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Substance K, a novel tachykinin injected bilaterally into the ventral tegmental area of rats increases behavioral response
Life Sciences, Vol. 37, pp. 2507-2514 Printed in the U.S.A.
Pergamon Press
S U B S T A N C E K, A N O V E L T A C H Y K I N I N I N J E C T E D B I L A T E R A L L Y INTO THEVENTRALTEGMm%'TALAREAOFRATS INCREASES BEHAVIORAL RESPONSE
Yukio
Takano,
Y a s u o Takeda, Katsushi Hiro-o Kamiya
Y a m a d a ~,
and
Department of Pharmacology, School of Pharmaceutical Sciences and *School of Medicine, Fukuoka University, Fukuoka 814-01, Japan. (Received in final form October 14, 1985) SUMMARY Novel tachykinins such as substance K and neuromedin K have been identified in the m a m m a l i a n CNS. Bilateral injection s of substance K and r e l a t e d p e p t i d e s as w e l l as of s u b s t a n c e P into the d o p a m i n e cell b o d y area of the v e n t r a l t e g m e n t a l a r e a c a u s e d dose-dependent i n c r e a s e s in l o c o m o t o r a c t i v i t y and rearing, in rats. As this b e h a v i o r w a s b l o c k e d by the p r e t r e a t m e n t w i t h h a l o p e r i d o l (0.1 m g / k g , i.p.), the activation of dopaminergic systems may be involved in the h y p e r m o t i l i t y i n d u c e d by the a d m i n i s t r a t i o n of substance K. S u b s t a n c e P (SP) is a m e m b e r of the t a c h y k i n i n f a m i l y of bioactive peptides including physalaemin, eledoisin and kassinin (I) and SP has a p u t a t i v e p h y s i o l o g i c a l a c t i o n in the CNS (2-4) These peptides share the c o m m o n C-terminal amino acid sequence Phe-X-Gly-Leu-Met-NH2, and exhibit similar biological activities in the p e r i p h e r a l organs. In a d d i t i o n to SP, t w o n o v e l t a c h y kinins have recently been identified in the m a m m a l i a n CNS, n a m e l y s u b s t a n c e K (SK)(or n e u r o k i n i n a) and n e u r o m e d i n K (or n e u r o kinin ~) (5,6). N a w a et al. (7) f o u n d that the b o v i n e s t r i a t u m contains two types of SP precursors, one termed a -preprotachykinin that contains the SP and the other, B -preprotachykinin that contains SP and SK (His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2)T h e r e s e e m to be d i f f e r e n c e s in the r e l a t i v e p o t e n c i e s of b i o logical activities between the two naturally occurring m a m m a l i a n t a c h y k i n i n s SP and SK in the p e r i p h e r a l s y s t e m s . T h e b i o l o g i c a l activities of SK more closely resemble those of kassinin than of SP or p h y s a l a e m i n (8 - 12). B ~ et el. (13) s t u d i e d the b i n d i n g characteristics of SK using I-Bolton-Hunter reagent-labelled SK, they o b t a i n e d e v i d e n c e for the e x i s t e n c e of t h r e e d i s t i n c t types of tachykinin receptors, SP-P (for physalaemin), SP-E (for eledoisin and kassinin), and SK. We examined the central physiological role of SK and found that bilateral injections of SK into the ventral tegmental area (VTA) in r a t s led to an i n c r e a s e in l o c o m o t o r a c t i v i t y and rearing, and that this hypermotility was blocked by haloperidol. 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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Substance K in the VTA of Rats
Vol. 37, No. 26, 1985
METHODS Animals
and s u r g e r y
M a l e W i s t a r r a t s (217 ± 6 g) p u r c h a s e d from Kyudo Animal Laboratory ( K u m a m o t o , J a p a n ) w e r e h o u s e d in a r o o m a t 20 - 25°C w i t h a 12 h l i g h t - d a r k c y c l e (light on at 7:00 am) and g i v e n free a c c e s s to c o m m e r c i a l food (CE-2, Clea Ltd., Japan) and tap water. T h e s e rats w e r e a n e s t h e t i z e d w i t h s o d i u m p e n t o b a r b i t a l (40 m g / k g , i.p.) a n d t h e n p l a c e d in a s t e r e o t a x i c apparatus. Guide cannulae (21 g a u g e s t a i n l e s s s t e e l ) w e r e i n s e r t e d b i l a t e r a l l y into the VTA. C o o r d i n a t e s w e r e (in mm); 4.8 p o s t e r i o r to Bregma, 0.7 l a t e r a l f r o m m i d l i n e , a n d 5.6 b e l o w t h e s k u l l s u r f a c e (14). The cannulae w e r e s e c u r e d to t h e s k u l l w i t h s t a i n l e s s - s t e e l s c r e w s and d e n t a l cement, and 2 6 - g a u g e s t a i n l e s s steel o b t u r a t o r s w e r e p l a c e d in e a c h c a n n u l a to m a i n t a i n p a t e n c y a n d to p r e v e n t infection. At t h e e n d of t h e e x p e r i m e n t , the brain was cut c o r o n a l l y at 100 ~ m w i t h a c r y o s t a t and the s e c t i o n s w e r e m o u n t e d on g l a s s slides. H i s t o l o g i c a l v e r i f i c a t i o n of e a c h i n j e c t i o n site was made, using a microscope to l o c a t e t h e m a l a c h i t e g r e e n as a marker. Behavioral
studies
A f t e r a 7 day r e c o v e r y period, s y n t h e t i c p e p t i d e s (0.1 - 2.0 ~g/1 ~i) d i s s o l v e d in s a l i n e w e r e i n j e c t e d bilaterally with i n j e c t i o n n e e d l e s (26 gauge) t h r o u g h g u i d e c a n n u l a to a p o i n t 8.6 mm below the skull surface over a 2 mins. The injection needles were removed I min after injection, and the rats were placed individually in a cage. A l l e x p e r i m e n t s were carried out from 8:00 a m to 1:00 p m in a t e m p e r a t u r e - c o n t r o l l e d (20-23°C), artificially lighted room. Locomotor activity (ambulation) and r e a r i n g w e r e m e a s u r e d u s i n g an o p e n - f i e l d a p p a r a t u s (15,16) 60 c m in d i a m e t e r a n d 53 c m in h e i g h t a n d t h e f l o o r w a s d i v i d e d i n t o 19 blocks. Animals w e r e l e f t a l o n e f o r 60 m i n to a d a p t to t h e o p e n field apparatus. T h e i r e x p l o r a t o r y b e h a v i o r w a s then o b s e r v e d 15 m i n b e f o r e a n d 15, 30, 60, 90, 120, a n d 180 m i n a f t e r t h e i n j e c t i o n s of d r u g s . T h e l o c o m o t o r s c o r e w a s e x p r e s s e d as t h e n u m b e r of b l o c k s o n t h e f l o o r t r a n s v e r s e d by t h e r a t d u r i n g 3 min. Statistical analysis was calculated using the two-tailed M a n n - W h i t n e y ' s U-test. Drugs SP a n d p h y s a l a e m i n were obtained from Peptide Research (Osaka, Japan) a n d k a s s i n i n w a s f r o m P e n i n s u l a Lab. (Belmont, CA, USA). SK a n d its a n a l o g u e , Arg-SK [I-10] were prepared in S h i o n o g i R e s e a r c h Lab. ( O s a k a , J a p a n ) b y c o n v e n t i o n a l solution methods. RESULTS Bilateral injections of SK i n t o t h e V T A c a u s e d a d o s e d e p e n d e n t i n c r e a s e in b o t h l o c o m o t o r a c t i v i t y and rearing. This SK-induced hypermotility l a s t e d f o r u p to 60 m i n in t h e c a s e of a l a r g e d o s e (2 ~ g p e r side) (Figs. 1A, B).
Vol. 37, No. 26, 1985
A lOO
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Substance K in the VTA of Rats
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Fig.1 D o s e - r e s p o n s e c u r v e of l o c o m o t o r a c t i v i t y (A) and r e a r i n g (B) after injection of substance K into the VTA. ~ p ~0.05, *~ p < 0.01, ~ * p < 0.001, the two-tailed Mann-Whitney's U-test, c o m p a r i n g all d o s e s of s u b s t a n c e K w i t h f i n d i n g s in the s a l i n e control. Each point represents the mean ± SEM conunts for 3 min.
To c o m p a r e the b e h a v i o r of rats r e s p o n d i n g to SK and o t h e r t a c h y k i n i n s , w e e x a m i n e d the e f f e c t s of v a r i o u s t a c h y k i n i n s on exploratory behavior. Bilateral injections of these peptides (2 ~g per side), SP, SK, A r g - S K [I-10], p h y s a l a e m i n and k a s s i n i n , also produced a significant increase in both locomotor activity and rearing (Figs. 2, 3). However, the response patterns obtained after SK, Arg-SK [I-10] and kassinin differed from those obtained a f t e r SP and p h y s a l a e m i n . The s h a r p r e s p o n s e p e a k w a s o b s e r v e d b e t w e e n 15 m i n a n d 30 m i n a f t e r the i n j e c t i o n of SK or S K - r e l a t e d peptides and the response peak after injection of SP or p h y s a l a e m i n appeared at around 90 min. The increases induced by SK i~ l o c o m o t i o n and r e a r i n g w e r e b l o c k e d by p r e t r e a t m e n t w i t h h a l o p e r i d o l g i v e n at a d o s e of 0.1 m g / k g (i.p.) w h i c h did not c a u s e c a t a l e p s y (Fig. 4). Arg-SK [I-10], which was synthesized based on the amino acid s e q u e n c e p r e d i c t e d f r o m the c l o n e d c D N A s e q u e n c e (7), a l s o increased the locomotor activity and rearing (Figs. 2, 3).
DISCUSSION The present study shows that bilateral injections of a novel tachykinin, SK, as well as of SP into the VTA led to increases in l o c o m o t o r a c t i v i t y and r e a r i n g in rats. T h e r e is a p p a r e n t l y a close relationship between the SP neurons and the dopamine (DA) n e u r o n s in the VTA. The a r e a of the b r a i n w h i c h r e c e i v e s SP innervation mainly from the medial habenular nucleus, contains
2510
Substance K zn the VTA of Rats
Vo]. 37, No. 26,
1985
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Time course of the effects of bilateral injections into the VTA of various tachykinin peptides (2 ~ g p e r s i d e ) o r i s o t o n i c saline on locomotor activity measured by the open-field method. Each point represents the mean ± SEM counts for 3 min.
the cell bodies of the DA neurons (17,18). D A n e u r o n s originating in the VTA terminate in the mesolimbic and mesocortical areas (19,20). Bannon e t a l . (21) r e p o r t e d that the function of SP in the VTA is related to activation of the mesocortical DA neurons in response to footshock stress. Additionally, behavioral studies
Vol. 37, No. 26, 1985
Substance K in the VTA of Rats
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T i m e c o u r s e of the e f f e c t s of b i l a t e r a l i n j e c t i o n s into the V T A of v a r i o u s t a c h y k i n i n p e p t i d e s (2 D g p e r side) o r i s o t o n i c saline on rearing. Each point represents the mean ± SEM counts for 3 min.
have s u g g e s t e d (2, 22).
an e x c i t a t o r y
effect
of SP on DA cells
in the V T A
T h e r e a r e o n l y a f e w r e p o r t s c o n c e r n i n g the d i s t r i b u t i o n of SK c o n t e n t s and SK b i n d i n g sites in the b r a i n (23,24). The neuroa n a t o m i c a l m e c h a n i s m s of S K - i n d u c e d h y p e r a c t i v i t y o b s e r v e d in the
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Substance K in the VTA of Rats
Vol. 37, No. 26, 1985
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Effect of h a l o p e r i d o l on the behavioral hyperactivity induced by s u b s t a n c e K. S u b s t a n c e K (2 ~ g per side) was injected 30 m i n i n t o t h e V T A a f t e r p r e t r e a t m e n t with haloperidol (0.1 m g / k g , i.p.) or s a l i n e . * p < 0.005, * p < 0.005, t h e t w o tailed Mann-Whitney's U-test was used for a statistical c o m p a r i s o n w i t h finding in the s a l i n e - i n j e c t e d group. Each point represents t h e m e a n ± S E M c o u n t s for 3 m i n of s i x s e p a r a t e experiments.
present study w e r e not clarified, however, w e p r o p o s e that activ a t i o n of the DA system, p r o b a b l y the m e s o l i m b i c DA s y s t e m m a y be involved in S K - i n d u c e d hypermotility, since haloperidol (0.1 mg/kg, i.p.) b l o c k e d this response. The diffusion of the microinjected peptides to t h e neighbouring structure h a s to be c o n s i d e r e d ; e.g. into the s u b s t a n t i a nigra (SN) located an about 1.5 m m lateral to the VTA site. K e l l e y a n d I v e r s e n (25) f o u n d t h a t m i c r o i n j e c t i o n of SP into the SN elicited a certain behavior, in p a r t i c u l a r , grooming. In our p r e l i m i n a r y e x p e r i m e n t s , we noted that injections of SK into the SN induced s n i f f i n g w h i c h is a s t e r e o t y p e d b e h a v i o r s in rats. It is o f i n t e r e s t that the response patterns of the locomotor activity and rearing differed between SK-related p e p t i d e s and the S P - r e l a t e d p e p t i d e s (Figs. 2, 3). The e x i s t e n c e of t w o d i f f e r e n t t a c h y k i n i n r e c e p t o r s has b e e n proposed; a SP-P r e c e p t o r (e.g. g u i n e a pig ileum) w h i c h is p r e f e r e n t i a l l y e x c i t e d by p h y s a l a e m i n and SP and a SP-E (e.g. rat vas deferens) w h i c h is preferentially excited by e l e d o i s i n and kassinin (26). SK r e c e p t o r s have b e e n d e t e c t e d in p e r i p h e r a l o r g a n s (13), however, t h e p r e s e n c e of SK r e c e p t o r s in t h e b r a i n is n o t y e t k n o w n . O u r r e s u l t s i n d i c a t e t h a t d i s t i n c t SK or S P - E t y p e r e c e p t o r s in t h e V T A m a y be l i n k e d t h e b e h a v i o r a l c h a n g e s i n d u c e d by SK a n d t h e r e l a t e d peptides.
Vol. 37, No. 26, 1985
Substance K in the VTA of Rats
2513
Acknowledgments We express our appreciation t o Dr. A r t h u r D. L o e w y (Washington University, St. L o u i s , MO.) for h e l p f u l d i s c u s s i o n s a n d t o D r s . M. D o t e u c h i a n d K. I n o u y e (Shionogi Research Laboratory, Osaka, Japan) for the SK and A r g - S K [I-10]. We thank Ms. K. T e t s u m i y a , S. H i r a k a w a , A. Y a m a d a , M. J i k u m a r u a n d Mr. A. N a g a s h i m a for technical assistance. This w o r k was supported by a G r a n t - i n Aid from the M i n i s t r y of Education, S c i e n c e and C u l t u r e of Japan (No. 59480420) and Kanae R e s e a r c h fund.
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Pergamon Press
S U B S T A N C E K, A N O V E L T A C H Y K I N I N I N J E C T E D B I L A T E R A L L Y INTO THEVENTRALTEGMm%'TALAREAOFRATS INCREASES BEHAVIORAL RESPONSE
Yukio
Takano,
Y a s u o Takeda, Katsushi Hiro-o Kamiya
Y a m a d a ~,
and
Department of Pharmacology, School of Pharmaceutical Sciences and *School of Medicine, Fukuoka University, Fukuoka 814-01, Japan. (Received in final form October 14, 1985) SUMMARY Novel tachykinins such as substance K and neuromedin K have been identified in the m a m m a l i a n CNS. Bilateral injection s of substance K and r e l a t e d p e p t i d e s as w e l l as of s u b s t a n c e P into the d o p a m i n e cell b o d y area of the v e n t r a l t e g m e n t a l a r e a c a u s e d dose-dependent i n c r e a s e s in l o c o m o t o r a c t i v i t y and rearing, in rats. As this b e h a v i o r w a s b l o c k e d by the p r e t r e a t m e n t w i t h h a l o p e r i d o l (0.1 m g / k g , i.p.), the activation of dopaminergic systems may be involved in the h y p e r m o t i l i t y i n d u c e d by the a d m i n i s t r a t i o n of substance K. S u b s t a n c e P (SP) is a m e m b e r of the t a c h y k i n i n f a m i l y of bioactive peptides including physalaemin, eledoisin and kassinin (I) and SP has a p u t a t i v e p h y s i o l o g i c a l a c t i o n in the CNS (2-4) These peptides share the c o m m o n C-terminal amino acid sequence Phe-X-Gly-Leu-Met-NH2, and exhibit similar biological activities in the p e r i p h e r a l organs. In a d d i t i o n to SP, t w o n o v e l t a c h y kinins have recently been identified in the m a m m a l i a n CNS, n a m e l y s u b s t a n c e K (SK)(or n e u r o k i n i n a) and n e u r o m e d i n K (or n e u r o kinin ~) (5,6). N a w a et al. (7) f o u n d that the b o v i n e s t r i a t u m contains two types of SP precursors, one termed a -preprotachykinin that contains the SP and the other, B -preprotachykinin that contains SP and SK (His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2)T h e r e s e e m to be d i f f e r e n c e s in the r e l a t i v e p o t e n c i e s of b i o logical activities between the two naturally occurring m a m m a l i a n t a c h y k i n i n s SP and SK in the p e r i p h e r a l s y s t e m s . T h e b i o l o g i c a l activities of SK more closely resemble those of kassinin than of SP or p h y s a l a e m i n (8 - 12). B ~ et el. (13) s t u d i e d the b i n d i n g characteristics of SK using I-Bolton-Hunter reagent-labelled SK, they o b t a i n e d e v i d e n c e for the e x i s t e n c e of t h r e e d i s t i n c t types of tachykinin receptors, SP-P (for physalaemin), SP-E (for eledoisin and kassinin), and SK. We examined the central physiological role of SK and found that bilateral injections of SK into the ventral tegmental area (VTA) in r a t s led to an i n c r e a s e in l o c o m o t o r a c t i v i t y and rearing, and that this hypermotility was blocked by haloperidol. 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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Substance K in the VTA of Rats
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METHODS Animals
and s u r g e r y
M a l e W i s t a r r a t s (217 ± 6 g) p u r c h a s e d from Kyudo Animal Laboratory ( K u m a m o t o , J a p a n ) w e r e h o u s e d in a r o o m a t 20 - 25°C w i t h a 12 h l i g h t - d a r k c y c l e (light on at 7:00 am) and g i v e n free a c c e s s to c o m m e r c i a l food (CE-2, Clea Ltd., Japan) and tap water. T h e s e rats w e r e a n e s t h e t i z e d w i t h s o d i u m p e n t o b a r b i t a l (40 m g / k g , i.p.) a n d t h e n p l a c e d in a s t e r e o t a x i c apparatus. Guide cannulae (21 g a u g e s t a i n l e s s s t e e l ) w e r e i n s e r t e d b i l a t e r a l l y into the VTA. C o o r d i n a t e s w e r e (in mm); 4.8 p o s t e r i o r to Bregma, 0.7 l a t e r a l f r o m m i d l i n e , a n d 5.6 b e l o w t h e s k u l l s u r f a c e (14). The cannulae w e r e s e c u r e d to t h e s k u l l w i t h s t a i n l e s s - s t e e l s c r e w s and d e n t a l cement, and 2 6 - g a u g e s t a i n l e s s steel o b t u r a t o r s w e r e p l a c e d in e a c h c a n n u l a to m a i n t a i n p a t e n c y a n d to p r e v e n t infection. At t h e e n d of t h e e x p e r i m e n t , the brain was cut c o r o n a l l y at 100 ~ m w i t h a c r y o s t a t and the s e c t i o n s w e r e m o u n t e d on g l a s s slides. H i s t o l o g i c a l v e r i f i c a t i o n of e a c h i n j e c t i o n site was made, using a microscope to l o c a t e t h e m a l a c h i t e g r e e n as a marker. Behavioral
studies
A f t e r a 7 day r e c o v e r y period, s y n t h e t i c p e p t i d e s (0.1 - 2.0 ~g/1 ~i) d i s s o l v e d in s a l i n e w e r e i n j e c t e d bilaterally with i n j e c t i o n n e e d l e s (26 gauge) t h r o u g h g u i d e c a n n u l a to a p o i n t 8.6 mm below the skull surface over a 2 mins. The injection needles were removed I min after injection, and the rats were placed individually in a cage. A l l e x p e r i m e n t s were carried out from 8:00 a m to 1:00 p m in a t e m p e r a t u r e - c o n t r o l l e d (20-23°C), artificially lighted room. Locomotor activity (ambulation) and r e a r i n g w e r e m e a s u r e d u s i n g an o p e n - f i e l d a p p a r a t u s (15,16) 60 c m in d i a m e t e r a n d 53 c m in h e i g h t a n d t h e f l o o r w a s d i v i d e d i n t o 19 blocks. Animals w e r e l e f t a l o n e f o r 60 m i n to a d a p t to t h e o p e n field apparatus. T h e i r e x p l o r a t o r y b e h a v i o r w a s then o b s e r v e d 15 m i n b e f o r e a n d 15, 30, 60, 90, 120, a n d 180 m i n a f t e r t h e i n j e c t i o n s of d r u g s . T h e l o c o m o t o r s c o r e w a s e x p r e s s e d as t h e n u m b e r of b l o c k s o n t h e f l o o r t r a n s v e r s e d by t h e r a t d u r i n g 3 min. Statistical analysis was calculated using the two-tailed M a n n - W h i t n e y ' s U-test. Drugs SP a n d p h y s a l a e m i n were obtained from Peptide Research (Osaka, Japan) a n d k a s s i n i n w a s f r o m P e n i n s u l a Lab. (Belmont, CA, USA). SK a n d its a n a l o g u e , Arg-SK [I-10] were prepared in S h i o n o g i R e s e a r c h Lab. ( O s a k a , J a p a n ) b y c o n v e n t i o n a l solution methods. RESULTS Bilateral injections of SK i n t o t h e V T A c a u s e d a d o s e d e p e n d e n t i n c r e a s e in b o t h l o c o m o t o r a c t i v i t y and rearing. This SK-induced hypermotility l a s t e d f o r u p to 60 m i n in t h e c a s e of a l a r g e d o s e (2 ~ g p e r side) (Figs. 1A, B).
Vol. 37, No. 26, 1985
A lOO
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Substance K in the VTA of Rats
Oai
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Fig.1 D o s e - r e s p o n s e c u r v e of l o c o m o t o r a c t i v i t y (A) and r e a r i n g (B) after injection of substance K into the VTA. ~ p ~0.05, *~ p < 0.01, ~ * p < 0.001, the two-tailed Mann-Whitney's U-test, c o m p a r i n g all d o s e s of s u b s t a n c e K w i t h f i n d i n g s in the s a l i n e control. Each point represents the mean ± SEM conunts for 3 min.
To c o m p a r e the b e h a v i o r of rats r e s p o n d i n g to SK and o t h e r t a c h y k i n i n s , w e e x a m i n e d the e f f e c t s of v a r i o u s t a c h y k i n i n s on exploratory behavior. Bilateral injections of these peptides (2 ~g per side), SP, SK, A r g - S K [I-10], p h y s a l a e m i n and k a s s i n i n , also produced a significant increase in both locomotor activity and rearing (Figs. 2, 3). However, the response patterns obtained after SK, Arg-SK [I-10] and kassinin differed from those obtained a f t e r SP and p h y s a l a e m i n . The s h a r p r e s p o n s e p e a k w a s o b s e r v e d b e t w e e n 15 m i n a n d 30 m i n a f t e r the i n j e c t i o n of SK or S K - r e l a t e d peptides and the response peak after injection of SP or p h y s a l a e m i n appeared at around 90 min. The increases induced by SK i~ l o c o m o t i o n and r e a r i n g w e r e b l o c k e d by p r e t r e a t m e n t w i t h h a l o p e r i d o l g i v e n at a d o s e of 0.1 m g / k g (i.p.) w h i c h did not c a u s e c a t a l e p s y (Fig. 4). Arg-SK [I-10], which was synthesized based on the amino acid s e q u e n c e p r e d i c t e d f r o m the c l o n e d c D N A s e q u e n c e (7), a l s o increased the locomotor activity and rearing (Figs. 2, 3).
DISCUSSION The present study shows that bilateral injections of a novel tachykinin, SK, as well as of SP into the VTA led to increases in l o c o m o t o r a c t i v i t y and r e a r i n g in rats. T h e r e is a p p a r e n t l y a close relationship between the SP neurons and the dopamine (DA) n e u r o n s in the VTA. The a r e a of the b r a i n w h i c h r e c e i v e s SP innervation mainly from the medial habenular nucleus, contains
2510
Substance K zn the VTA of Rats
Vo]. 37, No. 26,
1985
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Fig.
2
Time course of the effects of bilateral injections into the VTA of various tachykinin peptides (2 ~ g p e r s i d e ) o r i s o t o n i c saline on locomotor activity measured by the open-field method. Each point represents the mean ± SEM counts for 3 min.
the cell bodies of the DA neurons (17,18). D A n e u r o n s originating in the VTA terminate in the mesolimbic and mesocortical areas (19,20). Bannon e t a l . (21) r e p o r t e d that the function of SP in the VTA is related to activation of the mesocortical DA neurons in response to footshock stress. Additionally, behavioral studies
Vol. 37, No. 26, 1985
Substance K in the VTA of Rats
**
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• Substance K n=7 8
10
2511
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T i m e c o u r s e of the e f f e c t s of b i l a t e r a l i n j e c t i o n s into the V T A of v a r i o u s t a c h y k i n i n p e p t i d e s (2 D g p e r side) o r i s o t o n i c saline on rearing. Each point represents the mean ± SEM counts for 3 min.
have s u g g e s t e d (2, 22).
an e x c i t a t o r y
effect
of SP on DA cells
in the V T A
T h e r e a r e o n l y a f e w r e p o r t s c o n c e r n i n g the d i s t r i b u t i o n of SK c o n t e n t s and SK b i n d i n g sites in the b r a i n (23,24). The neuroa n a t o m i c a l m e c h a n i s m s of S K - i n d u c e d h y p e r a c t i v i t y o b s e r v e d in the
2512
Substance K in the VTA of Rats
Vol. 37, No. 26, 1985
~Sahne(i p.) + SK • HaloperzdoK,.p.) + SK a SaIIne(VTA)
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Effect of h a l o p e r i d o l on the behavioral hyperactivity induced by s u b s t a n c e K. S u b s t a n c e K (2 ~ g per side) was injected 30 m i n i n t o t h e V T A a f t e r p r e t r e a t m e n t with haloperidol (0.1 m g / k g , i.p.) or s a l i n e . * p < 0.005, * p < 0.005, t h e t w o tailed Mann-Whitney's U-test was used for a statistical c o m p a r i s o n w i t h finding in the s a l i n e - i n j e c t e d group. Each point represents t h e m e a n ± S E M c o u n t s for 3 m i n of s i x s e p a r a t e experiments.
present study w e r e not clarified, however, w e p r o p o s e that activ a t i o n of the DA system, p r o b a b l y the m e s o l i m b i c DA s y s t e m m a y be involved in S K - i n d u c e d hypermotility, since haloperidol (0.1 mg/kg, i.p.) b l o c k e d this response. The diffusion of the microinjected peptides to t h e neighbouring structure h a s to be c o n s i d e r e d ; e.g. into the s u b s t a n t i a nigra (SN) located an about 1.5 m m lateral to the VTA site. K e l l e y a n d I v e r s e n (25) f o u n d t h a t m i c r o i n j e c t i o n of SP into the SN elicited a certain behavior, in p a r t i c u l a r , grooming. In our p r e l i m i n a r y e x p e r i m e n t s , we noted that injections of SK into the SN induced s n i f f i n g w h i c h is a s t e r e o t y p e d b e h a v i o r s in rats. It is o f i n t e r e s t that the response patterns of the locomotor activity and rearing differed between SK-related p e p t i d e s and the S P - r e l a t e d p e p t i d e s (Figs. 2, 3). The e x i s t e n c e of t w o d i f f e r e n t t a c h y k i n i n r e c e p t o r s has b e e n proposed; a SP-P r e c e p t o r (e.g. g u i n e a pig ileum) w h i c h is p r e f e r e n t i a l l y e x c i t e d by p h y s a l a e m i n and SP and a SP-E (e.g. rat vas deferens) w h i c h is preferentially excited by e l e d o i s i n and kassinin (26). SK r e c e p t o r s have b e e n d e t e c t e d in p e r i p h e r a l o r g a n s (13), however, t h e p r e s e n c e of SK r e c e p t o r s in t h e b r a i n is n o t y e t k n o w n . O u r r e s u l t s i n d i c a t e t h a t d i s t i n c t SK or S P - E t y p e r e c e p t o r s in t h e V T A m a y be l i n k e d t h e b e h a v i o r a l c h a n g e s i n d u c e d by SK a n d t h e r e l a t e d peptides.
Vol. 37, No. 26, 1985
Substance K in the VTA of Rats
2513
Acknowledgments We express our appreciation t o Dr. A r t h u r D. L o e w y (Washington University, St. L o u i s , MO.) for h e l p f u l d i s c u s s i o n s a n d t o D r s . M. D o t e u c h i a n d K. I n o u y e (Shionogi Research Laboratory, Osaka, Japan) for the SK and A r g - S K [I-10]. We thank Ms. K. T e t s u m i y a , S. H i r a k a w a , A. Y a m a d a , M. J i k u m a r u a n d Mr. A. N a g a s h i m a for technical assistance. This w o r k was supported by a G r a n t - i n Aid from the M i n i s t r y of Education, S c i e n c e and C u l t u r e of Japan (No. 59480420) and Kanae R e s e a r c h fund.
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102 3 6 1 - 3 6 4 (1984). 25. A.E. K E L L E Y a n d S.D. I V E R S E N , 2 n d Eur. N e u r o s c i . Ass. A b s t . (Elsevier A m s t e r d a m ) (I 978). 26. C.M. LEE, L.L. I V E R S E N , M.R. H A N L E Y a n d B.E.B. S A N D B E R G , N a u n y n - S c h m i e d . A r c h P h a r m a c o l . 318 281-287 (1981).