Iodobenzamide for in vivo exploration of central dopamine receptros: Evaluation in animal models of supersensitivity

Life Sciences, Vol. 47, pp. 729-734 Printed in the U.S.A.

Pergamon Pres

IODOBENZAMIDE FOR IN VIVO EXPLORATION OF CENTRAL DOPAMINE RECEPTORS : EVALUATION IN ANIMAL MODELS OF SUPERSENSITIVITY

S. Chalon, C. Guimbal, D. Guilloteau, W. Mayo*, F. Huguet, M.-H. Schmitt, G. Desplanches**, J.-L. Baulieu and J.-C. B e s n a r d INSERM U316, L a b o r a t o i r e de B i o p h y s i q u e M6dicale, 2 bis Bd Tonnell6, 37032 Tours Cedex, FRANCE * : INSERM U259, rue Camille Saint-Sa~ns, 33077 B o r d e a u x cedex, FRANCE ** : Compagnie ORIS Industrie, BP 21, 91190 Gif s u r Yvette, FRANCE (Received in final form June 22, 1990)

Summary

l o d o b e n z a m i d e is a promising agent to i n v e s t i g a t e D 2 r e c e p t o r s by SPECT in living h u m a n brain. In this work, we h a v e e v a l u a t e d this radiolabeled compound in two animal models of D 2 r e c e p t o r s s u p e r s e n s i t i v i t y . In t h e first model, rats w e r e t r e a t e d chronically w i t h haloperidol during t h r e e weeks (S.C. injection of 0.5 m g / k g / d a y ) . One w e e k a f t e r t h e last d a y of t r e a t m e n t , t h e y w e r e I.V. injected w i t h 125I-IBZM. I_n_n vivo specific binding s t u d y s h o w e d a 45 p e r c e n t i n c r e a s e of 125I-IBZM fixation in the s t r i a t u m of t r e a t e d rats. In a second s t e p of experiments, animals w e r e u n i l a t e r a l l y lesioned by a s t e r e o t a x i c injection of 6-OHDA in t h e s u b s t a n t i a nigra, 23 d a y s before receiving 125I-IBZM. A u t o r a d i o g r a p h i c analysis of coronal brain sections s h o w e d a 38 p e r c e n t e n h a n c e m e n t of 125I-IBZM in vivo binding in the s t r i a t u m on the lesioned side as c o m p a r e d to t h e c o n t r o l a t e r a l i n t a c t side ; this increase o c c u r e d in s t r i a t a l lateral area. T h e s e d a t a d e m o n s t r a t e t h a t 125I-IBZM is c o n v e n i e n t to d e t e c t a l t e r a t i o n s of d o p a m i n e D 2 r e c e p t o r s in vivo in t h e rat. Thus IBZM labelled w i t h 12aI can be a v e r y useful imaging agent for t h e e x p l o r a t i o n of D 2 r e c e p t o r s in pathological situations. A l t e r a t i o n s of central d o p a m i n e r e c e p t o r s are involved in several neurologic and p s y c h i a t r i c d i s e a s e s such as P a r k i n s o n ' s disease and schizophrenia. E x p l o r a t i o n of t h e s e n e u r o r e c e p t o r s in t h e living h u m a n brain is of great i n t e r e s t for research, diagnostic p u r p o s e and t r e a t m e n t e v a l u a t i o n in t h e s e diseases. Specific radiolabeled c o m p o u n d s h a v e been r e p o r t e d for t h e imaging of D 2 r e c e p t o r s by p o s i t r o n emission t o m o g r a p h y (PET) : l l C - N - m e t h y l s p i p e r o n e (IIC-NMS) (1), l l C - r a c l o p r i d e (2), 18F-fluoroalkylspiperone (3), 7 ° B r - b r o m o s p i p e r o n e (4). In a p r i m a t e model of P a r k i n s o n ' s d i s e a s e induced by 1methyl-4-phenyl-1, 2, 3, 6 - t e t r a h y d r o p y r i d i n e (MPTP), a specific toxin of d o p a m i n e neurons (5), PET investigations w i t h llC-raclopride showed a post-synaptic s u p e r s e n s i t i v i t y of d o p a m i n e D 2 r e c e p t o r s (6). Nevertheless, PET e x p l o r a t i o n s in h u m a n p a r k i n s o n i a n subjects are h e t e r o g e n o u s . A r e c e n t s t u d y w i t h IlC-NMS s h o w e d no d i f f e r e n c e in t h e a v e r a g e of r e c e p t o r s d e n s i t y b e t w e e n p a r k i n s o n i a n and h e a l t h y control sujects (7). However, patients represented different stages of t h e disease ( h e m i p a r k i n s o n i s m to a d v a n c e d bilateral p a r k i n s o n i s m ) and t h e a u t h o r s p r o p o s e d t h a t

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a n h y p e r s e n s i t i v i t y m a y o c c u r e d o n l y in e a r l y d i s e a s e . To b e t t e r u n d e r s t a n d t h e p h y s i o p a t h o l o g i c m e c h a n i s m s a n d t h e e v o l u t i o n of s u c h a p a t h o l o g y it is n e c e s s a r y to i n c r e a s e t h e n u m b e r of i n v e s t i g a t i o n s a t d i f f e r e n t s t a g e s o f t h e d i s e a s e ; for t h i s p u r p o s e , single p h o t o n e m i s s i o n c o m p u t e d t o m o g r a p h y (SPECT) m a y b e s u i t a b l e for a w i d e s p r e a d clinical a p p l i c a t i o n . Thus, d o p a m i n e r e c e p t o r s imaging a g e n t s l a b e l e d w i t h single p h o t o n emitting radionuclides were recently developped. A benzamide derivative having a n t i d o p a m i n e r g i c p r o p e r t i e s (8) w a s i o d i n a t e d a n d p r o p o s e d f o r t h i s p u r p o s e (9, 10). The r e g i o n a l d i s t r i b u t i o n of 125I-IBZM, ( S ) - N - [ ( 1 - e t h y l - 2 - p y r r o l i d i n y l ) m e t h y l ] - 2 - h y d r o x y - 3 i o d o - 6 - m e t h o x y - b e n z a m i d e , in r a t b r a i n s h o w e d a l o c a l i z a t i o n a s s o c i a t e d w i t h D 2 r e c e p t o r s (9, 10). T h e n 123I-IBZM a l l o w e d to visualize t h e s e n e u r o r e e p t o r s in a m o n k e y b y SPECT (11). This s t u d y i n d i c a t e d t h a t t h i s r a d i o l a b e l e d c o m p o u n d w a s a p r o m i s i n g a g e n t to i n v e s t i g a t e D 2 r e c e p t o r s in living h u m a n b r a i n . T h e a i m of o u r p a p e r is to e v a l u a t e IBZM as a n in vivo m a r k e r of a l t e r e d c e n t r a l d o p a m i n e D 2 r e c e p t o r s . F o r this, w e t e s t e d 125I-IBZM in t w o a n i m a l m o d e l s in w h i c h a D 2 supersensitivity was induced : the first one by a chronic neuroleptic treatment, the s e c o n d o n e b y u n i l a t e r a l lesions of t h e n i g r o s t r i a t a l d o p a m i n e r g i c p a t h w a y w i t h 6h y d r o x y d o p a m i n e (6-OHDA).

Materials and methods P r e p a r a t i o n of 125I-IBZM : 125I-IBZM w a s p r e p a r e d b y a c h l o r a m i n e T m e t h o d a c c o r d i n g to K u n g et al. (10) w i t h m i n o r m o d i f i c a t i o n s . To 25 pl of a n e t h a n o l i c s o l u t i o n o f BZM, t h e p r e c u r s o r c o m p o u n d : ( S ) - N - [ ( 1 - e t h y l - 2 - p y r r o l i d i n y l ) m e t h y l ] - 2 - h y d r o x y - 6 - m e t h o x y b e n z a m i d e , (1 m g / m l ) w a s a d d e d 37 MBq of 1251Na (in 10 pl NaOH 0.1 N ; A m e r s h a m ) , t h e n 10 pl of a c h l o r a m i n e T s o l u t i o n (1 m g / m l ) a n d 5 pl of 0.2 N HC1. A f t e r 1 m i n a t 20°C in a sealed vial, 10 pl of s o d i u m d i s u l f i t e 0.1 N w a s a d d e d , followed b y 20 pl of a s o l u t i o n of NH4OH 1 N - NHaC1 1 N (1:1) a n d a CO 2 s t r e a m for n e u t r a l i z a t i o n . T h e p r o d u c t w a s e x t r a c t e d w i t h e t h y l a c e t a t e a n d d r i e d o n a Na2SO 4 column. E t h y l a c e t a t e w a s e v a p o r a t e d u n d e r N 2 atmospher. P u r i f i c a t i o n b y HPLC ( L i c h r o s o r b Si 60-5) e l u t e d w i t h a m i x t u r e of e t h y l a c e t a t e e t h a n o l - a m m o n i u m h y d r o x y d e (100:10:1) gave a p u r e p r o d u c t w i t h a specific a c t i v i t y of a b o u t 74 T B q / m m o l .

Animal models : - Chronic haloperidol treatment Male W i s t a r r a t s w e i g h i n g 200-250 g w e r e u s e d ; t h e y w e r e fed a c o m m e r c i a l diet a n d w a t e r ad libitum. A g r o u p of 5 a n i m a l s r e c e i v e d a daily s u b - c u t a n e o u s i n j e c t i o n of h a l o p e r i d o l (Haldol qg, J a n s s e n ) : 0.5 m g / k g b o d y w e i g h t d u r i n g 3 w e e k s . Five c o n t r o l r a t s w e r e t r e a t e d w i t h a s a l i n e s o l u t i o n in t h e s a m e c o n d i t i o n s . One w e e k a f t e r t h e l a s t d a y of t r e a t m e n t , all t h e a n i m a l s r e c e i v e d a n i n t r a - v e n o u s i n j e c t i o n of 125I-IBZM (0.37 MBq in 0.3 ml of 0.9 % NaCl). R a t s w e r e sacrificed b y d e c a p i t a t i o n 2 h o u r s a f t e r i n j e c t i o n as t h e r a t i o of I25I-IBZM c o n c e n t r a t i o n in s t r i a t u m / c e r e b e l l u m r e a c h e d a m a x i m u m v a l u e a t t h i s t i m e (10). D i f f e r e n t b r a i n a r e a (cerebellum, s t r i a t u m a n d f r o n t a l c o r t e x ) w e r e r e m o v e d a c c o r d i n g t o t h e m e t h o d of G l o w i n s k i a n d I v e r s e n (12), weighed, a n d t h e i r r a d i o a c t i v i t y w a s c o u n t e d u s i n g a g a m m a c o u n t e r (LKB 1260).

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- 6-OHDA l e s i o n s Unilateral lesions of nigrostriatal dopaminergic neurons were carried out on 8 rats b y s t e r e o t a x i c i n j e c t i o n of 6-OHDA (6 lJg/2 pl 0.9 % NaCI-0.2 % a s e o r b i c acid) i n t o t h e r i g h t S u b s t a n t i a Nigra a t t h e following c o o r d i n a t e s : AP = -4.8 ; L = -1.7 ; P = -8.4 ( a t l a s of P a x i n o s a n d W a t s o n ; 13). T w e n t y t w o d a y s a f t e r lesions, a n i m a l s r e c e i v e d a n i n t r a - v e n o u s i n j e c t i o n of 125IIBZM (2.96 MBq in 0.3 ml o f 0.9 % NaCl) a n d w e r e s a c r i f i c e d b y d e c a p i t a t i o n 2 h o u r s l a t e r . B r a i n s w e r e r e m o v e d a n d i m m e d i a t e l y frozen. C o n s e c u t i v e 20 IJm c o r o n a l s e c t i o n s w e r e c u t o n a c r y o s t a t m i c r o t o m e (IEC CFT), l a y e d d o w n o n m i c r o s c o p e slides, a n d a i r d r i e d a t r o o m t e m p e r a t u r e . Slides w e r e a p p o s e d to a n X - r a y film ( K o d a k SB film) w h i c h w a s d e v e l o p p e d 45 d a y s l a t e r ( r e v e l a t o r K o d a k LX24). A u t o r a d i o g r a p h i e s w e r e a n a l y s e d u s i n g a d e n s i t o m e t r i c image a n a l y z e r (BIOCOM RAG 500). D e n s i t o m e t r i c a n a l y s i s w e r e determined after selecting striatum and computing the average value within the defined area.

Results Regional b r a i n u p t a k e o f 125I-IBZM o n e w e e k a f t e r a c h r o n i c h a l o p e r i d o l t r e a t m e n t s h o w e d a 45 % i n c r e a s e in t h e r a t i o s t r i a t u m / c e r e b e l l u m as c o m p a r e d to c o n t r o l r a t s ( T a b l e 1). By c o n t r a s t , t h e r a t i o f r o n t a l c o r t e x / c e r e b e l l u m w a s u n c h a n g e d . TABLE I Regional B r a i n U p t a k e of 125I-IBZM a f t e r a C h r o n i c H a l o p e r i d o l T r e a t m e n t Str/Cer

Cx/Cer

Controls

7.55 _+ 1.48

1.67 _+ 0.14

Treated

10.97 + 0.35 (*)

1.79 _+ 0.29

S t r = S t r i a t u m ; Cx = F r o n t a l c o r t e x ; Cer = C e r e b e l l u m T r e a t e d a n i m a l s r e c e i v e d a daily S.C. i n j e c t i o n of h a l o p e r i d o l : 0.5 m g / k g b o d y w e i g h t d u r i n g 3 weeks. C o n t r o l r a t s w e r e t r e a t e d w i t h a s a l i n e s o l u t i o n in t h e s a m e conditions. One week after the last day of treatment, animals received an I.V. i n j e c t i o n of 125I-IBZM (0.37 MBq in 0.3 ml o f 0.9 % NaCl) a n d w e r e s a c r i f i c e d 2 h l a t e r . Cerebellum, s t r i a t u m a n d f r o n t a l c o r t e x w e r e r e m o v e d , weighed, a n d t h e i r r a d i o a c t i v i t y w a s c o u n t e d . R e s u l t s w e r e e x p r e s s e d as t h e r a t i o ( c p m / g t i s s u e ) / ( c p m / g c e r e b e l l u m ) _+ s t a n d a r d d e v i a t i o n a n d a n a l y z e d b y t t e s t f o r u n p a i r e d v a l u e s (*) : p ( 0.005 ; n = 5 for e a c h value.

T h e d e n s i t o m e t r i c a n a l y s i s o f c o r o n a l b r a i n s e c t i o n s 23 d a y s a f t e r u n i l a t e r a l 6OHDA i n d u c e d l e s i o n s s h o w e d a m e a n 38 % i n c r e a s e in 125I-IBZM in vivo b i n d i n g in s t r i a t u m ( T a b l e 2). A u t o r a d i o g r a p h i c s t u d i e s (FIG. 1) i n d i c a t e d t h a t t h i s i n c r e a s e w a s m a i n l y localized in d o r s o l a t e r a l a n d v e n t r o l a t e r a l regions of t h e s t r i a t u m .

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TABLE II D e n s i t o m e t r i c A n a l y s i s o f C o r o n a l B r a i n S e c t i o n s a f t e r 6-OHDA U n i l a t e r a l Lesions

Striatum

C o n t r o l side

L e s i o n e d side

% Increase

38.17 _+ 2.89

52.55(*) _+ 3.11

38.00 _+ 7.68

A n i m a l s w e r e u n i l a t e r a l y i n j e c t e d w i t h 6-OHDA i n t o t h e r i g h t s u b s t a n t i a n i g r a a n d r e c e i v e d a n I.V. i n j e c t i o n of 125I-IBZM 23 d a y s later. D e n s i t o m e t r i c a n a l y s i s w e r e p e r f o r m e d o n s t r i a t a l a r e a s of 20 p m c o r o n a l b r a i n s e c t i o n s . R e s u l t s a r e e x p r e s s e d in a r b i t r a r y u n i t as t h e m e a n i n t e n s i t y in t h e l e s i o n e d side as c o m p a r e d to t h e c o n t r o l side _+ s t a n d a r d d e v i a t i o n a n d a n a l y z e d b y p a i r e d t - t e s t ; ( * ) : p < 0.005. n = 8 for e a c h value.

FIG. 1 A u t o r a d i o g r a p h y of a c o r o n a l b r a i n s e c t i o n p e r f o r m e d 2 h a f t e r 125I-IBZM injection. N o t e t h e h i g h t u p t a k e in s t r i a t a l area, a n d t h e i n c r e a s e in 125IIBZM b i n d i n g o n t h e l a t e r a l s t r i a t a l regions o f t h e l e s i o n e d side (R) as c o m p a r e d to t h e c o n t r o l a t e r a l i n t a c t side (L).

Discussion B i o c h e m i c a l a n d physiological m e c h a n i s m s in t h e living h u m a n b r a i n a r e u n t i l r e c e n t l y e x p l o r e d b y PET. O n e a i m o f t h i s t e c h n i q u e is to i m a g e n e u r o r e c e p t o r s ' s d i s t r i b u t i o n a n d to q u a n t i f y t h e m (1, 2, 3, 4). P o t e n t i a l a p p l i c a t i o n s o f t h e s e i n v e s t i g a t i o n s a r e o f g r e a t i n t e r e s t s i n c e p e r t u r b a t i o n s o f t h e s e r e c e p t o r s a r e i n v o l v e d in d i f f e r e n t n e u r o p s y c h i a t r i c d i s e a s e s . H o w e v e r , for m u l t i p l e r e a s o n s a g r e a t e r n u m b e r of i n s t i t u t i o n s h a v e SPECT as c o m p a r e d to PET i n s t r u m e n t a t i o n . So T h e r e is a n e e d for imaging c o m p o u n d s l a b e l e d w i t h single p h o t o n e m i t t i n g r a d i o n u c l i d e s for e x t e n s i v e clinical e x p l o r a t i o n s w i t h SPECT.

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12aI-IBZM has s h o w n its ability to image in vivo c e n t r a l d o p a m i n e D 2 r e c e p t o r s in t h e p r i m a t e in n o r m a l conditions (11). H o w e v e r it is of m o s t i n t e r e s t to k n o w if r e c e p t o r a l t e r a t i o n s could be d e t e c t e d w i t h this radioligand. Haloperidol is a n e u r o l e p t i c drug which blocks c e n t r a l d o p a m i n e D 2 r e c e p t o r s and which is used for e x a m p l e in the t r e a t m e n t of schizophrenia. Chronic haloperidol t r e a t m e n t in r a t s h a v e a l r e a d y been used and s h o w e d to induce a 25 p e r c e n t increase in 3H-haloperidol binding in v i t r o on s t r i a t a l h o m o g e n a t e s (14). Such an increase was a t t r i b u t e d to an e n h a n c e d n u m b e r of p o s t - s y n a p t i c r e c e p t o r sites while in this s t u d y Kd w a s similar in t r e a t e d ans control animals. By using t h e s a m e e x p e r i m e n t a l animal model, w e o b s e r v e d a 45 p e r c e n t i n c r e a s e in t h e in v i v o binding of 125I-IBZM in the s t r i a t u m ( e x p r e s s e d as t h e ratio s t r i a t u m u p t a k e to cerebellum u p t a k e ) , w h e r e a s no i n c r e a s e occured in t h e frontal cortex. This result m a y be explained by an i n c r e a s e in the n u m b e r of in vivo binding sites for IBZM a n d / o r by an i n c r e a s e in affinity of this ligand for its r e c e p t o r sites. Difference of p e r c e n t increases in t h e t w o w o r k s could be explained by t h e difference b e t w e e n t h e two m e t h o d o l o g i e s a n d / o r by a d i f f e r e n t affinity or s e l e c t i v i t y for D 2 d o p a m i n e r e c e p t o r s for IBZM t h a n for haloperidol. Lesions of t h e nigrostriatal d o p a m i n e p a t h w a y by i n t r a c e r e b r a l injection of 6OHDA elicited a s t r i a t a l D 2 r e c e p t o r s u p e r s e n s i t i v i t y d e m o n s t r a t e d by in v i t r o binding studies w i t h aH-spiperone (15, 16) or by q u a n t i t a t i v e a u t o r a d i o g r a p h i c studies (17, 18) ; h o w e v e r results a r e v a r i a b l e (19, 20, 21) p r o b a b l y in relation w i t h t h e time of m e a s u r e m e n t a f t e r t h e lesion is introduced. In this e x p e r i m e n t a l m o d e l w e observed, 23 d a y s a f t e r 6-OHDA lesions of d o p a m i n e r g i c neurons, a 38 p e r c e n t e n h a n c e m e n t in the in vivo binding of 125I-IBZM in t h e s t r i a t u m on the lesioned side as c o m p a r e d to t h e control side. This result is in a g r e e m e n t with an u p r e g u l a t i o n of d o p a m i n e r g i c r e c e p t o r s induced by 6-OHDA lesions (15, 16, 17, 18). We can a s s u m e t h a t t h e s e u n i l a t e r a l lesions h a v e no effect on unspecific binding of IBZM since d e n s i t o m e t r i c analysis o f a u t o r a d i o g r a m s in t h e c o r t e x a r e a s h o w s identical values in the lesioned side as c o m p a r e d to t h e i n t a c t side (unpublished data). M o r e o v e r a u t o r a d i o g r a p h i c images s h o w e d t h a t IBZM fixation in s t r i a t a l regions s e e m e d localized in the lateral a r e a of t h e s t r i a t u m which agrees with a r e c e n t s t u d y with 3H-spiperone in the s a m e animal e x p e r i m e n t a l model (22). Thus it seems t h a t IBZM used in vivo is a suitable c o m p o u n d for t h e d e t e c t i o n of D 2 r e c e p t o r s s u p e r s e n s i t i v i t y . This d a t a v a l i d a t e s t h e use of 123I-IBZM as an in vivo m a r k e r of D 2 r e c e p t o r sites ; this c o m p o u n d may be a v e r y usefull tool to d e t e c t pathologic p e r t u r b a t i o n s of t h e s e r e c e p t o r s in t h e living h u m a n brain.

Acknowledgements This w o r k was realized as p a r t of the "R6seau INSERM N ° 489001". We t h a n k Dr T. d e Paulis for t h e gift of t h e IBZM precursor, Dr Y. B r o e r (INSERM U55) and M. Tafani (Facult6 de M6decine, Toulouse) for the d e n s i t o m e t r i c analysis o f a u t o r a d i o g r a p h i e s .

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