IV. Effects of bombesin on behavior

IV. Effects of bombesin on behavior

Life Sciences, Vol. 37, pp. 135-145 Printed in the U.S.A. Pergamon Press Current Concepts: IV. E F F E C T S A. Cowan, OF B O M B E S I N P. Khuna...

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Life Sciences, Vol. 37, pp. 135-145 Printed in the U.S.A.

Pergamon Press

Current Concepts: IV. E F F E C T S A. Cowan,

OF B O M B E S I N

P. Khunawat,

X.Zu

ON B E H A V I O R

Zhu and D.E.

Gmerek*

D e p a r t m e n t of P h a r m a c o l o g y , Temple U n i v e r s i t y School of M e d i c i n e P h i l a d e l p h i a , PA 19140, U.S.A. and * D e p a r t m e n t of P h a r m a c o l o g y U n i v e r s i t y of M i c h i g a n Medical School, Ann Arbor, MI 48109, U.S.A. Summary This r e p o r t d e s c r i b e s the i n f l u e n c e of b o m b e s i n on the gross b e h a v i o r of goldfish, frogs, mice, rats, g u i n e a pigs, rabbits, chicks, p i g e o n s and monkeys. Goldfish, frogs, chicks and pigeons w e r e o v e r t l y u n a f f e c t e d by bombesin given centrally and/or peripherally. Mice, rats, g u i n e a pigs, rabbits and monkeys r e s p o n d e d q u i c k l y to i n t r a c e r e b r o v e n t r i c u l a r (i.c.v.) and/or i n t r a t h e c a l (i.th.) a d m i n i s t r a t i o n of b o m b e s i n by d i s p l a y i n g a range of b e h a v i o r s s u g g e s t i v e of a l t e r e d skin sensation. In mice, b o m b e s i n was e s s e n t i a l l y e q u i p o t e n t as a s c r a t c h i n d u c e r by i.c.v, and i.th. routes (A~0=0.010-0.019 ~g) but 6800 times less p o t e n t i.p. In rats, b o m b e s i n i n d u c e d g r o o m i n g and s c r a t c h i n g b e h a v i o r s w e r e shown to be q u a l i t a t i v e l y d i f f e r e n t from those a s s o c i a t e d w i t h A C T H - ( I - 2 4 ) and t h y r o t r o p i n r e l e a s i n g hormone. Spantide and [D-Arg ~, D-Pro~ D-TrpT'~ Leu~']substance P (both at 0.20, 0.50 and 0.80 ~g i.c.v.), two p r o p o s e d b o m b e s i n r e c e p t o r a n t a g o n i s t s , did not m a r k e d l y i n f l u e n c e b o m b e s i n - i n d u c e d s c r a t c h i n g or h y p o t h e r m i a in rats. B o m b e s i n is a t e t r a d e c a p e p t i d e w h i c h was o r i g i n a l l y i s o l a t e d from frog skin (i). In k e e p i n g w i t h its d e s i g n a t i o n as a b r a i n gut peptide, b o m b e s i n p r o m o t e s h o m e o s t a s i s (2) by r e g u l a t i n g g a s t r o i n t e s t i n a l f u n c t i o n (3-5), food intake (6-8), b l o o d g l u c o s e levels (9,10) and body t e m p e r a t u r e (11-13). Mice and rats have been m a i n l y used in studies i n v o l v i n g b o m b e s i n and behavior. For example, central i n j e c t i o n of b o m b e s i n to rats i n c r e a s e s l o c o m o t o r a c t i v i t y w h e n the a n i m a l s are t e s t e d in small P l e x i g l a s c h a m b e r s (14). The i n c r e a s e d l o c o m o t i o n can be a n t a g o n i z e d if the animals are p r e t r e a t e d w i t h n e u r o l e p t i c s such as h a l o p e r i d o l or f l u p h e n a z i n e (15). The nature of the t e s t i n g e n v i r o n m e n t is i m p o r t a n t since b o m b e s i n s u p p r e s s e s a m b u l a t i o n (and rearing) if the rats are run in an o p e n - f i e l d a p p a r a t u s (16). The most obvious r e s p o n s e of mice (17-20) and rats (11,21-23) to i.c.v, b o m b e s i n is a r e m a r k a b l e d i s p l a y of g r o o m i n g and s c r a t c h ing d u r i n g w h i c h the animals seem p r e o c c u p i e d w i t h skin sensation. Does b o m b e s i n a f f e c t the o n g o i n g b e h a v i o r of other species in a similarly dramatic fashion? This q u e s t i o n p r o v i d e d the stimulus for the p r e s e n t i n v e s t i g a t i o n . A d d i t i o n a l l y , q u a n t i t a t i v e inform0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.

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a t i o n is p r o v i d e d on s c r a t c h i n g in m i c e w h e n b o m b e s i n is g i v e n by one of t h r e e r o u t e s - i.c.v., i.th. and i.p. Second, qualitative i n f o r m a t i o n is p r o v i d e d on s c r a t c h i n g in rats by c o m p a r i n g the behavioral syndromes associated with bombesin, adrenocorticotropin [specifically, ACTH-(I-24)] (24) and t h y r o t r o p i n r e l e a s i n g h o r m o n e (TRH)(25) . A n d third, two p r o p o s e d b o m b e s i n r e c e p t o r a n t a g o n i s t s , s p a n t i d e ([D-Arg~ D-Trp~,~ L e u ~ ] s u b s t a n c e P) {26) and D A P T L - S P ([D-Arg~ D-Pro~ D - T r p 7'9, L e u ~ ] s u b s t a n c e P (27) are t e s t e d a g a i n s t b o m b e s i n - i n d u c e d s c r a t c h i n g and h y p o t h e r m i a in rats. Materials

and M e t h o d s

Compounds N a l o x o n e HCl (kindly s u p p l i e d by Endo) was d i s s o l v e d in saline. The f o l l o w i n g p e p t i d e s w e r e d i s s o l v e d in d i s t i l l e d w a t e r immediately before injection: ACTH-(I-24) ( c o u r t e s y of H. Kuijs, O r g a n o n , H o l l a n d ) ; b o m b e s i n (Sigma); D A P T L - S P (Bachem) ; s p a n t i d e (Peninsula) and TRH (Sigma). Goldfish E i g h t g o l d f i s h of the c o m e t v a r i e t y , w e i g h i n g 3-5 g, w e r e o b t a i n e d f r o m a local s u p p l i e r . T h e y w e r e h o u s e d in a t a n k (under c o n s t a n t a e r a t i o n ) in a l a b o r a t o r y for 5 days. A standard l i g h t - d a r k c y c l e was m a i n t a i n e d w i t h a t i m e r - r e g u l a t e d l i g h t p e r i o d f r o m 07.00 hr to 19.00 hr. Experiments took place between i0.00 hr and noon. B o m b e s i n (5 pl) was i n j e c t e d s.c. in the b e l l ~ The d o s e s w e r e 0.i0 pg (n=2), 5 pg (n=2) and 25 ~g (8.3 m g / k g ; n=4) . The g o l d f i s h w e r e o b s e r v e d for 2 hr p o s t - i n j e c t i o n . Frogs M a l e f r o g s (Rana p i p i e n s ; 25-40 g) w e r e p u r c h a s e d f r o m W e s t J e r s e y B i o l o g i c a l S u p p l y Farm, W e n o n a h , N.J. Two d a y s b e f o r e e x p e r i m e n t a t i o n , t h e y w e r e w e i g h e d and p l a c e d in i n d i v i d u a l p l a s t i c o b s e r v a t i o n b o x e s (25 cm long; 20 cm wide; 15 cm high) c o n t a i n i n g w a t e r to a d e p t h of 1 cm. Environmental temperature was m a i n t a i n e d at 2 0 ± 0 . 5 ° C by m e a n s of an a u t o m a t i c t h e r m o s t a t . L i g h t s w e r e on b e t w e e n 9.00 hr and 18.00 hr. Bombesin was given i.th. at the a r t i c u l a t i o n b e t w e e n the s e v e n t h and e i g h t h v e r t e b r a e (28) (0.i0 ~g in a v o l u m e of 1 pl; n~6) or s.c. (0.25 m i / 2 5 g; i0 mg/kg; n=4). The f r o g s w e r e o b s e r v e d o v e r the f o l l o w i n g 1 hr. Mice M a l e ICR m i c e (22-28 g) (Temple U n i v e r s i t y S k i n and C a n c e r H o s p i t a l ) w e r e e a c h p l a c e d in an i n d i v i d u a l P l e x i g l a s o b s e r v a t i o n b o x (22 cm long; 18 cm wide; 25 cm high) and a l l o w e d to h a b i t u a t e for at l e a s t 15 min. T h e y w e r e i n j e c t e d w i t h b o m b e s i n i.th. (29) (5 pl) , i.c.v. (30) (5 ~i) or i.p. (0.25 m i / 2 5 g) and i m m e d i a t e l y o b s e r v e d (i.c.v. and i.th.) , or o b s e r v e d at +5 min (i.p.) , for 15 min. Four mice were scored simultaneously. The g r o o m i n g and s c r a t c h i n g s h o w n by e a c h a n i m a l was m o n i t o r e d for 5 sec out of e v e r y 20 sec. A p o s i t i v e s c o r e was g i v e n if the m o u s e g r o o m e d or scratched during each interval. R e s u l t s are p r e s e n t e d as p e r c e n t of the m a x i m u m (i.e., 45) p o s s i b l e n u m b e r of g r o o m i n g / s c r a t c h i n g e p i s o d e s (%MGE). F o u r m i c e w e r e u s e d per d o s e of b o m b e s i n . As0 v a l u e s w e r e e s t i m a t e d by l i n e a r r e g r e s s i o n a n a l y s i s (31). Experi m e n t s t o o k p l a c e b e t w e e n 14.00 hr and 18.00 hr. Rats M a l e S p r a g u e D a w l e y a l b i n o rats ( 1 8 0 - 2 0 0 g; Z i v i c - M i l l e r ) w e r e a n e s t h e t i z e d w i t h k e t a m i n e HCl (i00 m g / k g i.p.) and p l a c e d in a K o p f s t e r e o t a x i c frame. A p o l y e t h y l e n e c a n n u l a (PE i0) was p o s i t i o n e d in a l a t e r a l c e r e b r a l v e n t r i c l e of e a c h rat as p r e v i o u s l y d e s c r i b e d (32). A f t e r s u r g e r y , the a n i m a l s w e r e h o u s e d i n d i v i d u a l l y and a l l o w e d 5-7 d a y s for r e c o v e r y p r i o r to i.c.v.

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a d m i n i s t r a t i o n of A C T H - ( I - 2 4 ) , b o m b e s i n or T R H (each a g e n t in 5 W1 w a t e r t h e n a 3 Pl w a s h w i t h m o r e w a t e r ) . When appropriate, DAPTLSP, s p a n t i d e or w a t e r w a s g i v e n i.c.v. 5 m i n b e f o r e a s t a n d a r d d o s e of b o m b e s i n (0.i0 Pg i.c.v.) or w a t e r . B e h a v i o r of r a t s (n=10) w a s m o n i t o r e d ( b e g i n n i n g 5 m i n a f t e r e a c h b o m b e s i n i n j e c t ion) for 5 sec out of e v e r y 20 sec o v e r a 30 m i n o b s e r v a t i o n p e r i o d and q u a n t i t a t e d as p r e v i o u s l y d e s c r i b e d (33). Cannula p l a c e m e n t s w e r e v e r i f i e d a f t e r e a c h e x p e r i m e n t by i n j e c t i n g m e t h y l e n e b l u e and c h e c k i n g for d i s t r i b u t i o n w i t h i n the c e r e b r o ventricular system. A d d i t i o n a l r a t s w e r e i n f u s e d w i t h b o m b e s i n (0.18 w g / h r for 7 days) (23) to the c a u d a l r e g i o n of the S y l v i a n a q u e d u c t f r o m a s.c. i m p l a n t e d o s m o t i c m i n i p u m p (Alzet 2001) (34), t h e n c h a l l e n g e d w i t h D A P T L - S P (0.5 pg i . c . v . , n=3) and o b s e r v e d for 30 min. The p o s s i b i l i t y t h a t s p a n t i d e a n d / o r D A P T L - S P m i g h t p r e v e n t bombesin-induced hypothermia was studied. R a t s (n=5) w e r e e a c h p l a c e d in an i n d i v i d u a l P l e x i g l a s o b s e r v a t i o n b o x (26 cm long; 20 cm w i d e ; 30 cm high) and a l l o w e d to h a b i t u a t e to a t e m p e r a t u r e of 6 ± 2 ° C for 1 hr (ii). Rectal temperatures were taken several times w i t h a t h e r m i s t o r p r o b e to f a m i l i a r i z e the a n i m a l s w i t h the p r o c edure. The b a s e l i n e (zero time) t e m p e r a t u r e was n o t e d (with the p r o b e i n s e r t e d 6 cm i n t o the rectum) t h e n t e s t a g e n t or w a t e r was g i v e n i.c.v. 20 min b e f o r e b o m b e s i n (0.i0 pg i.c.v.) or w a t e r . T e m p e r a t u r e s w e r e t a k e n at 30, 60, 90 and 120 min. In a t o l e r a n c e e x p e r i m e n t , a d d i t i o n a l r a t s (n=6] r e c e i v e d a c o n t i n u o u s i.c.v, i n f u s i o n of w a t e r (i wl/hr) or b o m b e s i n (0.18 wg/hr) (23) f r o m a s.c. i m p l a n t e d A l z e t 2001 m i n i p u m p . After 7 days, t h e y w e r e c h a l l e n g e d w i t h b o m b e s i n (0.i0 ~g i.c.v.) and r e c t a l t e m p e r a t u r e s w e r e t a k e n at an a m b i e n t t e m p e r a t u r e of 6 ± 2 ° C as o u t l i n e d a b o v e . Guinea pigs M a l e a l b i n o g u i n e a p i g s (n:4) ( 1 8 0 - 2 0 0 g; C h a r l e s River) w e r e e a c h c a n n u l a t e d in the l a t e r a l c e r e b r a l v e n t r i c l e (35) in a m a n n e r s i m i l a r to t h a t d e s c r i b e d for rats. T h e y l i v e d in i n d i v i d u a l P l e x i g l a s b o x e s (26 cm long; 20 cm w i d e ; 30 cm high) and w e r e o b s e r v e d in t h e s e b o x e s d u r i n g the 60 min a f t e r i n j e c t i o n of b o m b e s i n (0.50 ~g in 5 W1 w a t e r ) . Rabbits T h r e e m a l e N e w Z e a l a n d a l b i n o r a b b i t s (2-4 kg; B i o s e a r c h , Philadelphia) were anesthetized with pentobarbital (30 m g / k g i.p.). A p o l y e t h y l e n e c a n n u l a was i n s e r t e d i n t o the left l a t e r a l c e r e b r o v e n t r i c l e (36) and b e h a v i o r a l e x p e r i m e n t s s t a r t e d 5 d a y s later. B o m b e s i n (0.i0 pg) was g i v e n i . c . v . , in i00 pl of w a t e r . This was f o l l o w e d by a i00 W1 w a s h w i t h m o r e w a t e r . R a b b i t s w e r e p l a c e d in t r a s h c a n s ( d i a m e t e r : 35 cm; h e i g h t : 55 cm) w i t h w h i c h t h e y h a d b e e n f a m i l i a r i z e d and t h e i r g r o s s b e h a v i o r was o b s e r v e d for 1 hr. Chicks G r o u p s of 5 w h i t e l e g h o r n c o c k e r e l s (2-3 d a y s old; M o y e r s C h i c k s , Q u a k e r t o w n , PA) w e r e p l a c e d in o p e n m a k r o l o n o b s e r v a t i o n boxes. The f l o o r of e a c h b o x w a s c o v e r e d w i t h s a w d u s t . W a t e r and f o o d c r u m b s w e r e m a d e a v a i l a b l e t h r o u g h o u t the e x p e r i m e n t . After a 30 min c o n t r o l p e r i o d , e a c h c h i c k w a s i n j e c t e d s.c. on one s i d e of the a b d o m e n w i t h w a t e r (0.25 m i / 2 5 g) or b o m b e s i n (i, 5 and 12.5 m g / k g ) . The o v e r t b e h a v i o r of the b i r d s was o b s e r v e d for 60 min. The s t u d y w a s r e p e a t e d w i t h d i f f e r e n t c h i c k s (n=5) w h e n t h e y w e r e ii d a y s old.

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Pigeons Two W h i t e C a r n e a u x p i g e o n s w e r e s e l e c t e d f r o m the g r o u p of b i r d s b e i n g u s e d in the e v a l u a t i o n of p s y c h o a c t i v e a g e n t s in the D e p a r t m e n t of P h a r m a c o l o g y , U n i v e r s i t y of M i c h i g a n . Both pigeons had p r e v i o u s l y r e c e i v e d a v a r i e t y of c o m p o u n d s . One d o s e of b o m b e s i n (I00 ~g) was g i v e n i.c.v. (37) in the p r e s e n t s t u d y and the b i r d s w e r e o b s e r v e d for the f o l l o w i n g 60 min. Monkeys F o u r r h e s u s m o n k e y s (4-6 kg) w e r e c h o s e n f r o m the c o l o n y of m o r p h i n e - d e p e n d e n t a n i m a l s b e i n g u s e d to a s s e s s the p h y s i c a l d e p e n d e n c e l i a b i l i t y of n e w a n a l g e s i c s at the U n i v e r s i t y of M i c h igan. A s t a i n l e s s s t e e l c a n n u l a was i m p l a n t e d in a l a t e r a l c e r e bral v e n t r i c l e of e a c h m o n k e y as p r e v i o u s l y d e s c r i b e d (38,39) . ( C a n n u l a p l a c e m e n t s w e r e s u b s e q u e n t l y v e r i f i e d by r a d i o g r a p h y ) . Two d o s e s of b o m b e s i n (750 pg and 1 mg) w e r e t e s t e d in the m o n k e y s (n=2) a b o u t 2 hr a f t e r t h e i r r e g u l a r m a i n t e n a n c e d o s e of m o r p h i n e (3 m g / k g s.c.). The a n i m a l s w e r e o b s e r v e d for the e n s u i n g 4 hr. Results B o m b e s i n c a u s e d no o v e r t e f f e c t s w h e n g i v e n to g o l d f i s h (0.i0, 5 and 25 pg s.c.), frogs (0.i0 ~g i.th. and i0 m g / k g s.c.), c h i c k s (i, 5 and 12.5 m g / k g s.c.) and p i g e o n s (i00 pg i.c.v.) . The m o s t o b v i o u s b e h a v i o r i n d u c e d by c e n t r a l l y a d m i n i s t e r e d b o m b e s i n in m i c e was an i m m e d i a t e and i n c e s s a n t s c r a t c h i n g of the f l a n k s and n e c k a r e a w i t h the h i n d p a w s (Fig. IA) . F a c i a l g r o o m i n g w i t h the f o r e p a w s was a l s o g r e a t l y i n c r e a s e d . Oral p r e e n i n g of the tail (Fig. 2A) was f r e q u e n t w h e n b o m b e s i n was g i v e n i.th.; less so w h e n g i v e n i.c.v.; and i n f r e q u e n t w h e n g i v e n .... S c r a t c h i n g / g r o o m i n g was d o s e - r e l a t e d (Fig. 3). AS0 v a l u e s (and 95% c o n f i d e n c e limits) for b o m b e s i n by i.c.v., i.th. and i.p. r o u t e s w e r e 0.010 ~g { 0 . 0 0 3 - 0 . 0 3 0 ) [i.e. 6 p m o l e (1.8-18)] , 0.019 ~g (0.010-0.30) and 68 ~g (32-147) , r e s p e c t i v e l y . In rats, b o m b e s i n (0.05, 0.i0 and 0.50 pg) c a u s e d e x c e s s i v e s c r a t c h i n g ~ the h e a d and n e c k a r e a by the h i n d p a w s w i t h i n i-3 min of i n j e c t i o n (Fig. IB). The rats f r e q u e n t l y l i c k e d t h e i r h i n d p a w s before scratching. F a c i a l g r o o m i n g (with f o r e p a w s ) , b o d y w a s h i n g (with t o n g u e and t e e t h ) , f o r e p a w t r e m o r , s t r e t c h i n g and w e t - d o g shaking also occurred. T h e s e b e h a v i o r s l a s t e d 45-60 min. TRH and A C T H - ( I - 2 4 ) a l s o c a u s e d J n c r e a s e d g r o o m i n g , f o r e p a w t r e m o r and b o d y s h a k i n g in rats b u t t h e s e s y n d r o m e s l a c k e d the c o m p u l s i v e s c r a t c h i n g c o m p o n e n t so c h a r a c t e r i s t i c of b o m b e s i n . TRH 13 and i0 ~g i.c.v.) p r o v o k e d e x t e n s i v e face w a s h i n g (Fig. iC), l i c k i n g the f r o n t paws and oral s t e r e o t y p i e s . These behaviors a p p e a r e d w i t h i n 5 min of i n j e c t i o n and l a s t e d 10-20 min. The increased grooming associated with ACTH-(I-24) (1.5 and 3 pg L ~ ) in rats was s i m i l a r to n o r m a l g r o o m i n g in t h a t the rats t h o r o u g h l y w a s h e d the face and e n t i r e b o d y (Fig. ID) but the b o u t s w e r e longer. O n s e t time was 12-17 min and the e f f e c t s l a s t e d a b o u t I hr. D A P T L - S P and s p a n t i d e (both at 0.20, 0.50 and 0.80 ~g i.c.v.) w e r e t e s t e d a g a i n s t the g r o o m i n g / s c r a t c h i n g c a u s e d by b o m b e s i n in rats. H i g h e r d o s e s of D A P T L - S P and s p a n t i d e (e.g. 2 ~g) e l i c i t e d behavioral effects which precluded further testing. These effects w e r e i m m e d i a t e p r o s t r a t i o n and b a r r e l r o t a t i o n . E v e n at 0.50 and 0.80 pg, b o t h a g e n t s p r o v o k e d , on o c c a s i o n , b a r r e l r o t a t i o n and motor impairment. These animals were rejected. Neither DAPTL-SP nor s p a n t i d e a t t e n u a t e d b o m b e s i n - i n d u c e d g r o o m i n g / s c r a t c h i n g .

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S c r a t c h i n g and g r o o m i n g b e h a v i o r in m o u s e (A) and rat (B-D) . (A) B o m b e s i n , 0.02 ~g i.th. (B) B o m b e s i n , 0.i0 ~g i.c.v. (C) TRH, i0 ~g i.c.v. (D) A C T H , 3 ~g i.c.v.

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Grooming/scratching i n d u c e d in m i c e (n=4) by i n t r a cerebroventricular, i n t r a t h e c a l and i n t r a p e r i t o n e a l a d m i n i s t r a t i o n of b o m b e s i n . %MGE is the p e r c e n t of the m a x i m u m p o s s i b l e n u m b e r of g r o o m i n g / s c r a t c h i n g e p i s o d e s in 15 min. D A P T L - S P (0.50 ~g i.c.v.} e v o k e d no o v e r t w h e n g i v e n to rats t h a t had r e c e i v e d a c e n t r a l for 7 days.

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D A P T L - S P and s p a n t i d e (both at 0.20, 0.50 and 0.80 ~g i.c.v.) w e r e a g a i n i n e f f e c t i v e as b o m b e s i n a n t a g o n i s t s a g a i n s t a s e c o n d e n d p o i n t - b o m b e s i n - i n d u c e d h y p o t h e r m i a in rats p l a c e d in an e n v i r o n m e n t of 6±2°C. It is k n o w n t h a t t o l e r a n c e d o e s not d e v e l o p to the s c r a t c h i n g a s s o c i a t e d w i t h b o m b e s i n a d m i n i s t r a t i o n to rats {23,40). T h i s was c o n f i r m e d in the p r e s e n t s t u d y and c o n t r a s t s w i t h the d e m o n s t r a t e d d e v e l o p m e n t of t o l e r a n c e to b o m b e s i n - i n d u c e d h y p o t h e r m i a (in the same rats) at 6 ± 2 ° C (Fig. 4). In g u i n e a pigs, b o m b e s i n (0.50 ~g i.c.v.} c a u s e d i m m e d i a t e behavioral activation. W i t h i n 3 min, all four a n i m a l s d i s p l a y e d e x c e s s i v e s c r a t c h i n g of the h e a d and n e c k a r e a w i t h the h i n d p a w s (Fig. 2B), f a c e w a s h i n g w i t h f o r e p a w s and w e t - d o g s h a k i n g . One g u i n e a pig s t r e t c h e d and w r i t h e d s e v e r a l t i m e s at +32 min. The s y n d r o m e l a s t e d 30-45 min; t h e r e a f t e r , the a n i m a l s w e r e q u i e t and all s h o w e d p t o s i s . B o m b e s i n (0.i0 ~g i.c.v.) c a u s e d i m m e d i a t e b e h a v i o r a l a g i t a t i o n and r e a r i n g in all t h r e e r a b b i t s s t u d i e d . W i t h i n 2 min, the animals displayed face washing with forepaws. B e t w e e n i0 and 30 min, f a c e w a s h i n g , s c r a t c h i n g of the f l a n k s and n e c k w i t h the h i n ~ paws, and w e t - d o g s h a k i n g , o c c u r r e d e v e r y 3-4 min, that is, in a r e g u l a r f a s h i o n but n ~ so f r e q u e n t l y as we o b s e r v e d in mice, rats and g u i n e a pigs.

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D e v e l o p m e n t of t o l e r a n c e to the h y p o t h e r m i c e f f e c t of b o m b e s i n . R a t s (n=6) r e c e i v e d an i.c.v, i n f u s i o n of w a t e r {.) or b o m b e s i n (0) (0.18 pg/hr) f r o m a s.c. o s m o t i c m i n i p u m p . A f t e r 7 days, b o t h g r o u p s r e c e i v e d b o m b e s i n (0.i0 pg i.c.v.) and r e c t a l t e m p e r a t u r e s w e r e t a k e n for 2 hr at 6±2°C. B o m b e s i n (750 pg and 1 mg i.c.v.) a l t e r e d the b e h a v i o r of f o u r r h e s u s m o n k e y s in a d r a m a t i c m a n n e r . D u r i n g i n j e c t i o n of the p e p t i d e (with the m o n k e y in a r e s t r a i n i n g c h a i r ) , a t r a n s i e n t redn e s s a p p e a r e d a b o u t the f a c e {and p a r t i c u l a r l y the eyes) of the animals. In the cage, t h e y i m m e d i a t e l y s p a t o u t f o o d t h a t w a s s t o r e d in t h e i r c h e e k s , b e c a m e a g i t a t e d and r e s t l e s s and b e g a n pacing. W i t h i n 2-5 min, the m o n k e y s r u b b e d t h e i r e y e s w i t h the b a c k of t h e i r w r i s t s . T h e r e a f t e r , t h e y s c r a t c h e d the face, ears, head, s o l e s of feet, and e n t i r e body; and r u b b e d t h e i r f a c e and p a l m s a l o n g the b a r s of the cage. On a v e r a g e , o n e s c r a t c h i n g / r u b b i n g e p i s o d e o c c u r r e d per m i n (Fig. 5). T h e r e was e v i d e n c e of a b d o m i n a l d i s c o m f o r t ( p r o t e c t i n g the s t o m a c h , r e t c h i n g , v o m i t i n g ) as w e l l as p i l o e r e c t i o n , a b n o r m a l p o s t u r e s , t o n g u e m o v e m e n t s and hypothermia. The s y n d r o m e l a s t e d at l e a s t 4 hr.

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S c r a t c h i n g / g r o o m i n g b e h a v i o r s in m o r p h i n e - d e p e n d e n t r h e s u s m o n k e y s 30-60 min after b o m b e s i n (750 pg i.c.v.) Discussion B o m b e s i n causes mice (17-20) and rats (11,21-23) to g r o o m and scratch. The p r e s e n t e x p e r i m e n t s have shown that b o m b e s i n is a p o t e n t i n d u c e r of s c r a t c h i n g / g r o o m i n g in c e r t a i n other a n i m a l s as well. Thus, mice, rats, g u i n e a pigs, r a b b i t s and m o n k e y s q u i c k l y r e s p o n d to small doses of c e n t r a l l y a d m i n i s t e r e d b o m b e s i n w i t h b e h a v i o r s which, o u t w a r d l y , are s u g g e s t i v e of c h a n g e s in skin sensation. The o v e r t b e h a v i o r of goldfish, frogs, chicks and p i g e o n s a p p e a r s to be u n a f f e c t e d by the peptide, at least under our e x p e r i m e n t a l c o n d i t i o n s . In this context, it is of i n t e r e s t that l o c o m o t o r a c t i v i t y of the t e l e o s t fish, C a t o s t o m u s c o m m e r s o n ~ is not m a r k e d l y i n f l u e n c e d by i.c.v, a p p l i c a t i o n of b o m b e s i n (5 p ~ g) (41). Also, De C a r o et al. ( 4 2 ) s t u d i e d b o m b e s i n in the p i g e o n at doses (0.01-I ~g i.c.v.) lower than we used (I00 ~g i.c.v.). These w o r k e r s found b o m b e s i n to be d i p s o g e n i c , and b e h a v i o r a l l y inactive, in this species. in mice, b o m b e s i n is e s s e n t i a l l y e q u i p o t e n t as a s c r a t c h inducer by i.c.v. (A50=0.010 pg) and i.th. (A50-0.019 pg) routes of a d m i n i s t r a t i o n . S c r a t c h i n g can be e l i c i t e d by i.p. i n j e c t i o n

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b u t a d o s e 6800 t i m e s g r e a t e r t h a n the i.c.v, d o s e is r e q u i r e d . O ' D o n o h u e et al. (20) s t u d i e d b o m b e s i n a f t e r i.th. a d m i n i s t r a t i o n to m i c e and c o n c l u d e d t h a t t h i s p e p t i d e , like s u b s t a n c e P, is a n e u r o t r a n s m i t t e r of p r i m a r y s e n s o r y a f f e r e n t s . Scratching induced by b o m b e s i n a n d s u b s t a n c e P in m i c e c a n be d i f f e r e n t i a t e d p h a r m a c o l o g i c a l l y s i n c e m o r p h i n e a t t e n u a t e s the b e h a v i o r a s s o c i a t e d w i t h s u b s t a n c e P (43) b u t not t h a t c a u s e d by b o m b e s i n (17). It r e m a i n s to be s e e n if a g o n i s t s w h i c h s h o w s e l e c t i v i t y for k a p p a opioid receptors will antagonize bombesin-induced s c r a t c h i n g in mice. T h i s is c e r t a i n l y the c a s e in r a t s w h e r e b o m b e s i n - i n d u c e d s c r a t c h i n g is a n t a g o n i z e d in a s t e r e o s p e c i f i c a n d d o s e - r e l a t e d m a n n e r by b e n z o m o r p h a n k a p p a a g o n i s t s b u t not by m a n y s t a n d a r d o p i o i d s and o p i o i d p e p t i d e s (44). As in mice, b o m b e s i n a c t s at s p i n a l , as w e l l as s u p r a s p i n a l , l e v e l s in r a t s to c a u s e s c r a t c h i n g (45). The i.c.v, and i.th. A50's for the p e p t i d e in m i c e and r a t s are c o m p a r a b l e (45). The b e h a v i o r a l e f f e c t s d i s p l a y e d by r a t s a f t e r b o m b e s i n c a n e a s i l y be d i s t i n g u i s h e d f r o m t h o s e p r o v o k e d by A C T H - ( I - 2 4 ) and TRH (see r e s u l t ) . W h a t m a k e s r a t s r e a c t in s u c h a s t a r t l i n g y e t d i s t i n c t i v e m a n n e r to t h e s e p e p t i d e s ? H o w and w h e r e d o e s the a n i m a l p e r c e i v e the s t i m u l u s t h a t t r i g g e r s the e x c e s s i v e b e h a v i o r s ? Why does tolerance not d e v e l o p to b o m b e s i n - i n d u c e d s c r a t c h i n g j u s t as it d o e s to the hypothermia (46)? We do not h a v e a n s w e r s to t h e s e q u e s t i o n s at the moment. The a v a i l a b i l i t y of b o m b e s i n a n t a g o n i s t s t h a t w o r k in v i v o w o u l d o b v i o u s l y help. The two a g e n t s t e s t e d in the p r e s e n t s t u d y s p a n t i d e (26) and D A P T L - S P (27) - p r o v e d d i s a p p o i n t i n g . We w e r e u n a b l e to d e m o n s t r a t e a n t a g o n i s m of b o m b e s i n - i n d u c e d s c r a t c h i n g or h y p o t h e r m i a o v e r the d o s e range, 0 . 2 0 - 0 . 8 0 ~g i.c.v. Higher doses c a u s e d b a r r e l r o t a t i o n (47) and m o t o r i m p a i r m e n t to s u c h an e x t e n t that behavioral experiments were rendered meaningless. In c o n t r a s t to our e x p e r i e n c e , Y a c h n i s et al. (48) r e c e n t l y u s e d a s a m p l e of s p a n t i d e (2 ~g i.c.v. - P e n i n s u l a ) t h a t g a v e no s i d e e f f e c t s and t h e y w e r e a b l e to p r e v e n t b o m b e s i n - i n d u c e d g r o o m i n g a n d h y p o t h e r m i a in rats. A 5 - m i n i n f u s i o n of b o m b e s i n (1-4 ~g/kg) h a d no m a r k e d e f f e c t on the g r o s s b e h a v i o r of b a b o o n s (49). We f o u n d t h a t b o m b e s i n c a u s e d e x t e n s i v e s c r a t c h i n g in n o n w i t h d r a w n m o r p h i n e - d e p e n d e n t r h e s u s m o n k e y s a f t e r i.c.v, a d m i n i s t r a t i o n of 750 and i000 ~g. T h i s b e h a v i o r has p r e v i o u s l y b e e n n o t e d in m o n k e y s u n d e r g o i n g w i t h d r a w a l f r o m c e r t a i n k a p p a a g o n i s t s (50). The o t h e r s i g n s and s y m p t o m s o b s e r v e d in the p r e s e n t s t u d y - r e s t l e s s n e s s , a b d o m i n a l d i s c o m f o r t , v o m i t i n g , p i l o e r e c t i o n , a b n o r m a l p o s t u r e s and h y p o t h e r m i a - m a y w e l l be a c u t e e f f e c t s of b o m b e s i n in t h i s s p e c i e s . They also constitute a passable morphine-like abstinence syndrome. Is b o m b e s i n p r e c i p i t a t i n g t r u e a b s t i n e n c e , q u a s i - a b s t i n e n c e (51) or a b i t of b o t h ? T e s t i n g the p e p t i d e in d r u g - n a i v e m o n k e y s s h o u l d p r o v i d e the a n s w e r s . Acknowledgements It is a p l e a s u r e to t h a n k Dr. R.J. K o s l o for his h e l p w i t h the i.th. i n j e c t i o n s in mice. The r e s e a r c h w a s s u p p o r t e d by G r a n t s DA 0 3 6 8 1 a n d DA 0 0 2 5 4 f r o m NIDA.

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References i. A. A N A S T A S I , V. E R S P A M E R and M. B U C C I , E x p e r i e n t i a 27 1 6 6 - 1 6 7 (1971) 2. Y. T A C H E and M. B R O W N , T r e n d s N e u r o s c i . 5 4 3 1 - 4 3 3 ( 1 9 8 2 ) 3 M. E R S P A M E R a n d P. M E L C H I O R R I , In: G a s t r o i n t e s t i n a l Hormones, J.C. T h o m p s o n , Ed., p. 575, Univ. of T e x a s P r e s s , A u s t i n ( 1 9 7 9 4 E.A. M A Y E R , J. E L A S H O F F a n d J.H. W A L S H , Am. J. P h y s i o l . 243 GI41-GI47(1982) 5 C. S C A R P I G N A T O , B. M I C A L I , F. V I T U L O , G. Z I M B A R O and G. B E R T A C C I N I , D i g e s t i o n 23 1 2 8 - 1 3 1 ( 1 9 8 2 ) 6 J. G I B B S , D.J. F A U S E R , E.A. ROWE, B.J. R O L L S , E.T. R O L L S a n d S . P . M A D D I S O N , N a t u r e 282 2 0 8 - 2 1 0 ( 1 9 7 9 ) 7 P.J. K U L K O S K Y , L. GRAY, J. G I B B S and G.P. SMITH, P e p t i d e s 2 6 ~ 64(1981) 8 J.E. M O R L E Y and A.S. L E V I N E , P h a r m a c o l . B i o c h e m . B e h a v . 14 1 4 ~ 151(1981) 9 M.R. B R O W N , J. R I V I E R and W.W. V A L E , L i f e Sci. 21 1 7 2 9 - 1 7 3 4 (1977) I0 M.R. B R O W N , Y. T A C H E and D. F I S H E R , E n d o c r i n o l o g y 105 6 6 0 - 6 6 5 (1979) ii M.R. B R O W N , J. R I V I E R and W. V A L E , S c i e n c e 196 9 9 8 - 1 0 0 0 ( 1 9 7 7 ) 12. Q.J. P I T T M A N , Y. T A C H E a n d M.R. BROWN, L i f e Sci. 26 7 2 5 - 7 3 0 (1980) 13. D.D. A V E R Y , M.F. H A W K I N S and B.A. W U N D E R , N e u r o p h a r m a c o l o g y 20 23-27(1981) 14. A. PERT, T.W. M O O D Y , C.B. PERT, L.A. D E W A L D a n d J. R I V I E R , B r a i n Res. 193 2 0 9 - 2 2 0 ( 1 9 8 0 ) 15. Z. M E R A L I , S. J O H N S T O N a n d S. Z A L C M A N , P e p t i d e s 4 693-697{1983) 16. TJ.B. V A N W I M E R S M A G R E I D A N U S , J.A. S C H I J F F , J.L. N O T E B O O M , M.C. SPIT, L. B R U I N S , M. V A N Z U M M E R E N and G . J . E . R I N K E L , P h a r m a c o l . B i o c h e m . B e h a v . 21 1 9 7 - 2 0 2 ( 1 9 8 4 } 17. R. KATZ, N e u r o p h a r m a c o l o g y 19 1 4 3 - 1 4 6 ( 1 9 8 0 ) 18. D. L U T T I N G E R , C.B. N E M E R O F F , G.A. M A S O N , G.D. FRYE, G.R. B R E E S E and A.J. P R A N G E , N e u r o p h a r m a c o l o g y 20 3 0 5 - 3 0 9 ( 1 9 8 1 ) 19. G. M E I S E N B E R G , N e u r o p h a r m a c o l o g y 21 3 0 9 - 3 1 6 ( 1 9 8 2 ) 20. T.L. O ' D O N O H U E , V.J. M A S S A R I , C.J. P A Z O L E S , B.M. C H R O N W A L L , C.W. S H U L T S , R. Q U I R I O N , T.N. C H A S E a n d T.W. M O O D Y , J. N e u r o sci. 4 2 9 5 6 - 2 9 6 2 ( 1 9 8 4 ) 21. P.J. K U L K O S K Y , J. G I B B S a n d G.P. S M I T H , P h y s i o l . B e h a v . 28 5 0 ~ 512(1982) 22. F. G I R A R D , C. AUBE, S. S T - P I E R R E and F.B. J O L I C O E U R , N e u r o peptides 3 443-452(1983) 23. D.E. G M E R E K a n d A. C O W A N , P e p t i d e s 4 9 0 7 - 9 1 3 ( 1 9 8 3 ) 24. W.H. G I S P E N and R.L. I S A A C S O N , P h a r m a c o l . Ther. 12 2 0 9 - 2 4 6 (1981) 25. M.A. G O U J E T , P. S I M O N , R. C H E R M A T a n d J.R. B O I S S I E R , P s y c h o pharmacologia 45 8 7 - 9 2 ( 1 9 7 5 ) 26. R.T. J E N S E N , S.W. J O N E S and J.D. G A R D N E R , Dig. Dis. Sci. 29 (Suppl.) 4 0 S ( 1 9 8 4 ) 27. R.T. J E N S E N , S.W. J O N E S , K. F O L K E R S a n d J.D. G A R D N E R , N a t u r e 309 6 1 - 6 3 ( 1 9 8 4 ) 28. C.W. S T E V E N S and P.D. P E Z A L L A , L i f e Sci. 33 2 0 9 7 - 2 1 0 3 ( 1 9 8 3 ) 29. J . L . K . H Y L D E N and G.L. W I L C O X , Eur. J. P h a r m a c o l . 67 3 1 3 - 3 1 6 (1980) 30. N.W. P E D I G O , W.L. D E W E Y and L.S. H A R R I S , J. P h a r m a c o l . exp. Ther. 193 8 4 5 - 8 5 2 ( 1 9 7 5 ) 31. R.J. T A L L A R I D A and R.B. M U R R A Y , M a n u a l of P h a r m a c o l o g i c Calcu~ ations with Computer Programs, Springer-Verlag, N.Y. (1981)

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32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46.

47. 48. 49. 50. 51.

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F.C. T O R T E L L A , A. C O W A N and M.W. A D L E R , L i f e Sci. 29 1 0 3 9 - 1 0 4 5 (1981) D.E. G M E R E K and A. C O W A N , Eur. J. P h a r m a c o l . 88 3 3 9 - 3 4 6 ( 1 9 8 3 ) E.T. WEI, J. P h a r m a c o l . exp. Ther. 216 1 2 - 1 8 ( 1 9 8 1 ) T.J. L U P A R E L L O , S t e r e o t a x i c A t l a s of the F o r e b r a i n of the G u i n e a Piq, K a r g e r , B a s e l ( 1 9 6 7 ) Y. C H I N G - C H E N G , L. T A - H S I E N , W. S H I H - H S I N U G a n d T. KANG, Sci. S i n i c a 20 1 1 3 - 1 2 4 ( 1 9 7 5 ) H.J. K A R T E N a n d W. H O O D S , A S t e r e o t a x i c A t l a s of the B r a i n of the P i g e o n , J o h n s H o p k i n s U n i v e r s i t y P r e s s , B a l t i m o r e (1968) D.E. G M E R E K , J.L. KATZ, C.P. F R A N C I S and J.H. W O O D S , L i f e Sci. 3 3 ( S u p p l . I) 3 6 1 - 3 6 4 ( 1 9 8 3 ) R.S. S N I D E R a n d J.C. LEE, A S t e r e o t a x i c A t l a s of the M o n k e y B r a i n (Macaca m u l a t t a ) , Univ. of C h i c a g o P r e s s , C h i c a g o ( 1 9 6 1 ) P.J. K U L K O S K Y , J. G I B B S and G.P. S M I T H , B r a i n Res. 242 1 9 4 - 1 9 6 (1982) M. K A V A L I E R S and M.F. H A W K I N S , L i f e Sci. 28 1 3 6 1 - 1 3 6 4 ( 1 9 8 1 ) G. DE CARO, M. M A S S I and L.G. M I C O S S I , Neuropharmacology 19 867-870(1980) N.N. S H A R E and A. R A C K H A M , B r a i n Res. 211 3 7 9 - 3 8 5 ( 1 9 8 1 ) D.E. G M E R E K and A. C O W A N , J. P h a r m a c o l . exp. Ther. 230 1 1 0 - 1 1 5 (1984) D.E. G M E R E K , A. C O W A N and J.L. V A U G H T , Eur. J. P h a r m a c o l . 94 141-143(1983) M. B R O W N , Y. T A C H E , J. R I V I E R and Q. P I T T M A N , In: N e u r o s e c r e t i o n and B r a i n P e p t i d e s , J.B. M a r t i n , S. R e i c h l i n a n d K.L. Bick, Eds., p. 397, R a v e n P r e s s , N e w Y o r k ( 1 9 8 1 ) R.E. B U R K E and S. FAHN, Reg. P e p t i d e s 7 2 0 7 - 2 2 0 ( 1 9 8 3 ) A.T. Y A C H N I S , J.N. C R A W L E Y , R.T. J E N S E N , M.M. M c G R A N E and T.W. M O O D Y , L i f e Sci. 35 1 9 6 3 - 1 9 6 9 ( 1 9 8 4 ) S.C. W O O D S , L.J. S T E I N , D.P. F I G L E W I C Z and D. P O R T E , P e p t i d e s 4 687-691(1983) A. C O W A N , Adv. B i o c h e m . P s y c h o p h a r m . 8 4 2 7 - 4 3 8 ( 1 9 7 3 ) A. C O W A N , Fed. Proc. 40 1 4 9 7 - 1 5 0 1 ( 1 9 8 T )