Pharmacology Biochemistry & Behavior, Vol. 7, pp. 501--506. Copyright © 1977 by ANKHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.
Role of Brain Norepinephrine in Neuroleptic-Induced Catalepsy in Rats T. H O N M A A N D H. F U K U S H I M A
Research and Development Center, Pharmaceuticals Division, Sumitomo Chemical Co., Ltd. 2-1, 4-Chome, Takatsukasa, Takarazuka, Hyogo, 665, Japan ( R e c e i v e d 13 May 1 9 7 7 ) HONMA, T. AND H. FUKUSHIMA. Role of brain norepinephrine in neuroleptic-induced catalepsy in rats. PHARMAC. BIOCHEM. BEHAV. 7(6) 5 0 1 - 5 0 6 , 1977. - Bis (4-methyl-l-homopiperazinylthiocarbonyl) disulfide (FLA 63) and diethyldithiocarbamate enhanced the catalepsy induced by haloperidol in rats. The norepinephrine (NE) content of the diencephalon in rats treated with haloperidol and FLA 63 was less than that in rats treated with haloperidol alone. The dopamine (DA) decrease in the striatum of haloperidol-treated rats was not altered by FLA 63. Intraperitoneally or intraventricularly administered phentolamine enhanced the catalepsy induced by haloperidol, and clonidine counteracted this effect. Propranolol failed to change haloperidol-induced catalepsy. FLA 63 enhanced the catalepsy induced by ID-4708 (a new butyrophenone compound) by an extent greater than that caused by haloperidol. ID-4708 was more potent in increasing the NE turnover than haloperidol. The combined administration of ID-4708 and FLA 63 produced a greater decrease in the NE content than the combination of haloperidol and FLA 63. These results suggest that the neuroleptic-induced catalepsy would be facilitated when the activity of the NE neurons is lowered or when a-receptors are blocked. A decrease in the activity of DA neurons or the blockade of the DA receptors is essential to elicite catalepsy in rats, and the function of NE neurons would play a role in regulating the degree of catalepsy. Catalepsy Dopamine Diethyldithiocarbamate
Norepiniphrine
Haloperidol
N E U R O L E P T I C S i n d u c e catalepsy in l a b o r a t o r y animals, a n d this activity o f n e u r o l e p t i c s m a y be an i n d e x for t h e e x t r a p y r a m i d a l side effects elicited b y n e u r o l e p t i c s in m e n [ 1 9 , 2 9 ] . High doses o f n e u r o l e p t i c s decrease striatal d o p a m i n e ( D A ) c o n t e n t in rats [ 1 4 , 2 3 ] , a n d this decrease appears t o be related to t h e i n t e n s i t y a n d t i m e - c o u r s e o f catalepsy [ 1 7 ] . It is k n o w n t h a t P a r k i n s o n ' s disease is associated w i t h a m a r k e d decrease of striatal D A c o n t e n t [ 4 , 1 2 ] , t h u s e x t r a p y r a m i d a l s y m p t o m s in m e n and t h e catalepsy in e x p e r i m e n t a l a n i m a l s m a y be d u e to a d e f i c i e n c y o f striatal d o p a m i n e r g i c f u n c t i o n . T h e c a t a l e p s y i n d u c e d b y n e u r o l e p t i c s is e n h a n c e d by a - m e t h y l p a r a t y r o sine [9, 10, 2 6 ] . Since a - m e t h y l p a r a t y r o s i n e reduces t h e c o n t e n t o f b o t h DA a n d NE [ 8 , 2 7 ] , it is n o t clear w h e t h e r t h e decrease of D A or t h a t of NE e n h a n c e s t h e n e u r o l e p t i c i n d u c e d catalepsy. In this paper, we have s h o w n t h a t drugs w h i c h relatively m o d i f y t h e f u n c t i o n of NE n e u r o n s m o d i f y t h e n e u r o l e p t i c - i n d u c e d catalepsy in rats.
Assessment of Catalepsy rats
FLA 63
Rats were d e c a p i t a t e d w i t h a guillotine. T h e b r a i n was rapidly r e m o v e d a n d o n an ice-cold b e a k e r dissected i n t o 5 parts: s t r i a t u m , d i e n c e p h a l o n c o n t a i n i n g t h a l a m u s a n d h y p o t h a l a m u s , c e r e b e l l u m , c o r t e x a n d r e m a i n i n g brain. D A a n d NE c o n t e n t s in these b r a i n regions e x c e p t t h e r e m a i n ing b r a i n were m e a s u r e d a f t e r h o m o g e n i z a t i o n . T h e r e m a i n ing b r a i n was discarded. C a t e c h o l a m i n e s were e x t r a c t e d a n d estimated quantitatively with spectrophotofluorometric assay [ 6 ] . Recoveries o f t h e s e c a t e c h o l a m i n e s a d d e d to tissue h o m o g e n a t e ranged b e t w e e n 60 a n d 80% t h r o u g h o u t t h e e x p e r i m e n t s . The c a t e c h o l a m i n e c o n t e n t s of t h e drugt r e a t e d rats were r e p r e s e n t e d as p e r c e n t a g e s of t h e m e a n c a t e c h o l a m i n e c o n t e n t of t h e c o n t r o l g r o u p . Doses a n d t i m e schedules are specified in the R E S U L T S .
Male a l b i n o rats (Wistar strain), weighing 1 9 0 - 2 0 0 g, were used. T h e y were allowed free access to f o o d a n d w a t e r e x c e p t d u r i n g t h e testing p r o c e d u r e s . All e x p e r i m e n t s were carried o u t in a r o o m at 26 ± I ° C a n d 55 _+ 5% h u m i d i t y . 4-8
Phentolamine
Measurement of Catecholamine Content
METHOD
of
Clonidine
catalepsy [ 1 7 , 3 0 ] . B o t h paws of t h e rat were p u t o n a h o r i z o n t a l m e t a l bar, 8.8 cm in height, a n d t h e rat was f o r c e d to rest o n h i n d legs only. T h e d u r a t i o n of this a b n o r m a l p o s i t i o n was m e a s u r e d , a n d t h e d u r a t i o n was r e g a r d e d as an i n t e n s i t y of catalepsy. W h e n catalepsy lasted for m o r e t h a n 3 0 0 sec, t h e d u r a t i o n was r e c o r d e d as 3 0 0 sec. T h e m e a n a n d SEM of the d u r a t i o n of catalepsy were calculated. Doses a n d t i m e schedules are specified in the RESULTS.
Animals and Experimental Conditions
Groups
ID-4708
Microin/ection Experiments were
used
for
assessment
of
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502
H O N M A AND F U K U S H I M A
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FIG. 1. Effects of diethyldithiocarbamate (upper graph) and FLA 63 (lower graph) on the duration of catalepsy induced by haloperidol in rats. FLA 63 (FLA, 2 or 10 mg/kg) or diethylditlriocarbamate (DDC, 80 or 320 mg/kg) was given IP to rats at 15 rain after the SC administration of haloperidol (HPD, 5 mg/kg). Instead of FLA and DDC, a 5% gum arabic solution was given to control rats pretreated with HPD alone. The duration of catalepsy was measured at 2, 4 and 8 hr after the administration of haloperidol. The mean and SEM of the duration of catalepsy were calculated. The vertical bars represent ± SEM. The statistical significances of the differences between the groups of rats treated with HPD + gum arabic and HPD + FLA or DDC were calculated using Student's t-test. *: 0.05>p. **: 0.01>p.
u n d e r s o d i u m p e n t o b a r b i t a l (40 m g / k g IP) anesthesia. The skull was e x p o s e d , and a guide cannula m a d e o f 20 gauge stainless steel was c h r o n i c a l l y i m p l a n t e d on t h e right lateral ventricle: a n t e r i o r 7.8 m m , lateral 1.2 m m , a c c o r d i n g to t h e c o o r d i n a t e s o f De G r o o t [ 1 1 ] , and 4.0 m m ventral to t h e brain surface. The guide cannula was f i x e d to the skull b o n e w i t h d e n t a l c e m e n t . A t least 24 hr were allowed for r e c u p e r a t i o n b e f o r e tests were m a d e . A 10 ~g p h e n t o l a m i n e was dissolved in 5 ul physiological saline and i n j e c t e d to t h e right lateral ventricle t h r o u g h an i n j e c t i o n cannula c o n s i s t e d o f 27 gauge stainless steel pipe, w h i c h p r o t r u d e d a distance o f 0.6 m m b e l o w the t o p o f the guide cannula. The i n j e c t i o n cannula was a t t a c h e d to a m i c r o s y r i n g e with p o l y e t h y l e n e tubing. The same v o l u m e o f saline was i n t r a v e n t r i c u l a r l y given to c o n t r o l rats. The rate o f i n f u s i o n was 1 u l / m i n . A f t e r i n j e c t i o n , t h e i n j e c t i o n cannula r e m a i n e d in the place for 1 min.
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FIG. 2. Effects of FLA 63 and haloperidol (HPD) on the CA contents in 4 regions of rat brain. FLA 63 (2 mg/kg IP) was given to rats at 15 min after the administration of HPD (5 mg/kg SC). Rats were decapitated at 4 hr after the administration of HPD. CA contents are expressed as percentages of control groups. C: control. F: FLA 63. H: HPD. H+F: HPD + FLA 63. The vertical bars represent SEM. Statistical comparison was performed between H group and H+F group with t-test. NS: not significant. Mean DA contents of the striatum, cortex, diencepbalon and cerebellum of control group were 7.10, 0.134, 0.353 and 0.075 ug/g tissue, respectively. Mean NE contents of these 4 regions of control group were 0.213, 0.379, 0.720 and 0.274 ug/g tissue, respectively.
Drugs The following drugs were m i c r o n i z e d prior to use and s u s p e n d e d in 5% gum arabic solution: Haloperidol, ID-4708 (a n e w b u t y r o p h e n o n e c o m p o u n d , 4 - [ 4 - h y d r o x y - 4 - ( 3 - t r i fl u o r o m e t h y l p h e n y l ) - p i p e r i d i n o ] - 2 ' - a m i n o - 4 ' - f l u o r o b u t y r o p h e n o n e . H C 1 ) [18] w h i c h were s y n t h e s i z e d in o u r laborat o r y , and F L A 63 (K and K laboratories). The following drugs were dissolved in physiological saline: D i e t h y l d i t h i o c a r b a m a t e (Nakarai Kagaku), p h e n t o l a m i n e (Ciba-Geigy), clonidine (Tanabe Seiyaku) and p r o p r a n o l o l (ICI). All drugs were given to rats in a v o l u m e o f 1 m l / 1 0 0 g b o d y weight e x c e p t for the intraventricular a d m i n i s t r a t i o n . Doses o f the drug are e x p r e s s e d in t e r m s o f base.
Statistics Statistical c o m p a r i s o n was p e r f o r m e d with S t u d e n t ' s t-test.
CATALEPSY AND BRAIN NOREPINEPHRINE
503
RESULTS
Effects o f FLA 63 and Diethyldithiocarbamate on the Catalepsy Induced by Haloperidol
Q. . . .
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G r o u p s of 8 rats were given haloperidol (5 m g / k g SC), and 15 min later received F L A 63 or diethyldithiocarbamate (DDC) IP. The duration of catalepsy was measured at 2, 4 and 8 hr after the administration of haloperidol. The mean durations of catalepsy in rats are shown in Fig. 1. The catalepsy induced by haloperidol was significantly enhanced by F L A 63 or DDC. F L A 63 at 10 m g / k g was more effective than at 2 mg/kg in enhancing the catalepsy. The e n h a n c e m e n t of catalepsy by 320 mg/kg DDC was greater than that by 80 mg/kg DDC.
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Effects o f Haloperidol and FLA 63 on the Catecholamine Contents in the Rat Brain Each group of rats received haloperidol (5 m g / k g SC), and 15 min later was injected with F L A 63 (2 mg/kg IP). Rats were decapitated at 4 hr after the administration of haloperidol, and the c o n t e n t s of DA and NE in f o u r parts of the brain were estimated. The mean catecholamine (CA) c o n t e n t of vehicle-treated control group was arbitrarily set to 100%. The mean CA c o n t e n t s of the drug-treated groups are shown in Fig. 2 as percentages of CA contents of control group. The administration o f F L A 63 alone increased DA contents in the cortex, diencephalon and cerebellum by 4 2 - 6 1 % and reduced NE c o n t e n t s in these 3 areas to 8 0 - 8 9 % of CA contents of control animals. F L A 63 p r o d u c e d a 5% increase in DA and a 5% decrease in NE in the striatum c o m p a r e d to the CA c o n t e n t s of control rats. The fall in contents of DA and NE caused by haloperidol alone was the greatest in the striatum a m o n g the 4 brain regions. T r e a t m e n t with F L A 63 did not alter the DA c o n t e n t in the striatum c o m p a r e d to that of animals given haloperidol alone. However, F L A 63 elevated DA levels in the cortex, diencephalon and cerebellum by 4 1 - 7 4 % c o m p a r e d to those of haloperidol-treated animals. The c o m b i n e d administration of haloperidol and F L A 63 caused a greater fall in the NE contents in the 4 brain regions than the administration of haloperidol alone.
Effects o f Clonidine, Phentolamine and Propranolol on the Catalepsy Induced by Haloperidol Groups of 8 rats were given haloperidol (2 mg/kg SC), and 4, 5 and 6 hr later the duration of catalepsy was measured. Clonidine (0.002 mg/kg), p h e n t o l a m i n e (10 mg/kg) or propranolol (10 mg/kg) was given IP to rats at 30 min before the first assessment of catalepsy. Other groups of 4 rats with a guide cannula implanted in the right ventricle were injected with haloperidol (2 m g / k g SC), and 4, 5 and 6 hr later the duration of catalepsy was measured. A dose of 10 ug p h e n t o l a m i n e was intraventricularly (IC) given to rats at 15 rain before the first assessment of catalepsy. The mean durations of catalepsy are shown in Fig. 3. Clonidine significantly c o u n t e r a c t e d the catalepsy induced by haloperidol. On the o t h e r hand, p h e n t o l a m i n e enhanced the catalepsy. P h e n t o l a m i n e given IC also enhanced the catalepsy, but the duration of its effect was short. Propranolol did not affect the catalepsy.
EfJect o f FLA 63 on the Catalepsy Induced by ID-4708 Groups of 8 rats were given ID-4708 or haloperidol (2
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mg/kg SC), and 15 min later received F L A 63 (2 mg/kg IP). The mean durations of catalepsy are shown in Fig. 4. The duration of catalepsy in rats treated w i t h ID-4708 was slightly longer than that in haloperidol-treated rats at 2 and 4 hr after the administration of neuroleptics. However, the order of the intensity of catalepsy induced by these two neuroleptics was reversed at 8 hr after the administration. F L A 63 enhanced the catalepsy induced by neuroleptics at each time of the assessment of catalepsy. Especially at 8 hr after the administration, F L A 63 caused a greater enhancement of catalepsy in rats pretreated with ID-4708 compared to haloperidol-treated rats.
Effects o f Combined Administration o f ID-4708 and FLA 63 on the Catecholamine Contents in the Rat Brain Groups of 6 rats were given ID-4708 or haloperidol (2 mg/kg SC), and 15 min later received F L A 63 (2 mg/kg IP). Rats were decapitated for the m e a s u r e m e n t of DA in the striatum and NE in the diencephalon at 8 hr after the administration of neuroleptics. The mean CA contents are shown in Fig. 5 as percentages of those of control rats. Haloperidol p r o d u c e d a slightly greater fall in DA level in
504
HONMA AND FUKUSHIMA
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FIG. 4. Effects of FLA 63 (FLA) on the duration of catalepsy induced by haloperidol (HPD) and ID-4708 (4708). FLA (2 mg/kg IP) was given to rats at 15 min after the administration of HPD (2 mg/kg SC) or 4708 (2 mg/kg SC). Instead of FLA, its vehicle (5% gum arabic solution) was given to groups pretreated with HPD or 4708 alone. Differences between the groups (HPD vs. HPD + FLA, 4708 vs. 4708 + FLA) were tested with t-test. *: 0.05>p. **: 0.01>p. ***: 0.001 >p. t h e s t r i a t u m t h a n ID-4708. H a l o p e r i d o l a n d I D - 4 7 0 8 decreased t h e N E c o n t e n t in t h e d i e n c e p h a l o n to t h e same e x t e n t . T h e D A level in the s t r i a t u m of rats t r e a t e d w i t h each o f these t w o n e u r o l e p t i c s was n o t a f f e c t e d by F L A 63. T h e c o m b i n e d a d m i n i s t r a t i o n o f F L A 63 a n d ID-4708 caused a greater fall in t h e N E c o n t e n t s c o m p a r e d to t h e c o m b i n a t i o n of F L A 63 a n d h a l o p e r i d o l . DISCUSSION T h e p r e s e n t e x p e r i m e n t s s h o w t h a t t h e catalepsy ind u c e d b y h a l o p e r i d o l was e n h a n c e d b y F L A 63 a n d d i e t h y l d i t h i o c a r b a m a t e (DDC). B o t h F L A 63 a n d DDC i n h i b i t t h e activity of d o p a m i n e - ~ - h y d r o x y l a s e (DBH). I n j e c t i o n of t h e s e c o m p o u n d s r e d u c e s t h e b r a i n N E c o n t e n t s in mice a n d rats [5, 7, 2 8 ] . In o u r p r e l i m i n a r y e x p e r i m e n t s , F L A 63 (2 m g / k g ) and DDC (80 m g / k g ) did n o t i n h i b i t t h e e x p l o r a t o r y b e h a v i o r of rats in open-field test [ u n p u b l i s h e d o b s e r v a t i o n s ] . However, these doses of t h e drugs e n h a n c e d t h e catalepsy i n d u c e d b y h a l o p e r i d o l . T h e s e findings s h o w t h a t t h e sedative a c t i o n of D B H i n h i b i t o r s is n o t a cause o f t h e e n h a n c e m e n t of catalepsy. T h e a d m i n i s t r a t i o n of F L A 63 caused an increase in DA a n d a r e d u c t i o n o f NE c o n t e n t in t h e c o r t e x , d i e n c e p h a l o n a n d c e r e b e l l u m . F L A 63 possessed n e i t h e r NE u p t a k e b l o c k i n g activity n o r a n y i n h i b i t o r y effects o n m o n o a m i n e oxidase activity [ 7 ] . T h e s e results i n d i c a t e NE s y n t h e s i s is i n h i b i t a t e d b y F L A 63 in vivo. F L A 63 p r o d u c e d little or n o c h a n g e of CA c o n t e n t s in t h e s t r i a t u m . This ineffectiveness of F L A 63 w o u l d be due to t h e p r e s e n c e of endogeneous DBH inhibitors which strongly inhibit the DBH activity in t h e s t r i a t u m [ 2 2 ] . O u r data suggest t h a t the fall in the activity of NE n e u r o n s e n h a n c e s t h e catalepsy i n d u c e d by h a l o p e r i d o l . T h e a d m i n i s t r a t i o n o f F L A 63 or p - c h l o r o p h e n y l a l a n i n e did n o t p r o d u c e catalepsy in rats [1 7 ] , whereas reserpine p r o d u c e s m o n o a m i n e d e p l e t i o n a n d catalepsy. These results suggest t h a t t h e fall in t h e activity only in NE or s e r o t o n i n n e u r o n s does n o t
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FIG. 5. Effects of combined administration of haloperidol (ttPD) or ID-4708 (4708) and FLA 63 (FLA) on the contents of DA in the striatum and NE in the diencephalon in rats. t"LA (2 mg/kg IP) was given to rats at 15 min after the administration of tlPD (2 mg/kg SC) or 4708 (2 mg/kg SC). Rats were decapitated at 8 hr after the administration of neuroleptics. The mean contents of DA and NF of control rats were 9.15 and 0.725 ug/g tissue, respectively. Differences between control and drug-treated groups were tested with t-test and its results are shown above the column. Differences between the groups (HPD vs. HPD + FLA, 4708 vs. 4708 + t:LA) were tested with t-test and its results are shown in the column. NS: not significant. p r o d u c e catalepsy. N e u r o l e p t i c s are t h o u g h t to b l o c k the D A r e c e p t o r s in t h e s t r i a t u m [ 1 ]. A f t e r t h e 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 , t h e s y n t h e s i s a n d release of DA are a c c e l e r a t e d p r o b a b l y t h r o u g h a f e e d b a c k a c t i v a t i o n [ 2 ] . If t h e s y n t h e s i s o f DA is n o t a c c e l e r a t e d c o m p a r a b l y to t h e a c c e l e r a t i o n o f its release and d e s t r u c t i o n , t h e D A s t o r e d in t h e nerve t e r m i n a l s will be r e d u c e d gradually. This w o u l d be t h e r e a s o n for the r e d u c t i o n o f D A by t h e a d m i n i s t r a t i o n of relatively high doses of h a l o p e r i d o l in rats. However, a f u r t h e r investigation is r e q u i r e d for t h e e x p l a n a t i o n o f this p o i n t . The h a l o p e r i d o l - i n d u c e d catelepsy was e n h a n c e d by p h e n t o l a m i n e a n d a n t a g o n i z e d by clonidine. P r o p r a n o l o l h a d n o effect on it. As d e m o n s t r a t e d previously, c l o n i d i n e possesses a c e n t r a l a - s y m p a t h o m i m e t i c a c t i o n , a n d p h e n t o l -
CATALEPSY AND BRAIN NOREPINEPHRINE
505
a m i n e a n t a g o n i z e s the e f f e c t o f clonidine on E E G and b e h a v i o r in animals [15, 16, 2 5 ] . There are data indicating t h a t p r o p r a n o l o l m a y possess an a c t i o n in the central nervous s y s t e m s [ 13, 20, 2 1 ] . T h e r e f o r e , our o b s e r v a t i o n s suggest that t h e catalepsy i n d u c e d b y h a l o p e r i d o l is e n h a n c e d b y the b l o c k a d e o f the central a-adrenergic r e c e p t o r s , and a n t a g o n i z e d by t h e s t i m u l a t i o n o f t h e m . A specific d e p l e t i o n of cerebral NE by 6 - h y d r o x y d o p a m i n e significantly e n h a n c e d the catalepsy i n d u c e d b y h a l o p e r i d o l in rats [ 2 4 ] . This result s u p p o r t s t h e idea t h a t t h e f u n c t i o n of NE n e u r o n s regulates the degree o f catalepsy. F r o m the lack o f e f f e c t o f p r o p r a n o l o l on the catalepsy, the t3-adrenergic r e c e p t o r s m a y n o t be involved in t h e regulation o f catalepsy. F L A 63 e n h a n c e d the catalepsy i n d u c e d b y ID-4708 b y an e x t e n t greater t h a n t h a t caused by h a l o p e r i d o l . At the same t i m e , the decrease in NE c o n t e n t in rats t r e a t e d w i t h F L A 63 and ID-4708 was greater t h a n that in rats t r e a t e d w i t h F L A 63 and h a l o p e r i d o l . The rapid d i s a p p e a r a n c e o f NE in rats t r e a t e d w i t h F L A 63 in c o m b i n a t i o n w i t h n e u r o l e p t i c s is due to the a c c e l e r a t e d t u r n o v e r o f NE w h i c h
is caused by n e u r o l e p t i c s [ 3 ] . ID-4708 w o u l d be m o r e p o t e n t in accelerating the t u r n o v e r o f NE in t h e rat brain t h a n h a l o p e r i d o l . This p r o p e r t y o f ID-4708 m a y be t h e cause o f r e m a r k a b l e decrease in NE c o n t e n t and the p r o n o u n c e d p o t e n t i a t i o n o f catalepsy w h e n a d m i n i s t e r e d t o rats with F L A 63. The d i f f e r e n t e f f e c t o f F L A 63 on the catalepsy i n d u c e d b y h a l o p e r i d o l and I D 4 7 0 8 s u p p o r t s the idea t h a t the n e u r o l e p t i c - i n d u c e d catalepsy is p o t e n t i a t e d by the decrease in the activity o f t h e NE n e u r o n s . F r o m our e x p e r i m e n t s , it was c o n c l u d e d that a decrease in the activity o f DA n e u r o n s or the b l o c k a d e o f DA r e c e p t o r s is essential to elicite catalepsy and t h a t t h e f u n c t i o n o f NE n e u r o n s play a role in regulating the degree o f catalepsy in rats.
ACKNOWLEDGEMENTS The authors thank Dr. H. Yamamoto for his advice in the course of this investigation and Miss Y. Moriyasu for excellent technical assistance.
REFERENCES
1. And6n, N. - E . , G. Bartholini, H. Corrodi, B. Csillik, E. De Robertis, A. Dresse, K. Fuxe, M. A. Gerebtzoff, J. Glowinski, E. Hansson, T. H6kfelt, J. Renson and B. E. Roos. Histological and molecular biochemistry. In: The Neuroleptics, edited by D. P. Bobon, P. A. J. Janssen and J. Bobon. Basel: S. Karger, 1970, pp. 1 11. 2. And6n, N. - E . , H. Butcher, H. Corrodi, K. Fuxe and U. Ungerstedt. Receptor activity and turnover of dopamine and norepinephrine after neuroleptics. Eur. J. Pharrnac. 11: 303-314, 1970. 3. Bartholini, G., H. H. Keller and A. Pletscher. Effects of neuroleptics on endogeneous norepinephrine in rat brain. Neuropharmacology 1 2 : 7 5 1 - 7 5 6 , 1973. 4. Bernheimer, H., W. Birkmayer and O. Hornykiewicz. Zur Biochemie des Parkinsonsyndroms des Menschen. Klin. Wschr. 41: 465-469, 1963. 5. Carlsson, A., M. Lindqvist, K. Fuxe and T. H6kfelt. Histochemical and biochemical effects of diethyldithiocarbamate on tissue catecholamines. J. Pharm. Pharmac. 18:60 62, 1966. 6. Chang, C. C. A sensitive method for spectrophotofluorometric assay of catecholamines. Int. J. Neuropharmac. 3 : 6 4 3 649, 1964. 7. Corrodi, H., K. Fuxe, B. Hamberger and A. Ljungdahl. Studies on central and peripheral noradrenaline neurons using a new dopamine-/3-hydroxylase inhibitor. Eur. J. Pharmac. 12: 145 155, 1970. 8. Corrodi, H. and L. C. F. Hanson. Central effects of an inhibitor o f t y r o s i n e hydroxylation. Psychopharmacologia 10: 116 125, 1966. 9. Costall, B. and R. J. Naylor. The importance of the ascending dopaminergic systems to the extrapyramidal and mesolimbic brain areas for the cataleptic action of the neuroleptics and cholinergic agents. Neuropharrnacology 13:353 364, 1974. 10. Costall, B. and R. J. Naylor. Detection of the neuroleptic properties of clozapine, sulpiride and thioridazine. Psychopharmacologia 43:69 74, 1975. 11. De Groot, J. The Rat Forebrain in Stereotaxic Coordinates. Amsterdam: N. V. Noord-Hollandsche Unitgevers Maatschappij, 1963. 12. Ehringer, H. and O. Hornykiewicz. Verteilung von Noradrenalin und ihr Verhalten bei Erkrankungen des extrapyramidalen Systems. Klin. Wschr. 38: 1236-1239, 1960.
13. Estler, C. - J . and H. P. T. Ammon. Der Einfluss yon Propranolol auf die durch Methamphetamin verursachten J~ndereungen yon Function und Stoffwechsel des Gehirns. jr. Neurochem. 14: 799-805, 1967. 14. Falck. B., L. Nordgren and E. Rosengren. Effect of haloperidol on monoamines in brain and heart. Int. J. Neuropharmac. 8: 631 634, 1969. 15. Finch. L., R. E. Buckingham, R. A. Moore and T. J. Bucher. Evidence for a central a-sympathomimetic action of clonidine in rats. J. Pharm. Pharmac. 27: 181-186, 1975. 16. Florio, V., L. Bianchi and V. G. Longo. A study of the central effects of sympathomimetic drugs: EEG and behavioural investigations on clonidine and naphazoline. Neuropharmacology 14:707 714, 1975. 17. Honma, T. and H. Fukushima. Correlation between catalepsy and dopamine decrease in the rat striatum induced by neuroleptics. Neuropharmacology 15 : 601-607, 1976. 18. Honma, T., K. Sasajima, K. Ono, S. Kitagawa, Sh. lnaba and H. Yamamoto. Synthesis and preliminary pharmacology of a novel butyrophenone derivative, ID-4708. Arzneimittel-Forseh. 24: 1248-1256, 1974. 19. Julou, L. On the interaction between neuroleptics and antiparkinson drugs. In: The Neuroleptics, edited by D. P. Bobon, P. A. J. Janssen and J. Bobon. Basel: S. Karger, 1970, p. 50-54. 20. Leszkovszky, G. and L. Tardos. Some effects of propranolol on the central nervous system. J. Pharm. Pharmac. 17:518 520, 1965. 21. Murmann, W., L. Almirante and M. Saccani-Guelfi. Central nervous system effects of four ~3-adrenergic receptor blocking agents. J. Pharm. Pharmac. 18:317 318, 1966. 22. Nagatsu, T., H. Kuzuya and H. Hidaka. Inhibition of dopamine-CNlydroxylase by sulfllydryl compounds and the nature of the natural inhibitors. Biochim. Biophys. Acta 139: 319 327, 1967. 23. O'Keeffe, R., D. F. Scharman and M. Vogt. Effect of drugs used in psychosis on cerebral dopamine metabolism. Br. J. Pharmac. 38: 287-304, 1970. 24. Pycock, C. Noradrenergic influences on dopamine-dependent behaviors in rats. Br. J. Pharrnae. 57: 424P, 1976. 25. Schmitt, H. Actions des alpha-sympathomim6tiqies sur les structures nerveuses. Actual. Pharmac. 24:93 131, 1971.
506 26. Shore, P. A. and R. L. Dorris. On a prime role for newly synthesized dopamine in striatal function. Eur. J. Pharmac. 30: 315-318, 1975. 27. Spector, S. A., A. Sjoerdsma and S. Udenfriend. Blockade of endogenous norepinephrine synthesis by a-methyltyrosine, an inhibitor of tyrosine hydroxylase. J. Pharmac. exp. Ther. 147: 86-95, 1965. 28. Svensson, T. H. and B. Waldeck. On the significance of central noradrenaline for motor activity: Experiments with a new dopamine-t3-hydroxylase inhibitor. Eur. J. Pharmac. 7: 278-282, 1969.
H O N M A AND F U K U S H I M A 29. Tedeschi, D. H. Criteria for the selection of pharmacological test procedures useful in the evaluation of neuroleptic drugs. In: The Present Status o f Psyehotropic Drugs, edited by A. Cerletti and F. J. Bov~. Amsterdam: Excerpta Medica, 1969, pp. 145-153. 30. Wirth, R., R. G6sswald, U. H6rlein, K1. H. Risse and It. Kreiskott. Zur Pharmacologie acylierter Phenothiazin-Derivat. Arehsint. Pharmaeodyn. ThOr. 115: 1- 31, 1958.
Role of Brain Norepinephrine in Neuroleptic-Induced Catalepsy in Rats T. H O N M A A N D H. F U K U S H I M A
Research and Development Center, Pharmaceuticals Division, Sumitomo Chemical Co., Ltd. 2-1, 4-Chome, Takatsukasa, Takarazuka, Hyogo, 665, Japan ( R e c e i v e d 13 May 1 9 7 7 ) HONMA, T. AND H. FUKUSHIMA. Role of brain norepinephrine in neuroleptic-induced catalepsy in rats. PHARMAC. BIOCHEM. BEHAV. 7(6) 5 0 1 - 5 0 6 , 1977. - Bis (4-methyl-l-homopiperazinylthiocarbonyl) disulfide (FLA 63) and diethyldithiocarbamate enhanced the catalepsy induced by haloperidol in rats. The norepinephrine (NE) content of the diencephalon in rats treated with haloperidol and FLA 63 was less than that in rats treated with haloperidol alone. The dopamine (DA) decrease in the striatum of haloperidol-treated rats was not altered by FLA 63. Intraperitoneally or intraventricularly administered phentolamine enhanced the catalepsy induced by haloperidol, and clonidine counteracted this effect. Propranolol failed to change haloperidol-induced catalepsy. FLA 63 enhanced the catalepsy induced by ID-4708 (a new butyrophenone compound) by an extent greater than that caused by haloperidol. ID-4708 was more potent in increasing the NE turnover than haloperidol. The combined administration of ID-4708 and FLA 63 produced a greater decrease in the NE content than the combination of haloperidol and FLA 63. These results suggest that the neuroleptic-induced catalepsy would be facilitated when the activity of the NE neurons is lowered or when a-receptors are blocked. A decrease in the activity of DA neurons or the blockade of the DA receptors is essential to elicite catalepsy in rats, and the function of NE neurons would play a role in regulating the degree of catalepsy. Catalepsy Dopamine Diethyldithiocarbamate
Norepiniphrine
Haloperidol
N E U R O L E P T I C S i n d u c e catalepsy in l a b o r a t o r y animals, a n d this activity o f n e u r o l e p t i c s m a y be an i n d e x for t h e e x t r a p y r a m i d a l side effects elicited b y n e u r o l e p t i c s in m e n [ 1 9 , 2 9 ] . High doses o f n e u r o l e p t i c s decrease striatal d o p a m i n e ( D A ) c o n t e n t in rats [ 1 4 , 2 3 ] , a n d this decrease appears t o be related to t h e i n t e n s i t y a n d t i m e - c o u r s e o f catalepsy [ 1 7 ] . It is k n o w n t h a t P a r k i n s o n ' s disease is associated w i t h a m a r k e d decrease of striatal D A c o n t e n t [ 4 , 1 2 ] , t h u s e x t r a p y r a m i d a l s y m p t o m s in m e n and t h e catalepsy in e x p e r i m e n t a l a n i m a l s m a y be d u e to a d e f i c i e n c y o f striatal d o p a m i n e r g i c f u n c t i o n . T h e c a t a l e p s y i n d u c e d b y n e u r o l e p t i c s is e n h a n c e d by a - m e t h y l p a r a t y r o sine [9, 10, 2 6 ] . Since a - m e t h y l p a r a t y r o s i n e reduces t h e c o n t e n t o f b o t h DA a n d NE [ 8 , 2 7 ] , it is n o t clear w h e t h e r t h e decrease of D A or t h a t of NE e n h a n c e s t h e n e u r o l e p t i c i n d u c e d catalepsy. In this paper, we have s h o w n t h a t drugs w h i c h relatively m o d i f y t h e f u n c t i o n of NE n e u r o n s m o d i f y t h e n e u r o l e p t i c - i n d u c e d catalepsy in rats.
Assessment of Catalepsy rats
FLA 63
Rats were d e c a p i t a t e d w i t h a guillotine. T h e b r a i n was rapidly r e m o v e d a n d o n an ice-cold b e a k e r dissected i n t o 5 parts: s t r i a t u m , d i e n c e p h a l o n c o n t a i n i n g t h a l a m u s a n d h y p o t h a l a m u s , c e r e b e l l u m , c o r t e x a n d r e m a i n i n g brain. D A a n d NE c o n t e n t s in these b r a i n regions e x c e p t t h e r e m a i n ing b r a i n were m e a s u r e d a f t e r h o m o g e n i z a t i o n . T h e r e m a i n ing b r a i n was discarded. C a t e c h o l a m i n e s were e x t r a c t e d a n d estimated quantitatively with spectrophotofluorometric assay [ 6 ] . Recoveries o f t h e s e c a t e c h o l a m i n e s a d d e d to tissue h o m o g e n a t e ranged b e t w e e n 60 a n d 80% t h r o u g h o u t t h e e x p e r i m e n t s . The c a t e c h o l a m i n e c o n t e n t s of t h e drugt r e a t e d rats were r e p r e s e n t e d as p e r c e n t a g e s of t h e m e a n c a t e c h o l a m i n e c o n t e n t of t h e c o n t r o l g r o u p . Doses a n d t i m e schedules are specified in the R E S U L T S .
Male a l b i n o rats (Wistar strain), weighing 1 9 0 - 2 0 0 g, were used. T h e y were allowed free access to f o o d a n d w a t e r e x c e p t d u r i n g t h e testing p r o c e d u r e s . All e x p e r i m e n t s were carried o u t in a r o o m at 26 ± I ° C a n d 55 _+ 5% h u m i d i t y . 4-8
Phentolamine
Measurement of Catecholamine Content
METHOD
of
Clonidine
catalepsy [ 1 7 , 3 0 ] . B o t h paws of t h e rat were p u t o n a h o r i z o n t a l m e t a l bar, 8.8 cm in height, a n d t h e rat was f o r c e d to rest o n h i n d legs only. T h e d u r a t i o n of this a b n o r m a l p o s i t i o n was m e a s u r e d , a n d t h e d u r a t i o n was r e g a r d e d as an i n t e n s i t y of catalepsy. W h e n catalepsy lasted for m o r e t h a n 3 0 0 sec, t h e d u r a t i o n was r e c o r d e d as 3 0 0 sec. T h e m e a n a n d SEM of the d u r a t i o n of catalepsy were calculated. Doses a n d t i m e schedules are specified in the RESULTS.
Animals and Experimental Conditions
Groups
ID-4708
Microin/ection Experiments were
used
for
assessment
of
The rat was placed in a s t a n d a r d s t e r e o t a x i c i n s t r u m e n t 501
502
H O N M A AND F U K U S H I M A
:3O0 O . . . .
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u o
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o
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lOO-
~.f"
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'~ ioo-
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o
2
hr
4
hr
8
hr
300
O[] . . . . HPO
u
,('1" 1,'
i
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~
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g "~ ioo-
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FIG. 1. Effects of diethyldithiocarbamate (upper graph) and FLA 63 (lower graph) on the duration of catalepsy induced by haloperidol in rats. FLA 63 (FLA, 2 or 10 mg/kg) or diethylditlriocarbamate (DDC, 80 or 320 mg/kg) was given IP to rats at 15 rain after the SC administration of haloperidol (HPD, 5 mg/kg). Instead of FLA and DDC, a 5% gum arabic solution was given to control rats pretreated with HPD alone. The duration of catalepsy was measured at 2, 4 and 8 hr after the administration of haloperidol. The mean and SEM of the duration of catalepsy were calculated. The vertical bars represent ± SEM. The statistical significances of the differences between the groups of rats treated with HPD + gum arabic and HPD + FLA or DDC were calculated using Student's t-test. *: 0.05>p. **: 0.01>p.
u n d e r s o d i u m p e n t o b a r b i t a l (40 m g / k g IP) anesthesia. The skull was e x p o s e d , and a guide cannula m a d e o f 20 gauge stainless steel was c h r o n i c a l l y i m p l a n t e d on t h e right lateral ventricle: a n t e r i o r 7.8 m m , lateral 1.2 m m , a c c o r d i n g to t h e c o o r d i n a t e s o f De G r o o t [ 1 1 ] , and 4.0 m m ventral to t h e brain surface. The guide cannula was f i x e d to the skull b o n e w i t h d e n t a l c e m e n t . A t least 24 hr were allowed for r e c u p e r a t i o n b e f o r e tests were m a d e . A 10 ~g p h e n t o l a m i n e was dissolved in 5 ul physiological saline and i n j e c t e d to t h e right lateral ventricle t h r o u g h an i n j e c t i o n cannula c o n s i s t e d o f 27 gauge stainless steel pipe, w h i c h p r o t r u d e d a distance o f 0.6 m m b e l o w the t o p o f the guide cannula. The i n j e c t i o n cannula was a t t a c h e d to a m i c r o s y r i n g e with p o l y e t h y l e n e tubing. The same v o l u m e o f saline was i n t r a v e n t r i c u l a r l y given to c o n t r o l rats. The rate o f i n f u s i o n was 1 u l / m i n . A f t e r i n j e c t i o n , t h e i n j e c t i o n cannula r e m a i n e d in the place for 1 min.
C FHH
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+
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FIG. 2. Effects of FLA 63 and haloperidol (HPD) on the CA contents in 4 regions of rat brain. FLA 63 (2 mg/kg IP) was given to rats at 15 min after the administration of HPD (5 mg/kg SC). Rats were decapitated at 4 hr after the administration of HPD. CA contents are expressed as percentages of control groups. C: control. F: FLA 63. H: HPD. H+F: HPD + FLA 63. The vertical bars represent SEM. Statistical comparison was performed between H group and H+F group with t-test. NS: not significant. Mean DA contents of the striatum, cortex, diencepbalon and cerebellum of control group were 7.10, 0.134, 0.353 and 0.075 ug/g tissue, respectively. Mean NE contents of these 4 regions of control group were 0.213, 0.379, 0.720 and 0.274 ug/g tissue, respectively.
Drugs The following drugs were m i c r o n i z e d prior to use and s u s p e n d e d in 5% gum arabic solution: Haloperidol, ID-4708 (a n e w b u t y r o p h e n o n e c o m p o u n d , 4 - [ 4 - h y d r o x y - 4 - ( 3 - t r i fl u o r o m e t h y l p h e n y l ) - p i p e r i d i n o ] - 2 ' - a m i n o - 4 ' - f l u o r o b u t y r o p h e n o n e . H C 1 ) [18] w h i c h were s y n t h e s i z e d in o u r laborat o r y , and F L A 63 (K and K laboratories). The following drugs were dissolved in physiological saline: D i e t h y l d i t h i o c a r b a m a t e (Nakarai Kagaku), p h e n t o l a m i n e (Ciba-Geigy), clonidine (Tanabe Seiyaku) and p r o p r a n o l o l (ICI). All drugs were given to rats in a v o l u m e o f 1 m l / 1 0 0 g b o d y weight e x c e p t for the intraventricular a d m i n i s t r a t i o n . Doses o f the drug are e x p r e s s e d in t e r m s o f base.
Statistics Statistical c o m p a r i s o n was p e r f o r m e d with S t u d e n t ' s t-test.
CATALEPSY AND BRAIN NOREPINEPHRINE
503
RESULTS
Effects o f FLA 63 and Diethyldithiocarbamate on the Catalepsy Induced by Haloperidol
Q. . . .
200= t- . . . . . .
G r o u p s of 8 rats were given haloperidol (5 m g / k g SC), and 15 min later received F L A 63 or diethyldithiocarbamate (DDC) IP. The duration of catalepsy was measured at 2, 4 and 8 hr after the administration of haloperidol. The mean durations of catalepsy in rats are shown in Fig. 1. The catalepsy induced by haloperidol was significantly enhanced by F L A 63 or DDC. F L A 63 at 10 m g / k g was more effective than at 2 mg/kg in enhancing the catalepsy. The e n h a n c e m e n t of catalepsy by 320 mg/kg DDC was greater than that by 80 mg/kg DDC.
O
4PD * Phentolomine
A u (I
o.
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÷ Propronolol
A ........... -~
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HPD+
,oo.
Clonidlne
g t*
2 .........
4 ..........
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0
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hr
5
hr
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hr
300
Effects o f Haloperidol and FLA 63 on the Catecholamine Contents in the Rat Brain Each group of rats received haloperidol (5 m g / k g SC), and 15 min later was injected with F L A 63 (2 mg/kg IP). Rats were decapitated at 4 hr after the administration of haloperidol, and the c o n t e n t s of DA and NE in f o u r parts of the brain were estimated. The mean catecholamine (CA) c o n t e n t of vehicle-treated control group was arbitrarily set to 100%. The mean CA c o n t e n t s of the drug-treated groups are shown in Fig. 2 as percentages of CA contents of control group. The administration o f F L A 63 alone increased DA contents in the cortex, diencephalon and cerebellum by 4 2 - 6 1 % and reduced NE c o n t e n t s in these 3 areas to 8 0 - 8 9 % of CA contents of control animals. F L A 63 p r o d u c e d a 5% increase in DA and a 5% decrease in NE in the striatum c o m p a r e d to the CA c o n t e n t s of control rats. The fall in contents of DA and NE caused by haloperidol alone was the greatest in the striatum a m o n g the 4 brain regions. T r e a t m e n t with F L A 63 did not alter the DA c o n t e n t in the striatum c o m p a r e d to that of animals given haloperidol alone. However, F L A 63 elevated DA levels in the cortex, diencephalon and cerebellum by 4 1 - 7 4 % c o m p a r e d to those of haloperidol-treated animals. The c o m b i n e d administration of haloperidol and F L A 63 caused a greater fall in the NE contents in the 4 brain regions than the administration of haloperidol alone.
Effects o f Clonidine, Phentolamine and Propranolol on the Catalepsy Induced by Haloperidol Groups of 8 rats were given haloperidol (2 mg/kg SC), and 4, 5 and 6 hr later the duration of catalepsy was measured. Clonidine (0.002 mg/kg), p h e n t o l a m i n e (10 mg/kg) or propranolol (10 mg/kg) was given IP to rats at 30 min before the first assessment of catalepsy. Other groups of 4 rats with a guide cannula implanted in the right ventricle were injected with haloperidol (2 m g / k g SC), and 4, 5 and 6 hr later the duration of catalepsy was measured. A dose of 10 ug p h e n t o l a m i n e was intraventricularly (IC) given to rats at 15 rain before the first assessment of catalepsy. The mean durations of catalepsy are shown in Fig. 3. Clonidine significantly c o u n t e r a c t e d the catalepsy induced by haloperidol. On the o t h e r hand, p h e n t o l a m i n e enhanced the catalepsy. P h e n t o l a m i n e given IC also enhanced the catalepsy, but the duration of its effect was short. Propranolol did not affect the catalepsy.
EfJect o f FLA 63 on the Catalepsy Induced by ID-4708 Groups of 8 rats were given ID-4708 or haloperidol (2
u
D--------O HPD+
IOhentolornlne
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iII
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HPD + saline
[c
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?, FIG. 3. Effects of clonidine, phentolamine and propranolol on the duration of catalepsy induced by haloperidol (HPD). Rats were given HPD (2 mg/kg SC) and 3.5 hr later injected with saline, clonidine (0.002 mg/kg IP), phentolamine (10 mg/kg IP) or propranolol (10 mg/kg IP). Other groups of rats were given HPD (2 mg/kg SC) and administered 10 ~g phentolamine or its solvent (saline) intraventricularly (IC) at 15 min before the first assessment of catalepsy. The duration of catalepsy was measured at 4, 5 and 6 hr after the administration of HPD. Differences between the treatments with HPD + saline and HPD + clonidine, phentolamine or propranolol were tested with t-test. *: 0.05>p. **: 0.01>p.
mg/kg SC), and 15 min later received F L A 63 (2 mg/kg IP). The mean durations of catalepsy are shown in Fig. 4. The duration of catalepsy in rats treated w i t h ID-4708 was slightly longer than that in haloperidol-treated rats at 2 and 4 hr after the administration of neuroleptics. However, the order of the intensity of catalepsy induced by these two neuroleptics was reversed at 8 hr after the administration. F L A 63 enhanced the catalepsy induced by neuroleptics at each time of the assessment of catalepsy. Especially at 8 hr after the administration, F L A 63 caused a greater enhancement of catalepsy in rats pretreated with ID-4708 compared to haloperidol-treated rats.
Effects o f Combined Administration o f ID-4708 and FLA 63 on the Catecholamine Contents in the Rat Brain Groups of 6 rats were given ID-4708 or haloperidol (2 mg/kg SC), and 15 min later received F L A 63 (2 mg/kg IP). Rats were decapitated for the m e a s u r e m e n t of DA in the striatum and NE in the diencephalon at 8 hr after the administration of neuroleptics. The mean CA contents are shown in Fig. 5 as percentages of those of control rats. Haloperidol p r o d u c e d a slightly greater fall in DA level in
504
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FIG. 4. Effects of FLA 63 (FLA) on the duration of catalepsy induced by haloperidol (HPD) and ID-4708 (4708). FLA (2 mg/kg IP) was given to rats at 15 min after the administration of HPD (2 mg/kg SC) or 4708 (2 mg/kg SC). Instead of FLA, its vehicle (5% gum arabic solution) was given to groups pretreated with HPD or 4708 alone. Differences between the groups (HPD vs. HPD + FLA, 4708 vs. 4708 + FLA) were tested with t-test. *: 0.05>p. **: 0.01>p. ***: 0.001 >p. t h e s t r i a t u m t h a n ID-4708. H a l o p e r i d o l a n d I D - 4 7 0 8 decreased t h e N E c o n t e n t in t h e d i e n c e p h a l o n to t h e same e x t e n t . T h e D A level in the s t r i a t u m of rats t r e a t e d w i t h each o f these t w o n e u r o l e p t i c s was n o t a f f e c t e d by F L A 63. T h e c o m b i n e d a d m i n i s t r a t i o n o f F L A 63 a n d ID-4708 caused a greater fall in t h e N E c o n t e n t s c o m p a r e d to t h e c o m b i n a t i o n of F L A 63 a n d h a l o p e r i d o l . DISCUSSION T h e p r e s e n t e x p e r i m e n t s s h o w t h a t t h e catalepsy ind u c e d b y h a l o p e r i d o l was e n h a n c e d b y F L A 63 a n d d i e t h y l d i t h i o c a r b a m a t e (DDC). B o t h F L A 63 a n d DDC i n h i b i t t h e activity of d o p a m i n e - ~ - h y d r o x y l a s e (DBH). I n j e c t i o n of t h e s e c o m p o u n d s r e d u c e s t h e b r a i n N E c o n t e n t s in mice a n d rats [5, 7, 2 8 ] . In o u r p r e l i m i n a r y e x p e r i m e n t s , F L A 63 (2 m g / k g ) and DDC (80 m g / k g ) did n o t i n h i b i t t h e e x p l o r a t o r y b e h a v i o r of rats in open-field test [ u n p u b l i s h e d o b s e r v a t i o n s ] . However, these doses of t h e drugs e n h a n c e d t h e catalepsy i n d u c e d b y h a l o p e r i d o l . T h e s e findings s h o w t h a t t h e sedative a c t i o n of D B H i n h i b i t o r s is n o t a cause o f t h e e n h a n c e m e n t of catalepsy. T h e a d m i n i s t r a t i o n of F L A 63 caused an increase in DA a n d a r e d u c t i o n o f NE c o n t e n t in t h e c o r t e x , d i e n c e p h a l o n a n d c e r e b e l l u m . F L A 63 possessed n e i t h e r NE u p t a k e b l o c k i n g activity n o r a n y i n h i b i t o r y effects o n m o n o a m i n e oxidase activity [ 7 ] . T h e s e results i n d i c a t e NE s y n t h e s i s is i n h i b i t a t e d b y F L A 63 in vivo. F L A 63 p r o d u c e d little or n o c h a n g e of CA c o n t e n t s in t h e s t r i a t u m . This ineffectiveness of F L A 63 w o u l d be due to t h e p r e s e n c e of endogeneous DBH inhibitors which strongly inhibit the DBH activity in t h e s t r i a t u m [ 2 2 ] . O u r data suggest t h a t the fall in the activity of NE n e u r o n s e n h a n c e s t h e catalepsy i n d u c e d by h a l o p e r i d o l . T h e a d m i n i s t r a t i o n o f F L A 63 or p - c h l o r o p h e n y l a l a n i n e did n o t p r o d u c e catalepsy in rats [1 7 ] , whereas reserpine p r o d u c e s m o n o a m i n e d e p l e t i o n a n d catalepsy. These results suggest t h a t t h e fall in t h e activity only in NE or s e r o t o n i n n e u r o n s does n o t
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FIG. 5. Effects of combined administration of haloperidol (ttPD) or ID-4708 (4708) and FLA 63 (FLA) on the contents of DA in the striatum and NE in the diencephalon in rats. t"LA (2 mg/kg IP) was given to rats at 15 min after the administration of tlPD (2 mg/kg SC) or 4708 (2 mg/kg SC). Rats were decapitated at 8 hr after the administration of neuroleptics. The mean contents of DA and NF of control rats were 9.15 and 0.725 ug/g tissue, respectively. Differences between control and drug-treated groups were tested with t-test and its results are shown above the column. Differences between the groups (HPD vs. HPD + FLA, 4708 vs. 4708 + t:LA) were tested with t-test and its results are shown in the column. NS: not significant. p r o d u c e catalepsy. N e u r o l e p t i c s are t h o u g h t to b l o c k the D A r e c e p t o r s in t h e s t r i a t u m [ 1 ]. A f t e r t h e 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 , t h e s y n t h e s i s a n d release of DA are a c c e l e r a t e d p r o b a b l y t h r o u g h a f e e d b a c k a c t i v a t i o n [ 2 ] . If t h e s y n t h e s i s o f DA is n o t a c c e l e r a t e d c o m p a r a b l y to t h e a c c e l e r a t i o n o f its release and d e s t r u c t i o n , t h e D A s t o r e d in t h e nerve t e r m i n a l s will be r e d u c e d gradually. This w o u l d be t h e r e a s o n for the r e d u c t i o n o f D A by t h e a d m i n i s t r a t i o n of relatively high doses of h a l o p e r i d o l in rats. However, a f u r t h e r investigation is r e q u i r e d for t h e e x p l a n a t i o n o f this p o i n t . The h a l o p e r i d o l - i n d u c e d catelepsy was e n h a n c e d by p h e n t o l a m i n e a n d a n t a g o n i z e d by clonidine. P r o p r a n o l o l h a d n o effect on it. As d e m o n s t r a t e d previously, c l o n i d i n e possesses a c e n t r a l a - s y m p a t h o m i m e t i c a c t i o n , a n d p h e n t o l -
CATALEPSY AND BRAIN NOREPINEPHRINE
505
a m i n e a n t a g o n i z e s the e f f e c t o f clonidine on E E G and b e h a v i o r in animals [15, 16, 2 5 ] . There are data indicating t h a t p r o p r a n o l o l m a y possess an a c t i o n in the central nervous s y s t e m s [ 13, 20, 2 1 ] . T h e r e f o r e , our o b s e r v a t i o n s suggest that t h e catalepsy i n d u c e d b y h a l o p e r i d o l is e n h a n c e d b y the b l o c k a d e o f the central a-adrenergic r e c e p t o r s , and a n t a g o n i z e d by t h e s t i m u l a t i o n o f t h e m . A specific d e p l e t i o n of cerebral NE by 6 - h y d r o x y d o p a m i n e significantly e n h a n c e d the catalepsy i n d u c e d b y h a l o p e r i d o l in rats [ 2 4 ] . This result s u p p o r t s t h e idea t h a t t h e f u n c t i o n of NE n e u r o n s regulates the degree o f catalepsy. F r o m the lack o f e f f e c t o f p r o p r a n o l o l on the catalepsy, the t3-adrenergic r e c e p t o r s m a y n o t be involved in t h e regulation o f catalepsy. F L A 63 e n h a n c e d the catalepsy i n d u c e d b y ID-4708 b y an e x t e n t greater t h a n t h a t caused by h a l o p e r i d o l . At the same t i m e , the decrease in NE c o n t e n t in rats t r e a t e d w i t h F L A 63 and ID-4708 was greater t h a n that in rats t r e a t e d w i t h F L A 63 and h a l o p e r i d o l . The rapid d i s a p p e a r a n c e o f NE in rats t r e a t e d w i t h F L A 63 in c o m b i n a t i o n w i t h n e u r o l e p t i c s is due to the a c c e l e r a t e d t u r n o v e r o f NE w h i c h
is caused by n e u r o l e p t i c s [ 3 ] . ID-4708 w o u l d be m o r e p o t e n t in accelerating the t u r n o v e r o f NE in t h e rat brain t h a n h a l o p e r i d o l . This p r o p e r t y o f ID-4708 m a y be t h e cause o f r e m a r k a b l e decrease in NE c o n t e n t and the p r o n o u n c e d p o t e n t i a t i o n o f catalepsy w h e n a d m i n i s t e r e d t o rats with F L A 63. The d i f f e r e n t e f f e c t o f F L A 63 on the catalepsy i n d u c e d b y h a l o p e r i d o l and I D 4 7 0 8 s u p p o r t s the idea t h a t the n e u r o l e p t i c - i n d u c e d catalepsy is p o t e n t i a t e d by the decrease in the activity o f t h e NE n e u r o n s . F r o m our e x p e r i m e n t s , it was c o n c l u d e d that a decrease in the activity o f DA n e u r o n s or the b l o c k a d e o f DA r e c e p t o r s is essential to elicite catalepsy and t h a t t h e f u n c t i o n o f NE n e u r o n s play a role in regulating the degree o f catalepsy in rats.
ACKNOWLEDGEMENTS The authors thank Dr. H. Yamamoto for his advice in the course of this investigation and Miss Y. Moriyasu for excellent technical assistance.
REFERENCES
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