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Characterization and manipulation of prefrontal cortex D2 dopamine receptors in the rat Alexander John, MacLennan 1986

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CHARACTERIZATION  AND  MANIPULATION  DOPAMINE R E C E P T O R S  OF P R E F R O N T A L IN THE  CORTEX  RAT  by ALEXANDER B.Sc,  The U n i v e r s i t y  M.A.,  A  JOHN  of British  The U n i v e r s i t y  THESIS THE  SUBMITTED  MACLENNAN  of Colorado,  IN PARTIAL  REQUIREMENTS DOCTOR OF  Columbia, 1983  F U L F I L L M E N T OF  FOR T H E D E G R E E  OF  PHILOSOPHY in  THE  F A C U L T Y O F GRADUATE Program  We  accept to  The  thesis  the,required  University  Alexander  as  John  conforming  standard  of British  November ©  STUDIES  i n Neuroscience  this  1979  Columbia  1986 MacLennan,  1986  D2  In  presenting  degree  at  this  the  freely available copying  of  department publication  of  in  partial  fulfilment  University  of  British  Columbia,  for  this or  thesis  reference  thesis by  this  for  his thesis  and study. scholarly  or for  her  Department  of  The University of British 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6(3/81)  Columbia  I further  purposes  the  requirements  I agree  gain shall  that  agree  may  representatives.  financial  permission.  of  It not  be is  that  the  Library  permission  granted  by  understood be  for  allowed  an  advanced  shall for  the that  without  make  it  extensive  head  of  my  copying  or  my  written  Abstract  A r a d i o l i g a n d b i n d i n g assay was developed t o measure p o s s i b l e D2 dopamine r e c e p t o r s i n the medial p r e f r o n t a l cortex  (mpfc) of the r a t .  competition  I t was demonstrated w i t h  s t u d i e s a t pH 7.9 t h a t these candidate  are, i n f a c t , D2 r e c e p t o r s .  A l l compounds without  sites activity  on dopaminergic systems were i n e f f e c t i v e competitors nanomolar c o n c e n t r a t i o n s . noradrenaline  Dopamine was more potent  which was more potent  than s e r o t o n i n .  nanomolar c o n c e n t r a t i o n s , a l l dopamine r e c e p t o r t h a t were t e s t e d competed f o r the D2 b i n d i n g .  at than At  antagonists The IC50's of  the dopamine r e c e p t o r a n t a g o n i s t s c o r r e l a t e d w i t h t h e i r i n vivo potencies.  The IC50's of a l l compounds  tested with  mpfc t i s s u e c o r r e l a t e d h i g h l y with t h e i r IC50's i n an i d e n t i c a l assay of the s t r i a t u m .  However, the dopamine  r e c e p t o r a g o n i s t s , apomorphine and ADTN, were more potent  i n the mpfc.  The c o m p e t i t i o n  significantly  curves of the  dopamine r e c e p t o r a g o n i s t s suggested t h a t the mpfc, when compared t o the s t r i a t u m , c o n t a i n s a higher p r o p o r t i o n of r e c e p t o r s w i t h a h i g h a f f i n i t y f o r dopamine r e c e p t o r agonists.  Reducing the pH of the assay from pH 7.9 t o  pH 6.2 e l i m i n a t e d the d i f f e r e n c e i n a g o n i s t a f f i n i t y but d i d not a f f e c t the IC50 c o r r e l a t i o n s between b r a i n r e g i o n s or the c o r r e l a t i o n s w i t h i n v i v o p o t e n c i e s . pH i n c r e a s e d the percent presumably reducing  The r e d u c t i o n i n  of D2 b i n d i n g i n the mpfc assay by  spirodecanone b i n d i n g .  Consequently the  pH 6.2 assay d i s p l a y e d a higher binding.  Chronic  sensitivity  t o changes i n D2  h a l o p e r i d o l a d m i n i s t r a t i o n f o r 21 weeks  i n c r e a s e d the D2 b i n d i n g i n the mpfc  (as measured a t pH  6.2)  by approximately 50% compared t o approximately 70% i n the striatum.  Footshock s t r e s s i n c r e a s e d the D2  binding  measured a t pH 6.2 by approximately 13% i n the mpfc and had no e f f e c t i n the s t r i a t u m .  Footshock s t r e s s i n c r e a s e d the  Bmax of the D2 b i n d i n g measured a t pH 7.9 by approximately 100% i n the mpfc and reduced the a f f i n i t y by approximately 70%.  The s t r i a t a l D2 b i n d i n g was u n a f f e c t e d by  s t r e s s as measured at pH 7.9.  footshock  iv  Table  of  Contents Page  Abstract  i i  List  of Tables  v i i  List  of Figures  v i i i  Abbreviations  x  Acknowledgements  .  General  Introduction  The  Mesocortical  Innervating Brain Medial D2  Experiment Assay  Cortex  Dopamine  System  Prefrontal  10  Cortex  Receptors  17  1: D e v e l o p m e n t a n d U s e D2  i n the Medial  of  an  Dopamine Prefrontal  of the Rat  20  Introduction  .  20  Methods  21  Results  34  Discussion  44  Experiment of  1  Receptors  to Characterize  Receptors  1  the P r e f r o n t a l Cortex  Dopamine  Dopamine  x i  Medial  Receptors  2:  Characterization  P r e f r o n t a l Cortex  D2  a t pH  6.2  Dopamine  i n the Rat  49  Introduction  49  Methods  50  V  page Results  51  Discussion  53  Experiment  3: T h e  Haloperidol Receptors Cortex  E f f e c t s of Chronic  Treatment  o n D2  i n the Medial  Dopamine  Prefrontal  of the Rat  56  Introduction  56  Methods  59  Results  61  Discussion  63  Experiment  4:  The  Effects of  Stress  o n D2  Dopamine  Medial  Prefrontal Cortex  Footshock  Receptors  i n the  of the Rat  67  Introduction The as  The as  67  E f f e c t s of Footshock Measured  a t pH  Stress  6.2  69  Methods  69  Results  70  Discussion  72  E f f e c t s of Footshock Measured  a t pH  Stress  7.9  74  Methods Results Discussion  75 .  75 78  vi  Page General  Discussion  82  A Hypothesis Dopamine  "Autoreceptor"  Interpretations Future References  83  Research  Research  o f pH-Dependent  85 Binding  96 97 101  vi i  List  of  Tables  Page Table  Table  1  2  I n h i b i t i o n of S p e c i f i c [ H]-Spiperone ( 2 5 pM) B i n d i n g a t pH 7.9  36  I n h i b i t i o n of S p e c i f i c [ H]-Spiperone ( 3 5 pM) B i n d i n g a t pH 6.2  52  3  3  vi i i  L i s t of F i g u r e s Page Figure 1  S t r i a t a l and mpfc t i s s u e used f o r assays  22  Figure 2  The amount of t o t a l t i s s u e b i n d i n g of [^H]-spiperone (25 pM) competed f o r by various concentrations of ketanserin t a r t r a t e i n the mpfc and the s t r i a t u m  27  The percent of s p e c i f i c [ - s p i p e r o n e (25 pM) b i n d i n g ( b i n d i n g competed f o r by 10~5 M s - ( - ) - s u l p i r i d e ) competed f o r by various concentrations of s - ( - ) - s u l p i r i d e and r - ( + ) - s u l p i r i d e i n the mpfc and the striatum  35  The r e l a t i o n s h i p between the a f f i n i t i e s of v a r i o u s dopamine r e c e p t o r a n t a g o n i s t s f o r mpfc D2 r e c e p t o r s and the a n t i p s y c h o t i c p o t e n c i e s of the same drugs  38  The r e l a t i o n s h i p between the a f f i n i t i e s of v a r i o u s dopamine r e c e p t o r a n t a g o n i s t s f o r mpfc D2 r e c e p t o r s and the a f f i n i t i e s of the same drugs f o r s t r i a t a l D2 receptors  39  The percent of s p e c i f i c [^H]-spiperone (25 pM) b i n d i n g ( b i n d i n g competed f o r by 10~5 M s - ( - ) - s u l p i r i d e ) competed f o r by v a r i o u s c o n c e n t r a t i o n s of dopamine i n the mpfc and the s t r i a t u m  40  The percent of s p e c i f i c [-^H]-spiperone (25 pM) b i n d i n g ( b i n d i n g competed f o r by 10~5 M s - ( - ) - s u l p i r i d e ) competed f o r by v a r i o u s c o n c e n t r a t i o n s o f apomorphine i n the mpfc and the s t r i a t u m  41  The percent of s p e c i f i c [^H]-spiperone • (25 pM) b i n d i n g ( b i n d i n g competed f o r by 10~5 M s - ( - ) - s u l p i r i d e ) competed f o r by v a r i o u s c o n c e n t r a t i o n s of ADTN i n the mpfc and the s t r i a t u m  42  Scatchard p l o t i l l u s t r a t i n g the e f f e c t s of c h r o n i c h a l o p e r i d o l a d m i n i s t r a t i o n on s t r i a t a l D2 r e c e p t o r s  62  Figure 3  Figure 4  Figure 5  Figure 6  Figure 7  Figure 8  Figure 9  ix  Page F i g u r e 10  F i g u r e 11  F i g u r e 12  F i g u r e 13  F i g u r e 14  F i g u r e 15  F i g u r e 16  Scatchard p l o t i l l u s t r a t i n g the e f f e c t s of c h r o n i c h a l o p e r i d o l a d m i n i s t r a t i o n on mpfc D2 r e c e p t o r s  64  Scatchard p l o t i l l u s t r a t i n g the e f f e c t s of footshock on mpfc D2 r e c e p t o r s assayed a t pH 6.2  71  Scatchard p l o t i l l u s t r a t i n g the e f f e c t s of footshock on s t r i a t a l D2 r e c e p t o r s assayed a t pH 6.2  73  Scatchard p l o t i l l u s t r a t i n g the e f f e c t s of footshock on mpfc D2 r e c e p t o r s assayed a t pH 7.9  76  Scatchard p l o t i l l u s t r a t i n g the e f f e c t s of footshock on s t r i a t a l D2 r e c e p t o r s assayed a t pH 7.9  77  An i l l u s t r a t i o n of how dopamine r e l e a s e d by m e s o c o r t i c a l dopamine neurons may i n h i b i t n o r a d r e n a l i n e s y n t h e s i s i n mpfc noradrenergic terminals  89  An i l l u s t r a t i o n of how m e s o c o r t i c a l dopaminergic a c t i v i t y may i n h i b i t s u b c o r t i c a l dopaminergic a c t i v i t y  95  X  Abbreviations  AMP DOPA  '.  adenosine  monophoshate  3,4-dihydroxyphenylalanine  DOPAC  dihydroxyphenylacetic  EDTA  ethylenediaminetetraacetic  GBL  gamma-butyrolactone  mpfc  medial  6-OHDA  prefrontal  6-hydroxydopamine  acid  cortex  acid  XI  Acknowledgement  I  would  like  Committee, defense. who  were  my  exposed  during  the research  Thanks  also  rapid  and  I would  Jakubovic phase  t o Nancy  who  was  like  there  not have  and  who  L e e who  my  typed  writing  the Chairman  Finlay draft  and of  Jim  phase this  of  my  Radke  this  comments.  I  would  of this  both project.  thesis  while  i s so ugly.  Most  gratitude  through the boring without.  Supervisory  made c o n t r i b u t i o n s  and w r i t i n g  to express  done  raw  helpful  i t t o h e r b e c a u s e my  all  could  Examiners  to a dangerously  to thank Alex  dictated  o f my  wish to thank Janet  y e t made  go  t h e members  University  I also  manuscript like  t o thank  to Stella  h o u r s and whose  I of  Atmadja  help  I  1  General  Introduction  B r a i n dopamine systems are thought to p l a y a r o l e i n a wide v a r i e t y of n e u r a l f u n c t i o n s i n c l u d i n g those f o r motor a c t i v i t y  (Marsden, 1984), f e e d i n g  (Heffner, Hartman and Seiden,  1980)  responsible  behavior  and endogenous reward  ( P h i l l i p s and F i b i g e r , 1978).  In a d d i t i o n , a b n o r m a l i t i e s  i n v o l v i n g dopamine systems may  be c r i t i c a l i n many  c o n d i t i o n s such as Parkinson's  disease  depression 1981;  (Antelman and Chiodo, 1981;  (Hornykiewicz, F i b i g e r and  B o r s i n i , P u l v i r e n t i and Samanin, 1985),  1973),  Phillips,  Tourette's  syndrome ( B u t l e r , 1984), some drug-induced psychoses (Snyder, Banerjee, schizophrenia  Yamamura and Greenberg, 1974)  and  ( M e l t z e r and S t a h l , 1976).  Information is substantial.  concerning  c e n t r a l dopaminergic  processes  This t h e s i s w i l l deal p r i m a r i l y with a  r e l a t i v e l y r e c e n t and  somewhat l i m i t e d area of t h i s body of  knowledge: the m e s o c o r t i c a l dopamine neurons i n n e r v a t i n g the prefrontal  cortex.  The M e s o c o r t i c a l Dopamine System I n n e r v a t i n g Prefrontal  In 1973 biochemical  the  Cortex  T h i e r r y and c o l l e a g u e s demonstrated w i t h and  l e s i o n s t u d i e s t h a t dopaminergic neurons  i n n e r v a t e the r a t p r e f r o n t a l c o r t e x S t i n u s and G l o w i n s k i ,  ( T h i e r r y , Blanc,  1973(a); T h i e r r y , S t i n u s , Blanc  Sobel, and  2  G l o w i n s k i , 1973(b)).  The p r e f r o n t a l c o r t e x a l s o r e c e i v e s a  dense n o r a d r e n e r g i c p r o j e c t i o n , and t h e r e f o r e u n t i l  the  r e s e a r c h of T h i e r r y e t a l . , dopamine i n t h i s b r a i n r e g i o n was  assumed t o r e p r e s e n t p r e c u r s o r pools f o r s y n t h e s i s of  noradrenaline.  T h i e r r y e t a l . (1973b) r u l e d out  p o s s i b i l i t y by demonstrating  t h a t dopamine c o n c e n t r a t i o n s i n  the p r e f r o n t a l c o r t e x were not reduced noradrenergic that  input.  by l e s i o n s of the  In a d d i t i o n , they were a b l e to show  [ H]-dopamine c o u l d be s y n t h e s i z e d from 3  ( i n v i v o and noradrenergic  this  [ H]-tyrosine 3  i n v i t r o ) i n p r e f r o n t a l c o r t i c e s d e p r i v e d of i n n e r v a t i o n ( T h i e r r y e t a l . , 1973a).  Furthermore, they found t h a t the p r e f r o n t a l c o r t e x c o n t a i n s high a f f i n i t y  [ H]-dopamine uptake s i t e s 3  Blanc and G l o w i n s k i , 1974).  (Tassin, Thierry,  I t has a l s o been demonstrated  (Pycock, C a r t e r and Kerwin, 1980)  through s i m i l a r  s t u d i e s t h a t t y r o s i n e hydroxylase  activity  l i m i t i n g enzyme f o r both dopamine and  lesion  (the r a t e  noradrenaline  s y n t h e s i s ) i s l o c a t e d i n non-noradrenergic  c e l l s innervating  the p r e f r o n t a l c o r t e x .  adenylate  Dopamine-sensitive  cyclase  has a l s o been i d e n t i f i e d i n the p r e f r o n t a l c o r t e x ( T a s s i n , Bockaert,  B l a n c , S t i n u s , T h i e r r y , L a v i e l l e , Premont  G l o w i n s k i , 1978).  and  F i n a l l y , the dopamine m e t a b o l i t e s ,  dihydroxyphenylacetic  acid  (HVA)  i n the p r e f r o n t a l c o r t e x (Bannon and  have been found  (DOPAC) and h o m o v a n i l l i c a c i d  Roth, 1983). The  above mentioned b i o c h e m i c a l i n d i c e s of dopaminergic  a c t i v i t y i n the p r e f r o n t a l c o r t e x i n d i c a t e t h a t the  3 dopaminergic p r o j e c t i o n to the p r e f r o n t a l c o r t e x i s q u i t e sparse compared to t h a t of s u b c o r t i c a l  dopaminergically  i n n e r v a t e d r e g i o n s such as the s t r i a t u m and the accumbens. are 1-5%  nucleus  Dopamine c o n c e n t r a t i o n s i n the p r e f r o n t a l c o r t e x  of those  i n the s t r i a t u m ( T a s s i n e t a l . , 1978;  Bannon and Roth, 1983). Dopamine turnover r a t e s i n the p r e f r o n t a l c o r t e x have been estimated both by the c o n c e n t r a t i o n of dopamine m e t a b o l i t e s and  the d e c l i n e i n dopamine content f o l l o w i n g  synthesis i n h i b i t i o n .  In both cases, dopamine turnover i n  the p r e f r o n t a l c o r t e x appears to be s i g n i f i c a n t l y than i n s u b c o r t i c a l r e g i o n s striatum)(Bannon Glowinski,  (approximately  and Roth, 1983;  higher  twice t h a t of  the  T h i e r r y , T a s s i n and  1984).  Anatomically,  the p r e f r o n t a l c o r t e x i s d e s c r i b e d as  those r e g i o n s of the f r o n t a l c o r t e x r e c e i v i n g d i r e c t p r o j e c t i o n s from the mediodorsal  nucleus  ( L i n d v a l l and B j o r k l u n d , 1984).  The  of the thalamus  dopaminergic  cells  i n n e r v a t i n g the p r e f r o n t a l c o r t e x can most e a s i l y be d i v i d e d i n t o two 1978).  subcategories The  ( L i n d v a l l , B j o r k l u n d and  Divac,  l a t e r a l or s u p r a r h i n a l system i n n e r v a t e s an  l o c a t e d on the d o r s a l bank of the r h i n a l s u l c u s . system i n n e r v a t e s the pregenual,  anteromedial  The  cortex.  v a s t m a j o r i t y of r e s e a r c h has been concerned w i t h the system.  area  medial The medial  In the r a t , the dopaminergic i n n e r v a t i o n of the  f r o n t a l c o r t e x appears to be r e s t r i c t e d to the p r e f r o n t a l c o r t e x ( L i n d v a l l e t a l . , 1978).  In primates,  the dopamine  4 i n n e r v a t i o n i s somewhat more e x t e n s i v e but s t i l l t o the p r e f r o n t a l c o r t e x  restricted  (Brown, Crane and Goldman, 1979;  L e v i t t , Rakic and Goldman-Rakic,  1984).  The p e r i k a r y a o f dopaminergic neurons t h a t p r o j e c t t o the p r e f r o n t a l c o r t e x are l o c a t e d i n the v e n t r a l tegmental area  (Fuxe, H o k f e l t , Johansson, Jonsson, L i d b r i n k and  Ljungdahl, 1974).  1974; H o k f e l t , Ljungdahl,  Fuxe and Johansson,  The medial system c e l l s a r e l o c a t e d p r i m a r i l y i n the  m e d i o - r o s t r a l p a r t o f the A10 group o f dopaminergic ( L i n d v a l l and B j o r k l u n d ,  1984; T h i e r r y e t a l . , 1984).  s u p r a r h i n a l system o r i g i n a t e s more l a t e r a l l y Bjorklund,  neurons The  ( L i n d v a l l and  1984; T h i e r r y e t a l . , 1984). Very few  m e s o c o r t i c a l dopamine c e l l s appear t o have c o l l a t e r a l s t o other b r a i n r e g i o n s  ( L i n d v a l l and B j o r k l u n d ,  1984).  The  dopamine f i b e r s of both systems i n n e r v a t i n g the p r e f r o n t a l c o r t e x a r e remarkably smooth w i t h few v a r i c o s i t i e s e t a l . , 1978).  (Lindvall  They are observed i n l a y e r s I I through VI  w i t h the h i g h e s t d e n s i t y i n l a y e r s V and VI ( L i n d v a l l and Bjorklund,  1984).  The dopaminergic m e s o c o r t i c a l neurons g e n e r a l l y d i s p l a y the low f i r i n g r a t e s and slow conduction t y p i c a l o f dopaminergic neurons  v e l o c i t i e s t h a t are  (Bunney and Chiodo,  1984).  However, dopaminergic c e l l s p r o j e c t i n g t o the p r e f r o n t a l c o r t e x f i r e a t much higher b a s a l r a t e s than s u b c o r t i c a l l y p r o j e c t i n g dopaminergic c e l l s or other m e s o c o r t i c a l dopamine c e l l s t h a t i n n e r v a t e other areas of the c o r t e x Chiodo, 1984).  (Bunney and  B u r s t i n g a c t i v i t y i s a l s o more frequent i n  5  the dopaminergic c e l l s t h a t p r o j e c t t o the p r e f r o n t a l c o r t e x (Bunney and Chiodo, 1984). Dopamine r e c e p t o r a g o n i s t s have been r e p o r t e d t o have both e x c i t a t o r y and i n h i b i t o r y e l e c t r o p h y s i o l o g i c a l a c t i o n s i n the p r e f r o n t a l c o r t e x 1984).  (Bunney and Chiodo, 1984; P h i l l i s ,  Both e f f e c t s a r e i n h i b i t e d by dopamine r e c e p t o r  antagonists.  Subchronic  treatment w i t h dopamine r e c e p t o r  a n t a g o n i s t s g r e a t l y reduces e l e c t r o p h y s i o l o g i c a l a c t i v i t y i n the mesolimbic and n i g r o s t r i a t a l dopamine systems (Chiodo and Bunney, 1983; White and Wang, 1983).  T h i s e f f e c t can be  r e v e r s e d by a d m i n i s t r a t i o n o f dopamine r e c e p t o r (Chiodo and Bunney, 1983; White and Wang, 1983). b e l i e v e d t h a t the subchronic receptor antagonists  agonists It i s  treatment w i t h the dopamine  f i r s t g r e a t l y i n c r e a s e s the f i r i n g  of the dopaminergic neurons (Bunney and Grace, 1978).  rate This  i s thought t o cause the c e l l s t o become so d e p o l a r i z e d t h a t t h e i r s p i k e g e n e r a t i n g mechanisms become i n a c t i v a t e d (Bunney and Grace, 1978; Bunney and Grace, 1986). receptor agonists are believed t o reverse  The dopamine this  " d e p o l a r i z a t i o n b l o c k " by h y p e r p o l a r i z i n g the dopaminergic neurons.  M e s o c o r t i c a l dopamine c e l l s a l s o d i s p l a y an  i n c r e a s e i n f i r i n g r a t e f o l l o w i n g acute a d m i n i s t r a t i o n o f dopamine r e c e p t o r a n t a g o n i s t s , but they do not become i n a c t i v a t e d by subchronic  treatment  (Chiodo and Bunney,  1983). Behavioural  r e s e a r c h on t h e m e s o c o r t i c a l dopamine  system has been very l i m i t e d .  T h i s i s probably  due t o two  6 factors.  First,  anatomical proper  behavioural studies require that  sufficient  and b i o c h e m i c a l data be a v a i l a b l e t o permit the  d e s i g n o f experiments.  s e l e c t i v e manipulation  Second, and more c r i t i c a l l y ,  o f the m e s o c o r t i c a l dopamine system  has n o t been achieved.  L e s i o n s o f the p r e f r o n t a l c o r t e x  w i t h 6-hydroxydopamine (6-OHDA) produce l a r g e decreases i n noradrenaline concentrations  (34-73%)in a d d i t i o n t o t h e i r  e f f e c t s on dopamine ( C a r t e r and Pycock, 1980; Pycock e t a l . , 1980;  Joyce,  S t i n u s and I v e r s e n , 1983).  C o n s i d e r i n g the  l a r g e l i t e r a t u r e i n d i c a t i n g t h a t dopamine and n o r a d r e n a l i n e i n t e r a c t e x t e n s i v e l y (Langer,  1974; Antelman and C a g g i u l a ,  1977), the b e h a v i o u r a l e f f e c t s o f p r e f r o n t a l c o r t e x 6-OHDA l e s i o n s cannot be regarded dopamine involvement.  as unequivocal  i n d i c a t i o n s of  P h a r m a c o l o g i c a l l y , no compounds have  been demonstrated t o be p a r t i c u l a r l y s e l e c t i v e a t d i r e c t l y a c t i v a t i n g o r b l o c k i n g dopamine r e c e p t o r s i n the p r e f r o n t a l cortex.  T h i s i s due,  a t l e a s t i n p a r t , t o the l a c k o f a  method t o measure p r e f r o n t a l c o r t e x dopamine r e c e p t o r s . D i r e c t c e r e b r a l a p p l i c a t i o n o f dopaminergic compounds seems, at  present, the only a v a i l a b l e research  tool.  U n f o r t u n a t e l y , the p r e f r o n t a l c o r t e x i s l o c a t e d very near other d o p a m i n e r g i c a l l y i n n e r v a t e d b r a i n r e g i o n s which possess many times the d e n s i t y o f dopamine r e c e p t o r s (see below).  T h e r e f o r e , even moderate degrees o f drug d i f f u s i o n  c o u l d produce m i s l e a d i n g Although  results.  6-OHDA l e s i o n s o f the A10 c e l l bodies have  been r e p o r t e d t o d i s r u p t the behaviour  o f r a t s t r a i n e d on a  7  s p a t i a l a l t e r n a t i o n task (Simon, S c a t t o n and Le Moal, 1980), i f the l e s i o n s a r e r e s t r i c t e d t o the medial f r o n t a l c o r t e x , no such r e s u l t i s observed ( I v e r s e n , 1984).  Rats w i t h  m e d i a l p r e f r o n t a l c o r t e x 6-OHDA l e s i o n s have been r e p o r t e d t o d i s p l a y more amphetamine-induced  stereotypy,  more  spontaneous a c t i v i t y , and l e s s apomorphine-induced s t e r e o t y p y than c o n t r o l s  ( C a r t e r and Pycock, 1980).  Carter  and Pycock (1980) suggest t h a t dopamine r e l e a s e i n the medial p r e f r o n t a l c o r t e x (mpfc) may i n h i b i t the a c t i v i t y o f the mesolimbic and n i g r o s t r i a t a l systems.  In support o f  t h i s , they found t h a t the c o n c e n t r a t i o n s o f dopamine and i t s m e t a b o l i t e s a r e i n c r e a s e d i n the s t r i a t u m and nucleus accumbens o f r a t s r e c e i v i n g 6-OHDA l e s i o n s o f the mpfc ( C a r t e r and Pycock, 1980; Pycock e t a l . ,  1980).  This  result  has r e c e n t l y been r e p l i c a t e d by M a r t i n - I v e r s o n , Szostak and Fibiger  (1986).  However, as p r e v i o u s l y noted, these e f f e c t s  are d i f f i c u l t t o i n t e r p r e t because the c o n c e n t r a t i o n o f mpfc n o r a d r e n a l i n e i s a l s o d r a m a t i c a l l y reduced by these l e s i o n s . The l e s i o n r e s u l t s p r e s e n t e d above have l e d most r e s e a r c h e r s t o agree w i t h C a r t e r and Pycock and t o suggest t h a t dopamine r e l e a s e i n the mpfc i n h i b i t s dopamine a c t i v i t y  subcortical  (Bannon and Roth, 1983; G l o w i n s k i , T a s s i n  and T h i e r r y , 1984; I v e r s e n , 1984).  U n f o r t u n a t e l y , the d a t a  a v a i l a b l e a r e f a r from c o n c l u s i v e . The m e s o c o r t i c a l dopamine neurons respond i n a unique f a s h i o n t o many e x p e r i m e n t a l m a n i p u l a t i o n s (Bannon and Roth, 1983; T h i e r r y e t a l . ,  1984).  F o r example,  a wide v a r i e t y o f  8  pharmacological metabolism their  i n the  effects  1983).  agents  Of  on  of  the  Moore, drugs  that  system acute mpfc  Inoue  these to  dopamine  concentrations than  (Bowers  in  and  nucleus  or  (as  mpfc  dopamine  than  those  (as  of  drugs  metabolites  required  to  (Matsumoto  antipsychotic-induced  can  increase et  dopamine  effects.  to  a  Thus,  increases in  the  greater  metabolism  amphetamine acceleration the  rate  inhibition  i s not  (Robinson the  of  decline  of  observed et  of  in  at  the  a l . , 1985).  concentrations  administered  lower  of  doses  s u b c o r t i c a l dopamine  a l . , 1983).  increases  suggest  increases  dopamine  increase  when  Shimomura,  administration  which  Becker,  studies  observed  after  striatum  and  mesocortical  i n d i c a t e d by  activity) the  These  enduring  dopamine  Roth,  antipsychotic  metabolites)  an  from  to  Yokoo,  Subchronic  produces  or  Antipsychotic  and  i n d i c a t e d by  striatal  1984).  hydroxylase  metabolites  clinically  and  Robinson,  Kim,  the  dopamine  turnover  accumbens  on  (Bannon  1984;  Hirano,  different  characteristic  1985)  phencyclidine  mpfc  also  are  amphetamine  Hoffman,  act  their  concentration  tyrosine  as  dopamine  neurons  Oomogari,1983).  metabolism of  i s the  Mittleman,  Hoffman,  dopamine  the  and  i t increases  administration mpfc  such  c o m p o u n d s may produce  that  dopamine  Uchimura,  and  amphetamine  extent  drugs  and  (Matsumoto,  Nakahara,  interest  (Bowers  Castaneda  on  s u b c o r t i c a l systems  mesocortical  psychosis-inducing phencyclidine  effects  prefrontal cortex  particular  sensitivity  produce  While  i n s u b c o r t i c a l dopamine  9  metabolites increases  diminish i n mpfc  relatively  less  al.,  Bannon  1979;  Since  susceptible  has  display  than  cells  subjected metabolites Gessa,  stressors.  (Fadda,  1978;  Bannon  (Reinhard  1984).  i n mpfc  tyrosine  hydroxylase  Glowinski,  1976;  noradrenergic  (Tissari,  Robinson  increase  Argiolas,  footshock-induced hydroxylase  Fadda,  decrease  inhibition  that  the  sensitive  dopamine  Tissari,  1982)  Onali  and  and mpfc  and  Fekete, dopamine  Kramarcy, Delaney  concentration  the rate observed  (Thierry,  e t a l . , 1985). o f t h e mpfc i n mpfc  do  and  of following  Tassin,  Blanc  Lesions  of the  not block  dopamine  and Gessa,  i n dopamine  (Thierry  their  animals  i n mpfc  accelerates  Serra  drugs  t o a c t i v a t i o n by  Melis,  inhibition  innervation  footshock-induced  groups  Herman, K a n y i c s k a  dopamine  to  t o produce  a r e much more  increases  also  e t a l . , 1983).  antipsychotic  several  e t a l . , 1982;  Footshock  decrease  by  and Roth,  et  antipsychotic  Thus, experimental  Argiolas,  be  e t a l . , 1979).  t h e mpfc  display  to  (Bacopoulos  resistance  neurons  systems  Szentendrei,  Reinhard,  synthesis Dunn,  been reported  footshock  Matsumoto  (Bacopoulos  innervating  tolerance  that  the  are reported  a parallel  dopamine  s u b c o r t i c a l dopamine  environmental  1980;  effects  also  treatment,  e f f e c t s of  i t has been suggested  antipsychotic  dopamine  1983;  beneficial  a c t on m e s o c o r t i c a l  It  t o such  and Roth,  reportedly  tolerance,  subchronic  dopamine m e t a b o l i t e s  the c l i n i c a l l y  treatment  may  during  and  the  metabolites 1979)  following  e t a l . , 1976).  or the tyrosine  Mpfc  10 dopamine m e t a b o l i t e s  are a l s o i n c r e a s e d by swim s t r e s s and  r e s t r a i n t s t r e s s (Yang, Knorn, Onel, Tarn, Deutch, Cubich and Roth, 1985) as w e l l as c o n d i t i o n e d Dantzer, Scatton,  f e a r (Herman,  Guilloneau,  Semerdjian-Rouquier and Le Moal, 1982).  A l l these s t u d i e s found t h a t s u b c o r t i c a l dopamine systems are much l e s s s e n s i t i v e t o s t r e s s . In summary, the m e s o c o r t i c a l  dopamine c e l l s  innervating  the mpfc d i s p l a y many c h a r a c t e r i s t i c s which i n d i c a t e t h a t they are f u n c t i o n a l l y d i s t i n c t from the mesolimbic and n i g r o s t r i a t a l dopamine c e l l s . higher  They d i s p l a y s i g n i f i c a n t l y  b a s a l r a t e s of f i r i n g , b u r s t i n g and t r a n s m i t t e r  turnover.  U n l i k e the s u b c o r t i c a l dopamine processes,  the  dopamine p r o c e s s e s i n n e r v a t i n g the p r e f r o n t a l c o r t e x are reported  t o be p a r t i c u l a r l y s e n s i t i v e t o s t r e s s and a wide  v a r i e t y of p h a r m a c o l o g i c a l agents, and r e s i s t a n t t o "depolarization block"  induced by n e u r o l e p t i c  treatment.  B r a i n Dopamine Receptors  As w i t h a l l p u t a t i v e n e u r o t r a n s m i t t e r s ,  dopamine i s  b e l i e v e d t o i n f l u e n c e neuronal a c t i v i t y by i n t e r a c t i n g w i t h s p e c i f i c neuronal p r o t e i n s or " r e c e p t o r s " . of b r a i n dopamine r e c e p t o r s  The p r o p e r t i e s  have been i n v e s t i g a t e d w i t h  various experimental protocols.  Research has i d e n t i f i e d  many compounds which can block and r e v e r s e  the b e h a v i o u r a l  e f f e c t s of dopamine (Janssen and Van Bever, 1975) as w e l l as compounds which mimic the b e h a v i o u r a l  e f f e c t s of dopamine  11 (Woodruff, 1981).  Many of these drugs are potent  nanomolar c o n c e n t r a t i o n s thereby high a f f i n i t y  f o r some c r i t i c a l  F u r t h e r evidence  at  suggesting t h a t they have a "receptor".  f o r the e x i s t e n c e of c e n t r a l dopamine  r e c e p t o r s comes from e l e c t r o p h y s i o l o g i c a l r e s e a r c h . I o n t o p h o r e t i c a l l y a p p l i e d dopamine can a f f e c t e l e c t r o p h y s i o l o g i c a l behavior  the  of neurons i n dopamine  i n n e r v a t e d r e g i o n s of the b r a i n .  S t u d i e s employing  e x t r a c e l l u l a r or i n t r a c e l l u l a r r e c o r d i n g techniques  have  found t h a t dopamine i n h i b i t s or e x c i t e s the a c t i v i t y of neurons i n the s t r i a t u m (Moore and Bloom,  1978;  Kitai,  dopamine both  1981).  e x c i t e s and  I t i s p r e s e n t l y u n c l e a r how  i n h i b i t s s t r i a t a l neurons.  t h a t dopamine may  I t has been proposed  produce i t s i n h i b i t o r y e f f e c t s by  i n h i b i t o r y s t r i a t a l interneurons  (Kitai,  exciting  1981). In any  case,  dopamine can produce s i g n i f i c a n t changes i n the a c t i v i t y of neurons l o c a t e d i n d o p a m i n e r g i c a l l y i n n e r v a t e d b r a i n regions.  T h e r e f o r e , the data suggest  t h a t dopamine  i n t e r a c t s w i t h some neuronal element ("receptor")  which i s  i n v o l v e d i n the e l e c t r o p h y s i o l o g i c a l e f f e c t s of dopamine. While e l e c t r o p h y s i o l o g i c a l and b e h a v i o u r a l experiments have p r o v i d e d important  i n f o r m a t i o n , n e i t h e r approach can  used to c h a r a c t e r i z e b r a i n dopamine r e c e p t o r s .  In order  c h a r a c t e r i z e a r e c e p t o r , the p o t e n c i e s of many compounds must be compared.  Such comparison r e q u i r e s p r e c i s e  knowledge of the r e l a t i v e c o n c e n t r a t i o n s of the compounds i n t e r a c t i n g w i t h the r e c e p t o r , and  such c o n t r o l cannot be  be to  12 achieved w i t h b e h a v i o u r a l or e l e c t r o p h y s i o l o g i c a l (Snyder and Bennett, 1976;  Woodruff, 1981).  In the e a r l y 1970's, i t was  d i s c o v e r e d t h a t dopamine  can s t i m u l a t e a "dopamine-sensitive" 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 1971).  The  c y c l i c AMP  adenylate c y c l a s e i n  (Kebabian and  Greengard,  produced by such s t i m u l a t i o n mediates  dopaminergic t r a n s m i s s i o n i n t h i s g a n g l i o n Schorderet  techniques  and Greengard, 1971;  (McAfee,  McAfee and Greengard, 1972).  Such data l e d these workers to conclude  t h a t the "dopamine  r e c e p t o r " i n the g a n g l i o n i s the dopamine b i n d i n g p o r t i o n of a dopamine-sensitive adenylate  adenylate  c y c l a s e was  of caudate n u c l e i  cyclase.  subsequently  (Kebabian,  A very  similar  i d e n t i f i e d i n homogenates  P e t z o l d and Greengard, 1972).  Dopamine and other dopamine r e c e p t o r a g o n i s t s were potent s t i m u l a t o r s of t h i s enzyme w h i l e dopamine r e c e p t o r a n t a g o n i s t s were potent  i n h i b i t o r s of  agonist-induced  stimulation. The  f i r s t attempts t o l a b e l b r a i n dopamine r e c e p t o r s  w i t h r a d i o a c t i v e compounds were r e p o r t e d i n 1975 Enna, Creese and Snyder, 1975; and Wong, 1975). l a b e l l e d by  (Burt,  Seeman, Chau-Wong, Tedesco  Research r e v e a l e d t h a t the b i n d i n g  sites  [ H ] - h a l o p e r i d o l d i s p l a y the c h a r a c t e r i s t i c s of 3  a dopamine r e c e p t o r w h i l e those s i t e s l a b e l l e d  by  [%]-dopamine do not d i s p l a y such c h a r a c t e r i s t i c s . the a f f i n i t i e s of dopaminergic and compounds f o r the  non-dopaminergic  [ H ] - h a l o p e r i d o l (but not 3  Thus,  the  [ H]-dopamine) l a b e l l e d s i t e s were h i g h l y c o r r e l a t e d with 3  13 t h e i r i n v i v o p o t e n c i e s as a n t a g o n i s t s of dopamine a g o n i s t induced 1975;  behaviors  i n experimental  animals (Seeman e t a l . ,  Creese, Burt and Snyder, 1976;  and Wong, 1976).  Seeman, Lee, Chau-Wong  These c o r r e l a t i o n s were much h i g h e r  analogous c o r r e l a t i o n s between c e n t r a l adenylate  dopamine-sensitive  c y c l a s e a c t i v i t y and the i n v i v o e f f e c t s of  dopaminergic compounds. dopamine-sensitive  T h i s suggested t h a t  adenylate  c y c l a s e may  dopamine t r a n s m i s s i o n i n the b r a i n . supported  than  not be r e l a t e d t o  This p o s s i b i l i t y  was  by r e p o r t s t h a t s u l p i r i d e d i s p l a c e s  t H ] - h a l o p e r i d o l b i n d i n g and b l o c k s the i n v i v o e f f e c t s of 3  dopamine r e c e p t o r a g o n i s t s , but does not dopamine-sensitive  adenylate  F r e s i a and Spano, 1975; and Spano, 1978; Marsden, 1978;  inhibit  c y c l a s e ( T r a b u c c h i , Longoni,  Garau, Govoni, S t e f a n i n i ,  Trabucchi  Jenner, Clow, R e a v i l l , Theodorou  and  Woodruff, 1981).  Based on these data, and data from r e s e a r c h p e r i p h e r a l dopamine r e c e p t o r s , Kebabian and proposed t h a t there are at l e a s t two  Calne (1979)  forms of dopamine  r e c e p t o r s ; r e c e p t o r s a s s o c i a t e d with adenylate r e c e p t o r s ) and (D2 r e c e p t o r s ) .  cyclase  r e c e p t o r s independent of adenylate The  on  (DI  cyclase  above mentioned data i n d i c a t e t h a t  r e c e p t o r s are of major importance i n mediating e f f e c t s of dopamine r e c e p t o r a g o n i s t s and Some data suggest t h a t DI and D2  D2  the c e n t r a l  antagonists.  r e c e p t o r s have  opposing e f f e c t s on dopamine s e n s i t i v e adenylate  cyclase  a c t i v i t y i n the s t r i a t u m (Stoof and Kebabian, 1981;  Sailer  14  and  Salama,  suggests  1986).  that  Radioligand  central  (Dumbrille-Ross,  DI  Niznik  behavioural  experiments  antagonists  suggest  dopamine  related  stereotypy (Iorio,  and  Mailman,  a  of  the  Arnt,  these  been  Stanford  and  but  Houser  Larsen  Staples,  and  activity on  of  i n s t e a d be  that  associated with  Stanford, also in  Wyrick  question  and  receptor 1984)  elicit agonists  although  in  activity 1983;  on  et  DeHaven, the  the  behaviours  similar  (Christensen produce  et  to  not  noted  be  adenylate of  i t s receptors  (Schulz,  available  activity  is  data involved  "Dl  receptor  those  elicited  a l . , 1984;  opposing  1985;  a l s o been  cyclase  the  Plantje,  Berkowitz,  dopamine  cyclase since  specificity  antagonist  c o m p o u n d may  The  1984).  specificity  DI  I t has  1985).  1984;  a l . , 1984;  consequence  adenylate  behaviours  they  involved  and  relied  adenylate  Mailman,  whether  be  Svendsen,  previously undetected  dopamine-related  agonists"  and  1985).  the  with  not  and  dopamine-sensitive  interactions are  agonists  Korduba,  (Hicks  this  addition,  DI  O h l s t e i n and  Mailman,  i t s effects may  1984;  In  apomorphine-induced  Rollema  have  also  interact  locomotor  and  questioned  Stoof,  in vivo  cyclase  as  "specific"  Schulz,  to  such  may  f r e q u e n t l y employed  Daus  related  "selective"  Recently,  Hansen,  the  of  1985).  receptors  studies  can  compounds.  (SCH23390) has  that  DI  Hyttel,  Lewis,  number  most  that  Leitz,  Schulz,  small  Seeman,  using  research  receptors  amphetamine-induced  Unfortunately, of  and  D2  behaviours  Barnett,  Christensen,  and  binding  effects  Mailman on  by  D2  et a l . ,  adenylate  15 cyclase a c t i v i t y Salama; 1986).  (Stoof and Kebabian, 1981; S a i l e r and Furthermore, e l e c t r o p h y s i o l o g i c a l data  i n d i c a t e t h a t dopamine can i n c r e a s e the amplitude o f C a ^  +  dependent somatic a c t i o n p o t e n t i a l s i n the s n a i l H e l i x aspersa  by d e c r e a s i n g  a K  +  c u r r e n t c o n t r o l l e d by c y c l i c  ( P a u p a r d i n - T r i t s c h , Colombaioni, Deterre 1985).  Gerschenfeld,  Dopamine can a l s o decrease the same a c t i o n  p o t e n t i a l s by d e c r e a s i n g AMP  and  AMP  Ca2  +  c u r r e n t independent of c y c l i c  ( P a u p a r d i n - T r i t s c h e t a l . , 1985). However, when both  e f f e c t s are found i n the same neuron the c y c l i c  AMP  independent decrease predominates ( P a u p a r d i n - T r i t s c h e t a l . , 1985).  Therefore,  a t the present  time, the f u n c t i o n s  by c e n t r a l DI dopamine r e c e p t o r s remain u n c l e a r .  served  This  issue  w i l l most l i k e l y be r e s o l v e d by s t u d i e s of s t r i a t a l t i s s u e where D2 and DI r e c e p t o r s are most numerous. T h i s t h e s i s was designed t o study mpfc dopamine receptors.  D2 r e c e p t o r s were examined because, i n c o n t r a s t  to the DI r e c e p t o r  l i t e r a t u r e , s t u d i e s of the D2 dopamine  r e c e p t o r have c l e a r l y i n d i c a t e d t h a t i t i s r e l a t e d t o many i n v i v o e f f e c t s of dopamine.  Almost a l l of t h i s  has made use of r a d i o l i g a n d b i n d i n g assays.  D2  research receptors  have been l a b e l l e d w i t h many compounds (Seeman, 1980), w i t h [ H]-haloperidol 3  and [ H ] - s p i p e r o n e having 3  been most  f r e q u e n t l y employed. The i n v i v o p o t e n c i e s behavioural  of dopaminergic compounds i n  experiments w i t h animals are h i g h l y c o r r e l a t e d  w i t h t h e i r a f f i n i t i e s f o r l a b e l l e d D2 r e c e p t o r s  (Creese e t  16  al.,  1976;  receptor  Seeman,  antagonists  correlated  with  al.,  Creese  1975;  1980).  The  of  to  1977;  subchronic  response Snyder,  a l . , 1976;  finding their  parallel dopamine  Seeman,  also  dopamine  1977;  Muller  neurons  display  applied  dopamine similar  dopamine The the  increases receptor  by  et  that  highly-  (Seeman  a l . , 1976; dopamine  et Seeman,  receptor  by  blocking  agonists  increases  (Moore  i n the  antagonists  and  of  of  D2  dopamine  i n vivo  concentrated  and  of  D2  Creese  and  These receptors  also  receptor  6-OHDA  lesions  striatal  to i o n t o p h o r e t i c a l l y  1978).  (Moore  and  behavioural  Striatal  s u p e r s e n s i t i v i t y following treatment  regional density  are  Bloom,  D2  receptor  number  (Burt,  i n D2  sensitivity  and  dopamine  i n the  1977).  following  in  Burt  ( s e v e r a l months)  agonists  Seeman,  (Creese,  with  increase  increases  behavioural  increase  receptor and  in  Chronic  an  increased  receptor  in vitro  receptors  also  produce  treatment  an  Thus,  regional density  Thus,  are  e l e c t r o p h y s i o l o g i c a l i n d i c e s of  supersensitivity.  a  Seeman  suggests  1980).  experimentally-induced  show  receptor  dopamine  antipsychotic effects  produces  accompanied to  parallel  D2  of  antipsychotic potencies  ( s e v e r a l weeks)  antagonists receptors  the  affinities  dopaminergic-neurons  which  sensitivity Snyder,  The  receptors.  Lesions receptors  et  produce  dopamine  for  their  latter  antagonists D2  1980).  and  Bloom,  receptors  have  i n areas  of  1978).  i n the  demonstrated the  with  is correlated  innervation  studies  neurons  with  brain. that  b r a i n which  are  D2  17  innervated Janssen  most  and  Leysen,  Differential site be  Leysen, D2  now  from  with  homogenates prepared can  be  labelling  any  (e.g.  surface receptor  the D2  and  the  positron  similar  D2  as  would  Janssen  emission  binding  labelled  ( e g . Klemm,  i n vivo.  or  brain  Murrin  and  Kuhar,  sites  (e.g. A l t a r  et  a l . , 1985)  over  1981).  In  ligand  properties to  been measured tomography  also or  D2  labelling  labelled receptors.  in living  (Farde,  humans  Hall,  Ehrin  1986).  Prefrontal  Cortex  have  dopamine-related  D2  been system  Dopamine  designed  Receptors  to determine  i s involved  mechanisms  be  vitro  vivo  the  the  can  concentrations  In of  Brain  slices  D2  Seeman,  when  techniques  The  in relation  r e c e p t o r s have  properties  a l . , 1978(a))  control  m e s o c o r t i c a l dopamine of  1985).  the  (Laduron,  for characterization.  Many e x p e r i m e n t s  number  be  assayed.  in slices  studied  means  the  can  et  pharmacokinetic  D2  Medial  that  structures  many r a d i o l i g a n d  sites  (e.g. L i s t  t o be  Sedvall,  (Laduron,  Marshall,  indicate  cell  subsequently  Recently of  and  "membrane-like"  Laduron  provides  the  Kim  with  of  The  i s essential  compounds  and  studies  receptors display  in vitro  homogenates  permits  Altar,  for autoradiography  labelled  that  1978(a);  neurons  1978(b)).  available.  1979)  a  dopamine  measured  dopaminergic  centrifugation  i s associated  expected  and  d e n s e l y by  i n the  reported to  how large  date.  by  18  Unfortunately,  despite  electrophysiological the  mesocortical  precluded of  D2  any  the  and  amount  anatomical  dopamine  definitive  dopamine  large  system,  data  i n the  biochemical,  gathered  concerning  t e c h n i c a l problems  demonstration  receptors  of  and c h a r a c t e r i z a t i o n  prefrontal cortex.  Therefore,  i n v e s t i g a t o r s have  been  prefrontal  cortex  regulation i s involved  dopamine-related limitation since,  as  mechanisms.-  i n the  implicated  sites  i n the  and  one  in this  potential  Mitrius  above,  great  antagonist  mpfc,  (Creese,  D2  and  al.,  1985;  Camus e t  dopamine  receptors  1978;  D2  dopamine  and  Murrin  assays  and  1975;  1980;  of  by  (as  the has  dopamine  been  strongly  receptor  be  Kuhar,  Quik  obtained  1979;  et  Howlett  to  of  detect  a l . , 1978; Seeman,  D2 any  a l . , 1984; Werf data  et  Engel,  1984;  Altar  et  a l . , 1986).  suggesting  that  prefrontal cortex  Fields and  Andorn,  1981;  Palacios,  i n the  Reisine,  density  failed  1984; et  low  innervation  prefrontal cortex  and  Vander  present  (Pedigo,  have  i n the  Morgan  have  dopamine  relatively  List  a l . , 1986;  may  a  sparse  Palacios,  1984;  studies  humans  in  processes  receptor  spiperone)  studies  binding  and  Wamsley,  other  rats  Many  Snyder,  However,  of  the  majority  how  constitutes a substantial dopamine  expect  U'Prichard,  and  determine  effects.  would  dopamine  to  mesocortical  relatively  region.  Burt and  the  Muller-Schweinitzer Gehlert  of  h a l o p e r i d o l and  Considering the  study  unable  This  i n radioligand binding  butyrophenones,  of  receptor  mentioned  labelled  agonist  D2  have  and  Yamamura,  Nahorski,  1980;  D2  19  Marchais,  Tassin  Rosengarten Liskowsky  and F r i e d h o f f ,  and P o t t e r ,  Bouthenet,  Sales,  Nevertheless, conclusively receptors. spiperone studies)  that  In  D2  their  true  that  D2  of the r a t . the possible  administration compare  these  effects  receptors  on  of  and a - a d r e n e r g i c  these  (Howlett, (Andorn  Considering  et the  i n the cortex  i t i s clear that  conclusions  that  Niemegeers,  spirodecanone  cortex.  reports  i n the p r e f r o n t a l cortex  1 and  2 of t h i s  which  dopamine  The  assay  effects  c a n be  large  and t h e any  must  be  reached  was  t h e s i s a new  subsequently  receptors was  then  of chronic  effects  t o those  assay  employed t o  are located  used  i n the  i n Experiment  3 to  haloperidol  t h e c h a r a c t e r i z e d mpfc  of footshock  were  by  dopamine  identity.  i s described  demonstrate  demonstrated  (Leysen,  of receptors,  receptors  Experiments  procedure  The  1979)  characterized before  regarding  study  1978),  i n the f r o n t a l  types  1985).  i n most  of p o t e n t i a l neurotransmitters  suspected  mpfc  has  Martres,  m e a s u r e d a r e D2  l i g a n d used  to serotonergic  sites  e t a l . , 1985;  i s further complicated  and Laduron,  corresponding  fully  being  Bohmaker,  e t a l . , 1982;  Schwartz,  studies  the s i t e s  and N a h o r s k i ,  number  and  Meller,  Mita  Martres  of these  (the labelled binds  al.,1980)  1985;  issue  1980;  1982;  Sokoloff  none  The  Tollenaere Morris  and Bockaert,  observed  s t r e s s on mpfc  D2  receptors  i n the and  i n v e s t i g a t e d i n Experiment  4.  and t o  striatum.  striatal  D2  20  Experiment 1: Development and Characterize  Use  of an Assay to  D2 Dopamine Receptors i n the M e d i a l P r e f r o n t a l Cortex of the  Rat  Introduction  As i n d i c a t e d i n the General I n t r o d u c t i o n , D2 dopamine r e c e p t o r s may  e x i s t i n the mpfc.  However, some  i n v e s t i g a t o r s have f a i l e d to f i n d any i n the mpfc.  In order  c l a s s of b i n d i n g  i n d i c a t i o n of D2  sites  t o demonstrate t h a t a p a r t i c u l a r  s i t e s represent  D2 dopamine r e c e p t o r s , i t  i s necessary t o demonstrate t h a t the b i n d i n g s i t e s d i s p l a y a number of c r i t i c a l p r o p e r t i e s .  Thus, compounds l a c k i n g  dopaminergic a c t i v i t y should be  i n e f f e c t i v e at d i s p l a c i n g  the b i n d i n g .  Dopaminergic compounds should be e f f e c t i v e  d i s p l a c e r s and  d i s p l a y potencies  vivo pharmacological a c t i v i t y . compounds t e s t e d should  p r o p o r t i o n a l to t h e i r i n The  potencies  of a l l  c o r r e l a t e with t h e i r potencies  the w e l l c h a r a c t e r i z e d s t r i a t a l D2 dopamine r e c e p t o r . potencies  of endogenous a g o n i s t s  should d i s p l a y  than s e r o t o n i n .  The  which should  s i t e s should  r e l a t i v e d e n s i t y should  be  be more potent  be s a t u r a b l e and  be at l e a s t roughly  The  the  c h a r a c t e r i s t i c r e l a t i o n s h i p such t h a t dopamine should more potent than n o r a d r e n a l i n e ,  on  their  r e l a t e d to  known d i s t r i b u t i o n of dopaminergic i n n e r v a t i o n .  the  Experiments  21 1 and 2 w i l l address a l l but the l a s t two c r i t e r i a , which w i l l be d i s c u s s e d i n Experiment  3.  The process of demonstrating  the presence  of a  p a r t i c u l a r form of r e c e p t o r f i r s t r e q u i r e s the development of a r e p r o d u c i b l e and accurate method of measuring the Once t h i s i s accomplished t e s t e d t o determine form of r e c e p t o r .  the c a n d i d a t e s i t e can then  whether i t i s , i n f a c t , the The method developed  site. be  proposed  t o measure the  site  must a l s o be r e a s o n a b l y e f f i c i e n t w i t h regards t o the amount of time and m a t e r i a l i t r e q u i r e s or e l s e  the subsequent  process of p h a r m a c o l o g i c a l c h a r a c t e r i z a t i o n w i l l not  be  feasible. The p r e s e n t p r o j e c t began w i t h a s e r i e s of designed t o determine  how  experiments  the standard dopamine r e c e p t o r  assays employed by i n v e s t i g a t o r s s t u d y i n g p r e f r o n t a l c o r t e x D2 dopamine r e c e p t o r s can be m o d i f i e d t o produce s u f f i c i e n t r e s o l u t i o n t o a l l o w f e a s i b l e c h a r a c t e r i z a t i o n of the p o t e n t i a l D2 s i t e s i n the mpfc.  Methods  Tissue Preparation  The  s t r i a t u m and the d o p a m i n e r g i c a l l y i n n e r v a t e d areas  of the mpfc were d i s s e c t e d on i c e from male Wistar  rats  (300-400g; i n d i v i d u a l l y housed f o r 4 weeks) as shown i n Figure la-Id.  Rats were k i l l e d by c e r v i c a l  dislocation.  22  F i g u r e 1. Shaded a r e a s r e p r e s e n t t i s s u e u s e d i n assays. C a u d a l s i d e s o f 1 mm t h i c k s l i c e s a r e shown ( a - c = mpfc; d = s t r i a t u m ) .  23 The  b r a i n s were irrunediately removed and  microtome w i t h CO2.  f r o z e n on a  Three s u c c e s s i v e , 1 mm  thick, coronal  s l i c e s were cut s t a r t i n g at the most r o s t r a l t i p of cortex.  The  s t r i a t a l t i s s u e was  thick s l i c e . such s l i c e s  F i g u r e 1(a)-(d)  a l s o d i s s e c t e d from a 1  ( a f t e r Konig and K l i p p e l , 1963)  procedure. of 0.32  maintained I t was  w i t h the shaded  at 4°C thoughout the  50 volumes  rehomogenized i n approximately prepared  TEAN b u f f e r (15 mM  ascorbate,  and  12.5  was  centrifuged  resulting pellet  10 volumes of  t r i s - H C l , 5 mM uM  nialamide;  was  used i n b i n d i n g  was  freshly  Na EDTA, 2  a d j u s t e d to pH  with NaOH) w i t h a t e f l o n homogenizer, then incubated t h i r t y minutes on i c e , and  5.5  x g f o r 15 minutes.  d i s c a r d e d and the supernatent  at 100,000 x g f o r 60 minutes. The  mM  preparation  homogenized i n approximately  f o r 20 seconds) and c e n t r i f u g e d a t 750  1.1  The  M sucrose with a Brinkman P o l y t r o n ( s e t t i n g of  The p e l l e t was  mm  i l l u s t r a t e s the caudal s i d e of  areas r e p r e s e n t i n g the t i s s u e used i n the assays. t i s s u e was  the  7.2  for  f i n a l l y s t o r e d at -80°C u n t i l i t  assays.  B i n d i n g Assays  C o n s t i t u e n t s of the B i n d i n g  The  Reaction  b i n d i n g r e a c t i o n s took p l a c e i n 25 ml Erlenmeyer  f l a s k s c o n t a i n i n g the f o l l o w i n g : (1) B u f f e r , (2) 150  u l of  [-^H]-spiperone d i s s o l v e d i n 95% e t h a n o l 25 pM),  (3) 150  y.1 of ascorbate  ( f i n a l c o n c e n t r a t i o n of 22.0 d i s s o l v e d i n ascorbate  (final  concentration  d i s s o l v e d i n d i s t i l l e d water  uM),  or competing drug  s o l u t i o n , (4) 150  u,l methanol or  competing drug d i s s o l v e d i n methanol, (5) 150  u.1 of  k e t a n s e r i n t a r t r a t e d i s s o l v e d i n 95% e t h a n o l  (final  c o n c e n t r a t i o n of 10 nM), The  (6) 150  u.1 of t i s s u e homogenate.  f i n a l volume of the r e a c t i o n was  15  ml.  Buffer  The The  pH was  noted. was  b u f f e r c o n s i s t e d of 50 mM a d j u s t e d to 7.9  The  t r i s - H C l and  100 mM  with NaOH except where  t r i s - H C l c o n c e n t r a t i o n i s standard  NaCl.  otherwise  and the NaCl  added because s u l p i r i d e b i n d i n g t o dopamine r e c e p t o r s  has been shown t o r e q u i r e N a  +  (Theodorou, H a l l , Jenner  and  Marsden, 1980).  [1H3-Spiperone  One  hundred and  f i f t y m i c r o l i t e r s of [ H ] - s p i p e r o n e  d i s s o l v e d i n 95% e t h a n o l Nuclear) was was  25 pM  3  (23-26 Ci/mmole, New  added to each f l a s k .  except where otherwise  c o n c e n t r a t i o n of spiperone  was  The noted.  final The  chosen f o r two  England concentration low reasons:  (1) P r e l i m i n a r y s a t u r a t i o n a n a l y s i s r e v e a l e d t h a t  spiperone  25 binds t o D2 s i t e s i n the s t r i a t u m ( b i n d i n g d i s p l a c e d by 10"5  M s - ( - ) - s u l p i r i d e ) w i t h an apparent  25 pM.  Kd of  approximately  To ensure accuracy, c o m p e t i t i o n s t u d i e s should be  performed  w i t h a c o n c e n t r a t i o n of l a b e l l e d l i g a n d a t or  below the Kd.  (2) Previous r e p o r t s (Howlett and  1980;  et a l . ,  Marchais  p r e s e n t l y confirmed)  1980)  have suggested  Nahorski,  (and i t was  t h a t the use of low c o n c e n t r a t i o n s of  spiperone i n c r e a s e s the percent of b i n d i n g t o p o t e n t i a l s i t e s i n the f r o n t a l c o r t e x .  Thus, the p o t e n t i a l D2  D2  sites  b e g i n t o s a t u r a t e a t h i g h e r c o n c e n t r a t i o n s w h i l e the n o n s p e c i f i c and f i l t e r b i n d i n g i n c r e a s e i n a l i n e a r f a s h i o n .  Ascorbate  Ascorbate was  employed as an a n t i o x i d a n t t o p r o t e c t the  drugs i n c l u d e d i n the r e a c t i o n mixture. f i f t y m i c r o l i t r e s of 1.1 mM  One-hundred and  a s c o r b a t e was  added t o each  r e a c t i o n t o produce a f i n a l c o n c e n t r a t i o n of 22.0 JJLM  from the a s c o r b a t e s o l u t i o n and 11.0  b u f f e r added w i t h the t i s s u e  u\M  (11.0  uM from the TEAN  [see above]).  The  ascorbate  s o l u t i o n a l s o a c t e d as the v e h i c l e f o r the water s o l u b l e compounds which were t e s t e d i n c o m p e t i t i o n s t u d i e s (see Experiments  1 and  2).  26  Methanol  Methanol acted as the v e h i c l e f o r the s p e c i f i c r e c e p t o r a n t a g o n i s t , s - ( - ) - s u l p i r i d e , as w e l l as  D2  the  m a j o r i t y of the compounds t e s t e d i n the c o m p e t i t i o n (see Experiments 1 and added t o each r e a c t i o n . 10~5  2).  Thus, 150  studies  u,l of methanol were  A f i n a l c o n c e n t r a t i o n of  M s - ( - ) - s u l p i r i d e was  used t o d e f i n e s p e c i f i c  binding  as i s p r e f e r r e d i n D2 dopamine r e c e p t o r assays ( L i s t  and  Seeman, 1981).  Ketanserin  As mentioned above, [^H]-spiperone binds t o s e r o t o n e r g i c S2 r e c e p t o r s i n the f r o n t a l c o r t e x .  In  order  t o i n c r e a s e the percent of [ H ] - s p i p e r o n e b i n d i n g to 3  p o t e n t i a l D2  r e c e p t o r s i n the mpfc, the S2  antagonist, ketanserin,  (Leysen, Awouters, Kennis, Laduron,  Vandenberk and Janssen, 1981) reactions.  receptor  was  i n c l u d e d i n the  binding  Competition s t u d i e s r e v e a l e d a s e l e c t i v i t y of  ketanserin for serotonergic  (S2) vs dopaminergic (D2)  Figure 2 i l l u s t r a t e s that ketanserin concentrations 1 nM  and  10 nM  the mpfc and 150  ul  sites.  between  i n h i b i t e d 30-35% of t o t a l t i s s u e b i n d i n g i n  l e s s than 5% i n the s t r i a t u m .  Therefore,  of k e t a n s e r i n t a r t r a t e , d i s s o l v e d i n 95%  ethanol,  27  K E T A N S E R I N  CONCENTRATION  (M)  F i g u r e 2. The amount o f t o t a l t i s s u e b i n d i n g of [^H]-spiperone (25 pM) competed f o r by v a r i o u s c o n c e n t r a t i o n s of k e t a n s e r i n t a r t r a t e i n the mpfc and the s t r i a t u m .  28 were i n c o r p o r a t e d i n t o each b i n d i n g r e a c t i o n at a c o n c e n t r a t i o n of 10 nM. f r o n t a l c o r t e x was  The  final  percentage of S2 b i n d i n g i n the  l e s s than t h a t r e p o r t e d by o t h e r s  e t a l , 1978).  T h i s was  probably  concentrations  of spiperone  due  (Leysen  to the lower  used i n the present  experiments  as w e l l as the l i m i t i n g of f r o n t a l c o r t e x t i s s u e samples to the dopamine i n n e r v a t e d  areas.  Tissue  For the b i n d i n g assays the t i s s u e was d i l u t e d w i t h f r e s h l y prepared thoroughly  thawed at 4°C  TEAN b u f f e r .  I t was  then  mixed on a v o r t e x mixer, to ensure  homogenization, and  incubated  on i c e f o r 45 minutes.  A l i q u o t s of the t i s s u e homogenate (150 u.1;  approximately  100-150 u,g of mpfc p r o t e i n or 50-75 u,g of s t r i a t a l were added l a s t to the Erlenmeyer f l a s k s to s t a r t reaction. r e g i o n s was  and  The  protein) the  amount of s p e c i f i c b i n d i n g i n both b r a i n  l i n e a r l y r e l a t e d t o the amount of t i s s u e i n the  i n c u b a t i o n s over the range of t i s s u e c o n c e n t r a t i o n s employed.  P r o t e i n c o n c e n t r a t i o n s were determined  t o Lowry, Rosebrough, F a r r and R a n d a l l ,  (1951).  according  29  T e r m i n a t i o n of the B i n d i n g R e a c t i o n  The f l a s k s were g e n t l y shaken f o r 2.75 hours a t room temperature. The r e a c t i o n s were terminated by vacuum filtration.  Filtration  Apparatus  P r e l i m i n a r y experiments r e v e a l e d t h a t the v a r i a n c e a s s o c i a t e d w i t h the f i l t r a t i o n procedure was g r e a t l y reduced when f i l t r a t i o n was performed under a h i g h and c o n s i s t e n t vacuum.  A Gelman vacuum pump (model 13152) was found t o be  more s a t i s f a c t o r y than the l a b o r a t o r y "house" vacuum. pump was a t t a c h e d t o a 500 ml sidearm Erlenmeyer f l a s k had a 50 ml Gelman f i l t e r  The which  f u n n e l secured t o the t o p w i t h a  rubber s t o p p e r . While more r e a c t i o n s c o u l d be terminated per u n i t o f time w i t h f i l t r a t i o n m a n i f o l d s , the f u n n e l - f l a s k apparatus reduced the v a r i a n c e a s s o c i a t e d w i t h the f i l t r a t i o n procedure. As a r e s u l t fewer r e a c t i o n s were r e q u i r e d i n the c h a r a c t e r i z a t i o n experiments.  T h e r e f o r e , the use of the  f u n n e l - f l a s k apparatus proved t o be the more e f f i c i e n t procedure.  Filters  The  r e a c t i o n s were f i l t e r e d through Whatman GF/B  filters.  Whatman GF/C f i l t e r s  were a l s o t e s t e d i n an  attempt t o reduce the b i n d i n g o f the [ H ] - s p i p e r o n e t o the 3  filters.  However, the p o t e n t i a l D2 b i n d i n g was  s i g n i f i c a n t l y lower when GF/C f i l t e r s suggesting  t h a t GF/C f i l t e r s  were used  thereby  do not t r a p as many r e c e p t o r s  as GF/B f i l t e r s .  Washes o f F i l t e r s  A f t e r the f i l t r a t i o n were washed.  of r e a c t i o n mixtures,  the f i l t e r s  T y p i c a l l y , r e c e p t o r b i n d i n g assays employ 1-4  r a p i d washes w i t h b u f f e r . employed i n b i n d i n g assays,  However, compared t o most l i g a n d [ H ] - s p i p e r o n e has a very 3  a f f i n i t y f o r the D2 dopamine r e c e p t o r . of experiments were r u n w i t h s t r i a t a l  Therefore,  high  a series  t i s s u e t o t e s t the  e f f e c t s o f m o d i f i c a t i o n s i n the standard wash procedures. was hoped t h a t these experiments would r e v e a l methods o f r e d u c i n g f i l t e r b i n d i n g and n o n s p e c i f i c b i n d i n g of [ H ] - s p i p e r o n e w h i l e not a f f e c t i n g the b i n d i n g of 3  [ H ] - s p i p e r o n e t o D2 r e c e p t o r s . 3  I n c r e a s i n g the number o f 5 ml washes reduced b i n d i n g and n o n s p e c i f i c b i n d i n g .  filter  An i n c r e a s e from 4 t o 6  washes had no e f f e c t on D2 b i n d i n g , however, i f 10 washes  I  31 were employed the D2 b i n d i n g was m a r g i n a l l y reduced. T h e r e f o r e , s i x 5 ml washes were used. I n c r e a s i n g the temperature of the washes from the standard of 4°C t o room temperature had no measurable on f i l t e r ,  n o n s p e c i f i c , or D2 b i n d i n g .  effect  Since the wash  b u f f e r was r o u t i n e l y s t o r e d a t 4°C, t h a t temperature  was  used f o r the wash procedure. Adding the d e t e r g e n t , T r i t o n X-100, t o the wash b u f f e r at  c o n c e n t r a t i o n s r a n g i n g from .025% t o .075% g r e a t l y  reduced D2 b i n d i n g w h i l e having l e s s e f f e c t on f i l t e r b i n d i n g and n o n s p e c i f i c b i n d i n g .  However, 10% e t h a n o l or  10% acetone i n the wash b u f f e r markedly reduced  filter  b i n d i n g w h i l e having no e f f e c t on n o n s p e c i f i c or D2 b i n d i n g . E t h a n o l was chosen over acetone due t o economic and s a f e t y considerations.  Filtration  Procedure  To f u r t h e r reduce the v a r i a b i l i t y a s s o c i a t e d w i t h the f i l t r a t i o n , a timed procedure was employed f o r each filtration:  (1) the f i l t e r was moistened w i t h 5 ml of  d i s t i l l e d water filtered  (2) 5 seconds l a t e r the b i n d i n g r e a c t i o n was  (3) 10 seconds l a t e r the f i l t e r was exposed t o s i x  5ml washes o f 4 °C b u f f e r  (9 p a r t s 50 mM  t r i s - H C l , pH 7.9 :  1 p a r t a b s o l u t e e t h a n o l ) a t a r a t e of one wash per second (4) 15 seconds a f t e r the l a s t wash the f i l t e r was removed  32 from the f u n n e l and p l a c e d i n a s c i n t i l l a t i o n v i a l .  The  vacuum remained on throughout the procedure.  Measurement of R a d i o a c t i v i t y  Approximately 1 hour a f t e r the f i l t r a t i o n , each  filter  was v i g o r o u s l y shaken f o r 5 minutes w i t h 15 ml of P i c o - F l u o r 15 (a h i g h e f f i c i e n c y s c i n t i l l a t i o n Packard I n s t r u m e n t s ) .  fluid;  S t a r t i n g a t l e a s t 24 hours l a t e r , the  r a d i o a c t i v i t y of the v i a l s was determined w i t h a Packard T r i - C a r b 4530 l i q u i d s c i n t i l l a t i o n counter a t an e f f i c i e n c y of approximately 50%.  An average of the v a l u e s of f o u r 20  minute r e c o r d i n g p e r i o d s was used i n a l l mpfc c a l c u l a t i o n s i n o r d e r t o minimize the v a r i a b i l i t y a s s o c i a t e d w i t h the measurement of r a d i o a c t i v i t y .  Only one 20 minute  was r e q u i r e d f o r each s t r i a t a l  vial.  reading  Competition S t u d i e s  V a r i o u s dopaminergic and nondopaminergic compounds were t e s t e d f o r t h e i r a b i l i t y t o compete f o r s p e c i f i c b i n d i n g of 25 pM [ H ] - s p i p e r o n e ( d e f i n e d by 10"^ M s - ( - ) - s u l p i r i d e ) i n 3  the s t r i a t u m and mpfc of the r a t .  I n c u b a t i o n s w i t h no  competing drug o r 10"^ M s - ( - ) - s u l p i r i d e were done i n q u a d r u p l i c a t e i n each mpfc assay and i n t r i p l i c a t e f o r each s t r i a t a l assay.  Competing drugs were run a t f i v e  c o n c e n t r a t i o n s i n d u p l i c a t e or t r i p l i c a t e i n mpfc assays and  33  in  duplicate  controls lO ^ -  in  M  i n striatal  were  also  run f o r each  s-(-)-sulpiride  t h e same b i n d i n g  competed  competing  drug  which  amount) linear  IC50's  reduces were  followed  by  in  1 a r e t h e mean  independent plotted  dopamine and  8,  multiple this  receptor drawn  binding  section)  by hand  comparisons  were  since  may  be  to  M of t o 50% o f  IC50's  listed  three  competition  curves  the  transformation  the l o g i t  curves  linear  f o r the  are plotted i n Figures  involved points  suggests  6,7  that  (see d i s c u s s i o n of are available f o r  curves.  Student's  samples  of l o g values.  expect  binding  The  evidence  of exact  made w i t h  or independent  coefficients  drug  t h e 10"^  logit  sulpiride from  included  s-(-)-sulpiride  analysis.  which  b u t t o o few d a t a  calculation  were  one would  than  competition  agonists,  statistical  dependent  The  Thus,  (the concentration  3 are derived  sites  drug  (± s.e.m.) o f a t l e a s t  The  blank  tested.  the specific  regression  equation.  were  then  c a l c u l a t e d by  experiments.  i n Figure  regression  binding,  and t h e 10"^ M  alone.  to  I f the competing  3  original  Table  compound  [ H]-spiperone binding  s-(-)-sulpiride  the  reaction.  of the drug  less  Additivity  and t h e competing  for nonspecific  combination produce  assays.  Statistical  two-tailed  or Pearson's  t-tests for  correlation  34  Results  As shown i n F i g u r e 3,  s - ( - ) - s u l p i r i d e (the  b i o c h e m i c a l l y and b e h a v i o r a l l y more a c t i v e isomer  [Jenner,  Clow, R e a v i l l , Theodorou and Marsden, 1980]) was more potent than r - ( + ) - s u l p i r i d e i n both the s t r i a t u m and the mpfc.  The  c o m p e t i t i o n curves were v e r y s i m i l a r i n the two b r a i n r e g i o n s when they were p l o t t e d as percent of s p e c i f i c binding.  The s - ( - ) - s u l p i r i d e c o m p e t i t i o n curve f o r mpfc  t i s s u e displayed a d i s t i n c t , intermediate  ( i n terms of t o t a l  t i s s u e b i n d i n g ) p l a t e a u i n the 10"^ M t o 10"^ M range.  This  result indicates that s - ( - ) - s u l p i r i d e i s a highly s e l e c t i v e l i g a n d f o r the s p e c i f i c b i n d i n g s i t e s .  The s p e c i f i c b i n d i n g  amounted t o 95-98% of t o t a l s t r i a t u m b i n d i n g and 20-25% of t o t a l mpfc b i n d i n g . The r e s u l t s of c o m p e t i t i o n s t u d i e s presented  i n Table 1  i l l u s t r a t e t h a t the s p e c i f i c b i n d i n g i n both b r a i n r e g i o n s d i s p l a y e d the c h a r a c t e r i s t i c s of D2 r e c e p t o r s . A l l nondopaminergic compounds t e s t e d were i n e f f e c t i v e a t nanomolar c o n c e n t r a t i o n s .  Dopamine was more potent  n o r a d r e n a l i n e which was more potent than s e r o t o n i n . nanomolar c o n c e n t r a t i o n s a l l the dopaminergic competed f o r the s p e c i f i c b i n d i n g s i t e s .  than At  antagonists  The IC50's of the  dopaminergic a n t a g o n i s t s i n the mpfc assays c o r r e l a t e d with t h e i r i n v i v o p o t e n c i e s as a n t a g o n i s t s of apomorphineinduced emesis i n the dog (r=.709; d/f=7; p<.05; Janssen  and  35  S U L P I R I D E 100  c->; S t r i a t u m  H  A  6  c+3; S t r i a t u m  i; Prefrontal Cortex  Z  o 2 CD  i; Prefrontal Cortex  80 -  O U.  O UJ Q. CO  60  O  40  H Z UJ DC UJ  20  H  0L  10  10  10  10  10  CONCENTRATION  (M)  F i g u r e 3. The p e r c e n t o f s p e c i f i c [ H ] - s p i p e r o n e (25 pM) b i n d i n g ( b i n d i n g c o m p e t e d f o r b y 1 0 " M s - ( - ) - s u l p i r i d e ) competed f o r b y v a r i o u s c o n c e n t r a t i o n s o f s - ( - ) - s u l p i r i d e and r - ( + ) - s u l p i r i d e i n t h e mpfc and the s t r i a t u m . J  5  Table  1  I n h i b i t i o n o f S p e c i f i c [ H ] - S p i p e r o n e (25 pM) 3  log Compound  1.  Pimozide  2.  Haloperidol  3. B e n p e r i d o l 4. S p i p e r o n e  Striatum  IC50  Binding  (M) ± s.e.m.  Medial  Prefrontal  -10. 31  +  .07  -9 .95  +  .16  -8. 73  +  .04  -8 .68  +  .09  -9. 35  + .04  -9 .68  + .09  -10. 20  + .08  -10 .02  +  .11  5. T h i o r i d a z i n e  -8. 79  +  .07  -8 .74  +  .06  6. P r o m a z i n e  -6. 99  +  .09  -6 .49  +  .02  7.  Clorpromazine  -8. 48  +  .08  -8 .85  +  .08  8. T r i f l u o p e r a z i n e  -9. 62  + .21  -9 .40  +  .43  9. ( - ) - s u l p i r i d e  -7. 77  +  .17  -7 .64  +  .12  10.  (+)-sulpiride  -5. 83  +  .16  -6 .04  +  .07  11.  Apomorphine  -7. 13  +  .08  -7 .70  +  .08  12.  ADTN  -7. 42  +  .20  -7 .76  +  .30  13.  Dopamine  -6. 28  +  .05  -6 .32  +  .15  14.  Noradrenaline  >  -5  >  -5  15.  Serotonin  >  -5  >  -5  16.  Baclofen  >  -5  >  -5  17.  Atropine  >  -5  >  -5  18.  Naloxone  >  -5  >  -5  19.  Phentolamine  >  -6  >  -6  20.  Clonazepam  >  -5  >  -5  Cortex  37  Van  Bever,  1975)  and  apomorphine-induced  (r=.752;  d/f=7;  p<.02; N i e m e g e e r s ,  Janssen,  1977).  In  receptor  a n t a g o n i s t s were  potencies 1980;  as  addition,  Creese  et  observed  with  The were  IC50's  IC50's  The  mpfc  IC50  correlate  of  r=.923; Although  striatal  correlate  receptors.  obtained  d/f=10;  p,.01;  a  correlation  high  Burt,  the  data  reveals that  the  the  dopaminergic  subclasses  of  Figures the  dopamine  antagonists  structurally 6,  displacing  7  and  receptor  as  such  related  8 present  the  5).  values  and  et a l . ,  Snyder,  1976).  between  striatum,  the  closer  dopaminergic  D2  binding  difference  a whole  with  (r=.917;  1978)  obtained  of  No  IC50  and  examination  striatum.  .01)  published  Creese  the  tissue  (Figure  Leysen  of  at  p<  p<.01;  and  more p o t e n t  the  Laduron,  was  those  mpfc  3  and  d/f=9;  i n the  with  also  [ H]-spiperone  mpfc  than  Seeman,  tissue(Creese et  other  Thus,  with  Gommeren  (r=.874;  with  the  mpfc  were  assays  of  are  their  p<.01;  d/f=ll;  values  agonists  dopamine  with  tested with  (r=.976;  the  also  Leysen,  3  IC50  from  those  p<.01;  rat  and  accordance  in striatal  a l l compounds  dopamine  with  Artois  the  correlations  receptors  values  [ H]-haloperidol 1978;  these  In  i n the  1980).  obtained  f o r D2  d/f=10;  D2  of  d/f=7;  (Figure 4).  highly correlated  the  values  the  Seeman,  very  IC50's  highly correlated  al.,1976)  r e p o r t s , a l l of  1976;  Lenaerts,  a n t i p s y c h o t i c s (r=.818;  previous  al.,  the  behavior  or  any  in  the  i s seen of  with  the  antagonists. competition  a g o n i s t s , dopamine,  curves  apomorphine  for and  38  F i g u r e 4. The r e l a t i o n s h i p b e t w e e n t h e a f f i n i t i e s o f v a r i o u s dopamine r e c e p t o r a n t a g o n i s t s f o r mpfc D2 r e c e p t o r s a n d t h e a n t i p s y c h o t i c p o t e n c i e s o f t h e same drugs. The numbers b e s i d e t h e d a t a p o i n t s r e f e r t o T a b l e 1.  39  F i g u r e 5. The r e l a t i o n s h i p between the a f f i n i t i e s of v a r i o u s dopamine r e c e p t o r a n t a g o n i s t s f o r mpfc D2 r e c e p t o r s and the a f f i n i t i e s o f the same drugs f o r s t r i a t a l D2 r e c e p t o r s . The numbers beside the data p o i n t s r e f e r t o Table 1.  D O P A M I N E  F i g u r e 6. The percent o f s p e c i f i c [ H ] - s p i p e r o n e (25 pM) b i n d i n g ( b i n d i n g competed f o r by IO"" M s - ( - ) - s u l p i r i d e ) competed f o r by v a r i o u s c o n c e n t r a t i o n s of dopamine i n the mpfc and the s t r i a t u m . J  5  A P O M O R P H I N E 100 -  C O N C E N T R A T I O N  (M)  F i g u r e 7. The percent o f s p e c i f i c [ •'H]-spiperone (25 pM) b i n d i n g ( b i n d i n g competed f o r by 10"-> M s - ( - ) - s u l p i r i d e ) competed f o r by v a r i o u s c o n c e n t r a t i o n s of apomorphine i n the mpfc and the s t r i a t u m .  42  Figure 8 . The percent of s p e c i f i c [ H ] - s p i p e r o n e (25 pM) b i n d i n g ( b i n d i n g competed f o r by IO'* M s - ( - ) - s u l p i r i d e ) competed f o r by v a r i o u s c o n c e n t r a t i o n s of ADTN i n the mpfc and the s t r i a t u m . J  43 ADTN.  Dopamine d i s p l a y s a tendency  to be more potent i n the  mpfc p a r t i c u l a r l y a t the lowest c o n c e n t r a t i o n (10 nM), t h i s d i f f e r e n c e f a i l s t o reach s t a t i s t i c a l Apomorphine was striatum.  significance.  more potent i n the mpfc than i n the  The mean IC50 of apomorphine was  significantly  lower i n the mpfc than i n the s t r i a t u m (t= 5.04; p<.005).  d/f=4;  The dopamine and apomorphine r e s u l t s prompted a  change i n the d e s i g n of the c o m p e t i t i o n experiments. ADTN was  but  t e s t e d i n f o u r independent  experiments  Thus,  i n which  s t r i a t a l t i s s u e and mpfc t i s s u e from the same r a t s were examined i n the same assay and t h e r e f o r e , under e x a c t l y the same assay c o n d i t i o n s .  The a p r i o r i p r e d i c t i o n t h a t ADTN  would be more potent i n the mpfc was  supported by the IC50  data (t=3.19; d/f=3; p<.05). Phentolamine was  i n e f f e c t i v e a t competing f o r mpfc  b i n d i n g i n the nanomolar range where i t i s known t o be a potent a - a d r e n e r g i c r e c e p t o r a n t a g o n i s t U ' P r i c h a r d , Greenberg and Snyder, 1977).  (Peroutka, As mentioned i n  the General I n t r o d u c t i o n , o t h e r s have r e p o r t e d t h a t spiperone can b i n d t o a - a d r e n e r g i c s i t e s i n the c o r t e x (Andorn e t a l . ,  1980).  t h i s i n the p r e s e n t data may c o n d i t i o n s or t i s s u e sources.  The be due  frontal  l a c k of any i n d i c a t i o n of t o d i f f e r e n c e s i n assay  However, i t i s a l s o p o s s i b l e  t h a t the k e t a n s e r i n , which has been demonstrated t o be  an  a - a d r e n e r g i c a n t a g o n i s t i n b i n d i n g s t u d i e s (Leysen e t a l . , 1981)  may  have occluded the a - a d r e n e r g i c  sites.  44  Discussion  The r e s u l t s o f the c o m p e t i t i o n  experiments i n d i c a t e  t h a t the s p e c i f i c b i n d i n g measured i n the mpfc d i s p l a y s the expected c h a r a c t e r i s t i c s of D2 dopamine r e c e p t o r s .  The high  c o r r e l a t i o n between the IC50 values of dopamine r e c e p t o r antagonists  i n the mpfc and the p o t e n c i e s of the same drugs  as a n t i p s y c h o t i c s , c o n s t i t u t e s the f i r s t evidence  from  l i g a n d b i n d i n g s t u d i e s t h a t the mpfc may be a s i t e of the t h e r a p e u t i c a c t i o n f o r these drugs.  However, s i n c e t h i s  same c o r r e l a t i o n has been observed w i t h D2 dopamine b i n d i n g i n s u b c o r t i c a l regions  (Seeman, 1980; Creese e t a l . , 1976),  the c o r r e l a t i o n a l data do not i n d i c a t e which dopaminergically  i n n e r v a t e d r e g i o n , or r e g i o n s , are the s i t e  or s i t e s a t which dopamine r e c e p t o r a n t a g o n i s t s  produce  their antipsychotic effects. The g r e a t e r potency of dopamine r e c e p t o r a g o n i s t s f o r the mpfc D2 r e c e p t o r s compared t o the s t r i a t a l D2 has a number of i n t e r e s t i n g i m p l i c a t i o n s . i n d i c a t e s t h a t although  receptors  This difference  the mpfc dopamine r e c e p t o r s are of  the D2 type they are not i d e n t i c a l t o the s t r i a t a l receptors.  A d i f f e r e n t r e c e p t o r p o p u l a t i o n i n the mpfc  c o u l d p o t e n t i a l l y be r e s p o n s i b l e f o r some of the unique c h a r a c t e r i s t i c s o f the m e s o c o r t i c a l dopamine system which were o u t l i n e d i n the General  Introduction.  Recent  on D2 dopamine r e c e p t o r s suggests t h a t the D2 p r o t e i n can be present  i n two i n t e r c h a n g a b l e  research  receptor conformations  45 (Creese, 1985).  S i b l e y and L e f f , 1984; G r i g o r i a d i s and Seeman, One of these conformations,  d i s p l a y s a high a f f i n i t y  the D2(high) s i t e  (Kd=l-10 nM) f o r dopamine r e c e p t o r  a g o n i s t s w h i l e the other conformation, d i s p l a y s a lower a f f i n i t y receptor agonists.  the D2(low) s i t e ,  (Kd*200-2,000 nM) f o r dopamine  Both s i t e s d i s p l a y e q u a l l y h i g h  f o r dopamine r e c e p t o r a n t a g o n i s t s .  In support  affinity  of t h i s  model, when dopamine r e c e p t o r a g o n i s t s compete with a r a d i o a c t i v e l y l a b e l l e d dopamine r e c e p t o r a n t a g o n i s t s i t e s , the c o m p e t i t i o n  curves  f o r D2  are not as steep as one would  expect i f a s i n g l e form of b i n d i n g s i t e were  labelled.  Under the same c o n d i t i o n s , dopamine r e c e p t o r  antagonists  d i s p l a y competition  curves  t h a t are steeper than those o f  the dopamine r e c e p t o r a g o n i s t s and c o n s i s t e n t w i t h a s i n g l e s i t e being l a b e l l e d .  The present  agreement w i t h these f i n d i n g s . s u l p i r i d e competition  curves  dopamine r e c e p t o r a g o n i s t s  r e s u l t s are i n complete  A comparison of the  ( F i g u r e 3) and those of the  ( F i g u r e s 5,6 and 7) r e v e a l s the  d i f f e r e n c e t h a t has p r e v i o u s l y been observed between a g o n i s t and a n t a g o n i s t c o m p e t i t i o n  curves  (Creese,  S i b l e y and L e f f ,  1984; G r i g o r i a d i s and Seeman, 1985). I f D2 r e c e p t o r s are present  i n D2(high) and D2(low)  s t a t e s , i t i s p o s s i b l e t h a t the mpfc possesses a higher p r o p o r t i o n of D2(high) s i t e s than i s present striatum.  i n the  T h i s would e x p l a i n why dopamine r e c e p t o r  a g o n i s t s , but not dopamine r e c e p t o r a n t a g o n i s t s , a r e more potent  i n the mpfc.  I t would a l s o e x p l a i n why the e f f e c t i s  46  much  less  pronounced  competing of  D2(high)  1985). of  drug  have  been  that  which  If sites be  the  obvious  been  greater  the  of  proportion  of  interact  with  higher  affinity  for  increased,  and the  D2(low)  greater sites  sites  the  Given  and  higher  receptor  be 10  not  then  studies  lower  are  agonist sites,  agonist agonist  (Creese  1985).  As  reported  receptor  agonists  1985)  the  at  agonist  the  to  D2(low)  sites  a l . ,  have  is  to  and  a  et  a l . ,  effects  of  the  receptors higher  data  for  1984;  D2(high)  concentrations present  a  1984;  saturation  results  a  (Creese  mpfc  a  Thus i t  D2(high)  of  to  should  of  affinity  due  D2(high)  affinity  The  be  of  concentration  lower.  may  sulpiride  low  the et  or  D2  the  concentrations,  towards  influence  1985)  might  employed.  because  tend  number  Seeman,  compared  the  the  difference  when  nM  agonists  for  this  agonists  Seeman,  present.  required  higher  and  observed  proportion  the  greatest  and  a proportion  reduce  a  to  Seeman,  ions  +  binding.  sites  begin  f o r dopamine  approximately  the  D2(high)  extent.  should  the  lower  specific  D2(high)  Seeman,  sites  Grigoriadis  are  competing at  Na  presently  striatum,  that  a  as  (Grigoriadis  were  i n competition  sites,  the  the  reported  Grigoriadis  ions  +  to  (Grigoriadis  ions  contains  to  concentrations has  Na  defines  mpfc  ADTN  differences +  i s employed  binds  that  sites  i f Na  the  compared  most  the  greater  to  indicate  D2(high)  Unfortunately, binding  dopamine  compared  data  detectable  suggesting  because  sites  Other  when d o p a m i n e  of  suggest  dopamine  proportion  of  that  47 D2(high) s i t e s i n the mpfc.  Thus, at c o n c e n t r a t i o n s of  10 nM or l e s s , apomorphine was  more potent i n the mpfc  i n the s t r i a t u m (t=5.17; d/f=4; p<.005).  In order  than  to  produce an e q u i v a l e n t r e d u c t i o n i n [ H ] - s p i p e r o n e b i n d i n g , 3  o n l y approximately apomorphine was  1/6  as h i g h a c o n c e n t r a t i o n of  r e q u i r e d i n mpfc b i n d i n g r e a c t i o n s compared  to s t r i a t a l r e a c t i o n s .  As h i g h e r c o n c e n t r a t i o n s of  apomorphine were employed, t h i s d i f f e r e n c e was ADTN data d i s p l a y e d a s i m i l a r p a t t e r n .  ADTN  reduced.  The  was  approximately  t h r e e times as potent at c o n c e n t r a t i o n s of  10 nM or l e s s  (t=18.70; d/f=3; p<.001) and t h i s d i f f e r e n c e  was  a l s o s m a l l e r at higher Low  concentrations.  doses of apomorphine, ADTN and other dopamine  r e c e p t o r a g o n i s t s , produce many b e h a v i o u r a l e f f e c t s are v e r y s i m i l a r  t o those observed  which  f o l l o w i n g the  a d m i n i s t r a t i o n of dopamine r e c e p t o r a n t a g o n i s t s .  For  example, low c o n c e n t r a t i o n s of dopamine r e c e p t o r a g o n i s t s can reduce spontaneous locomotor a c t i v i t y i n rodents (Bradbury,  C o s t a l l , Lim and N a y l o r ,  can cause s e d a t i o n , suppress  d y s k i n e t i c or  movements, p o t e n t i a t e parkinsonism, (Seeman, 1980).  1981).  In humans, they choreiform  and reduce p s y c h o s i s  These phenomena are commonly c o n s i d e r e d  be the r e s u l t of the dopamine r e c e p t o r a g o n i s t s activating neurons.  to  selectively  dopamine " a u t o r e c e p t o r s " l o c a t e d on dopaminergic "Autoreceptors"  are b e l i e v e d to be  selectively  a c t i v a t e d because dopamine r e c e p t o r a g o n i s t s are thought to have a h i g h e r a f f i n i t y  f o r dopamine a u t o r e c e p t o r s  than f o r  48  postsynaptic  dopamine  receptors.  dopamine  system  systems,  as has been proposed  then these in  the present effects  fact  be  receptors, doses  tonically  due  data  inhibits  raise  o f dopamine  t h e s u b c o r t i c a l dopamine  the p o s s i b i l i t y  to the selective on  action  Introduction), that  some o f  at autoreceptors  a c t i v a t i o n o f mpfc  nondopaminergic  receptor  mesocortical  (see General  a t t r i b u t e d t o an  located  I f the  agonists.  neurons,  by  may  D2 the  low  49  Experiment Prefrontal  2:  Characterization  Cortex  D2  Dopamine  at  pH  6.2  Receptors  of  in  Medial  the  Rat  Introduction  Spiperone sensitive,  has  by  compounds  tested  derivatives  and  nanomolar  of  the  Following  30  day  old  spirodecanone  sites  incubation  from  They  also  pH  reported  serotonergic This the pH  the  7.9  is the  resolution the  to  typical  that  sites that  binding  "nonspecific"  present  the  i f a  in  spirodecanone  of  of  the  sites  binding  assay.  experiment.  This  of  in  et a l . ,  and  a  a  to  a  6.2. and  affected.  assay  of  at  i n pH  increase  was  in  of  sites  reduction  possibility  pH  proportion  mpfc  thereby  tissue  shift  minimally  present  Bruinink  to  binding  significant  then  1,  by  range  only  not  many  forebrain  reduced  D2  is  heat  butyrophenone  spiperone  7.4-7.9  are  any  (Howlett  that  dopaminergic  binding  "nonspecific"  reported binding  and  which  Experiment  i s dramatically the  of  type of  saturable,  binding  spiperone  completion  f i n d i n g suggested  reduce  in  S2  by  (1984)  rats  display  affinity  spirodecanone the  to  concentrations  except  Lichtensteiger  from  reported  reversible, high  displaced  1979).  been  may the  investigated  50  Methods  I n i t i a l competition either  studies with tissue  samples from  the mpfc or the s t r i a t u m r e v e a l e d t h a t when  i n c u b a t i o n s were run a t pH 6.2 both isomers o f s u l p i r i d e were approximately  50 f o l d l e s s potent  s p e c i f i c b i n d i n g than a t pH 7.9.  a t competing f o r the  The c o m p e t i t i o n  curves a t  pH 6.2 were the same shape as curves o b t a i n e d a t pH 7.9 but simply clear  s h i f t e d t o the r i g h t from the present  (higher m o l a r i t i e s ) .  r e s u l t s whether t h i s change i n  s u l p i r i d e potencies represents interaction  a fundamental change i n the  between the s u l p i r i d e and the D2 r e c e p t o r or i s  simply due t o an a r t i f a c t u a l r e d u c t i o n i n the f r e e present  I t i s not  i n the b i n d i n g r e a c t i o n .  sulpiride  Thus, s u l p i r i d e may not be  as s t a b l e a t pH 6.2 as i t i s a t pH 7.9 or may not be as soluble.  In l i g h t o f these r e s u l t s ,  the s p e c i f i c D2 b i n d i n g  i n experiments run a t pH 6.2 was d e f i n e d as t h a t b i n d i n g i n h i b i t e d by 1 0 " M s - ( - ) - s u l p i r i d e . 4  T h i s was done i n order  to ensure t h a t the d e f i n i n g c o n c e n t r a t i o n of s u l p i r i d e occluded  as complete a p o p u l a t i o n o f D2 dopamine r e c e p t o r s  as p o s s i b l e .  The pH o f the stock s o l u t i o n  o f s u l p i r i d e was  a d j u s t e d t o pH 6.2 so t h a t i t would not i n f l u e n c e the pH of the b i n d i n g r e a c t i o n . has  T h i s was necessary  because t r i s - H C l  l e s s b u f f e r i n g c a p a c i t y a t pH 6.2 than a t pH 7.9.  order t o d i r e c t l y c h a r a c t e r i z e the s p e c i f i c s i t e s  In  labelled  51  at the  pH  6.2,  a  series  same d r u g s  as were  Saturation affinity brain The to  i s lower  (KD=75 pM)  [ H]-spiperone concentration  was  increased  3  pM.  With  a t pH  binding than  run  with  6.2,  the  i n  both  sites  a t pH  4.  amount  the  t o 35  that  i n Experiment  f o r the reduced  was  7.9.  revealed  for specific  are presented  compensate  experiments  t e s t e d a t pH  experiments  of spiperone  regions  data  of competition  7.9  (KD=25  Therefore,  of specific  i n order  binding,  i n the competition  the exception  mentioned  modifications the competition  performed  as d e s c r i b e d  i n Experiment  pM).  assays  o f t h e above  assays  were  1.  Results  The of  competition  t h e compounds  highly  studies demonstrated  t e s t e d i n t h e mpfc  correlated  with  their  of spiperone  potencies  the  IC50 v a l u e s  are  included  The  same p a t t e r n o f c o r r e l a t i o n s  Thus,  and both  i n the calculations  characterization  a t pH  IC50 v a l u e s  from  the  values  obtained  The  IC50  values  the  mpfc  correlated  7.9  same d r u g s  (r=.751;  correlated  with  was  t h e mpfc  with  a t pH isomers  7.9 of  observed  were  p<.01). with  a t pH  (r=.992; d / f = l l ; antagonists  In addition, the f o r D2  with  p<.01).  obtained  the antipsychotic potencies  values  the  6.2.  highly correlated  receptor  reported  when  sulpiride  observed  also  2) a r e  even  that  was  potencies  (Table  d/f=ll;  d/f=7; p<.02).  other  6.2  the  (r=.949;  i n the striatum  o f dopamine  a t pH  that  i n  of the values  receptors  as  Table 2 I n h i b i t i o n of S p e c i f i c [^H]-Spiperone (35 pM) B i n d i n g a t pH 6.2  log I C Compound  Striatum  5 0  (M) + s.e.m.  Medial P r e f r o n t a l  1. Pimozide  -9.98  -10.00  2. H a l o p e r i d o l  -8.69  -8.80  3. B e n p e r i d o l  -9.50  -9.43  4. Spiperone  -9.85  -9.80  5. T h i o r i d a z i n e  -8.75  -8.97  6. Promazine  -7.09  -7.30  7. Chlorpromazine  -8.52  -8.89  8. T r i f l u o p e r a z i n e  -9.60  -9.53  9. ( - ) - s u l p i r i d e  -6.42  -6.07  10. ( + ) - s u l p i r i d e  -4.75  -4.95  11. Apomorphine  -7.19  -7.03  12. ADTN  -6.87  -6.52  13. Dopamine  -6.20  -6.15  14. Noradrenaline  > -5  > -5  15. S e r o t o n i n  > -5  > -5  Cortex  l a b e l l e d with  [ H ] - s p i p e r o n e (r=.882; d/f=10; p<.01; Leysen J  e t a l . , 1978) or [ H ] - h a l o p e r i d o l (r=.927; d/f=9; p<.01; 3  Burt e t a l . , 1976; r=.794; d/f=10; p<.01; Leysen e t a l . , 1978).  However, i n c o n t r a s t w i t h the data o b t a i n e d a t pH  7.9, the dopamine r e c e p t o r a g o n i s t s were not more potent i n the mpfc than i n the s t r i a t u m . By f a r the most marked e f f e c t of the r e d u c t i o n i n pH was a l a r g e decrease i n the n o n s p e c i f i c b i n d i n g i n the mpfc. T h i s was probably  due t o the v i r t u a l e l i m i n a t i o n of  [ H ] - s p i p e r o n e b i n d i n g t o spirodecanone s i t e s which have 3  been r e p o r t e d t o be more numerous i n the f r o n t a l c o r t e x than i n the s t r i a t u m (Howlett  e t a l . , 1979).  Thus, n o n s p e c i f i c  mpfc b i n d i n g a t pH 6.2 was l e s s than 30% o f t h a t observed a t pH 7.9. Consequently, i n the assays performed a t pH 6.2, 5060% of the mpfc b i n d i n g was s p e c i f i c .  In the s t r i a t u m more  than 98% of the b i n d i n g was s p e c i f i c .  Discussion  The c h a r a c t e r i z a t i o n data i n d i c a t e t h a t the s p e c i f i c mpfc and s t r i a t a l s i t e s l a b e l l e d a t pH 6.2 a r e D2 dopamine receptors.  However, s i n c e the dopamine r e c e p t o r  when assayed a t pH 6.2, are not more potent  agonists,  i n the mpfc than  i n the s t r i a t u m , i t seems t h a t the mpfc p o p u l a t i o n  labelled  at pH 6.2 may not be i d e n t i c a l t o t h a t l a b e l l e d a t pH 7.9. The present data do not i n d i c a t e how these  different  p o p u l a t i o n s of r e c e p t o r s are i n v o l v e d i n the i n v i v o  54  f u n c t i o n of the m e s o c o r t i c a l dopamine system. r e s o l v e t h i s i s s u e i t w i l l be necessary e f f e c t s of v a r i o u s experimental  In order t o  t o compare the  manipulations  on the l i g a n d  b i n d i n g c h a r a c t e r i s t i c s of mpfc D2 r e c e p t o r s with the e f f e c t s t h a t the same manipulations functions.  have on i n v i v o dopamine  I t i s p o s s i b l e t h a t both the r e c e p t o r  population  l a b e l l e d a t pH 7.9 and the p o p u l a t i o n l a b e l l e d a t pH  6.2  t r u l y r e f l e c t a f u n c t i o n a l s t a t e of mpfc D2 dopamine receptors.  Thus, i t i s c o n c e i v a b l e t h a t the pH o f the  extremely s m a l l p o o l o f e x t r a c e l l u l a r f l u i d i n the s y n a p t i c c l e f t can be a f f e c t e d i n l o c a l areas by the neuronal of compounds  i n t o the c l e f t .  release  Such pH changes may a f f e c t the  l i g a n d b i n d i n g c h a r a c t e r i s t i c s of mpfc D2 r e c e p t o r s , as suggested by the present data, thereby  i n f l u e n c i n g the  s e n s i t i v i t y of mpfc D2 r e c e p t o r s t o dopamine r e c e p t o r agonists.  I f so, the present data suggest t h a t the neuronal  r e l e a s e of a c i d i c compounds may reduce the pH i n the r e g i o n of the p r e s y n a p t i c or p o s t s y n a p t i c membranes and thereby reduce the s e n s i t i v i t y of mpfc D2 r e c e p t o r s t o dopamine receptor agonists.  S i m i l a r l y , the neuronal  r e l e a s e of  a l k a l i n e compounds may i n c r e a s e the s e n s i t i v i t y of the mpfc D2 r e c e p t o r s t o dopamine r e c e p t o r a g o n i s t s . should be noted t h a t these p r o p o s a l s  However, i t  are extremely  s p e c u l a t i v e i n t h a t the l o c a l pH i n any p a r t i c u l a r area of the s y n a p t i c c l e f t i s p r e s e n t l y unknown. The primary m o t i v a t i o n f o r i n v e s t i g a t i n g the e f f e c t s of a r e d u c t i o n i n b i n d i n g r e a c t i o n pH was the hope t h a t the  55 n o n s p e c i f i c b i n d i n g would be lower at the lower pH. aspect of the experiments was b i n d i n g i n the mpfc was  successful.  g r e a t l y reduced.  The  nonspecific  This i s consistent  w i t h the f i n d i n g s of B r u i n i n k e t a l . (1984) who t h a t a drop i n pH to 6.2  reported  almost e l i m i n a t e d the b i n d i n g of  [ H ] - s p i p e r o n e t o spirodecanone s i t e s . 3  It i s also  c o n s i s t e n t w i t h r e p o r t s t h a t spirodecanone s i t e s numerous i n the f r o n t a l c o r t e x At pH 6.2,  (Howlett  are  e t a l . , 1979).  50-60% of the mpfc b i n d i n g was  dopamine r e c e p t o r s .  This  to  D2  Thus, a r e d u c t i o n i n pH g r e a t l y  i n c r e a s e d the " s i g n a l to n o i s e " r a t i o of the assay. s i g n i f i c a n t l y improves the accuracy s a t u r a t i o n s t u d i e s . The  of c o m p e t i t i o n  and  r e s u l t i n g enhanced r e s o l u t i o n of  assay i n c r e a s e s the f e a s i b i l i t y of c o m p e t i t i o n saturation analysis.  This  studies  the  and  56  Experiment Treatment  on  D2  3:  The  Dopamine  E f f e c t s of Receptors  Cortex  of  Chronic  i n the  the  Haloperidol  Medial  Prefrontal  dopamine  receptor  Rat  Introduction  Several antagonists acting  on  al.,  1979;  This  has  the  reports may  the  led  to  system  nigrostriatal  Robinson  et  psychotic  (displays dopamine  systems  very  antipsychotic metabolites  systems  similar  to  Held  the as  (Matsumoto  and  the  acute  and  the  mesocortical  mesolimbic  i n humans, some  for  in  et a l . ,  the  effects  of  and  drugs  Hoffman,  such 1984;  elicit  forms  of  1970;  Snyder,  schizophrenia 1980).  i s a l s o more s e n s i t i v e  mesolimbic activating  measured et  the  Oates,  system  abnormalities  that the  et  a l . , 1985).  responsible  (Bowers  can,  et  (Bacopoulos  fact  to  which  ED50) t h a n  drugs  the  by  (Bacopoulos  that  be  s e n s i t i v e than  dopamine  to  may  phencyclidine  Cavanaugh,  lower  system  include  a l . , 1985)  mesocortical  system Robinson  schizophrenia  i s more  states  (Griffith,  of  data  and  dopamine  related suggestion  dopamine  amphetamine  that  antipsychotic effects  a l . , 1983;  dopamine  symptoms  Pertinent  dopamine  The  the  et  suggested  their  mesocortical  Matsumoto  psychotic  as  produce  mesocortical  1979).  have  by  or  effects  increases  a l . , 1983).  nigrostriatal  in  of dopamine  Furthermore,  while  57  the  activation  systems  by a n t i p s y c h o t i c s  treatment, system  of mesolimbic  the activation  i s reported  and n i g r o s t r i a t a l  diminishes  tolerance  (Bacopoulos  Matsumoto  e t a l . , 1983; T h i e r r y  suggested  that  dopamine  system  clinically  1979;  reportedly  which  Matsumoto  studies  their  employing  have  ability  potencies  (Seeman,  binding  that  the  whereas t h e such  (Bacopoulos  et a l . ,  t h e mechanisms  through  Data  of these  dopamine  binding  receptors.  data  an i n c r e a s e  i n the pathology  i n D2  o f some  depends  on  Thus, the  their  clinical  correlated with  dopamine  have  the  that the  compounds  are highly  Experiments  have p r o v i d e d  indicate  f o r t h e D2  systems t o  effects.  techniques  1980 a n d s e e E x p e r i m e n t s  suggestion involved  D2  affinities  Radioligand  with  dopamine  beneficial  of antipsychotics  individual  since  antipsychotic  associated  influence  evidence.  property  to block  mesocortical  d o n o t show t o l e r a n c e  self-limiting  1983;  I t has been  of schizophrenia  investigated  may  radioligand  antipsychotic  the  of chronic  effects  clinically  conclusive  and Roth  e t a l . , 1983).  antipsychotics  produce  most  side  are frequently  Many  implicate  effects  t o such  e t a l . , 1984).  i n the pathology  parkinsonian-like treatment  findings  dopamine  resistant  e t a l . , 1979; Bannon  beneficial  administration  subchronic  of the mesocortical  t o be r e l a t i v e l y  these  with  dopamine  binding  their  site  1 and 2 ) .  also  l e d to the  dopamine forms  of  receptors  i s  schizophrenia  58 (Seeman, 1981). In postmortem s t u d i e s , b r a i n t i s s u e from s c h i z o p h r e n i c s has been found t o c o n t a i n a higher d e n s i t y of s u b c o r t i c a l D2 dopamine b i n d i n g s i t e s when compared t o c o n t r o l t i s s u e (Seeman, 1980).  However, s i n c e the  a d m i n i s t r a t i o n of a n t i p s y c h o t i c drugs i n c r e a s e s the d e n s i t y of s u b c o r t i c a l D2 r e c e p t o r s i n experimental al.,  animals (Owen e t  1980; Seeman, 1980), i t i s d i f f i c u l t t o determine how  much of the i n c r e a s e observed i n d r u g - t r e a t e d  schizophrenics  i s due t o the d i s e a s e and how much i s the r e s u l t of a n t i p s y c h o t i c treatment.  Unfortunately,  postmortem s t u d i e s  of drug f r e e p a t i e n t s are l i m i t e d t o very few s u b j e c t s and have produced c o n f l i c t i n g r e s u l t s .  B r a i n t i s s u e from drug  f r e e p a t i e n t s has been r e p o r t e d t o have h i g h e r  (Seeman,  1980), unchanged (Mackay, I v e r s e n , Rossor, Spokes, B i r d , A r r e g u i , Creese and Synder, 1982) or lower  (Reynolds,  R i e d e r e r , J e l l i n g e r and G a b r i e l , 1981) d e n s i t i e s of D2 dopamine r e c e p t o r s when compared t o c o n t r o l s . In 1982, M e l l e r e t a l . r e p o r t e d t h a t sulpiride-displaceable  [ H ] - s p i p e r o n e b i n d i n g i n the 3  p r e f r o n t a l c o r t e x o f the r a t i s not a f f e c t e d by treatment w i t h h a l o p e r i d o l (a dopamine r e c e p t o r a n t a g o n i s t and a commonly p r e s c r i b e d a n t i p s y c h o t i c d r u g ) .  These data  suggested t h a t i f an i n c r e a s e i n the D2 f r o n t a l  cortex  r e c e p t o r d e n s i t y of s c h i z o p h r e n i c s were documented i t c o u l d be d i s s o c i a t e d from a n t i p s y c h o t i c treatment.  Unfortunately,  M e l l e r e t a l . (1982) d i d not determine whether the b i n d i n g s i t e s d e t e c t e d by t h e i r assay procedure were, i n f a c t , D2  59  dopamine  receptors.  saturation  In  analyses.  haloperidol-induced a  Therefore, increase  haloperidol-induced  the  binding  sites  section).  Thus,  increased,  a  effect.  subchronic tissue  In  (Experiments sites  both  these  1  and  through  possible on  et  of  al.  2),  saturation  the  density  of  analysis. effect  mpfc  of  D2  [ H]-spiperone.  how  D2  receptors  chronic  treatment  masked rats  most  with  to of  this  may  have  this  postmortem receiving  several  months).  mpfc  receptor  the  method  mpfc  due  after  D2 to  of  study  these  i t became  experimental  i n the  of  sites  Therefore,  This  3  the  developed  and  been  affinity  patients  least  a  Results  tested  sites  for  by  was  of have  have  from (at  a method  the  investigate  of  weeks) whereas comes  perform  apparent (see  may  (1982)  not  may  c h a r a c t e r i z i n g the  measure  sites  the  density  treatment  to  influenced  the  (3-5  antipsychotic process  in  in affinity  Meller  did  absence  3  schizophrenics  the  the  [ H]-spiperone  although  treatment  from  chronic  for  they  i n binding  decrease  decrease  Also,  addition,  manipulations affinity  of  was  to  the  used rat  are  haloperidol.  Methods  Haloperidol solution the of  dark. stock  presented  at  a  base  dissolved  concentration  Stock  of  10  s o l u t i o n s were  s o l u t i o n were to  was  rats  as  0.5%  mg/ml a n d  prepared  diluted with  male Wistar  in a  lactic stored  monthly.  distilled  drinking  water  water  in  acid  at  4°C  in  Aliquots and light  60  protected basis of  bottles.  of pilot  The  concentration  results,  haloperidol/kg/day.  concentration for  21 w e e k s  free  access  250-350  of l a c t i c during  the  rats gained  The  hr On  from  between  3:00  from  protein The  measured  received The  housed chow.  The  colony  an  1.3-1.5  mg  equivalent  r a t s were  treated  4 per cage  with  They weighed  of the treatment  a t 2:00  and  350-450  the rate  r o o m was  g at  at which  maintained  r a t s was binding  England  Nuclear  on  a  various  final  assays  [ H]-spiperone  and from from  of specific  6.2  2 except  Amersham binding  striatum  150-250  u,g o f  as d e s c r i b e d  of  1.  mpfc  binding 3  3  from  89  f o r mpfc  Ci/mmole)  assays.  at  The  as h i g h  [ H]-spiperone 3  (New  5-450 pM f o r  d i d not produce as the  was  [ H]-spiperone.  o r Amersham  pM  i n  [ H]-spiperone  ranging  25-100  dislocation  saturation  that  concentrations  concentrations  lights  protein.  a t pH  Ci/mmole  were  i n Experiment  f o r each  reaction included  23-26  the  and the  reaction included  1 and Experiment  binding  by c e r v i c a l  as d e s c r i b e d  pooled  assayed  various  (6 h r s a f t e r  The mpfc  u,g o f s t r i a t a l  t i s s u e was  with  p.m.  p.m.  and processed  9-14  the drinking bottles  killed  a n d 6:00  o r 75-150  each  striatal  were  day o f treatment  p.m.  Each  Experiment  percent  they  laboratory  weight.  the cages  dissected  3  which  and t h e r a t s were  analysis.  Thus,  rats  on t h e  light/dark cycle.  came o n )  Tissue  Control  adjusted, consumed  haloperidol d i d not affect  the last  removed  were  the rats  g at the beginning  end.  12/12  so t h a t  acid vehicle.  to standard  the  was  a  obtained  61  from  New  England  activity  o f t h e Amersham  Throughout assay  were  However, product  the entire  procedures,  tissue) used  Nuclear.  t o determine  made  (from  tissue  treated  i n parallel.  Scatchard data)  (1/KD) o f t h e r e c e p t o r s f o r [ H ] - s p i p e r o n e 3  were  performed.  Student's  Three  Statistical  two-tailed  p r e p a r a t i o n and  and c o n t r o l  the saturation  (Bmax) o f t h e r e c e p t o r s .  specific  i t preferable.  treatment,  haloperidol  processed  the higher  independent  comparisons  rats (or  analysis  was  the a f f i n i t y  and t h e d e n s i t y replications  w e r e made  with  t-tests.  Results  Figure the  9 illustrates  haloperidol  treatment  striatal  D2 d o p a m i n e  d/f=2;  <  p  another  treatment Gamble  which  procedure  a n d Crow,  measuring 3  (Owen,  p  apparent  affinity  >  (Bmax) o f  Cross,  However,  70%  agreement  similar  that  (t=9.57; with  haloperidol  Waddington,  Poulter,  unlike  other  of haloperidol a very  most  treatment s m a l l and  reports  on insignificant  .05) h a l o p e r i d o l - i n d u c e d d e c r e a s e i n  o f t h e D2  detected.  The l a r g e  observed,  i s probably with  i s i n good a very  binding, only  d/f=2;  interfering  result  1980).  (t=2.21;  analysis  increased the density  employed  the effects  [ H]-spiperone  Scatchard  r e c e p t o r s by approximately  .02). This  study  through  sites  decrease  f o r [ H]-spiperone 3  i n affinity,  due t o r e s i d u a l  which  i s commonly  haloperidol  the binding of [ H]-spiperone 3  was  (Owen e t a l .  62  Control  a—o  B K »----• c "cu o  = m  a  x  3 0 4 2 fmoles/mg protein  =70-7 p M  D  Haloperidol B = 5 2 9 1 fmoles/mg protein m  a  x  K = 7308 p M D  Q.  E  UJ  tr u. o" z =>  o  CO  0  200 BOUND  400 (fmoles/mg  600 protein)  F i g u r e 9. S c a t c h a r d p l o t i l l u s t r a t i n g the e f f e c t s of c h r o n i c h a l o p e r i d o l a d m i n i s t r a t i o n on s t r i a t a l D2 receptors.  63 1980).  The absence of t h i s a r t i f a c t i n the present data  may  be the r e s u l t of e i t h e r the low t i s s u e c o n c e n t r a t i o n , the l o n g i n c u b a t i o n time, or both. F i g u r e 10 i l l u s t r a t e s t h a t the e f f e c t s of h a l o p e r i d o l on mpfc t i s s u e were very s i m i l a r t o those on s t r i a t a l tissue.  Thus, an approximately  50% higher d e n s i t y of D2  r e c e p t o r s was found i n the mpfc of h a l o p e r i d o l t r e a t e d r a t s (t=5.90; d/f=2; p < .03).  T h i s percent  i n c r e a s e was not  s i g n i f i c a n t l y d i f f e r e n t from t h a t found i n the s t r i a t u m (t=2.17; d/f=4; p > .05: independent t - t e s t of % i n c r e a s e s over c o n t r o l s comparing mpfc assays with s t r i a t a l  assays).  As w i t h the s t r i a t u m the medial p r e f r o n t a l c o r t e x d i s p l a y e d a s m a l l and i n s i g n i f i c a n t  (t=0.34; d/f=2; p > .05) r e d u c t i o n  i n apparent a f f i n i t y of spiperone  f o r the D2  sites.  Discussion  The present  r e s u l t s demonstrate t h a t the mpfc dopamine  r e c e p t o r s are s a t u r a b l e and t h e i r d e n s i t y i s p r e d i c t a b l e from i n d i c e s of mpfc dopamine i n n e r v a t i o n . D2 s i t e s s a t u r a t e with approximately  Thus, the mpfc  the same a f f i n i t y as  s t r i a t a l s i t e s and the c o n c e n t r a t i o n of D2 r e c e p t o r s i n the mpfc i s approximately  2% of t h a t i n the s t r i a t u m .  The r e s u l t s a l s o demonstrate t h a t c h r o n i c exposure t o the D2 dopamine r e c e p t o r a n t a g o n i s t , h a l o p e r i d o l , i n c r e a s e s D2 r e c e p t o r b i n d i n g i n the mpfc of the r a t .  These data are  the f i r s t t o i n d i c a t e t h a t mpfc D2 r e c e p t o r s can be  64  Control  o o  B  25  a>  o k_ E  UJ UJ  rr u. o" z  z>  O  x  = 9-3 fmoles/mg protein  D  Haloperidol  20 -  B K  a  O)  a  K = 62.2 p M  X +-»  m  m  D  a  x  = 13.8 fmoles/mg p r o t e i n  = 65 4 p M  15  10  5 -  03 0  2  4  BOUND  6  8  10  (fmoles/mg  12  14  16  protein)  Figure 10. Scatchard plot i l l u s t r a t i n g the e f f e c t s of chronic haloperidol administration on mpfc D2 receptors.  65  e x p e r i m e n t a l l y manipulated. due  The  e f f e c t i s l a r g e and  clearly  t o an i n c r e a s e i n the d e n s i t y of r e c e p t o r s . As noted i n the I n t r o d u c t i o n to t h i s s e c t i o n , b r a i n  t i s s u e from s c h i z o p h r e n i c s has been found to c o n t a i n a higher d e n s i t y of s u b c o r t i c a l D2 dopamine r e c e p t o r s compared t o c o n t r o l t i s s u e (Seeman, 1980). of t h i s f i n d i n g i s complicated  However, i n t e r p r e t a t i o n  by r e p o r t s t h a t a n t i p s y c h o t i c  treatment can i n c r e a s e the d e n s i t y of s u b c o r t i c a l D2 dopamine s i t e s i n experimental Seeman, 1980).  animals (Owen e t a l . , 1980;  In c o n t r a s t , M e l l e r e t a l . (1982) r e p o r t e d  t h a t h a l o p e r i d o l treatment d i d not sulpiride-displaceable  increase  [ -%J-spiperone  p r e f r o n t a l c o r t e x of the r a t .  b i n d i n g i n the  These data r a i s e d the  p o s s i b i l i t y t h a t any s c h i z o p h r e n i a r e l a t e d changes i n f r o n t a l c o r t e x D2  r e c e p t o r s c o u l d be d i s s o c i a t e d from the  e f f e c t s of a n t i p s y c h o t i c treatment. experimental  d e s i g n employed by M e l l e r e t a l . (1982)  inadequate i n s e v e r a l r e s p e c t s section).  The  present  these problems. any  However, the  (see I n t r o d u c t i o n to t h i s  experiment was  Unfortunately,  designed  to circumvent  the r e s u l t s i n d i c a t e t h a t  i n c r e a s e i n the d e n s i t y of f r o n t a l c o r t e x D2  r e c e p t o r s , which may  was  dopamine  be demonstrated i n f u t u r e s t u d i e s of  s c h i z o p h r e n i c s , cannot be e a s i l y d i s s o c i a t e d from the e f f e c t s of a n t i p s y c h o t i c treatment. The  h a l o p e r i d o l - i n d u c e d i n c r e a s e i n mpfc D2  r e c e p t o r s may  dopamine  be r e l a t e d t o the syndrome of t a r d i v e  p s y c h o s i s which i s observed i n some s c h i z o p h r e n i c p a t i e n t s  66  who are t r e a t e d w i t h a n t i p s y c h o t i c s (Chouinard 1980).  and Jones,  Thus, i f mpfc D2 dopamine r e c e p t o r s are i n v o l v e d i n  psychoses (see I n t r o d u c t i o n t o t h i s s e c t i o n ) then an antipsychotic-induced  i n c r e a s e i n these r e c e p t o r s may be  r e s p o n s i b l e f o r the r e t u r n of p s y c h o t i c symptoms w i t h p a t i e n t s r e c e i v i n g a n t i p s y c h o t i c s (Chouinard 1980).  observed and Jones,  67  Experiment Dopamine  4:  The  Receptors  E f f e c t s of  i n the  Footshock  Medial  Stress  on  P r e f r o n t a l Cortex  D2 of  the  Rat  Introduction  Dopaminergic schizophrenia psychosis (Angrist and of  1974;  to  sites  efficacy 1980).  i n reducing These  dopaminergic some brain  forms  tissue  increase  data  may  has  that be  schizophrenic  dopamine  agonist-induced  dopamine  agonists, (Crow,  and  1982).  and  of  1977).  an  their  activity  postsynaptic  D2  psychosis,  most  (Seeman,  pathology  postmortem  are most  s e n s i t i v e to  D2  clinical  g e n e r a l l y do  that  potency  in functional  i n the  associated  a  Sherwin  dopaminergic  increase  While  elicit  The  schizophrenia  involved  symptoms  can  of  schizophrenia  dopaminergic  been  positive  treatment  of  symptoms  Sathananthan,  Gershon,  schizophrenics  1980)  of  antagonists  suggest  i n presumed  Seeman,  and  schizophrenia. from  forms  correlated with  i n presynaptic  increase  1980;  as  1977)  Angrist,  symptoms  activity  of  some  Angrist  i s highly  exacerbate  Gershon,  1970;  a n t i p s y c h o t i c drugs  binding  an  similar  Gershon,  Gershon,  can  ( A n g r i s t and  very and  agonists  studies not  most  a  of  reveal  (Crow,  binding with  of  sites  1980), (Crow,  subclass similar  exacerbated dopamine  an  of  to by  antagonist  68  Heredity  appears  schizophrenia much  lower  indicates  theories  that  have  1981;  100%  proposed  become  of  dopamine Data  on  and  stress  eliciting  a  highly  from  et  as  animal stress  cells  can  The the  in  of  rats  Bustos, et  and  metabolism  schizophrenics  have  data  that  Spring,  dopaminergic (Dohrenwend ("Brief  with  and  Reactive  s i m i l a r to  those  abnormal  Spokes, to  and  various  activate  a l . , 1978;  (Matsumoto  a l . , 1979).  sufficient  1981;  system  abnormality and  has  drug  et  and  1982),  Furthermore, (Ingvar,  atypical neuronal  1982)  primates  including  regional suggest  activity  been  1979)  antipsychotic  Roth,  et  in  Roth,  a l . , 1983)  of  cortex  Szentendrei  dopamine  Bird  forms  the  prefrontal  primary  Redmond  to  associated  the  et  tolerance  (Bacopoulos glucose  the  (Bacopoulos,  administration  i s very  prefrontal cortex of  participate  etiological  with  syndrome  indicate  innervating  site  twins,  predisposed  As  selectively  Fadda  the  1980).  studies  a l . , 1976;  i t s lack  (Bacopoulos,  (Williams,  of  i s reportedly  most  function  also  Egri,  1982).  of  However,  when e x p o s e d  schizophrenia  schizophrenia based  Shields,  etiology  monozygotic  must  genetically  Dohrenwend  of  of  number  which  1980).  proposed  A  1980)  dopaminergic  al.,  factors  the  1982).  rate  (Williams,  environmental  (Thierry  and  capable  Psychosis") forms  that  in  Shields,  1982).  environmental  1981)  and  factor  schizophrenic  1972;  Gottesman  Egri,  a  concordance  Shields,  (Pollin,  agonists,  be  environmental  and  individuals stress  (Gottesman  than  (Gottesman  to  in  blood that the  man flow  69  frontal  cortex.  (footshock  or  dopaminergic (Antelman, Maier,  to  dopamine As  may  be  what  the  Kocan,  1 and  the  tissue  cortex  1979).  the was  and  prefrontal  footshock stress  2,  subsequent  MacLennan  presented i n Experiment  and  of  i n the r a t  1980;  Antelman,  i n t h e mpfc  stress  potentiation  through  and  effects  receptors  i n Experiments  pH  mediated  (Eichler  that  stereotypy  B l a c k and  experiments  determine  suggest  agonist-induced  system  The  data  tail-pinch)-induced  Eichler,  1983)  dopamine  Recent  4 were  has  on  striatum assayed  designed  D2  of  the r a t .  a t pH  6.2  and  7.9.  The  Effects  of  Footshock  Stress  as  Measured  a t pH  6.2  Methods  Male, for  3-4  been  Wistar  weeks  rats  prior  reported  to  (300-400 the  t o enhance dopamine  Lisoprowski,  G l o w i n s k i and  under 8:00  a a.m.  12/12 The  session  (one  between  12:00  a to  hr  .5  BRS-Foringer  p.m.  3 mA  and  SGS-003  a BRS-Foringer  rats  3:30 shock  operant  because  on  (Blanc,  Herve,  Simon,  1980). The  received  a  i n every  p.m.  The  Rats  lights 30  shock  generator which  chamber.  Between  was was 3:00  of  housed  came o n  sec  has  indices  were  minute 2.5  housed  isolation  stress  cycle.  shock  individually  of  Tassin,  light/dark  sec,  effect  activity  shocked  were  experiment  the  mesocortical  g)  at  footshock interval) delivered connected p.m.  and  by  70  5:30  p.m.  controls  the were  Experiment has  been  f o l l o w i n g day, killed  1.  used  This  and  dopamine  et  a l . , 1980;  MacLennan  assayed  as  Five brain  described  of  the  as  and  was  employed  Maier,  1983). 3  8-10  r a t s was  naive  because i t potentiation  The  (pH  the  in  stereotypy  (Antelman  tissue  was  6.2).  saturation analyses from  and  stress-induced  i n Experiment  Tissue  rats  described  agonist-induced  independent  region.  saturation  interval  i n studies  subsequent  shocked  dissected  time  of  the  were  run  for  each  pooled  for  each  analysis.  Results  Figure mpfc  11  tissue.  is a As  illustrated  showed  slightly  mpfc.  The  increase  effect  was  due  an  increase  more  to  effect  induced  of  change  concentrations concomitantly  D2 was  an  i n the  [%]-spiperone. the  Scatchard  too  was  of  assayed  receptor  obtained  from  footshocked  rats  binding  to determine  i n the the  observed  density  receptors  i f f o r each  [ H]-spiperone  the data  figure,  c a l c u l a t e d as  i n binding 3  of  the  small  increase  However,  of  by  dopamine  affinity  shock  plot  at  whether  of  this  receptors  for  saturation analysis, the  average  the  different  ( c a l c u l a t e d as  c o n t r o l s ) , then  i n the  shocked  shock  percent  rats  were  of  or  71  BOUND  (fmoles/mg  protein)  F i g u r e 11. Scatchard p l o t i l l u s t r a t i n g the e f f e c t s footshock on mpfc D2 r e c e p t o r s assayed a t pH 6.2.  of  72  found  to display  (t=2.97; No  d/f=4;  a  significant  increase  i n D2  binding  p<.05).  e f f e c t of  footshock  receptors  i n striatal  Figure  i s a Scatchard  12  13%  stress  tissue  was  observed  (t=.92; d/f=2;  plot  on  D2  p>.05).  of the s t r i a t a l  data.  Discussion  The  footshock  stress  increase  i n t h e amount  accuracy  of the present  would  expect  approaches indicate, increase  t h e amount  mechanisms However, studies  the data  sensitive  to stress.  From  a clinical  stress  may  contribute  psychoses  by  addition,  the present  further It  may  also  lead  striatal  t h e mpfc  to the onset t h e number  cortex  that  appear to  selectively The (possible  Discussion).  t h e dopamine  turnover  i s particularly  the data of  D2  suggest  that  schizophrenia-like  o f D2  paradigm should  to a better  data  can  the  one  binding  e f f e c t i s unknown  perspective,  of frontal  D2  i n t h e mpfc.  with  Given  tissue,  The  stress  i n the General  that  increasing  studies  binding  significant  i n t h e mpfc.  i n striatal  are consistent  which suggest  but  i n t h e mpfc.  for this  are discussed  small  footshock  o f D2  responsible  with  a change  that  a  binding  assay  of that  therefore,  mechanism  o f D2  to detect  the size  induced  mpfc  receptors.  prove  useful  binding  site  understanding  of the  In  i n dynamics.  Control B = 266-5 fmoles/mg protein m  BOUND  a  x  (f m o l e s/m g p r o t e i n )  F i g u r e 12. S c a t c h a r d p l o t i l l u s t r a t i n g the e f f e c t s f o o t s h o c k on s t r i a t a l D2 r e c e p t o r s assayed a t pH 6.  role  of stress  mental  i n the etiology of dopaminergically  diseases.  The  The  E f f e c t s of Footshock  t i s s u e from  pH  6.2  of  resolution at this  pH  6.2  while  because  t h e D2 that  on  D2  to  t h e mpfc.  related  receptors. mpfc,  affinity  may  D2  which  of  observed to  when is  observed.  2).  at  degree  The r e s u l t s  i s not.  i s increased  These  t h e mpfc  at  results  stress  effect  that  i s particular  the results  also  raise  may  b e , t o some  o f dopamine  compared  degree,  receptor  to the s t r i a t a l  a subpopulation for this  receptor  the  o f D2  area's  agonists,  may  agonists D2  receptors i n higher  be  a f f e c t e d by s t r e s s .  receptor 6.2.  be p r e d i c t e d assayed  a greater  7.9  property  i s responsible  demonstrated  a t pH  assayed  i n t h e mpfc  underlying  affinity  receptors  a t pH  i n Experiment agonists  increase  that  2, t h e g r e a t e r  f o r mpfc  Therefore,  the stress-induced  could  binding  some  first  possesses  the stress effect  f o r dopamine  dopamine  r a t s was  i n the striatum  Specifically,  differentially As  t h e D2  Consequently, that  a s M e a s u r e d a t pH  (see Experiment  involve  to the higher  t h e mpfc  pH  assay  t h e mechanism  binding  possibility  the  binding  Stress  footshocked  the present  indicate that  suggest  for  related  D2  i f this i n mpfc  the stress effect  7.9 w h e r e  In accordance  the agonist with  this  receptors  variable D2  affinity i s not  i s related  binding w o u l d be affinity  possibility,  then i t different difference the next  75 experiment was  designed  to t e s t the e f f e c t s of s t r e s s on  r e c e p t o r s assayed at pH  D2  7.9.  Methods  The  experiment was  performed as i n the  preceding  s e c t i o n except t h a t the b i n d i n g r e a c t i o n s were run at pH and a lower range of 50 pM)  was  [ H]-spiperone concentrations 3  used because at pH 7.9  higher a f f i n i t y f o r D2 s p e c i f i c binding.  7.9  (20 pM  [ H ] - s p i p e r o n e has 3  r e c e p t o r s and a lower percent  to  a of  Three independent r e p l i c a t i o n s were run  f o r each b r a i n r e g i o n .  Results  F i g u r e 13 i l l u s t r a t e s t h a t the footshock amount of D2 b i n d i n g i n the mpfc. i n d i c a t e s t h a t the d e n s i t y of D2 i n c r e a s e d by approximately [ H ] - s p i p e r o n e was 3  The  receptors  the decrease i n a f f i n i t y  analysis  (Bmax) was  30% of t h a t receptor density  (t=6.42; d/f=2; p<.025) as  was  (t=5.93; d/f=2; p<.03).  No changes were d e t e c t e d rats.  the  w h i l e the a f f i n i t y of  i n c r e a s e i n D2  statistically significant  of footshocked  Scatchard  reduced t o approximately  observed f o r c o n t r o l s . was  100%  The  increased  i n the s t r i a t a l D2  F i g u r e 14 i l l u s t r a t e s these  receptors results.  76  BOUND  (fmoles/mg  protein)  F i g u r e 13. Scatchard p l o t i l l u s t r a t i n g the e f f e c t s f o o t s h o c k on mpfc D2 r e c e p t o r s assayed at pH 7.9.  of  77  BOUND  (fmoles/mg  protein)  F i g u r e 14. Scatchard p l o t i l l u s t r a t i n g the e f f e c t s of footshock on s t r i a t a l D2 r e c e p t o r s assayed a t pH 7.9.  78  Discussion  Footshock in  the  about  mpfc. 100%  of  size  increased of  determine  the  as  the  Experiment  The  the  then  Scatchard appears  1).  I t was,  to  extrapolate  the  exact  The  apparent  of  resulting  from  substance  which  sites. effect  It  the a  reductions subcortical lesions Mouchet,  a  stressed  real  receptor  (Feuerstein,  Although  than  of  change have  in  Demenge,  amphetamine  a l l these  binding  is  increased to  lower,  limit  accurate  the  increase  in  increase  could  not  the  3  be  an  artifact  i n some  endogenous  [ H]-spiperone for  the  3  the  that D2  Caron,  (Howlett  the  increases by  and  may  in  Guerin  Nahorski,  antipsychotics  Similar  presynaptic  Barrette,  and  1979),  (Owen e t a l . ,  reflect  D2  affinity  receptors.  induced  effects  be  [ H]-spiperone for  accompanied  density  cannot  concentrations  is typical  increase  the  at  mpfc  specific  although  the  receptors  necessary  the  r a t s may  with  c h r o n i c a l l y administered  1980).  of  p o s s i b l e , however,  in affinity  1981),  of  stress-induced  i s also  D2  size  affinity  competes  reflects  more  Consequently,  determined.  receptors  i n the  higher  therefore, to  D2  i s estimated  3  conclusively  lower  of  [ H]-spiperone concentration  analyses.  large,  at  percent  3  and  density  assay  binding  [ H]-spiperone concentrations  range  and  D2  the  increase  control values.  3  (see  D2  this  [ H]-spiperone because  decreases  the  The  of  accurately  clearly  other  79 f a c t o r s , i t i s a l s o p o s s i b l e t h a t "new" s t i l l developing,  have a  spiperone.  experiments of t h i s s e c t i o n were prompted by  i d e a t h a t the i n c r e a s e i n the number of mpfc D2 may  while  or while o n l y t e m p o r a r i l y induced,  lower a f f i n i t y f o r The  receptors  the  receptors  be r e l a t e d to the higher a f f i n i t y of dopamine r e c e p t o r  a g o n i s t s f o r mpfc D2  receptors.  The present data  consistent with that p o s s i b i l i t y .  The  stress effect i s  l a r g e r at the pH at which the dopamine r e c e p t o r a f f i n i t y d i f f e r e n c e i s observed ( i . e . pH The  are  agonist  7.9).  s t r e s s - i n d u c e d i n c r e a s e i n r e c e p t o r s seems  counterintuitive. p r e f r o n t a l cortex  S t r e s s i n c r e a s e s dopamine r e l e a s e i n the ( T h i e r r y e t a l . , 1976).  Increased  agonist  s t i m u l a t i o n i s g e n e r a l l y thought t o reduce r e c e p t o r number. However, the l i t e r a t u r e i n d i c a t e s t h a t r e c e p t o r r e g u l a t i o n i s more complicated  than suggested by t h i s simple  view.  Thus, i n v i v o treatment w i t h dopamine r e c e p t o r  agonists  has been shown i n some cases t o i n c r e a s e dopamine-related receptor binding  (eg. Howlett and Nahorski,  and Fagan, 1979;  Robertson, 1983).  1979;  Taylor,  Ho  I t has a l s o been  r e p o r t e d t h a t i n v i t r o i n c u b a t i o n of dopamine or dopamine r e c e p t o r a g o n i s t s w i t h t i s s u e homogenates can d r a m a t i c a l l y i n c r e a s e dopamine-related b i n d i n g Seeman, 1978;  Robertson, 1980).  dopamine r e l e a s e d by s t r e s s may  (McManus, H a r t l e y  and  I t i s possible that i n t e r a c t with other dopamine  r e c e p t o r s which when a c t i v a t e d i n c r e a s e the number of receptors.  For example, r e c e n t data suggest t h a t  D2  80 s t i m u l a t i o n of DI b i n d i n g s i t e s can i n c r e a s e b i n d i n g at sites may  (Dumbrille-Ross  e t a l . , 1985).  D2  In a d d i t i o n , s t r e s s  promote the r e l e a s e of other t r a n s m i t t e r s which c o u l d  cause an i n c r e a s e i n D2 b i n d i n g .  For example,  c h o l e c y s t o k i n i n has been r e p o r t e d to i n c r e a s e the number of D2  r e c e p t o r s when administered  (Dumbrille-Ross  i n v i v o or i n v i t r o  and Seeman, 1984).  Research on a d r e n e r g i c r e c e p t o r s has t h a t r e c e p t o r i n t e r a c t i o n s may  supported  the  be r e s p o n s i b l e f o r  stress-induced increases i n receptors.  I t has been found  t h a t i n v i t r o treatment of r a t c e r e b r a l c o r t i c a l s l i c e s B-adrenergic  a g o n i s t s can decrease 3-adrenergic  b i n d i n g but i n c r e a s e c ^ - a d r e n e r g i c U'Prichard  and  Immobilization  Enna, 1980;  receptor binding  (Maggi,  Kitamura and Nomura, 1985).  e f f e c t s i n the r a t c e r e b r a l c o r t e x  noradrenaline  (U'Prichard  Thus, s t r e s s may  produce an i n c r e a s e i n a ^ - a d r e n e r g i c  these  and  release c o r t i c a l  which i n t e r a c t s w i t h B-adrenergic  analogous mechanism may  with  receptor  s t r e s s has been shown t o produce both  Kvetnansky, 1980).  idea  receptors  receptors.  to  An  be r e s p o n s i b l e f o r the p r e s e n t l y  reported s t r e s s e f f e c t . I t i s a l s o p o s s i b l e t h a t the i n c r e a s e i n mpfc  D2  r e c e p t o r s i s a compensatory response r e s u l t i n g from a s t r e s s - i n d u c e d decrease i n p r e s y n a p t i c dopamine f u n c t i o n d u r i n g the 28 hour i n t e r v a l between the footshock  session  and the t i s s u e p r e p a r a t i o n .  to suggest  There i s no evidence  t h a t such a decrease i n p r e s y n a p t i c f u n c t i o n f o l l o w s  footshock.  U n t i l evidence  i s a v a i l a b l e t o the c o n t r a r y ,  however, such a mechanism remains a p o s s i b i l i t y . As noted above, the dopamine r e c e p t o r s t h a t are i n c r e a s e d by s t r e s s may a l s o d i s p l a y a high a f f i n i t y f o r dopamine r e c e p t o r a g o n i s t s as w e l l as a pH dependent a f f i n i t y f o r [ H]-spiperone. 3  These p r o p e r t i e s were not  observed f o r the remainder of the mpfc r e c e p t o r s or the s t r i a t a l receptors.  I t i s p o s s i b l e t h a t these  receptors  a l s o q u i t e d i f f e r e n t from most dopamine r e c e p t o r s i n t h a t they i n c r e a s e i n number when s t i m u l a t e d by a g o n i s t s .  82  General  Discussion  The data i n t h i s t h e s i s i n d i c a t e t h a t D2 dopamine r e c e p t o r s a r e present  i n the mpfc of the r a t .  The  a f f i n i t i e s w i t h which v a r i o u s dopamine a n t a g o n i s t s b i n d t o these r e c e p t o r s are h i g h l y c o r r e l a t e d w i t h t h e i r f o r D2 r e c e p t o r s i n the s t r i a t u m .  affinities  I n c o n t r a s t , when  measured a t pH 7.9, dopamine r e c e p t o r a g o n i s t s d i s p l a y a higher a f f i n i t y receptors.  f o r mpfc D2 r e c e p t o r s than f o r s t r i a t a l D2  T h i s d i f f e r e n c e i s not observed a t pH 6.2.  Chronic h a l o p e r i d o l produced a l a r g e i n c r e a s e i n mpfc and s t r i a t a l D2 r e c e p t o r s measured a t pH 6.2.  Footshock s t r e s s  produced o n l y a s m a l l i n c r e a s e i n mpfc D2 r e c e p t o r s a t pH 6.2.  However, when measured a t pH 7.9, footshock  stress  produced a l a r g 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 and a l a r g e decrease i n a f f i n i t y of the r e c e p t o r s f o r [ H ] - s p i p e r o n e . 3  The s t r e s s d i d not a f f e c t D2 r e c e p t o r b i n d i n g i n the s t r i a t u m when measured a t e i t h e r pH 7.9 or pH 6.2. The General  D i s c u s s i o n of t h i s t h e s i s addresses f o u r  t o p i c s which a r e r e l a t e d t o the present data as a whole. First,  a hypothesis  i s presented  which attempts t o e x p l a i n  how the l i g a n d b i n d i n g c h a r a c t e r i s t i c s of D2 r e c e p t o r s may be r e l a t e d t o the manner i n which these r e c e p t o r s a r e a f f e c t e d by experimental dopamine a u t o r e c e p t o r s present d a t a .  manipulations.  Second, r e s e a r c h on  i s examined i n the context of the  T h i r d , p o s s i b l e i n t e r p r e t a t i o n s of  83  pH-dependent b i n d i n g a r e d i s c u s s e d . for  Fourth, the p r o s p e c t s  f u t u r e r e s e a r c h on mpfc D2 r e c e p t o r s are d i s c u s s e d .  A Hypothesis R e l a t i n g the B i n d i n g C h a r a c t e r i s t i c s of D2 Dopamine Receptors With T h e i r Response t o V a r i o u s Experimental  Manipulations  As d i s c u s s e d i n Experiment 4, the data i n t h i s t h e s i s r a i s e the p o s s i b i l i t y  t h a t the footshock-induced  changes i n  mpfc D2 r e c e p t o r s may be r e l a t e d t o the higher a f f i n i t y o f dopamine r e c e p t o r a g o n i s t s f o r mpfc D2 r e c e p t o r s compared t o s t r i a t a l D2 r e c e p t o r s .  Thus, the s t r e s s e f f e c t i s much  l a r g e r when measured a t the pH a t which the dopamine r e c e p t o r a g o n i s t a f f i n i t y d i f f e r e n c e i s observed ( i . e . pH 7.9).  The footshock  e f f e c t and the d i f f e r e n c e i n a g o n i s t  a f f i n i t y may be independent e f f e c t s which d i s p l a y s i m i l a r pH dependency. two  However, i t i s tempting t o s p e c u l a t e on how the  e f f e c t s may be r e l a t e d .  hypothesis  A c c o r d i n g l y , the f o l l o w i n g  i s proposed.  Perhaps the D2 r e c e p t o r p o p u l a t i o n measured i n the present  assays i s composed o f a t l e a s t two d i f f e r e n t forms  of D2 r e c e p t o r s .  One type  ( a r b i t r a r i l y designated  here as  the type A r e c e p t o r ) has a higher a f f i n i t y f o r dopamine r e c e p t o r a g o n i s t s than does the other type of r e c e p t o r ( a r b i t r a r i l y designated  here as the type B r e c e p t o r ) .  The  mpfc has a h i g h e r type A t o type B r a t i o .  At pH 7.9 both  type A and type B r e c e p t o r s are d e t e c t e d .  However, the D2  r e c e p t o r p o p u l a t i o n d e t e c t e d a t pH 6.2 i s p r i m a r i l y composed  84  of type B r e c e p t o r s .  Therefore,  at pH  a g o n i s t s d i s p l a y a higher a f f i n i t y compared to the s t r i a t a l D2  the D2  f o r the mpfc D2  The  receptors a  d i f f e r e n c e i n dopamine  i s not observed at pH 6.2  receptor population detected  p r i m a r i l y type  dopamine r e c e p t o r  r e c e p t o r s because the mpfc has  h i g h e r type A t o type B r a t i o . receptor agonist a f f i n i t y  7.9  because  i n both b r a i n r e g i o n s i s  B.  I t i s proposed t h a t t h e d e n s i t y of type A r e c e p t o r s i n c r e a s e s f o l l o w i n g an i n c r e a s e i n s y n a p t i c dopamine concentrations.  In a d d i t i o n , the a f f i n i t y of type  A  r e c e p t o r s f o r [ H ] - s p i p e r o n e i s reduced f o l l o w i n g an 3  i n c r e a s e i n s y n a p t i c dopamine c o n c e n t r a t i o n s . r e c e p t o r s are much l e s s s e n s i t i v e , or  Type B  completely  i n s e n s i t i v e , to such changes i n dopamine c o n c e n t r a t i o n . pH 7.9,  footshock  s t r i a t a l D2  may  a f f e c t mpfc D2  r e c e p t o r s but  r e c e p t o r s because footshock  At  not  selectively  i n c r e a s e s dopamine r e l e a s e (and t h e r e f o r e s y n a p t i c dopamine concentrations)  i n the mpfc ( T h i e r r y e t a l . , 1976).  Such  i n c r e a s e d s y n a p t i c dopamine c o n c e n t r a t i o n s produce the above mentioned changes i n mpfc type A r e c e p t o r s . r e c e p t o r s have very l i t t l e at pH 6.2  and,  experimental  The  present  manipulations  concentrations  i f any e f f e c t on b i n d i n g  t h e r e f o r e , the footshock  s m a l l e r at t h i s pH.  Type A  e f f e c t s are much  s p e c u l a t i o n s suggest t h a t  which i n c r e a s e s y n a p t i c dopamine  i n the s t r i a t u m should be able to i n c r e a s e  the number of type A r e c e p t o r s i n t h a t r e g i o n and their affinity  results  for [ H]-spiperone. 3  decrease  When assayed at pH  7.9  85 these changes would be expected to produce an i n c r e a s e i n d e t e c t e d D2 affinity  r e c e p t o r s and a decrease i n t h e i r apparent  for [ H]-spiperone.  A s i n g l e i n j e c t i o n of a l a r g e  3  dose of amphetamine, which should i n c r e a s e s y n a p t i c concentrations  of dopamine i n the s t r i a t u m , has  r e p o r t e d to produce (at pH 7.8)  been  these expected e f f e c t s on  r e c e p t o r s i n the s t r i a t u m (Howlett  and Nahorski,  D2  1979).  In summary, the higher a f f i n i t y of dopamine r e c e p t o r a g o n i s t s f o r mpfc D2 r e c e p t o r s compared to s t r i a t a l  D2  r e c e p t o r s may  A  be due  to a higher p r o p o r t i o n of type  r e c e p t o r s i n the mpfc. footshock may receptors.  be due  In a d d i t i o n , the e f f e c t s of  to s e l e c t i v e changes i n type A  I t i s a l s o p o s s i b l e t h a t both of these phenomena  are much l e s s pronounced at pH  6.2  r e c e p t o r s are d e t e c t e d at t h a t  pH.  Type A r e c e p t o r s may (Creese,  because many l e s s type  A  be the same as D2[high] r e c e p t o r s  S i b l e y and L e f f , 1984).  However, i t i s a l s o  p o s s i b l e t h a t they are o n l y a subset of the D2[high] r e c e p t o r p o p u l a t i o n which responds to manipulations  experimental  as d e s c r i b e d above.  Dopamine "Autoreceptor"  Roth and c o l l e a g u e s  Research  (Bannon, Michaud and Roth,  Bannon, Reinhard, Bunney and Roth, 1982; Roth, 1986)  have p r o v i d e d data suggesting  1981;  Galloway, Wolf  and  t h a t the  dopaminergic t e r m i n a l s i n the mpfc l a c k dopamine s e n s i t i v e  86 r e c e p t o r s t h a t i n h i b i t dopamine s y n t h e s i s modulating " a u t o r e c e p t o r s " ) .  ( i . e . synthesis  Thus, when the impulse flow of  mesolimbic or n i g r o s t r i a t a l dopaminergic c e l l s i s blocked e i t h e r by gamma-butyrolactone (GBL)  or axotomy, an  increase  i n dopamine s y n t h e s i s i s observed i n the s t r i a t u m and olfactory tubercle.  T h i s e f f e c t i s b e l i e v e d to be  r e s u l t of l e s s autoreceptor  s t i m u l a t i o n due  r e l a t e d r e l e a s e of dopamine.- The attenuated  the  the  to l e s s  impulse  increased synthesis i s  by the a d m i n i s t r a t i o n of low,  "autoreceptor"  doses of dopamine r e c e p t o r a g o n i s t s which are thought to produce t h i s a t t e n u a t i o n by a c t i n g on the a u t o r e c e p t o r s the mesolimbic and n i g r o s t r i a t a l c e l l s . and c o l l e a g u e s f i n d t h a t n e i t h e r GBL  of  In c o n t r a s t , Roth  nor axotomy are able to  i n c r e a s e dopamine s y n t h e s i s i n m e s o c o r t i c a l dopamine c e l l s (Bannon e t a l . , 1981;  Galloway e t a l . , 1986).  t h a t a l a c k of " a u t o r e c e p t o r s "  They conclude  c o u l d be r e s p o n s i b l e f o r the  above mentioned response c h a r a c t e r i s t i c s of the dopaminergic c e l l s have presented  (Bannon and Roth, 1983).  conflicting results  M e l i s , Fadda and Gessa, 1982; and R o s s e t t i , 1984).  However, o t h e r s  ( A r g i o l a s , Mereu, S e r r a ,  Fadda, Gessa, Marcou, Mosca  These authors  have r e p o r t e d t h a t  i n c r e a s e s dopamine s y n t h e s i s i n the mpfc and "autoreceptor"  mesocortical  that  GBL  low,  doses of dopamine r e c e p t o r a g o n i s t s reduce  mpfc dopamine s y n t h e s i s .  The  experimental  paradigms of Roth  and c o l l e a g u e s are s i m i l a r to those employed by Fadda e t a l . (1984).  Therefore,  i t is difficult  groups r e p o r t such d i f f e r e n t  to e x p l a i n why  results.  these  two  87 Both Fadda e t a l . (1984) and Galloway e t al.,(1986) determined dopamine s y n t h e s i s by measuring the accumulation of mpfc DOPA f o l l o w i n g DOPA decarboxylase  inhibition.  They  made the assumption t h a t t h i s measure r e f l e c t s dopamine synthesis.  T h i s was based on s t u d i e s of s u b c o r t i c a l  dopamine which were conducted i n b r a i n regions where the dopamine i n n e r v a t i o n i s much more dense than i n the mpfc. I t i s p r e s e n t l y suggested t h a t the mpfc DOPA  accumulation  measured by Fadda e t a l . (1984) may p r i m a r i l y r e f l e c t noradrenaline  s y n t h e s i s i n the n o r a d r e n e r g i c  i n n e r v a t e the mpfc. accumulation  t e r m i n a l s which  Fadda e t a l . (1984) r e p o r t t h a t DOPA  i s approximately  equal  i n the mpfc and the  d o r s o l a t e r a l f r o n t a l c o r t e x , an area which r e c e i v e s much l e s s dopaminergic i n n e r v a t i o n than the mpfc. these  r e s u l t s by s u g g e s t i n g  They e x p l a i n  t h a t most dopamine i n the  f r o n t a l cortex i s synthesized i n noradrenergic which are known t o be present c o r t e x t o the same degree.  terminals  i n both areas of the f r o n t a l  T h i s suggestion  i s incompatible  w i t h the many r e p o r t s t h a t l e s i o n s of the n o r a d r e n e r g i c i n p u t t o the mpfc do not reduce mpfc dopamine  concentrations  ( T h i e r r y e t a l . , 1973b; T i s s a r i e t a l . , 1979; C a r t e r and Pycock, 1980; Westerink and De V r i e s , 1985).  Therefore,  s i n c e DOPA i s a l s o a p r e c u r s o r o f n o r a d r e n a l i n e , al.'s  Fadda e t  (1984) method o f measuring mpfc dopamine s y n t h e s i s may  a c t u a l l y measure the r a t e of n o r a d r e n a l i n e noradrenergic  terminals.  s y n t h e s i s i n mpfc  Some u n i d e n t i f i e d d i f f e r e n c e i n  the paradigms of Roth's group and Fadda's group (perhaps  88 d i f f e r e n c e s i n t i s s u e p r e p a r a t i o n , t i s s u e d i s s e c t i o n or DOPA d e t e c t i o n ) may have l e d Fadda e t a l . (1984) t o d e t e c t mpfc noradrenaline  s y n t h e s i s not d e t e c t e d by Roth's group.  Fadda e t a l . (1984) a l s o assumed t h a t the e f f e c t s  they  observed were due t o changes i n dopamine s y n t h e s i s i n mpfc dopaminergic t e r m i n a l s because the low c o n c e n t r a t i o n s of dopamine r e c e p t o r a g o n i s t s w i t h which they reduced mpfc DOPA accumulation  were assumed t o . a f f e c t o n l y a u t o r e c e p t o r s on  dopaminergic  cells.  The present demonstration o f the h i g h a f f i n i t y of dopamine r e c e p t o r a g o n i s t s f o r mpfc r e c e p t o r s suggests an a l t e r n a t i v e explanation.  Thus, the GBL-induced blockade of  impulse flow i n m e s o c o r t i c a l dopamine c e l l s may reduce mpfc dopamine r e l e a s e .  T h i s may i n c r e a s e DOPA accumulation i n  mpfc n o r a d r e n e r g i c  t e r m i n a l s p o s s e s s i n g dopamine r e c e p t o r s  through which dopamine t o n i c a l l y i n h i b i t s s y n t h e s i s i n the mpfc n o r a d r e n e r g i c 15).  noradrenaline  terminals  (see F i g u r e  I f t h i s i s the case, then the GBL-induced i n c r e a s e i n  mpfc DOPA accumulation noradrenaline otherwise  r e f l e c t s the i n c r e a s e d s y n t h e s i s of  i n mpfc n o r a d r e n e r g i c  occur  t e r m i n a l s t h a t would  i f DOPA decarboxylase  The low, " a u t o r e c e p t o r "  concentrations  were not i n h i b i t e d . of dopamine r e c e p t o r  a g o n i s t s may reduce mpfc DOPA accumulation  by a c t i v a t i n g the  dopamine r e c e p t o r s on the mpfc n o r a d r e n e r g i c Therefore,  terminals.  these dopamine r e c e p t o r s would have t o be  s e n s i t i v e t o low, "autoreceptor" receptor agonists.  concentrations  of dopamine  The present demonstration t h a t dopamine  89  Dopamine Receptor ( I n h i b i t s n o r a d r e n a l i n e s y n t h e s i s when s t i m u l a t e d by a dopamine r e c e p t o r a g o n i s t )  G8L b l o c k s Impulse flow and decreases DA r e l e a s e a t t e r m i n a l s  F i g u r e 15. Dopamine r e l e a s e d i n t h e mpfc b y t h e m e s o c o r t i c a l dopamine n e u r o n s may i n h i b i t n o r a d r e n a l i n e s y n t h e s i s i n mpfc n o r a d r e n e r g i c t e r m i n a l s b y s t i m u l a t i n g i n h i b i t o r y dopamine r e c e p t o r s o n t h e noradrenergic terminals.  90 r e c e p t o r a g o n i s t s have a higher a f f i n i t y f o r mpfc dopamine r e c e p t o r s than s t r i a t a l dopamine r e c e p t o r s i s c o n s i s t e n t with t h i s p o s s i b i l i t y .  I f a t l e a s t some of the mpfc  dopamine r e c e p t o r s t h a t have a h i g h a f f i n i t y f o r dopamine r e c e p t o r a g o n i s t s are l o c a t e d on mpfc  noradrenergic  t e r m i n a l s , then the present data a l s o suggest t h a t s t r e s s may a f f e c t these neurons s i n c e the s t r e s s e f f e c t on mpfc dopamine r e c e p t o r s i s most pronounced a t a pH (7.9) where the h i g h e r a f f i n i t y s i t e s are best d e t e c t e d  (see above).  I t i s a l s o p o s s i b l e t h a t the GBL d i r e c t l y mpfc n o r a d r e n a l i n e the n o r a d r e n e r g i c  s y n t h e s i s by i n h i b i t i n g impulse flow i n c e l l s i n n e r v a t i n g the mpfc.  i n h i b i t i o n of impulse flow c o u l d reduce r e l e a s e i n the mpfc and consequently noradrenergic  autoreceptor  mpfc n o r a d r e n a l i n e  increases  The  noradrenaline  may r e s u l t i n l e s s  s t i m u l a t i o n , thereby  synthesis.  increasing  However, i t i s very  t h a t such a GBL-induced i n c r e a s e i n n o r a d r e n e r g i c i s i n v o l v e d i n the GBL e f f e c t s on mpfc DOPA t h a t were r e p o r t e d by Fadda e t a l . (1984). al.  unlikely synthesis  accumulation Thus, Fadda e t  (1984) found t h a t GBL d i d not i n c r e a s e DOPA  accumulation  i n the d o r s o l a t e r a l f r o n t a l c o r t e x which r e c e i v e s noradrenergic  i n p u t of s i m i l a r d e n s i t y t o t h a t i n n e r v a t i n g  the mpfc. The present hypothesis  p r e d i c t s t h a t a d m i n i s t r a t i o n of  dopaminergic drugs should a f f e c t n o r a d r e n a l i n e mpfc n o r a d r e n e r g i c  terminals.  synthesis i n  I t has been r e p o r t e d t h a t i n  s l i c e s t h a t i n c l u d e f r o n t a l , p a r i e t a l and a n t e r i o r c i n g u l a t e  areas of the c o r t e x , [ H ] - n o r a d r e n a l i n e J  formation i s  s i g n i f i c a n t l y a c c e l e r a t e d by a d m i n i s t r a t i o n of dopamine receptor antagonists  ( t h i o p r o p e r a z i n e and h a l o p e r i d o l ) and  s l i g h t l y reduced, a l b e i t  insignificantly,  by apomorphine  ( S c a t t o n , T h i e r r y , G l o w i n s k i and J u l o u , 1975; G l o w i n s k i and J u l o u , 1976).  Scatton,  I t i s p o s s i b l e t h a t i n the  s l i c e p r e p a r a t i o n employed by S c a t t o n e t a l . , endogenous levels  of dopamine occupied most of the proposed dopamine  r e c e p t o r s on n o r a d r e n e r g i c  terminals.  apomorphine would produce l i t t l e  I f t h i s were so,  a d d i t i o n a l s t i m u l a t i o n but  the dopamine r e c e p t o r a n t a g o n i s t would evoke a  significant  i n c r e a s e i n n o r a d r e n a l i n e s y n t h e s i s by r e d u c i n g inhibitory  i n f l u e n c e of endogenous dopamine.  dopamine r e c e p t o r s on n o r a d r e n e r g i c they may  the  When the  t e r m i n a l s are a c t i v a t e d  d i r e c t l y reduce n o r a d r e n a l i n e s y n t h e s i s or they  i n d i r e c t l y reduce n o r a d r e n a l i n e s y n t h e s i s by noradrenaline release.  may  reducing  A reduction i n noradrenaline release  should cause an i n c r e a s e i n n o r a d r e n a l i n e c o n c e n t r a t i o n s i n the mpfc n o r a d r e n e r g i c  terminals.  Such an i n c r e a s e would be  expected to decrease n o r a d r e n a l i n e s y n t h e s i s v i a end-product i n h i b i t i o n of t y r o s i n e hydroxylase 1982).  (Cooper, Bloom and  Roth,  I t i s , t h e r e f o r e , r e l e v a n t t h a t s e v e r a l groups have  i n v e s t i g a t e d the e f f e c t s of dopamine r e c e p t o r a g o n i s t s [ H]-noradrenaline 3  r e l e a s e i n the b r a i n and  Thus, dopamine does not a f f e c t  periphery.  [ H]-noradrenaline 3  from s l i c e s of the r a t o c c i p i t a l c o r t e x  on  release  (Taube, Starke  and  Borowski, 1977), but dopamine and other dopamine r e c e p t o r  a g o n i s t s do i n h i b i t the r e l e a s e of [ H ] - n o r a d r e n a l i n e J  from  s l i c e s of the d o r s a l hippocampus of the r a b b i t ( J a c k i s c h , Werle and H e r t t i n g , 1984; J a c k i s c h , M o l l , F e u e r s t e i n and H e r t t i n g , 1985).  Moreover,  the dopamine r e c e p t o r a g o n i s t s  produce t h i s e f f e c t by i n t e r a c t i n g w i t h D2 r e c e p t o r s ( J a c k i s c h e t a l . , 1985).  U n l i k e the o c c i p i t a l c o r t e x , the  d o r s a l hippocampus and the f r o n t a l c o r t e x are known t o be i n n e r v a t e d by dopaminergic t e r m i n a l s ( B i s c h o f f , S c a t t o n and K o r f , 1979).  Thus, the data c o l l e c t e d t o date suggest  that  c e n t r a l , p r e s y n a p t i c , n o r a d r e n a l i n e a c t i v i t y may be r e g u l a t e d by dopamine r e c e p t o r s , but o n l y i n b r a i n r e g i o n s t h a t r e c e i v e dopaminergic i n p u t .  I t i s important  t o note  t h a t these comparisons of d i f f e r e n t b r a i n r e g i o n s are p r e s e n t l y l i m i t e d t o those i n v o l v i n g data from studies.  Future r e s e a r c h should c o n c e n t r a t e  separate  on s t u d i e s  which employ i d e n t i c a l methods t o i n v e s t i g a t e dopamine r e g u l a t i o n of n o r a d r e n e r g i c regions.  I f evidence  noradrenergic  a c t i v i t y i n several brain  f o r dopamine r e c e p t o r s ( r e g u l a t i n g  a c t i v i t y ) i s found o n l y i n dopamine i n n e r v a t e d  b r a i n r e g i o n s then e i t h e r the dopamine r e c e p t o r s are l i m i t e d to noradrenergic  t e r m i n a l s i n those b r a i n r e g i o n s or  i n t e r n e u r o n s w i t h dopamine r e c e p t o r s may be i n v o l v e d . In the p e r i p h e r y , some dopamine r e c e p t o r s appear t o be l o c a t e d on n o r a d r e n e r g i c  nerve t e r m i n a l s .  they i n h i b i t the r e l e a s e of n o r a d r e n a l i n e G r i g o r i a d i s and Seeman, 1984).  When a c t i v a t e d , (Langer,  1974;  Dopamine r e c e p t o r a g o n i s t s  and a n t a g o n i s t s have v i r t u a l l y the i d e n t i c a l a f f i n i t i e s f o r  93 these r e c e p t o r s and D2(high) dopamine r e c e p t o r s i n the c e n t r a l nervous system ( G r i g o r i a d i s and Seeman, 1984).  If  c e n t r a l D2(high) r e c e p t o r s and the above mentioned p e r i p h e r a l dopamine r e c e p t o r s are i d e n t i c a l , as has  been  suggested ( G r i g o r i a d i s and Seeman, 1984), then i t i s p o s s i b l e t h a t these  r e c e p t o r s are s y n t h e s i z e d by some  p e r i p h e r a l and c e n t r a l n o r a d r e n e r g i c As d i s c u s s e d above, the,.present  neurons. data suggest t h a t the  higher a f f i n i t y of dopamine a g o n i s t s f o r the mpfc r e c e p t o r s compared to the s t r i a t a l D2 due  D2  receptors i s l i k e l y  to a h i g h e r p r o p o r t i o n of D2(high) s i t e s i n the mpfc.  I f a s i g n i f i c a n t number of D2(high) r e c e p t o r s are l o c a t e d on c e n t r a l noradrenergic may  e x p l a i n why  i n the mpfc.  terminals  (as proposed above), t h i s  they appear t o be r e l a t i v e l y more p r e v a l e n t  Thus, the r a t i o of n o r a d r e n e r g i c  innervation  to dopaminergic i n n e r v a t i o n i s much higher i n the mpfc than i n the s t r i a t u m (Brownstein,  Saavedra and P a l k o v i t s ,  1974;  C a r t e r and Pycock, 1980). I f t h e r e are mpfc dopamine r e c e p t o r s ,  on  nondopaminergic neurons, w i t h the h i g h a f f i n i t y f o r dopamine r e c e p t o r a g o n i s t s which i s c h a r a c t e r i s t i c of then many of the " a u t o r e c e p t o r "  autoreceptors,  r e s u l t s of Roth  and  c o l l e a g u e s are a l s o open to r e i n t e r p r e t a t i o n (Bannon e t a l . , 1981;  Bannon e t a l . , 1982;  C o n s i d e r i n g the evidence mpfc may  Galloway e t a l . , 1986).  t h a t dopaminergic a c t i v i t y i n the  i n h i b i t s u b c o r t i c a l dopaminergic a c t i v i t y ( l e s i o n s  of the mpfc dopamine i n n e r v a t i o n i n c r e a s e s u b c o r t i c a l  94 dopamine m e t a b o l i t e s  [ C a r t e r and Pycock, 1980; Pycock e t  al.,1980; M a r t i n - I v e r s o n  e t al.,1986] see General  Introduction f o r further d i s c u s s i o n ) , i t i s p o s s i b l e that the i n c r e a s e observed by Roth and c o l l e a g u e s i n s u b c o r t i c a l dopamine s y n t h e s i s f o l l o w i n g blockade of m e s o c o r t i c a l dopaminergic impulse flow may be l a r g e l y due t o reduced i n h i b i t i o n of s u b c o r t i c a l dopamine systems due t o l e s s dopamine r e l e a s e i n the mpfc-(see F i g u r e 16).  L i k e w i s e , the  r e d u c t i o n i n s u b c o r t i c a l dopamine s y n t h e s i s seen f o l l o w i n g low doses of dopamine r e c e p t o r a g o n i s t s may be due t o the s t i m u l a t i o n of mpfc h i g h a f f i n i t y , dopamine r e c e p t o r s l o c a t e d on nondopaminergic neurons.  However, the e x t e n t t o  which the p r o p e r t i e s of mpfc dopamine r e c e p t o r s are r e s p o n s i b l e f o r a l l dopamine " a u t o r e c e p t o r "  effects i s  p r e s e n t l y unknown. In summary, the dopamine a u t o r e c e p t o r  literature  be r e e v a l u a t e d due t o two l i n e s of r e c e n t evidence. the m e s o c o r t i c a l dopamine system may t o n i c a l l y s u b c o r t i c a l dopamine systems.  should First,  inhibit  Second, the mpfc may possess  a s i g n i f i c a n t number of dopamine r e c e p t o r s with a h i g h a f f i n i t y f o r dopamine r e c e p t o r a g o n i s t s . may be l o c a t e d on n o r a d r e n e r g i c reduce mpfc n o r a d r e n e r g i c  These r e c e p t o r s  t e r m i n a l s where they can  a c t i v i t y when a c t i v a t e d . They may  be a l s o i n v o l v e d i n the mechanism through which the m e s o c o r t i c a l dopamine system t o n i c a l l y i n h i b i t s s u b c o r t i c a l dopamine systems.  mpfc  F i g u r e 16. M e s o c o r t i c a l dopaminergic a c t i v i t y may t o n i c a l l y i n h i b i t s u b c o r t i c a l dopaminergic a c t i v i t y by i n f l u e n c i n g descending p r o j e c t i o n s . The mechanism r e s p o n s i b l e i s , as y e t , undetermined. The diagram i s not meant t o imply t h a t the i n t e r a c t i o n s i n the nucleus accumbens and the s t r i a t u m n e c e s s a r i l y i n v o l v e axo-axonic synapses. VTA = v e n t r a l tegmental area; SN = s u b s t a n t i a n i g r a ; (-) = i n h i b i t o r y i n f l u e n c e .  96  I n t e r p r e t a t i o n s of pH Dependent  D2 dopamine r e c e p t o r s  Binding  i n the mpfc  displayed  pH-dependent b i n d i n g c h a r a c t e r i s t i c s w i t h r e s p e c t t o both the e f f e c t s o f footshock and the a f f i n i t y of dopamine agonists.  While these e f f e c t s have been d i s c u s s e d  above,  t h i s s e c t i o n w i l l b r i e f l y review the d i f f i c u l t i e s i n i n t e r p r e t i n g these pH-dependent e f f e c t s . The f i r s t  issue regarding  have any i n v i v o r e l e v a n c e . agonists  these e f f e c t s i s whether they  I t i s possible that  dopamine  are degraded (or bound t o non-D2 s i t e s ) i n the  presence of s t r i a t a l t i s s u e a t pH 7.9, whereas mpfc t i s s u e may not have the same e f f e c t . reduction  At pH 6.2 t h i s  i n free agonist concentration  brain regions. concentrations  artifactual  may occur w i t h both  Such changes i n a c t u a l f r e e  agonist  i n b i n d i n g r e a c t i o n s c o u l d produce the  r e l a t i v e l y higher  "affinity"  f o r a g o n i s t s observed i n the  mpfc a t pH 7.9 but not pH 6.2. I t i s a l s o p o s s i b l e t h a t the pH-dependent e f f e c t s do accurately represent  the i n f l u e n c e t h a t changes i n pH whould  have i f they occured i n v i v o .  However, changes of the  magnitude employed here may not be present physiological conditions.  under  I f such l a r g e changes i n pH do  occur i n v i v o , and they have the e f f e c t s on D2 dopamine receptors receptor  i n d i c a t e d by the data i n t h i s t h e s i s , these e f f e c t s may s t i l l be i n s i g n i f i c a n t when compared t o  97 other pH-dependent changes. Pidoplichko  For i n s t a n c e ,  K r i s h t a l and  (1981) have demonstrated t h a t neurons i s o l a t e d  from the s p i n a l g a n g l i a and the g a n g l i o n of the t r i g e m i n a l nerve d i s p l a y an inward c u r r e n t i n response t o s m a l l pH changes i n e x t r a c e l l u l a r s o l u t i o n .  I f c e n t r a l neurons w i t h  dopamine D2 r e c e p t o r s are s i m i l a r l y s e n s i t i v e t o s m a l l changes i n pH then these e f f e c t s may mask any pH-dependent changes i n dopamine r e c e p t o r a f f i n i t y .  Future  Research  At l e a s t three areas of f u t u r e r e s e a r c h on mpfc D2 dopamine r e c e p t o r s would appear t o be worth F i r s t , i t w i l l be important  pursuing.  t o search f o r compounds t h a t  i n t e r a c t w i t h mpfc D2 r e c e p t o r s i n a h i g h l y s e l e c t i v e manner.  T h i s l i n e o f r e s e a r c h should be f a c i l i t a t e d by the  present development o f an assay t o measure mpfc D2 receptors.  I t i s a l s o encouraging t o note t h a t dopamine  r e c e p t o r a g o n i s t s d i s p l a y some s e l e c t i v i t y i n t h a t they a r e more potent  on mpfc D2 r e c e p t o r s than on s t r i a t a l D2  receptors. Second, the mechanisms by which mpfc D2 r e c e p t o r s are a f f e c t e d by v a r i o u s experimental investigated further. many behaviours  manipulations  should be  Dopamine i s thought t o be i n v o l v e d i n  and d i s e a s e s .  I f the p r e f r o n t a l c o r t e x  dopamine system i s f u n c t i o n a l l y and p h a r m a c o l o g i c a l l y distinct,  i t i s imperative  t h a t the f u n c t i o n of t h i s system  98 be d i r e c t l y e x p l o r e d .  E x t r a p o l a t i o n s from data c o l l e c t e d i n  s t u d i e s of s u b c o r t i c a l dopamine systems are not a c c e p t a b l e i n t h i s regard.  I t i s , therefore, s i g n i f i c a n t that i t i s  now p o s s i b l e t o d i r e c t l y i n v e s t i g a t e the f u n c t i o n of mpfc D2 r e c e p t o r s with the h e l p of the assay d e s c r i b e d i n t h i s thesis. T h i r d , w h i l e the present  assay i s capable  of measuring  the p r o p e r t i e s of D2 dopamine r e c e p t o r s i n the mpfc, i t i s important t h i s task.  t o continue  t o develop more and b e t t e r methods f o r  One such p o t e n t i a l new method may i n v o l v e the  use of [ l ^ S j j . g u i p j - i c i e .  IJ^Q cent r e  p u b l i c a t i o n s have  r e p o r t e d t h a t [ 1 2 5 j ] - s u l p r i d e l a b e l s p o t e n t i a l D2 dopamine r e l a t e d b i n d i n g s i t e s i n many b r a i n regions 1985; Martres,  (Martres e t a l . ,  S a l e s , Bouthenet and Schwartz, 1985).  i n the p a r i e t a l c o r t e x and cerebellum, d e n s i t y , seem t o be p h a r m a c o l o g i c a l l y s i t e s l a b e l l e d by [  1 2 5  although  Sites  a t very low  very s i m i l a r t o those  I ] - s u l p r i d e i n the s t r i a t u m .  The s i t e s l a b e l l e d by [ 1 2 5 j ] - s u l p r i d e i n the mpfc have not been p h a r m a c o l o g i c a l l y  characterized.  At pH 7.4  dopamine r e c e p t o r a g o n i s t s are no more potent for  a t competing  [ 1 2 5 j ] - s u l p r i d e b i n d i n g s i t e s i n the p a r i e t a l  cortex  than i n the s t r i a t u m (Martres, S a l e s , Bouthenet and Schwarts, 1985).  These r e s u l t s c o n t r a s t with the present  demonstration t h a t , a t pH 7.9, dopamine r e c e p t o r are more potent  agonists  a t competing f o r b i n d i n g s i t e s i n the mpfc  than i n the s t r i a t u m .  Therefore,  e i t h e r the [  1 2 5  I]-sulpride  l a b e l s a d i f f e r e n t p o p u l a t i o n of b i n d i n g s i t e s a t pH 7.4  than those p r e s e n t l y l a b e l l e d a t pH 7.9 by (as suggested  [ H]-spiperone J  above) o r the h i g h e r a f f i n i t y of dopamine  r e c e p t o r a g o n i s t s f o r c o r t i c a l D2 r e c e p t o r s i s r e l a t i v e l y s p e c i f i c t o the mpfc. As d i s c u s s e d above, there i s evidence t h a t the s i t e s l a b e l l e d by the butyrophenones, h a l o p e r i d o l and spiperone, are f u n c t i o n a l dopamine r e c e p t o r s because the b i n d i n g a f f i n i t i e s of the many dopaminergic  compounds t e s t e d are  highly c o r r e l a t e d with t h e i r i n vivo potencies. I f analogous  c o r r e l a t i o n s are o b t a i n e d w i t h [ 125j_]-sulpride  l a b e l l e d s i t e s then study of the mpfc w i t h t h i s  ligand  should f u r t h e r e l u c i d a t e the r o l e of D2 r e c e p t o r s i n the mpfc.  The h i g h s p e c i f i c a c t i v i t y and low n o n s p e c i f i c  b i n d i n g of t h i s l i g a n d make i t i d e a l f o r use i n b r a i n  areas  such as the mpfc, where the d e n s i t y of D2 r e c e p t o r s i s v e r y low.  However, i f f u t u r e r e s e a r c h i n d i c a t e s t h a t  [ 1 2 5 j ] - s u l p r i d e l a b e l s a d i f f e r e n t p o p u l a t i o n of r e c e p t o r s than t h a t l a b e l l e d by the butyrophenones then r e s e a r c h w i t h the butyrophenones should continue i n the mpfc. The p r e s e n t assay a t pH 6.2 had a h i g h e r degree of r e s o l u t i o n than t h a t a t pH 7.9 (see Experiments  1 and 2 ) .  T h i s i s v e r y l i k e l y due t o a near e l i m i n a t i o n of [ H]-spiperone 3  b i n d i n g t o spirodecanone  d i s c u s s e d above, the D2 b i n d i n g observed  sites.  However, as  i n the mpfc a t pH  7.9 has a number of i n t e r e s t i n g p r o p e r t i e s which make the pH 7.9 assay v e r y a t t r a c t i v e even though i t i s not as a c c u r a t e as the pH 6.2 assay.  I t has been demonstrated t h a t  solubilized  [ H ] - s p i p e r o n e b i n d i n g s i t e s from the s t r i a t u m J  c o n t a i n a very h i g h p r o p o r t i o n of spirodecanone (Laduron, 1981).  T h i s i s a problem not encountered w i t h  standard D2 b i n d i n g assays employing To d e a l w i t h t h i s problem, Pharmaceutica  sites  s t r i a t a l homogenates.  r e s e a r c h e r s a t Janssen  have developed compounds which  occlude the s o l u b i l i z e d  spirodecanone s i t e s  selectively (Laduron,  1981).  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