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Effects of haloperidol and sulpiride on dopamine-induced inhibition of nucleus accumbens neurons
Life Sciences, Vol. 32, pp. 2649-2653 Printed in the U.S.A.
EFFECTS
Pergamon Press
OF H A L O P E R I D O L AND S U L P I R I D E ON D O P A M I N E - I N D U C E D I N H I B I T I O N OF NUCLEUS A C C U M B E N S NEURONS
Akinori
Akaike,
Masashi
Sasa
and shuji
Takaori
D e p a r t m e n t of P h a r m a c o l o g y , F a c u l t y of Medicine, Kyoto University, Kyoto 606, J a p a n (Received in final form March 23, 1983) Summary M i c r o i o n t o p h o r e t i c study was p e r f o r m e d to e l u c i d a t e d o p a m i n e r g i c m e c h a n i s m in the nucleus accumbens (Acc) of rats a n e s t h e t i z e d w i t h chloral hydrate. Iontop h o r e t i c a l l y applied d o p a m i n e p r o d u c e d an i n h i b i t i o n of g l u t a m a t e - i n d u c e d firing in 28 (62 %) out of 45 Acc neurons tested. The d o p a m i n e - i n d u c e d i n h i b i t i o n of 14 Acc neurons was clearly a n t a g o n i z e d by simultaneous a p p l i c a t i o n of haloperidol, and a p a r t i a l antagonism by s u l p i r i d e was o b s e r v e d in 3 out of 10 Acc neurons. These results indicate that d o p a m i n e p r o d u c e s an i n h i b i t i o n of the Acc n e u r o n and that, compared to haloperidol, s u l p i r i d e is a less potent b l o c k e r of the p o s t s y n a p t i c d o p a m i n e r e c e p t o r involved in the d o p a m i n e - i n d u c e d inhibition. The nucleus accumbens (Acc) contains d o p a m i n e r g i c nerve term i n a l s d e r i v e d m a i n l y from the v e n t r a l t e g m e n t a l area (VTA) of the m e s e n c e p h a l o n (I-4). Several i n v e s t i g a t o r s (5-8) have shown that m i c r o i n j e c t i o n of d o p a m i n e and d i b u t y r y l cyclic 3"5"AMP in the Acc of rats results in i n c r e a s i n g the l o c o m o t o r a c t i v i t y and this effect is antagonized by d o p a m i n e antagonists such as haloperidol and sulpiride. W o o d r u f f et al. (9) have d e m o n s t r a t e d that i o n t o p h o r e t i c a p p l i c a t i o n of d o p a m i n e p r o d u c e s an i n h i b i t o r y effect on the Acc neurons d r i v e n by continuous a p p l i c a t i o n of h o m o c y s t e i c acid. The i n h i b i t o r y effects of d o p a m i n e on the Acc field p o t e n t i a l have been also reported by D e F r a n c e et al. (10). In previous works (11, 12), we found that V T A c o n d i t i o n i n g stimulation produces an i n h i b i t i o n of the spike g e n e r a t i o n of Acc neurons upon s t i m u l a t i o n of the p a r a f a s c i c u l a r nucleus of thalamus, s u g g e s t i n g that d o p a m i n e o r i g i n a t i n g in the V T A is an i n h i b i t o r y t r a n s m i t t e r or n e u r o m o d u l a t o r to the Acc neurons receiving input from the thalamus. Recently, Yim and Mogenson (13) have reported that d o p a m i n e applied i o n t o p h o r e t i c a l l y and s t i m u l a t i o n of the V T A attenuate the e x c i t a t o r y response of Acc neurons and this a t t e n u a t i n g effect is a b o l i s h e d by s y s t e m i c a d m i n i s t r a t i o n of haloperidol. Thus, further m i c r o i o n t o p h o r e t i c studies w e r e carried out in an attempt to d e t e r m i n e the role of d o p a m i n e and its antagonists on the Acc neurons.
0024-3205/83/232649-05503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
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Methods T w e n t y - t w o m a l e W i s t a r rats w e i g h i n g 300-500 g w e r e used. Under c h l o r a l h y d r a t e (300-400 m g / k g i.p.) a n e s t h e s i a , a t r a c h e a l c a n n u l a was i n s e r t e d and the a n i m a l was fixed in a s t e r e o t a x i c instrument. A f t e r c r a n i o t o m y , the a n i m a l was i m m o b i l i z e d w i t h g a l l a m i n e t r i e t h i o d i d e (80 m g / k g i.p.) and r e s p i r a t i o n was a r t i f i cially sustained. Body t e m p e r a t u r e was m a i n t a i n e d at 3 6 . 5 - 3 7 . 5 ° C w i t h a h e a t i n g pad. Single neuron activities were extracellularly r e c o r d e d in the Acc (A: 9.0-10.5, L: 1.0-2.0, H: 6.0-8.0 mm from c o r t i c a l surface) a c c o r d i n g to the b r a i n m a p of K~nig and K l i p p e l (14), using a g l a s s m i c r o e l e c t r o d e f i l l e d w i t h 2 M s o d i u m a c e t a t e c o n t a i n i n g Fast G r e e n FCF; the e l e c t r i c a l r e s i s t a n c e was 20-50 MQ. The r e c o r d i n g m i c r o e l e c t r o d e was a t t a c h e d a l o n g a s e v e n - b a r r e l e d m i c r o p i p e t t e the o u t e r d i a m e t e r of w h i c h was a p p r o x i m a t e l y 5 ~m. The d i s t a n c e b e t w e e n tips of the r e c o r d i n g e l e c t r o d e and m i c r o p i p e t t e was w i t h i n 15 ~m. The p i p e t t e was filled w i t h 2 M d o p a m i n e h y d r o c h l o r i d e (Sigma, pH 7.2), 30 m M h a l o p e r i d o l (Dainippon Pharm. Co., d i s s o l v e d in I % lactate, pH 4.5), 30 m M s u l p i r i d e (Fujisawa Pharm. Co., d i s s o l v e d in I N HCI; p H a d j u s t e d to 4.5-5.0 by a d d i n g 0 . 1 N NaOH), I M m o n o s o d i u m L - g l u t a m a t e (Sigma, p H 7.2) and 2 M NaCI; the c h e m i c a l s w e r e e j e c t e d to the i m m e d i a t e v i c i n i t y of the t a r g e t n e u r o n using a 4-ch M i c r o C o n s t a n t S u p p l y (Nihon Kohden, Model S - 5 1 2 5 S ) . L - g l u t a m a t e was a p p l i e d as an a n i o n and the o t h e r c h e m i c a l s as cation. S p o n t a n e o u s firing and g l u t a m a t e - i n d u c e d s p i k e s w e r e a m p l i f i e d and d i s p l a y e d on an o s c i l l o s c o p e (Nihon Kohden, V C - 9 ) , and t h e y w e r e s i m u l t a n e o u s l y c o u n t e d u s i n g a p u l s e c o u n t e r (Nihon Kohden, ET-612J) and a M u l t i c o r d e r (Watanabe, MC 6602) w i t h an i n t e g r a t i o n p e r i o d of I sec. The e f f e c t s of the drugs on g l u t a m a t e - i n d u c e d s p i k e s w e r e d e t e r m i n e d as p o s i t i v e w h e n the c h a n g e of firing r a t e / s e c was o v e r 50 % of the control. The r e c o r d i n g site w a s m a r k e d by p a s s i n g a d i r e c t c u r r e n t of 20 pA for 10 sec and it was h i s t o l o g i c a l l y c h e c k e d w i t h c r e s y l v i o l e t s t a i n after e v e r y e x p e r i m e n t . O t h e r d e t a i l s of the p r o c e d u r e s w e r e as p r e v i o u s l y d e s c r i b e d (11, 12, 15). Results H i s t o l o g i c a l e x a m i n a t i o n r e v e a l e d that 45 out of 53 n e u r o n s w e r e l o c a t e d in the Acc. S i n c e the m a j o r i t y o f Acc n e u r o n s (43 out of 45 n e u r o n s ) h a d a low s p o n t a n e o u s firing rate o f less t h a n I/sec, the e f f e c t s of the d r u g s w e r e e x a m i n e d on the f i r i n g prod u c e d by i o n t o p h o r e t i c a p p l i c a t i o n of g l u t a m a t e . The e j e c t i o n c u r r e n t and d u r a t i o n of g l u t a m a t e w e r e fixed in e a c h c e l l in the r a n g e s of 10-50 n A and 5-10 sec, r e s p e c t i v e l y , so as to o b t a i n c o n s i s t e n t f i r i n g rates of 2 0 - 6 0 / s e c d u r i n g the a p p l i c a t i o n . W h e n d o p a m i n e up to 50 n A was i o n t o p h o r e t i c a l l y applied, a d o s e d e p e n d e n t i n h i b i t i o n of the g l u t a m a t e - i n d u c e d s p i k e s was o b t a i n e d in 28 (62 %) out of 45 Acc neurons. In the r e m a i n i n g 17 n e u r o n s , the i n h i b i t i o n w i t h d o p a m i n e up to 50 nA of the g l u t a m a t e - i n d u c e d firing was less t h a n 50 %. Two t y p i c a l e x a m p l e s o f the d o p a m i n e i n d u c e d i n h i b i t i o n are d e m o n s t r a t e d in Figl IA and B. N o n e of 45 Acc n e u r o n s t e s t e d e x h i b i t e d an e x c i t a t i o n w i t h i o n t o p h o r e t i c app l i c a t i o n of d o p a m i n e or an a u g m e n t a t i o n of g l u t a m a t e - i n d u c e d spikes. The g l u t a m a t e - i n d u c e d firing was not a f f e c t e d by Na + e ~ e c t e d up to 50 n A (Fig. 1). E f f e c t s o f h a l o p e r i d o l and s u l p i r i d e w e r e e x a m i n e d on 14 and 10 Acc n e u r o n s r e s p e c t i v e l y , of w h i c h the g l u t a m a t e - i n d u c e d s p i k e s
Vol. 32, No. 23, 1983
30
Haloperidol and Sulpiride on Acc
2651
A
g ,x_
30
~1~/~ 20
DA 40
Na+ 40
~ ~/o1~ DoA Hal4~eri
DA 40
DA 40
DA 40
........~ Sulpiride 40
DA 40
~-~DA 40 ~ I rain
B
:iii i 40
DA 40
t
DA 40
DA 40
L1 rain'
40
FIG.
I
I n h i b i t o r y e f f e c t of d o p a m i n e (DA) on s p i k e s i n d u c e d by glutamate (G), and e f f e c t s of h a l o p e r i d o l and s u l p i r i d e on the D A - i n d u c e d i n h i b i t i o n of two n e u r o n s (A and B) in the n u c l e u s a c c u m b e n s . Periods during drug application are i n d i c a t e d by h o r i z o n t a l bars. N u m b e r s u n d e r the n a m e of d r u g s i n d i c a t e c u r r e n t in n A a p p l i e d .
TABLE
I
N u m b e r of N u c l e u s A c c u m b e n s N e u r o n s A f f e c t e d by D o p a m i n e (DA) in A b s e n c e and P r e s e n c e of H a l o p e r i d o l and S u l p i r i d e % Inhibition
below
DA alone
DA during haloperidol
25
0
9
25 - 50
0
51 - 75
2
over Total
75
DA alone
DA during sulpiride
0
I
2
0
2
3
2
I
12
0
8
6
14
14
10
10
w e r e i n h i b i t e d by d o p a m i n e . These dopamine antagonists were a p p l i e d for 2 m i n at the s a m e c u r r e n t as d o p a m i n e . One min after the o n s e t o f h a l o p e r i d o l or s u l p i r i d e a p p l i c a t i o n , d o p a m i n e was
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applied for I min. D u r i n g a p p l i c a t i o n of h a l o p e r i d o l up to 50 nA, s p o n t a n e o u s and g l u t a m a t e - i n d u c e d firing r e m a i n e d u n a l t e r e d but the i n h i b i t o r y effect of d o p a m i n e on the g l u t a m a t e - i n d u c e d spikes was a p p a r e n t l y a n t a g o n i z e d (Fig. I). As shown in T a b l e I, iontop h o r e t i c a p p l i c a t i o n of d o p a m i n e i n h i b i t e d over 75 % in 12 out of 14 Acc neurons of the g l u t a m a t e - i n d u c e d spikes, w h i l e w i t h the same dose of d o p a m i n e the i n h i b i t i o n of the g l u t a m a t e - i n d u c e d firing did not r e a c h this level in any of the 14 neurons during a p p l i c a t i o n of haloperidol. In c o n t r a s t to the effect of haloperidol, s u l p i r i d e up to 50 nA did not a n t a g o n i z e the i n h i b i t i o n by d o p a m i n e of the g l u t a m a t e - i n d u c e d spikes (Fig. IA). Iontophoretic a p p l i c a t i o n of d o p a m i n e i n h i b i t e d over 50 % of g l u t a m a t e - i n d u c e d spikes in all 10 neurons examined, and the i n h i b i t i o n by d o p a m i n e was still o b s e r v e d in 7 out of the same 10 neurons during application of sulpiride, a l t h o u g h p a r t i a l a n t a g o n i s m by s u l p i r i d e was found in the r e m a i n i n g 3 neurons (Table I). Discussion I o n t o p h o r e t i c a l l y applied d o p a m i n e p r o d u c e d an i n h i b i t i o n of the g l u t a m a t e - i n d u c e d spikes in about t w o - t h i r d s of the Acc neurons tested. These findings are in agreement w i t h the results o b t a i n e d by W o o d r u f f et al. (9) and Yim and M o g e n s o n (13) that d o p a m i n e blocked the firing d r i v e n by h o m o c y s t e i c acid and g l u t a m a t e in the Acc neurons. O u r previous papers (11, 12) have d e m o n s t r a t e d that c o n d i t i o n i n g s t i m u l a t i o n of the V T A i n h i b i t e d the Acc neurons r e c e i v i n g input from the p a r a f a s c i c u l a r nucleus of thalamus but not from the limbic structures such as a m y g d a l a and hippocampus. C o n v e r g e n c e of inputs to the Acc neurons was rarely found from the thalamus and limbic structures. Therefore, the r e m a i n i n g o n e - t h i r d of Acc neurons i n s e n s i t i v e to d o p a m i n e in the present study m a y be those receiving input from the limbic structures. K e b a b i a n and Calne (16) have c l a s s i f i e d d o p a m i n e receptors into two types: D-I and D-2 receptors. It is c o n s i d e r e d that the D-I receptor is linked with adenyl cyclase and is located in the p o s t s y n a p t i c m e m b r a n e of the n i g r o - s t r i a t a l d o p a m i n e r g i c system, w h i l e the D-2 r e c e p t o r is i n d e p e n d e n t from adenyl cyclase and is located on the p r e s y n a p t i c terminals of the d o p a m i n e r g i c fibers. In addition, it has been w e l l d o c u m e n t e d that h a l o p e r i d o l is a blocker of both D-I and D-2 receptors and s u l p i r i d e a s p e c i f i c blocker of the D-2 receptor (16). In the p r e s e n t study, h a l o p e ridol c l e a r l y a n t a g o n i z e d the i n h i b i t o r y effect of d o p a m i n e on the g l u t a m a t e - i n d u c e d firing, but s u l p i r i d e was m u c h less effective in b l o c k i n g the d o p a m i n e - i n d u c e d inhibition. These results indicate that d o p a m i n e produces an i n h i b i t i o n of the Acc neurons, and that, c o m p a r e d to haloperidol, s u l p i r i d e is a less potent blocker of the p o s t s y n a p t i c d o p a m i n e r e c e p t o r i n v o l v e d in the d o p a m i n e - i n d u c e d i n h i b i t i o n of g l u t a m a t e responses. Acknowledgements This w o r k was s u p p o r t e d in part by a G r a n t - i n - A i d for S c i e n t i f i c R e s e a r c h from the M i n i s t r y of Education, S c i e n c e Culture, J a p a n (No. 56480103).
and
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References I. J.H. F A L L O N and R.Y. MOORE, J. comp. Neurol. 180 545-580 (I 978). 2. R.Y. MOORE and F.E. BLOOM, Ann. Rev. Neurosci. I 129-169 (1978). 3. R.B. C H R O N I S T E R , R.W. SIKES, J. W O O D and J.F. D e F R A N C E , Neurosci. Lett. 17 231-235 (1980). 4. R. N E W M A N and S.S. W I N A N S , J. comp. Neurol. 191 167-192 (1 980). 5. A.J.J. P I J N E N B U R G and J.M. V A N ROSSUM, J. Pharm. Pharmac. 25 1 0 0 3 - 1 0 0 5 (1973) . 6. A.J.J. P I J N E N B U R G , W.M.M. H O N I G and J.M. V A N ROSSUM, Psychopharmac. (Berl.) 41 175-180 (1975). 7. D.M. JACKSON, N.-E. A N D E N and A. D A H L S T R O M , P s y c h o p h a r m . (Berl.) 45 139-149 (1975). 8 D.J. HEAL, A.G. PHILLIPS and A.R. GREEN, N e u r o p h a r m a c . 17 265-270 (I 978). 9 G.N. W O O D R U F F , P.S. M c C A R T H Y and R.J. WALKER, Brain R e s e a r c h 115 233-242 (1976). 10 J.F. D e F R A N C E , R.W. SIKES, G.C. P A L N E R and R.B. C H R O N I S T E R , Neurosci. Lett. 22 295-301 (I 981). 11 A. AKAIKE, M. S A S A and S. TAKAORI, Brain R e s e a r c h 225 189-194
(1 981 ) . 12
S. TAKAORI, M. SASA, A. A K A I K E and S. FUJIMOTO, Adv. Biosci. 37 341-355 (I 982). 13 C.Y. Y I M and G.J. MOGENSON, Brain R e s e a r c h 239 401-415 (1982). 14. J.F.R. K O N I G and R.A. KLIPPEL, The Rat Brain: A S t e r e o t a x i c Atlas, Krieger, H u n g t i n g t o n , N.Y. (1970). 15. M. SASA, S. FUJIMOTO, S. IGARASHI, K. M U N E K I Y O and S. TAKAORI, J. Pharmac. exp. Ther. 210 311-315 (1979). 16. J.W. K E B A B I A N and D.B. CLANE, N a t u r e 277 93-96 (1979).
EFFECTS
Pergamon Press
OF H A L O P E R I D O L AND S U L P I R I D E ON D O P A M I N E - I N D U C E D I N H I B I T I O N OF NUCLEUS A C C U M B E N S NEURONS
Akinori
Akaike,
Masashi
Sasa
and shuji
Takaori
D e p a r t m e n t of P h a r m a c o l o g y , F a c u l t y of Medicine, Kyoto University, Kyoto 606, J a p a n (Received in final form March 23, 1983) Summary M i c r o i o n t o p h o r e t i c study was p e r f o r m e d to e l u c i d a t e d o p a m i n e r g i c m e c h a n i s m in the nucleus accumbens (Acc) of rats a n e s t h e t i z e d w i t h chloral hydrate. Iontop h o r e t i c a l l y applied d o p a m i n e p r o d u c e d an i n h i b i t i o n of g l u t a m a t e - i n d u c e d firing in 28 (62 %) out of 45 Acc neurons tested. The d o p a m i n e - i n d u c e d i n h i b i t i o n of 14 Acc neurons was clearly a n t a g o n i z e d by simultaneous a p p l i c a t i o n of haloperidol, and a p a r t i a l antagonism by s u l p i r i d e was o b s e r v e d in 3 out of 10 Acc neurons. These results indicate that d o p a m i n e p r o d u c e s an i n h i b i t i o n of the Acc n e u r o n and that, compared to haloperidol, s u l p i r i d e is a less potent b l o c k e r of the p o s t s y n a p t i c d o p a m i n e r e c e p t o r involved in the d o p a m i n e - i n d u c e d inhibition. The nucleus accumbens (Acc) contains d o p a m i n e r g i c nerve term i n a l s d e r i v e d m a i n l y from the v e n t r a l t e g m e n t a l area (VTA) of the m e s e n c e p h a l o n (I-4). Several i n v e s t i g a t o r s (5-8) have shown that m i c r o i n j e c t i o n of d o p a m i n e and d i b u t y r y l cyclic 3"5"AMP in the Acc of rats results in i n c r e a s i n g the l o c o m o t o r a c t i v i t y and this effect is antagonized by d o p a m i n e antagonists such as haloperidol and sulpiride. W o o d r u f f et al. (9) have d e m o n s t r a t e d that i o n t o p h o r e t i c a p p l i c a t i o n of d o p a m i n e p r o d u c e s an i n h i b i t o r y effect on the Acc neurons d r i v e n by continuous a p p l i c a t i o n of h o m o c y s t e i c acid. The i n h i b i t o r y effects of d o p a m i n e on the Acc field p o t e n t i a l have been also reported by D e F r a n c e et al. (10). In previous works (11, 12), we found that V T A c o n d i t i o n i n g stimulation produces an i n h i b i t i o n of the spike g e n e r a t i o n of Acc neurons upon s t i m u l a t i o n of the p a r a f a s c i c u l a r nucleus of thalamus, s u g g e s t i n g that d o p a m i n e o r i g i n a t i n g in the V T A is an i n h i b i t o r y t r a n s m i t t e r or n e u r o m o d u l a t o r to the Acc neurons receiving input from the thalamus. Recently, Yim and Mogenson (13) have reported that d o p a m i n e applied i o n t o p h o r e t i c a l l y and s t i m u l a t i o n of the V T A attenuate the e x c i t a t o r y response of Acc neurons and this a t t e n u a t i n g effect is a b o l i s h e d by s y s t e m i c a d m i n i s t r a t i o n of haloperidol. Thus, further m i c r o i o n t o p h o r e t i c studies w e r e carried out in an attempt to d e t e r m i n e the role of d o p a m i n e and its antagonists on the Acc neurons.
0024-3205/83/232649-05503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
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Methods T w e n t y - t w o m a l e W i s t a r rats w e i g h i n g 300-500 g w e r e used. Under c h l o r a l h y d r a t e (300-400 m g / k g i.p.) a n e s t h e s i a , a t r a c h e a l c a n n u l a was i n s e r t e d and the a n i m a l was fixed in a s t e r e o t a x i c instrument. A f t e r c r a n i o t o m y , the a n i m a l was i m m o b i l i z e d w i t h g a l l a m i n e t r i e t h i o d i d e (80 m g / k g i.p.) and r e s p i r a t i o n was a r t i f i cially sustained. Body t e m p e r a t u r e was m a i n t a i n e d at 3 6 . 5 - 3 7 . 5 ° C w i t h a h e a t i n g pad. Single neuron activities were extracellularly r e c o r d e d in the Acc (A: 9.0-10.5, L: 1.0-2.0, H: 6.0-8.0 mm from c o r t i c a l surface) a c c o r d i n g to the b r a i n m a p of K~nig and K l i p p e l (14), using a g l a s s m i c r o e l e c t r o d e f i l l e d w i t h 2 M s o d i u m a c e t a t e c o n t a i n i n g Fast G r e e n FCF; the e l e c t r i c a l r e s i s t a n c e was 20-50 MQ. The r e c o r d i n g m i c r o e l e c t r o d e was a t t a c h e d a l o n g a s e v e n - b a r r e l e d m i c r o p i p e t t e the o u t e r d i a m e t e r of w h i c h was a p p r o x i m a t e l y 5 ~m. The d i s t a n c e b e t w e e n tips of the r e c o r d i n g e l e c t r o d e and m i c r o p i p e t t e was w i t h i n 15 ~m. The p i p e t t e was filled w i t h 2 M d o p a m i n e h y d r o c h l o r i d e (Sigma, pH 7.2), 30 m M h a l o p e r i d o l (Dainippon Pharm. Co., d i s s o l v e d in I % lactate, pH 4.5), 30 m M s u l p i r i d e (Fujisawa Pharm. Co., d i s s o l v e d in I N HCI; p H a d j u s t e d to 4.5-5.0 by a d d i n g 0 . 1 N NaOH), I M m o n o s o d i u m L - g l u t a m a t e (Sigma, p H 7.2) and 2 M NaCI; the c h e m i c a l s w e r e e j e c t e d to the i m m e d i a t e v i c i n i t y of the t a r g e t n e u r o n using a 4-ch M i c r o C o n s t a n t S u p p l y (Nihon Kohden, Model S - 5 1 2 5 S ) . L - g l u t a m a t e was a p p l i e d as an a n i o n and the o t h e r c h e m i c a l s as cation. S p o n t a n e o u s firing and g l u t a m a t e - i n d u c e d s p i k e s w e r e a m p l i f i e d and d i s p l a y e d on an o s c i l l o s c o p e (Nihon Kohden, V C - 9 ) , and t h e y w e r e s i m u l t a n e o u s l y c o u n t e d u s i n g a p u l s e c o u n t e r (Nihon Kohden, ET-612J) and a M u l t i c o r d e r (Watanabe, MC 6602) w i t h an i n t e g r a t i o n p e r i o d of I sec. The e f f e c t s of the drugs on g l u t a m a t e - i n d u c e d s p i k e s w e r e d e t e r m i n e d as p o s i t i v e w h e n the c h a n g e of firing r a t e / s e c was o v e r 50 % of the control. The r e c o r d i n g site w a s m a r k e d by p a s s i n g a d i r e c t c u r r e n t of 20 pA for 10 sec and it was h i s t o l o g i c a l l y c h e c k e d w i t h c r e s y l v i o l e t s t a i n after e v e r y e x p e r i m e n t . O t h e r d e t a i l s of the p r o c e d u r e s w e r e as p r e v i o u s l y d e s c r i b e d (11, 12, 15). Results H i s t o l o g i c a l e x a m i n a t i o n r e v e a l e d that 45 out of 53 n e u r o n s w e r e l o c a t e d in the Acc. S i n c e the m a j o r i t y o f Acc n e u r o n s (43 out of 45 n e u r o n s ) h a d a low s p o n t a n e o u s firing rate o f less t h a n I/sec, the e f f e c t s of the d r u g s w e r e e x a m i n e d on the f i r i n g prod u c e d by i o n t o p h o r e t i c a p p l i c a t i o n of g l u t a m a t e . The e j e c t i o n c u r r e n t and d u r a t i o n of g l u t a m a t e w e r e fixed in e a c h c e l l in the r a n g e s of 10-50 n A and 5-10 sec, r e s p e c t i v e l y , so as to o b t a i n c o n s i s t e n t f i r i n g rates of 2 0 - 6 0 / s e c d u r i n g the a p p l i c a t i o n . W h e n d o p a m i n e up to 50 n A was i o n t o p h o r e t i c a l l y applied, a d o s e d e p e n d e n t i n h i b i t i o n of the g l u t a m a t e - i n d u c e d s p i k e s was o b t a i n e d in 28 (62 %) out of 45 Acc neurons. In the r e m a i n i n g 17 n e u r o n s , the i n h i b i t i o n w i t h d o p a m i n e up to 50 nA of the g l u t a m a t e - i n d u c e d firing was less t h a n 50 %. Two t y p i c a l e x a m p l e s o f the d o p a m i n e i n d u c e d i n h i b i t i o n are d e m o n s t r a t e d in Figl IA and B. N o n e of 45 Acc n e u r o n s t e s t e d e x h i b i t e d an e x c i t a t i o n w i t h i o n t o p h o r e t i c app l i c a t i o n of d o p a m i n e or an a u g m e n t a t i o n of g l u t a m a t e - i n d u c e d spikes. The g l u t a m a t e - i n d u c e d firing was not a f f e c t e d by Na + e ~ e c t e d up to 50 n A (Fig. 1). E f f e c t s o f h a l o p e r i d o l and s u l p i r i d e w e r e e x a m i n e d on 14 and 10 Acc n e u r o n s r e s p e c t i v e l y , of w h i c h the g l u t a m a t e - i n d u c e d s p i k e s
Vol. 32, No. 23, 1983
30
Haloperidol and Sulpiride on Acc
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A
g ,x_
30
~1~/~ 20
DA 40
Na+ 40
~ ~/o1~ DoA Hal4~eri
DA 40
DA 40
DA 40
........~ Sulpiride 40
DA 40
~-~DA 40 ~ I rain
B
:iii i 40
DA 40
t
DA 40
DA 40
L1 rain'
40
FIG.
I
I n h i b i t o r y e f f e c t of d o p a m i n e (DA) on s p i k e s i n d u c e d by glutamate (G), and e f f e c t s of h a l o p e r i d o l and s u l p i r i d e on the D A - i n d u c e d i n h i b i t i o n of two n e u r o n s (A and B) in the n u c l e u s a c c u m b e n s . Periods during drug application are i n d i c a t e d by h o r i z o n t a l bars. N u m b e r s u n d e r the n a m e of d r u g s i n d i c a t e c u r r e n t in n A a p p l i e d .
TABLE
I
N u m b e r of N u c l e u s A c c u m b e n s N e u r o n s A f f e c t e d by D o p a m i n e (DA) in A b s e n c e and P r e s e n c e of H a l o p e r i d o l and S u l p i r i d e % Inhibition
below
DA alone
DA during haloperidol
25
0
9
25 - 50
0
51 - 75
2
over Total
75
DA alone
DA during sulpiride
0
I
2
0
2
3
2
I
12
0
8
6
14
14
10
10
w e r e i n h i b i t e d by d o p a m i n e . These dopamine antagonists were a p p l i e d for 2 m i n at the s a m e c u r r e n t as d o p a m i n e . One min after the o n s e t o f h a l o p e r i d o l or s u l p i r i d e a p p l i c a t i o n , d o p a m i n e was
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applied for I min. D u r i n g a p p l i c a t i o n of h a l o p e r i d o l up to 50 nA, s p o n t a n e o u s and g l u t a m a t e - i n d u c e d firing r e m a i n e d u n a l t e r e d but the i n h i b i t o r y effect of d o p a m i n e on the g l u t a m a t e - i n d u c e d spikes was a p p a r e n t l y a n t a g o n i z e d (Fig. I). As shown in T a b l e I, iontop h o r e t i c a p p l i c a t i o n of d o p a m i n e i n h i b i t e d over 75 % in 12 out of 14 Acc neurons of the g l u t a m a t e - i n d u c e d spikes, w h i l e w i t h the same dose of d o p a m i n e the i n h i b i t i o n of the g l u t a m a t e - i n d u c e d firing did not r e a c h this level in any of the 14 neurons during a p p l i c a t i o n of haloperidol. In c o n t r a s t to the effect of haloperidol, s u l p i r i d e up to 50 nA did not a n t a g o n i z e the i n h i b i t i o n by d o p a m i n e of the g l u t a m a t e - i n d u c e d spikes (Fig. IA). Iontophoretic a p p l i c a t i o n of d o p a m i n e i n h i b i t e d over 50 % of g l u t a m a t e - i n d u c e d spikes in all 10 neurons examined, and the i n h i b i t i o n by d o p a m i n e was still o b s e r v e d in 7 out of the same 10 neurons during application of sulpiride, a l t h o u g h p a r t i a l a n t a g o n i s m by s u l p i r i d e was found in the r e m a i n i n g 3 neurons (Table I). Discussion I o n t o p h o r e t i c a l l y applied d o p a m i n e p r o d u c e d an i n h i b i t i o n of the g l u t a m a t e - i n d u c e d spikes in about t w o - t h i r d s of the Acc neurons tested. These findings are in agreement w i t h the results o b t a i n e d by W o o d r u f f et al. (9) and Yim and M o g e n s o n (13) that d o p a m i n e blocked the firing d r i v e n by h o m o c y s t e i c acid and g l u t a m a t e in the Acc neurons. O u r previous papers (11, 12) have d e m o n s t r a t e d that c o n d i t i o n i n g s t i m u l a t i o n of the V T A i n h i b i t e d the Acc neurons r e c e i v i n g input from the p a r a f a s c i c u l a r nucleus of thalamus but not from the limbic structures such as a m y g d a l a and hippocampus. C o n v e r g e n c e of inputs to the Acc neurons was rarely found from the thalamus and limbic structures. Therefore, the r e m a i n i n g o n e - t h i r d of Acc neurons i n s e n s i t i v e to d o p a m i n e in the present study m a y be those receiving input from the limbic structures. K e b a b i a n and Calne (16) have c l a s s i f i e d d o p a m i n e receptors into two types: D-I and D-2 receptors. It is c o n s i d e r e d that the D-I receptor is linked with adenyl cyclase and is located in the p o s t s y n a p t i c m e m b r a n e of the n i g r o - s t r i a t a l d o p a m i n e r g i c system, w h i l e the D-2 r e c e p t o r is i n d e p e n d e n t from adenyl cyclase and is located on the p r e s y n a p t i c terminals of the d o p a m i n e r g i c fibers. In addition, it has been w e l l d o c u m e n t e d that h a l o p e r i d o l is a blocker of both D-I and D-2 receptors and s u l p i r i d e a s p e c i f i c blocker of the D-2 receptor (16). In the p r e s e n t study, h a l o p e ridol c l e a r l y a n t a g o n i z e d the i n h i b i t o r y effect of d o p a m i n e on the g l u t a m a t e - i n d u c e d firing, but s u l p i r i d e was m u c h less effective in b l o c k i n g the d o p a m i n e - i n d u c e d inhibition. These results indicate that d o p a m i n e produces an i n h i b i t i o n of the Acc neurons, and that, c o m p a r e d to haloperidol, s u l p i r i d e is a less potent blocker of the p o s t s y n a p t i c d o p a m i n e r e c e p t o r i n v o l v e d in the d o p a m i n e - i n d u c e d i n h i b i t i o n of g l u t a m a t e responses. Acknowledgements This w o r k was s u p p o r t e d in part by a G r a n t - i n - A i d for S c i e n t i f i c R e s e a r c h from the M i n i s t r y of Education, S c i e n c e Culture, J a p a n (No. 56480103).
and
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