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Studies involving somatostatin systems in the rodent central nervous system Radke, James Melvin 1987

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STUDIES INVOLVING SOMATOSTATIN SYSTEMS IN THE RODENT CENTRAL NERVOUS SYSTEM. by JAMES MELVIN RADKE B.Sc.(hon.) U n i v e r s i t y of B r i t i s h Columbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Neuroscience) We accept t h i s t h e s i s as conforming to the r e q u i r e d standards THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1987 c)James M e l v i n Radke, 1987 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. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) i i A B S T R A C T Somatostatin i s a neuropeptide found throughout the b r a i n . S e v e r a l s t u d i e s have e s t a b l i s h e d i t s anatomical d i s t r i b u t i o n as being q u i t e heterogenous w i t h r e l a t i v e l y h i g h c o n c e n t r a t i o n s appearing i n the l i m b i c and s t r i a t a l systems. P r e s e n t l y , very l i t t l e i s known about the f u n c t i o n s of s o m a t o s t a t i n systems i n the b r a i n and how they i n t e r a c t w i t h other t r a n s m i t t e r systems. The f o l l o w i n g r e p o r t i s a summary of experiments undertaken to assess the f u n c t i o n a l and chemical i n t e r a c t i o n s of s o m a t o s t a t i n w i t h other n e u r o t r a n s m i t t e r systems. P r e v i o u s s t u d i e s have e s t a b l i s h e d t h a t the dopaminergic i n p u t s to the b a s a l g a n g l i a are important f o r locomotor a c t i v i t y and reward. These systems have a l s o been i m p l i c a t e d i n s e v e r a l mental and n e u r a l d i s e a s e s such as s c h i z o p h r e n i a , d e p r e s s i o n , and P a r k i n s o n 1 s d i s e a s e . In the f i r s t experiment, i n t e r a c t i o n s between dopamine and s o m a t o s t a t i n systems were examined u s i n g paradigms i n v o l v i n g b e h a v i o u r a l responses to dopamine a g o n i s t s . D e p l e t i o n of s o m a t o s t a t i n l e v e l s by the drug cysteamine was found to attenuate amphetamine- and apomorphine-mediated motor behaviours but not the r e i n f o r c i n g aspects of amphetamine. The second experiment attempted to f u r t h e r c h a r a c t e r i z e the nature of the dopamine-s o m a t o s t a t i n i n t e r a c t i o n by examining the e f f e c t s of h a l o p e r i d o l , a dopamine a n t a g o n i s t , on c e n t r a l s o m a t o s t a t i n l e v e l s . Short term treatment w i t h h a l o p e r i d o l decreased s t r i a t a l s o m a t o s t a t i n i i i l e v e l s . Long term treatment (8 months) wi t h h a l o p e r i d o l f a i l e d t o a l t e r s o m a t o s t a t i n l e v e l s i n the caudate-putamen. Since s o m a t o s t a t i n l e v e l s appear t o be normal i n P a r k i n s o n i a n b r a i n s , the e f f e c t s of MPTP p o i s o n i n g i n mice on c e n t r a l s o m a t o s t a t i n l e v e l s was a l s o s t u d i e d to examine the accuracy of t h i s animal model of Parkinson's d i s e a s e and examine the e f f e c t s of dopaminergic l e s i o n s on so m a t o s t a t i n l e v e l s . The r e s u l t s of t h i s experiment i n d i c a t e t h a t MPTP causes a dose dependent i n c r e a s e i n n i g r a l s o m a t o s t a t i n l e v e l s without a l t e r i n g s t r i a t a l or c o r t i c a l l e v e l s . These r e s u l t s are i n p a r t i a l disagreement w i t h r e s u l t s o b t a i n e d from both post-mortem P a r k i n s o n i a n b r a i n s and primates g i v e n MPTP, thereby q u e s t i o n i n g the accuracy of t h i s mouse model of Parkinson's d i s e a s e . The f i n a l experiment examined the e f f e c t s of the a n t i c o n v u l s a n t - a n t i d e p r e s s a n t carbamazepine on c e n t r a l s o m a t o s t a t i n l e v e l s i n the r a t . Although the chemical mechanisms r e s p o n s i b l e f o r the t h e r a p e u t i c e f f e c t s of carbamazepine are unknown, p r e v i o u s s t u d i e s have suggested t h a t i t s e f f i c a c y i n the treatment of both manic-depression and e p i l e p s y may be a s s o c i a t e d w i t h the a b i l i t y of t h i s drug t o reduce the abnormal som a t o s t a t i n l e v e l s observed i n these d i s e a s e s . In t h i s experiment, n e i t h e r acute, c h r o n i c , nor withdrawal from c h r o n i c treatment w i t h carbamazepine were found t o a l t e r the l e v e l s of so m a t o s t a t i n i n r a t s . The l a c k of e f f e c t s of carbamazepine on b a s a l i v s o m a t o s t a t i n l e v e l s may i n d i c a t e s o m a t o s t a t i n c e l l s are s u s c e p t i b l e t o carbamazepine o n l y under p a t h o l o g i c a l s i t u a t i o n s . Together, these r e s u l t s are d i s c u s s e d i n the c o n t e x t of re c e n t o b s e r v a t i o n s of abnormal s o m a t o s t a t i n l e v e l s i n s e v e r a l d i s e a s e s of the c e n t r a l nervous system and p r o v i d e some i n s i g h t i n t o the i n t e r a c t i o n s and f u n c t i o n s of s o m a t o s t a t i n systems i n the normal and abnormal b r a i n . V TABLE OF CONTENTS A b s t r a c t i i Table of contents v L i s t of f i g u r e s v i i Acknowledgements i x INTRODUCTION 1 Somatostatin i n the b a s a l g a n g l i a 1 Dopamine-somatostatin i n t e r a c t i o n s 4 Somatostatin i n n e u r a l d i s e a s e 5 R a t i o n a l e 6 EXPERIMENTS 1. The e f f e c t s of cysteamine on s o m a t o s t a t i n immunoreactivity and on dopamine-mediated behaviours. I n t r o d u c t i o n 8 Methods 10 RIA methods 14 R e s u l t s 16 D i s c u s s i o n 24 2. The e f f e c t s of s h o r t and long term h a l o p e r i d o l treatment on so m a t o s t a t i n immunoreactivity. I n t r o d u c t i o n 31 Methods 32 R e s u l t s 34 D i s c u s s i o n 35 v i 3. The e f f e c t s of MPTP p o i s o n i n g on so m a t o s t a t i n immunoreactivity i n the mouse. I n t r o d u c t i o n 39 Methods 40 R e s u l t s 41 D i s c u s s i o n 42 4. The e f f e c t s of carbamazepine and desmethylimipramine (DMI) on s o m a t o s t a t i n immunoreactivity. I n t r o d u c t i o n 47 Methods 48 R e s u l t s 50 D i s c u s s i o n 50 GENERAL DISCUSSION Dopamine-somatostatin i n t e r a c t i o n s 55 Somatostatin i n n e u r o - p s y c h i a t r i c d i s e a s e s 57 The f u n c t i o n of so m a t o s t a t i n 61 REFERENCES 62 APPENDIX 88 v i i L I S T O F F I G U R E S F i g u r e 1: E f f e c t s of 4 hour pretreatment w i t h cysteamine on amphetamine-induced locomotor a c t i v i t y 18 F i g u r e 2: E f f e c t s of 7 hour pretreatment w i t h cysteamine on s t r i a t a l s o m a t o s t a t i n immunoreactivity 19 F i g u r e 3: E f f e c t s of 12 hour pretreatment w i t h cysteamine on apomorphine-induced s t e r e o t y p y 21 F i g u r e 4: E f f e c t s of 13 hour pretreatment w i t h cysteamine on s t r i a t a l s o m a t o s t a t i n immunoreactivity 22 F i g u r e 5: E f f e c t s of cysteamine pretreatment on amphetamine-induced p l a c e p r e f e r e n c e 25 F i g u r e 6: E f f e c t s of repeated cysteamine a d m i n i s t r a t i o n on s t r i a t a l s o m a t o s t a t i n immunoreactivity... 26 F i g u r e 7: E f f e c t s of s h o r t term 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 c e n t r a l s o m a t o s t a t i n immunoreactivity 35 F i g u r e 8: E f f e c t s of long term and withdrawal f o l l o w i n g long term 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 c e n t r a l s o m a t o s t a t i n immunoreactivity 36 F i g u r e 9: E f f e c t s of MPTP p o i s o n i n g on s t r i a t a l catecholamine l e v e l s i n mice 43 v i i i F i g u r e 10: E f f e c t s of MPTP p o i s o n i n g on c e n t r a l s o m a t o s t a t i n immunoreactivity 44 F i g u r e 11: E f f e c t s of carbamazepine a d m i n i s t r a t i o n on c e n t r a l s o m a t o s t a t i n immunoreactivity.... 51 F i g u r e 12: E f f e c t s of DMI a d m i n i s t r a t i o n on c e n t r a l s o m a t o s t a t i n immunoreactivity 52 F i g u r e 13: An example of a radioimmunoassay standard curve 89 i x ACKNOWLEDGEMENTS T h i s t h e s i s was w r i t t e n by me, but the c o l l e c t i o n and i n t e r p r e t a t i o n of data was performed by numerous people. I would l i k e t o thank Dr. A. J . MacLennan and Dr. H. C. F i b i g e r f o r p r o v i d i n g me wit h the t i s s u e samples f o r the h a l o p e r i d o l experiment. A l s o , the catecholamine a n a l y s i s w i t h the MPTP study was performed by Paul Cumming. The a s s i s t a n c e of Dr. M. T. M a r t i n - I v e r s o n was g r e a t l y a p p r e c i a t e d when I was t r y i n g t o understand what these b e h a v i o u r a l s t u d i e s are supposed t o mean. F i n a l l y , I would l i k e to express my deepest a p p r e c i a t i o n f o r the guidance p r o v i d e d by Dr. Steven V i n c e n t . His h e l p i n t r y i n g t o teach me to perform s c i e n c i f i c r e s e a r c h and to w r i t e about i t has not gone unnot i c e d . 1 INTRODUCTION Somatostatin i n the B a s a l G a n g l i a Somatostatin was f i r s t r e c o g n i z e d as a p h y s i o l o g i c a l l y a c t i v e p e p t i d e by Brazeau and h i s c o l l e g u e s (1973) who i s o l a t e d t h i s p e p t i d e from ovine hypothalami and found i t i n h i b i t e d somatotropin (growth hormone) r e l e a s e ; thus s o m a t o s t a t i n i s o f t e n r e f e r r e d to as SRIF (somatotropin r e l e a s e i n h i b i t i n g f a c t o r ) . Subsequent s t u d i e s have r e v e a l e d t h a t s o m a t o s t a t i n i s p r e s e n t i n v a r i o u s endocrine c e l l s , i n the p e r i p h e r a l nervous system, and throughout v a r i o u s r e g i o n s of the c e n t r a l nervous system (Beal e t a l . , 1986a; Brownstein e t a l . , 1975; F i n l e y e t a l . , 1981; Johansson e t a l . , 1984; Kobayashi e t a l . , 1977; V i n c e n t e t a l . , 1985). W i t h i n the b r a i n s o m a t o s t a t i n i s found i n h i g h c o n c e n t r a t i o n i n the hypothalamus and the l i m b i c system, i n t e r m e d i a t e l e v e l s i n the b a s a l g a n g l i a and c o r t e x , and low or u n d e t e c t a b l e amounts i n the cerebellum and o l f a c t o r y bulb. There appears to be at l e a s t two p h y s i o l o g i c a l l y a c t i v e s o m a t o s t a t i n p e p t i d e s . In the b r a i n s o m a t o s t a t i n i s mainly p r e s e n t as the 14 amino a c i d p e p t i d e , but a 28 amino a c i d p e p t i d e has a l s o been shown to e x i s t , which c o n t a i n s somatostatin-14 w i t h i n i t s s t r u c t u r e . C h a r a c t e r i z a t i o n of the s o m a t o s t a t i n gene has r e v e a l e d t h a t p r e p r o s o m a t o s t a t i n i s 116 amino a c i d s long and i s c l e a v e d to a 92 amino a c i d p r o s o m a t o s t a t i n (Epelbaum, 1986). I t appears t h a t somatostatin-14 can be c l e a v e d from e i t h e r the p r o s o m a t o s t a t i n p e p t i d e d i r e c t l y (Gomez e t a l . , 1985), or by a t r y p s i n - l i k e c o n v e r s i o n from somatostatin-28 (see review by 2 Epelbaum, 1986). S t u d i e s have suggested these two p e p t i d e s have d i f f e r e n t d i s t r i b u t i o n s i n the b r a i n ( P a t e l e t a l . , 1981) which may be due to d i f f e r e n t r e g i ons having d i f f e r e n t methods of p r o c e s s i n g somatostatin-28 to somatostatin-14 (Zingg and P a t e l , 1983). W i t h i n the r a t b a s a l g a n g l i a , s o m a t o s t a t i n i s found i n r e l a t i v e l y h i g h c o n c e n t r a t i o n s i n the nucleus accumbens and caudate-putamen, and low l e v e l s i n the s u b s t a n t i a n i g r a and v e n t r a l tegmental area (Beal e t a l . , 1986a; Brownstein e t a l . , 1975; Kobayashi e t a l . , 1977). B e a l and h i s c o l l e g u e s (1983; 1986a) have a l s o shown t h a t w i t h i n the caudate-putamen, the l e v e l s are q u i t e low i n the d o r s a l - l a t e r a l r e g i o n . L e s i o n s t u d i e s i n the r a t i n d i c a t e t h a t much of the s t r i a t a l (nucleus accumbens + caudate-putamen) soma t o s t a t i n i s p r e s e n t i n i n t r i n s i c neurons. E a r l y s t u d i e s suggested the hypothalamus as a source f o r some s t r i a t a l s o m a t o s t a t i n ( P a l k o v i t s e t a l . , 1980), however other s t u d i e s have f a i l e d t o support t h i s (Beal e t a l . , 1985; Crowley and T e r r y , 1980). Furthermore, l e s i o n s of the a f f e r e n t s to the s t r i a t u m from the c o r t e x , s u b s t a n t i a n i g r a , thalamus and amygdala do not reduce the s t r i a t a l l e v e l s of s o m a t o s t a t i n (Beal e t a l . , 1985). In c o n t r a s t , s t r i a t a l k a i n i c a c i d l e s i o n s , which d e s t r o y the s t r i a t a l neurons, reduce the l e v e l s of s o m a t o s t a t i n (Beal and M a r t i n , 1983). Immunohistochemical s t u d i e s have a l s o demonstrated the presence of neurons d i s p l a y i n g s o m a t o s t a t i n immunoreactivity w i t h i n the s t r i a t u m ( F i n l e y e t a l . , 1981; Johansson e t a l . , 1984; V i n c e n t e t a l . , 1985). U l t r a s t r u c t u r a l s t u d i e s i n d i c a t e t h a t 3 these c e l l s are medium-sized a s p i n y neurons ( D i F i g l i a and A r o n i n , 1982; Takagi e t a l . , 1983; V i n c e n t and Johansson, 1983; V i n c e n t e t a l . , 1982a). These c e l l s have an indented nucleus, and a r i c h cytoplasm c o n t a i n i n g w e l l developed G o l g i apparatus and rough endoplasmic r e t i c u l u m . Furthermore, t h i s p a r t i c u l a r p o p u l a t i o n of s t r i a t a l neurons has a l s o been shown to c o n t a i n an NADPH-diaphorase enzyme a c t i v i t y t h a t allows these c e l l s t o be s e l e c t i v e l y s t a i n e d (Vincent e t a l . , 1983a). T h i s l a t t e r c h a r a c t e r i s t i c of these c e l l s a llows them to be e a s i l y d i f f e r e n t i a t e d from the other major s t r i a t a l i n t e r n e u r o n p o p u l a t i o n , the c h o l i n e r g i c c e l l s , which are a l s o a s p i n y but do not d i s p l a y NADPH-diaphorase a c t i v i t y (Vincent e t a l . , 1983b). Immunohistochemical s t u d i e s have a l s o r e v e a l e d t h a t some of the s o m a t o s t a t i n c o n t a i n i n g c e l l s i n the s t r i a t u m a l s o c o n t a i n neuropeptide Y (Vincent e t a l . , 1982a; 1982b; 1983a). T h i s i s supported by radioimmunoassay s t u d i e s showing t h a t these two p e p t i d e s have a s i m i l a r d i s t r i b u t i o n i n the s t r i a t u m (Beal and M a r t i n , 1986a). Immunohistochemical s t u d i e s of the s y n a p t i c connections of s t r i a t a l s o m a t o s t a t i n c e l l s i n d i c a t e t h a t these neurons r e c e i v e most of t h e i r s y n a p t i c i n p u t on d e n d r i t e s , v i a both symmetric and asymmetric c o n t a c t s ( D i F i g l i a and A r o n i n , 1982; Takagi e t a l . , 1983). Somatostatin-immunoreactive t e r m i n a l s appear to form p r i m a r i l y symmetrical synapses w i t h d e n d r i t e s and spines of other s t r i a t a l neurons ( D i F i g l i a and A r o n i n , 1982; Takagi e t a l . , 1983). At p r e s e n t , the t r a n s m i t t e r s of the c e l l s w i t h which so m a t o s t a t i n c o n t a i n i n g i n t e r n e u r o n s make c o n t a c t i s unknown, but 4 may i n c l u d e the medium sp i n y neurons which are b e l i e v e d to be the major output neurons of the s t r i a t u m . Somatostatin-dopamine interactions The two main ascending dopaminergic systems i n the r a t are the n i g r o s t r i a t a l t r a c t t o the caudate-putamen o r i g i n a t i n g i n the s u b s t a n t i a n i g r a , and the mesolimbic p r o j e c t i o n to the nucleus accumbens, o l f a c t o r y t u b e r c l e , and the medial p r e f r o n t a l c o r t e x a r i s i n g from the v e n t r a l tegmental area. Although these systems have been e x t e n s i v e l y s t u d i e d , the s y n a p t i c connections they make are s t i l l p o o r l y understood. Dopamine t e r m i n a l s i n the s t r i a t u m have been shown to make both symmetric and asymmetric c o n t a c t s w i t h a wide v a r i e t y of c e l l s , forming a x o - d e n d r i t i c , axo-spinous, and axo-somatic c o n t a c t s ( A r l u i s o n e t a l . , 1984; Freund e t a l . , 1984; P i c k e l e t a l . , 1981). The neurochemical i d e n t i t i e s of these p o s t s y n a p t i c c e l l s are mostly unknown. There i s some evidence s u p p o r t i n g the view t h a t the a c t i v i t y of c e n t r a l dopamine neurons may be i n f l u e n c e d by s o m a t o s t a t i n . Both i n t r a v e n t r i c u l a r ( G a r c i a - S e v i l l a e t a l . , 1978) and i n t r a s t r i a t a l (Beal and M a r t i n , 1984a; 1986b) i n f u s i o n s of s o m a t o s t a t i n i n c r e a s e the turnover of s t r i a t a l dopamine. Somatostatin a l s o appears to i n c r e a s e the spontaneous ( C h e s s e l e t and R e i s i n e , 1983; S t a r r , 1982) and K +-evoked ( S t a r r , 1982) r e l e a s e of t r i t i a t e d dopamine from r a t s t r i a t a l s l i c e s . S i m i l a r r e s u l t s have a l s o been observed i n v i v o i n the c a t caudate nucleus ( C h e s s e l e t and R e i s i n e , 1983). 5 Some of the e f f e c t s of s o m a t o s t a t i n might be mediated through c y c l i c AMP. I n t r a v e n t r i c u l a r a d m i n i s t r a t i o n of s o m a t o s t a t i n produces an e l e v a t i o n i n s t r i a t a l c y c l i c AMP l e v e l s ( H erchl e t a l . , 1977). More r e c e n t s t u d i e s have i n d i c a t e d t h a t s o m a t o s t a t i n can i n h i b i t the s t i m u l a t i o n of s t r i a t a l adenylate c y c l a s e a c t i v i t y produced by dopamine (Chneiweiss e t a l . , 1985; Moser e t a l . , 1986) . The dopamine system a l s o appears to be able to modulate the s t r i a t a l s o m a t o s t a t i n neurons. In t h i s regard, c h r o n i c a d m i n i s t r a t i o n of n e u r o l e p t i c s produces a r e d u c t i o n i n the l e v e l s of s o m a t o s t a t i n i n the s t r i a t u m (Beal and M a r t i n , 1984c). Somatostatin i n n e u r a l d i s e a s e There has been c o n s i d e r a b l e i n t e r e s t i n the f u n c t i o n s of s o m a t o s t a t i n i n the p e r i p h e r y and i t s i m p i c a t i o n s i n endocrine d i s o r d e r s . For example so m a t o s t a t i n analogs are being t e s t e d f o r treatment of p a n c r e a t i c endocrine tumours (Wood e t a l . , 1985), d i a b e t e s ( G e r i c h , 1976), and acromegaly (Kingsnorth e t a l . , 1986). Rec e n t l y , s o m a t o s t a t i n l e v e l s i n the b r a i n have a l s o been shown to be a l t e r e d i n s e v e r a l neuronal d i s o r d e r s . In Huntington's d i s e a s e , which i s c h a r a c t e r i z e d by severe l o s s of s t r i a t a l t i s s u e , t h e r e appears to be a s p a r i n g of s o m a t o s t a t i n c e l l s (Dawborn e t a l . , 1985; F e r r a n t e e t a l . , 1985) r e s u l t i n g i n s t r i a t a l s o m a t o s t a t i n c o n c e n t r a t i o n s being g r e a t l y e l e v a t e d i n post mortem b r a i n s of Huntington's p a t i e n t s (Aronin e t a l . , 1983; B e a l e t a l . , 1984; Nemeroff e t a l . , 1983; Sagar e t a l . , 1984). 6 In c o n t r a s t , other d i s o r d e r s such as Parkinson's d i s e a s e (Agid and Javoy-Agid, 1985; Epelbaum e t a l . , 1983; Rinne e t a l . , 1984) and s c h i z o p h r e n i a (Nemeroff e t a l . , 1983) appear to have normal s t r i a t a l s o m a t o s t a t i n l e v e l s . Most c l i n i c a l s t u d i e s use lumbar punctures to study s o m a t o s t a t i n and have p r o v i d e d some i n t e r e s t i n g r e s u l t s . Included i n the l i s t of d i s e a s e s showing abnormal c e r e b r o s p i n a l f l u i d (CSF) s o m a t o s t a t i n l e v e l s are: Parkinson's d i s e a s e (Cramer e t a l . , 1985), Alzheimer's d i s e a s e (Gomez e t a l . , 1986a; 1986b; Raskind e t a l . , 1986), Huntington's d i s e a s e (Cramer e t a l . , 1981), d e p r e s s i o n (Agren and Lundqvist, 1984; Gerner and Yamada, 1982; Rubinow e t a l . , 1985; Rubinow, 1986), and p a t i e n t s w i t h d y s t o n i c syndromes (Cramer e t a l . , 1985; T h a i e t a l . , 1985). Drug regimens have a l s o been shown to a l t e r s o m a t o s t a t i n l e v e l s . As p r e v i o u s l y mentioned, n e u r o l e p t i c s reduce s t r i a t a l s o m a t o s t a t i n l e v e l s i n the r a t (Beal and M a r t i n , 1984c). In the c l i n i c a l s e t t i n g , n e u r o l e p t i c s have a l s o been shown to a l t e r CSF l e v e l s of somatostatin-14 and -28 (Gattez e t a l . , 1986). In a d d i t i o n , a l t e r e d CSF s o m a t o s t a t i n l e v e l s have been r e p o r t e d i n a f f e c t i v e l y i l l p a t i e n t s on carbamazepine, z i m e l i d i n e (ZMI), or f l u p h e n a z i n e treatment (Rubinow, 1986). With regard to carbamazepine, t h i s drug has been shown to lower s o m a t o s t a t i n l e v e l s when the l e v e l s are abnormal, i n both a f f e c t i v e l y i l l humans (Rubinow e t a l . , 1984; 1985; Rubinow, 1986) and i n k i n d l e d r a t s ( H i g uchi e t a l . , 1986). 7 The purpose of the f o l l o w i n g s e t of experiments was to examine f u r t h e r the p o s s i b l e i n t e r a c t i o n s between dopamine and s o m a t o s t a t i n i n the b a s a l g a n g l i a . The i n i t i a l experiments i n v o l v e a b e h a v i o u r a l approach to study the i n t e r a c t i o n . T h i s was f o l l o w e d by two experiments i n v o l v i n g the e f f e c t s of the drugs h a l o p e r i d o l and MPTP on s o m a t o s t a t i n l e v e l s . F i n a l l y , the e f f e c t s of the a n t i d e p r e s s a n t s carbamazepine and desmethylimipramine (DMI) on the l e v e l s of s o m a t o s t a t i n were examined to determine i f these c l i n i c a l l y important drugs have some of t h e i r e f f e c t s by a l t e r i n g s o m a t o s t a t i n systems. 8 EXPERIMENT 1. THE EFFECTS OF CYSTEAMINE ON SOMATOSTATIN IMMUNOREACTIVITY AND ON DOPAMINE MEDIATED BEHAVIOURS. INTRODUCTION Very l i t t l e i s p r e s e n t l y known about the f u n c t i o n of so m a t o s t a t i n i n v a r i o u s r e g i o n s of the b r a i n . I n f u s i o n s of so m a t o s t a t i n i n t o the v e n t r i c l e s have been shown to a l t e r open f i e l d a c t i v i t y ( H a v l i c e k e t a l . , 1976; P l o t n i k o f f e t a l . , 1982; Rezek e t a l . , 1976; V e c s e i e t a l . , 1983a; 1984), s t e r e o t y p i c behaviours (Rezek e t a l . , 1976), and e l e c t r i c a l s e l f - s t i m u l a t i o n of hypothalamic s i t e s ( V e c s e i e t al.,1982). D i r e c t i n f u s i o n s of so m a t o s t a t i n i n t o the s t r i a t u m have a l s o been shown to i n f l u e n c e behaviour, w i t h low doses i n c r e a s i n g locomotion and producing s t e r e o t y p i c s c r a t c h i n g and b i t i n g , w h i l e higher doses produce motor impairment (Rezek e t a l . , 1977). Numerous b e h a v i o u r a l s t u d i e s i n v o l v i n g the dopamine systems have e s t a b l i s h e d some of t h e i r f u n c t i o n s . Two common b e h a v i o u r a l t e s t s which appear to be dependent upon dopamine systems are 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 and locomotor a c t i v i t y . S t e r e o t y p i c behaviours, such as s n i f f i n g , chewing, and gnawing i n the r a t , appear to be mediated v i a dopaminergic r e c e p t o r s i n the caudate-putamen, w h i l e locomotor a c t i v i t y appears to i n v o l v e the mesolimbic pathway to the nucleus accumbens ( K e l l y e t a l . , 1975). The mesolimbic dopamine system has a l s o been shown to be i n v o l v e d i n reward mechanisms u s i n g d i f f e r e n t paradigms, i n c l u d i n g : e l e c t r i c a l s e l f s t i m u l a t i o n ( Corbett and Wise, 1980), c o n d i t i o n e d 9 r e i n f o r c e r s ( T a y l o r and Robbins, 1984), and p l a c e p r e f e r e n c e c o n d i t i o n i n g ( S p y r a k i e t a l . , 1983; see review by F i b i g e r and P h i l l i p s , 1986). Recent s t u d i e s u s i n g the p l a c e p r e f e r e n c e paradigm suggest t h a t the mesolimbic mediated reward and locomotor mechanisms may be d i f f e r e n t i a t e d ( D i S c a l a e t a l . , 1985; M a r t i n - I v e r s o n e t a l . , 1983; 1985; M i t h a n i e t a l . , 1986; Radke e t a l . , 1987a). A p o s s i b l e u s e f u l p h a r m a c o l o g i c a l t o o l f o r s t u d y i n g s o m a t o s t a t i n systems i s the a m i n o t h i o l cysteamine which appears to t e m p o r a r i l y d e p l e t e the l e v e l s of soma t o s t a t i n and p r o l a c t i n f o r approximately one week (Beal and M a r t i n , 1984b; Brown e t a l . , 1983; M i l l a r d e t a l . , 1982; P a l k o v i t s e t a l . , 1982; Sagar e t a l . , 1982; Szabo and R e i c h l i n , 1981). Cysteamine does not appear to a f f e c t other p e p t i d e s , i n c l u d i n g v a s o p r e s s i n , enkephalin, VIP, CCK, LHRH ( P a l k o v i t s e t a l . , 1982) and neuropeptide Y (Chattha and B e a l , 1987) and s i n c e p r o l a c t i n i s present i n ve r y low c o n c e n t r a t i o n s i n the b r a i n (Fuxe e t a l . , 1977), cysteamine may be used to study c e n t r a l s o m a t o s t a t i n systems. To understand b e t t e r the i n t e r a c t i o n s between dopamine and soma t o s t a t i n i n the b a s a l g a n g l i a , the e f f e c t s of r e d u c t i o n of som a t o s t a t i n by cysteamine on amphetamine-induced locomotor a c t i v i t y , apomorphine-induced s t e r e o t y p y , and amphetamine-induced p l a c e p r e f e r e n c e c o n d i t i o n i n g were examined. 10 METHODS AND MATERIALS S u b j e c t s Male Long Evans r a t s (Charles R i v e r s , weighing 275-350 g were used and housed 4-5 per cage under a 12 h l i g h t c y c l e (08:00 - 20:00) wi t h ad l i b i t u m access t o food and water. A l l r a t s were l e f t i n t h e i r home cages f o r a t l e a s t 1 week a f t e r a r r i v a l at the l a b o r a t o r y b e f o r e experiments were conducted. Drugs Cysteamine (p-mercaptoethylamine h y d r o c h l o r i d e , Sigma) was d i s s o l v e d i n 0.9% s a l i n e (100 mg/ml) and i n j e c t e d subcutaneously (s.c.) (1 ml/kg). D-amphetamine sulphate (Smith K l i n e and French) was d i s s o l v e d i n s a l i n e (1.5 mg/ml) and i n j e c t e d i n t r a p e r i t o n e a l l y ( i . p . ) (1 ml/kg). Apomorphine h y d r o c h l o r i d e (Sigma)(0.5mg/ml) was d i s s o l v e d i n 0.9% s a l i n e w i t h ascorbate (0.3 mg/ml) added as an a n t i o x i d a n t . T h i s s o l u t i o n was kept p r o t e c t e d from l i g h t and on i c e d u r i n g the i n j e c t i o n procedures. Rats were i n j e c t e d w i t h 1 ml/kg, s.c.. Procedure Experiment l a . Locomotor a c t i v i t y was measured i n c i r c u l a r (61 cm) a c t i v i t y cages (BRS/LVE), t r a n s e c t e d by 6 i n f r a r e d p h o t o c e l l beams. Photobeam i n t e r r u p t i o n s were recorded and analyzed w i t h a NOVA IV/X minicomputer (Data General) equipped w i t h a Manx i n t e r f a c e and software (GC C o n t r o l s ) . Rats were i n j e c t e d w i t h 11 cysteamine or s a l i n e (n= 11 per group) 4 hours p r i o r to amphetamine i n j e c t i o n s . One hour p r i o r to amphetamine i n j e c t i o n s , r a t s were p l a c e d i n t o the locomotor a c t i v i t y cages, and a c t i v i t y was recorded f o r 1 hour ( h a b i t u a t i o n ) . Amphetamine was then a d m i n i s t e r e d , and a c t i v i t y was recorded f o r an a d d i t i o n a l 3 hours. At the end of locomotor t e s t i n g the 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 , the b r a i n s were removed, the s t r i a t a d i s s e c t e d out on i c e and assayed f o r so m a t o s t a t i n immunoreactivity as d e s c r i b e d l a t e r (p. 14). Experiment l b . Stereotypy was observed while r a t s were i n s t e e l cages (35 x 40 x 18 cm) with a s t e e l mesh f l o o r , t i l t e d m i r r o r underneath and a c l e a r P l e x i g l a s door on one s i d e . Rats t h a t had been p r e t r e a t e d 11 hours p r e v i o u s l y w i t h cysteamine (n=19) or s a l i n e (n=20) were g i v e n 1 hour exposure t o the s t e r e o t y p y cages ( h a b i t u a t i o n ) . They were then (12 hours a f t e r pretreatment) i n j e c t e d w i t h apomorphine, and r e p l a c e d i n t o the s t e r e o t y p y cages. An observer b l i n d to the treatment of the r a t s r a t e d t h e i r behaviour d u r i n g 30 second p e r i o d s every 10 minutes from 5-65 minutes post-apomorphine i n j e c t i o n , u s i n g the 7-point r a t i n g s c a l e of K e l l y e t a l . , (1976). T h i s s c a l e has the f o l l o w i n g p o i n t s : 0 = a s l e e p or immobile; 1 = a c t i v e ; 2 = a c t i v e w i t h b u r s t s of s n i f f i n g ; 3 = a c t i v e w i t h continuous s n i f f i n g ; 4 = continuous s n i f f i n g i n one l o c a t i o n ; 5 = s n i f f i n g w i t h b u r s t s of l i c k i n g or gnawing; 6 = continuous l i c k i n g or gnawing. Rats were k i l l e d immediately f o l l o w i n g the l a s t o b s e r v a t i o n p e r i o d , t h e i r b r a i n s were removed, the s t r i a t a were d i s s e c t e d and the l e v e l s of 12 s o m a t o s t a t i n immunoreactivity determined by radioimmunoassay as d e s c r i b e d l a t e r (p. 14). Experiment l c . P l a c e p r e f e r e n c e c o n d i t i o n i n g and t e s t i n g was conducted w i t h f o u r s h u t t l e boxes (80 x 25 x 36 cm), each d i v i d e d i n t o 2 compartments (34 x 25 cm), connected by a t u n n e l (8 x 8 x 6 cm) which c o u l d be c l o s e d by g u i l l o t i n e doors. Each of the 2 compartments was d i s t i n c t i v e i n the c o l o u r of the w a l l s ( s o l i d brown or b l a c k w i t h white s t r i p e s ) and i n the type of f l o o r ( g r i d or mesh). T r a n s l u c e n t P l e x i g l a s l i d s allowed f o r d i f f u s e i l l u m i n a t i o n of the i n t e r i o r of the compartments. Each box was balanced on a fulcrum; the s h i f t i n g of a r a t ' s weight t i l t i n g the box such t h a t the time spent on each s i d e , and the number of c r o s s i n g s c o u l d be recorded w i t h e l e c t r o m e c h a n i c a l equipment. T h i s experiment c o n s i s t e d of t h r e e phases. During Phase 1 ( P r e t e s t ) , r a t s were p l a c e d i n one of the two compartments ( s t a r t s i d e ) with the doors open, and the time spent i n each compartment and the number of c r o s s i n g s from one compartment to the other recorded over a 15 minute p e r i o d . T h i s procedure was conducted on two c o n s e c u t i v e days. In Phase 2, r a t s were c o n f i n e d to the n o n - s t a r t compartment immediately a f t e r an i n j e c t i o n of amphetamine or v e h i c l e and 12-14 hours a f t e r an i n j e c t i o n of cysteamine or v e h i c l e . T h i s procedure was conducted f o r each of 4 days (Day 1, 3, 5 and 7 of Phase 2); each r a t was i n j e c t e d and p l a c e d w i t h i n the compartment at the same time each day f o r 3 0 minutes. On a l t e r n a t e days (Day 2, 4, 6 and 8), r a t s were c o n f i n e d to the s t a r t compartment immediately a f t e r i n j e c t i o n s of s a l i n e ; there were no i n j e c t i o n s 13 made 12-14 hours p r i o r to these i n j e c t i o n s . Rats were randomly a s s i g n e d to one of 4 treatment groups (SALINE + SALINE, SALINE + AMPHETAMINE, CYSTEAMINE + SALINE, CYSTEAMINE + AMPHETAMINE; the f i r s t drug i n each p a i r r e f e r s t o the 12-14 hour pretreatment; the second r e f e r s t o the treatment immediately b e f o r e compartment confinement; n = 10 per group). During Phase 3 ( T e s t ) , a s i n g l e 15 minute t e s t of p l a c e p r e f e r e n c e was conducted, as i n Phase 1. No i n j e c t i o n s preceded t h i s t e s t . Immediately a f t e r t h i s 15 minute t e s t , the r a t s were k i l l e d , t h e i r b r a i n s removed, and the s t r i a t a d i s s e c t e d out and the l e v e l s of so m a t o s t a t i n immunoreactivity determined by radioimmunoassay as d e s c r i b e d l a t e r (p. 14). S t a t i s t i c a l A n a l y s i s Locomotor a c t i v i t y was s u b j e c t e d t o a n a l y s i s of v a r i a n c e w i t h 2 f a c t o r s : 1 between f a c t o r (drug treatment) and 1 repeated f a c t o r (blocks of 10 min). I n d i v i d u a l planned comparisons were made f o l l o w i n g the method of Winer (1971). Peak, t o t a l and median s t e r e o t y p y scores were analysed s e p a r a t e l y w i t h one-way a n a l y s i s of v a r i a n c e . P l a c e p r e f e r e n c e data was s u b j e c t e d t o a n a l y s i s of v a r i a n c e w i t h 2 between f a c t o r s (cysteamine X s a l i n e and amphetamine X s a l i n e ) and one w i t h i n f a c t o r (second p r e t e s t X t e s t ) . Planned comparisons were conducted. B i o c h e m i c a l assay r e s u l t s were a l s o s u b j e c t e d t o a n a l y s i s of v a r i a n c e . 14 B i o c h e m i c a l Assays  T i s s u e P r e p a r a t i o n . 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 , the b r a i n s were removed, and the s t r i a t a , i n c l u d i n g both the caudate-putarr.en and NAS ( b i l a t e r a l ) were d i s s e c t e d out on i c e . T i s s u e f o r radioimmunoassay was weighed, b o i l e d f o r 10 minutes i n 2.0 N a c e t i c a c i d and then s o n i c a t e d . The samples were then c e n t r i f u g e d a t 4°C f o r 20 min a t 10,000 rpm, and l y o p h i l i z e d . Samples were s t o r e d a t -70°C u n t i l assayed. Radioimmunoassay. S o m a t o s t a t i n - l i k e immunoreactivity was measured u s i n g a radioimmunoassay which compared t i s s u e c o n c e n t r a t i o n s of s o m a t o s t a t i n - l i k e a c t i v i t y t o a standard curve formed by v a r y i n g c o n c e n t r a t i o n s of s y n t h e t i c s o m a t o s t a t i n i n assay b u f f e r . The b u f f e r s o l u t i o n used f o r the assay c o n t a i n e d 24 mM sodium b a r b i t a l , 3.4 mM sodium a c e t a t e , 43.6 mM sodium c h l o r i d e , 0.25 mM t h i m e r o s a l , 0.5% bovine serum albumin (BSA) and 500 KlU/ml a p r o t i n i n (pH 7.4), and was prepared f r e s h weekly. The antibody was a monoclonal (Ab 3) t h a t r e c o g n i z e s both s o m a t o s t a t i n 14 and 28 i n equimolar r a t i o s . The antibody was generously s u p p l i e d by Dr J . C. Brown, MRC Regulatory Peptide Group, Department of P h y s i o l o g y , U. B. C . Antibody s o l u t i o n s were prepared from a stock s o l u t i o n , c o n t a i n i n g 0.1% sodium a z i d e and 0.1% BSA a t a 1:1 d i l u t i o n and kept f r o z e n a t -70°C. The stock s o l u t i o n was f u r t h e r d i l u t e d 100 f o l d i n a 0.1% sodium azide/BSA s o l u t i o n and s t o r e d a t 4°C. On the day of the assay 15 t h i s was f u r t h e r d i l u t e d i n c o l d assay b u f f e r to a f i n a l c o n c e n t r a t i o n of 1:500,000. T y r o s i n e - l - s o m a t o s t a t i n ( P e n i s u l a Labs.) i o d i n a t e d (^^1) v i a the chloramine T method was generously s u p p l i e d by Dr. K. Kwok, Department of P h y s i o l o g y , U.B.C.., and s t o r e d at -20°C. On the day of an assay, the l a b e l l e d s o m a t o s t a t i n was d i s s o l v e d i n 0.1% t r i f l u o r o a c e t i c a c i d and p u r i f i e d w i t h a sep pak column (C^g c a r t r i d g e , Waters Assoc.) u s i n g an a c e t o n i t r i l e step g r a d i e n t ranging from 20% to 40% a c e t o n i t r i l e i n 2% steps of 1.0 ml washes. The p u r i f i e d s o m a t o s t a t i n was o b t a i n e d from the 28% a c e t o n i t r i l e wash. The p u r i f i e d l a b e l was d i l u t e d i n b u f f e r s o l u t i o n to o b t a i n a f i n a l c o n c e n t r a t i o n of approximately 2500 CPM/ 100 u l . The s y n t h e t i c somatostatin-14 ( P e n i s u l a Labs) was s t o r e d a t -70°C i n 10u.g a l i q u o t s i n a s o l u t i o n c o n t a i n i n g 140mM l a c t o s e , 40u.M BSA, lOmM c i t r i c a c i d , lOOmM form i c a c i d , and a p r o t i n i n (80,000 K l U / l i t e r ) . On the day of the assay, an a l i q u o t was s e r i a l l y d i l u t e d i n assay b u f f e r to o b t a i n a range of 0.975 to 500 picograms so m a t o s t a t i n per tube and analyzed i n t r i p l i c a t e . T i s s u e samples were d i l u t e d a p p r o p r i a t e l y i n assay b u f f e r t o o b t a i n r e s u l t s w i t h i n the s e n s i t i v i t y range of the standard curve (ED20 = 5.0-10.0 pg; ED80 = 70-100 pg) and analyzed i n d u p l i c a t e . The f i n a l volume of 0.4 ml per tube was incubated f o r 3 days at 4°C. On the t h i r d day, s e p a r a t i o n of the f r e e and bound l a b e l l e d s o m a t o s t a t i n was performed by adding dextran-T70 (Pharmacia)-coated c h a r c o a l ( N o r i t - n e u t r a l ; F i s h e r ) i n 0.5% phosphate b u f f e r w i t h 0.1% hormone f r e e plasma. The tubes were 16 c e n t r i f u g e d f o r 30 minutes a t 3,000 RPM, and the supernatant, c o n t a i n i n g the bound somat o s t a t i n , was removed. The r a d i o a c t i v i t y i n the c h a r c o a l p e l l e t c o n t a i n i n g the f r e e l a b e l l e d s o m a t o s t a t i n was counted on a gamma counter (Packard, M u l t i P r i a s 4) equipped w i t h PC-Data A c q u i s i t i o n and A n a l y s i s System (PC-DAAS) to analyze the data. R e s u l t s were expressed as picograms of s o m a t o s t a t i n per m i l l i g r a m t i s s u e wet weight. RESULTS  Experiment l a . Cysteamine i n j e c t i o n s g i v e n 3 hours p r i o r t o locomotor t e s t i n g s i g n i f i c a n t l y reduced locomotor a c t i v i t y d u r i n g the f i r s t 30 min of h a b i t u a t i o n ( F i g u r e 1), as i n d i c a t e d by a s i g n i f i c a n t treatment X time i n t e r a c t i o n (F(5,80) = 3.64, p < 0.01), and i n d i v i d u a l comparisons ( f i r s t 10 min b l o c k : F ( l , 1 6 ) = 83.15, p < 0.001; second 10 min b l o c k : F ( l , 1 6 ) = 23.11, p < 0.001; t h i r d 10 min b l o c k : F ( l , 1 6 ) = 5.21, p < 0.05). The a c t i v i t y of the 2 groups d i d not d i f f e r s i g n i f i c a n t l y d u r i n g the remaining b l o c k s of h a b i t u a t i o n ( F ( l , 1 6 ) < 1.0 f o r a l l 3 b l o c k s ) . F o l l o w i n g amphetamine i n j e c t i o n , the cysteamine p r e t r e a t e d group e x h i b i t e d l e s s s t i m u l a t i o n of locomotor a c t i v i t y than was seen i n the s a l i n e t r e a t e d group ( F i g u r e 1), w i t h the main e f f e c t of cysteamine treatment s i g n i f i c a n t ( F ( l , 1 6 ) = 7.38, p < 0.025). The i n t e r a c t i o n between treatment and time (blocks of 10 min) was not s i g n i f i c a n t . Planned comparisons r e v e a l e d t h a t d u r i n g the f i r s t and the t h i r d through to the n i n t h time b l o c k s the 17 cysteamine p r e t r e a t e d group d i f f e r e d s i g n i f i c a n t l y from the c o n t r o l s ( F ( l , 1 6 ) = 14.76, 10.47, 21.08, 17.94, 15.01, 14.92, 8.25, 7.68, r e s p e c t i v e l y , p < 0.025 i n a l l c a s e s ) . Examination of the s t r i a t a l s o m a t o s t a t i n l e v e l s f o r these two groups of r a t s r e v e a l e d a s i g n i f i c a n t r e d u c t i o n ( F ( l , 2 0 ) = 6.69, p < 0.02) i n the cysteamine group (38.12 ± 1.20 pg/mg) compared w i t h the c o n t r o l group (49.40 ± 2.54 pg/mg) ( F i g u r e 2). These r e s u l t s i n d i c a t e t h a t 4 hour pretreatment w i t h systemic i n j e c t i o n s of 100 mg/kg cysteamine, reduced both s t r i a t a l s o m a t o s t a t i n l e v e l s and amphetamine induced h y p e r a c t i v i t y . 18 900 -habituation amph 20 40 60 80 100 120 140 160 180 TIME (min) FIGURE 1. E f f e c t of 4 hour pretreatment with s a l i n e (1 ml/kg) or cysteamine (100 mg/kg, SC) on amphetamine (1.5 mg/kg, IP) induced locomotor a c t i v i t y . Data re p r e s e n t mean values of beam i n t e r u p t i o n s per 10 minute block f o r each group. * p < 0.05 compared to s a l i n e group's ten minute block a c t i v i t y . 19 FIGURE 2. E f f e c t of 7 hour pretreatment w i t h s a l i n e (1 ml/kg) or cysteamine (100 mg/kg, SC) on s t r i a t a l (caudate-putamen + nucleus accumbens) s o m a t o s t a t i n l e v e l s as measured by radioimmunoassay. The t i s s u e o b t a i n e d i n t h i s study were from r a t s immediately f o l l o w i n g . t h e locomotor t e s t i n g . Data r e p r e s e n t s mean ± SEM value s f o r each group. * p < 0.05 compared to s a l i n e group. 20 Experiment lb. As can be observed i n F i g u r e 3, the group of r a t s p r e t r e a t e d w i t h cysteamine 12 hours p r i o r t o apomorphine i n j e c t i o n s e x h i b i t e d s i g n i f i c a n t r e d u c t i o n s i n apomorphine-induced s t e r e o t y p y , r e l a t i v e to the s a l i n e p r e t r e a t e d group. T h i s r e d u c t i o n was apparent i n both the median (F ( l , 3 7 ) = 4.25, p < 0.05) and the t o t a l ( F ( l , 3 7 ) = 4.81, p < 0.05) s t e r e o t y p y s c o r e s , but apparent r e d u c t i o n s i n the peak s t e r e o t y p y scores were not s i g n i f i c a n t ( F ( l , 3 7 ) = 2.13, p > 0.1). Thus, i t appears t h a t although apomorphine produced a s i m i l a r degree (peak e f f e c t ) of s t e r e o t y p y i n cysteamine p r e t r e a t e d r a t s as i n c o n t r o l s , the st e r e o t y p y was of a s i g n i f i c a n t l y s h o r t e r d u r a t i o n . The cysteamine t r e a t e d r a t s i n t h i s experiment had s t r i a t a l s o m a t o s t a t i n l e v e l s h a l f t h a t of c o n t r o l s ( s a l i n e : 59.43 ± 8.61 pg/mg, n = 9; cysteamine: 30.38 ± 3.35 pg/mg, n = 8; F ( l , 1 5 ) = 6.28, p < 0.025) ( F i g u r e 4). Thus, systemic cysteamine pretreatment s i g n i f i c a n t l y d e p l e t e d s t r i a t a l s o m a t o s t a t i n and decreased apomorphine induced s t e r e o t y p y . 21 PEAK ' MEDIAN TOTAL FIGURE 3. E f f e c t of 12 hour pretreatment with s a l i n e (1 ml/kg) or cysteamine (100 mg/kg, SC) on apomorphine (0.5 mg/kg, SC) induced s t e r e o t y p y . T o t a l = sum a l l 7 scores taken a t ten minute i n t e r v a l s , and peak = the hig h e s t s i n g l e score obtained. Data r e p r e s e n t mean ± SEM f o r each group. * p < 0.05 compared to the s a l i n e group. 22 8 0 7 0 -I i SAL CYST FIGURE 4. E f f e c t of 13 hour pretreatment w i t h s a l i n e (1 ml/kg) or cysteamine (100 mg/kg, SC) on s t r i a t a l (caudate-putamen + nucleus accumbens) somatostatin l e v e l s as measured by radioimmunoassay. The t i s s u e o b t a i n e d i n t h i s study were from r a t s immediately f o l l o w i n g the s t e r e o t y p y t e s t i n g . Data r e p r e s e n t s mean ± SEM values f o r each group. * p < 0.05 compared to s a l i n e group. 23 Experiment l c . The i n f l u e n c e of cysteamine pretreatment on amphetamine-induced p l a c e p r e f e r e n c e s i s d e p i c t e d i n F i g u r e 5. A n a l y s i s of v a r i a n c e r e v e a l e d t h a t there was no i n t e r a c t i o n between cysteamine and amphetamine on the change i n p l a c e p r e f e r e n c e from p r e t e s t t o t e s t ( F ( l , 3 0 ) = 0.62, p > 0.1), but t h a t there was a s i g n i f i c a n t e f f e c t of amphetamine, i n c r e a s i n g the time spent i n the amphetamine-associated compartment on the t e s t day, r e l a t i v e t o the l a s t p r e t e s t (F(l,30) = 5.12, p < 0.05). Planned comparisons i n d i c a t e d t h a t w h i l e the amphetamine-treated groups e x h i b i t e d an i n c r e a s e i n time spent i n the c o n d i t i o n e d compartment ( F ( l , 3 0 ) = 5.63, p < 0.025), the s a l i n e t r e a t e d groups d i d not ( F ( l , 3 0 ) = 0.54, p < 0.1). There was no cysteamine X t e s t i n t e r a c t i o n e v i d e n t ( F ( l , 3 0 ) = 0.16, p > 0.1). Thus, repeated cysteamine pretreatment was without s i g n i f i c a n t e f f e c t on amphetamine c o n d i t i o n e d p l a c e p r e f e r e n c e s . Cysteamine pretreatment produced a s u b s t a n t i a l (65.2%) d e p l e t i o n of s o m a t o s t a t i n i n both s a l i n e and amphetamine c o n d i t i o n e d groups ( F ( l , 1 6 ) = 34.4, p < 0.005) ( F i g u r e 6). Somatostatin l e v e l s i n s a l i n e p r e - t r e a t e d r a t s were observed to be h i g h e r than those from r a t s i n the other experiments; t h i s may be due to the e x t e n s i v e h a n d l i n g the r a t s r e c e i v e d i n the p l a c e p r e f e r e n c e experiment r e l a t i v e t o the locomotor and s t e r e o t y p y experiments, or t o the i n f l u e n c e ( p o s s i b l y s t r e s s f u l ) of repeated i n j e c t i o n s . Amphetamine treatments had no s i g n i f i c a n t e f f e c t on s o m a t o s t a t i n l e v e l s i n the v e h i c l e p r e t r e a t e d group ( F ( l , 1 6 ) = 24 1.27, p > 0.1), nor d i d i t i n t e r a c t s i g n i f i c a n t l y w i t h cysteamine treatments (F(1,16) = 2.58, p > 0.1). DISCUSSION The p r e s e n t r e s u l t s i n d i c a t e t h a t s t r i a t a l s o m a t o s t a t i n d e p l e t i o n s produced w i t h cysteamine are a s s o c i a t e d w i t h an a t t e n u a t i o n of the motor e f f e c t s of dopamine a g o n i s t s . V e c s e i e t a l . (1984) have p r e v i o u s l y r e p o r t e d t h a t i n t r a v e n t r i c u l a r i n j e c t i o n s of cysteamine 4 hours b e f o r e b e h a v i o u r a l t e s t i n g reduces spontaneous locomotor a c t i v i t y . In agreement w i t h t h i s , cysteamine g i v e n subcutaneously 3 hours b e f o r e t e s t i n g reduced locomotor a c t i v i t y d u r i n g h a b i t u a t i o n ( F i g u r e 1). Amphetamine induced motor s t i m u l a t i o n was a l s o a ttenuated by t h i s treatment 4 hours a f t e r cysteamine. That t h i s l a t t e r a t t e n u a t i o n i s a r e s u l t of a modulation of the a c t i o n s of c e n t r a l dopamine a c t i v i t y , and not merely a r e f l e c t i o n of lower b a s a l a c t i v i t y l e v e l s , i s supported by a study performed i n c o l l a b o r a t i o n w i t h Drs. M a r t i n -I v e r s o n and V i n c e n t . In t h i s study ( M a r t i n - I v e r s o n e t a l . , 1986) r a t s r e c e i v i n g 11 hour, i n s t e a d of 3 hour, pretreatment w i t h cysteamine (100 mg/kg, s.c.) showed no change i n h a b i t u a t i o n a c t i v i t y but d i d show a s i g n i f i c a n t r e d u c t i o n i n amphetamine-induced locomotor a c t i v i t y . Furthermore, intra-accumbens 25 o n us Q tn o UJ z g Q Z o o UJ sal+sal sal+amp sal+cys cys+amp FIGURE 5. E f f e c t of 12 hour pretreatment with s a l i n e (1 ml/kg) or cysteamine (100 mg/kg, SC) d u r i n g c o n d i t i o n i n g , on c o n d i t i o n e d p l a c e p r e f e r e n c e with s a l i n e or amphetamine (1.5 mg/kg, I P ) . Data r e p r e s e n t mean values ± SEM of time spent by each group i n a s h u t t l e box compartment before ( p r e t e s t ) and a f t e r ( t e s t ) the c o n d i t i o n i n g . * i n d i c a t e s the t e s t times were s i g n i f i c a n t l y d i f f e r e n t compared t o the p r e t e s t times, p < 0.05. 2 6 180 170 -sal+sal sal-i-amp sal+cys cys+amp FIGURE 6. E f f e c t of f o u r i n j e c t i o n s of s a l i n e or cysteamine (100 mg/kg, SC) g i v e n over e i g h t days f o l l o w e d by 3 days of no i n j e c t i o n s , on the s t r i a t a l s omatostatin l e v e l s as measured by radioimmunoassay. T i s s u e samples were o b t a i n e d from r a t s immediately f o l l o w i n g the f i n a l t e s t s e s s i o n of p l a c e p r e f e r e n c e c o n d i t i o n i n g . Data r e p r e s e n t mean value s ± SEM f o r each group. * p < 0.05 compared to s a l i n e groups. 27 cysteamine i n j e c t i o n s were a l s o found to attenuate amphetamine-induced locomotor a c t i v i t y without a l t e r i n g h a b i t u a t i o n a c t i v i t y ( M a r t i n - I v e r s o n e t a l . , 1986). These r e s u l t s i n d i c a t e t h a t s o m a t o s t a t i n r e d u c t i o n s by e i t h e r systemic or intra-accumbens cysteamine i n j e c t i o n s c o r r e l a t e w i t h a r e d u c t i o n i n dopamine mediated locomotor a c t i v i t y . The observed r e d u c t i o n i n h a b i t u a t i o n a c t i v i t y ( F i g u r e 1) a f t e r 3 hour pretreatment but not a f t e r 11 hour pretreatment ( M a r t i n - I v e r s o n e t al.,1986) i s s i m i l a r to the r e s u l t s observed by V e c s e i e t a l . (1984) showing 4 hour, but not 24 hour, pretreatment w i t h cysteamine to reduce open f i e l d a c t i v i t y , even though cysteamine reduces s o m a t o s t a t i n l e v e l s f o r over 72 hours (Beal and M a r t i n , 1984b; M a r t i n - I v e r s o n e t a l . , 1986). The e a r l y change i n a c t i v i t y may r e f l e c t a s t r e s s f u l f a c t o r a s s o c i a t e d w i t h the i n i t i a l changes i n s o m a t o s t a t i n l e v e l s . In a d d i t i o n to cysteamine a l t e r i n g amphetamine induced locomotor a c t i v i t y , p e r i p h e r a l a d m i n i s t r a t i o n of cysteamine, which reduces s t r i a t a l s o m a t o s t a t i n , attenuates the s t e r e o t y p y produced by apomorphine ( F i g u r e 3). As apomorphine i s a d i r e c t dopamine r e c e p t o r a g o n i s t , t h i s r e s u l t suggests t h a t s o m a t o s t a t i n may modulate the e f f e c t s of dopamine a c t i v i t y p o s t - s y n a p t i c to dopamine-releasing t e r m i n a l s i n the s t r i a t u m . The f i n d i n g t h a t n e i t h e r p e r i p h e r a l nor c e n t r a l a d m i n i s t r a t i o n of cysteamine a l t e r e d dopamine or dopamine m e t a b o l i t e l e v e l s (Beal and Martin,1984a; B e a l and M a r t i n 1984b; M a r t i n - I v e r s o n e t a l . , 1986) i s c o n s i s t e n t w i t h t h i s s u g g e s t i o n . In c o n t r a s t w i t h these r e s u l t s are the o b s e r v a t i o n s t h a t s o m a t o s t a t i n i n f u s i o n s a l t e r 28 s t r i a t a l dopamine turnover (Beal and Martin,1984a; G a r c i a - S e v i l l a et.al.,1978) and r e l e a s e ( C h e s s e l e t and Reisine,1983), and t h a t h a l o p e r i d o l can b l o c k the motor e f f e c t s of somat o s t a t i n ( V e c s e i e t a l . , 1983b). These s t u d i e s suggests t h a t i n a d d i t i o n t o soma t o s t a t i n a c t i n g p o s t s y n a p t i c t o dopamine t e r m i n a l s , there may a l s o be a feedback mechanism t o the dopamine t e r m i n a l s when s t r i a t a l s o m a t o s t a t i n c e l l s are a c t i v e . While cysteamine treatment a t t e n u a t e d the motor e f f e c t s of dopamine a g o n i s t s , the rewarding a c t i o n of amphetamine, as r e v e a l e d by the c o n d i t i o n e d p l a c e p r e f e r e n c e procedure, was not reduced. T h i s f i n d i n g supports p r e v i o u s work sugges t i n g t h a t the motor and r e i n f o r c i n g a c t i o n s of dopamine a g o n i s t s are independent of each other ( D i S c a l a et.al.,1985; M a r t i n - I v e r s o n et.al.,1985; M i t h a n i e t a l . , 1987; Radke e t a l . , 1987a). For example, a GABA a g o n i s t (SL 76002) attenuated locomotor a c t i v i t y , but had no e f f e c t on p l a c e p r e f e r e n c e c o n d i t i o n i n g induced by amphetamine ( D i S c a l i et.al.,1985) s u g g e s t i n g GABA and soma t o s t a t i n c e l l s i n the b a s a l g a n g l i a may have o p p o s i t e a c t i o n s on the motor s t i m u l a n t p r o p e r t i e s of dopaminergic drugs, but n e i t h e r appears t o be e s s e n t i a l f o r the rewarding p r o p e r t i e s of amphetamine. While both dopamine-mediated a c t i v i t y ( K e l l y e t a l . , 1977) and r e i n f o r c e m e n t (Corbett and Wise, 1984; F i b i g e r and P h i l l i p s , 1986; S p y r a k i e t a l . , 1983; T a y l o r and Robbins, 1984) appear to be dependent on the mesolimbic dopamine t r a c t , the observed d i f f e r e n t i a t i o n of these two behaviours by drug m a n i p u l a t i o n s 29 i n d i c a t e s f u r t h e r work on t h i s aspect of dopamine f u n c t i o n i s e s s e n t i a l . I t i s i n t e r e s t i n g t o note t h a t s o m a t o s t a t i n was never d e p l e t e d more than 65% i n the p r e s e n t study even w i t h repeated cysteamine treatments. Previous r e p o r t s have a l s o noted t h a t d e p l e t i o n s of c e n t r a l s o m a t o s t a t i n produced by s i n g l e i n j e c t i o n s of cysteamine are not complete (Beal and M a r t i n 1984b; Brown et.al.,1975; P a l k o v i t s et.al.,1982; Sagar et.al.,1982; S t r i k a n t and Patel,1984). T h i s suggests t h a t s o m a t o s t a t i n may be d i f f e r e n t i a l l y compartmentalized; the somatostatin i n o n l y c e r t a i n compartments being s u s c e p t i b l e to the a c t i o n s of cysteamine. T h i s hypothesis i s supported by immunohistochemical r e s u l t s ( M a r t i n - I v e r s o n e t al.,1986) i n which cysteamine d e p l e t e d s o m a t o s t a t i n immunoreactivity from nucleus accumbens t e r m i n a l f i e l d s , but not c e l l b o d i e s . A study by B a k h i t e t a l . , (1983) has shown cysteamine to have no e f f e c t on somatostatin-28 l e v e l s s u g g e s t i n g the s o m a t o s t a t i n p o o l i n c e l l bodies has more somatostatin-28, and the t e r m i n a l s have more somatostatin-14. Cysteamine appears to be a u s e f u l p h a r m a c o l o g i c a l agent w i t h which to study the c e n t r a l a c t i o n s of s o m a t o s t a t i n . P r i o r t o t h i s study, cysteamine has been used mostly to study p e r i p h e r a l s o m a t o s t a t i n but i t has been used to study c e n t r a l s o m a t o s t a t i n changes i n k i n d l i n g s e i z u r e s (Higuchi et.al.,1983; see experiment 3 f o r d e t a i l e d d i s c u s s i o n ) . I t i s apparent from the p r e s e n t study u s i n g cysteamine, t h a t s o m a t o s t a t i n may p l a y a r o l e i n modulating the motor e f f e c t s , but not the r e i n f o r c i n g a c t i o n s , of dopamine a g o n i s t s . Furthermore, 30 i t i s l i k e l y t h a t t h i s modulation occurs p o s t s y n a p t i c t o the dopamine-releasing t e r m i n a l s i n the s t r i a t u m , s i n c e s o m a t o s t a t i n d e p l e t i o n s reduce the motor e f f e c t s of both i n d i r e c t and d i r e c t a c t i n g dopamine r e c e p t o r a g o n i s t s , w h i l e not appearing to a l t e r dopamine metabolism. To i n v e s t i g a t e f u r t h e r the p o s s i b i l i t y of s t r i a t a l s o m a t o s t a t i n c e l l s b e i n g under the i n f l u e n c e of dopamine a f f e r e n t s , the e f f e c t s of c h r o n i c h a l o p e r i d o l on s o m a t o s t a t i n l e v e l s was i n v e s t i g a t e d (experiment 2 ) . 31 EXPERIMENT 2 . THE EFFECTS OF HALOPERIDOL ON SOMATOSTATIN IMMUNOREACTIVITY. INTRODUCTION As mentioned i n the d i s c u s s i o n of experiment 1, there i s growing evidence t h a t s o m a t o s t a t i n c o n t a i n i n g neurons i n the s t r i a t u m have t h e i r major p h y s i o l o g i c a l a c t i o n s p o s t s y n a p t i c to dopamine t e r m i n a l s . T h e r e f o r e i t was of i n t e r e s t t o examine the e f f e c t s of c l i n i c a l l y p r e s c r i b e d drugs, which have been shown to a f f e c t dopaminergic systems, on som a t o s t a t i n l e v e l s . One important drug i s h a l o p e r i d o l , a d i r e c t dopamine a n t a g o n i s t , which i s commonly used as an a n t i s c h i z o p h r e n i c drug. S e v e r a l s t u d i e s have examined the e f f e c t s of n e u r o l e p t i c treatment on non-dopaminergic systems, i n c l u d i n g those c o n t a i n i n g GABA (Gunne and HaggstrOm, 1983), substance P (Hanson e t a l . , 1980), n e u r o t e n s i n (Govoni e t a l . , 1980), c h o l e c y s t o k i n i n (Frey, 1983), o p i o i d s (Hong e t a l . , , 1978), as w e l l as som a t o s t a t i n (Beal and M a r t i n , 1984c). These s t u d i e s are u s e f u l f o r a b e t t e r understanding of dopamine i n t e r a c t i o n s w i t h other t r a n s m i t t e r systems, but they a l l share a common problem, the l e n g t h of treatment. In the c l i n i c a l s e t t i n g , n e u r o l e p t i c treatment l a s t s f o r s e v e r a l months or ye a r s , whereas these s t u d i e s are g e n e r a l l y of a s h o r t d u r a t i o n , l a s t i n g t h r e e or fou r weeks. Examination of long term treatment i n animals would t h e r e f o r e p r o v i d e a b e t t e r i n s i g h t i n t o the neuronal mechanisms i n v o l v e d i n maintenance of treatment as w e l l as i n the development of the d y s k i n e t i c 32 movements o f t e n a s s o c i a t e d w i t h long term n e u r o l e p t i c treatment and withdrawal. The purpose of the f o l l o w i n g s e t of experiments was to examine and compare the e f f e c t s of h a l o p e r i d o l treatment f o r both th r e e weeks ( s h o r t term) and e i g h t months (long term) on the l e v e l s of so m a t o s t a t i n immunoreactivity i n the r a t b r a i n . Furthermore, the e f f e c t of withdrawal (2 months) of h a l o p e r i d o l a f t e r long term a d m i n i s t r a t i o n were a l s o examined. METHODS AND MATERIALS Sub j e c t s . Male Wistar r a t s (Woodlyn) were group housed 4 per cage under a 12 hour l i g h t / d a r k c y c l e w i t h standard l a b o r a t o r y r a t chow p r o v i d e d ad l i b i t u m . For the long term s t u d i e s (8 months), r a t s began the experiment a t a young age, while i n the s h o r t term experiment, aged r a t s were used t o c o n t r o l f o r the e f f e c t s of aging on neurochemistry. Drugs. H a l o p e r i d o l base 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 (10 mg/ml) and s t o r e d a t 4°C i n the dark. F r e s h stock s o l u t i o n s were made monthly. A l i q u o t s of the stock 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 water and g i v e n t o the animals as d r i n k i n g water. Based on a p i l o t study performed by A. J . MacLennan, the r a t s consumed 1.3-1.5 mg of h a l o p e r i d o l / k g / d a y . 33 Procedure. T h i s study c o n s i s t e d of t h r e e experiments. In experiment 2a ( s h o r t term tre a t m e n t ) , r a t s were g i v e n 3 weeks of h a l o p e r i d o l s u p p l i e d i n t h e i r d r i n k i n g water (1.3-1.5 mg/kg/day) (n=12) or v e h i c l e s o l u t i o n (n=12). In experiment 2b (long term tre a t m e n t ) , r a t s r e c e i v e d 8 months of h a l o p e r i d o l (n=10) or v e h i c l e (n=8). In experiment 2c (withdrawal f o l l o w i n g long term treatment) r a t s r e c e i v e d 8 months of h a l o p e r i d o l (n=8) or v e h i c l e (n=6), f o l l o w e d by 2 months of water. The animals were s a c r i f i c e d by c e r v i c a l d i s l o c a t i o n between 1:00 and 3:00 pm. T h e i r b r a i n s were removed and samples of the nucleus accumbens, caudate-putamen, a c o r t i c a l sample d o r s a l to the s t r i a t u m , medial p r e f r o n t a l c o r t e x , o l f a c t o r y t u b e r c l e , s u b s t a n t i a n i g r a , and the v e n t r a l tegmental area were d i s s e c t e d from s e c t i o n s cut on a f r e e z i n g microtome, and samples processed f o r radioimmunoassay as d e s c r i b e d i n experiment 1, except t h a t c o l d 0.1 N HCl was used i n s t e a d of b o i l i n g samples i n 2.0 N a c e t i c a c i d . P r e l i m i n a r y s t u d i e s r e v e a l e d the use of c o l d 0.1 N HCl d i d not e f f e c t s o m a t o s t a t i n immunoreactivity compared to p r e p a r i n g t i s s u e w i t h 2.0 N a c e t i c a c i d . S t a t i s t i c s . Data was expressed as picograms of s o m a t o s t a t i n immunoreactivity per m i l l i g r a m t i s s u e wet weight. Mean (± S.E.M) val u e s f o r each b r a i n r e g i o n w i t h i n each group were c a l c u l a t e d and a n a l y s i s between treatment and c o n t r o l groups f o r each i n d i v i d u a l b r a i n r e g i o n was performed u s i n g Student's t - t e s t . 34 RESULTS As shown i n F i g u r e 7, 3 week treatment w i t h h a l o p e r i d o l C s i g n i f i c a n t l y reduced s o m a t o s t a t i n l e v e l s i n the nucleus accumbens (t(22)=2.55; p <0.025) caudate-putamen (t(22)=3.46; p <0.005), and the v e n t r a l tegmental area (t(22)=2.34; p <0.05). Long term treatment however o n l y reduced s o m a t o s t a t i n l e v e l s i n the nucleus accumbens (t(16)=2.2; p <0.05) w i t h l e v e l s i n a l l other r e g i o n s examined not being s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s ( F i g u r e 8 ) . F o l l o w i n g withdrawal from long term h a l o p e r i d o l treatment s o m a t o s t a t i n l e v e l s were not d i f f e r e n t from c o n t r o l l e v e l s i n any of the r e g i o n s examined ( F i g u r e 8). DISCUSSION The e f f i c a c y of n e u r o l e p t i c treatment appears to be c o r r e l a t e d w i t h the a n t a g o n i s t i c potency of these drugs on dopamine r e c e p t o r s (Creese et a l . , 1976). The t h e r a p e u t i c e f f e c t s of a n t i p s y c h o t i c s r e q u i r e s e v e r a l days to develop, and animal s t u d i e s i n v o l v i n g n e u r o l e p t i c s have begun to p r o v i d e some i n s i g h t i n t o the mechanisms i n v o l v e d i n t h i s d e l a y . A popular theory i s t h a t continuous h a l o p e r i d o l treatment r e s u l t s i n a slow development of d e p o l a r i z a t i o n i n a c t i v a t i o n of dopamine c e l l s (Bunney, 1984; White and Wang, 1983). Although t h i s i s a p l a u s i b l e e x p l a n a t i o n , r e c e n t s t u d i e s have shown t h a t n e u r o l e p t i c s can a f f e c t s e v e r a l non-dopaminergic systems 35 300 - r 280 -260 -x: C T X C - P N A S m P F C OT S N VTA FIGURE 7 . E f f e c t of s h o r t term ( 3 weeks) treatment w i t h v e h i c l e ( 2 2 ) or h a l o p e r i d o l ( £ 3 ) ( 1 . 3 - 1 . 5 mg/kg/day) on the l e v e l s of c e n t r a l s o m a t o s t a t i n immunoreactivity as measured by radioimmunoassay. The b r a i n regions examined i n c l u d e : CTX = c o r t i c a l sample d o r s a l to the s t r i a t u m , C-P = caudate putamen, NAS = nucleus accumbens s e p t i , mPFC = medial p r e f r o n t a l c o r t e x , OT = o l f a c t o r y t u b e r c l e , SN = s u b s t a n t i a n i g r a , and VTA = v e n t r a l tegmental area. Data r e p r e s e n t s mean values ± SEM f o r each group. *.p < 0.05 compared to c o n t r o l group. 36 JZ v cn E \ cn c I o o m P F C V T A FIGURE 8. E f f e c t of long term (6* months) treatment w i t h v e h i c l e ( J72 ) or h a l o p e r i d o l ( £ 5 1 ) (1.3-1.5 mg/kg/day) and the e f f e c t s of withdrawal from long term treatment w i t h v e h i c l e ( ^ ) or h a l o p e r i d o l ( ggg ) on the l e v e l s of c e n t r a l somatostatin immunoreactivity as measured by radioimmunoassay. The b r a i n r e g i o n s examined i n c l u d e : CTX = c o r t i c a l sample d o r s a l to the s t r i a t u m , C-P = caudate putamen, NAS = nucleus accumbens s e p t i , mPFC = medial p r e f r o n t a l c o r t e x , OT = o l f a c t o r y t u b e r c l e , SN = s u b s t a n t i a n i g r a , and VTA = v e n t r a l tegmental area. Data r e p r e s e n t s mean valu e s + SEM f o r each group. * p < 0.05 compared to a p p r o p r i a t e c o n t r o l group. 37 i n c l u d i n g those c o n t a i n i n g GABA (Gunne and HaggstrOm, 1983), substance P (Hanson e t a l . , 1080; Radke e t a l . , 1987b), o p i o i d s (Hong e t a l . , 1978), s o m a t o s t a t i n (Beal and M a r t i n , 1984c), c h o l e c y s t o k i n i n (CCK) (Frey, 1983; Radke e t a l . , 1987c), and n e u r o t e n s i n (Govoni e t a l . , 1980; Radke e t a l . , 1987c). These changes may a l s o be important i n the delayed onset of the c l i n i c a l e f f e c t s of n e u r o l e p t i c s , and i n the s i d e e f f e c t s of c h r o n i c treatment. To p r o v i d e a more complete understanding of the neurochemical changes o c c u r r i n g d u r i n g n e u r o l e p t i c treatment, the e f f e c t s of v a r i o u s lengths of 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 the l e v e l s of s o m a t o s t a t i n were examined. The r e s u l t s showed t h a t s h o r t term o r a l treatment of r a t s w i t h h a l o p e r i d o l s i g n i f i c a n t l y reduced s o m a t o s t a t i n l e v e l s i n the nucleus accumbens and caudate-putamen. T h i s agrees w i t h the o b s e r v a t i o n s of B e a l and M a r t i n (1984c) who gave r a t s h a l o p e r i d o l i n j e c t i o n s f o r 3 weeks and found decreases i n the caudate-putamen and the nucleus accumbens. A s l i g h t decrease i n s o m a t o s t a t i n l e v e l s i n the v e n t r a l tegmental area was a l s o found a f t e r s h o r t term h a l o p e r i d o l treatment. These r e s u l t s support the i d e a s t a t e d i n the d i s c u s s i o n of experiment 1, t h a t dopamine and s o m a t o s t a t i n neurons i n t e r a c t i n the b a s a l g a n g l i a . T h e r e f o r e a decrease i n dopamine r e c e p t o r a c t i v a t i o n by h a l o p e r i d o l may decrease the a c t i v a t i o n of s o m a t o s t a t i n neurons, which i n t u r n c o u l d e x p l a i n the r e d u c t i o n i n s t r i a t a l s o m a t o s t a t i n l e v e l s observed. Another p o s s i b i l i t y would be t h a t s o m a t o s t a t i n r e l e a s e i s enhanced a f t e r h a l o p e r i d o l treatment to compensate f o r the decreased dopamine a c t i v i t y , thereby r e d u c i n g the measurable 38 l e v e l s of s o m a t o s t a t i n . I t should be mentioned t h a t t h i s i s very s p e c u l a t i v e and more d e t a i l e d examination of the e f f e c t s of h a l o p e r i d o l on s o m a t o s t a t i n s y n t h e s i s (mRNA l e v e l s ) or s o m a t o s t a t i n r e l e a s e are r e q u i r e d t o f u l l y understand the mechanisms i n v o l v e d i n r e d u c i n g s o m a t o s t a t i n l e v e l s . In c o n t r a s t t o the s h o r t term study, long term treatment (8 months) o n l y decreased s o m a t o s t a t i n l e v e l s i n the nucleus accumbens. Somatostatin l e v e l s i n the caudate-putamen and v e n t r a l tegmental area were s i m i l a r t o c o n t r o l l e v e l s . These f i n d i n g s suggest t h a t s o m a t o s t a t i n c o n t a i n i n g neurons i n the caudate-putamen, but not the nucleus accumbens, adapt over time to the a l t e r a t i o n s i n dopamine r e c e p t o r blockade. S i m i l a r changes have a l s o been observed w i t h n e u r o t e n s i n and substance P (Radke e t a l . , 1987b: 1987c), which are a l t e r e d a f t e r s h o r t term treatment but not a f t e r long term treatment. In summary, the e f f e c t s of s h o r t term h a l o p e r i d o l treatment on s o m a t o s t a t i n l e v e l s i n the b a s a l g a n g l i a are c o n s i s t e n t w i t h p r e v i o u s s t u d i e s . In c o n t r a s t , f o l l o w i n g long term h a l o p e r i d o l treatment these a l t e r e d l e v e l s r e t u r n t o c o n t r o l v a l u e s except i n the nucleus accumbens where s o m a t o s t a t i n l e v e l s remained reduced. Furthermore, f o l l o w i n g withdrawal from long term h a l o p e r i d o l treatment no s i g n i f i c a n t changes i n s o m a t o s t a t i n l e v e l s were d e t e c t e d i n any of the b r a i n r e g i o n s examined. These r e s u l t s p r o v i d e f u r t h e r evidence f o r an i n t e r a c t i o n of dopamine w i t h s o m a t o s t a t i n i n the b a s a l g a n g l i a , and suggest t h a t s o m a t o s t a t i n systems may be important i n the c l i n i c a l 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 long term n e u r o l e p t i c treatment. 39 EXPERIMENT 3. THE EFFECTS OF MPTP ON SOMATOSTATIN IMMUNOREACTIVITY IN THE MOUSE. INTRODUCTION Le s i o n s of dopamine t r a c t s by 6-hydroxydopamine (6-OHDA) have been shown to a l t e r s e v e r a l t r a n s m i t t e r systems, i n c l u d i n g GABA, a c e t y l c h o l i n e (Kim, 1973), c h o l e c y s t o k i n i n (Chang e t a l . , 1983), and substance P (Hanson e t a l . , 1981). 6-OHDA l e s i o n s do not appear t o a l t e r s t r i a t a l s o m a t o s t a t i n l e v e l s i n the r a t (Beal and M a r t i n , 1983). T h i s l a t t e r f i n d i n g c o n f l i c t s w i t h the changes observed i n s t r i a t a l s o m a t o s t a t i n l e v e l s f o l l o w i n g s h o r t term h a l o p e r i d o l treatment (Beal and M a r t i n , 1984c; experiment 3), but i s c o n s i s t e n t with s t u d i e s i n v o l v i n g long term n e u r o l e p t i c treatment (experiment 3), as w e l l as w i t h o b s e r v a t i o n s i n Parkinson's d i s e a s e where s t r i a t a l s o m a t o s t a t i n l e v e l s are normal (Agid and Javoy-Agid, 1985; Epelbaum e t a l . , 1983; Rinne e t a l . , 1984). T h i s suggests t h a t 6-OHDA l e s i o n s may be a u s e f u l model f o r Parkinson's d i s e a s e , w i t h r e s p e c t to non-dopaminergic aspects of t h i s d i s e a s e and t h e i r i m p l i c a t i o n s f o r treatment. R e c e n t l y , the n e u r o t o x i n MPTP (1-methyl-4-phenyl-1,2,3,6-t e t r a h y d r o p y r i d i n e ) has been shown to cause P a r k i n s o n - l i k e symptoms i n humans (Langston e t a l . , 1983) and has been used as a model of t h i s d i s e a s e i n both primates (Burns e t a l . , 1983) and mice (Hallman e t a l . , 1984; H e i k i l l a e t a l . , 1983). MPTP has been shown to a f f e c t s e v e r a l non-dopaminergic systems, i n c l u d i n g v a r i o u s neuropeptides ( A l l e n e t a l . , 1986; Zamir e t a l . , 1984). 40 Since s e v e r a l non-dopaminergic systems are a l t e r e d i n Parkinson's d i s e a s e (Agid and Javoy-Agid, 1985; Epelbaum e t a l . , 1983; Rinne e t a l . , 1984), as w e l l as wit h 6-OHDA l e s i o n s (Chang e t al.,1983; Hanson e t al.,1981; Kim e t a l . , 1973) and MPTP p o i s o n i n g ( A l l e n e t a l . , 1986; Zamir e t a l . , 1984), a comparison of the nondopaminergic changes a f t e r MPTP and 6-OHDA l e s i o n s w i t h those found i n Parkinson's d i s e a s e may e s t a b l i s h which of these two drugs produces a more a c c u r a t e model of Parkinson's d i s e a s e . The purpose of the f o l l o w i n g experiment was to examine the e f f e c t s of MPTP a d m i n i s t r a t i o n i n C57 mice on the l e v e l s of soma t o s t a t i n immunoreactivity t o determine i f MPTP produces an accurate model of the neurochemical changes observed i n Parkinsonism. T h i s study a l s o f u r t h e r examined the i n t e r a c t i o n s between dopamine and som a t o s t a t i n i n the b r a i n . METHODS AND MATERIALS  Subjects. Male C57 mice (Charles R i v e r s ) weighing 20-25 grams were group housed (5-6/cage) i n po l y p r o p y l e n e cages and maintained w i t h i n a fume hood f o r the d u r a t i o n of the experiment. Food and water were p r o v i d e d ad l i b i t u m . Drugs. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine h y d r o c h l o r i d e (MPTP) (Research B i o c h e m i c a l s Inc.) was d i s s o l v e d i n s a l i n e a t c o n c e n t r a t i o n s of 40 mg/ml, and 50 mg/ml and i n j e c t e d i . p . , (l.Oml/lOOg). 41 Procedure. C57 male mice were randomly assig n e d t o one of three groups; a c o n t r o l group (n=7) r e c e i v i n g a d a i l y i n j e c t i o n of s a l i n e (1.0 ml/100 g) f o r two days, a second group (n=7) r e c e i v i n g two i n j e c t i o n s of 40 mg/kg MPTP, and a t h i r d (n=6) g i v e n two i n j e c t i o n s of 50 mg/kg MPTP. Fourteen days a f t e r the l a s t i n j e c t i o n , animals were s a c r i f i c e d by d e c a p i t a t i o n and samples of the c o r t e x , caudate-putamen, and the s u b s t a n t i a n i g r a were d i s s e c t e d on i c e , and prepared f o r radioimmunoassay as d e s c r i b e d i n experiment 1. In a d d i t i o n , samples of the caudate-putamen were processed f o r catecholamine l e v e l s u s i n g r e v e r s e phase h i g h performance l i q u i d chromatography (HPLC) wit h e l e c t r o c h e m i c a l d e t e c t i o n (Cumming e t a l . , 1986). RESULTS As shown i n F i g u r e 9, MPTP p o i s o n i n g r e s u l t e d i n s i g n i f i c a n t r e d u c t i o n s i n s t r i a t a l dopamine l e v e l s . For the 2x40 mg/kg group, the r e d u c t i o n was 75 %, and the r e d u c t i o n w i t h the 2x50 mg/kg group was 77 %. There were a l s o decreases i n s t r i a t a l h o m o v a n i l l i c a c i d (HVA), 3,4-dihydroxy-phenyl-acetic a c i d (DOPAC), and no r e p i n e p h r i n e , but no changes were observed i n s t r i a t a l s e r o t o n i n l e v e l s . F i g u r e 10 i l l u s t r a t e s the e f f e c t s of MPTP p o i s o n i n g on the som a t o s t a t i n l e v e l s i n v a r i o u s b r a i n r e g i o n s . Somatostatin immunoreactivity i n the c o r t e x and s t r i a t u m was not changed by MPTP p o i s o n i n g . Instead, there was a s i g n i f i c a n t i n c r e a s e i n 42 s o m a t o s t a t i n l e v e l s i n the s u b s t a n t i a n i g r a t h a t appeared to be dose dependent, w i t h the 2x40 group having a s i g n i f i c a n t i n c r e a s e of 56 % from c o n t r o l v a l u e s (t(12) = 3.33, p < 0.025), and the 2x50 group an i n c r e a s e of 72 % ( t ( l l ) = 3.48, p < 0.025). D I S C U S S I O N Recent o b s e r v a t i o n s of a l t e r e d p e p t i d e l e v e l s i n post-mortem b r a i n t i s s u e s from Alzheimer's d i s e a s e (Davies e t a l . , 1980), Huntington's chorea (Aronin e t a l . , 1983; B e a l e t a l . , 1984; Nemeroff e t a l . , 1983; Sagar e t al.,1984) and Parkinson's d i s e a s e (Agid and Javoy-Agid, 1985; Epelbaum e t a l . , 1983; Rinne e t al.,1984) have i l l u s t r a t e d the importance of neuropeptides i n pathology. To develop an accurate animal model of a d i s e a s e , the neurochemical a l t e r a t i o n s which may be secondary t o the b e l i e v e d major neurochemical d y s f u n c t i o n , should a l s o be c o n s i s t e n t w i t h the observed neurochemical changes found i n the d i s e a s e . In post-mortem s t u d i e s of P a r k i n s o n i a n b r a i n s no changes i n n i g r a l s o m a t o s t a t i n l e v e l s have been observed (Agid and Javoy-Agid, 1985; Epelbaum e t a l . , 1983; Rinne e t al.,1984). However, so m a t o s t a t i n l e v e l s are reduced i n the f r o n t a l c o r t e x and hippocampus of demented P a r k i n s o n i a n p a t i e n t s (Agid and Javoy-Agid, 1985; Epelbaum e t a l . , 1983; Rinne e t al.,1984). In c o n t r a s t w i t h these f i n d i n g s , our study has shown MPTP p o i s o n i n g t o i n c r e a s e n i g r a l somatostatin l e v e l s as the l e v e l s of catecholamines decreased. The l e v e l s of s o m a t o s t a t i n i n the c o r t e x and s t r i a t u m were unchanged. 43 200 - r 190 -180 -170 -160 -FIGURE 9. E f f e c t s of s a l i n e ( 2 x 1 ml/lOOg) ( Q ) or MPTP a t doses of 2 x 40 mg/kg ( ) and 2 x 50 mg/kg, IP ( ) o n s t r i a t a l catecholamine l e v e l s i n the mouse. The r e s u l t s are expressed as a precentage of c o n t r o l , w i t h the ab s o l u t e v a l u e s of the c o n t r o l groups being, dopamine (DA) = 11,145 ± 324 ng/g wet weight, 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 (DOPAC) = 1,323 ± 59 ng/g, h o m o v a n i l l i c a c i d (HVA) = 1,812 ± 137 ng/g, norepinephrine (NE) = 337 ± 69 ng/g, s e r o t o n i n (5-HT) = 480 ± 15 ng/g. * p < 0.05 compared t o c o n t r o l group. 44 100 -r 90 -C T X C - P S N FIGURE 10. E f f e c t s of s a l i n e ( 2 x 1 ml/lOOg) ( JZ2 > o r M P T P a t doses of 2 x 40 mg/kg ( £\] ) and 2 x 50 mg/kg, SC ( jggj ) on the l e v e l s of c e n t r a l s omatostatin immunoreactivity as measured by radioimmunoassay. B r a i n r e g i o n s examined i n c l u d e ; CTX = c o r t i c a l sample d o r s a l to the s t r i a t u m , C-P = caudate putamen, SN = s u b s t a n t i a n i g r a . Data r e p r e s e n t s mean values ± SEM f o r each group. * p < 0.05 compared t o c o n t r o l group. 45 These r e s u l t s are i n p a r t i a l disagreement w i t h p r e v i o u s MPTP s t u d i e s i n primates. Both A l l e n e t al.(1986) and Zamir e t a l . (1984), found MPTP to have no e f f e c t on n i g r a l s o m a t o s t a t i n l e v e l s i n primates. The p r e s e n t r e s u l t s w i t h MPTP are s i m i l a r t o e a r l i e r s t u d i e s u s i n g 6-OHDA, another proposed animal model f o r Parkinson's d i s e a s e . 6-OHDA l e s i o n s do not a f f e c t s t r i a t a l s o m a t o s t a t i n l e v e l s i n the r a t (Beal and Martin,1983). B e a l and M a r t i n (1983) however d i d not measure n i g r a l s o m a t o s t a t i n l e v e l s i n t h e i r study, so i t i s p r e s e n t l y unknown whether n i g r a l s o m a t o s t a t i n l e v e l s are i n c r e a s e d as w e l l , f o l l o w i n g 6-OHDA l e s i o n s . A p o s s i b l e e x p l a n a t i o n f o r the d i s c r e p a n c y between the r e s u l t s of t h i s experiment and s t u d i e s i n other animals g i v e n MPTP ( A l l e n e t a l . , 1986; Zamir e t a l . , 1984) i s the anatomy of soma t o s t a t i n systems i n the mouse. As shown i n F i g u r e 10, the n i g r a l s o m a t o s t a t i n l e v e l s i n the mouse were f a i r l y h igh, whereas i n the primate and r a t , the l e v e l s i n the s u b s t a n t i a n i g r a are q u i t e low (see F i g u r e 7, experiment 2). The reason why h a l o p e r i d o l does a f f e c t s t r i a t a l s o m a t o s t a t i n l e v e l s i n the r a t (experiment 2; Be a l and M a r t i n , 1984c) but n e i t h e r MPTP ( F i g u r e 10; A l l e n e t a l . , 1984; Zamir e t a l . , 1986) nor 6-OHDA l e s i o n s (Beal and M a r t i n , 1983) a l t e r s t r i a t a l s o m a t o s t a t i n l e v e l s i s p r e s e n t l y unknown but may i n d i c a t e t h a t changes i n dopamine r e c e p t o r s , but not dopamine l e v e l s , have a g r e a t e r i n f l u e n c e on som a t o s t a t i n neurons. In summary, mice g i v e n MPTP show i n c r e a s e s i n n i g r a l s o m a t o s t a t i n l e v e l s . These o b s e r v a t i o n s are not c o n s i s t e n t w i t h 46 e i t h e r MPTP s t u d i e s i n primates, or wit h f i n d i n g s from p o s t -mortem Parkinson's b r a i n s . However, the present i n v e s t i g a t i o n does support these e a r l i e r s t u d i e s i n showing no r e d u c t i o n s i n s t r i a t a l s o m a t o s t a t i n l e v e l s . The changes observed i n n i g r a l s o m a t o s t a t i n l e v e l s f o l l o w i n g MPTP may r e f l e c t d i f f e r e n c e s i n t r a n s m i t t e r i n t e r a c t i o n s i n d i f f e r e n t s p e c i e s . F u r t h e r work i n v o l v i n g the i n t e r a c t i o n s of dopamine and somat o s t a t i n i n d i f f e r e n t s p e c i e s may h e l p i n the i n t e r p r e t a t i o n of these r e s u l t s . F i n a l l y , these r e s u l t s expand the l i s t of non-ca t e c h o l a m i n e r g i c t r a n s m i t t e r s a f f e c t e d by MPTP. F u r t h e r work u s i n g MPTP should r e c o g n i z e these changes when examining the a c t i o n s of t h i s t o x i n . 47 EXPERIMENT 4, THE EFFECTS OF CARBAMAZEPINE AND DESMETHYLIMIPRAMINE (DMI) ON SOMATOSTATIN IMMUNOREACTIVITY. INTRODUCTION As shown i n experiments 2 and 3, drugs which d i s r u p t dopaminergic systems appear to a l t e r s o m a t o s t a t i n l e v e l s . In the c l i n i c a l s e t t i n g a n t i d e p r e s s a n t s , a n t i c o n v u l s a n t s (Rubinow e t a l . , 1984; Rubinow, 1986) and n e u r o l e p t i c s (Gattaz e t a l . , 1986), have been shown to a l t e r human CSF s o m a t o s t a t i n l e v e l s d u r i n g treatment. In experiment 2, n e u r o l e p t i c s were shown to a l s o a f f e c t s o m a t o s t a t i n l e v e l s i n the r a t b r a i n , and i t i s of i n t e r e s t whether s o m a t o s t a t i n l e v e l s can be a l t e r e d by other c l i n i c a l l y important drugs. One drug of i n t e r e s t i s carbamazepine which has been shown to be u s e f u l i n the treatment of manic-depression, paroxysmal p a i n d i s o r d e r s , and e p i l e p s y (see reviews by Post e t a l . , 1983 and Rubinow, 1986). C l i n i c a l l y , carbamazepine treatment has been shown to lower CSF s o m a t o s t a t i n l e v e l s of p a t i e n t s w i t h a f f e c t i v e i l l n e s s , an o b s e r v a t i o n which i s f a i r l y unique to t h i s drug s i n c e desmethylimipramine (DMI) and l i t h i u m do not appear to a l t e r s o m a t o s t a t i n l e v e l s (Rubinow, 1986). Human s t u d i e s have a l s o shown a c o r r e l a t i o n between both carbamazepine treatment and low s o m a t o s t a t i n l e v e l s w i t h escape from the dexamethasone s u p p r e s s i o n t e s t (Rubinow, 1986). Whether or not carbamazepine has i t s c l i n i c a l e f f i c a c y v i a a d i r e c t i n f l u e n c e on c e n t r a l s o m a t o s t a t i n systems i s p r e s e n t l y unknown. 48 The a n t i c o n v u l s a n t p r o p e r t i e s of carbamazepine might a l s o be due to i t s i n t e r a c t i o n s w i t h c e n t r a l s o m a t o s t a t i n systems. C o r t i c a l s o m a t o s t a t i n l e v e l s are i n c r e a s e d i n animal models of e p i l e p s y ( H i g uchi e t a l . , 1984; 1986; Kato e t a l . , 1983), and i n p a t i e n t s w i t h i n t r a c t a b l e e p i l e p s y (Nadi e t a l . , 1986). Rec e n t l y , H i g u c h i e t a l . (1986) have shown t h a t carbamazepine decreases s o m a t o s t a t i n l e v e l s i n k i n d l e d r a t s , but o n l y i n the b r a i n areas showing s e i z u r e - a s s o c i a t e d e l e v a t i o n s i n the l e v e l s of t h i s p e p t i d e . T h i s suggests t h a t carbamazepine might reduce so m a t o s t a t i n l e v e l s o n l y i f they are abnormal, as i n k i n d l e d r a t s (Higuchi e t a l . , 1984; 1986; Kato e t a l . , 1983), or a f f e c t i v e i l l n e s s (Rubinow e t a l . , 1984; Rubinow, 1986). The purpose of the present study was to determine i f acute or c h r o n i c a d m i n i s t r a t i o n of carbamazepine a l t e r s b a s a l s o m a t o s t a t i n l e v e l s i n the r a t . The a d m i n i s t r a t i o n of acute and c h r o n i c DMI was a l s o examined as a c o n t r o l group s i n c e DMI does not appear to a l t e r CSF s o m a t o s t a t i n l e v e l s i n p a t i e n t s w i t h a f f e c t i v e d i s o r d e r s . METHODS AND MATERIALS  Subj e c t s . Male Long Evans r a t s (Charles R i v e r ) weighing 250-300 grams were group housed 4 per cage under a 12 hour l i g h t / d a r k c y c l e w i t h food and water p r o v i d e d ad l i b i t u m . 49 Drugs. Carbamazepine (Sigma) was d i s s o l v e d i n 70% g l y c e r o l s o l u t i o n (20 mg/ml) and i n j e c t e d i . p . (1.0 ml/kg). Desmethylimipramine h y d r o c h l o r i d e (DMI) (Merr e l Dow) was d i s s o l v e d i n d i s t i l l e d water (5.0 mg/ml) and i n j e c t e d i . p . (1.0 ml/kg). Procedure. Experiment 4a. Rats were randomly assi g n e d t o one of f o u r groups; 1) c o n t r o l group (n=8) g i v e n 2 d a i l y i n j e c t i o n s of v e h i c l e (70% g l y c e r o l , i .p.) f o r 12 c o n s e c u t i v e days, 2) acute group (n=7) g i v e n 11 days of v e h i c l e i n j e c t i o n s (2/day) f o l l o w e d by one i n j e c t i o n of carbamazepine (20 mg/kg) on day 12, 3) c h r o n i c group (n=8) r e c e i v i n g 12 days of carbamazepine (2 x 20 mg/kg), 4) c h r o n i c withdrawal group (n=8) r e c e i v i n g 10 days of carbamazepine f o l l o w e d by 2 days of v e h i c l e i n j e c t i o n s . A l l r a t s r e c e i v e d t h e i r l a s t i n j e c t i o n 30 minutes p r i o r t o being s a c r i f i c e d . The hippocampus, hypothalamus, nucleus accumbens, caudate-putamen, and a c o r t i c a l sample d o r s a l t o the s t r i a t u m were d i s s e c t e d from s e c t i o n s c u t on a f r e e z i n g microtome and prepared f o r radioimmunoassay as d e s c r i b e d i n experiment 1. Experiment 4b. Rats were randomly assi g n e d t o one of thr e e groups; 1) c o n t r o l group (n=9) r e c e i v i n g 14 days of two d a i l y i n j e c t i o n s of v e h i c l e (dB^O, i . p . ) , 2) acute group (n=9) r e c e i v i n g 12 days of v e h i c l e s o l u t i o n f o l l o w e d by 2 days of DMI (2 x 5.0 mg/mg, i . p . ) , 3) c h r o n i c group (n=7) r e c e i v i n g 14 days of DMI (2 x 5.0 mg/kg, i . p . ) . A l l r a t s r e c e i v e d t h e i r l a s t i n j e c t i o n 12 hours p r i o r to being s a c r i f i c e d and the nucleus accumbens, caudate-putamen, and a c o r t e x sample d o r s a l t o the 50 s t r i a t u m d i s s e c t e d from s e c t i o n s c u t on a f r e e z i n g microtome and prepared f o r radioimmunoassay, as d e s c r i b e d i n experiment 1. RESULTS As shown i n F i g u r e 11, n e i t h e r acute, c h r o n i c , nor withdrawal f o l l o w i n g c h r o n i c a d m i n i s t r a t i o n of carbamazepine s i g n i f i c a n t l y changed the l e v e l s of soma t o s t a t i n i n any of the b r a i n r e g i o n s examined, when compared to c o n t r o l group mean v a l u e s . S i m i l a r l y , acute, as w e l l as c h r o n i c DMI a d m i n i s t r a t i o n d i d not a l t e r s o m a t o s t a t i n l e v e l s i n any b r a i n r e g i o n examined (F i g u r e 12). DISCUSSION The l a c k of e f f e c t of carbamazepine on b a s a l s o m a t o s t a t i n l e v e l s ( F i g u r e 11) would agree wi t h the r e s u l t s of H i g u c h i e t a l . (1986) who showed t h a t i n re g i o n s of the b r a i n w i t h normal s o m a t o s t a t i n l e v e l s a f t e r k i n d l i n g ( i e . hypothalamus, st r i a t u m ) the s o m a t o s t a t i n l e v e l s were u n a f f e c t e d by carbamazepine treatment. However, H i g u c h i e t a l . (1986) found t h a t the k i n d l i n g - i n d u c e d e l e v a t i o n i n c o r t i c a l s o m a t o s t a t i n was reduced by carbamazepine u s i n g a s i m i l a r dosage. In the pr e s e n t study, carbamazepine was without e f f e c t on b a s a l c o r t i c a l s o m a t o s t a t i n 51-N A c c C - P H I P P O C FIGURE 11. E f f e c t of v e h i c l e ( [73 ) , acute ( Q ), c h r o n i c (ggl) , and withdrawal f o l l o w i n g c h r o n i c ( £j^J ) treatment w i t h carbamazepine (2 x 25 mg/kg, IP) on the l e v e l s of c e n t r a l s o matostatin immunoreactivity as measured by radioimmunoassay. B r a i n r e g ions examined i n c l u d e ; N Acc = nucleus accumbens s e p t i , C-P = caudate putamen, CTX = c o r t i c a l sample d o r s a l t o the s t r i a t u m , HIPPOC = hippocampus, HT = hypothalamus. Data r e p r e s e n t s mean va l u e s ± SEM of each group. 52 200 190 -180 -C T X C - P N A S FIGURE 12. E f f e c t s of s a l i n e ( (7/1 ) , acute ( [£J ) , and c h r o n i c (£22) DMI treatment on the l e v e l s of c e n t r a l s o m a t o s t a t i n immunoreactivity as measured by radioimmunoassay. B r a i n regions examined i n c l u d e ; CTX = c o r t i c a l sample d o r s a l to the s t r i a t u m , C-P = caudate putamen, NAS = nucleus accumbens s e p t i . Data r e p r e s e n t s mean v a l u e s + SEM of each group. 53 l e v e l s . R e c e n t l y , Nagaki e t a l . (1985) found t h a t carbamazepine a t a dose of 50 mg/kg d a i l y (acute and c h r o n i c treatment) d i d not a l t e r c e n t r a l s o m a t o s t a t i n l e v e l s , however they d i d observe very s l i g h t decreases i n somat o s t a t i n l e v e l s w i t h a ve r y h i g h dose (200 mg/kg) i n the c o r t e x (acute) and hippocampus ( c h r o n i c t r e a t m e n t ) . Carbamazepine has been shown t o a f f e c t c e n t r a l catecholamines (Kowalik e t a l . 1984; Post e t a l . , 1985; Purdy e t a l . , 1987; Waldmeier e t a l . 1984), a c e t y l c h o l i n e , t h y r o i d hormones, c y c l i c n u c l e o t i d e s , and GABA (Post e t a l . 1983). Thus, i t i s p o s s i b l e t h a t the carbamazepine-induced r e d u c t i o n s i n soma t o s t a t i n l e v e l s observed by H i g u c h i e t a l . (1986) and Rubinow (1986) are the r e s u l t of carbamazepine's a c t i o n s on these other systems. A prime candidate might be GABA, s i n c e GABA and soma t o s t a t i n c o e x i s t i n v a r i o u s r e g i o n s , i n c l u d i n g the c o r t e x (Hendry e t a l . 1984), and c o r t i c a l l e v e l s of GABA are i n c r e a s e d by carbamazepine i n k i n d l e d r a t s ( H i g uchi e t a l . 1986). Th e r e f o r e i t i s p o s s i b l e t h a t i n c r e a s e s i n c o r t i c a l GABA may be a s s o c i a t e d w i t h the s u s c e p t i b i l i t y of so m a t o s t a t i n c e l l s t o carbamazepine's a c t i o n s . With r e g a r d t o DMI, Rubinow (1986) has shown DMI to have no e f f e c t on CSF so m a t o s t a t i n l e v e l s i n a f f e c t i v e l y i l l p a t i e n t s . In agreement w i t h t h i s , r a t s g i v e n DMI d i d not show any a l t e r a t i o n s i n c e n t r a l s o m a t o s t a t i n l e v e l s ( F i g u r e 12). A study by M a r t i n - I v e r s o n e t a l . (1983) has shown DMI to enhance dopamine mediated behaviours, p o s s i b l y by a non-dopaminergic mechanism. Based on r e s u l t s from experiment 1, showing cysteamine t o 54 attenuate dopamine mediated behaviours, a p o s s i b l e mechanism f o r DMI to enhance dopamine mediated behaviours would be by-i n c r e a s i n g s t r i a t a l s o m a t o s t a t i n a c t i v i t y . The r e s u l t s of t h i s experiment i n d i c a t e DMI i s probably not having i t s e f f e c t s on dopamine-mediated behaviours v i a a s o m a t o s t a t i n mechanism. F u r t h e r a n a l y s i s i n v o l v i n g the e f f e c t s of DMI on other t r a n s m i t t e r s known to a f f e c t dopamine-mediated behaviours, such as a c e t y l c h o l i n e (Radke e t a l . , 1987a), GABA ( D i S c a l i e t a l . , 1985), substance P ( K e l l e y e t a l . , 1979) or n e u r o t e n s i n (Nemeroff, 1986) may p r o v i d e some answers to t h i s problem. In summary, carbamazepine and DMI do not appear to a l t e r b a s a l s o m a t o s t a t i n l e v e l s i n the r a t b r a i n . T h i s suggests t h a t carbamazepine may a c t i n d i r e c t l y t o reduce abnormal s o m a t o s t a t i n l e v e l s . Furthermore, the l a c k of e f f e c t of DMI on s o m a t o s t a t i n l e v e l s supports c l i n i c a l o b s e r v a t i o n s but f a i l s t o e s t a b l i s h the mechanisms by which DMI has i t s e f f e c t s on dopamine-mediated behaviours. For both drugs, f u r t h e r work to e s t a b l i s h the mechanism of a c t i o n i s c l e a r l y warranted. 55 GENERAL DISCUSSION Dopamine-somatostatin interactions The e f f e c t s of s o m a t o s t a t i n d e p l e t i o n by cysteamine on dopamine-mediated behaviours (experiment 1; M a r t i n - I v e r s o n e t a l . , 1986) suggests t h a t s t r i a t a l s o m a t o s t a t i n neurons may p l a y a r o l e i n modulating the motor, but not the r e i n f o r c i n g a c t i o n s , of dopamine a g o n i s t s . Furthermore, i t i s l i k e l y t h a t t h i s modulation occurs p o s t s y n a p t i c t o dopamine-releasing t e r m i n a l s i n the s t r i a t u m , s i n c e s o m a t o s t a t i n d e p l e t i o n s by cysteamine reduce the motor e f f e c t s of both i n d i r e c t (amphetamine-induced h y p e r a c t i v i t y ) and d i r e c t (apomorphine-induced st e r e o t y p y ) dopamine a g o n i s t s . T h i s i s f u r t h e r supported by the o b s e r v a t i o n s t h a t cysteamine does not appear to a l t e r s t r i a t a l dopamine metabolism (Beal and M a r t i n , 1984a; 1984b; M a r t i n - I v e r s o n e t a l . , 1986). However, soma t o s t a t i n i n f u s i o n s i n t o the s t r i a t u m do a l t e r s t r i a t a l dopamine turnover (Beal and M a r t i n , 1984a; G a r c i a -S e v i l l a e t a l . , 1978) and s o m a t o s t a t i n enhances both b a s a l ( C h e s s e l e t and R e i s i n e , 1983; S t a r r , 1982) and K + - i n d u c e d ( S t a r r , 1982) -^-dopamine r e l e a s e from r a t s t r i a t a l s l i c e s , s u g g e s t i n g t h a t s o m a t o s t a t i n can a c t on dopaminergic t e r m i n a l s . T h i s i s f u r t h e r supported by a study by V e c s e i e t a l . (1983b) showing t h a t h a l o p e r i d o l can b l o c k the i n c r e a s e i n motor a c t i v i t y caused by v e n t r i c u l a r s o m a t o s t a t i n i n f u s i o n s . Since s o m a t o s t a t i n i n f u s i o n s appear to a l t e r dopamine a c t i v i t y , but cysteamine does not, i t i s p o s s i b l e t h a t s o m a t o s t a t i n has both p r e s y n a p t i c and 56 p o s t s y n a p t i c i n t e r a c t i o n s w i t h dopamine t e r m i n a l s . The p r e s y n a p t i c s o m a t o s t a t i n a c t i o n s may be o n l y e f f e c t i v e d u r i n g i n c r e a s e d s o m a t o s t a t i n a c t i v i t y s i n c e cysteamine does not a l t e r dopamine metabolism, which would be expected i f s o m a t o s t a t i n had a t o n i c e f f e c t on dopamine t e r m i n a l s . Based on these o b s e r v a t i o n s i t would be expected t h a t d i s r u p t i o n of s t r i a t a l dopaminergic a c t i v i t y should a f f e c t the a c t i v i t y of s t r i a t a l s o m a t o s t a t i n neurons. T h i s appears to be the case. When animals are t r e a t e d w i t h h a l o p e r i d o l f o r 3 weeks (F i g u r e 7), s t r i a t a l s o m a t o s t a t i n l e v e l s are reduced. These o b s e r v a t i o n s c o u l d be i n t e r p r e t e d as dopamine r e c e p t o r blockade ca u s i n g e i t h e r a r e d u c t i o n i n s o m a t o s t a t i n s y n t h e s i s or an i n c r e a s e i n s o m a t o s t a t i n r e l e a s e . Future s t u d i e s i n v o l v i n g the e f f e c t s of dopaminergic drugs on the r e l e a s e and/or s y n t h e s i s of s o m a t o s t a t i n i n the s t r i a t u m may p r o v i d e a more p r e c i s e i n t e r p r e t a t i o n of these r e s u l t s . The r e t u r n of s o m a t o s t a t i n l e v e l s i n the caudate-putamen to c o n t r o l v a l u e s a f t e r long term n e u r o l e p t i c treatment i s i n t e r e s t i n g i n l i g h t of the o b s e r v a t i o n s of long term n e u r o l e p t i c treatment being a s s o c i a t e d w i t h t a r d i v e d y s k i n e s i a (Casey and G e r l a c h , 1984). T a r d i v e d y s k i n e s i a has been suggested to be due to e i t h e r dopaminergic s u p e r s e n s i t i v y and/or c h o l i n e r g i c h y p o f u n c t i o n (Casey and G e r l a c h , 1984), s i n c e a n t i c h o l i n e r g i c s appear to enhance or s t i m u l a t e t a r d i v e d y s k i n e s i a i n humans. Another th e o r y s t a t e d by F i b i g e r and L l o y d (1984) i s t h a t t a r d i v e d y s k i n e s i a may be the r e s u l t of n e u r o l e p t i c - i n d u c e d damage to s t r i a t a l GABA neurons. The proposed s y n e r g i s t i c r e l a t i o n s h i p 57 between dopamine and s o m a t o s t a t i n i n the s t r i a t u m i s o p p o s i t e to the b e l i e v e d i n t e r a c t i o n of dopaminergic neurons w i t h s t r i a t a l a c e t y l c h o l i n e (McGeer e t a l . , 1961; Radke e t a l . , 1987a) or GABA ( D i S c a l i e t a l . , 1985) systems. T h e r e f o r e the observed r e t u r n of s o m a t o s t a t i n t o c o n t r o l l e v e l s i n the caudate-putamen a f t e r long term treatment may be r e l a t e d t o the development of t a r d i v e d y s k i n e s i a by i n c r e a s i n g the imbalance between n e u r o t r a n s m i t t e r systems, not observed d u r i n g s h o r t term treatment. Somatostatin i n N e u r o - p s y c h i a t r i c d i s e a s e As mentioned i n the i n t r o d u c t i o n , CSF s o m a t o s t a t i n l e v e l s appear to be abnormal i n s e v e r a l n e u r a l d i s e a s e s , i n c l u d i n g Alzheimer's (Gomez e t a l . , 1986a; 1986b; Rasind e t a l . , 1986), Huntington's (Cramer e t a l . , 1981), and Parkinson's (Cramer e t a l . , 1985) d i s e a s e , d e p r e s s i o n (Agren and Lundqvist, 1984; Gerner and Yamada, 1982; Rubinow e t a l . , 1985; Rubinow, 1986), and p a t i e n t s w i t h d y s t o n i c syndromes (Cramer e t a l . , 1985; T h a i e t a l . , 1985). CSF s o m a t o s t a t i n l e v e l s are a l s o a l t e r e d when me n t a l l y i l l p a t i e n t s are t r e a t e d w i t h carbamazepine (Rubinow e t a l . , 1984; Rubinow, 1986) or n e u r o l e p t i c s (Gattez e t a l . , 1986). One problem w i t h measurements of p e p t i d e l e v e l s i n the CSF i s the d i f f i c u l t y of i n t e r p r e t a t i o n . At p r e s e n t the o r i g i n s of CSF s o m a t o s t a t i n are u n c l e a r (Sorenson e t a l . , 1981). In regard to a f f e c t i v e i l l n e s s , t here i s growing evidence t h a t s o m a t o s t a t i n i s an important element i n t h i s d i s e a s e . The reduced CSF s o m a t o s t a t i n l e v e l s i n a f f e c t i v e l y i l l humans has 58 been c o r r e l a t e d w i t h the l e v e l s of both n o r a d r e n a l i n e (Rubinow e t a l . , 1984) and n o r a d r e n a l i n e m e t a b o l i t e s (Agren and L u n d q v i s t , 1984). Furthermore, reduced CSF s o m a t o s t a t i n l e v e l s are c o r r e l a t e d w i t h escape from the dexamethasone s u p p r e s s i o n t e s t (Rubinow, 1986). Treatment w i t h carbamazepine, which has been shown to lower abnormal s o m a t o s t a t i n l e v e l s i n both a f f e c t i v e l y i l l humans (Rubinow e t a l . , 1984; 1986; Rubinow, 1986) and k i n d l e d r a t s ( H i g u chi e t a l . , 1986) has a l s o been c o r r e l a t e d to escape from the dexamethasone s u p p r e s s i o n t e s t . These o b s e r v a t i o n s would suggest t h a t carbamazepine c o u l d be r e d u c i n g hypothalamic s o m a t o s t a t i n l e v e l s t o have i t s e f f e c t on the dexamethasone s u p p r e s s i o n t e s t s i n c e s o m a t o s t a t i n has been shown to i n h i b i t ACTH s e c r e t i o n (Brown e t a l . , 1984; L i t v i n e t a l . , 1986; R e i s i n e , 1985; Richardson e t a l . , 1983). The r e s u l t s of experiment 4 i n d i c a t e t h a t carbamazepine does not a l t e r the b a s a l l e v e l s of s o m a t o s t a t i n i n the r a t . The term 'basal" i s important i n d i s c u s s i n g these r e s u l t s because carbamazepine appears to reduce s o m a t o s t a t i n l e v e l s when these l e v e l s are abnormal (Higuchi e t a l . , 1986; Rubinow, 1986). T h i s suggests t h a t carbamazepine i s not having a d i r e c t e f f e c t on s o m a t o s t a t i n . Carbamazepine has been shown to a f f e c t other neurochemical systems, i n c l u d i n g GABA, a c e t y l c h o l i n e , v a s o p r e s s i n , c y c l i c n u c l e o t i d e s (see review by Post e t a l . , 1983), substance P (Jones e t a l . , 1985) and catecholamines (Kowalik e t a l . , 1984; Post e t a l . , 1985; Purdy e t a l . , 1977; Waldmeier e t a l . , 1984). Carbamazepine's e f f e c t s on s o m a t o s t a t i n may i n v o l v e one of these 59 other t r a n s m i t t e r systems s i n c e s e v e r a l of them have been shown to a f f e c t hypothalamic s o m a t o s t a t i n r e l e a s e (Epelbaum e t a l . , 1979; Richardson e t a l . , 1980; Sheppard e t a l . , 1979). Another p o s s i b i l i t y , however, would i n v o l v e a d i r e c t e f f e c t of carbamazepine on s o m a t o s t a t i n neurons d u r i n g s t r e s s . A study by A r a n c i b i a e t a l . (1984) has shown hypothalamic s o m a t o s t a t i n r e l e a s e to double when animals are under s t r e s s . Since carbamazepine does not appear to a l t e r b a s a l s o m a t o s t a t i n l e v e l s , i t s a b i l i t y to lower abnormal s o m a t o s t a t i n (Higuchi e t a l . , 1986; Rubinow e t a l . , 1984; Rubinow, 1986) may i n v o l v e changes i n the s u s c e p t i b i l i t y of s o m a t o s t a t i n c e l l s t o drugs when under s t r e s s , i n c l u d i n g the s t r e s s i n v o l v e d i n a f f e c t i v e i l l n e s s or k i n d l i n g . Two d i s o r d e r s i n which c e n t r a l s o m a t o s t a t i n l e v e l s are s i g n i f i c a n t l y a l t e r e d , t h a t are p e r t i n e n t to t h i s r e p o r t , are Huntington's d i s e a s e and e p i l e p s y . In Huntington's d i s e a s e t h e r e appears to be a s p a r i n g of s t r i a t a l s o m a t o s t a t i n c e l l s (Dawbarn e t a l . , 1985; F e r r a n t e e t a l . , 1985) and s t r i a t a l s o m a t o s t a t i n l e v e l s are e l e v a t e d (Aronin e t a l . , 1986; B e a l e t a l . , 1984a; Nemeroff e t a l . , 1983; Sagar e t a l . , 1984). Re c e n t l y , experiments i n the r a t have shown q u i n o l i n i c a c i d l e s i o n s to spare s t r i a t a l s o m a t o s t a t i n neurons w h i l e k i l l i n g other s t r i a t a l c e l l s (Beal e t a l . , 1986c), thus p r o v i d i n g a u s e f u l model of t h i s d i s e a s e . Based on the r e s u l t s of the b e h a v i o u r a l study presented i n t h i s r e p o r t (experiment 1), the i n c r e a s e i n s o m a t o s t a t i n l e v e l s c o u l d c o n t r i b u t e to the motor problems seen i n Huntington's 60 d i s e a s e . Treatment w i t h cysteamine might t h e r e f o r e be u s e f u l i n r e - e s t a b l i s h i n g a balanced neurochemical environment w i t h i n the d i s e a s e d s t r i a t u m . In f a c t , cysteamine has been r e c e n t l y e v a l u a t e d as a p o s s i b l e treatment ( S h u l t s e t a l . , 1986). U n f o r t u n a t e l y , the treatment regimen used f a i l e d t o r e l i e v e the motor a b n o r m a l i t i e s i n t h i s group of p a t i e n t s . T h i s treatment a l s o f a i l e d to a l t e r CSF l e v e l s of soma t o s t a t i n s u g g e s t i n g t h a t the dose of cysteamine used was too low. Cysteamine, or s i m i l a r drugs, might s t i l l prove u s e f u l i n Huntington's p a t i e n t s i n f u t u r e s t u d i e s . In i n t r a c t a b l e e p i l e p s y , c o r t i c a l s o m a t o s t a t i n l e v e l s appear to be i n c r e a s e d i n f o c a l e p i l e p t i c t i s s u e (Nadi e t a l . , 1986). T h i s i s c o n s i s t e n t w i t h experiments i n v o l v i n g animal models of e p i l e p s y which a l s o show an i n c r e a s e i n c o r t i c a l s o m a t o s t a t i n l e v e l s ( H i g u chi e t a l . , 1983; H i g u c h i e t a l . , 1986; Kato e t a l . , 1983). In the animal models, cysteamine has been shown to decrease both s o m a t o s t a t i n l e v e l s and s e i z u r e a c t i v i t y ( A s s o u l i n e e t a l . , 1984; H i g u c h i e t a l . , 1983) sugges t i n g t h a t the i n c r e a s e d s o m a t o s t a t i n l e v e l s may be an important f a c t o r i n e p i l e p s y . Carbamazepine a l s o reduces the i n c r e a s e d s o m a t o s t a t i n l e v e l s and s e i z u r e s i n k i n d l e d r a t s ( H i g u chi e t a l . , 1986), f u r t h e r s u g g e s t i n g s o m a t o s t a t i n i s i n v o l v e d i n e p i l e p t i c b ehaviours. Since both carbamazepine and cysteamine attenuate k i n d l i n g , but o n l y cysteamine reduces b a s a l s o m a t o s t a t i n l e v e l s , t h i s suggests t h a t carbamazepine i s a c t i n g i n d i r e c t l y to decrease s o m a t o s t a t i n i n k i n d l e d animals, but so m a t o s t a t i n i s a very important f a c t o r i n k i n d l i n g s i n c e cysteamine i n h i b i t s s e i z u r e s . Future s t u d i e s 61 i n v o l v i n g s o m a t o s t a t i n i n k i n d l i n g experiments may p r o v i d e a b e t t e r understanding of the c l i n i c a l importance of so m a t o s t a t i n i n e p i l e p s y and i t s treatment. The functions of somatostatin systems The f u n c t i o n s of so m a t o s t a t i n systems i n the b r a i n are o b v i o u s l y d i f f e r e n t f o r d i f f e r e n t b r a i n r e g i o n s . In the s t r i a t u m , s o m a t o s t a t i n may be important i n Huntington's d i s e a s e and t a r d i v e d y s k i n e s i a . In the hippocampus and c o r t e x , s o m a t o s t a t i n may be a f a c t o r i n e p i l e p s y . In the hypothalamus, so m a t o s t a t i n may c o n t r i b u t e t o the hormonal imbalance o f t e n o c c u r r i n g i n mental i l l n e s s . S ince s o m a t o s t a t i n l e v e l s appear t o be abnormal i n s e v e r a l d i s e a s e s , they may be a u s e f u l marker f o r pathology as f i r s t suggested by P a t e l e t a l . (1977). But what does s o m a t o s t a t i n do i n the normal b r a i n ? What are so m a t o s t a t i n systems c o n t r o l l i n g i n the s t r i a t u m , c o r t e x , hypothalamus, et c . ? P r e s e n t l y , these q u e s t i o n s do not have answers. Future s t u d i e s measuring s o m a t o s t a t i n r e l e a s e i n the behaving animal may p r o v i d e some answers. 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Y., (1983) Comparison of the e f f e c t s of c h r o n i c h a l o p e r i d o l treatment on A9 and A10 dopamine neurons i n the r a t . L i f e S c i . , 32: 983-993. Winer, B. J . , (1971) S t a t i s t i c a l P r i n c i p l e s i n Experimental Design, 2nd E d i t i o n . New York: McGraw-Hill. Wood, S. M., K r a e n z l i n , M. E., A d r i a n , T. E. and Bloom, S. R., (1985) Treatment of p a t i e n t s w i t h p a n c r e a t i c endocrine tumours u s i n g a new l o n g - a c t i n g s o m a t o s t a t i n analogue symptomatic and pep t i d e response. Gut, 26: 438-444. Zamir, N., S k o f i t s c h , G., Bannon, M. J . , Helke, C. J . Kopin, I. J . and Jacobowitz, D. M., (1984) Primate model of Parkinson's d i s e a s e : a l t e r a t i o n s i n m u l t i p l e o p i o i d systems i n the b a s a l g a n g l i a . B r a i n Res., 322: 356- 360. Zingg,H. H. and P a t e l , Y. C , (1983) P r o c e s s i n g of so m a t o s t a t i n -28 t o somatostatin-14 by r a t hypothalamic synaptosomal membranes. L i f e S c i . , 33: 1241-1247. 88 APPENDIX 1. Somatostatin radioimmunoassay The standard curve developed to measure the l e v e l s of s o m o s t o s t a t i n immunoreactivity i n t i s s u e was d i s c u s s e d i n the methods s e c t i o n of experiment 1 (p.14). For a more complete d e s c r i p t i o n of t h i s procedure an i l l u s t r a t i o n of the assay and of a t y p i c a l standard cureve i s shown below. P r o t o c o l f o r s o m a t o s t a t i n RIA B u f f e r AB T r a c e r Standard CPM %Bound SOM-IR (u.1) ( u l ) ( u l ) (lOOul) (mean) ( p g / m g ) 200 300 100 100 100 100 0.987pg 1.975pg 3.950pg 7.825pg 15.62pg 31.25pg 62.50pg 125.Opg 250.Opg 500.Opg 1215 2500 1277 1415 1443 1532 1698 1953 2266 2416 2446 2442 51.4 48, 43, 42, 38, 32, 21, 9, 3, 2, 2, Samples #101(0.82mg) 2207 11.7 65.0 #201(0.66mg) 2012 19.5 54.0 89 Q Z ZD O m 7.8 SOMATOSTATIN (pg) 500 F i g u r e 13. An example of a standard curve used t o measure somatostatin immunoreactivity i n t i s s u e s . The X-axis represents i n c r e a s i n g l e v e l s of s y n t h e t i c somatostatin-14 used. The Y-axis i s the percent of s y n t h e t i c somatostatin-14 bound to the antibody which can be c a l c u l a t e d u s i n g the formula: %B = t o t a l counts standard or sample c o u n t s x iQO. 

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