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Interaction between the effects of tremorine and harmine and of other drugs in chicks
EUROPEAN
JOUFINAL OF PHARMACOLOGY
:3 (1968) 1 0 6 - 1 1 1 .
NOtlTH-HOLLAND
PUBL. COMP..
AMSTEIll)AM
I N T E R A C T I O N B E T W E E N THE EFFECTS OF T R E M O R I N E A N D H A R M I N E A N D OF O T H E R D R U G S I N CHICKS W. C. B O W M A N a n d G. OSUIDE Dcpa~tn~ents of Pharmacology. University of Slralhclydc. Glasgow, C. 1. U.K. and Uniz,ersily of lbadan. Nit~cria ~ Received 5 December 1967
Accepted 11 Marcia 196S
W. C. B O W M A N and G. O S U I D E , Interaction
between lhe ef/~'cls o f l r e m o r i n e and h a r m i n c and of o l h c t
drugs in chicks. European J. Pharmaeol. 3 (1968) 106-t11. l~esponses to t r e m o r i n e and harminc were r e c o r d e d in conscious chicks. The t r e m o r r e s p o n s e to t r e m o r i n c was d e p r e s s e d by atropine, hyoscine, propantheline, orphenadrine, benzhcxol, c h l o r p r o m a zinc, morphine, hemicholinium, O/-methyldopa, 5-hydroxytrypt'unine and 5-hydroxytryptophan. and was potentiatedl)y amphetamine, dopumine, dopa and physostigmhlc. Small doses of deaner potentiated and large doses d e p r e s s e d the response. Acute administration of r e s e r p i n e or tetrabenazine d e p r e s s e d whereas chronic administration potentiated the response. Chlorpromazine, 5-hydroxytryptamine. 5-hydroxytryptophan, amphetamine, dopamim~ dopa, r e serpine and tetrabenazine affected the t r e m o r r e s p o n s e to harminc in a s i m i l a r way to that of t r e m o r inc.
Responses to t r e m o r i n e and harmine, effects ol drugs on
1. I N T R O D U C T I O N Tremorine produces tremor, rigidity, akinesia and parasympathetic symptoms in y o u n g c h i c k s ( B o w m a n a n d O s u i d e , 1967) a s it d o e s in some mammalian species (see Friedman and E v e r e t t , 1964 f o r r e f s . ) . H a r m i n e a l s o p r o d u c e s t r e m o r a n d s o m e a m n e s i a in t h e c h i c k a n d in s o m e m a m m a l s , but p a r a s y m p a t h e t i c e f f e c t s a r e w e a k o r a b s e n t ( F r i e d m a n a n d E v e r e t t , 1964; V e r n i e r , 1964; B o w m a n a n d O s u i d e , 1967). T h i s p a p e r d e s c r i b e s e x p e r i m e n t s in w h i c h the e f f e c t s of o t h e r d r u g s , i n c l u d i n g s o m e u s e d in P a r k i n s o n ' s d i s e a s e , on r e s p o n s e s to t r e m o r i n e a n d harmine were studied.
2. M E T H O D S All e x p e r i m e n t s w e r e p e r f o r m e d on m a l e d o m e s t i c fowl c h i c k s ( S i l v e r Link) a g e d 1 to 4 days, which is the optimal age for d e m o n s t r a t i n g t h e e f f e c t s of t r e m o r i n e ( B o w m a n a n d O s u i d e , 1967). E n v i r o n m e n t a l c o n d i t i o n s w e r e s t a n d a r d i s e d by c a r r y i n g out t h e e x p e r i m e n t s in a q u i e t . T h i s w o r k w a s b e g u n 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 . S c h o o l of P h a r m a c y , U n i v e r s i t y of L o n d o n .
a r t i f i c i a l l y l i g h t e d r o o m a t a t e m p e r a t u r e of 1 9 2 1 o c . T r e m o r w a s r e c o r d e d on a n o s c i l l o s c o p e and its frequency counted electronically as des c r i b e d p r e v i o u s l y ( B o w m a n a n d O s u i d e , 1967). T h e d e g r e e of a k i u e s i a p r o d u c e d by t r e m o r i n e was arbitrarily assessed as described previously ( B o w m a n a n d O s u i d e , 1967). In a s s e s s i n g the e f f e c t s of o t h e r d r u g s on t h e t r e m o r r e s p o n s e s to t r e m o r i n e a n d h a r m i n e , the following procedure was usually used. Each c h i c k w a s i n j e c t e d w i t h 20 m g / k g of t r e m o r i n e o r 20 m g k g h a r m i n e i n t r a p e r i t o n e a l l y a n d t h e tremor produced was counted for 1 hour after i n j e c t i o n . T h r e e to f o u r h o u r s a f t e r t h e f i r s t i n j e c t i o n of t r e m o r i n e o r h a r m i n e , t h e c h i c k s w e r e injected i n t r a p e r i t o n e a l l y with a t e s t drug, and 15 rain l a t e r t h e s a m e d o s e (20 m g / k g ) of t r e m o r i n e o r h a r m i n e w a s i n j e c t e d . T h e e f f e c t of the t e s t d r u g w a s d e t e r m i n e d by c o m p a r i n g the e f f e c t of the s e c o n d i n j e c t i o n o f t r e m o r i n e o r b a r m i n e w i t h that p r o d u c e d by t h e f i r s t , s o t h a t e a c h c h i c k a c t e d a s i t s own c o n t r o l . T h e e f f e c t of e a c h d o s e of a t e s t d r u g w a s d e t e r m i n e d a s the m e a n e f f e c t on at l e a s t 3 c h i c k s . C o n t r o l e x p e r i m e n t s s h o w e d t h a t a s e c o n d d o s e of t r e m o r i n e o r b a r m i n e g i v e n 3 - 4 h r a f t e r t h e f i r s t , but in the a b s e n c e of any o t h e r d r u g s , p r o d u c e d a t r e m o r r e s p o n s e only s l i g h t l y g r e a t e r (1-5%) t h a n t h a t
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an i m m e d i a t e s h o r t - l a s t i n g (5-10 min) block of the t w i t c h e s of the t i b i a l i s a n t e r i o r m u s c l e . Benzhexol (7.5 m g / k g ) slightly d e p r e s s e d (12-20%) the t r e m o r count p r o d u c e d by h a r m i n e , but in the d o s e s e f f e c t i v e a g a i n s t t r e m o r i n e , none of the o t h e r d r u g s a l t e r e d the r e s p o n s e s to harmine. 3.2. Hemicholinium (HC-3) In d o s e s of 12.5 g g / k g and 25 ~zg/kg, HC-3 p r o d u c e d 5-10% and 25-40% r e d u c t i o n r e s p e c t i v e l y in the t r e m o r count p r o d u c e d by t r e m o r inc. The l a r g e r dose u s u a l l y p r o d u c e d h e a d - d r o p but the chicks r e m a i n e d standing. A k i n e s i a and parasympathetic effects were also reduced. L a r g e r d o s e s of HC-3 (50 p g / k g ) p r o d u c e d , a f t e r a latent p e r i o d of 7-10 min, c o m p l e t e p a r a l y s i s of the chicks which l a s t e d for 30-50 min. No t r e m o r was evident during the p a r a l y s i s . In s u b - p a r a l y t i c d o s e s , HC-3 was without effect on r e s p o n s e s to h a r m i n e . Do s es of HC-3 up to 100 ~ g / k g w e r e without effect on m a x i m a l t w i t c h e s of the t i b i a l i s a n t e r i o r m u s c l e evoked by s t i m u l a t i o n of the s c i a t i c n e r v e at a f r e q u e n c y of 1 or 2 / s e c . 3.3. Morphine In a dose of 10 m g / k g , m o r p h i n e p r o d u c e d a 35-45% d e p r e s s i o n of the t r e m o r count in r e sponse to t r e m o r i n e . A k i n e s i a and p a r a s y m p a thetic e f f e c t s w e r e a l s o s l i g h t l y inhibited. The r e s p o n s e to h a r m i n e w a s unchanged by this dose of m o r p h i n e . 3.4. Physostigmine In d o s e s of 0.5 m g / k g and above, p h y s o s t i g mine i t s e l f p r o d u c e d t r e m o r , f a s c i c u l a t i o n s , s a l i v a t i o n and d e f a e c a t i o n within 2 min a f t e r i n j e c t i o n . S m a l l e r d o s e s w e r e without this effect. In a dose of 0.25 m g / k g , p h y s o s t i g m i n e p o t e n tiated the t r e m o r count p r o d u c e d by t r e m o r i n e by 30-40% and a u g m e n t e d the a k i n e s i a and p a r a s y m p a t h e t i c s y m p t o m s . P h y s o s t i g m i n e did not a u g m e n t the t r e m o r r e s p o n s e to h a r m i n e . 3.5. DeangJ" The e f f e c t s of d e a n e r on r e s p o n s e s to t r e m o r i n e v a r i e d with the dose of d e a n e r u s e d and the p e r i o d of p r e - t r e a t m e n t . When t r e m o r i n e w as i n j e c t e d 15 rain a f t e r 2 m g / k g d e a n e r , the t r e m or count was a u g m e n t e d by 20-25% but t h e r e was no d e t e c t a b l e change in the d e g r e e of a k i n e s i a . When t r e m o r i n e w a s i n j e c t e d 1 hr a f t e r a dose of 20 m g / k g d e a n e r , the t r e m o r count was d e p r e s s e d by 30-40% and a k i n e s i a was augmented. D e a n e r in d o s e s of 2-20 m g / k g w a s without e f -
f e c t on the r e s p o n s e to h a r m i n e . When t r e m o r ine was i n j e c t e d 2 hr a f t e r a dose of 200 m g / k g d e a n e r , t r e m o r w as c o m p l e t e l y s u p p r e s s e d and a k i n e s i a was f u r t h e r enhanced. This dose of d e a n e r slightly d e p r e s s e d (24-33%) the t r e m o r r e s p o n s e to h a r m i n e . P a r a s y m p a t h e t i c e f f e c t s of t r e m o r i n e w e r e enhanced by d e a n e r , e s p e c i a l l y in high d o s e s . 3.6. 5 - H y d r o x y t r y p t a m i n e (5-HT) and 5-hydroxy-
tryptophan (5 -HTP) In d o s e s of 10-20 m g / k g , 5-HT produced a 20-50% d e p r e s s i o n of the t r e m o r count produced by t r e m o r i n e but did not change the a k i n e s i a or p a r a s y m p a t h e t i c effects. 5 - H T P in a dose of 100 m g / k g produced s e d a tion. T r e m o r i n e , i n j e c t e d when the s e d a t i v e e f fect of 5 - H T P had a p p a r e n t l y d i s a p p e a r e d , p r o duced a t r e m o r count which was 60-80% s m a l l e r than the control. O t h er e f f e c t s of t r e m o r i n e w e r e not r e d u c e d by 5 - H T P . In the s a m e d o s e s , 5-HT and 5 - H T P d e p r e s s e d the t r e m o r count p r o d u c e d by h a r m i n e by 30-40%. The 5-HT an t ag o n i st , b r o m o l y s e r g i c a c i d d i e t h y l a m i d e , in d o s e s up to 25 m g / k g did not a l t e r the e f f e c t s of t r e m o r i n e or h a r m i n e . 3.7. Dopamine and dopa In d o s e s of 50 m g / k g , dopamine produced a s m a l l potentiation (about 30%) of the t r e m o r count but did not change the o t h er a c t i o n s of tremorine. Dopa, in a dose of 100 m g / k g i n j e c t e d 1 to 2 hr p r e v i o u s l y , a l m o s t doubled the t r e m o r count p r o d u c e d by t r e m o r i n e , and a u g m e n t e d the d e g r e e of a k i n e s i a . No d e t e c t a b l e changes in the p a r a s y m p a t h e t i c r e s p o n s e s to t r e m o r i n e w e r e produced. In the s a m e d o s e s , dopamine and dopa p r o duced s i m i l a r i n c r e a s e s in the t r e m o r count p r o d u c e d by h a r m i n e . In the d o s e s used, dopa alone did not p r o d u ce obvious b e h a v i o u r a l changes a f t e r i n t r a p e r i t o neal i n j ect i o n in chicks and did not produce a k i n e s i a . Spooner and W i n t e r s (1964), using l a r g e r d o s e s (300 m g / k g ) , did find it to p r o d u ce a k i n e si a in chicks, and a c c o r d i n g to Sourkes (1964) and L e v y and M i c h e l - B e r (1964) dopa p r o d u c e s r i g i d i t y and a k i n e s i a in m i c e . 3.8. Amphetamine In d o s e s of 2-5 mg//kg, a m p h e t a m i n e a u g m e n t e d the t r e m o r count by 80-100% but did not change the o t h e r r e s p o n s e s to t r e m o r i n e . In a dose of 15 mg/~kg, a m p h e t a m i n e i t s e l f produced
TREMORINE AND HARMINE ataxia and the chicks r e s t e d t h e i r s t e r n u m s on the base of the r e c o r d i n g a p p a r a t u s making it i m p o s s i b l e to count the t r e m o r a c c u r a t e l y a l though a s far a s could be d e t e r m i n e d by v i s u a l o b s e r v a t i o n the t r e m o r was s u p p r e s s e d . This l a r g e dose of a m p h e t a m i n e s t i m u l a t e d chirping (see also Z a i m i s 1960a,b). In doses of 2-5 m g / k g , a m p h e t a m i n e p r o duced a s i m i l a r a u g m e n t a t i o n of the t r e m o r count produced by h a r m i n e . 3.9. Reserpine and tetrabenazine R e s e r p i n e (3 mg/kg) or t e t r a b e n a z i n e (3 m g / kg) i n j e c t e d 30 m i n before t r e m o r i n e or h a r m i n e d e p r e s s e d the t r e m o r count by 30-50% but slightly a u g m e n t e d the a k i n e s i a produced by t r e m o r i n e . When the s a m e dose of r e s e r p i n e or t e t r a b e n a z i n e was injected 24 hr before the test dose of t r e m o r i n e or h a r m i n e , a k i n e s i a was u n a l t e r e d , but the t r e m o r count was now i n c r e a s e d by 12-40%. Salivation in r e s p o n s e to t r e m o r i n e was i n c r e a s e d after r e s e r p i n e p r e t r e a t m e n t but this effect was not detected after t e t r a b e n a z i n e . 3.10. c~-Methyldopa In a dose of 800 m g / k g i n j e c t e d 1 hr before t r e m o r i n e , c~-methyldopa prolonged the latent p e r i o d between injection and onset of t r e m o r by 50-80%, r e d u c e d the t r e m o r count by 25-40% and a u g m e n t e d the d e g r e e of a m n e s i a . S m a l l e r doses of ~ - m e t h y l d o p a w e r e without effect on the r e sponse to t r e m o r i n e . (~-Methyldopa was without effect on r e s p o n s e s to h a r m i n e . 3.11. Phenoxybenzamine In doses of 25 m g / k g , p h e n o x y b e n z a m i n e p r o duced a s m a l l r e d u c t i o n (10-20%) in the t r e m o r count produced by t r e m o r i n e and slightly r e duced the degree of a k i n e s i a . P a r a s y m p a t h e t i c effects of t r e m o r i n e were abolished. P h e n o x y b e n z a m i n e was without detectable effect on r e s p o n s e s to h a r m i n e . 3.12. Pronethalol In s u b - l e t h a l doses (< 80 mg/kg) pronethalol did not affect the t r e m o r or a k i n e s i a produced by t r e m o r i n e or h a r m i n e , but the p a r a s y m p a t h e t i c effects of t r e m o r i n e were antagonised. 3.13. Chlorpromazine In doses of 2.5 m g / k g , c h l o r p r o m a z i n e p r o duced a 50-60% r e d u c t i o n in the t r e m o r counts produced both by t r e m o r i n e and by h a r m i n e but did not produce a detectable change in the degree of a k i n e s i a . P a r a s y m p a t h e t i c effects of t r e m o r -
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ine w e r e abolished by this dose of c h l o r p r o m a zine. 3.14. Mepyramine and diphenhydramine M e p y r a m i n e (25 mg/kg) p r e v e n t e d the p a r a s y m p a t h e t i c effects of t r e m o r i n e and r e d u c e d the t r e m o r count by 20%. D i p h e n h y d r a m i n e (5 m g / kg) produced s i m i l a r effects. Neither drug changed the r e s p o n s e s to h a r m i n e . 3.15. Mephenesin Mephenesin (40 mg/kg) was without effect on the r e s p o n s e s to t r e m o r i n e or h a r m i n e injected 15 m i n l a t e r . However, when i n j e c t e d d u r i n g the effects of t r e m o r i n e or h a r m i n e , the same dose of m e p h e n e s i n produced a t e m p o r a r y (5-10 min) c e s s a t i o n of the t r e m o r r e s p o n s e s . 3.16. Pentobarbitone sodium In s u b - h y p n o t i c doses (5 m g / k g and below) pentobarbitone was without effect on r e s p o n s e s to t r e m o r i n e or h a r m i n e . In a dose of 7.5 m g / kg, pentobarbitone itself produced slight ataxia and r e d u c e d the t r e m o r r e s p o n s e s to t r e m o r i n e or h a r m i n e by 25-35%. The p a r a s y m p a t h e t i c r e sponses to t r e m o r i n e were unchanged. 3.17. Other substances G a m m a a m i n o b u t y r i c acid (250 mg/kg), 4 - h y d r o x y b u t y r a t e (50 mg/kg), t y r a m i n e (100 mg/kg), and ergothioneine (100 mg/kg) were all without effect on the r e s p o n s e s to t r e m o r i n e and h a r mine.
4. DISCUSSION T r e m o r i n e i n c r e a s e s b r a i n levels of 5-HT and r e d u c e s those of n o r a d r e n a l i n e both in mice ( F r i e d m a n , 1963; F r i e d m a n , Aylesworth and F r i e d m a n , 1963) and in chicks (Bowman and Osuide, 1967). Injection of 5 - H T P r a i s e s the b r a i n level of 5-HT in chicks (Osuide, 1966). The d i r e c t i o n of change in b r a i n 5-HT is t h e r e fore the s a m e as that produced by t r e m o r i n e , yet the t r e m o r effect of t r e m o r i n e was reduced by 5 - H T P . Injection of dopa i n c r e a s e s the b r a i n l e v e l s of n o r a d r e n a l i n e in chicks (Osuide, 1966) so that the depleting effect of t r e m o r i n e would be r e v e r s e d . N e v e r t h e l e s s , dopa augmented the t r e m o r and a k i n e s i a produced by t r e m o r i n e . T h e s e r e s u l t s make it unlikely that the t r e m o r produced by t r e m o r i n e is a consequence of its a l t e r i n g b r a i n l e v e l s of n o r a d r e n a l i n e or 5-HT. Holmstedt (1964) r e p o r t e d an i n c r e a s e in the b r a i n l e v e l s of a c e t y l c h o l i n e produced by t r e m -
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o r i n e in r a t s and showed that t h e r e was a c o r r e lation between this effect and the extent and dur a t i o n of the t r e m o r produced. A c c o r d i n g to A p r i s o n and T a k a h a s h i (1965) c h o l i n e r g i c m e c h a n i s m s a r e e s p e c i a l l y i m p o r t a n t in a v i a n b r a i n , and an i n c r e a s e in b r a i n l e v e l s of a c e t y l c h o l i n e p r o d u c e d by t r e m o r i n e was a l s o d e t e c t e d in chicks (Bowman and Osuide, 1967). The p r e s e n t r e s u l t s showed that o th e r d r u g s known to influe n c e acetylcholine m e t a b o l i s m modified the t r e m o r r e s p o n s e to t r e m o r i n e in a m a n n e r c o n s i s t e n t with H o l m s t e d t ' s (1964) c o n c l u s i o n that the b a s i c action of t r e m o r i n e is on b r a i n a c e t y l choline l e v e l s . Thus, the c h o l i n e s t e r a s e i n h i b i t o r , p h y s o s t i g m i n e , p o t e n ti a t e d t r e m o r . D e a n e r , which is m e t a b o l i s e d into a c e t y l c h o l i n e in the b r a i n ( K i p l i n g e r , Swain and Brody, 1958; Groth, Bain and P f e i f f e r , 1958) potentiated t r e m o r in s m a l l d o s e s and d e p r e s s e d it in l a r g e d o s e s ; a c e t y l c h o l i n e is known to s t i m u l a t e c h o l i n o c e p t i v e s i t e s in s m a l l d o s e s but to block in l a r g e d o s e s . Morphine inhibits choline a c e t y l t r a n s f e r a s e (Balotin and Coon, 1960), and h e m i c h o l i n i u m inhibits the t r a n s p o r t of choline to its a c e t y l a t i o n s i t e s , so that both d r u g s inhibit a c e t y l c h o l i n e s y n t h e s i s and both d e p r e s s e d the t r e m o r r e s p o n s e to t r e m o r i n e . F i n a l l y , d r u g s with an a t r o p i n e - l i k e action ( a t r o p i n e , h y o s c i n e , b e n z hexol, p r o p a n t h e l i n e and o r p h e n a d r i n e ) blocked the t r e m o r p r o d u c e d by t r e m o r i n e . The weak a n t i - t r e m o r e f f e c t s of p h e n o x y b e n z a m i n e and the a n t i - h i s t a m i n e s may a l s o have been the r e s u l t of a weak a t r o p i n e - l i k e action. It was of i n t e r e s t that the a n t i - t r e m o r e f f e c t s of the q u a t e r n a r y compounds, h e m i c h o l i n i u m and p r o p a n t h e l i n e , like those of the other a n t a g o n i s t s , a p p e a r e d to be due to a c e n t r a l action and this probably r e f l e c t s the u n d e r d e v e l o p m e n t of the b l o o d - b r a i n b a r r i e r in young chicks (Waelsch, 1955; Z a i m i s , 1960a, Key and M a r l e y , 1962). H e m i c h o l i n i u m w a s e f f e c t i v e a g a i n s t t r e m o r p r o d u c e d by t r e m o r i n e in d o s e s which w e r e too s m a l l to affect p e r i p h e r a l n e u r o m u s c u l a r t r a n s m i s s i o n , and a d ditional a s yet unpublished e x p e r i m e n t s by one of us (G. O.) c o n f i r m that, in the chick, s m a l l d o s e s of h e m i c h o l i n i u m s e l e c t i v e l y affect c h o l i n e r g i c m e c h a n i s m s in the b r a i n . P r o p a n t h e l i n e did p r o duce a t r a n s i e n t block of c o n t r a c t i o n s of the t i b i a l i s a n t e r i o r m u s c l e evoked by m o t o r n e r v e s t i m u l a t i o n but this effect did not a p p e a r to c o n t r i b u t e to i t s a n t i - t r e m o r action. P r o p a n t h e l i n e w a s a l w a y s i n j e c t e d 15 min b e f o r e the t r e m o r ine, so that by the t i m e t r e m o r i n e was i n j e c t e d any t r a n s i e n t n e u r o m u s c u l a r blocking action would have d i s a p p e a r e d . T h e r e was no e v i d e n c e in c o n s c i o u s chicks of any m u s c u l a r w e a k n e s s
p r o d u c e d by p r o p a n t h e l i n e in the d o s e s used. F u r t h e r m o r e , the t r e m o r i n e count was continued for an hour a f t e r its injection, and any t r a n s i e n t n e u r o m u s c u l a r blocking action would have made a n e g l i g i b l e d i f f e r e n c e . P r o p a n t h e l i n e does not affect r e s p o n s e s to t r e m o r i n e in m i c e (Spencer, 1965), p r e s u m a b l y b e c a u s e of its inability to p e n e t r a t e the b l o o d - b r a i n b a r r i e r in this s p e c i e s . N e i t h e r h e m i c h o l i n i u m nor p r o p an t h el i n e w e r e e f f e c t i v e a g a i n s t t r e m o r p r o d u c e d by h a r m i n e, yet a p e r i p h e r a l a c t i o n of t h ese d r u g s would be e x p e c t e d to affect d i f f e r e n t types of c e n t r a l l y - i n d u c e d t r e m o r in the s a m e way. A k i n e s i a p r o d u c e d by t r e m o r i n e a l s o a p p e a r e d to depend on a c e n t r a l c h o l i n e r g i c m e c h a n i s m . Thus, a k i n e s i a was r e d u c e d by a t r o p i n e like d r u g s , m o r p h i n e and h e m i c h o l i n i u m , and enhanced by p h y s o s t i g m i n e and d e a n e r . The l a r g e d o s e s of d e a n e r which d e p r e s s e d t r e m o r , enhanced a k i n e s i a . This d i f f e r e n c e may be e x plained if it is a s s u m e d that t r e m o r is the r e s u l t of a c t i v a t i o n of c h o l i n e r g i c s y n a p s e s by s t i m ulant l e v e l s of a c e t y l c h o l i n e (and hence is d e p r e s s e d by e x c e s s a c e t y l c h o l i n e ) , w h e r e a s a k i n e s i a is the r e s u l t of block of c h o l i n e r g i c s y n a p s e s by e x c e s s a c e t y l c h o l i n e (and hence is a u g m e n t e d by an additional e x c e s s of a c e t y l c h o l i n e ) . Although t r e m o r and a m n e s i a p r o d u c e d by t r e m o r i n e did not a p p e a r to be d i r e c t l y a t t r i b u t able to changes in c a t e c h o l a m i n e l e v e l s , they w e r e n e v e r t h e l e s s influenced by changes in t h ese l e v e l s . Injection of dopa a u g m e n t e d t r e m o r and a k i n e s i a , p r e s u m a b l y as a r e s u l t of its c o n v e r s i o n to n o r a d r e n a l i n e . R e s e r p i n e , t e t r a b e n a zine and ~ - m e t h y l d o p a i n i t i a l l y cau se the r e l e a s e of c a t e c h o l a m i n e s , and t h e r e f o r e r a i s e the l e v e l of f r e e a m i n e s . When t r e m o r i n e was i n j e c t e d soon a f t e r one of t h e s e d r u g s , t r e m o r w as r e d u c e d and a k i n e s i a enhanced. When 24 hr e l a p s e d between i n j ect i o n of r e s e r p i n e and i n j e c tion of t r e m o r i n e , catecholamine depletion would be e x p e c t e d to have o c c u r r e d ; t r e m o r was now enhanced. T r e m o r was i n i t i a l l y enhanced by a m p h e t a m i n e , p o s s i b l y through c a t e c h o l a m i n e r e l e a s e . Some of t h e s e i n t e r a c t i o n s may be e x plained by the known a c t i o n s of c a t e c h o l a m i n e s on c h o l i n e r g i c s y n a p s e s , e s p e c i a l l y if it is a s s u m e d that the a c c e s s i b i l i t y of the s y n a p s e s i n v o l v e d in t r e m o r d i f f e r s f r o m that of the s y n a p s e s involved in a k i n e s i a and that t h ese s y n a p s e s a r e n o r m a l l y modulated by c a t e c h o l a m i n e s . Small a m o u n t s of a d r e n a l i n e have been shown to f a c i l i t a t e c h o l i n e r g i c synaptic t r a n s m i s s i o n (Biilbring and Burn, 1942) by i n c r e a s i n g the r e l e a s e of a c e t y l c h o l i n e f r o m p r e s y n a p t i c n e r v e endings ( B i r k s and Macintosh, 1961).
T R E M O R I N E AND HARMINE Larger amounts depress synaptic transmission (Marazzi, 1939) by hyperpolarizing the post-synaptic cell membranes (De Groat and Volle, 1965).
ACKNOWLEDGEMENTS T h i s w o r k w a s s u p p o r t e d by a g r a n t f r o m the B r i t i s h Egg M a r k e t i n g B o a r d . We a r e g r a t e f u l to A b b o t t ' s L a b o r a t o r i e s L i m i t e d for g i f t s of t r e m o r i n e .
REFERENCES A p r i s o n , M . H . and R. T a k a h a s h i , 1965, B i o c h e m i s t r y of the a v i a n c e n t r a l n e r v o u s s y s t e m - II. 5 - H y droxytryptamine, acetylcholine, 3, 4 - d i h y d r o x y p h e n y l n y l e t h y l a m i n e and n o r e p i n e p h r i n e in s e v e r a l d i s c r e t e a r e a s of the p i g e o n b r a i n , J. N e u r o c h e m . 12, 221. Balotin, H . M . a n d J. M. Coon, 1960, T h e a n t a g o n i s m of s o m e a c t i o n s of t e t r a e t h y l p y r o p h o s p h a t e by m o r phine, A r c h s . Int. P h a r m a e o d y n . T h e r . 123, 395. B i r k s , R . L and F . C . M a c I n t o s h , 196], A c e t y l c h o l i n e m e t a b o l i s m of a s y m p a t h e t i c g a n g l i o n , Can. J. B i o c h e m . P h y s i o l . 39, 787. B o w m a n , W, C., B . A . C a l l i n g h a m and G. O s u i d e , 1964, E f f e c t s of t y r a m i n e on a s p i n a l r e f l e x in the a n a e s t h e t i s e d chick, J. P h a r m . P h a r m a c . 16, 505. B o w m a n , W . C . a n d G . O s u i d e , 1967, E f f e c t s of t r e m o r i n e and h a r m i n e in the c h i c k , E u r o p e a n J. P h a r m a c o l . 1, 71. Bfilbring, E. and J . H . B u r n , 1942, An a c t i o n of a d r e n aline on t r a n s m i s s i o n in a s y m p a t h e t i c g a n g l i o n w h i c h m a y play a p a r t in s h o c k , J. P h y s i o l . 101, 289. F r i e d m a n , A . H . , 1963, N o r e p i n e p h r i n e d e p l e t i o n in the b r a i n s t e m by t r e m o r i n e , F e d n . P r o c . 22, 272. Friedman, A.H., R.J.Aylesworth and G. F r i e d m a n , 1963, T r e m o r i n e : E f f e c t s on a m i n e s of the c e n t r a l n e r v o u s s y s t e m , S c i e n c e 141, 1188. F r i e d m a n , A . H . and G . M . E v e r e t t , 1964, P h a r m a c o l o g i c a l a s p e c t s of P a r k i n s o n i s m , A d v a n c e s in P h a r m a c o l o g y 3, e d s . S. G a r a t t i n i and P . A . S h o r e (Acad e m i c P r e s s , New York) p. 83, De G r o a t , W . C . and R. L. Volle, 1965, G a n g l i o n i c a c tions of c a t e e h o l a m i n e s , P h a r m a c o l o g i s t 7, 158. G r o t h , D . P . , J . A . B a i n and C . C . P f e i f f e r , 1958, The c o m p a r a t i v e d i s t r i b u t i o n of C14 l a b e l l e d 2 - d i m e t h -
111
y l - a m i n o e t h a n o l a n d c h o l i n e in the m o u s e , J. P h a r m a c . Exp. T h e r . 122, 28A. H o h n s t e d t , B., 1964, P r o c e e d i n g s of the 2rid I n t e r n a tional P h a r m a c o l o g i c a l C o n g r e s s ( P e r g a m o n P r e s s , Oxford) p. 128. Key, B . J . and E. M a r l e y , 1962, T h e e f f e c t of s y m p a t h o m i m e t i c a m i n e s on b e h a v i o u r and e l e c t r o c o r t i c a l a c t i v i t y of c h i c k e n s , E l e c t r o n . N e u r o p h y s i o l . 14, 90. K i p l i n g e r , G . F . , H . H . Swain and T . M . Bordy, 1958, T h e a c t i o n of d i m e t h y l a m i n o e t h a n o l on the i s o l a t e d h e a r t , J. P h a r m a c . Exp. T h e r . 122, 37A. Levy,, J. and E. M i c h e l - B e r , 1964, De l ' a n t a g o n i s m c v i s - h - v i s de d i f f 6 r e n t s e f f e c t s de la t r S m o r i n e c h e z la s o u r i s , in: P r o c e e d i n g s of the s e c o n d i n t e r n a tional p h a r m a c o l o g i c a l m e e t i n g , e d s . E. T r a b u c c h i , R. P a o l e t t i and N. C a n a l ( P e r g a m o n P r e s s , O,,dord) 2, 109. M a r a z z i , A . S . , 1939, E l e c t r i c a l s t u d i e s on t h e p h a r m a c o l o g y of a u t o n o m i c s y n a p s e s . II. T h e a c t i o n of a s y m p a t h o m i m e t i c d r u g (epinephrine) on s y m p a thetic g a n g l i a , J. P h a r m a c . Exp. T h e r . 65, 395. O s u i d e , G., 1966, P h a r m a c o l o g i c a l l y a c t i v e s u b s t a n c e s in the c e n t r a l n e r v o u s s y s t e m of the chick, P h . D . T h e s i s , U n i v e r s i t y of London. Pale'6ek, F., 1963, A s i m p l e r e s p i r a t o r f o r l a b o r a t o r y u s e , J. Appl. P h y s i o l . 18, 443. S o u r k e s , T . L . , 1964, A c t i o n s of dopa and d o p a m i n e in r e l a t i o n to f u n c t i o n of the c e n t r a l n e r v o u s s y s t e m , in: P r o c e e d i n g s of the s e c o n d i n t e r n a t i o n a l p h a r m a c o l o g i c a l m e e t i n g , e d s , E. T r a b u c c h i , R. P a o l e t t i and N. C a n a l ( P e r g a m o n P r e s s , Oxford) 2, 35. S p e n c e r , P . S . J . , 1965, A c t i v i t y of c e n t r a l l y a c t i n g and o t h e r d r u g s a g a i n s t t r e m o r and h y p o t h e r m i a ind u c e d in m i c e by t r e m o r i n e , B r i t . J. P h a r m a c . C h e m o t h e r . 25, 442. S p o o n e r , C . E . and W. D. W i n t e r s , 1964, B e h a v i o u r a l , eeg, and blood p r e s s u r e e f f e c t s of c e n t r a l l y a c t i n g d r u g s in the chick, P h a r m a c o l o g i s t 6, 171. V e r n i e r , U . G . , 1964, A n t i - P a r k i n s o n a g e n t s : E v a l u atio~ of d r u g a c t i v i t i e s : P h a r m a c o m e t r i c s , eds. D. 1l. L a u r e n c e and A, L. B a c h a r a c h (Academic P r e s s , New York and London) p, 301. W a e l s c h , H., 1955, T h e t u r n o v e r of c o m p o n e n t s of the d e v e l o p i n g b r a i n ; the blood b r a i n b a r r i e r , B i o c h e m . i s t r y of the d e v e l o p i n g n e r v o u s s y s t e m ( A c a d e m i c P r e s s , New York) p. 187. Z a i m i s , E., 1960a, Ciba F o u n d a t i o n S y m p o s i u m on A d r e n e r g i c M e c h a n i s m s ( C h u r c h i l l , London) p. 562. Z a i m i s , E., 1960b, S i m u l t a n e o u s r e c o r d i n g of a c t i v i t y and r a t e of c h i r p i n g in the y o u n g chick, J. P h y s i o l . (London) 152, 11P.
JOUFINAL OF PHARMACOLOGY
:3 (1968) 1 0 6 - 1 1 1 .
NOtlTH-HOLLAND
PUBL. COMP..
AMSTEIll)AM
I N T E R A C T I O N B E T W E E N THE EFFECTS OF T R E M O R I N E A N D H A R M I N E A N D OF O T H E R D R U G S I N CHICKS W. C. B O W M A N a n d G. OSUIDE Dcpa~tn~ents of Pharmacology. University of Slralhclydc. Glasgow, C. 1. U.K. and Uniz,ersily of lbadan. Nit~cria ~ Received 5 December 1967
Accepted 11 Marcia 196S
W. C. B O W M A N and G. O S U I D E , Interaction
between lhe ef/~'cls o f l r e m o r i n e and h a r m i n c and of o l h c t
drugs in chicks. European J. Pharmaeol. 3 (1968) 106-t11. l~esponses to t r e m o r i n e and harminc were r e c o r d e d in conscious chicks. The t r e m o r r e s p o n s e to t r e m o r i n c was d e p r e s s e d by atropine, hyoscine, propantheline, orphenadrine, benzhcxol, c h l o r p r o m a zinc, morphine, hemicholinium, O/-methyldopa, 5-hydroxytrypt'unine and 5-hydroxytryptophan. and was potentiatedl)y amphetamine, dopumine, dopa and physostigmhlc. Small doses of deaner potentiated and large doses d e p r e s s e d the response. Acute administration of r e s e r p i n e or tetrabenazine d e p r e s s e d whereas chronic administration potentiated the response. Chlorpromazine, 5-hydroxytryptamine. 5-hydroxytryptophan, amphetamine, dopamim~ dopa, r e serpine and tetrabenazine affected the t r e m o r r e s p o n s e to harminc in a s i m i l a r way to that of t r e m o r inc.
Responses to t r e m o r i n e and harmine, effects ol drugs on
1. I N T R O D U C T I O N Tremorine produces tremor, rigidity, akinesia and parasympathetic symptoms in y o u n g c h i c k s ( B o w m a n a n d O s u i d e , 1967) a s it d o e s in some mammalian species (see Friedman and E v e r e t t , 1964 f o r r e f s . ) . H a r m i n e a l s o p r o d u c e s t r e m o r a n d s o m e a m n e s i a in t h e c h i c k a n d in s o m e m a m m a l s , but p a r a s y m p a t h e t i c e f f e c t s a r e w e a k o r a b s e n t ( F r i e d m a n a n d E v e r e t t , 1964; V e r n i e r , 1964; B o w m a n a n d O s u i d e , 1967). T h i s p a p e r d e s c r i b e s e x p e r i m e n t s in w h i c h the e f f e c t s of o t h e r d r u g s , i n c l u d i n g s o m e u s e d in P a r k i n s o n ' s d i s e a s e , on r e s p o n s e s to t r e m o r i n e a n d harmine were studied.
2. M E T H O D S All e x p e r i m e n t s w e r e p e r f o r m e d on m a l e d o m e s t i c fowl c h i c k s ( S i l v e r Link) a g e d 1 to 4 days, which is the optimal age for d e m o n s t r a t i n g t h e e f f e c t s of t r e m o r i n e ( B o w m a n a n d O s u i d e , 1967). E n v i r o n m e n t a l c o n d i t i o n s w e r e s t a n d a r d i s e d by c a r r y i n g out t h e e x p e r i m e n t s in a q u i e t . T h i s w o r k w a s b e g u n 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 . S c h o o l of P h a r m a c y , U n i v e r s i t y of L o n d o n .
a r t i f i c i a l l y l i g h t e d r o o m a t a t e m p e r a t u r e of 1 9 2 1 o c . T r e m o r w a s r e c o r d e d on a n o s c i l l o s c o p e and its frequency counted electronically as des c r i b e d p r e v i o u s l y ( B o w m a n a n d O s u i d e , 1967). T h e d e g r e e of a k i u e s i a p r o d u c e d by t r e m o r i n e was arbitrarily assessed as described previously ( B o w m a n a n d O s u i d e , 1967). In a s s e s s i n g the e f f e c t s of o t h e r d r u g s on t h e t r e m o r r e s p o n s e s to t r e m o r i n e a n d h a r m i n e , the following procedure was usually used. Each c h i c k w a s i n j e c t e d w i t h 20 m g / k g of t r e m o r i n e o r 20 m g k g h a r m i n e i n t r a p e r i t o n e a l l y a n d t h e tremor produced was counted for 1 hour after i n j e c t i o n . T h r e e to f o u r h o u r s a f t e r t h e f i r s t i n j e c t i o n of t r e m o r i n e o r h a r m i n e , t h e c h i c k s w e r e injected i n t r a p e r i t o n e a l l y with a t e s t drug, and 15 rain l a t e r t h e s a m e d o s e (20 m g / k g ) of t r e m o r i n e o r h a r m i n e w a s i n j e c t e d . T h e e f f e c t of the t e s t d r u g w a s d e t e r m i n e d by c o m p a r i n g the e f f e c t of the s e c o n d i n j e c t i o n o f t r e m o r i n e o r b a r m i n e w i t h that p r o d u c e d by t h e f i r s t , s o t h a t e a c h c h i c k a c t e d a s i t s own c o n t r o l . T h e e f f e c t of e a c h d o s e of a t e s t d r u g w a s d e t e r m i n e d a s the m e a n e f f e c t on at l e a s t 3 c h i c k s . C o n t r o l e x p e r i m e n t s s h o w e d t h a t a s e c o n d d o s e of t r e m o r i n e o r b a r m i n e g i v e n 3 - 4 h r a f t e r t h e f i r s t , but in the a b s e n c e of any o t h e r d r u g s , p r o d u c e d a t r e m o r r e s p o n s e only s l i g h t l y g r e a t e r (1-5%) t h a n t h a t
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an i m m e d i a t e s h o r t - l a s t i n g (5-10 min) block of the t w i t c h e s of the t i b i a l i s a n t e r i o r m u s c l e . Benzhexol (7.5 m g / k g ) slightly d e p r e s s e d (12-20%) the t r e m o r count p r o d u c e d by h a r m i n e , but in the d o s e s e f f e c t i v e a g a i n s t t r e m o r i n e , none of the o t h e r d r u g s a l t e r e d the r e s p o n s e s to harmine. 3.2. Hemicholinium (HC-3) In d o s e s of 12.5 g g / k g and 25 ~zg/kg, HC-3 p r o d u c e d 5-10% and 25-40% r e d u c t i o n r e s p e c t i v e l y in the t r e m o r count p r o d u c e d by t r e m o r inc. The l a r g e r dose u s u a l l y p r o d u c e d h e a d - d r o p but the chicks r e m a i n e d standing. A k i n e s i a and parasympathetic effects were also reduced. L a r g e r d o s e s of HC-3 (50 p g / k g ) p r o d u c e d , a f t e r a latent p e r i o d of 7-10 min, c o m p l e t e p a r a l y s i s of the chicks which l a s t e d for 30-50 min. No t r e m o r was evident during the p a r a l y s i s . In s u b - p a r a l y t i c d o s e s , HC-3 was without effect on r e s p o n s e s to h a r m i n e . Do s es of HC-3 up to 100 ~ g / k g w e r e without effect on m a x i m a l t w i t c h e s of the t i b i a l i s a n t e r i o r m u s c l e evoked by s t i m u l a t i o n of the s c i a t i c n e r v e at a f r e q u e n c y of 1 or 2 / s e c . 3.3. Morphine In a dose of 10 m g / k g , m o r p h i n e p r o d u c e d a 35-45% d e p r e s s i o n of the t r e m o r count in r e sponse to t r e m o r i n e . A k i n e s i a and p a r a s y m p a thetic e f f e c t s w e r e a l s o s l i g h t l y inhibited. The r e s p o n s e to h a r m i n e w a s unchanged by this dose of m o r p h i n e . 3.4. Physostigmine In d o s e s of 0.5 m g / k g and above, p h y s o s t i g mine i t s e l f p r o d u c e d t r e m o r , f a s c i c u l a t i o n s , s a l i v a t i o n and d e f a e c a t i o n within 2 min a f t e r i n j e c t i o n . S m a l l e r d o s e s w e r e without this effect. In a dose of 0.25 m g / k g , p h y s o s t i g m i n e p o t e n tiated the t r e m o r count p r o d u c e d by t r e m o r i n e by 30-40% and a u g m e n t e d the a k i n e s i a and p a r a s y m p a t h e t i c s y m p t o m s . P h y s o s t i g m i n e did not a u g m e n t the t r e m o r r e s p o n s e to h a r m i n e . 3.5. DeangJ" The e f f e c t s of d e a n e r on r e s p o n s e s to t r e m o r i n e v a r i e d with the dose of d e a n e r u s e d and the p e r i o d of p r e - t r e a t m e n t . When t r e m o r i n e w as i n j e c t e d 15 rain a f t e r 2 m g / k g d e a n e r , the t r e m or count was a u g m e n t e d by 20-25% but t h e r e was no d e t e c t a b l e change in the d e g r e e of a k i n e s i a . When t r e m o r i n e w a s i n j e c t e d 1 hr a f t e r a dose of 20 m g / k g d e a n e r , the t r e m o r count was d e p r e s s e d by 30-40% and a k i n e s i a was augmented. D e a n e r in d o s e s of 2-20 m g / k g w a s without e f -
f e c t on the r e s p o n s e to h a r m i n e . When t r e m o r ine was i n j e c t e d 2 hr a f t e r a dose of 200 m g / k g d e a n e r , t r e m o r w as c o m p l e t e l y s u p p r e s s e d and a k i n e s i a was f u r t h e r enhanced. This dose of d e a n e r slightly d e p r e s s e d (24-33%) the t r e m o r r e s p o n s e to h a r m i n e . P a r a s y m p a t h e t i c e f f e c t s of t r e m o r i n e w e r e enhanced by d e a n e r , e s p e c i a l l y in high d o s e s . 3.6. 5 - H y d r o x y t r y p t a m i n e (5-HT) and 5-hydroxy-
tryptophan (5 -HTP) In d o s e s of 10-20 m g / k g , 5-HT produced a 20-50% d e p r e s s i o n of the t r e m o r count produced by t r e m o r i n e but did not change the a k i n e s i a or p a r a s y m p a t h e t i c effects. 5 - H T P in a dose of 100 m g / k g produced s e d a tion. T r e m o r i n e , i n j e c t e d when the s e d a t i v e e f fect of 5 - H T P had a p p a r e n t l y d i s a p p e a r e d , p r o duced a t r e m o r count which was 60-80% s m a l l e r than the control. O t h er e f f e c t s of t r e m o r i n e w e r e not r e d u c e d by 5 - H T P . In the s a m e d o s e s , 5-HT and 5 - H T P d e p r e s s e d the t r e m o r count p r o d u c e d by h a r m i n e by 30-40%. The 5-HT an t ag o n i st , b r o m o l y s e r g i c a c i d d i e t h y l a m i d e , in d o s e s up to 25 m g / k g did not a l t e r the e f f e c t s of t r e m o r i n e or h a r m i n e . 3.7. Dopamine and dopa In d o s e s of 50 m g / k g , dopamine produced a s m a l l potentiation (about 30%) of the t r e m o r count but did not change the o t h er a c t i o n s of tremorine. Dopa, in a dose of 100 m g / k g i n j e c t e d 1 to 2 hr p r e v i o u s l y , a l m o s t doubled the t r e m o r count p r o d u c e d by t r e m o r i n e , and a u g m e n t e d the d e g r e e of a k i n e s i a . No d e t e c t a b l e changes in the p a r a s y m p a t h e t i c r e s p o n s e s to t r e m o r i n e w e r e produced. In the s a m e d o s e s , dopamine and dopa p r o duced s i m i l a r i n c r e a s e s in the t r e m o r count p r o d u c e d by h a r m i n e . In the d o s e s used, dopa alone did not p r o d u ce obvious b e h a v i o u r a l changes a f t e r i n t r a p e r i t o neal i n j ect i o n in chicks and did not produce a k i n e s i a . Spooner and W i n t e r s (1964), using l a r g e r d o s e s (300 m g / k g ) , did find it to p r o d u ce a k i n e si a in chicks, and a c c o r d i n g to Sourkes (1964) and L e v y and M i c h e l - B e r (1964) dopa p r o d u c e s r i g i d i t y and a k i n e s i a in m i c e . 3.8. Amphetamine In d o s e s of 2-5 mg//kg, a m p h e t a m i n e a u g m e n t e d the t r e m o r count by 80-100% but did not change the o t h e r r e s p o n s e s to t r e m o r i n e . In a dose of 15 mg/~kg, a m p h e t a m i n e i t s e l f produced
TREMORINE AND HARMINE ataxia and the chicks r e s t e d t h e i r s t e r n u m s on the base of the r e c o r d i n g a p p a r a t u s making it i m p o s s i b l e to count the t r e m o r a c c u r a t e l y a l though a s far a s could be d e t e r m i n e d by v i s u a l o b s e r v a t i o n the t r e m o r was s u p p r e s s e d . This l a r g e dose of a m p h e t a m i n e s t i m u l a t e d chirping (see also Z a i m i s 1960a,b). In doses of 2-5 m g / k g , a m p h e t a m i n e p r o duced a s i m i l a r a u g m e n t a t i o n of the t r e m o r count produced by h a r m i n e . 3.9. Reserpine and tetrabenazine R e s e r p i n e (3 mg/kg) or t e t r a b e n a z i n e (3 m g / kg) i n j e c t e d 30 m i n before t r e m o r i n e or h a r m i n e d e p r e s s e d the t r e m o r count by 30-50% but slightly a u g m e n t e d the a k i n e s i a produced by t r e m o r i n e . When the s a m e dose of r e s e r p i n e or t e t r a b e n a z i n e was injected 24 hr before the test dose of t r e m o r i n e or h a r m i n e , a k i n e s i a was u n a l t e r e d , but the t r e m o r count was now i n c r e a s e d by 12-40%. Salivation in r e s p o n s e to t r e m o r i n e was i n c r e a s e d after r e s e r p i n e p r e t r e a t m e n t but this effect was not detected after t e t r a b e n a z i n e . 3.10. c~-Methyldopa In a dose of 800 m g / k g i n j e c t e d 1 hr before t r e m o r i n e , c~-methyldopa prolonged the latent p e r i o d between injection and onset of t r e m o r by 50-80%, r e d u c e d the t r e m o r count by 25-40% and a u g m e n t e d the d e g r e e of a m n e s i a . S m a l l e r doses of ~ - m e t h y l d o p a w e r e without effect on the r e sponse to t r e m o r i n e . (~-Methyldopa was without effect on r e s p o n s e s to h a r m i n e . 3.11. Phenoxybenzamine In doses of 25 m g / k g , p h e n o x y b e n z a m i n e p r o duced a s m a l l r e d u c t i o n (10-20%) in the t r e m o r count produced by t r e m o r i n e and slightly r e duced the degree of a k i n e s i a . P a r a s y m p a t h e t i c effects of t r e m o r i n e were abolished. P h e n o x y b e n z a m i n e was without detectable effect on r e s p o n s e s to h a r m i n e . 3.12. Pronethalol In s u b - l e t h a l doses (< 80 mg/kg) pronethalol did not affect the t r e m o r or a k i n e s i a produced by t r e m o r i n e or h a r m i n e , but the p a r a s y m p a t h e t i c effects of t r e m o r i n e were antagonised. 3.13. Chlorpromazine In doses of 2.5 m g / k g , c h l o r p r o m a z i n e p r o duced a 50-60% r e d u c t i o n in the t r e m o r counts produced both by t r e m o r i n e and by h a r m i n e but did not produce a detectable change in the degree of a k i n e s i a . P a r a s y m p a t h e t i c effects of t r e m o r -
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ine w e r e abolished by this dose of c h l o r p r o m a zine. 3.14. Mepyramine and diphenhydramine M e p y r a m i n e (25 mg/kg) p r e v e n t e d the p a r a s y m p a t h e t i c effects of t r e m o r i n e and r e d u c e d the t r e m o r count by 20%. D i p h e n h y d r a m i n e (5 m g / kg) produced s i m i l a r effects. Neither drug changed the r e s p o n s e s to h a r m i n e . 3.15. Mephenesin Mephenesin (40 mg/kg) was without effect on the r e s p o n s e s to t r e m o r i n e or h a r m i n e injected 15 m i n l a t e r . However, when i n j e c t e d d u r i n g the effects of t r e m o r i n e or h a r m i n e , the same dose of m e p h e n e s i n produced a t e m p o r a r y (5-10 min) c e s s a t i o n of the t r e m o r r e s p o n s e s . 3.16. Pentobarbitone sodium In s u b - h y p n o t i c doses (5 m g / k g and below) pentobarbitone was without effect on r e s p o n s e s to t r e m o r i n e or h a r m i n e . In a dose of 7.5 m g / kg, pentobarbitone itself produced slight ataxia and r e d u c e d the t r e m o r r e s p o n s e s to t r e m o r i n e or h a r m i n e by 25-35%. The p a r a s y m p a t h e t i c r e sponses to t r e m o r i n e were unchanged. 3.17. Other substances G a m m a a m i n o b u t y r i c acid (250 mg/kg), 4 - h y d r o x y b u t y r a t e (50 mg/kg), t y r a m i n e (100 mg/kg), and ergothioneine (100 mg/kg) were all without effect on the r e s p o n s e s to t r e m o r i n e and h a r mine.
4. DISCUSSION T r e m o r i n e i n c r e a s e s b r a i n levels of 5-HT and r e d u c e s those of n o r a d r e n a l i n e both in mice ( F r i e d m a n , 1963; F r i e d m a n , Aylesworth and F r i e d m a n , 1963) and in chicks (Bowman and Osuide, 1967). Injection of 5 - H T P r a i s e s the b r a i n level of 5-HT in chicks (Osuide, 1966). The d i r e c t i o n of change in b r a i n 5-HT is t h e r e fore the s a m e as that produced by t r e m o r i n e , yet the t r e m o r effect of t r e m o r i n e was reduced by 5 - H T P . Injection of dopa i n c r e a s e s the b r a i n l e v e l s of n o r a d r e n a l i n e in chicks (Osuide, 1966) so that the depleting effect of t r e m o r i n e would be r e v e r s e d . N e v e r t h e l e s s , dopa augmented the t r e m o r and a k i n e s i a produced by t r e m o r i n e . T h e s e r e s u l t s make it unlikely that the t r e m o r produced by t r e m o r i n e is a consequence of its a l t e r i n g b r a i n l e v e l s of n o r a d r e n a l i n e or 5-HT. Holmstedt (1964) r e p o r t e d an i n c r e a s e in the b r a i n l e v e l s of a c e t y l c h o l i n e produced by t r e m -
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W.C. BOWMAN and G. OSUIDE
o r i n e in r a t s and showed that t h e r e was a c o r r e lation between this effect and the extent and dur a t i o n of the t r e m o r produced. A c c o r d i n g to A p r i s o n and T a k a h a s h i (1965) c h o l i n e r g i c m e c h a n i s m s a r e e s p e c i a l l y i m p o r t a n t in a v i a n b r a i n , and an i n c r e a s e in b r a i n l e v e l s of a c e t y l c h o l i n e p r o d u c e d by t r e m o r i n e was a l s o d e t e c t e d in chicks (Bowman and Osuide, 1967). The p r e s e n t r e s u l t s showed that o th e r d r u g s known to influe n c e acetylcholine m e t a b o l i s m modified the t r e m o r r e s p o n s e to t r e m o r i n e in a m a n n e r c o n s i s t e n t with H o l m s t e d t ' s (1964) c o n c l u s i o n that the b a s i c action of t r e m o r i n e is on b r a i n a c e t y l choline l e v e l s . Thus, the c h o l i n e s t e r a s e i n h i b i t o r , p h y s o s t i g m i n e , p o t e n ti a t e d t r e m o r . D e a n e r , which is m e t a b o l i s e d into a c e t y l c h o l i n e in the b r a i n ( K i p l i n g e r , Swain and Brody, 1958; Groth, Bain and P f e i f f e r , 1958) potentiated t r e m o r in s m a l l d o s e s and d e p r e s s e d it in l a r g e d o s e s ; a c e t y l c h o l i n e is known to s t i m u l a t e c h o l i n o c e p t i v e s i t e s in s m a l l d o s e s but to block in l a r g e d o s e s . Morphine inhibits choline a c e t y l t r a n s f e r a s e (Balotin and Coon, 1960), and h e m i c h o l i n i u m inhibits the t r a n s p o r t of choline to its a c e t y l a t i o n s i t e s , so that both d r u g s inhibit a c e t y l c h o l i n e s y n t h e s i s and both d e p r e s s e d the t r e m o r r e s p o n s e to t r e m o r i n e . F i n a l l y , d r u g s with an a t r o p i n e - l i k e action ( a t r o p i n e , h y o s c i n e , b e n z hexol, p r o p a n t h e l i n e and o r p h e n a d r i n e ) blocked the t r e m o r p r o d u c e d by t r e m o r i n e . The weak a n t i - t r e m o r e f f e c t s of p h e n o x y b e n z a m i n e and the a n t i - h i s t a m i n e s may a l s o have been the r e s u l t of a weak a t r o p i n e - l i k e action. It was of i n t e r e s t that the a n t i - t r e m o r e f f e c t s of the q u a t e r n a r y compounds, h e m i c h o l i n i u m and p r o p a n t h e l i n e , like those of the other a n t a g o n i s t s , a p p e a r e d to be due to a c e n t r a l action and this probably r e f l e c t s the u n d e r d e v e l o p m e n t of the b l o o d - b r a i n b a r r i e r in young chicks (Waelsch, 1955; Z a i m i s , 1960a, Key and M a r l e y , 1962). H e m i c h o l i n i u m w a s e f f e c t i v e a g a i n s t t r e m o r p r o d u c e d by t r e m o r i n e in d o s e s which w e r e too s m a l l to affect p e r i p h e r a l n e u r o m u s c u l a r t r a n s m i s s i o n , and a d ditional a s yet unpublished e x p e r i m e n t s by one of us (G. O.) c o n f i r m that, in the chick, s m a l l d o s e s of h e m i c h o l i n i u m s e l e c t i v e l y affect c h o l i n e r g i c m e c h a n i s m s in the b r a i n . P r o p a n t h e l i n e did p r o duce a t r a n s i e n t block of c o n t r a c t i o n s of the t i b i a l i s a n t e r i o r m u s c l e evoked by m o t o r n e r v e s t i m u l a t i o n but this effect did not a p p e a r to c o n t r i b u t e to i t s a n t i - t r e m o r action. P r o p a n t h e l i n e w a s a l w a y s i n j e c t e d 15 min b e f o r e the t r e m o r ine, so that by the t i m e t r e m o r i n e was i n j e c t e d any t r a n s i e n t n e u r o m u s c u l a r blocking action would have d i s a p p e a r e d . T h e r e was no e v i d e n c e in c o n s c i o u s chicks of any m u s c u l a r w e a k n e s s
p r o d u c e d by p r o p a n t h e l i n e in the d o s e s used. F u r t h e r m o r e , the t r e m o r i n e count was continued for an hour a f t e r its injection, and any t r a n s i e n t n e u r o m u s c u l a r blocking action would have made a n e g l i g i b l e d i f f e r e n c e . P r o p a n t h e l i n e does not affect r e s p o n s e s to t r e m o r i n e in m i c e (Spencer, 1965), p r e s u m a b l y b e c a u s e of its inability to p e n e t r a t e the b l o o d - b r a i n b a r r i e r in this s p e c i e s . N e i t h e r h e m i c h o l i n i u m nor p r o p an t h el i n e w e r e e f f e c t i v e a g a i n s t t r e m o r p r o d u c e d by h a r m i n e, yet a p e r i p h e r a l a c t i o n of t h ese d r u g s would be e x p e c t e d to affect d i f f e r e n t types of c e n t r a l l y - i n d u c e d t r e m o r in the s a m e way. A k i n e s i a p r o d u c e d by t r e m o r i n e a l s o a p p e a r e d to depend on a c e n t r a l c h o l i n e r g i c m e c h a n i s m . Thus, a k i n e s i a was r e d u c e d by a t r o p i n e like d r u g s , m o r p h i n e and h e m i c h o l i n i u m , and enhanced by p h y s o s t i g m i n e and d e a n e r . The l a r g e d o s e s of d e a n e r which d e p r e s s e d t r e m o r , enhanced a k i n e s i a . This d i f f e r e n c e may be e x plained if it is a s s u m e d that t r e m o r is the r e s u l t of a c t i v a t i o n of c h o l i n e r g i c s y n a p s e s by s t i m ulant l e v e l s of a c e t y l c h o l i n e (and hence is d e p r e s s e d by e x c e s s a c e t y l c h o l i n e ) , w h e r e a s a k i n e s i a is the r e s u l t of block of c h o l i n e r g i c s y n a p s e s by e x c e s s a c e t y l c h o l i n e (and hence is a u g m e n t e d by an additional e x c e s s of a c e t y l c h o l i n e ) . Although t r e m o r and a m n e s i a p r o d u c e d by t r e m o r i n e did not a p p e a r to be d i r e c t l y a t t r i b u t able to changes in c a t e c h o l a m i n e l e v e l s , they w e r e n e v e r t h e l e s s influenced by changes in t h ese l e v e l s . Injection of dopa a u g m e n t e d t r e m o r and a k i n e s i a , p r e s u m a b l y as a r e s u l t of its c o n v e r s i o n to n o r a d r e n a l i n e . R e s e r p i n e , t e t r a b e n a zine and ~ - m e t h y l d o p a i n i t i a l l y cau se the r e l e a s e of c a t e c h o l a m i n e s , and t h e r e f o r e r a i s e the l e v e l of f r e e a m i n e s . When t r e m o r i n e was i n j e c t e d soon a f t e r one of t h e s e d r u g s , t r e m o r w as r e d u c e d and a k i n e s i a enhanced. When 24 hr e l a p s e d between i n j ect i o n of r e s e r p i n e and i n j e c tion of t r e m o r i n e , catecholamine depletion would be e x p e c t e d to have o c c u r r e d ; t r e m o r was now enhanced. T r e m o r was i n i t i a l l y enhanced by a m p h e t a m i n e , p o s s i b l y through c a t e c h o l a m i n e r e l e a s e . Some of t h e s e i n t e r a c t i o n s may be e x plained by the known a c t i o n s of c a t e c h o l a m i n e s on c h o l i n e r g i c s y n a p s e s , e s p e c i a l l y if it is a s s u m e d that the a c c e s s i b i l i t y of the s y n a p s e s i n v o l v e d in t r e m o r d i f f e r s f r o m that of the s y n a p s e s involved in a k i n e s i a and that t h ese s y n a p s e s a r e n o r m a l l y modulated by c a t e c h o l a m i n e s . Small a m o u n t s of a d r e n a l i n e have been shown to f a c i l i t a t e c h o l i n e r g i c synaptic t r a n s m i s s i o n (Biilbring and Burn, 1942) by i n c r e a s i n g the r e l e a s e of a c e t y l c h o l i n e f r o m p r e s y n a p t i c n e r v e endings ( B i r k s and Macintosh, 1961).
T R E M O R I N E AND HARMINE Larger amounts depress synaptic transmission (Marazzi, 1939) by hyperpolarizing the post-synaptic cell membranes (De Groat and Volle, 1965).
ACKNOWLEDGEMENTS T h i s w o r k w a s s u p p o r t e d by a g r a n t f r o m the B r i t i s h Egg M a r k e t i n g B o a r d . We a r e g r a t e f u l to A b b o t t ' s L a b o r a t o r i e s L i m i t e d for g i f t s of t r e m o r i n e .
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y l - a m i n o e t h a n o l a n d c h o l i n e in the m o u s e , J. P h a r m a c . Exp. T h e r . 122, 28A. H o h n s t e d t , B., 1964, P r o c e e d i n g s of the 2rid I n t e r n a tional P h a r m a c o l o g i c a l C o n g r e s s ( P e r g a m o n P r e s s , Oxford) p. 128. Key, B . J . and E. M a r l e y , 1962, T h e e f f e c t of s y m p a t h o m i m e t i c a m i n e s on b e h a v i o u r and e l e c t r o c o r t i c a l a c t i v i t y of c h i c k e n s , E l e c t r o n . N e u r o p h y s i o l . 14, 90. K i p l i n g e r , G . F . , H . H . Swain and T . M . Bordy, 1958, T h e a c t i o n of d i m e t h y l a m i n o e t h a n o l on the i s o l a t e d h e a r t , J. P h a r m a c . Exp. T h e r . 122, 37A. Levy,, J. and E. M i c h e l - B e r , 1964, De l ' a n t a g o n i s m c v i s - h - v i s de d i f f 6 r e n t s e f f e c t s de la t r S m o r i n e c h e z la s o u r i s , in: P r o c e e d i n g s of the s e c o n d i n t e r n a tional p h a r m a c o l o g i c a l m e e t i n g , e d s . E. T r a b u c c h i , R. P a o l e t t i and N. C a n a l ( P e r g a m o n P r e s s , O,,dord) 2, 109. M a r a z z i , A . S . , 1939, E l e c t r i c a l s t u d i e s on t h e p h a r m a c o l o g y of a u t o n o m i c s y n a p s e s . II. T h e a c t i o n of a s y m p a t h o m i m e t i c d r u g (epinephrine) on s y m p a thetic g a n g l i a , J. P h a r m a c . Exp. T h e r . 65, 395. O s u i d e , G., 1966, P h a r m a c o l o g i c a l l y a c t i v e s u b s t a n c e s in the c e n t r a l n e r v o u s s y s t e m of the chick, P h . D . T h e s i s , U n i v e r s i t y of London. Pale'6ek, F., 1963, A s i m p l e r e s p i r a t o r f o r l a b o r a t o r y u s e , J. Appl. P h y s i o l . 18, 443. S o u r k e s , T . L . , 1964, A c t i o n s of dopa and d o p a m i n e in r e l a t i o n to f u n c t i o n of the c e n t r a l n e r v o u s s y s t e m , in: P r o c e e d i n g s of the s e c o n d i n t e r n a t i o n a l p h a r m a c o l o g i c a l m e e t i n g , e d s , E. T r a b u c c h i , R. P a o l e t t i and N. C a n a l ( P e r g a m o n P r e s s , Oxford) 2, 35. S p e n c e r , P . S . J . , 1965, A c t i v i t y of c e n t r a l l y a c t i n g and o t h e r d r u g s a g a i n s t t r e m o r and h y p o t h e r m i a ind u c e d in m i c e by t r e m o r i n e , B r i t . J. P h a r m a c . C h e m o t h e r . 25, 442. S p o o n e r , C . E . and W. D. W i n t e r s , 1964, B e h a v i o u r a l , eeg, and blood p r e s s u r e e f f e c t s of c e n t r a l l y a c t i n g d r u g s in the chick, P h a r m a c o l o g i s t 6, 171. V e r n i e r , U . G . , 1964, A n t i - P a r k i n s o n a g e n t s : E v a l u atio~ of d r u g a c t i v i t i e s : P h a r m a c o m e t r i c s , eds. D. 1l. L a u r e n c e and A, L. B a c h a r a c h (Academic P r e s s , New York and London) p, 301. W a e l s c h , H., 1955, T h e t u r n o v e r of c o m p o n e n t s of the d e v e l o p i n g b r a i n ; the blood b r a i n b a r r i e r , B i o c h e m . i s t r y of the d e v e l o p i n g n e r v o u s s y s t e m ( A c a d e m i c P r e s s , New York) p. 187. Z a i m i s , E., 1960a, Ciba F o u n d a t i o n S y m p o s i u m on A d r e n e r g i c M e c h a n i s m s ( C h u r c h i l l , London) p. 562. Z a i m i s , E., 1960b, S i m u l t a n e o u s r e c o r d i n g of a c t i v i t y and r a t e of c h i r p i n g in the y o u n g chick, J. P h y s i o l . (London) 152, 11P.