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Increased muscarinic cholinergic receptors in prefrontal cortices of medicated schizophrenics
Life Sciences, Vol. 33, pp. 2187-2196 Printed in the U.S.A.
INCREASED PREFRONTAL
M U S C A R I N I C C H O L I N E R G I C R E C E P T O R S IN C O R T I C E S OF M E D I C A T E D S C H I Z O P H R E N I C S
Shuzo Watanabe, Mizuo Takashima Division Mental
Pergamon Press
Toru Nishikawa, and M i c h i o T o r u
of P s y c h o b i o l o g y , N a t i o n a l C e n t e r for Nervous, and M u s c u l a r D i s o r d e r s , 2620 O g a w a - H i g a s h i , K o d a i r a , Tokyo, 187, J a p a n (Received in final form September 12, 1983) Summary
A l t e r a t i o n s in 3 H - q u i n u c l i d i n y l b e n z i l a t e b i n d ing sites a s s o c i a t e d w i t h m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s w e r e i n v e s t i g a t e d in o r b i t o - f r o n t a l and m e d i a l frontal c o r t i c e s from 12 s c h i z o p h r e n i c s , 6 o n - d r u g and 6 o f f - d r u g cases, and from I0 controls. S i g n i f i c a n t l y lower a f f i n i t i e s of the sites w e r e found in b o t h areas of s c h i z o p h r e n i c s than controls. An i n c r e a s e in r e c e p t o r n u m b e r was shown only in the o r b i t o - f r o n t a l c o r t e x from s c h i z o p h r e n i c s . On-drug g r o u p of s c h i z o p h r e n i c s did, however, s h o w a sign i f i c a n t i n c r e a s e in r e c e p t o r n u m b e r and a s i g n i f i cant d e c r e a s e in a f f i n i t y in b o t h areas, w h i l e there w e r e no s i g n i f i c a n t d i f f e r e n c e s in any b i n d i n g p a r a m e t e r s of o f f - d r u g s c h i z o p h r e n i c s from controls. A l s o in the c a u d a t e the s i m i l a r results w e r e o b t a i n e d . It is, thus, c o n c l u d e d that a l t e r a t i o n s in m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s of s c h i z o p h r e n i c p a t i e n t s result from l o n g - t e r m m e d i c a t i o n w i t h a n t i m u s c a r i n i c actions. A n t i p s y c h o t i c drugs have the a n t i c h o l i n e r g i c p r o p e r t y in g r e a t e r or lesser degree w h i c h are t h o u g h t to c o m p e n s a t e for their i n t r i n s i c e x t r a p y r a m i d a l e f f e c t s c a u s e d by the d o p a m i n e r e c e p t o r blockade. It has b e e n shown that their a n t i c h o l i n e r g i c e f f e c t s r e s u l t e d from the d i r e c t action to m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s (i) . In animal e x p e r i m e n t s , the d i r e c t d o p a m i n e r e c e p t o r b l o c k a d e by l o n g - t e r m n e u r o l e p t i c s has been d e m o n s t r a t e d to lead to an i n c r e a s e in the n u m b e r of d o p a m i n e r g i c a n t a g o n i s t b i n d i n g sites, or the r e c e p t o r s u p e r s e n s i t i v i t y (2,3,4). It seems to be r e a s o n a b l e on the a n a l o g y of the d o p a m i n e r e c e p t o r s that the l o n g - t e r m 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 w i t h the a n t i c h o l i n e r g i c action also r e s u l t s in the s u p e r s e n s i t i v i t y of the m u s c a r i n i c receptors. P r e v i o u s p a p e r s c o n c e r n i n g the m u s c a r i n i c r e c e p t o r s of s c h i z o p h r e n i c p o s t m o r t e m b r a i n s r e p o r t e d no a l t e r a t i o n s in s p e c i f i c 3 H - q u i n u c l i d i n y l b e n z i l a t e (QNB) b i n d i n g to the c a u d a t e and the p u t a m e n (5) or the t e n d e n c y for r e d u c e d s p e c i f i c b i n d i n g to the frontal c o r t e x from p a t i e n t s w i t h s c h i z o p h r e n i a (6). In the p r e s e n t study, S c a t c h a r d a n a l y s i s of 3H-QNB b i n d i n g a s s o c i a t e d w i t h m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s in two s u b a r e a s of prefrontal c o r t e x w h i c h r e c e i v e d i s c r e t e p r o j e c t i o n s from the parts 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
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of m e d i o d o r s a l n u c l e u s in the thalamus r e v e a l e d no a l t e r a t i o n s in the o f f - d r u g group of s c h i z o p h r e n i c s , w h i l e m e d i c a t e d schizop h r e n i c s showed h i g h e r a p p a r e n t K D and B m a x than o f f - d r u g schizop h r e n i c s or controls. A l s o in the caudate the same d i r e c t i o n of the r e c e p t o r a l t e r a t i o n s s e e m i n g l y induced by the drugs was displayed. Materials
and M e t h o d s
P o s t m o r t e m b r a i n s w e r e o b t a i n e d from 12 i n d i v i d u a l s w i t h s c h i z o p h r e n i a h a v i n g an average age of 60.4 years (41-75) and I0 control i n d i v i d u a l s w i t h no r e c o r d e d h i s t o r y of n e u r o l o g i c or p s y c h i a t r i c illness h a v i n g an average age of 66.7 years (52-74). The d i a g n o s i s of s c h i z o p h r e n i a in these p a t i e n t s were a s c e r t a i n e d by their c a t a m n e s e s and c a t e g o r i z e d a c c o r d i n g to DSM-I[[. Briefly s u m m a r i z e d b a c k g r o u n d s of s c h i z o p h r e n i c s and controls are as follows. S c h i z o p h r e n i c - l : Age at death; 50, Sex; M, Type c l a s s i f i e d a c c o r d i n g to DSM-]II categories; d i s o r g a n i z e d type, in remission, Cause of death; m y o c a r d i a l infarction, P s y c h o t r o p i c m e d i c a t i o n b e f o r e death; c h l o r d i a z e p o x i d e I0 mg for 7.5 months b e f o r e death (off-drug case). S-2: 62, F, u n d i f f e r e n t i a t e d type, s u b c h r o n i c w i t h acute e x a c e r b a t i o n , m y o c a r d i a l infarction, c h l o r d i a z e p o x i d e i0 mg and p h e n o b a r b i t a l 60 mg only for 5 days before death (offdrug case). S-3: 41, M, c a t a t o n i c type, c h r o n i c w i t h acute e x a c e r b a t i o n , pneumonia, a n t i p s y c h o t i c m e d i c a t i o n free for 3 m o n t h s prior to death e x c e p t n i t r a z e p a m i0 mg, g l u t e t h i m i d e 500 mg and a m i t r i p t y l i n e i0 mg at b e d t i m e till 3 days b e f o r e death (offdrug case). S-5: 46, F, p a r a n o i d type, chronic, sudden death, s p i p e r o n e 6 mg, p i p a m p e r o n e 200 mg, t h i o p r o p e r a z i n e 30 mg, t r i h e x y p h e n i d y l 8 mg and h y d r o x y z i n e 200 mg till the m o r n i n g of the day of death (on-drug case). S-6: 64, F, d i s o r g a n i z e d type, chronic, cardiac thrombosis, h a l o p e r i d o l 4.5 mg, a m i t r i p t y l i n e 30 mg and p r o m e t h a z i n e 75 mg till 2 days b e f o r e death (on-drug case). T h e s e are the cases p r e v i o u s l y reported in regard to the d o p a m i n e m e t a b o l i s m in basal g a n g l i a of s c h i z o p h r e n i c p a t i e n t s (7). S-7: 54, M, u n d i f f e r e n t i a t e d type, chronic, p a n p e r i t o n i t i s , chlorp r o m a z i n e 50 mg, p i p a m p e r o n e 75 mg and m e t o c l o p r a m i d e 30 mg till 18 days b e f o r e death and then c h l o r p r o m a z i n e 50 mg till 5 days b e f o r e death (on-drug case). S-II: 70, M, d i s o r g a n i z e d type, chronic, m y o c a r d i a l infarction, drug free for 7 years (off-drug case). S-12: 69, M, d i s o r g a n i z e d type, chronic, c a r d i a c failure, h a l o p e r i d o l 3 mg and t r i h e x y p h e n i d y l 3 mg till 2 days before death (on-drug case). S-13: 73, M, u n d i f f e r e n t i a t e d type, chronic, stomach cancer, h a l o p e r i d o l 3 mg and t r i h e x y p h e n i d y l 3 mg till 5.5 months and then d i a z e p a m 5 mg till 1.5 months b e f o r e death (offdrug case). S-14: 53, F, d i s o r g a n i z e d type, chronic, o v a r i a n cancer, o x y p e r t i n e 90 mg, c l o c a p r a m i n e 200 mg, a m i t r i p t y l i n e 50 mg and m e t h i x e n e i0 mg till 16 days before death (on-drug case). S-15: 68, M, u n d i f f e r e n t i a t e d type, chronic, suffocation, h a l o p e r i d o l 3 mg, l e v o m e p r o m a z i n e 25 mg, p r o m e t h a z i n e 25 mg and n i t r a z e p a m i0 mg just b e f o r e death (on-drug case). S-16: 75, F, d i s o r g a n i z e d type, chronic, p a n c r e a t i c cancer, h a l o p e r i d o l 1.5 mg till 40 days and c l o t i a z e p a m 20 mg till 33 days before death (offdrug case). C o n t r o l s consist of 7 males and 3 females w h o s e cause of death is sudden death, cancer in the lung or the d i g e s t i v e tract, ileus or d i s s e m i n a t e d i n t r a v a s c u l a r coagulation. The m i s s i n g numbers of p a t i e n t s listed above and in figures in "Results"
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i n d i c a t e that their b r a i n c o r t i c e s w e r e u n s u i t a b l e for r e c e p t o r a n a l y s i s by b i n d i n g t e c h n i q u e o w i n g to the d e h y d r a t i o n y i e l d e d d u r i n g frozen s t o r a g e or that the tissues p r e p a r e d w e r e e x h a u s t e d in o t h e r b i o c h e m i c a l analyses. The b r a i n s taken at a u t o p s y in the h o s p i t a l s w h e r e those p a t i e n t s h a d d i e d w e r e put into -20°C or -40°C f r e e z e r until t r a n s f o r m a t i o n to our l a b o r a t o r y (0.5 h - 4 m o n t h s , a v e r a g e 31 days). The i n t e r v a l s b e t w e e n d e a t h and f r e e z i n g of the b r a i n are 2 - 13 h (average 5.2 h) for c o n t r o l s and 3 - 22 h (average 10.2 h) for s c h i z o p h r e n i c s . T h e y w e r e s t o r e d in a i r t i g h t p a c k a g e s in our l a b o r a t o r y at -80°C until d i s s e c t i o n (20 days - 32 m o n t h s , a v e r a g e 15.6 months). On the b a s i s of the p r o j e c t i o n from d i s c r e t e areas of m e d i o d o r s a l n u c l e u s of the t h a l a m u s (8,9) and the d i f f e r e n c e s in the c o r t i c a l c y t o a r c h i t e c t u r e and in the p u r p o r t e d f u n c t i o n (10), the p r e f r o n t a l c o r t e x w a s d e v i d e d into 4 areas; m e d i a l frontal cortex, o r b i t o - frontal cortex, o r b i t a l c o r t e x and frontal eye field c o r r e s p o n d i n g to B r o d m a n n areas 9.10-46, 45-47, 11-12 and 8, respectively. F r o n t a l parts of the b r a i n s w e r e p l a c e d at -15°C for a b o u t 12 h p r i o r to d i s s e c t i o n to a l l o w serial c o r o n a l c u t t i n g in 1.5 m m t h i c k n e s s w i t h criotome. F r o m e a c h slice, the g r a y m a t t e r of d i s c r e t e p r e f r o n t a l areas w a s d i s s e c t e d out in a c o l d b o x k e p t at -15°C a c c o r d i n g to their l a n d m a r k s p u t b e f o r e dissection. The d i s s e c t i o n of the c a u d a t e was d e s c r i b e d p r e v i o u s ly (7). D i s s e c t e d t i s s u e s w e r e h o m o g e n i z e d in 3 vol. of i c e - c o l d 0.32 M s u c r o s e w i t h a P o l y t r o n . A l i q u o t s of 1.5 ml of the h o m o g e n a t e w e r e d i s p e n s e d into small p l a s t i c tubes and s t o r e d at -80°C until b i o c h e m i c a l and p h a r m a c o l o g i c a l analyses. " O f f - d r u g " g r o u p of s c h i z o p h r e n i c s (6 out of 12 s c h i z o p h r e n ics) had b e e n a d m i n i s t e r e d no a n t i p s y c h o t i c s for at least 40 days b e f o r e d e a t h (5 out of 6 cases h a d r e c e i v e d no a n t i p s y c h o t i c s for m o r e than 3 m o n t h s ) . The a n a l y s i s of 3H-QNB b i n d i n g in all cases w a s c a r r i e d out u s i n g m a i n two out of four s u b a r e a s d e s c r i b e d above. T h e y are the m e d i a l frontal c o r t e x r e c e i v i n g the p r o j e c t i o n from pars p a r v o c e l l u l a r i s of the m e d i o d o r s a l n u c l e u s of t h a l a m u s and the o r b i t o frontal c o r t e x p r o j e c t e d from pars m a g n o c e l l u l a r i s of the nucleus. 3H-QNB b i n d i n g to the m e m b r a n e p r e p a r a t i o n s from two d i s c r e t e p r e f r o n t a l c o r t i c e s and the c a u d a t e w a s p e r f o r m e d in t r i p l i c a t e by a m o d i f i c a t i o n of the r a p i d f i l t r a t i o n m e t h o d of Y a m a m u r a and S n y d e r (ii). The t h a w e d s u c r o s e h o m o g e n a t e d i l u t e d three times w i t h 50 m M T r i s . H C l b u f f e r , pH 7.4, c o n t a i n i n g i0 m M M g C l 2 was c e n t r i f u g e d for 10 m i n at 1,000 xg and the r e s u l t i n g s u p e r n a t a n t w a s c e n t r i f u g e d at 45,000 xg for 20 min. The r e s u l t i n g p e l l e t was s u s p e n d e d in the T r i s . H C l b u f f e r to get the p r o t e i n c o n c e n t r a t i o n of 0.2 - 0.4 mg per ml. To a s s a y the s p e c i f i c b i n d i n g of 3H-QNB to the t i s s u e p r e p a r a t i o n , 0.2 ml of this p r e p a r a t i o n w e r e incub a t e d in 12 x 75 m m glass tube at 21°C for 180 m i n w i t h 0.2 ml of 3 x 10 -6 M a t r o p i n e d i l u t e d w i t h the b u f f e r from 3 x 10 -3 M a l c o h o l i c s o l u t i o n and 0.2 ml of v a r y i n g c o n c e n t r a t i o n s (0.06 1.2 nM) of 3H-QNB (33.1 C i / m m o l , NEN) d i l u t e d w i t h buffer. The b i n d i n g r e a c t i o n w a s t e r m i n a t e d b y f i l t e r i n g 0.5 ml a l i q u o t of an a s s a y m i x t u r e of 0.6 ml t h r o u g h a G F / B glass fiber f i l t e r (Whatman) u n d e r v a c u u m and e a c h filter w a s r i n s e d twice w i t h i0 ml of ice-cold buffer. The filters w e r e c o u n t e d for 3H in a P a c k a r d s c i n t i l l a t i o n s p e c t r o m e t e r w i t h 10 ml of A q u a s o l (NEN). P r o t e i n w a s d e t e r m i n e d b y the m e t h o d of L o w r y et al. w i t h b o v i n e s e r u m a l b u m i n as s t a n d a r d (12). B i n d i n g p a r a m e t e r s w e r e c a l c u l a t e d by least s q u a r e s r e g r e s -
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sion analysis of S c a t c h a r d plots. The added 3H-QNB minus the total b i n d i n g was taken as the free c o n c e n t r a t i o n of the ligand. The p a r a m e t e r values o b t a i n e d for controls, o n - d r u g and o f f - d r u g s c h i z o p h r e n i c s were c o m p a r e d by two t a i l e d M a n n - W h i t n e y U test. Results There w e r e no s i g n i f i c a n t d i f f e r e n c e s in the age d i s t r i b u t i o n b e t w e e n controls, o n - d r u g and o f f - d r u g s c h i z o p h r e n i c s (MannW h i t n e y U test), as the a g e - d e p e n d e n t loss in the n u m b e r of m u s c a r i n i c c h o l i n e r g i c receptors in human and animal brains has been r e p o r t e d (13, 14, 15). The s i g n i f i c a n t c o r r e l a t i o n s b e t w e e n the interval from death to freezing of the b r a i n or the d u r a t i o n of frozen storage and the b i n d i n g p a r a m e t e r s did not exist in any groups (Spearman Rank C o r r e l a t i o n test). 3H-QNB b i n d i n g to the m e m b r a n e p r e p a r a t i o n of the human prefrontal cortex and the caudate using atropine as b a s e l i n e could be r e g a r d e d as m o n o p h a s i c w i t h a Hill c o e f f i c i e n t of 1.0, as e x a m i n e d in p r e l i m i n a r y s a t u r a t i o n isotherms using 10 - 12 ligand concentrations. Thus, 5 - 6 c o n c e n t r a t i o n s of 3H-QNB were e m p l o y e d in the s t a n d a r d assay. The Hill c o e f f i c i e n t s of o r b i t o - f r o n t a l cortices for 3H-QNB were a p p r o x i m a t e l y 1.0; 1.02 • 0.04 for controls and 0.97 ± 0.08 for s c h i z o p h r e n i c s as a whole, and
J05 -
[
CONTROLS (S)
~CHIZOPHRLNICS ( ± 2 )
KD: 3.015±0.301~C~'~ BMAX: 789.3'27.8~ ,: 0.592*0.003
KD: 0.073t0.037~" EMAX:1023.7~C3.2~ :: 0.990±0.003 OFF-DR.G (6)
\ \
0N-DRuG (6)
!:D:0.02Gt0.003"I~ BMAX: 844.8,43.3~ ~ 0.994±0.002 [
%: 0.126t0.070t'~ EMAX: 1202.6,53.7::#:~ ,: 0.q8710.004
5x104" m
"
11
c
500
1000
500
i000
500
10GU
B ( FMOL/MG PROT, )
Fig. Scatchard
1
a n a l y s i s of 3H-QNB b i n d i n g o r b i t o - f r o n t a l cortex
to
3H-QNB (33.1 Ci/mmol) r a n g e d from 0.02 to 0.4 nM. S p e c i f i c b i n d i n g was that i n h i b i t e d by 10 -6 M atropine. The tissue used in the assay was 0.079 ± 0.003 mg p r o t e i n per tube. V a l u e s of b i n d i n g p a r a m e t e r s are m e a n ± S.E.M. K D (nM) and B m a x (fmol/mg protein) values w e r e d e t e r m i n e d by linear r e g r e s s i o n analysis of S c a t c h a r d p l o t data c o n s i s t i n g of six points in this area. A c o r r e l a t i o n c o e f f i c i e n t of the plots is i n d i c a t e d by r.
.,.,.:
p<0.02, liE: p<0.002, ~ , ~ : p<0.001
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f u r t h e r m o r e , no d i f f e r e n c e b e t w e e n the o n - d r u g g r o u p (0.96 ± 0.05) and the o f f - d r u g g r o u p of s c h i z o p h r e n i c s (0.98 ± 0.11) was found. The c o e f f i c i e n t s in m e d i a l frontal c o r t e x w e r e e s s e n t i a l l y the same as in o r b i t o - f r o n t a l cortex. As shown in Fig. i, the a p p a r e n t K D of 3H-QNB b i n d i n g to the o r b i t o - f r o n t a l c o r t e x p r e p a r a t i o n in o n - d r u g s c h i z o p h r e n i c s was s i g n i f i c a n t l y h i g h e r than c o n t r o l s (p<0.001) or o f f - d r u g s c h i z o p h r e n i c s (p<0.02). T h e r e was no d i f f e r e n c e in the a p p a r e n t K D b e t w e e n c o n t r o l s and o f f - d r u g s c h i z o p h r e n i c s . Similarly, a s i g n i f i c a n t l y h i g h e r a p p a r e n t K D was found in the m e d i a l frontal cortex of o n - d r u g s c h i z o p h r e n i c s than o f f - d r u g g r o u p or c o n t r o l s as shown in Fig. 2 (p<0.002) . In b o t h cortices, the m e a n K D for all s c h i z o p h r e n i c s was also h i g h e r than that for c o n t r o l s (p<0.02 in the o r b i t o - f r o n t a l c o r t e x and p < 0 . 0 5 in the m e d i a l frontal cortex), o w i n g to h i g h e r K D for o n - d r u ~ g r o u p of s c h i z o p h r e n i c s . The a l t e r a t i o n in the d e n s i t y of ~ H - Q N B b i d d i n g to the m e m b r a n e p r e p a r a t i o n from e a c h s u b a r e a was also s i m i l a r to that in KD; a h i g h e r B m a x in o n - d r u g s c h i z o p h r e n i c s than in c o n t r o l s (p<0.002 for b o t h areas) or o f f - d r u g s c h i z o p h r e n i c s (p<0.002 for b o t h areas) w i t h no d i f f e r e n c e b e t w e e n the latter two. In c o n c l u sion, only o n - d r u g cases of all s c h i z o p h r e n i c s s h o w e d h i g h e r K D and Bmax of 3H-QNB b i n d i n g to the p r e f r o n t a l c o r t e x than c o n t r o l s or o f f - d r u g s c h i z o p h r e n i c s . In the a d d i t i o n a l a n a l y s i s of 3H-QNB b i n d i n g to the caudate p r e p a r a t i o n done in o r d e r to check w h e t h e r the drug t r e a t m e n t ~(HITOPIIRF'dI( ~ ~1~?'.
CONTROL.'; (10) KD:
ElF
0.016.C.001~-~
KD: ~.07!*0.32B BMAX: iiR7.5*7:F.g :: 3 . 9 g ' , : 0 . 9 9 3
:i
~k '4
OFF ~RlJ(i (()::
dr,-~':!, F !( )
KD: 0.019,().:):)4 5~AX: 993.1~C3.9
• L:
X,~
590
]Qr]O
9.985"-0. 005
5r0
15OC
10[:3
[~ ( F~IOL/"I~: FPOT.
Fig. Scatchard
.12h%.'~4 , " . ~ '
1
2
a n a l y s i s of 3H-QNB b i n d i n g m e d i a l frontal c o r t e x
to
3H-QNB (33.1 Ci/mmol) r a n g e d from 0.02 to 0.32 nM. The tissue used in the assay was 0.061 ± 0.003 mg p r o t e i n per tube. The assay was done in the same w a y as in o r b i t o - f r o n t a l c o r t e x e x c e p t that Scatchard plot data c o n s i s t of five p o i n t s in this area. A c o r r e l a t i o n c o e f f i c i e n t of the p l o t s is i n d i c a t e d by r. ~: p<0.05, ~ : p<0.01, ~: p<0.005, ~, ~: p<0.002
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e f f e c t s all b r a i n r e g i o n s or is s e l e c t i v e for the p r e f r o n t a l c o r tex, the a l t e r a t i o n of b i n d i n g p a r a m e t e r s w a s in the s a m e d i r e c t i o n as in the p r e f r o n t a l c o r t e x , as m e a s u r e d u s i n g the c a s e s whose caudate membrane preparations were available. The K D values (nM) w e r e 0 . 0 2 2 ~ 0 . 0 0 1 for c o n t r o l s (C-II, -12, -14, -15 a n d -16), 0 . 0 2 9 ± 0 . 0 0 5 for o f f - d r u g p a t i e n t s (S-l, -2, -3, -ii a n d -16) and 0 . 1 5 9 ± 0 . 0 9 5 for o n - d r u g p a t i e n t s (S-5, -7, -14 and -15). The l a s t one w a s s i g n i f i c a n t l y d i f f e r e n t f r o m the f o r m e r t w o (p<0.05) b e t w e e n w h i c h no d i f f e r e n c e w a s found. The B m a x v a l u e s ( f m o l / m g p r o t e i n ) w e r e 1 5 3 0 . 5 ± 99.0 for c o n t r o l s , 1 8 0 2 . 6 ± 1 1 1 . 5 for o f f d r u g p a t i e n t s a n d 2 1 5 4 . 6 ± 81.8 for o n - d r u g p a t i e n t s . The f i r s t one w a s s i g n i f i c a n t l y l o w e r t h a n the l a s t one (p<0.02) w h i l e it s h o w e d no d i f f e r e n c e f r o m the s e c o n d o n e (p>0.1) .
Total
Bindlnq
cpm/~11tcr
I00
~0
80
6O 440
4O
2O
0
i0
9
8
7
-logl01
Druq
Fig. Drug i n h i b i t i o n frontal
cortex
of
6
] ( M
5
4
)
3
3H-QNB b i n d i n g
from a control
to the medial patient (C-l)
0.1 n M of 3H-QNB ( 3 3 . 1 C i / m m o l ) w a s u s e d i n t h i s study. T h e t i s s u e in the a s s a y w a s o.i mg p r o t e i n p e r tube.
Drugs were diluted alcoholic solution.
with
the
buffer
from
3 x 10 - 3 M
As s h o w n in Fig. 3, a n t i m u s c a r i n i c p o t e n c i e s of the d r u g s i n d i c a t e d in the d r u g i n h i b i t i o n of 3 H - Q N B b i n d i n g to the m e d i a l f r o n t a l c o r t e x of a c o n t r o l p a t i e n t (C-l) w e r e e s s e n t i a l l y s i m i l a r to p r e v i o u s r e p o r t s by o t h e r s (i, 16). The a p p a r e n t K D of 3 H - Q N B b i n d i n g to the 1 , 0 0 0 xg s u p e r n a t a n t or to the s u s p e n s i o n s of rep e a t e d l y w a s h e d 4 5 , 0 0 0 xg p e l l e t of the s u p e r n a t a n t r e m a i n e d unchanged in s a t u r a t i o n i s o t h e r m s in w h i c h the o r b i t o - f r o n t a l c o r t e x f r o m a t y p i c a l s c h i z o p h r e n i c p a t i e n t (S-15) a d m i n i s t e r e d l e v o m e p r o m a z i n e , h a l o p e r i d o l and p r o m e t h a z i n e for a long time w a s u s e d (Fig. 4). Discussion The
prefrontal
cortex
develops
most
exceedingly
in h u m a n
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QNB Binding Sites in Schizophrenic Brain
WASHINO KD
15000-
• • • O
0X ix 2X 3X
0.062 0.067 0.071 0.069
2193
BMAX 425.6 916.3 1180.6 1099.5
~ o
10000-
5000"
!
I000 B ( FMOL/MG PROTEIN )
Fig. Effect
4
of w e s h i n ~ of the tissue on JH-QNB b i n d i n g
preparation
The 1,000 xg s u p e r n a t a n t ( • ) of the h o m o g e n a t e of the o r b i t o - f r o n t a l cortex from a s c h i z o p h r e n i c p a t i e n t (S-15) was c e n t r i f u g e d at 45,000 xg for 20 min and the p e l l e t was s u s p e n d e d in the b u f f e r ( • ) . The suspension was c e n t r i f u g e d at 45,000 xg once more and the p e l l e t was s u s p e n d e d ( • ) . It was further c e n t r i f u g e d at 45,000 xg and the r e s u l t i n g p e l l e t was s u s p e n d e d (O). The p r o t e i n in the assay r a n g e d from 0.06 to 0.i mg per tube at r e s p e c t i v e steps of washing. The final c o n c e n t r a t i o n s of 3H-QNB w e r e from 0.02 to 0.4 nM. KD: nM, Bmax: fmol/mg p r o t e i n
among primates. By the injury in this area, h i g h e r functions of h u m a n brain, such as attention, c o g n i t i o n and creation, may be affected, and the r e d u c t i o n of s p o n t a n e i t y and the a l t e r a t i o n of a f f e c t i v i t y m a y o c c u r (7). F r o m such a viewpoint, this area is t h o u g h t to be one of the areas of the b r a i n w o r t h y of the i n v e s t i gation for the b i o c h e m i s t r y of s c h i z o p h r e n i a . Only two reports c o n c e r n i n g m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s in s c h i z o p h r e n i c postm o r t e m b r a i n s can be found as yet; the t e n d e n c y for r e d u c e d levels of s p e c i f i c 3H-QNB b i n d i n g to w h o l e frontal cortex m e m b r a n e p r e p a ration r e p o r t e d by B e n n e t t et al. (6) and no a l t e r a t i o n in s p e c i f ic 3H-QNB b i n d i n g to the m e m b r a n e from s c h i z o p h r e n i c caudate and p u t a m e n a s s e r t e d b y C r o w et al. (5). A n a l y s e s of the frontal
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c o r t e x of s c h i z o p h r e n i c s have been p e r f o r m e d as to neurotransmitter r e c e p t o r s i n v o l v i n g the m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s by B e n n e t t et al. and o p i a t e and n e u r o l e p t i c r e c e p t o r s by R e i s i n e et al. (17). The area used in the study by B e n n e t t et al., w h i c h i n v o l v e d B r o d m a n n areas 6, 8 - ii and 44 - 47, implies the w h o l e frontal c o r t e x a p p r o x i m a t e l y . In the report by R e i s i n e et al. is there no d e t a i l e d d e s c r i p t i o n about the area u s e d as the frontal cortex. The s u b a r e a s of the p r e f r o n t a l c o r t e x are not h o m o g e n e ous, but show a v a r i e t y of d i f f e r e n c e s in their neural c o n n e c t i o n s w i t h o t h e r b r a i n areas, their c y t o a r c h i t e c t u r e s and their p u r p o r t ed f u n c t i o n s (7). However, in r e g a r d to m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s in two m a i n s u b a r e a s e x a m i n e d here, the o r b i t o - f r o n t a l cortex m e r e l y s h o w e d s l i g h t l y lower d e n s i t y than that in the m e d i a l frontal cortex. T h e r e w e r e no s i g n i f i c a n t d i f f e r e n c e s in the a f f i n i t y to 3H-QNB b e t w e e n the areas in control, o n - d r u g and o f f - d r u g s c h i z o p h r e n i c groups. In the studies p r e v i o u s l y r e p o r t e d (5, 6), only one c o n c e n t r a t i o n of 3H-QNB was used for a s s e s s m e n t of the a n t a g o n i s t b i n d ing ability to m u s c a r i n i c r e c e p t o r s in p r e p a r a t i o n s . However, the a n a l y s i s of s a t u r a t i o n i s o t h e r m s will be n e c e s s a r y for the k n o w l edge of the a m o u n t or the d e n s i t y of m u s c a r i n i c r e c e p t o r s in h u m a n b r a i n s unless the a f f i n i t i e s of the r e c e p t o r s in p r e p a r a t i o n s to the ligand are i d e n t i c a l . A f t e r c h r o n i c a t r o p i n e t r e a t m e n t to rats, W e s t l i n d et al. o b t a i n e d an increase in the n u m b e r of m u s c a r i n i c a n t a g o n i s t b i n d i n g sites in h i p p o c a m p u s and the l o w e r e d a f f i n i t y for 3 H - a n t a g o n i s t s in dose d e p e n d e n t m a n n e r (18). Thus, also in the s c h i z o p h r e n i c p a t i e n t s e x a m i n e d in the p r e s e n t study, the l o n g - t e r m 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 and a n t i p a r k i n s o n i a n drugs w i t h a n t i m u s c a r i n i c actions used t o g e t h e r w i t h them will be e x p e c t e d to lead to the l o w e r e d a f f i n i t y of the r e c e p t o r s to 3HQNB. The p r e s e n t study d e m o n s t r a t e d that this s p e c u l a t i o n was the case and the m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s in the p r e f r o n t a l cortex of o f f - d r u g s c h i z o p h r e n i c p a t i e n t s h a d no d i f f e r e n c e s in the b i n d i n g p a r a m e t e r s as c o m p a r e d w i t h those of the control patients. J u d g i n g from the e f f e c t of w a s h i n g the tissue p r e p a r a tion from a p a t i e n t (S-15) a d m i n i s t e r e d l e v o m e p r o m a z i n e , h a l o p e r i dol and p r o m e t h a z i n e for a long p e r i o d (Fig. 4), the lowered a f f i n i t y of the r e c e p t o r s in o n - d r u g s c h i z o p h r e n i c s does not appear to r e s u l t from the r e m a i n i n g a m o u n t of drugs in the p r e p a r a t i o n s used in the assay. M o s t amount of n e u r o l e p t i c s in tissues is, however, e x p e c t e d to be b i n d i n g w i t h or s o l u b i l i z e d in the m e m b r a n e and not to be r e l e a s e d into the s u p e r n a t a n t by c e n t r i f u g a t i o n used here. The c h r o n i c a t r o p i n e t r e a t m e n t to rats by W e s t l i n d et al. in w h i c h the last i n j e c t i o n was done at 24 h p r i o r to s a c r i f i c e led to a d e c r e a s e in the a f f i n i t y of m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s in P2 fraction to 3H-QNB (18). The p o s s i b i l i t y of r e s i d u a l a t r o p i n e p r e s e n t in the b r a i n tissue was v e r i f i e d by their 3 H - a t r o p i n e t r a c e r findings. A l s o in this study, residual drugs w i t h a n t i m u s c a r i n i c p o t e n c y p r e s e n t in memb r a n e p r e p a r a t i o n s may p o s s i b l y e x p l a i n the low a f f i n i t y of 3H-QNB b i n d i n g sites only in o n - d r u g group of s c h i z o p h r e n i c s . Exceptional cases, i.e. r e l a t i v e l y low a f f i n i t y in S-3 of o f f - d r u g group w h o had been a d m i n i s t e r e d a low dose of p o t e n t a n t i m u s c a r i n i c agent, a m i t r i p t y l i n e , till 3 days b e f o r e d e a t h and r e l a t i v e l y high a f f i n i t y in S-14 of o n - d r u g group w h o had b e e n free of all neuroleptics for 15 days b e f o r e death, may p o s s i b l y be e x p l a i n e d by the r e s i d u a l amount of n e u r o l e p t i c s with a n t i m u s c a r i n i c potency. A n o t h e r e x c e p t i o n a l case (S-12) w i t h s i m i l a r b i n d i n g p a r a m e t e r s to those of S-14 had b e e n a d m i n i s t e r e d 3 mg of h a l o p e r i d o l and 3 mg
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QNB Binding Sites in Schizophrenic Brain
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of t r i h e x y p h e n i d y l till 2 days b e f o r e his death. Antimuscarinic p o t e n c y of h a l o p e r i d o l is very w e a k as shown in Fig. 3. Though t r i h e x y p h e n i d y l c o a d m i n i s t e r e d to h i m is a p o t e n t a n t i m u s c a r i n i c a g e n t (Fig. 4), the low dose of it may e x p l a i n the r e l a t i v e l y h i g h a f f i n i t y of the sites in his s p e c i m e n among o n - d r u g cases. It was, thus, c o n c l u d e d that there w e r e no a l t e r a t i o n s in m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s in the p r e f r o n t a l c o r t e x of s c h i z o p h r e n i c p a t i e n t s and that l o n g - t e r m n e u r o l e p t i c s and a n t i p a r k i n s o n i a n drugs w i t h a n t i m u s c a r i n i c actions used t o g e t h e r w i t h t h e m led to an i n c r e a s e in r e c e p t o r n u m b e r and a d e c r e a s e in affinity. Furt h e r m o r e , the s u p p l e m e n t a l data o b t a i n e d using the caudate tissues s u g g e s t that the drug t r e a t m e n t has the same e f f e c t on the m u s c a rinic c h o l i n e r g i c r e c e p t o r s in all b r a i n r e g i o n s as in the prefrontal cortex. In addition, an i n c r e a s e in n u m b e r of m u s c a r i n i c r e c e p t o r s , or s u p e r s e n s i t i v i t y of the receptors, found in o n - d r u g cases of s c h i z o p h r e n i c s m a y be one of the a t t r i b u t a b l e factors in the l o w e r e d t h r e s h o l d a g a i n s t c o n v u l s i o n in the p a t i e n t s a d m i n i s t e r e d neuroleptics especially with potent anticholinergic properties. Acknowled@ement We thank Drs. T. Kanaya, M. Naito, H. Y a m a z u m i , H. Nomura, T. F u j i m o r i , O. Matsuda, A. Ando, S. T a k a h a s h i , H. Shimada, H. M o r i o k a , T. N u m a k u r a and J. S e m b a for t h e i r h e l p in o b t a i n i n g b r a i n s p e c i mens and Prof. T. M a e d a of S h i g a M e d i c a l C o l l e g e for his advice in s u b d i v i d i n g the p r e f r o n t a l cortex. We also a p p r e c i a t e T. O h u c h i and Y. M a s u o for their t e c h n i c a l a s s i s t a n c e . This study w a s s u p p o r t e d b y the G r a n t No. 82-09 from N a t i o n a l C e n t e r for Nervous, M e n t a l and M u s c u l a r D i s o r d e r s of the M i n i s t r y of H e a l t h and W e l f a r e , Japan. References i. S. SNYDER, D. G R E E N B E R G and H. I. Y A M A M U R A , Arch. Gen. P s y c h i at. 3 1 5 8 - 6 1 (1974) 2. R. P. EBSTEIN, D. P I C K H O L Z and R. H. B E L M A K E R , J. Pharm. Pharmacol. 3 1 5 5 8 - 5 5 9 (1979) 3. F. OWEN, A. J. CROSS, J. L. W A D D I N G T O N , M . P O U L T E R , S. J. G A M B L E and T. J. CROW, L i f e Sci. 2 6 5 5 - 5 9 (1980) 4. A. CLOW, A. T H E O D O R O U , P. J E N N E R and C. D. MARSDEN, Eur. J. Pharmacol. 63145-157 (1979) 5. T. J. CROW, F. OWEN, A. J. CROSS, E. C. J O H N S T O N E , M. H. J O S E P H and A. LONGDEN, in E n z y m e s and N e u r o t r a n s m i t t e r s in M e n t a l Disease. E. Usdin, T. L. S o u r k e s and M. B. H. Youdim, Eds., p p . 5 5 9 - 5 7 2 , J o h n W i l e y & Sons, C h i c h e s t e r (1980) 6. J. P. B E N N E T T , J r , S. J. ENNA, D. B. BYLUND, J. C. GILLIN, R. J. W Y A T T and S. H. SNYDER, Arch. Gen. Psychiat. 3_66927-934 (1979) 7. M. TORU, T. N I S H I K A W A , N. M A T A G A and M. T A K A S H I M A , J. N e u r a l Transmission 54181-191 (1982) 8. J. M. FUSTER, The p r e f r o n t a l Cortex, R a v e n Press, N e w York (1980) 9. K. AKERT, in The F r o n t a l G r a n u l a r C o r t e x and B e h a v i o r , J. M. W a r r e n and K. Akert, Eds., p p . 3 7 2 - 3 9 6 , M c G r a w - H i l l , N e w Y o r k (1964) i0. F. SANIDES, Die A r c h i t e k t o n i k des M e n s c h l i c h e n S t i r n h i r n s , S p r i n g e r - V e r l a g , B e r l i n (1962) ii. H. I. Y A M A M U R A and S. H. SNYDER, Proc. Nat. Acad. Sci. U S A 711725-1729 (1974)
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12. O. H. LOWRY, N. J. R O S E B R O U G H , A. L. F A R R and R. J. RANDALL, J. Biol. Chem. 193 2 6 5 - 2 7 5 (1951) 13. A. N O R D B E R G and B. W I N B L A D , L i f e Sci. 29 1 9 3 7 - 1 9 4 4 (1981) 14. R. C. SMITH, H. S. SHELAT, J. S A M M E T A and C. H. MISRA, in B r a i n N e u r o t r a n s m i t t e r s and R e c e p t o r s in A @ i n ~ and A @ e - R e l a t e d D i s o r d e r s , S. J. Enna, T. S a m o r a j s k i , B. Beer, Eds., p p . 2 3 1 243, R a v e n Press, N e w Y o r k (1981) 15. G. F R E U N D , L i f e Sci. 26 371-375 (1980) 16. R. J. M I L L E R and C. R. HILEY, N a t u r e 248 5 9 6 - 5 9 7 (1974) 17. T. D. RE ISINE, M. ROSSOR, E. SPOKES, L. L. I V E R S E N and H. I. Y A M A M U R A , in R e c e p t o r s for N e u r o t r a n s m i t t e r s and P e p t i d e H o r m o n e s , G. Pepeu, M. K u h a r and S. J. Enna, Eds., p p . 4 4 3 - 4 5 0 , R a v e n Press, N e w Y o r k (1980) 18. A. W E S T L I N D , M. G R Y N F A R B , B. H E D L U N D , T. B A R T F A I and K. FUXE, B r a i n Res. 225 1 3 1 - 1 4 1 (1981)
INCREASED PREFRONTAL
M U S C A R I N I C C H O L I N E R G I C R E C E P T O R S IN C O R T I C E S OF M E D I C A T E D S C H I Z O P H R E N I C S
Shuzo Watanabe, Mizuo Takashima Division Mental
Pergamon Press
Toru Nishikawa, and M i c h i o T o r u
of P s y c h o b i o l o g y , N a t i o n a l C e n t e r for Nervous, and M u s c u l a r D i s o r d e r s , 2620 O g a w a - H i g a s h i , K o d a i r a , Tokyo, 187, J a p a n (Received in final form September 12, 1983) Summary
A l t e r a t i o n s in 3 H - q u i n u c l i d i n y l b e n z i l a t e b i n d ing sites a s s o c i a t e d w i t h m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s w e r e i n v e s t i g a t e d in o r b i t o - f r o n t a l and m e d i a l frontal c o r t i c e s from 12 s c h i z o p h r e n i c s , 6 o n - d r u g and 6 o f f - d r u g cases, and from I0 controls. S i g n i f i c a n t l y lower a f f i n i t i e s of the sites w e r e found in b o t h areas of s c h i z o p h r e n i c s than controls. An i n c r e a s e in r e c e p t o r n u m b e r was shown only in the o r b i t o - f r o n t a l c o r t e x from s c h i z o p h r e n i c s . On-drug g r o u p of s c h i z o p h r e n i c s did, however, s h o w a sign i f i c a n t i n c r e a s e in r e c e p t o r n u m b e r and a s i g n i f i cant d e c r e a s e in a f f i n i t y in b o t h areas, w h i l e there w e r e no s i g n i f i c a n t d i f f e r e n c e s in any b i n d i n g p a r a m e t e r s of o f f - d r u g s c h i z o p h r e n i c s from controls. A l s o in the c a u d a t e the s i m i l a r results w e r e o b t a i n e d . It is, thus, c o n c l u d e d that a l t e r a t i o n s in m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s of s c h i z o p h r e n i c p a t i e n t s result from l o n g - t e r m m e d i c a t i o n w i t h a n t i m u s c a r i n i c actions. A n t i p s y c h o t i c drugs have the a n t i c h o l i n e r g i c p r o p e r t y in g r e a t e r or lesser degree w h i c h are t h o u g h t to c o m p e n s a t e for their i n t r i n s i c e x t r a p y r a m i d a l e f f e c t s c a u s e d by the d o p a m i n e r e c e p t o r blockade. It has b e e n shown that their a n t i c h o l i n e r g i c e f f e c t s r e s u l t e d from the d i r e c t action to m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s (i) . In animal e x p e r i m e n t s , the d i r e c t d o p a m i n e r e c e p t o r b l o c k a d e by l o n g - t e r m n e u r o l e p t i c s has been d e m o n s t r a t e d to lead to an i n c r e a s e in the n u m b e r of d o p a m i n e r g i c a n t a g o n i s t b i n d i n g sites, or the r e c e p t o r s u p e r s e n s i t i v i t y (2,3,4). It seems to be r e a s o n a b l e on the a n a l o g y of the d o p a m i n e r e c e p t o r s that the l o n g - t e r m 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 w i t h the a n t i c h o l i n e r g i c action also r e s u l t s in the s u p e r s e n s i t i v i t y of the m u s c a r i n i c receptors. P r e v i o u s p a p e r s c o n c e r n i n g the m u s c a r i n i c r e c e p t o r s of s c h i z o p h r e n i c p o s t m o r t e m b r a i n s r e p o r t e d no a l t e r a t i o n s in s p e c i f i c 3 H - q u i n u c l i d i n y l b e n z i l a t e (QNB) b i n d i n g to the c a u d a t e and the p u t a m e n (5) or the t e n d e n c y for r e d u c e d s p e c i f i c b i n d i n g to the frontal c o r t e x from p a t i e n t s w i t h s c h i z o p h r e n i a (6). In the p r e s e n t study, S c a t c h a r d a n a l y s i s of 3H-QNB b i n d i n g a s s o c i a t e d w i t h m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s in two s u b a r e a s of prefrontal c o r t e x w h i c h r e c e i v e d i s c r e t e p r o j e c t i o n s from the parts 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
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of m e d i o d o r s a l n u c l e u s in the thalamus r e v e a l e d no a l t e r a t i o n s in the o f f - d r u g group of s c h i z o p h r e n i c s , w h i l e m e d i c a t e d schizop h r e n i c s showed h i g h e r a p p a r e n t K D and B m a x than o f f - d r u g schizop h r e n i c s or controls. A l s o in the caudate the same d i r e c t i o n of the r e c e p t o r a l t e r a t i o n s s e e m i n g l y induced by the drugs was displayed. Materials
and M e t h o d s
P o s t m o r t e m b r a i n s w e r e o b t a i n e d from 12 i n d i v i d u a l s w i t h s c h i z o p h r e n i a h a v i n g an average age of 60.4 years (41-75) and I0 control i n d i v i d u a l s w i t h no r e c o r d e d h i s t o r y of n e u r o l o g i c or p s y c h i a t r i c illness h a v i n g an average age of 66.7 years (52-74). The d i a g n o s i s of s c h i z o p h r e n i a in these p a t i e n t s were a s c e r t a i n e d by their c a t a m n e s e s and c a t e g o r i z e d a c c o r d i n g to DSM-I[[. Briefly s u m m a r i z e d b a c k g r o u n d s of s c h i z o p h r e n i c s and controls are as follows. S c h i z o p h r e n i c - l : Age at death; 50, Sex; M, Type c l a s s i f i e d a c c o r d i n g to DSM-]II categories; d i s o r g a n i z e d type, in remission, Cause of death; m y o c a r d i a l infarction, P s y c h o t r o p i c m e d i c a t i o n b e f o r e death; c h l o r d i a z e p o x i d e I0 mg for 7.5 months b e f o r e death (off-drug case). S-2: 62, F, u n d i f f e r e n t i a t e d type, s u b c h r o n i c w i t h acute e x a c e r b a t i o n , m y o c a r d i a l infarction, c h l o r d i a z e p o x i d e i0 mg and p h e n o b a r b i t a l 60 mg only for 5 days before death (offdrug case). S-3: 41, M, c a t a t o n i c type, c h r o n i c w i t h acute e x a c e r b a t i o n , pneumonia, a n t i p s y c h o t i c m e d i c a t i o n free for 3 m o n t h s prior to death e x c e p t n i t r a z e p a m i0 mg, g l u t e t h i m i d e 500 mg and a m i t r i p t y l i n e i0 mg at b e d t i m e till 3 days b e f o r e death (offdrug case). S-5: 46, F, p a r a n o i d type, chronic, sudden death, s p i p e r o n e 6 mg, p i p a m p e r o n e 200 mg, t h i o p r o p e r a z i n e 30 mg, t r i h e x y p h e n i d y l 8 mg and h y d r o x y z i n e 200 mg till the m o r n i n g of the day of death (on-drug case). S-6: 64, F, d i s o r g a n i z e d type, chronic, cardiac thrombosis, h a l o p e r i d o l 4.5 mg, a m i t r i p t y l i n e 30 mg and p r o m e t h a z i n e 75 mg till 2 days b e f o r e death (on-drug case). T h e s e are the cases p r e v i o u s l y reported in regard to the d o p a m i n e m e t a b o l i s m in basal g a n g l i a of s c h i z o p h r e n i c p a t i e n t s (7). S-7: 54, M, u n d i f f e r e n t i a t e d type, chronic, p a n p e r i t o n i t i s , chlorp r o m a z i n e 50 mg, p i p a m p e r o n e 75 mg and m e t o c l o p r a m i d e 30 mg till 18 days b e f o r e death and then c h l o r p r o m a z i n e 50 mg till 5 days b e f o r e death (on-drug case). S-II: 70, M, d i s o r g a n i z e d type, chronic, m y o c a r d i a l infarction, drug free for 7 years (off-drug case). S-12: 69, M, d i s o r g a n i z e d type, chronic, c a r d i a c failure, h a l o p e r i d o l 3 mg and t r i h e x y p h e n i d y l 3 mg till 2 days before death (on-drug case). S-13: 73, M, u n d i f f e r e n t i a t e d type, chronic, stomach cancer, h a l o p e r i d o l 3 mg and t r i h e x y p h e n i d y l 3 mg till 5.5 months and then d i a z e p a m 5 mg till 1.5 months b e f o r e death (offdrug case). S-14: 53, F, d i s o r g a n i z e d type, chronic, o v a r i a n cancer, o x y p e r t i n e 90 mg, c l o c a p r a m i n e 200 mg, a m i t r i p t y l i n e 50 mg and m e t h i x e n e i0 mg till 16 days before death (on-drug case). S-15: 68, M, u n d i f f e r e n t i a t e d type, chronic, suffocation, h a l o p e r i d o l 3 mg, l e v o m e p r o m a z i n e 25 mg, p r o m e t h a z i n e 25 mg and n i t r a z e p a m i0 mg just b e f o r e death (on-drug case). S-16: 75, F, d i s o r g a n i z e d type, chronic, p a n c r e a t i c cancer, h a l o p e r i d o l 1.5 mg till 40 days and c l o t i a z e p a m 20 mg till 33 days before death (offdrug case). C o n t r o l s consist of 7 males and 3 females w h o s e cause of death is sudden death, cancer in the lung or the d i g e s t i v e tract, ileus or d i s s e m i n a t e d i n t r a v a s c u l a r coagulation. The m i s s i n g numbers of p a t i e n t s listed above and in figures in "Results"
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i n d i c a t e that their b r a i n c o r t i c e s w e r e u n s u i t a b l e for r e c e p t o r a n a l y s i s by b i n d i n g t e c h n i q u e o w i n g to the d e h y d r a t i o n y i e l d e d d u r i n g frozen s t o r a g e or that the tissues p r e p a r e d w e r e e x h a u s t e d in o t h e r b i o c h e m i c a l analyses. The b r a i n s taken at a u t o p s y in the h o s p i t a l s w h e r e those p a t i e n t s h a d d i e d w e r e put into -20°C or -40°C f r e e z e r until t r a n s f o r m a t i o n to our l a b o r a t o r y (0.5 h - 4 m o n t h s , a v e r a g e 31 days). The i n t e r v a l s b e t w e e n d e a t h and f r e e z i n g of the b r a i n are 2 - 13 h (average 5.2 h) for c o n t r o l s and 3 - 22 h (average 10.2 h) for s c h i z o p h r e n i c s . T h e y w e r e s t o r e d in a i r t i g h t p a c k a g e s in our l a b o r a t o r y at -80°C until d i s s e c t i o n (20 days - 32 m o n t h s , a v e r a g e 15.6 months). On the b a s i s of the p r o j e c t i o n from d i s c r e t e areas of m e d i o d o r s a l n u c l e u s of the t h a l a m u s (8,9) and the d i f f e r e n c e s in the c o r t i c a l c y t o a r c h i t e c t u r e and in the p u r p o r t e d f u n c t i o n (10), the p r e f r o n t a l c o r t e x w a s d e v i d e d into 4 areas; m e d i a l frontal cortex, o r b i t o - frontal cortex, o r b i t a l c o r t e x and frontal eye field c o r r e s p o n d i n g to B r o d m a n n areas 9.10-46, 45-47, 11-12 and 8, respectively. F r o n t a l parts of the b r a i n s w e r e p l a c e d at -15°C for a b o u t 12 h p r i o r to d i s s e c t i o n to a l l o w serial c o r o n a l c u t t i n g in 1.5 m m t h i c k n e s s w i t h criotome. F r o m e a c h slice, the g r a y m a t t e r of d i s c r e t e p r e f r o n t a l areas w a s d i s s e c t e d out in a c o l d b o x k e p t at -15°C a c c o r d i n g to their l a n d m a r k s p u t b e f o r e dissection. The d i s s e c t i o n of the c a u d a t e was d e s c r i b e d p r e v i o u s ly (7). D i s s e c t e d t i s s u e s w e r e h o m o g e n i z e d in 3 vol. of i c e - c o l d 0.32 M s u c r o s e w i t h a P o l y t r o n . A l i q u o t s of 1.5 ml of the h o m o g e n a t e w e r e d i s p e n s e d into small p l a s t i c tubes and s t o r e d at -80°C until b i o c h e m i c a l and p h a r m a c o l o g i c a l analyses. " O f f - d r u g " g r o u p of s c h i z o p h r e n i c s (6 out of 12 s c h i z o p h r e n ics) had b e e n a d m i n i s t e r e d no a n t i p s y c h o t i c s for at least 40 days b e f o r e d e a t h (5 out of 6 cases h a d r e c e i v e d no a n t i p s y c h o t i c s for m o r e than 3 m o n t h s ) . The a n a l y s i s of 3H-QNB b i n d i n g in all cases w a s c a r r i e d out u s i n g m a i n two out of four s u b a r e a s d e s c r i b e d above. T h e y are the m e d i a l frontal c o r t e x r e c e i v i n g the p r o j e c t i o n from pars p a r v o c e l l u l a r i s of the m e d i o d o r s a l n u c l e u s of t h a l a m u s and the o r b i t o frontal c o r t e x p r o j e c t e d from pars m a g n o c e l l u l a r i s of the nucleus. 3H-QNB b i n d i n g to the m e m b r a n e p r e p a r a t i o n s from two d i s c r e t e p r e f r o n t a l c o r t i c e s and the c a u d a t e w a s p e r f o r m e d in t r i p l i c a t e by a m o d i f i c a t i o n of the r a p i d f i l t r a t i o n m e t h o d of Y a m a m u r a and S n y d e r (ii). The t h a w e d s u c r o s e h o m o g e n a t e d i l u t e d three times w i t h 50 m M T r i s . H C l b u f f e r , pH 7.4, c o n t a i n i n g i0 m M M g C l 2 was c e n t r i f u g e d for 10 m i n at 1,000 xg and the r e s u l t i n g s u p e r n a t a n t w a s c e n t r i f u g e d at 45,000 xg for 20 min. The r e s u l t i n g p e l l e t was s u s p e n d e d in the T r i s . H C l b u f f e r to get the p r o t e i n c o n c e n t r a t i o n of 0.2 - 0.4 mg per ml. To a s s a y the s p e c i f i c b i n d i n g of 3H-QNB to the t i s s u e p r e p a r a t i o n , 0.2 ml of this p r e p a r a t i o n w e r e incub a t e d in 12 x 75 m m glass tube at 21°C for 180 m i n w i t h 0.2 ml of 3 x 10 -6 M a t r o p i n e d i l u t e d w i t h the b u f f e r from 3 x 10 -3 M a l c o h o l i c s o l u t i o n and 0.2 ml of v a r y i n g c o n c e n t r a t i o n s (0.06 1.2 nM) of 3H-QNB (33.1 C i / m m o l , NEN) d i l u t e d w i t h buffer. The b i n d i n g r e a c t i o n w a s t e r m i n a t e d b y f i l t e r i n g 0.5 ml a l i q u o t of an a s s a y m i x t u r e of 0.6 ml t h r o u g h a G F / B glass fiber f i l t e r (Whatman) u n d e r v a c u u m and e a c h filter w a s r i n s e d twice w i t h i0 ml of ice-cold buffer. The filters w e r e c o u n t e d for 3H in a P a c k a r d s c i n t i l l a t i o n s p e c t r o m e t e r w i t h 10 ml of A q u a s o l (NEN). P r o t e i n w a s d e t e r m i n e d b y the m e t h o d of L o w r y et al. w i t h b o v i n e s e r u m a l b u m i n as s t a n d a r d (12). B i n d i n g p a r a m e t e r s w e r e c a l c u l a t e d by least s q u a r e s r e g r e s -
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sion analysis of S c a t c h a r d plots. The added 3H-QNB minus the total b i n d i n g was taken as the free c o n c e n t r a t i o n of the ligand. The p a r a m e t e r values o b t a i n e d for controls, o n - d r u g and o f f - d r u g s c h i z o p h r e n i c s were c o m p a r e d by two t a i l e d M a n n - W h i t n e y U test. Results There w e r e no s i g n i f i c a n t d i f f e r e n c e s in the age d i s t r i b u t i o n b e t w e e n controls, o n - d r u g and o f f - d r u g s c h i z o p h r e n i c s (MannW h i t n e y U test), as the a g e - d e p e n d e n t loss in the n u m b e r of m u s c a r i n i c c h o l i n e r g i c receptors in human and animal brains has been r e p o r t e d (13, 14, 15). The s i g n i f i c a n t c o r r e l a t i o n s b e t w e e n the interval from death to freezing of the b r a i n or the d u r a t i o n of frozen storage and the b i n d i n g p a r a m e t e r s did not exist in any groups (Spearman Rank C o r r e l a t i o n test). 3H-QNB b i n d i n g to the m e m b r a n e p r e p a r a t i o n of the human prefrontal cortex and the caudate using atropine as b a s e l i n e could be r e g a r d e d as m o n o p h a s i c w i t h a Hill c o e f f i c i e n t of 1.0, as e x a m i n e d in p r e l i m i n a r y s a t u r a t i o n isotherms using 10 - 12 ligand concentrations. Thus, 5 - 6 c o n c e n t r a t i o n s of 3H-QNB were e m p l o y e d in the s t a n d a r d assay. The Hill c o e f f i c i e n t s of o r b i t o - f r o n t a l cortices for 3H-QNB were a p p r o x i m a t e l y 1.0; 1.02 • 0.04 for controls and 0.97 ± 0.08 for s c h i z o p h r e n i c s as a whole, and
J05 -
[
CONTROLS (S)
~CHIZOPHRLNICS ( ± 2 )
KD: 3.015±0.301~C~'~ BMAX: 789.3'27.8~ ,: 0.592*0.003
KD: 0.073t0.037~" EMAX:1023.7~C3.2~ :: 0.990±0.003 OFF-DR.G (6)
\ \
0N-DRuG (6)
!:D:0.02Gt0.003"I~ BMAX: 844.8,43.3~ ~ 0.994±0.002 [
%: 0.126t0.070t'~ EMAX: 1202.6,53.7::#:~ ,: 0.q8710.004
5x104" m
"
11
c
500
1000
500
i000
500
10GU
B ( FMOL/MG PROT, )
Fig. Scatchard
1
a n a l y s i s of 3H-QNB b i n d i n g o r b i t o - f r o n t a l cortex
to
3H-QNB (33.1 Ci/mmol) r a n g e d from 0.02 to 0.4 nM. S p e c i f i c b i n d i n g was that i n h i b i t e d by 10 -6 M atropine. The tissue used in the assay was 0.079 ± 0.003 mg p r o t e i n per tube. V a l u e s of b i n d i n g p a r a m e t e r s are m e a n ± S.E.M. K D (nM) and B m a x (fmol/mg protein) values w e r e d e t e r m i n e d by linear r e g r e s s i o n analysis of S c a t c h a r d p l o t data c o n s i s t i n g of six points in this area. A c o r r e l a t i o n c o e f f i c i e n t of the plots is i n d i c a t e d by r.
.,.,.:
p<0.02, liE: p<0.002, ~ , ~ : p<0.001
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f u r t h e r m o r e , no d i f f e r e n c e b e t w e e n the o n - d r u g g r o u p (0.96 ± 0.05) and the o f f - d r u g g r o u p of s c h i z o p h r e n i c s (0.98 ± 0.11) was found. The c o e f f i c i e n t s in m e d i a l frontal c o r t e x w e r e e s s e n t i a l l y the same as in o r b i t o - f r o n t a l cortex. As shown in Fig. i, the a p p a r e n t K D of 3H-QNB b i n d i n g to the o r b i t o - f r o n t a l c o r t e x p r e p a r a t i o n in o n - d r u g s c h i z o p h r e n i c s was s i g n i f i c a n t l y h i g h e r than c o n t r o l s (p<0.001) or o f f - d r u g s c h i z o p h r e n i c s (p<0.02). T h e r e was no d i f f e r e n c e in the a p p a r e n t K D b e t w e e n c o n t r o l s and o f f - d r u g s c h i z o p h r e n i c s . Similarly, a s i g n i f i c a n t l y h i g h e r a p p a r e n t K D was found in the m e d i a l frontal cortex of o n - d r u g s c h i z o p h r e n i c s than o f f - d r u g g r o u p or c o n t r o l s as shown in Fig. 2 (p<0.002) . In b o t h cortices, the m e a n K D for all s c h i z o p h r e n i c s was also h i g h e r than that for c o n t r o l s (p<0.02 in the o r b i t o - f r o n t a l c o r t e x and p < 0 . 0 5 in the m e d i a l frontal cortex), o w i n g to h i g h e r K D for o n - d r u ~ g r o u p of s c h i z o p h r e n i c s . The a l t e r a t i o n in the d e n s i t y of ~ H - Q N B b i d d i n g to the m e m b r a n e p r e p a r a t i o n from e a c h s u b a r e a was also s i m i l a r to that in KD; a h i g h e r B m a x in o n - d r u g s c h i z o p h r e n i c s than in c o n t r o l s (p<0.002 for b o t h areas) or o f f - d r u g s c h i z o p h r e n i c s (p<0.002 for b o t h areas) w i t h no d i f f e r e n c e b e t w e e n the latter two. In c o n c l u sion, only o n - d r u g cases of all s c h i z o p h r e n i c s s h o w e d h i g h e r K D and Bmax of 3H-QNB b i n d i n g to the p r e f r o n t a l c o r t e x than c o n t r o l s or o f f - d r u g s c h i z o p h r e n i c s . In the a d d i t i o n a l a n a l y s i s of 3H-QNB b i n d i n g to the caudate p r e p a r a t i o n done in o r d e r to check w h e t h e r the drug t r e a t m e n t ~(HITOPIIRF'dI( ~ ~1~?'.
CONTROL.'; (10) KD:
ElF
0.016.C.001~-~
KD: ~.07!*0.32B BMAX: iiR7.5*7:F.g :: 3 . 9 g ' , : 0 . 9 9 3
:i
~k '4
OFF ~RlJ(i (()::
dr,-~':!, F !( )
KD: 0.019,().:):)4 5~AX: 993.1~C3.9
• L:
X,~
590
]Qr]O
9.985"-0. 005
5r0
15OC
10[:3
[~ ( F~IOL/"I~: FPOT.
Fig. Scatchard
.12h%.'~4 , " . ~ '
1
2
a n a l y s i s of 3H-QNB b i n d i n g m e d i a l frontal c o r t e x
to
3H-QNB (33.1 Ci/mmol) r a n g e d from 0.02 to 0.32 nM. The tissue used in the assay was 0.061 ± 0.003 mg p r o t e i n per tube. The assay was done in the same w a y as in o r b i t o - f r o n t a l c o r t e x e x c e p t that Scatchard plot data c o n s i s t of five p o i n t s in this area. A c o r r e l a t i o n c o e f f i c i e n t of the p l o t s is i n d i c a t e d by r. ~: p<0.05, ~ : p<0.01, ~: p<0.005, ~, ~: p<0.002
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e f f e c t s all b r a i n r e g i o n s or is s e l e c t i v e for the p r e f r o n t a l c o r tex, the a l t e r a t i o n of b i n d i n g p a r a m e t e r s w a s in the s a m e d i r e c t i o n as in the p r e f r o n t a l c o r t e x , as m e a s u r e d u s i n g the c a s e s whose caudate membrane preparations were available. The K D values (nM) w e r e 0 . 0 2 2 ~ 0 . 0 0 1 for c o n t r o l s (C-II, -12, -14, -15 a n d -16), 0 . 0 2 9 ± 0 . 0 0 5 for o f f - d r u g p a t i e n t s (S-l, -2, -3, -ii a n d -16) and 0 . 1 5 9 ± 0 . 0 9 5 for o n - d r u g p a t i e n t s (S-5, -7, -14 and -15). The l a s t one w a s s i g n i f i c a n t l y d i f f e r e n t f r o m the f o r m e r t w o (p<0.05) b e t w e e n w h i c h no d i f f e r e n c e w a s found. The B m a x v a l u e s ( f m o l / m g p r o t e i n ) w e r e 1 5 3 0 . 5 ± 99.0 for c o n t r o l s , 1 8 0 2 . 6 ± 1 1 1 . 5 for o f f d r u g p a t i e n t s a n d 2 1 5 4 . 6 ± 81.8 for o n - d r u g p a t i e n t s . The f i r s t one w a s s i g n i f i c a n t l y l o w e r t h a n the l a s t one (p<0.02) w h i l e it s h o w e d no d i f f e r e n c e f r o m the s e c o n d o n e (p>0.1) .
Total
Bindlnq
cpm/~11tcr
I00
~0
80
6O 440
4O
2O
0
i0
9
8
7
-logl01
Druq
Fig. Drug i n h i b i t i o n frontal
cortex
of
6
] ( M
5
4
)
3
3H-QNB b i n d i n g
from a control
to the medial patient (C-l)
0.1 n M of 3H-QNB ( 3 3 . 1 C i / m m o l ) w a s u s e d i n t h i s study. T h e t i s s u e in the a s s a y w a s o.i mg p r o t e i n p e r tube.
Drugs were diluted alcoholic solution.
with
the
buffer
from
3 x 10 - 3 M
As s h o w n in Fig. 3, a n t i m u s c a r i n i c p o t e n c i e s of the d r u g s i n d i c a t e d in the d r u g i n h i b i t i o n of 3 H - Q N B b i n d i n g to the m e d i a l f r o n t a l c o r t e x of a c o n t r o l p a t i e n t (C-l) w e r e e s s e n t i a l l y s i m i l a r to p r e v i o u s r e p o r t s by o t h e r s (i, 16). The a p p a r e n t K D of 3 H - Q N B b i n d i n g to the 1 , 0 0 0 xg s u p e r n a t a n t or to the s u s p e n s i o n s of rep e a t e d l y w a s h e d 4 5 , 0 0 0 xg p e l l e t of the s u p e r n a t a n t r e m a i n e d unchanged in s a t u r a t i o n i s o t h e r m s in w h i c h the o r b i t o - f r o n t a l c o r t e x f r o m a t y p i c a l s c h i z o p h r e n i c p a t i e n t (S-15) a d m i n i s t e r e d l e v o m e p r o m a z i n e , h a l o p e r i d o l and p r o m e t h a z i n e for a long time w a s u s e d (Fig. 4). Discussion The
prefrontal
cortex
develops
most
exceedingly
in h u m a n
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QNB Binding Sites in Schizophrenic Brain
WASHINO KD
15000-
• • • O
0X ix 2X 3X
0.062 0.067 0.071 0.069
2193
BMAX 425.6 916.3 1180.6 1099.5
~ o
10000-
5000"
!
I000 B ( FMOL/MG PROTEIN )
Fig. Effect
4
of w e s h i n ~ of the tissue on JH-QNB b i n d i n g
preparation
The 1,000 xg s u p e r n a t a n t ( • ) of the h o m o g e n a t e of the o r b i t o - f r o n t a l cortex from a s c h i z o p h r e n i c p a t i e n t (S-15) was c e n t r i f u g e d at 45,000 xg for 20 min and the p e l l e t was s u s p e n d e d in the b u f f e r ( • ) . The suspension was c e n t r i f u g e d at 45,000 xg once more and the p e l l e t was s u s p e n d e d ( • ) . It was further c e n t r i f u g e d at 45,000 xg and the r e s u l t i n g p e l l e t was s u s p e n d e d (O). The p r o t e i n in the assay r a n g e d from 0.06 to 0.i mg per tube at r e s p e c t i v e steps of washing. The final c o n c e n t r a t i o n s of 3H-QNB w e r e from 0.02 to 0.4 nM. KD: nM, Bmax: fmol/mg p r o t e i n
among primates. By the injury in this area, h i g h e r functions of h u m a n brain, such as attention, c o g n i t i o n and creation, may be affected, and the r e d u c t i o n of s p o n t a n e i t y and the a l t e r a t i o n of a f f e c t i v i t y m a y o c c u r (7). F r o m such a viewpoint, this area is t h o u g h t to be one of the areas of the b r a i n w o r t h y of the i n v e s t i gation for the b i o c h e m i s t r y of s c h i z o p h r e n i a . Only two reports c o n c e r n i n g m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s in s c h i z o p h r e n i c postm o r t e m b r a i n s can be found as yet; the t e n d e n c y for r e d u c e d levels of s p e c i f i c 3H-QNB b i n d i n g to w h o l e frontal cortex m e m b r a n e p r e p a ration r e p o r t e d by B e n n e t t et al. (6) and no a l t e r a t i o n in s p e c i f ic 3H-QNB b i n d i n g to the m e m b r a n e from s c h i z o p h r e n i c caudate and p u t a m e n a s s e r t e d b y C r o w et al. (5). A n a l y s e s of the frontal
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c o r t e x of s c h i z o p h r e n i c s have been p e r f o r m e d as to neurotransmitter r e c e p t o r s i n v o l v i n g the m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s by B e n n e t t et al. and o p i a t e and n e u r o l e p t i c r e c e p t o r s by R e i s i n e et al. (17). The area used in the study by B e n n e t t et al., w h i c h i n v o l v e d B r o d m a n n areas 6, 8 - ii and 44 - 47, implies the w h o l e frontal c o r t e x a p p r o x i m a t e l y . In the report by R e i s i n e et al. is there no d e t a i l e d d e s c r i p t i o n about the area u s e d as the frontal cortex. The s u b a r e a s of the p r e f r o n t a l c o r t e x are not h o m o g e n e ous, but show a v a r i e t y of d i f f e r e n c e s in their neural c o n n e c t i o n s w i t h o t h e r b r a i n areas, their c y t o a r c h i t e c t u r e s and their p u r p o r t ed f u n c t i o n s (7). However, in r e g a r d to m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s in two m a i n s u b a r e a s e x a m i n e d here, the o r b i t o - f r o n t a l cortex m e r e l y s h o w e d s l i g h t l y lower d e n s i t y than that in the m e d i a l frontal cortex. T h e r e w e r e no s i g n i f i c a n t d i f f e r e n c e s in the a f f i n i t y to 3H-QNB b e t w e e n the areas in control, o n - d r u g and o f f - d r u g s c h i z o p h r e n i c groups. In the studies p r e v i o u s l y r e p o r t e d (5, 6), only one c o n c e n t r a t i o n of 3H-QNB was used for a s s e s s m e n t of the a n t a g o n i s t b i n d ing ability to m u s c a r i n i c r e c e p t o r s in p r e p a r a t i o n s . However, the a n a l y s i s of s a t u r a t i o n i s o t h e r m s will be n e c e s s a r y for the k n o w l edge of the a m o u n t or the d e n s i t y of m u s c a r i n i c r e c e p t o r s in h u m a n b r a i n s unless the a f f i n i t i e s of the r e c e p t o r s in p r e p a r a t i o n s to the ligand are i d e n t i c a l . A f t e r c h r o n i c a t r o p i n e t r e a t m e n t to rats, W e s t l i n d et al. o b t a i n e d an increase in the n u m b e r of m u s c a r i n i c a n t a g o n i s t b i n d i n g sites in h i p p o c a m p u s and the l o w e r e d a f f i n i t y for 3 H - a n t a g o n i s t s in dose d e p e n d e n t m a n n e r (18). Thus, also in the s c h i z o p h r e n i c p a t i e n t s e x a m i n e d in the p r e s e n t study, the l o n g - t e r m 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 and a n t i p a r k i n s o n i a n drugs w i t h a n t i m u s c a r i n i c actions used t o g e t h e r w i t h them will be e x p e c t e d to lead to the l o w e r e d a f f i n i t y of the r e c e p t o r s to 3HQNB. The p r e s e n t study d e m o n s t r a t e d that this s p e c u l a t i o n was the case and the m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s in the p r e f r o n t a l cortex of o f f - d r u g s c h i z o p h r e n i c p a t i e n t s h a d no d i f f e r e n c e s in the b i n d i n g p a r a m e t e r s as c o m p a r e d w i t h those of the control patients. J u d g i n g from the e f f e c t of w a s h i n g the tissue p r e p a r a tion from a p a t i e n t (S-15) a d m i n i s t e r e d l e v o m e p r o m a z i n e , h a l o p e r i dol and p r o m e t h a z i n e for a long p e r i o d (Fig. 4), the lowered a f f i n i t y of the r e c e p t o r s in o n - d r u g s c h i z o p h r e n i c s does not appear to r e s u l t from the r e m a i n i n g a m o u n t of drugs in the p r e p a r a t i o n s used in the assay. M o s t amount of n e u r o l e p t i c s in tissues is, however, e x p e c t e d to be b i n d i n g w i t h or s o l u b i l i z e d in the m e m b r a n e and not to be r e l e a s e d into the s u p e r n a t a n t by c e n t r i f u g a t i o n used here. The c h r o n i c a t r o p i n e t r e a t m e n t to rats by W e s t l i n d et al. in w h i c h the last i n j e c t i o n was done at 24 h p r i o r to s a c r i f i c e led to a d e c r e a s e in the a f f i n i t y of m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s in P2 fraction to 3H-QNB (18). The p o s s i b i l i t y of r e s i d u a l a t r o p i n e p r e s e n t in the b r a i n tissue was v e r i f i e d by their 3 H - a t r o p i n e t r a c e r findings. A l s o in this study, residual drugs w i t h a n t i m u s c a r i n i c p o t e n c y p r e s e n t in memb r a n e p r e p a r a t i o n s may p o s s i b l y e x p l a i n the low a f f i n i t y of 3H-QNB b i n d i n g sites only in o n - d r u g group of s c h i z o p h r e n i c s . Exceptional cases, i.e. r e l a t i v e l y low a f f i n i t y in S-3 of o f f - d r u g group w h o had been a d m i n i s t e r e d a low dose of p o t e n t a n t i m u s c a r i n i c agent, a m i t r i p t y l i n e , till 3 days b e f o r e d e a t h and r e l a t i v e l y high a f f i n i t y in S-14 of o n - d r u g group w h o had b e e n free of all neuroleptics for 15 days b e f o r e death, may p o s s i b l y be e x p l a i n e d by the r e s i d u a l amount of n e u r o l e p t i c s with a n t i m u s c a r i n i c potency. A n o t h e r e x c e p t i o n a l case (S-12) w i t h s i m i l a r b i n d i n g p a r a m e t e r s to those of S-14 had b e e n a d m i n i s t e r e d 3 mg of h a l o p e r i d o l and 3 mg
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QNB Binding Sites in Schizophrenic Brain
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of t r i h e x y p h e n i d y l till 2 days b e f o r e his death. Antimuscarinic p o t e n c y of h a l o p e r i d o l is very w e a k as shown in Fig. 3. Though t r i h e x y p h e n i d y l c o a d m i n i s t e r e d to h i m is a p o t e n t a n t i m u s c a r i n i c a g e n t (Fig. 4), the low dose of it may e x p l a i n the r e l a t i v e l y h i g h a f f i n i t y of the sites in his s p e c i m e n among o n - d r u g cases. It was, thus, c o n c l u d e d that there w e r e no a l t e r a t i o n s in m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s in the p r e f r o n t a l c o r t e x of s c h i z o p h r e n i c p a t i e n t s and that l o n g - t e r m n e u r o l e p t i c s and a n t i p a r k i n s o n i a n drugs w i t h a n t i m u s c a r i n i c actions used t o g e t h e r w i t h t h e m led to an i n c r e a s e in r e c e p t o r n u m b e r and a d e c r e a s e in affinity. Furt h e r m o r e , the s u p p l e m e n t a l data o b t a i n e d using the caudate tissues s u g g e s t that the drug t r e a t m e n t has the same e f f e c t on the m u s c a rinic c h o l i n e r g i c r e c e p t o r s in all b r a i n r e g i o n s as in the prefrontal cortex. In addition, an i n c r e a s e in n u m b e r of m u s c a r i n i c r e c e p t o r s , or s u p e r s e n s i t i v i t y of the receptors, found in o n - d r u g cases of s c h i z o p h r e n i c s m a y be one of the a t t r i b u t a b l e factors in the l o w e r e d t h r e s h o l d a g a i n s t c o n v u l s i o n in the p a t i e n t s a d m i n i s t e r e d neuroleptics especially with potent anticholinergic properties. Acknowled@ement We thank Drs. T. Kanaya, M. Naito, H. Y a m a z u m i , H. Nomura, T. F u j i m o r i , O. Matsuda, A. Ando, S. T a k a h a s h i , H. Shimada, H. M o r i o k a , T. N u m a k u r a and J. S e m b a for t h e i r h e l p in o b t a i n i n g b r a i n s p e c i mens and Prof. T. M a e d a of S h i g a M e d i c a l C o l l e g e for his advice in s u b d i v i d i n g the p r e f r o n t a l cortex. We also a p p r e c i a t e T. O h u c h i and Y. M a s u o for their t e c h n i c a l a s s i s t a n c e . This study w a s s u p p o r t e d b y the G r a n t No. 82-09 from N a t i o n a l C e n t e r for Nervous, M e n t a l and M u s c u l a r D i s o r d e r s of the M i n i s t r y of H e a l t h and W e l f a r e , Japan. References i. S. SNYDER, D. G R E E N B E R G and H. I. Y A M A M U R A , Arch. Gen. P s y c h i at. 3 1 5 8 - 6 1 (1974) 2. R. P. EBSTEIN, D. P I C K H O L Z and R. H. B E L M A K E R , J. Pharm. Pharmacol. 3 1 5 5 8 - 5 5 9 (1979) 3. F. OWEN, A. J. CROSS, J. L. W A D D I N G T O N , M . P O U L T E R , S. J. G A M B L E and T. J. CROW, L i f e Sci. 2 6 5 5 - 5 9 (1980) 4. A. CLOW, A. T H E O D O R O U , P. J E N N E R and C. D. MARSDEN, Eur. J. Pharmacol. 63145-157 (1979) 5. T. J. CROW, F. OWEN, A. J. CROSS, E. C. J O H N S T O N E , M. H. J O S E P H and A. LONGDEN, in E n z y m e s and N e u r o t r a n s m i t t e r s in M e n t a l Disease. E. Usdin, T. L. S o u r k e s and M. B. H. Youdim, Eds., p p . 5 5 9 - 5 7 2 , J o h n W i l e y & Sons, C h i c h e s t e r (1980) 6. J. P. B E N N E T T , J r , S. J. ENNA, D. B. BYLUND, J. C. GILLIN, R. J. W Y A T T and S. H. SNYDER, Arch. Gen. Psychiat. 3_66927-934 (1979) 7. M. TORU, T. N I S H I K A W A , N. M A T A G A and M. T A K A S H I M A , J. N e u r a l Transmission 54181-191 (1982) 8. J. M. FUSTER, The p r e f r o n t a l Cortex, R a v e n Press, N e w York (1980) 9. K. AKERT, in The F r o n t a l G r a n u l a r C o r t e x and B e h a v i o r , J. M. W a r r e n and K. Akert, Eds., p p . 3 7 2 - 3 9 6 , M c G r a w - H i l l , N e w Y o r k (1964) i0. F. SANIDES, Die A r c h i t e k t o n i k des M e n s c h l i c h e n S t i r n h i r n s , S p r i n g e r - V e r l a g , B e r l i n (1962) ii. H. I. Y A M A M U R A and S. H. SNYDER, Proc. Nat. Acad. Sci. U S A 711725-1729 (1974)
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QNB Binding Sites in Schizophrenic Brain
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