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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 OF PREFRONTAL CORTEX D2 DOPAMINE RECEPTORS IN THE RAT by ALEXANDER JOHN MACLENNAN B . S c , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1979 M.A., The U n i v e r s i t y o f C o l o r a d o , 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES P r o g r a m i n N e u r o s c i e n c e We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e , r e q u i r e d s t a n d a r d The U n i v e r s i t y o f B r i t i s h C o l u m b i a November 1986 © A l e x a n d e r J o h n MacLennan, 1986 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Department of DE-6(3/81) Abstract A radioligand binding assay was developed to measure possible D2 dopamine receptors i n the medial pr e f r o n t a l cortex (mpfc) of the rat. It was demonstrated with competition studies at pH 7.9 that these candidate s i t e s are, i n fa c t , D2 receptors. A l l compounds without a c t i v i t y on dopaminergic systems were i n e f f e c t i v e competitors at nanomolar concentrations. Dopamine was more potent than noradrenaline which was more potent than serotonin. At nanomolar concentrations, a l l dopamine receptor antagonists that were tested competed for the D2 binding. The IC50's of the dopamine receptor antagonists correlated with t h e i r i n  vivo potencies. The IC50's of a l l compounds tested with mpfc tissue correlated highly with t h e i r IC50's i n an i d e n t i c a l assay of the striatum. However, the dopamine receptor agonists, apomorphine and ADTN, were s i g n i f i c a n t l y more potent i n the mpfc. The competition curves of the dopamine receptor agonists suggested that the mpfc, when compared to the striatum, contains a higher proportion of receptors with a high a f f i n i t y for dopamine receptor agonists. Reducing the pH of the assay from pH 7.9 to pH 6.2 eliminated the difference i n agonist a f f i n i t y but did not a f f e c t the IC50 correlations between brain regions or the correlations with i n vivo potencies. The reduction i n pH increased the percent of D2 binding i n the mpfc assay by presumably reducing spirodecanone binding. Consequently the pH 6.2 assay displayed a higher s e n s i t i v i t y to changes i n D2 binding. Chronic haloperidol administration for 21 weeks increased the D2 binding i n the mpfc (as measured at pH 6.2) by approximately 50% compared to approximately 70% i n the striatum. Footshock stress increased the D2 binding measured at pH 6.2 by approximately 13% i n the mpfc and had no e f f e c t i n the striatum. Footshock stress increased the Bmax of the D2 binding measured at 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 binding was unaffected by footshock stress as measured at pH 7.9. i v T a b l e o f C o n t e n t s Page A b s t r a c t i i L i s t o f T a b l e s v i i L i s t o f F i g u r e s v i i i A b b r e v i a t i o n s x Acknowledgements . x i G e n e r a l I n t r o d u c t i o n 1 The M e s o c o r t i c a l Dopamine S y s t e m I n n e r v a t i n g t h e P r e f r o n t a l C o r t e x 1 B r a i n Dopamine R e c e p t o r s 10 M e d i a l 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 17 E x p e r i m e n t 1: D e v e l o p m e n t and Use o f an A s s a y t o C h a r a c t e r i z e D2 Dopamine R e c e p t o r s i n t h e M e d i a l P r e f r o n t a l C o r t e x o f t h e R a t 20 I n t r o d u c t i o n . 20 Methods 21 R e s u l t s 34 D i s c u s s i o n 44 E x p e r i m e n t 2: C h a r a c t e r i z a t i o n a t pH 6.2 o f M e d i a l 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 i n t h e R a t 49 I n t r o d u c t i o n 49 Methods 50 V page R e s u l t s 51 D i s c u s s i o n 53 E x p e r i m e n t 3: The E f f e c t s o f C h r o n i c H a l o p e r i d o l T r e a t m e n t on D2 Dopamine R e c e p t o r s i n t h e M e d i a l P r e f r o n t a l C o r t e x o f t h e R a t 56 I n t r o d u c t i o n 56 Methods 59 R e s u l t s 61 D i s c u s s i o n 63 E x p e r i m e n t 4: The E f f e c t s o f F o o t s h o c k S t r e s s on D2 Dopamine R e c e p t o r s i n t h e M e d i a l P r e f r o n t a l C o r t e x o f t h e R a t 67 I n t r o d u c t i o n 67 The E f f e c t s o f F o o t s h o c k S t r e s s as M e a s u r e d a t pH 6.2 69 Methods 69 R e s u l t s 70 D i s c u s s i o n 72 The E f f e c t s o f F o o t s h o c k S t r e s s as M e a s u r e d a t pH 7.9 74 Methods 75 R e s u l t s . 75 D i s c u s s i o n 78 v i Page G e n e r a l D i s c u s s i o n 82 A H y p o t h e s i s 83 Dopamine " A u t o r e c e p t o r " R e s e a r c h 85 I n t e r p r e t a t i o n s o f pH-Dependent B i n d i n g 96 F u t u r e R e s e a r c h 97 R e f e r e n c e s 101 v i i L i s t o f T a b l e s Page T a b l e 1 I n h i b i t i o n o f S p e c i f i c [ 3 H ] - S p i p e r o n e (25 pM) B i n d i n g a t pH 7.9 36 T a b l e 2 I n h i b i t i o n o f S p e c i f i c [ 3 H ] - S p i p e r o n e (35 pM) B i n d i n g a t pH 6.2 52 v i i i L i s t of Figures Figure 1 S t r i a t a l and mpfc tissue used for assays Figure 2 The amount of t o t a l tissue binding of [^H]-spiperone (25 pM) competed for by various concentrations of ketanserin t a r t r a t e i n the mpfc and the striatum Figure 3 The percent of s p e c i f i c [ - s p i p e r o n e (25 pM) binding (binding competed for by 10~5 M s - ( - ) - s u l p i r i d e ) competed for by various concentrations of s - ( - ) - s u l p i r i d e and r-(+)-sulpiride i n the mpfc and the striatum Figure 4 The re l a t i o n s h i p between the a f f i n i t i e s of various dopamine receptor antagonists for mpfc D2 receptors and the a n t i -psychotic potencies of the same drugs Figure 5 The re l a t i o n s h i p between the a f f i n i t i e s of various dopamine receptor antagonists for mpfc D2 receptors and the a f f i n i t i e s of the same drugs for s t r i a t a l D2 receptors Figure 6 The percent of s p e c i f i c [^H]-spiperone (25 pM) binding (binding competed for by 10~5 M s - ( - ) - s u l p i r i d e ) competed for by various concentrations of dopamine i n the mpfc and the striatum Figure 7 The percent of s p e c i f i c [-^H]-spiperone (25 pM) binding (binding competed for by 10~5 M s - ( - ) - s u l p i r i d e ) competed for by various concentrations of apomorphine i n the mpfc and the striatum Figure 8 The percent of s p e c i f i c [^H]-spiperone • (25 pM) binding (binding competed for by 10~5 M s - ( - ) - s u l p i r i d e ) competed for by various concentrations of ADTN i n the mpfc and the striatum Figure 9 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 s t r i a t a l D2 receptors Page 22 27 35 38 39 40 41 42 62 i x Page Figure 10 Scatchard plo t 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 64 Figure 11 Scatchard plo 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 receptors assayed at pH 6.2 71 Figure 12 Scatchard plot 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 receptors assayed at pH 6.2 73 Figure 13 Scatchard plo 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 receptors assayed at pH 7.9 76 Figure 14 Scatchard plot 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 receptors assayed at pH 7.9 77 Figure 15 An i l l u s t r a t i o n of how dopamine released by mesocortical dopamine neurons may i n h i b i t noradrenaline synthesis i n mpfc noradrenergic terminals 89 Figure 16 An i l l u s t r a t i o n of how mesocortical 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 A b b r e v i a t i o n s AMP '. a d e n o s i n e monophoshate DOPA 3 , 4 - d i h y d r o x y p h e n y l a l a n i n e DOPAC d i h y d r o x y p h e n y l a c e t i c a c i d EDTA e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d GBL g a m m a - b u t y r o l a c t o n e mpfc m e d i a l p r e f r o n t a l c o r t e x 6-OHDA 6-hydroxydopamine XI Acknowledgement I w o u l d l i k e t o t h a n k t h e members o f my S u p e r v i s o r y Committee, my U n i v e r s i t y E x a m i n e r s and t h e C h a i r m a n o f my d e f e n s e . I a l s o w i s h t o t h a n k J a n e t F i n l a y and J i m Radke who were e x p o s e d t o a d a n g e r o u s l y raw d r a f t o f t h i s m a n u s c r i p t y e t made r a p i d and h e l p f u l comments. I w o u l d l i k e t o t h a n k A l e x J a k u b o v i c who made c o n t r i b u t i o n s b o t h d u r i n g t h e r e s e a r c h p h a s e and w r i t i n g p h a s e o f t h i s p r o j e c t . T h a n k s a l s o go t o Nancy Lee who t y p e d t h i s t h e s i s w h i l e I d i c t a t e d i t t o h e r b e c a u s e my w r i t i n g i s s o u g l y . Most o f a l l I w o u l d l i k e t o e x p r e s s my g r a t i t u d e t o S t e l l a A t m a d j a who was t h e r e t h r o u g h t h e b o r i n g h o u r s and whose h e l p I c o u l d n o t have done w i t h o u t . 1 General Introduction Brain dopamine systems are thought to play a role i n a wide var i e t y of neural functions including those responsible for motor a c t i v i t y (Marsden, 1984), feeding behavior (Heffner, Hartman and Seiden, 1980) and endogenous reward ( P h i l l i p s and F i b i g e r , 1978). In addition, abnormalities involving dopamine systems may be c r i t i c a l i n many conditions such as Parkinson's disease (Hornykiewicz, 1973), depression (Antelman and Chiodo, 1981; Fibiger and P h i l l i p s , 1981; B o r s i n i , P u l v i r e n t i and Samanin, 1985), Tourette's syndrome (Butler, 1984), some drug-induced psychoses (Snyder, Banerjee, Yamamura and Greenberg, 1974) and schizophrenia (Meltzer and Stahl, 1976). Information concerning central dopaminergic processes i s substantial. This thesis w i l l deal primarily with a r e l a t i v e l y recent and somewhat lim i t e d area of t h i s body of knowledge: the mesocortical dopamine neurons innervating the prefrontal cortex. The Mesocortical Dopamine System Innervating the  Prefrontal Cortex In 1973 Thierry and colleagues demonstrated with biochemical and l e s i o n studies that dopaminergic neurons innervate the rat prefrontal cortex (Thierry, Blanc, Sobel, Stinus and Glowinski, 1973(a); Thierry, Stinus, Blanc and 2 Glowinski, 1973(b)). The p r e f r o n t a l cortex also receives a dense noradrenergic projection, and therefore u n t i l the research of Thierry et a l . , dopamine i n t h i s brain region was assumed to represent precursor pools for synthesis of noradrenaline. Thierry et a l . (1973b) ruled out t h i s p o s s i b i l i t y by demonstrating that dopamine concentrations i n the prefrontal cortex were not reduced by lesions of the noradrenergic input. In addition, they were able to show that [3H]-dopamine could be synthesized from [ 3H]-tyrosine (in vivo and 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 deprived of noradrenergic innervation (Thierry et a l . , 1973a). Furthermore, they found that the prefrontal cortex contains high a f f i n i t y [3H]-dopamine uptake s i t e s (Tassin, Thierry, Blanc and Glowinski, 1974). I t has also been demonstrated (Pycock, Carter and Kerwin, 1980) through si m i l a r l e s i o n studies that tyrosine hydroxylase a c t i v i t y (the rate l i m i t i n g enzyme for both dopamine and noradrenaline synthesis) i s located i n non-noradrenergic c e l l s innervating the p r e f r o n t a l cortex. Dopamine-sensitive adenylate cyclase has also been i d e n t i f i e d i n the p r e f r o n t a l cortex (Tassin, Bockaert, Blanc, Stinus, Thierry, L a v i e l l e , Premont and Glowinski, 1978). F i n a l l y , the dopamine metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) have been found i n the p r e f r o n t a l cortex (Bannon and Roth, 1983). The above mentioned biochemical indices of dopaminergic a c t i v i t y i n the prefrontal cortex indicate that the 3 dopaminergic projection to the prefrontal cortex i s quite sparse compared to that of subcortical dopaminergically innervated regions such as the striatum and the nucleus accumbens. Dopamine concentrations i n the p r e f r o n t a l cortex are 1-5% of those i n the striatum (Tassin et a l . , 1978; Bannon and Roth, 1983). Dopamine turnover rates i n the prefrontal cortex have been estimated both by the concentration of dopamine metabolites and the decline i n dopamine content following 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 cortex appears to be s i g n i f i c a n t l y higher than i n s u b c o r t i c a l regions (approximately twice that of the striatum)(Bannon and Roth, 1983; Thierry, Tassin and Glowinski, 1984). Anatomically, the prefrontal cortex i s described as those regions of the f r o n t a l cortex receiving d i r e c t projections from the mediodorsal nucleus of the thalamus (Lin d v a l l and Bjorklund, 1984). The dopaminergic c e l l s innervating the prefrontal cortex can most e a s i l y be divided into two subcategories ( L i n d v a l l , Bjorklund and Divac, 1978). The l a t e r a l or suprarhinal system innervates an area located on the dorsal bank of the r h i n a l sulcus. The medial system innervates the pregenual, anteromedial cortex. The vast majority of research has been concerned with the medial system. In the r a t , the dopaminergic innervation of the f r o n t a l cortex 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 cortex ( L i n d v a l l et a l . , 1978). In primates, the dopamine 4 innervation i s somewhat more extensive but s t i l l r e s t r i c t e d to the pre f r o n t a l cortex (Brown, Crane and Goldman, 1979; L e v i t t , Rakic and Goldman-Rakic, 1984). The perikarya of dopaminergic neurons that project to the p r e f r o n t a l cortex are located i n the ventral tegmental area (Fuxe, Hokfelt, Johansson, Jonsson, Lidbrink and Ljungdahl, 1974; Hokfelt, Ljungdahl, Fuxe and Johansson, 1974). The medial system c e l l s are located primarily i n the medio-rostral part of the A10 group of dopaminergic neurons (Li n d v a l l and Bjorklund, 1984; Thierry et a l . , 1984). The suprarhinal system originates more l a t e r a l l y ( L i n d v a l l and Bjorklund, 1984; Thierry et a l . , 1984). Very few mesocortical dopamine c e l l s appear to have c o l l a t e r a l s to other brain regions (Lindvall and Bjorklund, 1984). The dopamine f i b e r s of both systems innervating the prefrontal cortex are remarkably smooth with few v a r i c o s i t i e s ( L i n d v a l l et a l . , 1978). They are observed i n layers II through VI with the highest density i n layers V and VI (Lindvall and Bjorklund, 1984). The dopaminergic mesocortical neurons generally display the low f i r i n g rates and slow conduction v e l o c i t i e s that are t y p i c a l of dopaminergic neurons (Bunney and Chiodo, 1984). However, dopaminergic c e l l s projecting to the prefrontal cortex f i r e at much higher basal rates than s u b c o r t i c a l l y projecting dopaminergic c e l l s or other mesocortical dopamine c e l l s that innervate other areas of the cortex (Bunney and Chiodo, 1984). Bursting a c t i v i t y i s also more frequent i n 5 the dopaminergic c e l l s that project to the prefrontal cortex (Bunney and Chiodo, 1984). Dopamine receptor agonists have been reported to have both excitatory 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 actions i n the pref r o n t a l cortex (Bunney and Chiodo, 1984; P h i l l i s , 1984). Both e f f e c t s are i n h i b i t e d by dopamine receptor antagonists. Subchronic treatment with dopamine receptor antagonists greatly 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). This e f f e c t can be reversed by administration of dopamine receptor agonists (Chiodo and Bunney, 1983; White and Wang, 1983). I t i s believed that the subchronic treatment with the dopamine receptor antagonists f i r s t greatly increases the f i r i n g rate of the dopaminergic neurons (Bunney and Grace, 1978). This i s thought to cause the c e l l s to become so depolarized that t h e i r spike generating mechanisms become inactivated (Bunney and Grace, 1978; Bunney and Grace, 1986). The dopamine receptor agonists are believed to reverse t h i s "depolarization block" by hyperpolarizing the dopaminergic neurons. Mesocortical dopamine c e l l s also display an increase i n f i r i n g rate following acute administration of dopamine receptor antagonists, but they do not become inactivated by subchronic treatment (Chiodo and Bunney, 1983). Behavioural research on the mesocortical dopamine system has been very l i m i t e d . This i s probably due to two 6 factors. F i r s t , behavioural studies require that s u f f i c i e n t anatomical and biochemical data be available to permit the proper design of experiments. Second, and more c r i t i c a l l y , s e l e c t i v e manipulation of the mesocortical dopamine system has not been achieved. Lesions of the prefrontal cortex with 6-hydroxydopamine (6-OHDA) produce large decreases i n noradrenaline concentrations (34-73%)in addition to t h e i r e f f e c t s on dopamine (Carter and Pycock, 1980; Pycock et a l . , 1980; Joyce, Stinus and Iversen, 1983). Considering the large l i t e r a t u r e i n d i c a t i n g that dopamine and noradrenaline inter a c t extensively (Langer, 1974; Antelman and Caggiula, 1977), the behavioural e f f e c t s of prefrontal cortex 6-OHDA lesions cannot be regarded as unequivocal indications of dopamine involvement. Pharmacologically, no compounds have been demonstrated to be p a r t i c u l a r l y s e l e c t i v e at d i r e c t l y a c t i v a t i n g or blocking dopamine receptors i n the prefrontal cortex. This i s due, at least i n part, to the lack of a method to measure prefrontal cortex dopamine receptors. Direct cerebral application of dopaminergic compounds seems, at present, the only available research t o o l . Unfortunately, the prefrontal cortex i s located very near other dopaminergically innervated brain regions which possess many times the density of dopamine receptors (see below). Therefore, even moderate degrees of drug d i f f u s i o n could produce misleading r e s u l t s . Although 6-OHDA lesions of the A10 c e l l bodies have been reported to disrupt the behaviour of rats trained on a 7 s p a t i a l a l t e r n a t i o n task (Simon, Scatton and Le Moal, 1980), i f the lesions are r e s t r i c t e d to the medial f r o n t a l cortex, no such r e s u l t i s observed (Iversen, 1984). Rats with medial prefrontal cortex 6-OHDA lesions have been reported to display more amphetamine-induced stereotypy, more spontaneous a c t i v i t y , and less apomorphine-induced stereotypy than controls (Carter and Pycock, 1980). Carter and Pycock (1980) suggest that dopamine release i n the medial pre f r o n t a l cortex (mpfc) may i n h i b i t the a c t i v i t y of the mesolimbic and n i g r o s t r i a t a l systems. In support of t h i s , they found that the concentrations of dopamine and i t s metabolites are increased i n the striatum and nucleus accumbens of rats receiving 6-OHDA lesions of the mpfc (Carter and Pycock, 1980; Pycock et a l . , 1980). This r e s u l t has recently been r e p l i c a t e d by Martin-Iverson, Szostak and Fibiger (1986). However, as previously noted, these e f f e c t s are d i f f i c u l t to interpret because the concentration of mpfc noradrenaline i s also dramatically reduced by these lesions. The l e s i o n r e s u l t s presented above have led most researchers to agree with Carter and Pycock and to suggest that dopamine release i n the mpfc i n h i b i t s s u bcortical dopamine a c t i v i t y (Bannon and Roth, 1983; Glowinski, Tassin and Thierry, 1984; Iversen, 1984). Unfortunately, the data available are fa r from conclusive. The mesocortical dopamine neurons respond i n a unique fashion to many experimental manipulations (Bannon and Roth, 1983; Thierry et a l . , 1984). For example, a wide var i e t y of 8 p h a r m a c o l o g i c a l a g e n t s p r o d u c e e f f e c t s on dopamine m e t a b o l i s m i n t h e p r e f r o n t a l c o r t e x t h a t a r e d i f f e r e n t f r o m t h e i r e f f e c t s on t h e s u b c o r t i c a l s y s t e m s (Bannon and R o t h , 1 9 8 3 ) . Of p a r t i c u l a r i n t e r e s t i s t h e c h a r a c t e r i s t i c s e n s i t i v i t y o f m e s o c o r t i c a l dopamine n e u r o n s t o p s y c h o s i s - i n d u c i n g d r u g s s u c h as amphetamine and p h e n c y c l i d i n e (Bowers and Hoffman, 1984; R o b i n s o n , B e c k e r , Moore, C a s t a n e d a and M i t t l e m a n , 1985) and a n t i p s y c h o t i c d r u g s (Matsumoto, U c h i m u r a , H i r a n o , Kim, Yokoo, Shimomura, N a k a h a r a , I n o u e and O o m o g a r i , 1 9 8 3 ) . T h e s e s t u d i e s s u g g e s t t h a t t h e s e compounds may a c t on t h e m e s o c o r t i c a l dopamine s y s t e m t o p r o d u c e t h e i r c l i n i c a l l y o b s e r v e d e f f e c t s . T h u s , a c u t e amphetamine o r p h e n c y c l i d i n e a d m i n i s t r a t i o n i n c r e a s e s mpfc dopamine m e t a b o l i s m ( a s i n d i c a t e d by i n c r e a s e s i n t h e c o n c e n t r a t i o n s o f mpfc dopamine m e t a b o l i t e s ) t o a g r e a t e r e x t e n t t h a n i t i n c r e a s e s s t r i a t a l dopamine m e t a b o l i s m (Bowers and H o f f man, 1 9 8 4 ) . S u b c h r o n i c amphetamine a d m i n i s t r a t i o n a l s o p r o d u c e s an e n d u r i n g a c c e l e r a t i o n o f mpfc dopamine t u r n o v e r ( a s i n d i c a t e d by t h e r a t e o f d e c l i n e i n t h e c o n c e n t r a t i o n o f dopamine a f t e r i n h i b i t i o n o f t y r o s i n e h y d r o x y l a s e a c t i v i t y ) w h i c h i s n o t o b s e r v e d i n t h e n u c l e u s accumbens o r t h e s t r i a t u m ( R o b i n s o n e t a l . , 1 9 8 5 ) . A n t i p s y c h o t i c d r u g s c a n i n c r e a s e t h e c o n c e n t r a t i o n s o f mpfc dopamine m e t a b o l i t e s when a d m i n i s t e r e d a t l o w e r d o s e s t h a n t h o s e r e q u i r e d t o i n c r e a s e s u b c o r t i c a l dopamine m e t a b o l i t e s (Matsumoto e t a l . , 1 9 8 3 ) . W h i l e a n t i p s y c h o t i c - i n d u c e d i n c r e a s e s i n s u b c o r t i c a l dopamine 9 m e t a b o l i t e s d i m i n i s h d u r i n g s u b c h r o n i c t r e a t m e n t , t h e i n c r e a s e s i n mpfc dopamine m e t a b o l i t e s a r e r e p o r t e d t o be r e l a t i v e l y l e s s s u s c e p t i b l e t o s u c h t o l e r a n c e ( B a c o p o u l o s e t a l . , 1979; Bannon and R o t h , 1983; Matsumoto e t a l . , 1 9 8 3 ) . S i n c e t h e c l i n i c a l l y b e n e f i c i a l e f f e c t s o f a n t i p s y c h o t i c t r e a t m e n t r e p o r t e d l y d i s p l a y a p a r a l l e l r e s i s t a n c e t o t o l e r a n c e , i t has b e e n s u g g e s t e d t h a t a n t i p s y c h o t i c d r u g s may a c t on m e s o c o r t i c a l dopamine n e u r o n s t o p r o d u c e t h e i r a n t i p s y c h o t i c e f f e c t s ( B a c o p o u l o s e t a l . , 1 9 7 9 ) . I t h a s a l s o b e e n r e p o r t e d by s e v e r a l g r o u p s t h a t t h e dopamine c e l l s i n n e r v a t i n g t h e mpfc a r e much more s e n s i t i v e t h a n s u b c o r t i c a l dopamine s y s t e m s t o a c t i v a t i o n b y e n v i r o n m e n t a l s t r e s s o r s . T h u s , e x p e r i m e n t a l a n i m a l s s u b j e c t e d f o o t s h o c k d i s p l a y i n c r e a s e s i n mpfc dopamine m e t a b o l i t e s ( F a d d a , A r g i o l a s , M e l i s , T i s s a r i , O n a l i and G e s s a , 1978; S z e n t e n d r e i , Herman, K a n y i c s k a and F e k e t e , 1980; R e i n h a r d , Bannon and R o t h , 1982) and mpfc dopamine s y n t h e s i s ( R e i n h a r d e t a l . , 1982; K r a m a r c y , D e l a n e y and Dunn, 1 9 8 4 ) . F o o t s h o c k a l s o a c c e l e r a t e s t h e r a t e o f d e c r e a s e i n mpfc dopamine c o n c e n t r a t i o n o b s e r v e d f o l l o w i n g t y r o s i n e h y d r o x y l a s e i n h i b i t i o n ( T h i e r r y , T a s s i n , B l a n c and G l o w i n s k i , 1976; R o b i n s o n e t a l . , 1 9 8 5 ) . L e s i o n s o f t h e n o r a d r e n e r g i c i n n e r v a t i o n o f t h e mpfc do n o t b l o c k t h e f o o t s h o c k - i n d u c e d i n c r e a s e i n mpfc dopamine m e t a b o l i t e s ( T i s s a r i , A r g i o l a s , F a d d a , S e r r a and G e s s a , 1979) o r t h e f o o t s h o c k - i n d u c e d d e c r e a s e i n dopamine f o l l o w i n g t y r o s i n e h y d r o x y l a s e i n h i b i t i o n ( T h i e r r y e t a l . , 1 9 7 6 ) . Mpfc 10 dopamine metabolites are also increased by swim stress and r e s t r a i n t stress (Yang, Knorn, Onel, Tarn, Deutch, Cubich and Roth, 1985) as well as conditioned fear (Herman, Guilloneau, Dantzer, Scatton, Semerdjian-Rouquier and Le Moal, 1982). A l l these studies found that subcortical dopamine systems are much less s e n s i t i v e to stress. In summary, the mesocortical dopamine c e l l s innervating the mpfc display many c h a r a c t e r i s t i c s which indicate that 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 . They display s i g n i f i c a n t l y higher basal rates of f i r i n g , bursting and transmitter turnover. Unlike the subcortical dopamine processes, the dopamine processes innervating the prefrontal cortex are reported to be p a r t i c u l a r l y sensitive to stress and a wide vari e t y of pharmacological agents, and re s i s t a n t to "depolarization block" induced by neuroleptic treatment. Brain Dopamine Receptors As with a l l putative neurotransmitters, dopamine i s believed to influence neuronal a c t i v i t y by i n t e r a c t i n g with s p e c i f i c neuronal proteins or "receptors". The properties of brain dopamine receptors have been investigated with various experimental protocols. Research has i d e n t i f i e d many compounds which can block and reverse the behavioural e f f e c t s of dopamine (Janssen and Van Bever, 1975) as well as compounds which mimic the behavioural e f f e c t s of dopamine 11 (Woodruff, 1981). Many of these drugs are potent at nanomolar concentrations thereby suggesting that they have a high a f f i n i t y for some c r i t i c a l "receptor". Further evidence for the existence of central dopamine receptors comes from ele c t r o p h y s i o l o g i c a l research. Iontophoretically applied dopamine can a f f e c t the e l e c t r o p h y s i o l o g i c a l behavior of neurons i n dopamine innervated regions of the brain. Studies 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 recording techniques have found that dopamine i n h i b i t s or excites the a c t i v i t y of neurons i n the striatum (Moore and Bloom, 1978; K i t a i , 1981). I t i s presently unclear how dopamine both excites and i n h i b i t s s t r i a t a l neurons. I t has been proposed that dopamine may produce i t s i n h i b i t o r y e f f e c t s by e x c i t i n g i n h i b i t o r y s t r i a t a l interneurons ( K i t a i , 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 located i n dopaminergically innervated brain regions. Therefore, the data suggest that dopamine inter a c t s with some neuronal element ("receptor") which i s involved 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 behavioural experiments have provided important information, neither approach can be used to characterize brain dopamine receptors. In order to characterize a receptor, the potencies of many compounds must be compared. Such comparison requires precise knowledge of the r e l a t i v e concentrations of the compounds int e r a c t i n g with the receptor, and such control cannot be 12 achieved with behavioural or e l e c t r o p h y s i o l o g i c a l techniques (Snyder and Bennett, 1976; Woodruff, 1981). In the early 1970's, i t was discovered that dopamine can stimulate a "dopamine-sensitive" adenylate cyclase i n the superior c e r v i c a l ganglion (Kebabian and Greengard, 1971). The c y c l i c AMP produced by such stimulation mediates dopaminergic transmission i n t h i s ganglion (McAfee, Schorderet and Greengard, 1971; McAfee and Greengard, 1972). Such data led these workers to conclude that the "dopamine receptor" i n the ganglion i s the dopamine binding portion of a dopamine-sensitive adenylate cyclase. A very s i m i l a r adenylate cyclase was subsequently i d e n t i f i e d i n homogenates of caudate nuclei (Kebabian, Petzold and Greengard, 1972). Dopamine and other dopamine receptor agonists were potent stimulators of t h i s enzyme while dopamine receptor antagonists were potent i n h i b i t o r s of agonist-induced stimulation. The f i r s t attempts to l a b e l brain dopamine receptors with radioactive compounds were reported i n 1975 (Burt, Enna, Creese and Snyder, 1975; Seeman, Chau-Wong, Tedesco and Wong, 1975). Research revealed that the binding s i t e s l a b e l l e d by [ 3H]-haloperidol display the c h a r a c t e r i s t i c s of a dopamine receptor while those s i t e s l a b e l l e d by [%]-dopamine do not display such c h a r a c t e r i s t i c s . Thus, the a f f i n i t i e s of dopaminergic and non-dopaminergic compounds for the [ 3H]-haloperidol (but not the [3H]-dopamine) l a b e l l e d s i t e s were highly correlated with 13 t h e i r i n vivo potencies as antagonists of dopamine agonist induced behaviors i n experimental animals (Seeman et a l . , 1975; Creese, Burt and Snyder, 1976; Seeman, Lee, Chau-Wong and Wong, 1976). These correlations were much higher than analogous correlations between central dopamine-sensitive adenylate cyclase a c t i v i t y and the i n vivo e f f e c t s of dopaminergic compounds. This suggested that dopamine-sensitive adenylate cyclase may not be related to dopamine transmission i n the brain. This p o s s i b i l i t y was supported by reports that s u l p i r i d e displaces t 3H]-haloperidol binding and blocks the i n vivo e f f e c t s of dopamine receptor agonists, but does not i n h i b i t dopamine-sensitive adenylate cyclase (Trabucchi, Longoni, Fresia and Spano, 1975; Garau, Govoni, S t e f a n i n i , Trabucchi and Spano, 1978; Jenner, Clow, R e a v i l l , Theodorou and Marsden, 1978; Woodruff, 1981). Based on these data, and data from research on peripheral dopamine receptors, Kebabian and Calne (1979) proposed that there are at least two forms of dopamine receptors; receptors associated with adenylate cyclase (DI receptors) and receptors independent of adenylate cyclase (D2 receptors). The above mentioned data indicate that D2 receptors are of major importance i n mediating the c e n t r a l e f f e c t s of dopamine receptor agonists and antagonists. Some data suggest that DI and D2 receptors have opposing e f f e c t s on dopamine sens i t i v e adenylate cyclase a c t i v i t y i n the striatum (Stoof and Kebabian, 1981; S a i l e r 14 and S a l a m a, 1 9 8 6 ) . R a d i o l i g a n d b i n d i n g r e s e a r c h a l s o s u g g e s t s t h a t c e n t r a l DI and D2 r e c e p t o r s c a n i n t e r a c t ( D u m b r i l l e - R o s s , N i z n i k and Seeman, 1 9 8 5 ) . I n a d d i t i o n , b e h a v i o u r a l e x p e r i m e n t s u s i n g " s e l e c t i v e " DI a g o n i s t s and a n t a g o n i s t s s u g g e s t t h a t DI r e c e p t o r s may be i n v o l v e d i n dopamine r e l a t e d b e h a v i o u r s s u c h as a p o m o r p h i n e - i n d u c e d s t e r e o t y p y and a m p h e t a m i n e - i n d u c e d l o c o m o t o r a c t i v i t y ( I o r i o , B a r n e t t , L e i t z , H o u s e r and K o r d u b a , 1983; C h r i s t e n s e n , A r n t , H y t t e l , L a r s e n and S v e n d s e n , 1984; M a i l m a n , S c h u l z , L e w i s , S t a p l e s , R o l l e m a and DeHaven, 1 9 8 4 ) . U n f o r t u n a t e l y , t h e s e s t u d i e s h a v e r e l i e d on t h e s p e c i f i c i t y o f a s m a l l number o f compounds. R e c e n t l y , t h e s p e c i f i c i t y o f t h e most f r e q u e n t l y e m p l o y e d " s p e c i f i c " DI a n t a g o n i s t (SCH23390) ha s b e e n q u e s t i o n e d ( H i c k s e t a l . , 1984; P l a n t j e , Hansen, Daus and S t o o f , 1984; O h l s t e i n and B e r k o w i t z , 1985; S c h u l z , S t a n f o r d and M a i l m a n , 1 9 8 5 ) . I t has a l s o b e e n n o t e d t h a t t h e i n v i v o a c t i v i t y o f t h i s compound may n o t be r e l a t e d t o i t s e f f e c t s on d o p a m i n e - s e n s i t i v e a d e n y l a t e c y c l a s e b u t may i n s t e a d be t h e c o n s e q u e n c e o f i t s i n t e r a c t i o n s w i t h p r e v i o u s l y u n d e t e c t e d dopamine r e c e p t o r s t h a t a r e n o t a s s o c i a t e d w i t h a d e n y l a t e c y c l a s e ( S c h u l z , S t a n f o r d , W y r i c k and M a i l m a n , 1 9 8 5 ) . The a v a i l a b l e d a t a a l s o q u e s t i o n w h e t h e r a d e n y l a t e c y c l a s e a c t i v i t y i s i n v o l v e d i n d o p a m i n e - r e l a t e d b e h a v i o u r s s i n c e t h e " D l r e c e p t o r a g o n i s t s " e l i c i t b e h a v i o u r s s i m i l a r t o t h o s e e l i c i t e d by D2 r e c e p t o r a g o n i s t s ( C h r i s t e n s e n e t a l . , 1984; M a i l m a n e t a l . , 1984) a l t h o u g h t h e y p r o d u c e o p p o s i n g e f f e c t s on a d e n y l a t e 15 cyclase a c t i v i t y (Stoof and Kebabian, 1981; S a i l e r and Salama; 1986). Furthermore, e l e c t r o p h y s i o l o g i c a l data indicate that dopamine can increase the amplitude of Ca^ + dependent somatic action potentials i n the s n a i l Helix  aspersa by decreasing a K + current controlled by c y c l i c AMP (Paupardin-Tritsch, Colombaioni, Deterre and Gerschenfeld, 1985). Dopamine can also decrease the same action potentials by decreasing Ca2 + current independent of c y c l i c AMP (Paupardin-Tritsch et 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 (Paupardin-Tritsch et a l . , 1985). Therefore, at the present time, the functions served by central DI dopamine receptors remain unclear. This issue w i l l most l i k e l y be resolved by studies of s t r i a t a l tissue where D2 and DI receptors are most numerous. This thesis was designed to study mpfc dopamine receptors. D2 receptors were examined because, i n contrast to the DI receptor l i t e r a t u r e , studies of the D2 dopamine receptor have c l e a r l y indicated that i t i s related to many i n vivo e f f e c t s of dopamine. Almost a l l of t h i s research has made use of radioligand binding assays. D2 receptors have been l a b e l l e d with many compounds (Seeman, 1980), with [ 3H]-haloperidol and [ 3H]-spiperone having been most frequently employed. The i n vivo potencies of dopaminergic compounds i n behavioural experiments with animals are highly correlated with t h e i r a f f i n i t i e s for l a b e l l e d D2 receptors (Creese et 16 a l . , 1976; Seeman, 1 9 8 0 ) . The a f f i n i t i e s o f dopamine r e c e p t o r a n t a g o n i s t s f o r t h e D2 r e c e p t o r a r e a l s o 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 n t i p s y c h o t i c p o t e n c i e s (Seeman e t a l . , 1975; C r e e s e e t a l . , 1976; Seeman e t a l . , 1976; Seeman, 1 9 8 0 ) . The l a t t e r f i n d i n g s u g g e s t s t h a t dopamine r e c e p t o r a n t a g o n i s t s p r o d u c e t h e i r a n t i p s y c h o t i c e f f e c t s by b l o c k i n g D2 dopamine r e c e p t o r s . L e s i o n s o f d o p a m i n e r g i c - n e u r o n s p r o d u c e i n c r e a s e s i n D2 r e c e p t o r s w h i c h p a r a l l e l i n c r e a s e s i n b e h a v i o u r a l s e n s i t i v i t y t o dopamine r e c e p t o r a g o n i s t s ( C r e e s e , B u r t and S n y d e r , 1977; Seeman, 1 9 8 0 ) . C h r o n i c ( s e v e r a l months) and s u b c h r o n i c ( s e v e r a l weeks) t r e a t m e n t w i t h dopamine r e c e p t o r a n t a g o n i s t s a l s o p r o d u c e s an i n c r e a s e i n t h e number o f D2 r e c e p t o r s a c c o m p a n i e d by an i n c r e a s e i n t h e b e h a v i o u r a l r e s p o n s e t o dopamine r e c e p t o r a g o n i s t s ( B u r t , C r e e s e and S n y d e r , 1977; M u l l e r and Seeman, 1 9 7 7 ) . T h e s e e x p e r i m e n t a l l y - i n d u c e d i n c r e a s e s i n D2 r e c e p t o r s a l s o p a r a l l e l 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 o f r e c e p t o r s u p e r s e n s i t i v i t y . T h u s , f o l l o w i n g 6-OHDA l e s i o n s s t r i a t a l n e u r o n s d i s p l a y i n c r e a s e d s e n s i t i v i t y t o 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 (Moore and Bloom, 1 9 7 8 ) . S t r i a t a l n e u r o n s show a s i m i l a r s u p e r s e n s i t i v i t y f o l l o w i n g t r e a t m e n t w i t h dopamine r e c e p t o r a n t a g o n i s t s (Moore and Bloom, 1 9 7 8 ) . The r e g i o n a l d e n s i t y o f D2 r e c e p t o r s i s c o r r e l a t e d w i t h t h e r e g i o n a l d e n s i t y o f dopamine i n n e r v a t i o n i n t h e b r a i n . T h u s , i n v i t r o and i n v i v o s t u d i e s h a v e d e m o n s t r a t e d t h a t D2 r e c e p t o r s a r e c o n c e n t r a t e d i n a r e a s o f t h e b r a i n w h i c h a r e 17 i n n e r v a t e d most d e n s e l y b y d o p a m i n e r g i c n e u r o n s ( L a d u r o n , J a n s s e n and L e y s e n , 1 9 7 8 ( a ) ; A l t a r , Kim and M a r s h a l l , 1 9 8 5 ) . D i f f e r e n t i a l c e n t r i f u g a t i o n s t u d i e s i n d i c a t e t h a t t h e D2 s i t e i s a s s o c i a t e d w i t h " m e m b r a n e - l i k e " s t r u c t u r e s as w o u l d be e x p e c t e d f r o m a c e l l s u r f a c e r e c e p t o r ( L a d u r o n , J a n s s e n and L e y s e n , 1 9 7 8 ( b ) ) . D2 dopamine r e c e p t o r s d i s p l a y s i m i l a r p r o p e r t i e s when m e a s u r e d w i t h any o f t h e many r a d i o l i g a n d b i n d i n g t e c h n i q u e s now a v a i l a b l e . The D2 s i t e s c a n be l a b e l l e d i n v i v o . B r a i n homogenates ( e . g . L a d u r o n e t a l . , 1 9 7 8 ( a ) ) o r b r a i n s l i c e s p r e p a r e d f o r a u t o r a d i o g r a p h y ( e g . Klemm, M u r r i n and K u h a r , 1979) c a n be s u b s e q u e n t l y a s s a y e d . The D2 s i t e s c a n a l s o be l a b e l l e d i n v i t r o i n s l i c e s ( e . g . A l t a r e t a l . , 1985) o r homogenates ( e . g . L i s t and Seeman, 1 9 8 1 ) . I n v i t r o l a b e l l i n g p r o v i d e s t h e c o n t r o l o v e r l i g a n d c o n c e n t r a t i o n s t h a t i s e s s e n t i a l f o r c h a r a c t e r i z a t i o n . I n v i v o l a b e l l i n g p e r m i t s t h e p h a r m a c o k i n e t i c p r o p e r t i e s o f t h e l a b e l l e d compounds t o be s t u d i e d i n r e l a t i o n t o t h e D2 r e c e p t o r s . R e c e n t l y D2 r e c e p t o r s h a v e b e e n m e a s u r e d i n l i v i n g humans by means o f p o s i t r o n e m i s s i o n t o m o g r a p h y ( F a r d e , H a l l , E h r i n and S e d v a l l , 1 9 8 6 ) . M e d i a l 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 Many e x p e r i m e n t s have b e e n d e s i g n e d t o d e t e r m i n e how t h e m e s o c o r t i c a l dopamine s y s t e m i s i n v o l v e d i n t h e l a r g e number o f d o p a m i n e - r e l a t e d mechanisms r e p o r t e d t o d a t e . 18 U n f o r t u n a t e l y , d e s p i t e t h e l a r g e amount o f b i o c h e m i c a l , e l e c t r o p h y s i o l o g i c a l and a n a t o m i c a l d a t a g a t h e r e d c o n c e r n i n g t h e m e s o c o r t i c a l dopamine s y s t e m , t e c h n i c a l p r o b l e m s h ave p r e c l u d e d any d e f i n i t i v e d e m o n s t r a t i o n and c h a r a c t e r i z a t i o n o f D2 dopamine r e c e p t o r s i n t h e p r e f r o n t a l c o r t e x . T h e r e f o r e , i n v e s t i g a t o r s have b e e n u n a b l e t o d e t e r m i n e how p r e f r o n t a l c o r t e x D2 r e c e p t o r r e g u l a t i o n i s i n v o l v e d i n d o p a m i n e - r e l a t e d mechanisms.- T h i s c o n s t i t u t e s a s u b s t a n t i a l l i m i t a t i o n i n t h e s t u d y o f m e s o c o r t i c a l dopamine p r o c e s s e s s i n c e , as m e n t i o n e d above, t h e dopamine D2 r e c e p t o r ( a s l a b e l l e d i n r a d i o l i g a n d b i n d i n g a s s a y s by t h e b u t y r o p h e n o n e s , h a l o p e r i d o l and s p i p e r o n e ) h a s b e e n s t r o n g l y i m p l i c a t e d i n t h e g r e a t m a j o r i t y o f dopamine r e c e p t o r a g o n i s t and a n t a g o n i s t e f f e c t s . C o n s i d e r i n g t h e r e l a t i v e l y s p a r s e dopamine i n n e r v a t i o n o f t h e mpfc, one w o u l d e x p e c t a r e l a t i v e l y low d e n s i t y o f D2 s i t e s i n t h i s r e g i o n . Many s t u d i e s have f a i l e d t o d e t e c t any p o t e n t i a l D2 dopamine b i n d i n g i n t h e p r e f r o n t a l c o r t e x ( C r e e s e , B u r t and S n y d e r , 1975; Q u i k e t a l . , 1978; A n d o r n , M i t r i u s and U ' P r i c h a r d , 1980; L i s t and Seeman, 1981; E n g e l , M u l l e r - S c h w e i n i t z e r and P a l a c i o s , 1984; P a l a c i o s , 1984; G e h l e r t and Wamsley, 1984; Morgan e t a l . , 1984; A l t a r e t a l . , 1985; Camus e t a l . , 1986; V a n d e r Werf e t a l . , 1 9 8 6 ) . However, o t h e r s t u d i e s have o b t a i n e d d a t a s u g g e s t i n g t h a t D2 dopamine r e c e p t o r s may be p r e s e n t i n t h e p r e f r o n t a l c o r t e x o f r a t s and humans ( P e d i g o , R e i s i n e , F i e l d s and Yamamura, 1978; M u r r i n and K u h a r , 1979; H o w l e t t and N a h o r s k i , 1980; 19 M a r c h a i s , T a s s i n and B o c k a e r t , 1980; M e l l e r , Bohmaker, R o s e n g a r t e n and F r i e d h o f f , 1982; M i t a e t a l . , 1982; L i s k o w s k y and P o t t e r , 1985; M a r t r e s e t a l . , 1985; M a r t r e s , B o u t h e n e t , S a l e s , S o k o l o f f and S c h w a r t z , 1 9 8 5 ) . N e v e r t h e l e s s , none o f t h e s e s t u d i e s h a s d e m o n s t r a t e d c o n c l u s i v e l y t h a t t h e s i t e s b e i n g m e a s u r e d a r e D2 dopamine r e c e p t o r s . The i s s u e i s f u r t h e r c o m p l i c a t e d by r e p o r t s t h a t s p i p e r o n e ( t h e l a b e l l e d l i g a n d u s e d i n most o f t h e s e s t u d i e s ) b i n d s t o s e r o t o n e r g i c ( L e y s e n , N i e m e g e e r s , T o l l e n a e r e and L a d u r o n , 1 9 7 8 ) , s p i r o d e c a n o n e ( H o w l e t t , M o r r i s and N a h o r s k i , 1979) and a - a d r e n e r g i c ( A n d o r n e t a l . , 1 9 8 0 ) s i t e s i n t h e f r o n t a l c o r t e x . C o n s i d e r i n g t h e l a r g e number o f p o t e n t i a l n e u r o t r a n s m i t t e r s i n t h e c o r t e x and t h e c o r r e s p o n d i n g t y p e s o f r e c e p t o r s , i t i s c l e a r t h a t any s u s p e c t e d D2 r e c e p t o r s i n t h e p r e f r o n t a l c o r t e x must be f u l l y c h a r a c t e r i z e d b e f o r e c o n c l u s i o n s c a n be r e a c h e d r e g a r d i n g t h e i r t r u e i d e n t i t y . I n E x p e r i m e n t s 1 and 2 o f t h i s t h e s i s a new a s s a y p r o c e d u r e i s d e s c r i b e d w h i c h was s u b s e q u e n t l y e m p l o y e d t o d e m o n s t r a t e t h a t D2 dopamine r e c e p t o r s a r e l o c a t e d i n t h e mpfc o f t h e r a t . The a s s a y was t h e n u s e d i n E x p e r i m e n t 3 t o s t u d y t h e p o s s i b l e e f f e c t s o f 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 t h e c h a r a c t e r i z e d mpfc D2 r e c e p t o r s and t o compare t h e s e e f f e c t s t o t h o s e o b s e r v e d i n t h e s t r i a t u m . The e f f e c t s o f f o o t s h o c k s t r e s s on mpfc and s t r i a t a l D2 r e c e p t o r s were i n v e s t i g a t e d i n E x p e r i m e n t 4. 20 Experiment 1: Development and Use of an Assay to  Characterize D2 Dopamine Receptors i n the Medial Prefrontal Cortex of the Rat Introduction As indicated i n the General Introduction, D2 dopamine receptors may e x i s t i n the mpfc. However, some investigators have f a i l e d to f i n d any i n d i c a t i o n of D2 s i t e s i n the mpfc. In order to demonstrate that a p a r t i c u l a r class of binding s i t e s represent D2 dopamine receptors, i t i s necessary to demonstrate that the binding s i t e s display a number of c r i t i c a l properties. Thus, compounds lacking 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 binding. Dopaminergic compounds should be e f f e c t i v e displacers and display potencies proportional to t h e i r i n  vivo pharmacological a c t i v i t y . The potencies of a l l compounds tested should correlate with t h e i r potencies on the well characterized s t r i a t a l D2 dopamine receptor. The potencies of endogenous agonists should display 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 that dopamine should be more potent than noradrenaline, which should be more potent than serotonin. The s i t e s should be saturable and t h e i r r e l a t i v e density should be at least roughly related to the known d i s t r i b u t i o n of dopaminergic innervation. 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 discussed i n Experiment 3. The process of demonstrating the presence of a p a r t i c u l a r form of receptor f i r s t requires the development of a reproducible and accurate method of measuring the s i t e . Once t h i s i s accomplished the candidate s i t e can then be tested to determine whether i t i s , i n f a c t , the proposed form of receptor. The method developed to measure the s i t e must also be reasonably e f f i c i e n t with regards to the amount of time and material i t requires or else the subsequent process of pharmacological characterization w i l l not be f e a s i b l e . The present project began with a series of experiments designed to determine how the standard dopamine receptor assays employed by investigators studying prefrontal cortex D2 dopamine receptors can be modified to produce s u f f i c i e n t r esolution to allow f e a s i b l e characterization of the po t e n t i a l D2 s i t e s i n the mpfc. Methods Tissue Preparation The striatum and the dopaminergically innervated areas of the mpfc were dissected on ice from male Wistar rats (300-400g; i n d i v i d u a l l y housed for 4 weeks) as shown i n Figure la - I d . Rats were k i l l e d by c e r v i c a l d i s l o c a t i o n . 22 F i g u r e 1. Shaded areas r e p r e s e n t t i s s u e used i n assays. Caudal s i d e s of 1 mm t h i c k s l i c e s are shown (a-c = mpfc; d = s t r i a t u m ) . 23 The brains were irrunediately removed and frozen on a microtome with CO2. Three successive, 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 the cortex. The s t r i a t a l tissue was also dissected from a 1 mm thick s l i c e . Figure 1(a)-(d) i l l u s t r a t e s the caudal side of such s l i c e s (after Konig and K l i p p e l , 1963) with the shaded areas representing the tissue used i n the assays. The tissue was maintained at 4°C thoughout the preparation procedure. I t was homogenized i n approximately 50 volumes of 0.32 M sucrose with a Brinkman Polytron (setting of 5.5 for 20 seconds) and centrifuged at 750 x g for 15 minutes. The p e l l e t was discarded and the supernatent was centrifuged at 100,000 x g for 60 minutes. The r e s u l t i n g p e l l e t was rehomogenized i n approximately 10 volumes of f r e s h l y prepared TEAN buffer (15 mM t r i s - H C l , 5 mM Na2EDTA, 1.1 mM ascorbate, and 12.5 uM nialamide; adjusted to pH 7.2 with NaOH) with a t e f l o n homogenizer, then incubated for t h i r t y minutes on i c e , and f i n a l l y stored at -80°C u n t i l i t was used i n binding assays. Binding Assays Constituents of the Binding Reaction The binding reactions took place i n 25 ml Erlenmeyer flas k s containing the following: (1) Buffer, (2) 150 u l of [-^H]-spiperone dissolved i n 95% ethanol ( f i n a l concentration 25 pM), (3) 150 y.1 of ascorbate dissolved i n d i s t i l l e d water ( f i n a l concentration of 22.0 uM), or competing drug dissolved i n ascorbate solution, (4) 150 u,l methanol or competing drug dissolved i n methanol, (5) 150 u.1 of ketanserin t a r t r a t e dissolved i n 95% ethanol ( f i n a l concentration of 10 nM), (6) 150 u.1 of tissue homogenate. The f i n a l volume of the reaction was 15 ml. Buffer The buffer consisted of 50 mM t r i s - H C l and 100 mM NaCl. The pH was adjusted to 7.9 with NaOH except where otherwise noted. The t r i s - H C l concentration i s standard and the NaCl was added because s u l p i r i d e binding to dopamine receptors has been shown to require Na + (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 [ 3H]-spiperone dissolved i n 95% ethanol (23-26 Ci/mmole, New England Nuclear) was added to each f l a s k . The f i n a l concentration was 25 pM except where otherwise noted. The low concentration of spiperone was chosen for two reasons: (1) Preliminary saturation analysis revealed that spiperone 25 binds to D2 s i t e s i n the striatum (binding displaced by 10"5 M s - ( - ) - s u l p i r i d e ) with an apparent Kd of approximately 25 pM. To ensure accuracy, competition studies should be performed with a concentration of l a b e l l e d ligand at or below the Kd. (2) Previous reports (Howlett and Nahorski, 1980; Marchais et a l . , 1980) have suggested (and i t was presently confirmed) that the use of low concentrations of spiperone increases the percent of binding to p o t e n t i a l D2 s i t e s i n the f r o n t a l cortex. Thus, the pot e n t i a l D2 s i t e s begin to saturate at higher concentrations while the nonspecific and f i l t e r binding increase i n a l i n e a r fashion. Ascorbate Ascorbate was employed as an antioxidant to protect the drugs included i n the reaction mixture. One-hundred and f i f t y m i c r o l i t r e s of 1.1 mM ascorbate was added to each reaction to produce a f i n a l concentration of 22.0 u\M (11.0 JJLM from the ascorbate solution and 11.0 uM from the TEAN buffer added with the tissue [see above]). The ascorbate solution also acted as the vehicle for the water soluble compounds which were tested i n competition studies (see Experiments 1 and 2). 26 Methanol Methanol acted as the vehicle for the s p e c i f i c D2 receptor antagonist, s - ( - ) - s u l p i r i d e , as well as the majority of the compounds tested i n the competition studies (see Experiments 1 and 2). Thus, 150 u,l of methanol were added to each reaction. A f i n a l concentration of 10~5 M s - ( - ) - s u l p i r i d e was used to define s p e c i f i c binding as i s preferred i n D2 dopamine receptor assays ( L i s t and Seeman, 1981). Ketanserin As mentioned above, [^H]-spiperone binds to serotonergic S2 receptors i n the f r o n t a l cortex. In order to increase the percent of [ 3H]-spiperone binding to p o t e n t i a l D2 receptors i n the mpfc, the S2 receptor antagonist, ketanserin, (Leysen, Awouters, Kennis, Laduron, Vandenberk and Janssen, 1981) was included i n the binding reactions. Competition studies revealed a s e l e c t i v i t y of ketanserin for serotonergic (S2) vs dopaminergic (D2) s i t e s . Figure 2 i l l u s t r a t e s that ketanserin concentrations between 1 nM and 10 nM i n h i b i t e d 30-35% of t o t a l tissue binding i n the mpfc and less than 5% i n the striatum. Therefore, 150 ul of ketanserin t a r t r a t e , dissolved i n 95% ethanol, 27 K E T A N S E R I N C O N C E N T R A T I O N ( M ) Figure 2. The amount of t o t a l tissue binding of [^H]-spiperone (25 pM) competed for by various concentrations of ketanserin t a r t r a t e i n the mpfc and the striatum. 28 were incorporated into each binding reaction at a f i n a l concentration of 10 nM. The percentage of S2 binding i n the f r o n t a l cortex was less than that reported by others (Leysen et a l , 1978). This was probably due to the lower concentrations of spiperone used i n the present experiments as well as the l i m i t i n g of f r o n t a l cortex tissue samples to the dopamine innervated areas. Tissue For the binding assays the tissue was thawed at 4°C and d i l u t e d with f r e s h l y prepared TEAN buffer. I t was then thoroughly mixed on a vortex mixer, to ensure homogenization, and incubated on ice for 45 minutes. Aliquots of the tissue homogenate (150 u.1; approximately 100-150 u,g of mpfc protein or 50-75 u,g of s t r i a t a l protein) were added l a s t to the Erlenmeyer flasks to s t a r t the reaction. The amount of s p e c i f i c binding i n both brain regions was l i n e a r l y related to the amount of tissue i n the incubations over the range of tissue concentrations employed. Protein concentrations were determined according to Lowry, Rosebrough, Farr and Randall, (1951). 29 Termination of the Binding Reaction The flasks were gently shaken for 2.75 hours at room temperature. The reactions were terminated by vacuum f i l t r a t i o n . F i l t r a t i o n Apparatus Preliminary experiments revealed that the variance associated with the f i l t r a t i o n procedure was greatly reduced when f i l t r a t i o n was performed under a high and consistent vacuum. A Gelman vacuum pump (model 13152) was found to be more s a t i s f a c t o r y than the laboratory "house" vacuum. The pump was attached to a 500 ml sidearm Erlenmeyer f l a s k which had a 50 ml Gelman f i l t e r funnel secured to the top with a rubber stopper. While more reactions could be terminated per unit of time with f i l t r a t i o n manifolds, the funnel-flask apparatus reduced the variance associated with the f i l t r a t i o n procedure. As a r e s u l t fewer reactions were required i n the characterization experiments. Therefore, the use of the funnel-flask apparatus proved to be the more e f f i c i e n t procedure. F i l t e r s The reactions were f i l t e r e d through Whatman GF/B f i l t e r s . Whatman GF/C f i l t e r s were also tested i n an attempt to reduce the binding of the [ 3H]-spiperone to the f i l t e r s . However, the po t e n t i a l D2 binding 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 were used thereby suggesting that GF/C f i l t e r s do not trap as many receptors as GF/B f i l t e r s . Washes of F i l t e r s After the f i l t r a t i o n of reaction mixtures, the f i l t e r s were washed. T y p i c a l l y , receptor binding assays employ 1-4 rapid washes with buffer. However, compared to most ligand employed i n binding assays, [ 3H]-spiperone has a very high a f f i n i t y for the D2 dopamine receptor. Therefore, a series of experiments were run with s t r i a t a l tissue to tes t the eff e c t s of modifications i n the standard wash procedures. I was hoped that these experiments would reveal methods of reducing f i l t e r binding and nonspecific binding of [ 3H]-spiperone while not a f f e c t i n g the binding of [ 3H]-spiperone to D2 receptors. Increasing the number of 5 ml washes reduced f i l t e r binding and nonspecific binding. An increase from 4 to 6 washes had no e f f e c t on D2 binding, however, i f 10 washes 31 were employed the D2 binding was marginally reduced. Therefore, s i x 5 ml washes were used. Increasing the temperature of the washes from the standard of 4°C to room temperature had no measurable e f f e c t on f i l t e r , nonspecific, or D2 binding. Since the wash buffer was routinely stored at 4°C, that temperature was used for the wash procedure. Adding the detergent, T r i t o n X-100, to the wash buffer at concentrations ranging from .025% to .075% greatly reduced D2 binding while having less e f f e c t on f i l t e r binding and nonspecific binding. However, 10% ethanol or 10% acetone i n the wash buffer markedly reduced f i l t e r binding while having no e f f e c t on nonspecific or D2 binding. Ethanol was chosen over acetone due to economic and safety considerations. F i l t r a t i o n Procedure To further reduce the v a r i a b i l i t y associated with the f i l t r a t i o n , a timed procedure was employed for each f i l t r a t i o n : (1) the f i l t e r was moistened with 5 ml of d i s t i l l e d water (2) 5 seconds l a t e r the binding reaction was f i l t e r e d (3) 10 seconds l a t e r the f i l t e r was exposed to s i x 5ml washes of 4 °C buffer (9 parts 50 mM t r i s - H C l , pH 7.9 : 1 part absolute ethanol) at a rate 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 funnel and placed 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 Radioactivity Approximately 1 hour after the f i l t r a t i o n , each f i l t e r was vigorously shaken for 5 minutes with 15 ml of Pico-Fluor 15 (a high e f f i c i e n c y s c i n t i l l a t i o n f l u i d ; Packard Instruments). Starting at least 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 with a Packard Tri-Carb 4530 l i q u i d s c i n t i l l a t i o n counter at an e f f i c i e n c y of approximately 50%. An average of the values of four 20 minute recording periods was used i n a l l mpfc calculations i n order to minimize the v a r i a b i l i t y associated with the measurement of r a d i o a c t i v i t y . Only one 20 minute reading was required for each s t r i a t a l v i a l . Competition Studies Various dopaminergic and nondopaminergic compounds were tested for t h e i r a b i l i t y to compete for s p e c i f i c binding of 25 pM [ 3H]-spiperone (defined by 10"^ M s - ( - ) - s u l p i r i d e ) i n the striatum and mpfc of the r a t . Incubations with no competing drug or 10"^ M s - ( - ) - s u l p i r i d e were done i n quadruplicate i n each mpfc assay and i n t r i p l i c a t e f or each s t r i a t a l assay. Competing drugs were run at f i v e concentrations i n duplicate or t r i p l i c a t e i n mpfc assays and 33 i n d u p l i c a t e i n s t r i a t a l a s s a y s . A d d i t i v i t y t o b l a n k c o n t r o l s were a l s o r u n f o r e a c h compound t e s t e d . T h u s , l O - ^ M s - ( - ) - s u l p i r i d e and t h e c o m p e t i n g d r u g were i n c l u d e d i n t h e same b i n d i n g r e a c t i o n . I f t h e c o m p e t i n g d r u g competed f o r n o n s p e c i f i c b i n d i n g , t h e n one w o u l d e x p e c t t h e c o m b i n a t i o n o f t h e d r u g and t h e 10"^ M s - ( - ) - s u l p i r i d e t o p r o d u c e l e s s [ 3 H ] - s p i p e r o n e b i n d i n g t h a n t h e 10"^ M s - ( - ) - s u l p i r i d e a l o n e . I C 5 0 ' s ( t h e c o n c e n t r a t i o n o f c o m p e t i n g d r u g w h i c h r e d u c e s t h e s p e c i f i c b i n d i n g t o 50% o f t h e o r i g i n a l amount) were c a l c u l a t e d by l o g i t t r a n s f o r m a t i o n f o l l o w e d by l i n e a r r e g r e s s i o n a n a l y s i s . The I C 5 0 ' s l i s t e d i n T a b l e 1 a r e t h e mean (± s.e.m.) o f a t l e a s t t h r e e i n d e p e n d e n t e x p e r i m e n t s . The s u l p i r i d e c o m p e t i t i o n c u r v e s p l o t t e d i n F i g u r e 3 a r e d e r i v e d f r o m t h e l o g i t l i n e a r r e g r e s s i o n e q u a t i o n . The c o m p e t i t i o n c u r v e s f o r t h e dopamine r e c e p t o r a g o n i s t s , w h i c h a r e p l o t t e d i n F i g u r e s 6,7 and 8, were drawn b y hand s i n c e e v i d e n c e s u g g e s t s t h a t m u l t i p l e b i n d i n g s i t e s may be i n v o l v e d ( s e e d i s c u s s i o n o f t h i s s e c t i o n ) b u t t o o few d a t a p o i n t s a r e a v a i l a b l e f o r s t a t i s t i c a l c a l c u l a t i o n o f e x a c t c u r v e s . S t a t i s t i c a l c o m p a r i s o n s were made w i t h S t u d e n t ' s t w o - t a i l e d t - t e s t s f o r d e p e n d e n t o r i n d e p e n d e n t s a m p l e s o r P e a r s o n ' s c o r r e l a t i o n c o e f f i c i e n t s o f l o g v a l u e s . 34 Results As shown i n Figure 3, s - ( - ) - s u l p i r i d e (the biochemically and behaviorally more active isomer [Jenner, Clow, R e a v i l l , Theodorou and Marsden, 1980]) was more potent than r-(+)-sulpiride i n both the striatum and the mpfc. The competition curves were very s i m i l a r i n the two brain regions when they were plotted as percent of s p e c i f i c binding. The s - ( - ) - s u l p i r i d e competition curve for mpfc tissue displayed a d i s t i n c t , intermediate (in terms of t o t a l tissue binding) plateau i n the 10"^ M to 10"^ M range. This r e s u l t 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 ligand for the s p e c i f i c binding s i t e s . The s p e c i f i c binding amounted to 95-98% of t o t a l striatum binding and 20-25% of t o t a l mpfc binding. The r e s u l t s of competition studies presented i n Table 1 i l l u s t r a t e that the s p e c i f i c binding i n both brain regions displayed the c h a r a c t e r i s t i c s of D2 receptors. A l l nondopaminergic compounds tested were i n e f f e c t i v e at nanomolar concentrations. Dopamine was more potent than noradrenaline which was more potent than serotonin. At nanomolar concentrations a l l the dopaminergic antagonists competed for the s p e c i f i c binding s i t e s . The IC50's of the dopaminergic antagonists i n the mpfc assays correlated with t h e i r i n vivo potencies as antagonists of apomorphine-induced emesis i n the dog (r=.709; d/f=7; p<.05; Janssen and 35 S U L P I R I D E 1 0 0 H Z o 2 8 0 -CD O U . O 6 0 UJ Q. CO O 4 0 H Z UJ DC 2 0 H U J 0 L 1 0 A 6 c->; S t r i a t u m c+3; S t r i a t u m i; Prefrontal Cortex i; Prefrontal Cortex 10 10 1 0 C O N C E N T R A T I O N ( M ) 1 0 F i g u r e 3. The pe r c e n t of s p e c i f i c [ J 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 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 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 s t r i a t u m . Table 1 I n h i b i t i o n of S p e c i f i c [ 3H]-Spiperone (25 pM) B i n d i n g l o g I C 5 0 (M) ± s.e.m. Compound Str i a t u m Medial P r e f r o n t a l Cortex 1. Pimozide -10. 31 + .07 -9 .95 + .16 2. H a l o p e r i d o l -8. 73 + .04 -8 .68 + .09 3. B e n p e r i d o l -9. 35 + .04 -9 .68 + .09 4. Spiperone -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. Promazine -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. ( + ) - s u l p i r i d e -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. S e r o t o n i n > -5 > -5 16. B a c l o f e n > -5 > -5 17. A t r o p i n e > -5 > -5 18. Naloxone > -5 > -5 19. Phentolamine > -6 > -6 20. Clonazepam > -5 > -5 37 Van B e v e r , 1975) and a p o m o r p h i n e - i n d u c e d b e h a v i o r i n t h e r a t (r=.752; d/f=7; p<.02; N i e m e g e e r s , L e n a e r t s , A r t o i s and J a n s s e n , 1 9 7 7 ) . I n a d d i t i o n , t h e I C 5 0 ' s o f t h e dopamine r e c e p t o r a n t a g o n i s t s were 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 p o t e n c i e s as a n t i p s y c h o t i c s (r=.818; d/f=7; p<.01; Seeman, 1980; C r e e s e e t a l . , 1 9 7 6 ) ( F i g u r e 4 ) . I n a c c o r d a n c e w i t h p r e v i o u s r e p o r t s , a l l o f t h e s e c o r r e l a t i o n s were a l s o o b s e r v e d w i t h t h e D2 r e c e p t o r s i n s t r i a t a l t i s s u e ( C r e e s e e t a l . , 1976; Seeman, 1 9 8 0 ) . The I C 5 0 ' s o f a l l compounds t e s t e d w i t h mpfc t i s s u e were v e r y h i g h l y c o r r e l a t e d (r=.976; d / f = l l ; p< .01) w i t h t h e I C 5 0 ' s o b t a i n e d f r o m t h e s t r i a t a l a s s a y s ( F i g u r e 5 ) . The mpfc IC50 v a l u e s a l s o c o r r e l a t e w i t h o t h e r p u b l i s h e d v a l u e s f o r D2 dopamine r e c e p t o r s . T h u s , t h e IC50 v a l u e s c o r r e l a t e w i t h t h o s e o b t a i n e d w i t h [ 3 H ] - s p i p e r o n e (r=.917; d/f=10; p<.01; L e y s e n , Gommeren and L a d u r o n , 1978) and [ 3 H ] - h a l o p e r i d o l (r=.874; d/f=10; p<.01; L e y s e n e t a l . , 1978; r=.923; d/f=9; p,.01; B u r t , C r e e s e and S n y d e r , 1 9 7 6 ) . A l t h o u g h a h i g h c o r r e l a t i o n was o b t a i n e d between t h e IC50 v a l u e s o f t h e mpfc and t h o s e o f t h e s t r i a t u m , c l o s e r e x a m i n a t i o n o f t h e d a t a r e v e a l s t h a t t h e d o p a m i n e r g i c a g o n i s t s a r e more p o t e n t a t d i s p l a c i n g D2 b i n d i n g i n t h e mpfc t h a n i n t h e s t r i a t u m . No s u c h d i f f e r e n c e i s s e e n w i t h t h e d o p a m i n e r g i c a n t a g o n i s t s as a w h o l e o r any o f t h e s u b c l a s s e s o f s t r u c t u r a l l y r e l a t e d a n t a g o n i s t s . F i g u r e s 6, 7 and 8 p r e s e n t t h e c o m p e t i t i o n c u r v e s f o r t h e dopamine r e c e p t o r a g o n i s t s , dopamine, a p o m o r p h i n e and 3 8 F i g u r e 4. 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. The numbers b e s i d e the data p o i n t s r e f e r t o Tabl e 1. 39 Figure 5. The re l a t i o n s h i p between the a f f i n i t i e s of various dopamine receptor antagonists for mpfc D2 receptors and the a f f i n i t i e s of the same drugs for s t r i a t a l D2 receptors. The numbers beside the data points r e f e r to Table 1. D O P A M I N E Figure 6. The percent of s p e c i f i c [ JH]-spiperone (25 pM) binding (binding competed for by IO""5 M s - ( - ) - s u l p i r i d e ) competed for by various concentrations of dopamine i n the mpfc and the striatum. A P O M O R P H I N E 1 0 0 -C O N C E N T R A T I O N ( M ) Figure 7. The percent of s p e c i f i c [ •'H]-spiperone (25 pM) binding (binding competed for by 10"-> M s - ( - ) - s u l p i r i d e ) competed for by various concentrations of apomorphine i n the mpfc and the striatum. 42 Figure 8 . The percent of s p e c i f i c [ JH]-spiperone (25 pM) binding (binding competed for by IO'* M s - ( - ) - s u l p i r i d e ) competed for by various concentrations of ADTN i n the mpfc and the striatum. 43 ADTN. Dopamine displays a tendency to be more potent i n the mpfc p a r t i c u l a r l y at the lowest concentration (10 nM), but t h i s difference f a i l s to reach s t a t i s t i c a l s i g n i f i c a n c e . Apomorphine was more potent i n the mpfc than i n the striatum. The mean IC50 of apomorphine was s i g n i f i c a n t l y lower i n the mpfc than i n the striatum (t= 5.04; d/f=4; p<.005). The dopamine and apomorphine results prompted a change i n the design of the competition experiments. Thus, ADTN was tested i n four independent experiments i n which s t r i a t a l tissue and mpfc tissue from the same rats were examined i n the same assay and therefore, under exactly the same assay conditions. The a p r i o r i prediction that 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 at competing for mpfc binding i n the nanomolar range where i t i s known to be a potent a-adrenergic receptor antagonist (Peroutka, U'Prichard, Greenberg and Snyder, 1977). As mentioned i n the General Introduction, others have reported that spiperone can bind to a-adrenergic s i t e s i n the f r o n t a l cortex (Andorn et a l . , 1980). The lack of any i n d i c a t i o n of t h i s i n the present data may be due to differences i n assay conditions or tissue sources. However, i t i s also possible that the ketanserin, which has been demonstrated to be an a-adrenergic antagonist i n binding studies (Leysen et a l . , 1981) may have occluded the a-adrenergic s i t e s . 44 Discussion The r e s u l t s of the competition experiments indicate that the s p e c i f i c binding measured i n the mpfc displays the expected c h a r a c t e r i s t i c s of D2 dopamine receptors. The high c o r r e l a t i o n between the IC50 values of dopamine receptor antagonists i n the mpfc and the potencies of the same drugs as antipsychotics, constitutes the f i r s t evidence from ligand binding studies that the mpfc may be a s i t e of the therapeutic action f o r these drugs. However, since t h i s same c o r r e l a t i o n has been observed with D2 dopamine binding i n s u b c o r t i c a l regions (Seeman, 1980; Creese et a l . , 1976), the c o r r e l a t i o n a l data do not indicate which dopaminergically innervated region, or regions, are the s i t e or s i t e s at which dopamine receptor antagonists produce t h e i r antipsychotic e f f e c t s . The greater potency of dopamine receptor agonists for the mpfc D2 receptors compared to the s t r i a t a l D2 receptors has a number of i n t e r e s t i n g implications. This difference indicates that although the mpfc dopamine receptors are of the D2 type they are not i d e n t i c a l to the s t r i a t a l receptors. A d i f f e r e n t receptor population i n the mpfc could p o t e n t i a l l y be responsible for some of the unique c h a r a c t e r i s t i c s of the mesocortical dopamine system which were outlined i n the General Introduction. Recent research on D2 dopamine receptors suggests that the D2 receptor protein can be present i n two interchangable conformations 45 (Creese, Sibley and Le f f , 1984; Grigoriadis and Seeman, 1985). One of these conformations, the D2(high) s i t e displays a high a f f i n i t y (Kd=l-10 nM) for dopamine receptor agonists while the other conformation, the D2(low) s i t e , displays a lower a f f i n i t y (Kd*200-2,000 nM) for dopamine receptor agonists. Both s i t e s display equally high a f f i n i t y for dopamine receptor antagonists. In support of t h i s model, when dopamine receptor agonists compete with a ra d i o a c t i v e l y l a b e l l e d dopamine receptor antagonist for D2 s i t e s , the competition curves are not as steep as one would expect i f a single form of binding s i t e were l a b e l l e d . Under the same conditions, dopamine receptor antagonists display competition curves that are steeper than those of the dopamine receptor agonists and consistent with a single s i t e being l a b e l l e d . The present r e s u l t s are i n complete agreement with these findings. A comparison of the s u l p i r i d e competition curves (Figure 3) and those of the dopamine receptor agonists (Figures 5,6 and 7) reveals the difference that has previously been observed between agonist and antagonist competition curves (Creese, Sibley and Leff, 1984; Grigoriadis and Seeman, 1985). If D2 receptors are present i n D2(high) and D2(low) states, i t i s possible that the mpfc possesses a higher proportion of D2(high) s i t e s than i s present i n the striatum. This would explain why dopamine receptor agonists, but not dopamine receptor antagonists, are more potent i n the mpfc. I t would also explain why the e f f e c t i s 46 much l e s s p r o n o u n c e d when dopamine i s e m p l o y e d as a c o m p e t i n g d r u g b e c a u s e dopamine b i n d s t o a l o w e r p r o p o r t i o n o f D 2 ( h i g h ) s i t e s compared t o ADTN ( G r i g o r i a d i s and Seeman, 1 9 8 5 ) . O t h e r d a t a i n d i c a t e t h a t N a + i o n s r e d u c e t h e number o f d e t e c t a b l e D 2 ( h i g h ) s i t e s ( G r i g o r i a d i s and Seeman, 1985) s u g g e s t i n g t h a t t h e d i f f e r e n c e s p r e s e n t l y o b s e r v e d m i g h t h a ve b e e n g r e a t e r i f N a + i o n s were n o t p r e s e n t . U n f o r t u n a t e l y , t h e N a + i o n s a r e r e q u i r e d f o r t h e s u l p i r i d e b i n d i n g w h i c h d e f i n e s t h e s p e c i f i c b i n d i n g . I f t h e mpfc c o n t a i n s a h i g h e r p r o p o r t i o n o f D 2 ( h i g h ) s i t e s compared t o t h e s t r i a t u m , t h e n t h i s d i f f e r e n c e s h o u l d be most o b v i o u s i n c o m p e t i t i o n s t u d i e s when low c o n c e n t r a t i o n s o f c o m p e t i n g a g o n i s t s a r e e m p l o y e d . Thus i t h a s b e e n r e p o r t e d t h a t a t l o w e r a g o n i s t c o n c e n t r a t i o n s , a g r e a t e r p r o p o r t i o n o f D 2 ( h i g h ) s i t e s , compared t o D 2(low) s i t e s , i n t e r a c t w i t h t h e a g o n i s t b e c a u s e t h e s i t e s have a h i g h e r a f f i n i t y f o r t h e a g o n i s t ( C r e e s e e t a l . , 1984; G r i g o r i a d i s and Seeman, 1 9 8 5 ) . As t h e c o n c e n t r a t i o n i s i n c r e a s e d , t h e D 2 ( h i g h ) s i t e s t e n d t o w a r d s s a t u r a t i o n and t h e D 2(low) s i t e s b e g i n t o i n f l u e n c e t h e r e s u l t s t o a g r e a t e r e x t e n t . G i v e n t h e r e p o r t e d a f f i n i t y o f D 2 ( h i g h ) s i t e s f o r dopamine r e c e p t o r a g o n i s t s ( C r e e s e e t a l . , 1984; G r i g o r i a d i s and Seeman, 1985) t h e e f f e c t s o f t h e D 2 ( h i g h ) s i t e s s h o u l d be g r e a t e s t a t a g o n i s t c o n c e n t r a t i o n s o f a p p r o x i m a t e l y 10 nM o r l o w e r . The p r e s e n t d a t a s u g g e s t t h a t t h e h i g h e r a f f i n i t y o f mpfc D2 r e c e p t o r s f o r dopamine r e c e p t o r a g o n i s t s may be due t o a h i g h e r p r o p o r t i o n o f 47 D2(high) s i t e s i n the mpfc. Thus, at concentrations of 10 nM or l e s s , apomorphine was more potent i n the mpfc than i n the striatum (t=5.17; d/f=4; p<.005). In order to produce an equivalent reduction i n [ 3H]-spiperone binding, only approximately 1/6 as high a concentration of apomorphine was required i n mpfc binding reactions compared to s t r i a t a l reactions. As higher concentrations of apomorphine were employed, t h i s difference was reduced. The ADTN data displayed a si m i l a r pattern. ADTN was approximately three times as potent at concentrations of 10 nM or less (t=18.70; d/f=3; p<.001) and t h i s difference was also smaller at higher concentrations. Low doses of apomorphine, ADTN and other dopamine receptor agonists, produce many behavioural e f f e c t s which are very s i m i l a r to those observed following the administration of dopamine receptor antagonists. For example, low concentrations of dopamine receptor agonists 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 Naylor, 1981). In humans, they can cause sedation, suppress dyskinetic or choreiform movements, potentiate parkinsonism, and reduce psychosis (Seeman, 1980). These phenomena are commonly considered to be the r e s u l t of the dopamine receptor agonists s e l e c t i v e l y a c t i v a t i n g dopamine "autoreceptors" located on dopaminergic neurons. "Autoreceptors" are believed to be s e l e c t i v e l y activated because dopamine receptor agonists are thought to have a higher a f f i n i t y for dopamine autoreceptors than for 48 p o s t s y n a p t i c dopamine r e c e p t o r s . I f t h e m e s o c o r t i c a l dopamine s y s t e m t o n i c a l l y i n h i b i t s t h e s u b c o r t i c a l dopamine s y s t e m s , as h a s b e e n p r o p o s e d ( s e e G e n e r a l I n t r o d u c t i o n ) , t h e n t h e p r e s e n t d a t a r a i s e t h e p o s s i b i l i t y t h a t some o f t h e s e e f f e c t s a t t r i b u t e d t o an a c t i o n a t a u t o r e c e p t o r s may i n f a c t be due t o t h e s e l e c t i v e a c t i v a t i o n o f mpfc D2 r e c e p t o r s , l o c a t e d on n o n d o p a m i n e r g i c n e u r o n s , by t h e low d o s e s o f dopamine r e c e p t o r a g o n i s t s . 49 E x p e r i m e n t 2: C h a r a c t e r i z a t i o n a t pH 6.2 o f M e d i a l  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 i n t h e R a t I n t r o d u c t i o n S p i p e r o n e h a s b e e n r e p o r t e d t o d i s p l a y s a t u r a b l e , h e a t s e n s i t i v e , r e v e r s i b l e , h i g h a f f i n i t y b i n d i n g w h i c h i s n o t d i s p l a c e d b y n a n o m o l a r c o n c e n t r a t i o n s o f any o f many compounds t e s t e d e x c e p t b y s p i p e r o n e and b u t y r o p h e n o n e d e r i v a t i v e s o f t h e s p i r o d e c a n o n e t y p e ( H o w l e t t e t a l . , 1 9 7 9 ) . F o l l o w i n g t h e c o m p l e t i o n o f E x p e r i m e n t 1, B r u i n i n k and L i c h t e n s t e i g e r (1984) r e p o r t e d t h a t i n f o r e b r a i n t i s s u e f r o m 30 d a y o l d r a t s t h e b i n d i n g o f s p i p e r o n e t o s p i r o d e c a n o n e s i t e s i s d r a m a t i c a l l y r e d u c e d by a s h i f t i n i n c u b a t i o n pH f r o m t h e t y p i c a l 7.4-7.9 r a n g e t o a pH o f 6.2. T h e y a l s o r e p o r t e d t h a t d o p a m i n e r g i c D2 b i n d i n g s i t e s and s e r o t o n e r g i c S2 b i n d i n g s i t e s a r e o n l y m i n i m a l l y a f f e c t e d . T h i s f i n d i n g s u g g e s t e d t h a t i f a s i g n i f i c a n t p r o p o r t i o n o f t h e " n o n s p e c i f i c " b i n d i n g i n t h e p r e s e n t mpfc a s s a y a t pH 7.9 i s t o s p i r o d e c a n o n e s i t e s t h e n a r e d u c t i o n i n pH may r e d u c e t h e " n o n s p e c i f i c " b i n d i n g and t h e r e b y i n c r e a s e t h e r e s o l u t i o n o f t h e a s s a y . T h i s p o s s i b i l i t y was i n v e s t i g a t e d i n t h e p r e s e n t e x p e r i m e n t . 50 Methods I n i t i a l competition studies with tissue samples from either the mpfc or the striatum revealed that when incubations were run at pH 6.2 both isomers of s u l p i r i d e were approximately 50 f o l d less potent at competing for the s p e c i f i c binding than at pH 7.9. The competition curves at pH 6.2 were the same shape as curves obtained at pH 7.9 but simply s h i f t e d to the ri g h t (higher m o l a r i t i e s ) . I t i s not clear from the present 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 a fundamental change i n the int e r a c t i o n between the s u l p i r i d e and the D2 receptor or i s simply due to an a r t i f a c t u a l reduction i n the free s u l p i r i d e present i n the binding reaction. Thus, s u l p i r i d e may not be as stable at pH 6.2 as i t i s at pH 7.9 or may not be as soluble. In l i g h t of these r e s u l t s , the s p e c i f i c D2 binding i n experiments run at pH 6.2 was defined as that binding i n h i b i t e d by 10" 4 M s - ( - ) - s u l p i r i d e . This was done i n order to ensure that the defining concentration of s u l p i r i d e occluded as complete a population of D2 dopamine receptors as possible. The pH of the stock solution of s u l p i r i d e was adjusted to pH 6.2 so that i t would not influence the pH of the binding reaction. This was necessary because t r i s - H C l has less buffering capacity at pH 6.2 than at pH 7.9. In order to d i r e c t l y characterize the s p e c i f i c s i t e s l a b e l l e d 51 a t pH 6.2, a s e r i e s o f c o m p e t i t i o n e x p e r i m e n t s was r u n w i t h t h e same d r u g s as were t e s t e d a t pH 7.9. S a t u r a t i o n e x p e r i m e n t s r e v e a l e d t h a t a t pH 6.2, t h e a f f i n i t y o f s p i p e r o n e f o r s p e c i f i c b i n d i n g s i t e s i n b o t h b r a i n r e g i o n s i s l o w e r (KD=75 pM) t h a n a t pH 7.9 (KD=25 pM). The d a t a a r e p r e s e n t e d i n E x p e r i m e n t 4. T h e r e f o r e , i n o r d e r t o c o m p e n s a t e f o r t h e r e d u c e d amount o f s p e c i f i c b i n d i n g , t h e [ 3 H ] - s p i p e r o n e c o n c e n t r a t i o n i n t h e c o m p e t i t i o n a s s a y s was i n c r e a s e d t o 35 pM. W i t h t h e e x c e p t i o n o f t h e above m e n t i o n e d m o d i f i c a t i o n s t h e c o m p e t i t i o n a s s a y s were p e r f o r m e d as d e s c r i b e d i n E x p e r i m e n t 1. R e s u l t s The c o m p e t i t i o n s t u d i e s d e m o n s t r a t e d t h a t t h e p o t e n c i e s o f t h e compounds t e s t e d i n t h e mpfc a t pH 6.2 ( T a b l e 2) a r e 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 p o t e n c i e s a t pH 7.9 e v e n when t h e IC50 v a l u e s o f s p i p e r o n e and b o t h i s o m e r s o f s u l p i r i d e a r e i n c l u d e d i n t h e c a l c u l a t i o n s (r=.949; d / f = l l ; p<.01). The same p a t t e r n o f c o r r e l a t i o n s t h a t was o b s e r v e d w i t h t h e c h a r a c t e r i z a t i o n a t pH 7.9 was a l s o o b s e r v e d a t pH 6.2. T h u s , IC50 v a l u e s f r o m t h e mpfc were h i g h l y c o r r e l a t e d w i t h t h e v a l u e s o b t a i n e d i n t h e s t r i a t u m (r=.992; d / f = l l ; p<.01). The IC50 v a l u e s o f dopamine r e c e p t o r a n t a g o n i s t s o b t a i n e d i n t h e mpfc c o r r e l a t e d w i t h 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 d r u g s (r=.751; d/f=7; p<.02). I n a d d i t i o n , t h e v a l u e s c o r r e l a t e d w i t h o t h e r r e p o r t e d v a l u e s f o r D2 r e c e p t o r s as Table 2 Inhibition of Spec i f i c [^H]-Spiperone (35 pM) Binding at pH 6.2 log I C 5 0 (M) + s.e.m. Compound Striatum Medial Prefrontal Cortex 1. Pimozide -9.98 -10.00 2. Haloperidol -8.69 -8.80 3. Benperidol -9.50 -9.43 4. Spiperone -9.85 -9.80 5. Thioridazine -8.75 -8.97 6. Promazine -7.09 -7.30 7. Chlorpromazine -8.52 -8.89 8. Trifluoperazine -9.60 -9.53 9. (-)-sulpiride -6.42 -6.07 10. (+)-sulpiride -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. Serotonin > -5 > -5 l a b e l l e d with [ JH]-spiperone (r=.882; d/f=10; p<.01; Leysen et a l . , 1978) or [ 3H]-haloperidol (r=.927; d/f=9; p<.01; Burt et a l . , 1976; r=.794; d/f=10; p<.01; Leysen et a l . , 1978). However, i n contrast with the data obtained at pH 7.9, the dopamine receptor agonists were not more potent i n the mpfc than i n the striatum. By f a r the most marked e f f e c t of the reduction i n pH was a large decrease i n the nonspecific binding i n the mpfc. This was probably due to the v i r t u a l elimination of [ 3H]-spiperone binding to spirodecanone s i t e s which have been reported to be more numerous i n the f r o n t a l cortex than i n the striatum (Howlett et a l . , 1979). Thus, nonspecific mpfc binding at pH 6.2 was less than 30% of that observed at pH 7.9. Consequently, i n the assays performed at pH 6.2, 50-60% of the mpfc binding was s p e c i f i c . In the striatum more than 98% of the binding was s p e c i f i c . Discussion The characterization data indicate that 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 at pH 6.2 are D2 dopamine receptors. However, since the dopamine receptor agonists, when assayed at pH 6.2, are not more potent i n the mpfc than i n the striatum, i t seems that the mpfc population l a b e l l e d at pH 6.2 may not be i d e n t i c a l to that l a b e l l e d at pH 7.9. The present data do not indicate how these d i f f e r e n t populations of receptors are involved i n the i n vivo 54 function of the mesocortical dopamine system. In order to resolve t h i s issue i t w i l l be necessary to compare the ef f e c t s of various experimental manipulations on the ligand binding c h a r a c t e r i s t i c s of mpfc D2 receptors with the ef f e c t s that the same manipulations have on i n vivo dopamine functions. I t i s possible that both the receptor population l a b e l l e d at pH 7.9 and the population l a b e l l e d at pH 6.2 t r u l y r e f l e c t a functional state of mpfc D2 dopamine receptors. Thus, i t i s conceivable that the pH of the extremely small pool of e x t r a c e l l u l a r f l u i d i n the synaptic c l e f t can be affected i n l o c a l areas by the neuronal release of compounds into the c l e f t . Such pH changes may a f f e c t the ligand binding c h a r a c t e r i s t i c s of mpfc D2 receptors, as suggested by the present data, thereby influencing the s e n s i t i v i t y of mpfc D2 receptors to dopamine receptor agonists. If so, the present data suggest that the neuronal release of a c i d i c compounds may reduce the pH i n the region of the presynaptic or postsynaptic membranes and thereby reduce the s e n s i t i v i t y of mpfc D2 receptors to dopamine receptor agonists. S i m i l a r l y , the neuronal release of al k a l i n e compounds may increase the s e n s i t i v i t y of the mpfc D2 receptors to dopamine receptor agonists. However, i t should be noted that these proposals are extremely speculative i n that the l o c a l pH i n any p a r t i c u l a r area of the synaptic c l e f t i s presently unknown. The primary motivation for investigating the e f f e c t s of a reduction i n binding reaction pH was the hope that the 55 nonspecific binding would be lower at the lower pH. This aspect of the experiments was successful. The nonspecific binding i n the mpfc was greatly reduced. This i s consistent with the findings of Bruinink et a l . (1984) who reported that a drop i n pH to 6.2 almost eliminated the binding of [ 3H]-spiperone to spirodecanone s i t e s . I t i s also consistent with reports that spirodecanone s i t e s are numerous i n the f r o n t a l cortex (Howlett et a l . , 1979). At pH 6.2, 50-60% of the mpfc binding was to D2 dopamine receptors. Thus, a reduction i n pH greatly increased the "signal to noise" r a t i o of the assay. This s i g n i f i c a n t l y improves the accuracy of competition and saturation studies. The r e s u l t i n g enhanced resolution of the assay increases the f e a s i b i l i t y of competition studies and saturation analysis. 56 E x p e r i m e n t 3: The E f f e c t s o f C h r o n i c H a l o p e r i d o l  T r e a t m e n t on D2 Dopamine R e c e p t o r s i n t h e M e d i a l P r e f r o n t a l C o r t e x o f t h e R a t I n t r o d u c t i o n S e v e r a l r e p o r t s have s u g g e s t e d t h a t dopamine r e c e p t o r a n t a g o n i s t s may p r o d u c e t h e i r a n t i p s y c h o t i c e f f e c t s b y a c t i n g on t h e m e s o c o r t i c a l dopamine s y s t e m ( B a c o p o u l o s e t a l . , 1979; Matsumoto e t a l . , 1983; R o b i n s o n e t a l . , 1 9 8 5 ) . T h i s has l e d t o t h e r e l a t e d s u g g e s t i o n t h a t a b n o r m a l i t i e s i n t h e m e s o c o r t i c a l dopamine s y s t e m may be r e s p o n s i b l e f o r t h e p s y c h o t i c symptoms o f s c h i z o p h r e n i a ( B a c o p o u l o s e t a l . , 1 9 7 9 ) . P e r t i n e n t d a t a i n c l u d e t h e f a c t t h a t t h e m e s o c o r t i c a l dopamine s y s t e m i s more s e n s i t i v e t h a n t h e m e s o l i m b i c and n i g r o s t r i a t a l dopamine s y s t e m s t o t h e e f f e c t s o f d r u g s s u c h as amphetamine and p h e n c y c l i d i n e (Bowers and Hoffman, 1984; R o b i n s o n e t a l . , 1985) w h i c h c a n , i n humans, e l i c i t p s y c h o t i c s t a t e s v e r y s i m i l a r t o some fo r m s o f s c h i z o p h r e n i a ( G r i f f i t h , C avanaugh, H e l d and O a t e s , 1970; S n y d e r , 1 9 8 0 ) . The m e s o c o r t i c a l dopamine s y s t e m i s a l s o more s e n s i t i v e ( d i s p l a y s l o w e r ED50) t h a n t h e m e s o l i m b i c o r n i g r o s t r i a t a l dopamine s y s t e m s t o t h e a c u t e a c t i v a t i n g e f f e c t s o f a n t i p s y c h o t i c d r u g s as m e a s u r e d by i n c r e a s e s i n dopamine m e t a b o l i t e s (Matsumoto e t a l . , 1 9 8 3 ) . F u r t h e r m o r e , w h i l e 57 t h e a c t i v a t i o n o f m e s o l i m b i c and n i g r o s t r i a t a l dopamine s y s t e m s b y a n t i p s y c h o t i c s d i m i n i s h e s w i t h s u b c h r o n i c t r e a t m e n t , t h e a c t i v a t i o n o f t h e m e s o c o r t i c a l dopamine s y s t e m i s r e p o r t e d t o be r e l a t i v e l y r e s i s t a n t t o s u c h t o l e r a n c e ( B a c o p o u l o s e t a l . , 1979; Bannon and R o t h 1983; Matsumoto e t a l . , 1983; T h i e r r y e t a l . , 1 9 8 4 ) . I t has b e e n s u g g e s t e d t h a t t h e s e f i n d i n g s i m p l i c a t e t h e m e s o c o r t i c a l dopamine s y s t e m i n t h e p a t h o l o g y o f s c h i z o p h r e n i a s i n c e t h e c l i n i c a l l y b e n e f i c i a l e f f e c t s o f c h r o n i c a n t i p s y c h o t i c a d m i n i s t r a t i o n r e p o r t e d l y do n o t show t o l e r a n c e whereas t h e p a r k i n s o n i a n - l i k e s i d e e f f e c t s a s s o c i a t e d w i t h s u c h t r e a t m e n t a r e f r e q u e n t l y s e l f - l i m i t i n g ( B a c o p o u l o s e t a l . , 1979; Matsumoto e t a l . , 1 9 8 3 ) . Many s t u d i e s have i n v e s t i g a t e d t h e mechanisms t h r o u g h w h i c h a n t i p s y c h o t i c s may i n f l u e n c e dopamine s y s t e m s t o p r o d u c e t h e i r c l i n i c a l l y b e n e f i c i a l e f f e c t s . E x p e r i m e n t s e m p l o y i n g r a d i o l i g a n d b i n d i n g t e c h n i q u e s have p r o v i d e d t h e most c o n c l u s i v e e v i d e n c e . D a t a i n d i c a t e t h a t t h e a n t i p s y c h o t i c p r o p e r t y o f t h e s e compounds depends on t h e i r a b i l i t y t o b l o c k D2 dopamine r e c e p t o r s . T h u s , t h e c l i n i c a l p o t e n c i e s o f a n t i p s y c h o t i c s a r e 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 i n d i v i d u a l a f f i n i t i e s f o r t h e D2 dopamine b i n d i n g s i t e (Seeman, 1980 and s e e E x p e r i m e n t s 1 and 2 ) . R a d i o l i g a n d b i n d i n g d a t a have a l s o l e d t o t h e s u g g e s t i o n t h a t an i n c r e a s e i n D2 dopamine r e c e p t o r s i s i n v o l v e d i n t h e p a t h o l o g y o f some f o r m s o f s c h i z o p h r e n i a 58 (Seeman, 1981). In postmortem studies, brain tissue from schizophrenics has been found to contain a higher density of sub c o r t i c a l D2 dopamine binding s i t e s when compared to control tissue (Seeman, 1980). However, since the administration of antipsychotic drugs increases the density of subcortical D2 receptors i n experimental animals (Owen et a l . , 1980; Seeman, 1980), i t i s d i f f i c u l t to determine how much of the increase observed i n drug-treated schizophrenics i s due to the disease and how much i s the r e s u l t of antipsychotic treatment. Unfortunately, postmortem studies of drug free patients are lim i t e d to very few subjects and have produced c o n f l i c t i n g r e s u l t s . Brain tissue from drug free patients has been reported to have higher (Seeman, 1980), unchanged (Mackay, Iversen, Rossor, Spokes, Bird, Arregui, Creese and Synder, 1982) or lower (Reynolds, Riederer, J e l l i n g e r and Gabriel, 1981) densities of D2 dopamine receptors when compared to controls. In 1982, Meller et a l . reported that sulpiride-displaceable [ 3H]-spiperone binding i n the prefrontal cortex of the rat i s not affected by treatment with haloperidol (a dopamine receptor antagonist and a commonly prescribed antipsychotic drug). These data suggested that i f an increase i n the D2 f r o n t a l cortex receptor density of schizophrenics were documented i t could be dissociated from antipsychotic treatment. Unfortunately, Meller et a l . (1982) d i d not determine whether the binding s i t e s detected by t h e i r assay procedure were, i n f a c t , D2 59 dopamine r e c e p t o r s . I n a d d i t i o n , t h e y d i d n o t p e r f o r m s a t u r a t i o n a n a l y s e s . T h e r e f o r e , t h e a b s e n c e o f a 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 b i n d i n g may have b e e n due t o a 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 t h e a p p a r e n t a f f i n i t y o f t h e b i n d i n g s i t e s f o r [ 3 H ] - s p i p e r o n e ( s e e R e s u l t s o f t h i s s e c t i o n ) . T h u s , a l t h o u g h t h e d e n s i t y o f t h e s i t e s may have i n c r e a s e d , a d e c r e a s e i n a f f i n i t y may have masked t h i s e f f e c t . A l s o , M e l l e r e t a l . (1982) t e s t e d r a t s a f t e r s u b c h r o n i c t r e a t m e n t (3-5 weeks) whereas most p o s t m o r t e m t i s s u e f r o m s c h i z o p h r e n i c s comes f r o m p a t i e n t s r e c e i v i n g c h r o n i c a n t i p s y c h o t i c t r e a t m e n t ( a t l e a s t s e v e r a l m o n t h s ) . I n t h e p r o c e s s o f c h a r a c t e r i z i n g t h e mpfc D2 r e c e p t o r ( E x p e r i m e n t s 1 and 2 ) , a method was d e v e l o p e d t o s t u d y t h e s e s i t e s t h r o u g h s a t u r a t i o n a n a l y s i s . T h e r e f o r e , i t became p o s s i b l e t o measure t h e e f f e c t o f 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 on b o t h t h e d e n s i t y o f mpfc D2 s i t e s and t h e a f f i n i t y o f t h e s e s i t e s f o r [ 3 H ] - s p i p e r o n e . T h i s method was u s e d t o i n v e s t i g a t e how D2 r e c e p t o r s i n t h e mpfc o f t h e r a t a r e i n f l u e n c e d b y c h r o n i c t r e a t m e n t w i t h h a l o p e r i d o l . M ethods H a l o p e r i d o l b a s e was d i s s o l v e d i n a 0.5% l a c t i c a c i d s o l u t i o n a t a c o n c e n t r a t i o n o f 10 mg/ml and s t o r e d a t 4°C i n t h e d a r k . S t o c k s o l u t i o n s were p r e p a r e d m o n t h l y . A l i q u o t s o f s t o c k s o l u t i o n were d i l u t e d w i t h d i s t i l l e d w a t e r and p r e s e n t e d t o male W i s t a r r a t s as d r i n k i n g w a t e r i n l i g h t 60 p r o t e c t e d b o t t l e s . The c o n c e n t r a t i o n was a d j u s t e d , on t h e b a s i s o f p i l o t r e s u l t s , so t h a t t h e r a t s consumed 1.3-1.5 mg o f h a l o p e r i d o l / k g / d a y . C o n t r o l r a t s r e c e i v e d an e q u i v a l e n t c o n c e n t r a t i o n o f l a c t i c a c i d v e h i c l e . The r a t s were t r e a t e d f o r 21 weeks d u r i n g w h i c h t h e y were h o u s e d 4 p e r c a g e w i t h f r e e a c c e s s t o s t a n d a r d l a b o r a t o r y chow. They w e i g h e d 250-350 g a t t h e b e g i n n i n g o f t h e t r e a t m e n t and 350-450 g a t t h e e nd. The h a l o p e r i d o l d i d n o t a f f e c t t h e r a t e a t w h i c h t h e r a t s g a i n e d w e i g h t . The c o l o n y room was m a i n t a i n e d on a 12/12 h r l i g h t / d a r k c y c l e . On t h e l a s t d a y o f t r e a t m e n t t h e d r i n k i n g b o t t l e s were removed f r o m t h e c a g e s a t 2:00 p.m. (6 h r s a f t e r t h e l i g h t s came on) and t h e r a t s were k i l l e d by c e r v i c a l d i s l o c a t i o n between 3:00 p.m. and 6:00 p.m. The mpfc and t h e s t r i a t u m were d i s s e c t e d and p r o c e s s e d as d e s c r i b e d i n E x p e r i m e n t 1. T i s s u e f r o m 9-14 r a t s was p o o l e d f o r e a c h s a t u r a t i o n a n a l y s i s . E a c h b i n d i n g r e a c t i o n i n c l u d e d 150-250 u,g o f mpfc p r o t e i n o r 75-150 u,g o f s t r i a t a l p r o t e i n . The t i s s u e was a s s a y e d a t pH 6.2 as d e s c r i b e d i n E x p e r i m e n t 1 and E x p e r i m e n t 2 e x c e p t t h a t b i n d i n g was m e a s u r e d w i t h v a r i o u s c o n c e n t r a t i o n s o f [ 3 H ] - s p i p e r o n e . T h u s , e a c h b i n d i n g r e a c t i o n i n c l u d e d [ 3 H ] - s p i p e r o n e (New E n g l a n d N u c l e a r 23-26 Ci/mmole o r Amersham 89 Ci/mmole) a t v a r i o u s f i n a l c o n c e n t r a t i o n s r a n g i n g f r o m 5-450 pM f o r s t r i a t a l a s s a y s and f r o m 25-100 pM f o r mpfc a s s a y s . The [ 3 H ] - s p i p e r o n e f r o m Amersham d i d n o t p r o d u c e as h i g h a p e r c e n t o f s p e c i f i c b i n d i n g as t h e [ 3 H ] - s p i p e r o n e o b t a i n e d 61 f r o m New E n g l a n d N u c l e a r . However, t h e h i g h e r s p e c i f i c a c t i v i t y o f t h e Amersham p r o d u c t made i t p r e f e r a b l e . T h r o u g h o u t t h e e n t i r e t r e a t m e n t , t i s s u e p r e p a r a t i o n and a s s a y p r o c e d u r e s , h a l o p e r i d o l t r e a t e d and c o n t r o l r a t s ( o r t i s s u e ) were p r o c e s s e d i n p a r a l l e l . S c a t c h a r d a n a l y s i s was u s e d t o d e t e r m i n e ( f r o m t h e s a t u r a t i o n d a t a ) t h e a f f i n i t y (1/KD) o f t h e r e c e p t o r s f o r [ 3 H ] - s p i p e r o n e and t h e d e n s i t y (Bmax) o f t h e r e c e p t o r s . T h r e e i n d e p e n d e n t r e p l i c a t i o n s were p e r f o r m e d . S t a t i s t i c a l c o m p a r i s o n s were made w i t h S t u d e n t ' s t w o - t a i l e d t - t e s t s . R e s u l t s F i g u r e 9 i l l u s t r a t e s t h r o u g h S c a t c h a r d a n a l y s i s t h a t t h e h a l o p e r i d o l t r e a t m e n t i n c r e a s e d t h e d e n s i t y (Bmax) o f s t r i a t a l D2 dopamine r e c e p t o r s by a p p r o x i m a t e l y 70% (t=9.57; d/f=2; p < . 0 2 ) . T h i s r e s u l t i s i n good agreement w i t h a n o t h e r s t u d y w h i c h e m p l o y e d a v e r y s i m i l a r h a l o p e r i d o l t r e a t m e n t p r o c e d u r e (Owen, C r o s s , W a d d i n g t o n , P o u l t e r , Gamble and Crow, 1 9 8 0 ) . However, u n l i k e most o t h e r r e p o r t s m e a s u r i n g t h e e f f e c t s o f h a l o p e r i d o l t r e a t m e n t on [ 3 H ] - s p i p e r o n e b i n d i n g , o n l y a v e r y s m a l l and i n s i g n i f i c a n t ( t = 2.21; d/f=2; p > .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 a p p a r e n t a f f i n i t y o f t h e D2 s i t e s f o r [ 3 H ] - s p i p e r o n e was d e t e c t e d . The l a r g e d e c r e a s e i n a f f i n i t y , w h i c h i s commonly o b s e r v e d , i s p r o b a b l y due t o r e s i d u a l h a l o p e r i d o l i n t e r f e r i n g w i t h t h e b i n d i n g o f [ 3 H ] - s p i p e r o n e (Owen e t a l . 62 c "cu o Q. E UJ tr u. o" z => o CO a—o C o n t r o l B m a x = 3 0 4 2 fmoles/mg protein K D =70-7 p M »----• H a l o p e r i d o l B m a x = 5291 fmoles/mg protein K D = 7 3 0 8 p M 0 200 400 600 B O U N D ( f m o l e s / m g p r o t e i n ) Figure 9. 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 chronic haloperidol administration 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 either the low tissue concentration, the long incubation time, or both. Figure 10 i l l u s t r a t e s that the e f f e c t s of haloperidol on mpfc tissue were very s i m i l a r to those on s t r i a t a l t i s s u e . Thus, an approximately 50% higher density of D2 receptors was found i n the mpfc of haloperidol treated rats (t=5.90; d/f=2; p < .03). This percent increase 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 that found i n the striatum (t=2.17; d/f=4; p > .05: independent t - t e s t of % increases over controls comparing mpfc assays with s t r i a t a l assays). As with the striatum the medial prefrontal cortex displayed a small and i n s i g n i f i c a n t (t=0.34; d/f=2; p > .05) reduction i n apparent a f f i n i t y of spiperone for the D2 s i t e s . Discussion The present r e s u l t s demonstrate that the mpfc dopamine receptors are saturable and t h e i r density i s predictable from indices of mpfc dopamine innervation. Thus, the mpfc D2 s i t e s saturate with approximately 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 concentration of D2 receptors i n the mpfc i s approximately 2% of that i n the striatum. The r e s u l t s also demonstrate that chronic exposure to the D2 dopamine receptor antagonist, haloperidol, increases D2 receptor binding i n the mpfc of the r a t . These data are the f i r s t to indicate that mpfc D2 receptors can be 64 o o X a> +-» o k_ a O) E UJ UJ rr u. o" z z> O 03 25 20 -15 10 5 -C o n t r o l B m a x = 9-3 fmoles/mg protein K D = 62.2 p M H a l o p e r i d o l B m a x = 13.8 fmoles/mg protein K D = 6 5 4 p M 0 2 4 6 8 10 12 14 16 B O U N D ( f m o l e s / m g p r o t e i n ) Figure 10. Scatchard plot i l lus t ra t ing the effects of chronic haloperidol administration on mpfc D2 receptors. 65 experimentally manipulated. The e f f e c t i s large and c l e a r l y due to an increase i n the density of receptors. As noted i n the Introduction to t h i s section, brain tissue from schizophrenics has been found to contain a higher density of subcortical D2 dopamine receptors compared to control tissue (Seeman, 1980). However, in t e r p r e t a t i o n of t h i s f i n d i n g i s complicated by reports that antipsychotic treatment can increase the density of subcortical D2 dopamine s i t e s i n experimental animals (Owen et a l . , 1980; Seeman, 1980). In contrast, Meller et a l . (1982) reported that haloperidol treatment did not increase sulpiride-displaceable [ -%J-spiperone binding i n the pr e f r o n t a l cortex of the rat. These data raised the p o s s i b i l i t y that any schizophrenia related changes i n f r o n t a l cortex D2 receptors could be dissociated from the e f f e c t s of antipsychotic treatment. However, the experimental design employed by Meller et a l . (1982) was inadequate i n several respects (see Introduction to t h i s section). The present experiment was designed to circumvent these problems. Unfortunately, the r e s u l t s indicate that any increase i n the density of f r o n t a l cortex D2 dopamine receptors, which may be demonstrated i n future studies of schizophrenics, cannot be e a s i l y dissociated from the e f f e c t s of antipsychotic treatment. The haloperidol-induced increase i n mpfc D2 dopamine receptors may be related to the syndrome of tardive psychosis which i s observed i n some schizophrenic patients 66 who are treated with antipsychotics (Chouinard and Jones, 1980). Thus, i f mpfc D2 dopamine receptors are involved i n psychoses (see Introduction to t h i s section) then an antipsychotic-induced increase i n these receptors may be responsible for the return of psychotic symptoms observed with patients receiving antipsychotics (Chouinard and Jones, 1980). 67 E x p e r i m e n t 4: The E f f e c t s o f F o o t s h o c k S t r e s s on D2  Dopamine R e c e p t o r s i n t h e M e d i a l P r e f r o n t a l C o r t e x o f t h e R a t I n t r o d u c t i o n D o p a m i n e r g i c a g o n i s t s c a n e x a c e r b a t e symptoms o f s c h i z o p h r e n i a ( A n g r i s t and G e r s h o n , 1977) and c a n e l i c i t a p s y c h o s i s v e r y s i m i l a r t o some f o r m s o f s c h i z o p h r e n i a ( A n g r i s t and G e r s h o n , 1970; A n g r i s t , S a t h a n a n t h a n , S h e r w i n and G e r s h o n , 1974; A n g r i s t and G e r s h o n , 1 9 7 7 ) . The p o t e n c y o f a n t i p s y c h o t i c d r u g s as a n t a g o n i s t s o f d o p a m i n e r g i c D2 b i n d i n g s i t e s i s 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 c l i n i c a l e f f i c a c y i n r e d u c i n g symptoms o f s c h i z o p h r e n i a (Seeman, 1 9 8 0 ) . T h e s e d a t a s u g g e s t t h a t an i n c r e a s e i n f u n c t i o n a l d o p a m i n e r g i c a c t i v i t y may be i n v o l v e d i n t h e p a t h o l o g y o f some f o r m s o f s c h i z o p h r e n i a . W h i l e p o s t m o r t e m s t u d i e s o f b r a i n t i s s u e f r o m s c h i z o p h r e n i c s g e n e r a l l y do n o t r e v e a l an i n c r e a s e i n p r e s y n a p t i c d o p a m i n e r g i c a c t i v i t y (Crow, 1 9 8 0 ) , an i n c r e a s e i n presumed p o s t s y n a p t i c D2 b i n d i n g s i t e s (Crow, 1980; Seeman, 1980) h a s b e e n a s s o c i a t e d w i t h a s u b c l a s s o f p o s i t i v e s c h i z o p h r e n i c symptoms t h a t a r e most s i m i l a r t o dopamine a g o n i s t - i n d u c e d p s y c h o s i s , most e x a c e r b a t e d by dopamine a g o n i s t s , and most s e n s i t i v e t o dopamine a n t a g o n i s t t r e a t m e n t (Crow, 1 9 8 2 ) . 68 H e r e d i t y a p p e a r s t o be a f a c t o r i n t h e e t i o l o g y o f s c h i z o p h r e n i a ( G o t t e s m a n and S h i e l d s , 1 9 82). However, t h e much l o w e r t h a n 100% c o n c o r d a n c e r a t e o f m o n o z y g o t i c t w i n s , i n d i c a t e s t h a t e n v i r o n m e n t a l f a c t o r s must a l s o p a r t i c i p a t e ( G o t t e s m a n and S h i e l d s , 1 9 8 2 ) . A number o f e t i o l o g i c a l t h e o r i e s h a v e p r o p o s e d t h a t g e n e t i c a l l y p r e d i s p o s e d i n d i v i d u a l s become s c h i z o p h r e n i c when e x p o s e d t o s u f f i c i e n t s t r e s s ( P o l l i n , 1972; Dohrenwend and E g r i , 1981; S p r i n g , 1981; G o t t e s m a n and S h i e l d s , 1 9 8 2 ) . As w i t h d o p a m i n e r g i c a g o n i s t s , e n v i r o n m e n t a l s t r e s s i s r e p o r t e d l y (Dohrenwend and E g r i , 1981) c a p a b l e o f e l i c i t i n g a syndrome ( " B r i e f R e a c t i v e P s y c h o s i s " ) ( W i l l i a m s , 1980) w h i c h i s v e r y s i m i l a r t o t h o s e f o r m s o f s c h i z o p h r e n i a most h i g h l y a s s o c i a t e d w i t h a b n o r m a l dopamine f u n c t i o n ( W i l l i a m s , 1 9 8 0 ) . D a t a f r o m a n i m a l s t u d i e s i n d i c a t e t h a t v a r i o u s f o r m s o f e n v i r o n m e n t a l s t r e s s c a n s e l e c t i v e l y a c t i v a t e t h e d o p a m i n e r g i c c e l l s i n n e r v a t i n g t h e p r e f r o n t a l c o r t e x ( T h i e r r y e t a l . , 1976; F a d d a e t a l . , 1978; S z e n t e n d r e i e t a l . , 1 9 8 0 ) . The p r e f r o n t a l c o r t e x dopamine s y s t e m h a s b e e n p r o p o s e d as t h e s i t e o f t h e p r i m a r y a b n o r m a l i t y i n s c h i z o p h r e n i a ( B a c o p o u l o s , S p o k e s , B i r d and R o t h , 1979) b a s e d on i t s l a c k o f t o l e r a n c e t o a n t i p s y c h o t i c d r u g a d m i n i s t r a t i o n i n r a t s (Matsumoto e t a l . , 1983) and p r i m a t e s ( B a c o p o u l o s , B u s t o s , Redmond and R o t h , 1 9 8 2 ) , i n c l u d i n g man ( B a c o p o u l o s e t a l . , 1 9 7 9 ) . F u r t h e r m o r e , r e g i o n a l b l o o d f l o w and g l u c o s e m e t a b o l i s m d a t a ( I n g v a r , 1982) s u g g e s t t h a t s c h i z o p h r e n i c s h a v e a t y p i c a l n e u r o n a l a c t i v i t y i n t h e 69 f r o n t a l c o r t e x . R e c e n t d a t a s u g g e s t t h a t t h e s t r e s s ( f o o t s h o c k o r t a i l - p i n c h ) - i n d u c e d p o t e n t i a t i o n o f s u b s e q u e n t d o p a m i n e r g i c a g o n i s t - i n d u c e d s t e r e o t y p y i n t h e r a t ( A n t e l m a n , E i c h l e r , B l a c k and K o c a n , 1980; MacLennan and M a i e r , 1983) may be m e d i a t e d t h r o u g h t h e p r e f r o n t a l c o r t e x dopamine s y s t e m ( E i c h l e r and A n t e l m a n , 1 9 7 9 ) . The e x p e r i m e n t s p r e s e n t e d i n E x p e r i m e n t 4 were d e s i g n e d t o d e t e r m i n e what e f f e c t s f o o t s h o c k s t r e s s has on D2 dopamine r e c e p t o r s i n t h e mpfc and t h e s t r i a t u m o f t h e r a t . As i n E x p e r i m e n t s 1 and 2, t i s s u e was a s s a y e d a t pH 6.2 and pH 7.9. The E f f e c t s o f F o o t s h o c k S t r e s s as M e a s u r e d a t pH 6.2  Methods M a l e , W i s t a r r a t s (300-400 g) were i n d i v i d u a l l y h o u s e d f o r 3-4 weeks p r i o r t o t h e e x p e r i m e n t b e c a u s e i s o l a t i o n has b e e n r e p o r t e d t o e n h a n c e t h e e f f e c t o f s t r e s s on i n d i c e s o f m e s o c o r t i c a l dopamine a c t i v i t y ( B l a n c , H e r v e , Simon, L i s o p r o w s k i , G l o w i n s k i and T a s s i n , 1 9 8 0 ) . R a t s were h o u s e d u n d e r a 12/12 h r l i g h t / d a r k c y c l e . The l i g h t s came on a t 8:00 a.m. The s h o c k e d r a t s r e c e i v e d a 30 m i n u t e f o o t s h o c k s e s s i o n (one .5 s e c , 3 mA s h o c k i n e v e r y 2.5 s e c i n t e r v a l ) b e t w e e n 12:00 p.m. and 3:30 p.m. The s h o c k was d e l i v e r e d by a B R S - F o r i n g e r SGS-003 s h o c k g e n e r a t o r w h i c h was c o n n e c t e d t o a B R S - F o r i n g e r o p e r a n t chamber. Between 3:00 p.m. and 70 5:30 p.m. t h e f o l l o w i n g day, t h e s h o c k e d r a t s and t h e n a i v e c o n t r o l s were k i l l e d and d i s s e c t e d as d e s c r i b e d i n E x p e r i m e n t 1. T h i s t i m e i n t e r v a l was e m p l o y e d b e c a u s e i t has b e e n u s e d i n s t u d i e s o f t h e s t r e s s - i n d u c e d p o t e n t i a t i o n o f s u b s e q u e n t dopamine a g o n i s t - i n d u c e d s t e r e o t y p y ( A n t e l m a n e t a l . , 1980; MacLennan and M a i e r , 1 9 8 3 ) . The t i s s u e was a s s a y e d as d e s c r i b e d i n E x p e r i m e n t 3 (pH 6.2). F i v e i n d e p e n d e n t s a t u r a t i o n a n a l y s e s were r u n f o r e a c h b r a i n r e g i o n . T i s s u e f r o m 8-10 r a t s was p o o l e d f o r e a c h s a t u r a t i o n a n a l y s i s . R e s u l t s F i g u r e 11 i s a S c a t c h a r d p l o t o f t h e d a t a o b t a i n e d f r o m mpfc t i s s u e . As i l l u s t r a t e d b y t h e f i g u r e , f o o t s h o c k e d r a t s showed s l i g h t l y more D2 dopamine r e c e p t o r b i n d i n g i n t h e mpfc. The i n c r e a s e was t o o s m a l l t o d e t e r m i n e w h e t h e r t h i s e f f e c t was due t o an i n c r e a s e i n t h e d e n s i t y o f r e c e p t o r s o r an i n c r e a s e i n t h e a f f i n i t y o f t h e r e c e p t o r s f o r [ % ] - s p i p e r o n e . However, i f f o r e a c h s a t u r a t i o n a n a l y s i s , t h e e f f e c t o f s h o c k was c a l c u l a t e d as t h e a v e r a g e s h o c k i n d u c e d c h a n g e i n b i n d i n g o b s e r v e d a t t h e d i f f e r e n t c o n c e n t r a t i o n s o f [ 3 H ] - s p i p e r o n e ( c a l c u l a t e d as p e r c e n t o f c o n c o m i t a n t l y a s s a y e d c o n t r o l s ) , t h e n s h o c k e d r a t s were 71 B O U N D ( f m o l e s / m g p r o t e i n ) Figure 11. Scatchard plo 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 receptors assayed at pH 6.2. 72 f o u n d t o d i s p l a y a s i g n i f i c a n t 13% i n c r e a s e i n D2 b i n d i n g (t=2.97; d/f=4; p<.05). No e f f e c t o f f o o t s h o c k s t r e s s was o b s e r v e d on D2 r e c e p t o r s i n s t r i a t a l t i s s u e (t=.92; d/f=2; p>.05). F i g u r e 12 i s a S c a t c h a r d p l o t o f t h e s t r i a t a l d a t a . D i s c u s s i o n The f o o t s h o c k s t r e s s i n d u c e d a s m a l l b u t s i g n i f i c a n t i n c r e a s e i n t h e amount o f D2 b i n d i n g i n t h e mpfc. G i v e n t h e a c c u r a c y o f t h e p r e s e n t a s s a y w i t h s t r i a t a l t i s s u e , one w o u l d e x p e c t t o d e t e c t a change i n s t r i a t a l D2 b i n d i n g t h a t a p p r o a c h e s t h e s i z e o f t h a t i n t h e mpfc. The d a t a a p p e a r t o i n d i c a t e , t h e r e f o r e , t h a t f o o t s h o c k s t r e s s c a n s e l e c t i v e l y i n c r e a s e t h e amount o f D2 b i n d i n g i n t h e mpfc. The mechanism r e s p o n s i b l e f o r t h i s e f f e c t i s unknown ( p o s s i b l e mechanisms a r e d i s c u s s e d i n t h e G e n e r a l D i s c u s s i o n ) . However, t h e d a t a a r e c o n s i s t e n t w i t h t h e dopamine t u r n o v e r s t u d i e s w h i c h s u g g e s t t h a t t h e mpfc i s 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 . From a c l i n i c a l p e r s p e c t i v e , t h e d a t a s u g g e s t t h a t s t r e s s may c o n t r i b u t e t o t h e o n s e t o f s c h i z o p h r e n i a - l i k e p s y c h o s e s b y i n c r e a s i n g t h e number o f D2 mpfc r e c e p t o r s . I n a d d i t i o n , t h e p r e s e n t p a r a d i g m s h o u l d p r o v e u s e f u l i n f u r t h e r s t u d i e s o f f r o n t a l c o r t e x D2 b i n d i n g s i t e d y n a m i c s . I t may a l s o l e a d t o a b e t t e r u n d e r s t a n d i n g o f t h e C o n t r o l B m a x = 266-5 fmoles/mg protein B O U N D (f m o l e s/m g p r o t e in) Figure 12. 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 s t r i a t a l D2 receptors assayed at pH 6. r o l e o f s t r e s s i n t h e e t i o l o g y o f d o p a m i n e r g i c a l l y r e l a t e d m e n t a l d i s e a s e s . The E f f e c t s o f F o o t s h o c k S t r e s s as M e a s u r e d a t pH 7.9 The t i s s u e f r o m f o o t s h o c k e d r a t s was f i r s t a s s a y e d a t pH 6.2 b e c a u s e t h e p r e s e n t a s s a y p o s s e s s e s a g r e a t e r d e g r e e o f r e s o l u t i o n a t t h i s pH ( s e e E x p e r i m e n t 2 ) . The r e s u l t s a t pH 6.2 i n d i c a t e t h a t t h e D2 b i n d i n g i n t h e mpfc i s i n c r e a s e d w h i l e t h e D2 b i n d i n g i n t h e s t r i a t u m i s n o t . T h e s e r e s u l t s s u g g e s t t h a t t h e mechanism u n d e r l y i n g t h e mpfc s t r e s s e f f e c t on D2 b i n d i n g may i n v o l v e some p r o p e r t y t h a t i s p a r t i c u l a r t o t h e mpfc. C o n s e q u e n t l y , t h e r e s u l t s a l s o r a i s e t h e p o s s i b i l i t y t h a t t h e s t r e s s e f f e c t may be, t o some d e g r e e , r e l a t e d t o t h e h i g h e r 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 t h e mpfc D2 r e c e p t o r s compared t o t h e s t r i a t a l D2 r e c e p t o r s . S p e c i f i c a l l y , a s u b p o p u l a t i o n o f D2 r e c e p t o r s i n t h e mpfc, w h i c h i s r e s p o n s i b l e f o r t h i s a r e a ' s h i g h e r 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 d i f f e r e n t i a l l y a f f e c t e d b y s t r e s s . As d e m o n s t r a t e d i n E x p e r i m e n t 2, t h e g r e a t e r 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 i s n o t o b s e r v e d a t pH 6.2. T h e r e f o r e , i f t h i s v a r i a b l e i s r e l a t e d t o t h e s t r e s s - i n d u c e d i n c r e a s e i n mpfc D2 b i n d i n g t h e n i t c o u l d be p r e d i c t e d t h a t t h e s t r e s s e f f e c t w o u l d be d i f f e r e n t when a s s a y e d a t pH 7.9 where t h e 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 o b s e r v e d . I n a c c o r d a n c e w i t h t h i s p o s s i b i l i t y , t h e n e x t 75 experiment was designed to test the e f f e c t s of stress on D2 receptors assayed at pH 7.9. Methods The experiment was performed as i n the preceding section except that the binding reactions were run at pH 7.9 and a lower range of [ 3H]-spiperone concentrations (20 pM to 50 pM) was used because at pH 7.9 [ 3H]-spiperone has a higher a f f i n i t y for D2 receptors and a lower percent of s p e c i f i c binding. Three independent r e p l i c a t i o n s were run for each brain region. Results Figure 13 i l l u s t r a t e s that the footshock increased the amount of D2 binding i n the mpfc. The Scatchard analysis indicates that the density of D2 receptors (Bmax) was increased by approximately 100% while the a f f i n i t y of [ 3H]-spiperone was reduced to approximately 30% of that observed for controls. The increase i n D2 receptor density was s t a t i s t i c a l l y s i g n i f i c a n t (t=6.42; d/f=2; p<.025) as was the decrease i n a f f i n i t y (t=5.93; d/f=2; p<.03). No changes were detected i n the s t r i a t a l D2 receptors of footshocked r a t s . Figure 14 i l l u s t r a t e s these r e s u l t s . 76 B O U N D ( f m o l e s / m g p r o t e i n ) Figure 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 of footshock on mpfc D2 receptors assayed at pH 7.9. 77 B O U N D ( f m o l e s / m g p r o t e i n ) Figure 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 receptors assayed at pH 7.9. 78 D i s c u s s i o n F o o t s h o c k c l e a r l y i n c r e a s e d t h e d e n s i t y o f D2 r e c e p t o r s i n t h e mpfc. The s i z e o f t h i s i n c r e a s e i s e s t i m a t e d a t a b o u t 100% o f c o n t r o l v a l u e s . The a s s a y i n t h e mpfc c a n n o t a c c u r a t e l y d e t e r m i n e t h e D2 b i n d i n g a t h i g h e r c o n c e n t r a t i o n s o f [ 3 H ] - s p i p e r o n e b e c a u s e t h e p e r c e n t o f s p e c i f i c b i n d i n g d e c r e a s e s as t h e [ 3 H ] - s p i p e r o n e c o n c e n t r a t i o n i s i n c r e a s e d ( s e e E x p e r i m e n t 1 ) . I t was, t h e r e f o r e , n e c e s s a r y t o l i m i t t h e [ 3 H ] - s p i p e r o n e c o n c e n t r a t i o n s t o t h e l o w e r , a c c u r a t e r a n g e and t h e n t o e x t r a p o l a t e more t h a n i s t y p i c a l i n S c a t c h a r d a n a l y s e s . C o n s e q u e n t l y , a l t h o u g h t h e i n c r e a s e a p p e a r s l a r g e , t h e e x a c t s i z e o f t h e i n c r e a s e c o u l d n o t be c o n c l u s i v e l y d e t e r m i n e d . The l o w e r a p p a r e n t a f f i n i t y o f [ 3 H ] - s p i p e r o n e f o r t h e D2 r e c e p t o r s o f t h e s t r e s s e d r a t s may be an a r t i f a c t r e s u l t i n g f r o m a s t r e s s - i n d u c e d i n c r e a s e i n some endogenous s u b s t a n c e w h i c h competes w i t h t h e [ 3 H ] - s p i p e r o n e f o r t h e D2 s i t e s . I t i s a l s o p o s s i b l e , however, t h a t t h e a f f i n i t y e f f e c t r e f l e c t s a r e a l c h ange i n t h e D2 r e c e p t o r s . S i m i l a r r e d u c t i o n s i n a f f i n i t y have a c c o m p a n i e d i n c r e a s e s i n s u b c o r t i c a l D2 r e c e p t o r d e n s i t y i n d u c e d b y p r e s y n a p t i c l e s i o n s ( F e u e r s t e i n , Demenge, C a r o n , B a r r e t t e , G u e r i n and M ouchet, 1 9 8 1 ) , amphetamine ( H o w l e t t and N a h o r s k i , 1 9 7 9 ) , and c h r o n i c a l l y a d m i n i s t e r e d a n t i p s y c h o t i c s (Owen e t a l . , 1 9 8 0 ) . A l t h o u g h a l l t h e s e e f f e c t s may r e f l e c t o t h e r 79 factors, i t i s also possible that "new" receptors while s t i l l developing, or while only temporarily induced, have a lower a f f i n i t y for spiperone. The experiments of t h i s section were prompted by the idea that the increase i n the number of mpfc D2 receptors may be related to the higher a f f i n i t y of dopamine receptor agonists for mpfc D2 receptors. The present data are consistent with that p o s s i b i l i t y . The stress e f f e c t i s larger at the pH at which the dopamine receptor agonist a f f i n i t y difference i s observed ( i . e . pH 7.9). The stress-induced increase i n receptors seems count e r i n t u i t i v e . Stress increases dopamine release i n the pr e f r o n t a l cortex (Thierry et a l . , 1976). Increased agonist stimulation i s generally thought to reduce receptor number. However, the l i t e r a t u r e indicates that receptor regulation i s more complicated than suggested by t h i s simple view. Thus, i n vivo treatment with dopamine receptor agonists has been shown i n some cases to increase dopamine-related receptor binding (eg. Howlett and Nahorski, 1979; Taylor, Ho and Fagan, 1979; Robertson, 1983). I t has also been reported that i n v i t r o incubation of dopamine or dopamine receptor agonists with tissue homogenates can dramatically increase dopamine-related binding (McManus, Hartley and Seeman, 1978; Robertson, 1980). I t i s possible that dopamine released by stress may i n t e r a c t with other dopamine receptors which when activated increase the number of D2 receptors. For example, recent data suggest that 80 stimulation of DI binding s i t e s can increase binding at D2 s i t e s (Dumbrille-Ross et a l . , 1985). In addition, stress may promote the release of other transmitters which could cause an increase i n D2 binding. For example, cholecystokinin has been reported to increase the number of D2 receptors when administered i n vivo or i n v i t r o (Dumbrille-Ross and Seeman, 1984). Research on adrenergic receptors has supported the idea that receptor interactions may be responsible for stress-induced increases i n receptors. I t has been found that i n v i t r o treatment of rat cerebral c o r t i c a l s l i c e s with B-adrenergic agonists can decrease 3-adrenergic receptor binding but increase c^-adrenergic receptor binding (Maggi, U'Prichard and Enna, 1980; Kitamura and Nomura, 1985). Immobilization stress has been shown to produce both these e f f e c t s i n the rat cerebral cortex (U'Prichard and Kvetnansky, 1980). Thus, stress may release c o r t i c a l noradrenaline which interacts with B-adrenergic receptors to produce an increase i n a^-adrenergic receptors. An analogous mechanism may be responsible for the presently reported stress e f f e c t . I t i s also possible that the increase i n mpfc D2 receptors i s a compensatory response r e s u l t i n g from a stress-induced decrease i n presynaptic dopamine function during the 28 hour i n t e r v a l between the footshock session and the tissue preparation. There i s no evidence to suggest that such a decrease i n presynaptic function follows footshock. U n t i l evidence i s available to the contrary, however, such a mechanism remains a p o s s i b i l i t y . As noted above, the dopamine receptors that are increased by stress may also display a high a f f i n i t y for dopamine receptor agonists as well as a pH dependent a f f i n i t y for [ 3H]-spiperone. These properties were not observed for the remainder of the mpfc receptors or the s t r i a t a l receptors. I t i s possible that these receptors also quite d i f f e r e n t from most dopamine receptors i n that they increase i n number when stimulated by agonists. 82 General Discussion The data i n t h i s thesis indicate that D2 dopamine receptors are present i n the mpfc of the ra t . The a f f i n i t i e s with which various dopamine antagonists bind to these receptors are highly correlated with t h e i r a f f i n i t i e s for D2 receptors i n the striatum. In contrast, when measured at pH 7.9, dopamine receptor agonists display a higher a f f i n i t y for mpfc D2 receptors than for s t r i a t a l D2 receptors. This difference i s not observed at pH 6.2. Chronic haloperidol produced a large increase i n mpfc and s t r i a t a l D2 receptors measured at pH 6.2. Footshock stress produced only a small increase i n mpfc D2 receptors at pH 6.2. However, when measured at pH 7.9, footshock stress produced a large increase i n receptor density and a large decrease i n a f f i n i t y of the receptors for [ 3H]-spiperone. The stress d i d not a f f e c t D2 receptor binding i n the striatum when measured at either pH 7.9 or pH 6.2. The General Discussion of t h i s thesis addresses four topics which are related to the present data as a whole. F i r s t , a hypothesis i s presented which attempts to explain how the ligand binding c h a r a c t e r i s t i c s of D2 receptors may be related to the manner i n which these receptors are affected by experimental manipulations. Second, research on dopamine autoreceptors i s examined i n the context of the present data. Third, possible interpretations of 83 pH-dependent binding are discussed. Fourth, the prospects for future research on mpfc D2 receptors are discussed. A Hypothesis Relating the Binding Characteristics of D2 Dopamine Receptors With Their Response to Various  Experimental Manipulations As discussed i n Experiment 4, the data i n t h i s thesis rais e the p o s s i b i l i t y that the footshock-induced changes i n mpfc D2 receptors may be related to the higher a f f i n i t y of dopamine receptor agonists for mpfc D2 receptors compared to s t r i a t a l D2 receptors. Thus, the stress e f f e c t i s much larger when measured at the pH at which the dopamine receptor agonist a f f i n i t y difference i s observed ( i . e . pH 7.9). The footshock e f f e c t and the difference i n agonist a f f i n i t y may be independent e f f e c t s which display s i m i l a r pH dependency. However, i t i s tempting to speculate on how the two e f f e c t s may be related. Accordingly, the following hypothesis i s proposed. Perhaps the D2 receptor population measured i n the present assays i s composed of at least two d i f f e r e n t forms of D2 receptors. One type ( a r b i t r a r i l y designated here as the type A receptor) has a higher a f f i n i t y for dopamine receptor agonists than does the other type of receptor ( a r b i t r a r i l y designated here as the type B receptor). The mpfc has a higher type A to type B r a t i o . At pH 7.9 both type A and type B receptors are detected. However, the D2 receptor population detected at pH 6.2 i s primarily composed 84 of type B receptors. Therefore, at pH 7.9 dopamine receptor agonists display a higher a f f i n i t y for the mpfc D2 receptors compared to the s t r i a t a l D2 receptors because the mpfc has a higher type A to type B r a t i o . The difference i n dopamine receptor agonist a f f i n i t y i s not observed at pH 6.2 because the D2 receptor population detected i n both brain regions i s primarily type B. It i s proposed that t h e d e n s i t y of type A receptors increases following an increase i n synaptic dopamine concentrations. In addition, the a f f i n i t y of type A receptors for [ 3H]-spiperone i s reduced following an increase i n synaptic dopamine concentrations. Type B receptors are much less s e n s i t i v e , or completely i n s e n s i t i v e , to such changes i n dopamine concentration. At pH 7.9, footshock may a f f e c t mpfc D2 receptors but not s t r i a t a l D2 receptors because footshock s e l e c t i v e l y increases dopamine release (and therefore synaptic dopamine concentrations) i n the mpfc (Thierry et a l . , 1976). Such increased synaptic dopamine concentrations produce the above mentioned changes i n mpfc type A receptors. Type A receptors have very l i t t l e i f any e f f e c t on binding r e s u l t s at pH 6.2 and, therefore, the footshock e f f e c t s are much smaller at t h i s pH. The present speculations suggest that experimental manipulations which increase synaptic dopamine concentrations i n the striatum should be able to increase the number of type A receptors i n that region and decrease t h e i r a f f i n i t y for [ 3H]-spiperone. When assayed at pH 7.9 85 these changes would be expected to produce an increase i n detected D2 receptors and a decrease i n t h e i r apparent a f f i n i t y for [ 3H]-spiperone. A single i n j e c t i o n of a large dose of amphetamine, which should increase synaptic concentrations of dopamine i n the striatum, has been reported to produce (at pH 7.8) these expected e f f e c t s on D2 receptors i n the striatum (Howlett and Nahorski, 1979). In summary, the higher a f f i n i t y of dopamine receptor agonists for mpfc D2 receptors compared to s t r i a t a l D2 receptors may be due to a higher proportion of type A receptors i n the mpfc. In addition, the e f f e c t s of footshock may be due to s e l e c t i v e changes i n type A receptors. I t i s also possible that both of these phenomena are much less pronounced at pH 6.2 because many less type A receptors are detected at that pH. Type A receptors may be the same as D2[high] receptors (Creese, Sibley and L e f f , 1984). However, i t i s also possible that they are only a subset of the D2[high] receptor population which responds to experimental manipulations as described above. Dopamine "Autoreceptor" Research Roth and colleagues (Bannon, Michaud and Roth, 1981; Bannon, Reinhard, Bunney and Roth, 1982; Galloway, Wolf and Roth, 1986) have provided data suggesting that the dopaminergic terminals i n the mpfc lack dopamine sensitive 86 receptors that i n h i b i t dopamine synthesis ( i . e . synthesis modulating "autoreceptors"). 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 either by gamma-butyrolactone (GBL) or axotomy, an increase i n dopamine synthesis i s observed i n the striatum and the o l f a c t o r y tubercle. This e f f e c t i s believed to be the r e s u l t of less autoreceptor stimulation due to less impulse related release of dopamine.- The increased synthesis i s attenuated by the administration of low, "autoreceptor" doses of dopamine receptor agonists which are thought to produce t h i s attenuation by acting on the autoreceptors of the mesolimbic and n i g r o s t r i a t a l c e l l s . In contrast, Roth and colleagues f i n d that neither GBL nor axotomy are able to increase dopamine synthesis i n mesocortical dopamine c e l l s (Bannon et a l . , 1981; Galloway et a l . , 1986). They conclude that a lack of "autoreceptors" could be responsible for the above mentioned response c h a r a c t e r i s t i c s of the mesocortical dopaminergic c e l l s (Bannon and Roth, 1983). However, others have presented c o n f l i c t i n g r e s u l t s (Argiolas, Mereu, Serra, Melis, Fadda and Gessa, 1982; Fadda, Gessa, Marcou, Mosca and Rossetti, 1984). These authors have reported that GBL increases dopamine synthesis i n the mpfc and that low, "autoreceptor" doses of dopamine receptor agonists reduce mpfc dopamine synthesis. The experimental paradigms of Roth and colleagues are s i m i l a r to those employed by Fadda et a l . (1984). Therefore, i t i s d i f f i c u l t to explain why these two groups report such d i f f e r e n t r e s u l t s . 87 Both Fadda et a l . (1984) and Galloway et al.,(1986) determined dopamine synthesis by measuring the accumulation of mpfc DOPA following DOPA decarboxylase i n h i b i t i o n . They made the assumption that t h i s measure r e f l e c t s dopamine synthesis. This was based on studies of subcortical dopamine which were conducted i n brain regions where the dopamine innervation i s much more dense than i n the mpfc. It i s presently suggested that the mpfc DOPA accumulation measured by Fadda et a l . (1984) may primarily r e f l e c t noradrenaline synthesis i n the noradrenergic terminals which innervate the mpfc. Fadda et a l . (1984) report that DOPA accumulation i s approximately equal i n the mpfc and the dorsola t e r a l f r o n t a l cortex, an area which receives much less dopaminergic innervation than the mpfc. They explain these r e s u l t s by suggesting that most dopamine i n the f r o n t a l cortex i s synthesized i n noradrenergic terminals which are known to be present i n both areas of the f r o n t a l cortex to the same degree. This suggestion i s incompatible with the many reports that lesions of the noradrenergic input to the mpfc do not reduce mpfc dopamine concentrations (Thierry et a l . , 1973b; T i s s a r i et a l . , 1979; Carter and Pycock, 1980; Westerink and De Vries , 1985). Therefore, since DOPA i s also a precursor of noradrenaline, Fadda et al . ' s (1984) method of measuring mpfc dopamine synthesis may act u a l l y measure the rate of noradrenaline synthesis i n mpfc noradrenergic terminals. Some uni d e n t i f i e d difference i n the paradigms of Roth's group and Fadda's group (perhaps 88 differences i n tissue preparation, tissue d i s s e c t i o n or DOPA detection) may have led Fadda et a l . (1984) to detect mpfc noradrenaline synthesis not detected by Roth's group. Fadda et a l . (1984) also assumed that the ef f e c t s they observed were due to changes i n dopamine synthesis i n mpfc dopaminergic terminals because the low concentrations of dopamine receptor agonists with which they reduced mpfc DOPA accumulation were assumed t o . a f f e c t only autoreceptors on dopaminergic c e l l s . The present demonstration of the high a f f i n i t y of dopamine receptor agonists for mpfc receptors suggests an alte r n a t i v e explanation. Thus, the GBL-induced blockade of impulse flow i n mesocortical dopamine c e l l s may reduce mpfc dopamine release. This may increase DOPA accumulation i n mpfc noradrenergic terminals possessing dopamine receptors through which dopamine t o n i c a l l y i n h i b i t s noradrenaline synthesis i n the mpfc noradrenergic terminals (see Figure 15). I f t h i s i s the case, then the GBL-induced increase i n mpfc DOPA accumulation r e f l e c t s the increased synthesis of noradrenaline i n mpfc noradrenergic terminals that would otherwise occur i f DOPA decarboxylase were not i n h i b i t e d . The low, "autoreceptor" concentrations of dopamine receptor agonists may reduce mpfc DOPA accumulation by a c t i v a t i n g the dopamine receptors on the mpfc noradrenergic terminals. Therefore, these dopamine receptors would have to be sensitive to low, "autoreceptor" concentrations of dopamine receptor agonists. The present demonstration that dopamine 89 Dopamine Receptor (Inh ib i ts noradrenaline synthesis when stimulated by a dopamine receptor agonist) G8L blocks Impulse flow and decreases DA release at terminals F i g u r e 15. Dopamine r e l e a s e d i n the mpfc by the 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 n o r a d r e n e r g i c t e r m i n a l s by 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 on the n o r a d r e n e r g i c t e r m i n a l s . 90 receptor agonists have a higher a f f i n i t y for mpfc dopamine receptors than s t r i a t a l dopamine receptors i s consistent with t h i s p o s s i b i l i t y . If at least some of the mpfc dopamine receptors that have a high a f f i n i t y for dopamine receptor agonists are located on mpfc noradrenergic terminals, then the present data also suggest that stress may a f f e c t these neurons since the stress e f f e c t on mpfc dopamine receptors i s most pronounced at a pH (7.9) where the higher a f f i n i t y s i t e s are best detected (see above). It i s also possible that the GBL d i r e c t l y increases mpfc noradrenaline synthesis by i n h i b i t i n g impulse flow i n the noradrenergic c e l l s innervating the mpfc. The i n h i b i t i o n of impulse flow could reduce noradrenaline release i n the mpfc and consequently may r e s u l t i n less noradrenergic autoreceptor stimulation, thereby increasing mpfc noradrenaline synthesis. However, i t i s very u n l i k e l y that such a GBL-induced increase i n noradrenergic synthesis i s involved i n the GBL e f f e c t s on mpfc DOPA accumulation that were reported by Fadda et a l . (1984). Thus, Fadda et a l . (1984) found that GBL did not increase DOPA accumulation i n the dor s o l a t e r a l f r o n t a l cortex which receives noradrenergic input of similar density to that innervating the mpfc. The present hypothesis predicts that administration of dopaminergic drugs should a f f e c t noradrenaline synthesis i n mpfc noradrenergic terminals. I t has been reported that i n s l i c e s that include f r o n t a l , p a r i e t a l and anterior cingulate areas of the cortex, [ JH]-noradrenaline formation i s s i g n i f i c a n t l y accelerated by administration of dopamine receptor antagonists (thioproperazine and haloperidol) and s l i g h t l y reduced, a l b e i t i n s i g n i f i c a n t l y , by apomorphine (Scatton, Thierry, Glowinski and Julou, 1975; Scatton, Glowinski and Julou, 1976). I t i s possible that i n the s l i c e preparation employed by Scatton et a l . , endogenous level s of dopamine occupied most of the proposed dopamine receptors on noradrenergic terminals. If t h i s were so, apomorphine would produce l i t t l e a dditional stimulation but the dopamine receptor antagonist would evoke a s i g n i f i c a n t increase i n noradrenaline synthesis by reducing the i n h i b i t o r y influence of endogenous dopamine. When the dopamine receptors on noradrenergic terminals are activated they may d i r e c t l y reduce noradrenaline synthesis or they may i n d i r e c t l y reduce noradrenaline synthesis by reducing noradrenaline release. A reduction i n noradrenaline release should cause an increase i n noradrenaline concentrations i n the mpfc noradrenergic terminals. Such an increase would be expected to decrease noradrenaline synthesis v i a end-product i n h i b i t i o n of tyrosine hydroxylase (Cooper, Bloom and Roth, 1982). I t i s , therefore, relevant that several groups have investigated the e f f e c t s of dopamine receptor agonists on [ 3H]-noradrenaline release i n the brain and periphery. Thus, dopamine does not a f f e c t [ 3H]-noradrenaline release from s l i c e s of the rat o c c i p i t a l cortex (Taube, Starke and Borowski, 1977), but dopamine and other dopamine receptor agonists do i n h i b i t the release of [ JH]-noradrenaline from s l i c e s of the dorsal hippocampus of the rabbit (Jackisch, Werle and Hertting, 1984; Jackisch, Moll, Feuerstein and Hertting, 1985). Moreover, the dopamine receptor agonists produce t h i s e f f e c t by i n t e r a c t i n g with D2 receptors (Jackisch et a l . , 1985). Unlike the o c c i p i t a l cortex, the dorsal hippocampus and the f r o n t a l cortex are known to be innervated by dopaminergic terminals (Bischoff, Scatton and Korf, 1979). Thus, the data c o l l e c t e d to date suggest that c e n t r a l , presynaptic, noradrenaline a c t i v i t y may be regulated by dopamine receptors, but only i n brain regions that receive dopaminergic input. I t i s important to note that these comparisons of d i f f e r e n t brain regions are presently l i m i t e d to those involving data from separate studies. Future research should concentrate on studies which employ i d e n t i c a l methods to investigate dopamine regulation of noradrenergic a c t i v i t y i n several brain regions. If evidence for dopamine receptors (regulating noradrenergic a c t i v i t y ) i s found only i n dopamine innervated brain regions then either the dopamine receptors are lim i t e d to noradrenergic terminals i n those brain regions or interneurons with dopamine receptors may be involved. In the periphery, some dopamine receptors appear to be located on noradrenergic nerve terminals. When activated, they i n h i b i t the release of noradrenaline (Langer, 1974; Grigoriadis and Seeman, 1984). Dopamine receptor agonists and antagonists 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 for 93 these receptors and D2(high) dopamine receptors i n the c e n t r a l nervous system (Grigoriadis and Seeman, 1984). If c e n t r a l D2(high) receptors and the above mentioned peripheral dopamine receptors are i d e n t i c a l , as has been suggested (Grigoriadis and Seeman, 1984), then i t i s possible that these receptors are synthesized by some peripheral and c e n t r a l noradrenergic neurons. As discussed above, the,.present data suggest that the higher a f f i n i t y of dopamine agonists for the mpfc D2 receptors compared to the s t r i a t a l D2 receptors i s l i k e l y due to a higher proportion of D2(high) s i t e s i n the mpfc. If a s i g n i f i c a n t number of D2(high) receptors are located on central noradrenergic terminals (as proposed above), t h i s may explain why they appear to be r e l a t i v e l y more prevalent i n the mpfc. Thus, the r a t i o of noradrenergic innervation to dopaminergic innervation i s much higher i n the mpfc than i n the striatum (Brownstein, Saavedra and Palkovits, 1974; Carter and Pycock, 1980). If there are mpfc dopamine receptors, on nondopaminergic neurons, with the high a f f i n i t y for dopamine receptor agonists which i s c h a r a c t e r i s t i c of autoreceptors, then many of the "autoreceptor" r e s u l t s of Roth and colleagues are also open to rei n t e r p r e t a t i o n (Bannon et a l . , 1981; Bannon et a l . , 1982; Galloway et a l . , 1986). Considering the evidence that dopaminergic a c t i v i t y i n the mpfc may i n h i b i t s u b cortical dopaminergic a c t i v i t y (lesions of the mpfc dopamine innervation increase subcortical 94 dopamine metabolites [Carter and Pycock, 1980; Pycock et al.,1980; Martin-Iverson et al.,1986] see General Introduction for further discussion), i t i s possible that the increase observed by Roth and colleagues i n subcortical dopamine synthesis following blockade of mesocortical dopaminergic impulse flow may be larg e l y due to reduced i n h i b i t i o n of subcortical dopamine systems due to less dopamine release i n the mpfc-(see Figure 16). Likewise, the reduction i n subcortical dopamine synthesis seen following low doses of dopamine receptor agonists may be due to the stimulation of mpfc high a f f i n i t y , dopamine receptors located on nondopaminergic neurons. However, the extent to which the properties of mpfc dopamine receptors are responsible for a l l dopamine "autoreceptor" e f f e c t s i s presently unknown. In summary, the dopamine autoreceptor l i t e r a t u r e should be reevaluated due to two l i n e s of recent evidence. F i r s t , the mesocortical dopamine system 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 dopamine systems. Second, the mpfc may possess a s i g n i f i c a n t number of dopamine receptors with a high a f f i n i t y for dopamine receptor agonists. These receptors may be located on noradrenergic terminals where they can reduce mpfc noradrenergic a c t i v i t y when activated. They may be also involved i n the mechanism through which the mesocortical dopamine system t o n i c a l l y i n h i b i t s subcortical dopamine systems. mpfc Figure 16. Mesocortical 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 cortical dopaminergic a c t i v i t y by influencing descending projections. The mechanism responsible i s , as yet, undetermined. The diagram i s not meant to imply that the interactions i n the nucleus accumbens and the striatum necessarily involve axo-axonic synapses. VTA = ventral tegmental area; SN = substantia nigra; (-) = i n h i b i t o r y influence. 96 Interpretations of pH Dependent Binding D2 dopamine receptors i n the mpfc displayed pH-dependent binding c h a r a c t e r i s t i c s with respect to both the e f f e c t s of 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 discussed above, th i s section 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 inter 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 these e f f e c t s i s whether they have any i n vivo relevance. I t i s possible that dopamine agonists are degraded (or bound to non-D2 sites) i n the presence of s t r i a t a l tissue at pH 7.9, whereas mpfc tissue may not have the same e f f e c t . At pH 6.2 t h i s a r t i f a c t u a l reduction i n free agonist concentration may occur with both brain regions. Such changes i n actual free agonist concentrations i n binding reactions could produce the r e l a t i v e l y higher " a f f i n i t y " f or agonists observed i n the mpfc at pH 7.9 but not pH 6.2. It i s also possible that the pH-dependent e f f e c t s do accurately represent the influence that changes i n pH whould have i f they occured i n vivo. However, changes of the magnitude employed here may not be present under physi o l o g i c a l conditions. If such large changes i n pH do occur i n vivo, and they have the e f f e c t s on D2 dopamine receptors indicated by the data i n t h i s thesis, these receptor 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 to 97 other pH-dependent changes. For instance, K r i s h t a l and Pidoplichko (1981) have demonstrated that neurons i s o l a t e d from the spin a l ganglia and the ganglion of the trigeminal nerve display an inward current i n response to small pH changes i n e x t r a c e l l u l a r solution. If central neurons with dopamine D2 receptors are s i m i l a r l y sensitive to small changes i n pH then these e f f e c t s may mask any pH-dependent changes i n dopamine receptor a f f i n i t y . Future Research At least three areas of future research on mpfc D2 dopamine receptors would appear to be worth pursuing. F i r s t , i t w i l l be important to search for compounds that i n t e r a c t with mpfc D2 receptors i n a highly s e l e c t i v e manner. This l i n e of research should be f a c i l i t a t e d by the present development of an assay to measure mpfc D2 receptors. I t i s also encouraging to note that dopamine receptor agonists display some s e l e c t i v i t y i n that they are more potent on mpfc D2 receptors than on s t r i a t a l D2 receptors. Second, the mechanisms by which mpfc D2 receptors are affected by various experimental manipulations should be investigated further. Dopamine i s thought to be involved i n many behaviours and diseases. If the prefrontal cortex dopamine system i s fu n c t i o n a l l y and pharmacologically d i s t i n c t , i t i s imperative that the function of t h i s system 98 be d i r e c t l y explored. Extrapolations from data c o l l e c t e d i n studies of su b c o r t i c a l dopamine systems are not acceptable 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 possible to d i r e c t l y investigate the function of mpfc D2 receptors with the help of the assay described i n t h i s thesis. Third, while the present assay i s capable of measuring the properties of D2 dopamine receptors i n the mpfc, i t i s important to continue to develop more and better methods for t h i s task. One such p o t e n t i a l new method may involve the use of [l^Sjj.guipj-icie. I J ^ Q r e c e n t publications have reported that [125j]-sulpride labels p o t e n t i a l D2 dopamine related binding s i t e s i n many brain regions (Martres et a l . , 1985; Martres, Sales, Bouthenet and Schwartz, 1985). Sites i n the p a r i e t a l cortex and cerebellum, although at very low density, seem to be pharmacologically very s i m i l a r to those s i t e s l a b e l l e d by [ 1 2 5 I ] - s u l p r i d e i n the striatum. The s i t e s l a b e l l e d by [125j]-sulpride i n the mpfc have not been pharmacologically characterized. At pH 7.4 dopamine receptor agonists are no more potent at competing for [125j]-sulpride binding s i t e s i n the p a r i e t a l cortex than i n the striatum (Martres, Sales, Bouthenet and Schwarts, 1985). These re s u l t s contrast with the present demonstration that, at pH 7.9, dopamine receptor agonists are more potent at competing for binding s i t e s i n the mpfc than i n the striatum. Therefore, either the [ 1 2 5 I ] - s u l p r i d e labels a d i f f e r e n t population of binding s i t e s at pH 7.4 than those presently l a b e l l e d at pH 7.9 by [ JH]-spiperone (as suggested above) or the higher a f f i n i t y of dopamine receptor agonists for c o r t i c a l D2 receptors i s r e l a t i v e l y s p e c i f i c to the mpfc. As discussed above, there i s evidence that the s i t e s l a b e l l e d by the butyrophenones, haloperidol and spiperone, are functional dopamine receptors because the binding a f f i n i t i e s of the many dopaminergic compounds tested are highly correlated with t h e i r i n vivo potencies. If analogous correlations are obtained with [ 125j_]-sulpride l a b e l l e d s i t e s then study of the mpfc with t h i s ligand should further elucidate the role of D2 receptors i n the mpfc. The high s p e c i f i c a c t i v i t y and low nonspecific binding of t h i s ligand make i t i d e a l for use i n brain areas such as the mpfc, where the density of D2 receptors i s very low. However, i f future research indicates that [125j]-sulpride labels a d i f f e r e n t population of receptors than that l a b e l l e d by the butyrophenones then research with the butyrophenones should continue i n the mpfc. The present assay at pH 6.2 had a higher degree of resolution than that at pH 7.9 (see Experiments 1 and 2). This i s very l i k e l y due to a near elimination of [ 3H]-spiperone binding to spirodecanone s i t e s . However, as discussed above, the D2 binding observed i n the mpfc at pH 7.9 has a number of in t e r e s t i n g properties which make the pH 7.9 assay very a t t r a c t i v e even though i t i s not as accurate as the pH 6.2 assay. I t has been demonstrated that s o l u b i l i z e d [ JH]-spiperone binding s i t e s from the striatum contain a very high proportion of spirodecanone s i t e s (Laduron, 1981). This i s a problem not encountered with standard D2 binding assays employing s t r i a t a l homogenates. To deal with t h i s problem, researchers at Janssen Pharmaceutica have developed compounds which s e l e c t i v e l y occlude the s o l u b i l i z e d spirodecanone s i t e s (Laduron, 1981). 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P., K a n y i c s k a , B. & F e k e t e , M. I . K. S t r e s s i n d u c e d c h a n g e s i n dopamine m e t a b o l i s m o f r a t b r a i n c o r t e x as w e l l as p l a s m a c o r t i c o s t e r o n e and p r o l a c t i n l e v e l s - e f f e c t s o f d i a z e p a m and t o f i s o p a m . I n U s d i n / K v e t r i a n s k y / K o p i n ( E d s . ) , C a t e c h o l a m i n e s and s t r e s s : R e c e n t A d v a n c e s . H o l l a n d : E l s e v i e r N o r t h H o l l a n d , I n c . , 1980. T a s s i n , J . P., B o c k a e r t , J . , B l a n c , G., S t i n u s , L., T h i e r r y , A. M., L a v i e l l e , S., Premont, J . & G l o w i n s k i , J . T o p o g r a p h i c a l d i s t r i b u t i o n o f d o p a m i n e r g i c i n n e r v a t i o n and d o p a m i n e r g i c r e c e p t o r s o f t h e a n t e r i o r c e r e b r a l c o r t e x o f t h e r a t . B r a i n R e s e a r c h , 1978, 154, 241-251. T a s s i n , J . P., T h i e r r y , A. 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C a t i o n r e g u l a t i o n d i f f e r e n t i a t e s s p e c i f i c b i n d i n g o f [ 3 H ] s u l p i r i d e and [ 3 H ] s p i p e r o n e t o r a t s t r i a t a l p r e p a r a t i o n s . J o u r n a l o f Pharmacy and P h a r m a c o l o g y , 1980, 32, 441. T h i e r r y , A. M., B l a n c , G., S o b e l , A., S t i n u s , L. & G l o w i n s k i , J . D o p a m i n e r g i c t e r m i n a l s i n t h e r a t c o r t e x . S c i e n c e , 1973a, 182, 499-501. T h i e r r y , A. M., S t i n u s , L., B l a n c , G. & G l o w i n s k i , J . Some e v i d e n c e f o r t h e e x i s t e n c e o f d o p a m i n e r g i c n e u r o n s i n t h e r a t c o r t e x . B r a i n R e s e a r c h , 1973b, 50, 230-234. T h i e r r y , A. M., T a s s i n , J . P., B l a n c , G. & G l o w i n s k i , J . S e l e c t i v e a c t i v a t i o n o f t h e m e s o c o r t i c a l DA s y s t e m by s t r e s s . N a t u r e , 1976, 263, 242-244. T h i e r r y , A. M., T a s s i n , J . P. & G l o w i n s k i , J . B i o c h e m i c a l and e l e c t r o p h y s i o l o g i c a l s t u d i e s o f t h e m e s o c o r t i c a l dopamine s y s t e m . I n L. D e s c a r r i e s , T. R. R e a d e r & H. H. J a s p e r ( E d s . ) , Monoamine i n n e r v a t i o n o f c e r e b r a l  c o r t e x . New Y o r k : A l a n R. L i s s , I n c . , 1984. T i s s a r i , A. H., A r g i o l a s , A., F a d d a , F., S e r r a , G. & G e s s a , G. L. F o o t - s h o c k s t r e s s a c c e l e r a t e s n o n - s t r i a t a l dopamine s y n t h e s i s w i t h o u t a c t i v a t i n g t y r o s i n e h y d r o x y l a s e . N a u n y n - S c h m i e d e b e r g ' s A r c h i v e s  o f P h a r m a c o l o g y , 1979, 308, 155-157. T r a b u c c h i , M., L o n g o n i , R., F r e s i a , P. & Spano, P. F. S u l p i r i d e : A s t u d y o f t h e e f f e c t s on dopamine r e c e p t o r s i n r a t n e o s t r i a t u m and l i m b i c f o r e b r a i n . 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D i f f e r e n t i a l e f f e c t s o f c l a s s i c a l and a t y p i c a l a n t i p s y c h o t i c d r u g s on A9 and A10 dopamine n e u r o n s . S c i e n c e , 1983, 221, 1054-1057. W i l l i a m s , J . B. W. D i a g n o s t i c and s t a t i s t i c a l m a nual o f  m e n t a l d i s o r d e r s . New Y o r k : A m e r i c a n P s y c h i a t r i c A s s o c i a t i o n , 1980. W o o d r u f f , G. N. P l e n a r y l e c t u r e on dopamine r e c e p t o r s . I n M. K o h s a k a , T. S h o h m o r i , Y. T s u k a d a & G. N. W o o d r u f f ( E d s . ) , A d v a n c e s i n dopamine r e s e a r c h . New Y o r k : Pergamon P r e s s , 1981. Yang, J . X., K n o r r , A. M., O n e l , K., Tarn, S. Y., D e u t c h , A. Y., L u b i c h , L. & R o t h , R. H. E f f e c t s o f d i f f e r e n t s t r e s s p a r a d i g m s on c e n t r a l dopamine and n o r e p i n e p h r i n e m e t a b o l i s m . 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Ma ie r , S . F . , Drugan, R.C., Grau, J .W., Hyson, R., MacLennan, A . J . , Madden, J . and Barchas, J .D . Op io id and nonopio id mechanisms of s t r e s s - i nduced a n a l g e s i a . J_n Advances i n Endogenous and Exogenous Op io i d s , Proceedings of the I n t e r na t i ona l Na r co t i c Research Conference, Kyoto, Japan, J u l y 26-30, 1981. MacLennan, A . J . , Drugan, R.C., Hyson, R.L., Ma ie r , S . F . , Madden, J . and Barchas, J .D . (1982) Co r t i c o s t e r one : A c r i t i c a l f a c t o r i n an op i o i d form o f s t r e s s - i nduced a n a l g e s i a . Sc i ence , V o l . 215, ppl530-1532. MacLennan, A . J . , Drugan, R.C., Hyson, R.L. , Ma ie r , S . F . , Madden, J . and Barchas, J .D . (1982) D i s s o c i a t i o n of long-term ana lges ia and the shu t t l e -box escape d e f i c i t caused by inescapable shock. Journal o f Comparative and Phy s i o l o g i c a l Psychology, V o l . 96, pp904-912. Ma ie r , S . F . , Drugan, R.C., Grau, J.W., Hyson, R.L., MacLennan, A . J . , Moye, T. , Madden, J . and Barchas, J .D . Learned he l p l e s sne s s , pain i n h i b i t i o n , and the endogenous o p i a t e s . I_n Advances i n Ana l y s i s o f Behav io r , V o l . 4, (Eds. Z e i l e r , M.D. and Harzem, P. ) New York: John Wi ley and Sons, 1983. MacLennan, A . J . , Drugan, R.C. and Ma ie r , S .F . (1983) Long-term s t r e s s -induced ana lges i a b locked by scopolamine. Psychopharmacology, V o l . 80, pp267-268. MacLennan, A . J . .and Ma ie r , S .F . (1983) Coping and the s t r e s s - i nduced p o t e n t i a t i o n o f s t imu l an t s te reotypy i n the r a t . Sc i ence , V o l . 219, ppl091-1093. MacLennan, A . J . , Jakubov ic , A. and F i b i g e r , H.C. Cha r a c t e r i z a t i o n of D2 dopamine receptors i n the medial f r o n t a l co r tex of the r a t . ( i n p r epa r a t i o n ) . 

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