s h o r t - t e r m animals s l o p e o f the curve. i n j e c t e d animals  and a c o n s i d e r a b l e change i n the  T h i s change i n s l o p e was l e s s marked i n the long-term  and occurs  as the dose-response curve moves i n r e l a t i o n t o  the range o f NA c o n c e n t r a t i o n s a t which n e u r o n a l long-term  uptake i s s a t u r a t e d .  The  i n j e c t e d r a t s a l s o e x h i b i t e d a marked i n c r e a s e i n the maximum  response as w e l l as an augmented b a s e l i n e l e v e l .  I t i s i n t e r e s t i n g to f i n d  t h a t t h i s e f f e c t was e v i d e n t a f t e r 10 weeks p o s t - i n j e c t i o n and t h i s i s the f i r s t p a r t i a l e v i d e n c e t h a t 6-OHDA-induced s u p e r s e n s i t i v i t y i s permanent. I t i s not p o s s i b l e t o exclude the changes, a l t h o u g h PDE i n t e r f e r e n c e .  a decrease i n PDE a c t i v i t y as an e x p l a n a t i o n f o r  the i n c u b a t i o n c o n d i t i o n s were chosen t o m i n i m i z e any  -  However, the data on the long-term  81  -  animals, p a r t i c u l a r l y the h o r i z o n t a l  s h i f t to the l e f t o f the dose-response c u r v e , i s c o n s i s t e n t w i t h the i n d u c t i o n o f s u p e r s e n s i t i v i t y i n the a d r e n e r g i c r e c e p t o r s . numerous times  that disuse of neuronal  I t has been demonstrated  systems, i . e . f u n c t i o n a l d e n e r v a t i o n ,  i s u s u a l l y accompanied by an i n c r e a s e i n t h e number o f 3 r e c e p t o r  binding  s i t e s (90,91).  The time course  parallels  the time course  o f development o f i n c r e a s e cAMP s e n s i t i v i t y t o NA.  The  of t h i s increase i n b i n d i n g s i t e s  f a i l u r e to demonstrate s u p e r s e n s i t i v i t y o f the c e n t r a l n o r a d r e n e r g i c  system i n the s h o r t - t e r m i n j e c t e d animals  i n p u z z l i n g s i n c e an i n c r e a s e i n t h e  number o f 8 b i n d i n g s i t e s has been observed to occur as r a p i d l y as 2 hours a f t e r disuse o f neural input (198).  I t i s q u i t e p o s s i b l e t h a t the h i g h e r  maximum s t i m u l a t i o n and the s m a l l s h i f t i n the curve were i n d i c a t i o n s o f a t r e n d towards s u p e r s e n s i t i v i t y o f the n o r a d r e n e r g i c  r e c e p t o r which might have  been r e v e a l e d had they been assayed a t 2 weeks p o s t - i n j e c t i o n r a t h e r than 1 week.  Anterograde degeneration  i s complete a t 15 days and i t i s p o s s i b l e t h a t  i t would take t h a t l o n g or longer to develop a s u p e r s e n s i t i v e response. I t i s a l s o p o s s i b l e t h a t the d e g e n e r a t i v e days was an u n f o r t u n a t e  c h o i c e o f time.  processes  were slow and t h a t 7  K a l i s k e r e_t a l . (89) has shown a  b i p h a s i c s e n s i t i v i t y change f o l l o w i n g i n t r a v e n t r i c u l a r 6-OHDA  treatment.  Perhaps 7 days i s i n t e r m e d i a t e between the development o f the e a r l y p r e s y n a p t i c e f f e c t and the l a t t e r p o s t s y n a p t i c The  present  changes.  i n v e s t i g a t i o n was l i m i t e d o n l y to the e f f e c t on the NA-  s e n s i t i v e cAMP g e n e r a t i n g  system.  P o s t j u n c t i o n a l s u p e r s e n s i t i v i t y i n smooth  muscle p e r i p h e r a l system has been thought to be n o n - s p e c i f i c , w h i l e the s i t u a t i o n i n t h e CNS i s l e s s c l e a r .  Reserpine-induced  s u p e r s e n s i t i v i t y i n the  - 82 -  CNS does appear to be n o n - s p e c i f i c , w h i l e 6-OHDA i n j e c t i o n s r e s u l t i n r e c e p t o r s e n s i t i v i t y changes i n response to catecholamines  only (96).  S u p e r s e n s i t i v i t y o f the CNS c o u l d r e s u l t from any one, or a  combination,  of the f o l l o w i n g mechanisms: 1)  an i n c r e a s e i n the number o f r e c e p t o r s  2)  a change i n the p r o p e r t i e s o f r e c e p t o r s  3)  a decrease i n PDE  4)  an i n c r e a s e i n the amount o f a d e n y l a t e  5)  an i n c r e a s e i n a c t i v i t y o f a d e n y l a t e  6)  an i n c r e a s e i n the e f f i c i e n c y o f c o u p l i n g between r e c e p t o r and cellular  activity cyclase  cyclase intra-  mechanisms  Recent b i n d i n g s t u d i e s p o i n t to the p o s s i b i l i t y t h a t i n a s u p e r s e n s i t i v e p r e p a r a t i o n t h e r e are more 8 b i n d i n g s i t e s . novo or they c o u l d be unmasked  i f they e x i s t e d i n a sequestered  c o u l d simply move i n a f l u i d membrane. excluded, (71).  These c o u l d be s y n t h e s i z e d de s t a t e or they  De novo s y n t h e s i s can p r o b a b l y  be  as p r o t e i n s y n t h e s i s i s not r e q u i r e d f o r s u p e r s e n s i t i v i t y to develop  Decreased PDE a c t i v i t y has a l s o been suggested as a p o s s i b l e mechanism  of s u p e r s e n s i t i v i t y .  But i n t h i s p r e p a r a t i o n i t c o u l d p r o b a b l y not be the  case, as i n c u b a t i o n i s t e r m i n a t e d b e f o r e the a c t i o n o f PDE becomes e v i d e n t . I n a s u b s e n s i t i v e p r e p a r a t i o n t h i s s i t u a t i o n c o u l d obscure the i n t e r p r e t a t i o n o f r e s u l t s , and i t would be p r e f e r a b l e i n such a study  to i n c l u d e a PDE  i n h i b i t o r i n the i n c u b a t i o n medium. The c o u p l i n g mechanisms understood.  i n v o l v e d i n the cAMP system are not  completely  However, i f more r e c e p t o r s are a c t i v a t e d and t h e r e i s a one-to-  one transmembranal c o u p l i n g between the r e g u l a t o r y (R) s u b u n i t on the outer f a c e o f the membrane and the c a t a l y t i c (C) s u b u n i t on t h e i n n e r aspect o f t h e  -  83  -  membrane, an i n c r e a s e i n the amount of cAMP produced would r e s u l t . if  Similarly,  one r e c e p t o r ' s c o n f i g u r a t i o n changed to become a one-to-two ( o r more)  c o u p l i n g , a s u p e r s e n s i t i v e r e c e p t o r would s t i m u l a t e cAMP p r o d u c t i o n by the i n c r e a s e d number o f a c t i v a t e d a d e n y l a t e c y c l a s e m o l e c u l e s . The  c h o i c e o f p r e p a r a t i o n employed always c a r r i e s both advantages  advantages.  and  dis-  B r a i n s l i c e p r e p a r a t i o n s are always used when s t u d y i n g NA-  s t i m u l a t e d e f f e c t s on a d e n y l a t e c y c l a s e , s i n c e the enzyme does not respond NA i n homogenates. o f the enzyme.  T h i s i s , however, not the case f o r the D A - s t i m u l a t e d  to  form  B r a i n s l i c e p r e p a r a t i o n s are a l s o i n t u i t i v e l y c l o s e r to the  b i o l o g i c a l s i t u a t i o n w i t h at l e a s t some of the a n a t o m i c a l f e a t u r e s i n t a c t i n the c l o s e s t v i c i n i t y of the neurons, such as s u p p o r t i n g a s t r o c y t e s and d e n d r o c y t e s , a l t h o u g h many axons and d e n d r i t e s are severed i n b r a i n  oligo-  slice  preparations too. However, the s l i c e p r e p a r a t i o n s are p r o b a b l y one of the most s e r i o u s sources o f the v a r i a b i l i t y seen i n these types o f e x p e r i m e n t s .  Homogenates,  on the o t h e r hand, always y i e l d v e r y u n i f o r m and r e p r o d u c i b l e r e s u l t s , p r o b a b l y because the homogenate p r e p a r a t i o n i s v e r y u n i f o r m from assay t o assay. was  T h i s i s not p o s s i b l e i n s l i c e p r e p a r a t i o n s .  A l t h o u g h the utmost care  taken to e l i m i n a t e a l l w h i t e m a t t e r , t h e r e are always some f i b e r s  spersed i n the grey m a t t e r . t h i c k n e s s was  And  a l t h o u g h the d i s s e c t i o n of s l a b s of u n i f o r m  attempted, i t i s not p o s s i b l e always  of the grey c o r t i c a l m a t t e r .  inter-  If different cell  p r o p o r t i o n s , the cAMP response may  differ  to i n c l u d e a l l s i x lamina  types are p r e s e n t i n d i f f e r e n t  too.  When s t u d y i n g the e f f e c t of a n e u r o t r a n s m i t t e r on cAMP a c c u m u l a t i o n , the " s t i m u l u s " and " r e s p o n s e " are removed s e v e r a l s t e p s .  I d e a l l y , pharmacological  - 84 responses should be measured d i r e c t l y , s i n c e the more i n t e r v e n i n g steps are p r e s e n t ,  that  the more l i k e l y the r e s u l t s are to show v a r i a b i l i t y .  When the work on t h i s t h e s i s was s t a r t e d , cAMP s t u d i e s r e p r e s e n t e d v e r y few approaches to r e c e p t o r  studies.  Since  then, new r a d i o l i g a n d  one o f binding  t e c h n i q u e s have been developed a l l o w i n g f o r q u a n t i t a t i v e measurements o f receptor  s e n s i t i v i t y changes.  However, d a t a from r e c e p t o r b i n d i n g assays must  be r e c o n c i l e d w i t h r e s u l t s o f cAMP a c c u m u l a t i o n s t u d i e s , and t h i s has not y e t been a c c o m p l i s h e d . What can be s t a t e d w i t h c e r t a i n t y i s t h a t cAMP i s a major component o f the postsynaptic established.  e v e n t s , but the importance o f i t s r o l e i s not y e t c l e a r l y  - 85 V.  REFERENCES  1.  Anden NE, D a h l s t r o m A, Fuxe K, L a r s o n K, O l s s o n K, U n g e r s t e d t U, A c t a P h y s i o l . Scand. 67:313, 1966.  2.  F a l c k B, H i l l a r p NA, J . Histochem.  3.  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Sporn JR, Harden TK, Wolfe BB, S c i e n c e 194:624, 1976.  TABLE I  <•  The s t i m u l a t i o n o f cAMP f o r m a t i o n by NA i n a n t e r i o r and p o s t e r i o r c o r t e x o f k i n d l e d and c o n t r o l rats.  £  a H  x  C o r t i c a l region  cAMP pmoles/mg p r o t e i n Baseline  Anterior -kindled -control  33.7+5.0 37.4+4.5  10 uMNA  62.0+9.98 62.9+9.5  % stimulation (100 uMNA)  NA ug/gm wet weight  100 UNA  66.3+6.3 71.9+15.6  196.7 192.3  0.471+0.054 0.453+0.108  , <D  Posterior -kindled - control  1  30.5+4.2 30.9 + 4.1  56.6+7.8 48.8 + 4.9  67.3+8.8 66.6 + 10.1  220.7 215.5  0.364+0.031 0.268 + 0.052  TABLE I I The s t i m u l a t i o n o f cAMP f o r m a t i o n by NA i n 6-OHDA i n j e c t e d and c o n t r o l r a t s .  % Stimulation  cAMP pmoles/mg p r o t e i n  Treatment  EC N (uM MA) 5 0  g e o m e t r i c mean 95% C. I .  Baseline  luMNA  3UMNA  lOuMNA  30 uMNA  Control  31.0+0.99  35.0+2.19  38.7+1.69  55.3+2.65  65.2+2.65  59.4+5.73  225.3  6.81 (5.58-8. 31)  Mocki n j ected  25 .9+0.96  26.4+1.8  34.2+2.2  47.1+2.1  50.4+0.93  50.2+2.7  202.3  4.44 (3.78-5. 22)  6-0HDA1 week  32.0+0.95  39.6+2.94  51.7+3.9  75.3+4.3  89.5+7.1  88.8+5.2  291.3  4.97 (4/30-5. 76)  6-0HDA10 weeks  39.3+2.6  48.7+4.3  69.1+4.8  78.3+5.6  87.9+6.0  94.2+5.0  253.7  3.31 (2.60-4. 24)  125.7+9.5  264.4  5.85 (4.88-7. 02)  100 uMNA  The s t i m u l a t i o n o f cAMP < f o r m a t i o n by NA i n 15 day o l d anda d u l t r a t s  15 day  50.7+2.16  58.5+5.1  74.2+6.47  111.2+12.73  124.7+5.86  

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