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Effects of tremorine and harmine in the chick
EUROPEAN JOURNAL OF PHARMACOLOGY 1 (1967) 71-80. NORTH-HOLLAND PUBL. COMP., AMSTERDAM
EFFECTS OF T R E M O R I N E A N D H A R M I N E I N THE CHICK W. C. BOWMAN and G. OSUIDE *
Department of Pharmacology, School of Pharmacy, University of London and University of Strathclyde, Glasgow, C. 1, U.K. Accepted 14 November 1966
W. C. BOWMAN and G. OSUIDE, Effects oftremorine and harmine in the chick, European J. Pharmacol. 1 (1967) 71-80. T r e m o r i n e produced pronounced t r e m o r , akinesia, hypothermia and parasympathetic effects in young chicks. T r e m o r was r e c o r d e d quantitatively and 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 was found to diminish with increasing age and with repeated dosage. The optimal age of the chicks for obtaining a t r e m o r r e s p o n s e to t r e m o r i n e was 3 to 4 days. Harmine also produced t r e m o r , and some akinesia and hypothermia. The harmine effect did not diminish with age or with repeated dosage. T r e m o r i n e produced a significant i n c r e a s e in the brain levels of acetylcholine, and a d e c r e a s e in the brain levels of noradrenaline. It did not affect the levels of tyramine, dopamine or ergothioneine. Harmine produced an i n c r e a s e in the brain levels of noradrenaline but did not affect the levels of a c e tylcholine, tyramine or ergothioneine.
Acetylcholine effect of harmine on chick brain levels effect of t r e m o r i n e on chick brain levels
Dopamine effect of harmine on chick brain levels effect of t r e m o r i n e on chick brain levels
Ergothioneine effect of harmine on chick brain levels effect of t r e m o r i n e on chick brain levels
Harmine akinesia in the chick produced by hypothermia in the chick produced by t r e m o r in the chick produced by
5- Hydroxy trypta mine effect of harmine on chick brain levels effect of t r e m o r i n e on chick brain levels
Noradrenaline effect of harmine on chick brain levels effect of t r e m o r i n e on chick brain levels
Tremorine akinesia in the chick produced by hypothermia in the chick produced by parasympathetic effects in the chick produced by t r e m o r in the chick produced by
Tyramine effect of harmine on chick brain levels effect of t r e m o r i n e on chick brain levels
I. INTRODUCTION
H a r m i n e is an alkaloid obtained f r o m Pegahum harmala a n d f r o m Banisteria caapi. It too
T r e m o r i n e p r o d u c e s t r e m o r a n d a k i n e s i a in m i c e , a n d in s o m e o t h e r m a m m a l s , by a n a c t i o n b e l i e v e d to b e e x e r t e d in t h e b r a i n s t e m r e t i c u l a r f o r m a t i o n ( E v e r e t t , 1956; E v e r e t t , B l o c k u s a n d S h e p p e r d , 1956; B l o c k u s a n d E v e r e t t , 1957; S t e r n , R a d o v i c a n d B u l j u b a s i c , 1965). T r e m o r i n e o w e s i t s a c t i v i t y to a m e t a b o l i t e , o x o t r e m o r i n e , f o r m e d in t h e l i v e r m i c r o s o m e s (Kocsis and W e l c h , 1960; Cho, H a s l e t t a n d J e n d e n , 1961). T h e s y m p t o m s of t r e a t m e n t w i t h t r e m o r i n e in animals superficially resemble s o m e of t h e s y m p t o m s of P a r k i n s o n ' s d i s e a s e a n d m a n y a n t i P a r k i n s o n d r u g s a r e e f f e c t i v e in a n t a g o n i s i n g t h e a c t i o n s of t r e m o r i n e .
p r o d u c e s t r e m o r in s o m e a n i m a l s p e c i e s by a n a c t i o n in t h e e x t r a - p y r a m i d a l s y s t e m ( H a r a , 1953; H a r a a n d K a w a m o r i , 1954). H o w e v e r , a n t i P a r k i n s o n d r u g s u s u a l l y do not a n t a g o n i s e the t r e m o r r e s p o n s e p r o d u c e d by h a r m i n e ( E v e r e t t , 1956; Z e t l e r , ]957; 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). A c c o r d i n g to A r i e n s - K a p p e r s (1929) and to Patten, Sakamoto, Van Woert, P a p a v a s i l i o u and C o t z i a s (1964), t h e c o r p u s s t r i a t u m of b i r d s (and r e p t i l e s ) i s w e l l d e v e l o p e d a n d i s the h i g h e s t c e n t r e controlling m o t o r activity. During the f i r s t few d a y s a f t e r h a t c h i n g , t h e b l o o d - b a r r i e r of t h e c h i c k i s d e f e c t i v e , at l e a s t w i t h r e s p e c t to s o m e s u b s t a n c e s ( W a e l s c h , 1955; Z a i m i s , 1960a; K e y a n d M a r l e y , 1962). T h e s e f a c t s s u g g e s t e d that young chicks might p r o v i d e useful t e s t ani-
* P r e s e n t a d d r e s s : Department of Pharmacology, University of Ibadan, Nigeria
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m a l s for studying the actions of d r u g s acting on the e x t r a - p y r a m i d a l s y s t e m , including antiP a r k i n s o n drugs. T h i s p a p e r d e s c r i b e s e x p e r i me nts in which the e f f e c ts of t r e m o r i n e and h a r m i n e w e r e studied as a p r e l i m i n a r y to i n v e s tigating the i n t e r a c t i o n of a n t i - P a r k i n s o n drugs with them.
2. METHODS The 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 d o m e s tic fowl chicks (Silver link) aged f r o m 1-25 days a f t e r hatching.
2.1. Spinal reflexes Crossed extensor reflexes were recorded f r o m one limb of chicks lightly a n a e s t h e t i s e d with c h l o r a l o s e (30-60 m g / k g ) i n j e c t e d i n t r a p e r i t o n e a l l y . The method used was that des c r i b e d by Bowman, Callingham and Osuide (1964). Artificial v e n t il a t io n was applied throughout by the method d e s c r i b e d by P a le ~ e k (1963), and drugs w e r e i n j e c t e d through a cannula in a j u g u l ar vein. 2.2. Recording of tremor Chicks w e r e p l a c e d individually in a jiggle cage made f r o m w i r e m e s h with a r u b b e r base.
A t h r e a d fixed to the r u b b e r b a s e was attached to a s t r a i n gauge made f r o m a RCA 5734 m e c h a n o e l e c t r i c t r a n s d u c e r v a l v e (Btllbring, 1955), the output of which was co n n ect ed to a T e k t r o n i x (type 502) double b e a m o s c i l l o s c o p e through a b ack i n g - o f f p o t e n t i o m e t e r used to find the null point of the s t r a i n gauge. The input of a C o s s e r p r e - a m p l i f i e r (model 1440) was connected in p a r a l l e l to the input of the o s c i l l o s c o p e . A m p l i fied signals w e r e fed into a T e k t r o n i x (type 161) pulse g e n e r a t o r . The p u l s e s p r o d u c e d during t r e m o r of the chick w e r e counted by a Panax Counter (type SA 102) connected to the pulse g e n e r a t o r . Fig. 1 i l l u s t r a t e s a t r e m o r g r a m r e c o r d e d on the o s c i l l o s c o p e f r o m a chick t r e a t e d with t r e m o r i n e . Chicks u su al l y do not exhibit e x p l o r a t o r y m o v e m e n t s and they usually r e mained s t a t i o n a r y in the middle of the jiggle cage. This was e s p e c i a l l y so a f t e r t r e a t m e n t with t r e m o r i n e which e x e r t s a s e d a t i v e effect. Consequently, g r o s s m o v e m e n t a r t i f a c t s did not usually c o m p l i c a t e the t r e m o r count and the few chicks that did exhibit spontaneous m o v e m e n t s w e r e d i s c a r d e d . E a c h chick was p l a c e d in the jiggle cage and r e m a i n e d t h e r e f o r 1 hr b e f o r e drug t r e a t m e n t . During this p e r i o d the s e n s i t i v i ty of the s t r a i n gauge was adjusted to avoid counting the c h i c k ' s r e s p i r a t o r y m o v e m e n t s and h e a r t beat. Under t h e s e conditions, the total count during 1 hr did not e x c e e d 1% of the total
1 HR 45 MIN
|
0.5 sec
I
Fig. 1. Tremorgram recorded on a cathode ray oscilloscope from a chick injected with 20 mg/kg tremorine intraperitoneally. These signals were electronically counted as described in the test. The times denote the times after injection.
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count produced in 1 hr by the s m a l l e s t dose of t r e m o r i n e used.
w e r e pooled to make each sample, and 12 s a m ples w e r e used for the d e t e r m i n a t i o n s .
2.3. Body temperature The body t e m p e r a t u r e of chicks was r e c o r d e d by m e a n s of a s m a l l t h e r m i s t o r (Grant I n s t r u m e n t s Ltd., Cambridge) i n s e r t e d into the cloaca.
2.6. Acetylcholine assay I m m e d i a t e l y a f t e r r e m o v a l , b r a i n s from 12 control chicks and f r o m 12 chicks t r e a t e d either with t r e m o r i n e or with h a r m i n e were placed s e p a r a t e l y in McIlwan's buffer at pH4 and homogeni'sed by grinding with sand in a glass m o r tar. The buffer i n a c t i v a t e s the c h o l i n e s t e r a s e and acetylcholine is stable at this pH. The ext r a c t i o n method was that d e s c r i b e d by Beani and Bianchi (1963). I m m e d i a t e l y before a s s a y the test solutions were n e u t r a l i s e d to pH 7.0 by the dropwise addition of HC1 and then a s s a y e d against s t a n d a r d acetylcholine solutions on the s e n s i t i s e d guinea-pig ileum (Blaber and Cuthb e r t , 1961). Tyrode solution containing m e p y r a m i n e 10 -7 was used to bathe the ileum, and before the a s s a y was c o m m e n c e d the i l e u m was r e n d e r e d i n s e n s i t i v e to the s t i m u l a n t action of t r e m o r i n e by applying repeated s m a l l doses of t r e m o r i n e until complete tachyphylaxis was p r o duced. The substance p r e s e n t was identified as an a c e t y l c h o l i n e - l i k e substance by the facts that its action was completely blocked by atropine (10 n g / m l ) and it r e q u i r e d s e n s i t i s a t i o n of the i l e u m with dyflos before its p r e s e n c e could be demonstrated.
2.4. Spectrophotofluorornetric assays Chicks aged 3 days w e r e killed by cutting through the thorax into the h e a r t and allowing them to bleed to death. The b r a i n s were r e moved, d r i e d with blotting paper to remove any blood and kept at - 1 0 o c until the a s s a y was b e gun. Whole b r a i n s from control groups of chicks and from groups t r e a t e d e i t h e r with t r e m o r i n e or with h a r m i n e w e r e a s s a y e d for n o r a d r e n a l i n e , dopamine, t y r a m i n e and 5 - h y d r o x y t r y p t a m i n e , u s i n g an A m i n c o - B o w m a n s p e c t r o p h o t o f l u o r i m eter. In the a s s a y s of the a m i n e s , each sample c o m p r i s e d a single b r a i n , and 12 s a m p l e s were used for each d e t e r m i n a t i o n except in the case of 5 - h y d r o x y t r y p t a m i n e w h e r e 24 s a m p l e s were used. Noradrenaline was e x t r a c t e d and a s s a y e d by the method of Shore and Olin (1958) as modified by Cass and Spriggs (1961) and Callingham and Cass (1963). Doparnine was e x t r a c t e d by the method of Shore and Olin (1958) as modified by Callingham and Cass (1963). The s a m p l e s w e r e a s s a y e d a c c o r d ing to the method of C a r l s s o n and Waldeck (1958) and C a r l s s o n (1959). Tyrarnine was e x t r a c t e d and a s s a y e d according to the method of Spector, Melmon, Lovenberg and S j o e r d s m a (1962, 1963) except that the p o s s i bility of loss of a m i n e due to heat generated d u r ing the p r e p a r a t i o n of the t i s s u e s for e x t r a c t i o n was avoided by u s i n g liquid n i t r o g e n (Callingham and Cass, 1963). 5-Hydroxytryptarnine was extracted and a s s a y e d a c c o r d i n g to the method of Udenfriend, W e i s s bach and Clark (1955) with the exception that the t i s s u e was p r e p a r e d for e x t r a c t i o n using liquid n i t r o g e n (Callingham and Cass, 1963). 2.5. Ergothioneine assay The b r a i n s of chicks aged 3 days were r e moved as d e s c r i b e d above. Ergothioneine was a s s a y e d in c o n t r o l b r a i n s and in b r a i n s from chicks t r e a t e d with t r e m o r i n e or h a r m i n e a c c o r d i n g to the method of C r o s s l a n d , Mitchell and Woodruff (1964, 1966), which is s i m i l a r to the methods of Melville and H o r n e r (1953) and Melville, H o r n e r and Lubschez (1954), using a Hilger H810 Biochem A b s o r p t i o m e t e r . T h r e e b r a i n s
2.7. Drugs The drugs used were t r e m o r i n e hydrochloride (Abbott's l a b o r a t o r i e s ) , h a r m i n e hydrochloride (L. Light and Co.), acetylcholine chloride (Roche), b r o m o l y s e r g i c acid diethylamide (Sandoz), atropine sulphate (British Drug Houses), m e p y r a m i n e m a l e a t e (May and Baker Ltd.) and dyflos (DFP, Boots).
3. RESULTS
3.1. Anaesthetised chicks. Crossed extensor r e flex In m i n i m a l effective doses (20-25 mg/kg) i n jected i n t r a v e n o u s l y , t r e m o r i n e slightly augm e n t e d t h e reflex c o n t r a c t i o n s and converted the single r e s p o n s e to each s t i m u l u s into a double or t r i p l e c o n t r a c t i o n (fig. 2). Occasional c o n t r a c tions u n r e l a t e d to the e l e c t r i c a l s t i m u l a t i o n also o c c u r r e d u n d e r the influence of t r e m o r i n e . The effects lasted 15-45 min and after the f i r s t injection t h e r e was always a latent period of 10-15 rain before the onset of the effect. With s u b s e quent doses the latent period b e c a m e s h o r t e r . After s e v e r a l doses of t r e m o r i n e , the effect
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Fig. 2. Chick aged 3 days and weighing 50 g. C r o s s e d extensor reflex elicited e v e r y 15 sec; contractions downwards. At T, 1.5 mg t r e m o r i n e intravenously before (above) and 15 min a f t e r (below) the intravenous injection of 50 ttg atropine. These injections were the 3rd and 4th injections of the same dose of t r e m o r i n e in this e x p e r i m e n t so that the latent period between injection and effect was brief. The double contractions to which t r e m o r i n e gives r i s e can be distinguished by the thickening of the contraction r e c o r d s in the upper panel. Atropine blocked the effects of tremorine.
c h a n g e d to a d e p r e s s i o n of t h e e v o k e d c o n t r a c tions, but the double or triple responses and the c o n t r a c t i o n s not e v o k e d b y e l e c t r i c a l s t i m u l a t i o n persisted. After sectioning the spinal chord at t h e l e v e l of t h e 10th c e r v i c a l v e r t e b r a ( s e e B o w m a n et al., 1964) a s e c o n d d o s e of t r e m o r i n e p r o d u c e d a n e f f e c t s i m i l a r to, b u t m u c h w e a k e r than, the effect produced by the first dose given to t h e i n t a c t c h i c k , i n d i c a t i n g t h a t t h e s i t e of a c t i o n of t r e m o r i n e i s m a i n l y , t h o u g h n o t e n t i r e l y , w i t h i n the b r a i n . In d o s e s of 2 . 5 - 1 0 m g / k g i n t r a v e n o u s l y , h a r mine slightly potentiated the reflex contractions in c h i c k s w i t h i n t a c t c e n t r a l n e r v o u s s y s t e m a n d produced vigorous muscular activity which was u n r e l a t e d to t h e e l e c t r i c a l s t i m u l a t i o n (fig. 3). The second and succeeding doses were more effective than the first. After sectioning the spinal c o r d at t h e l e v e l of C10, h a r m i n e w a s w i t h o u t e f f e c t on the r e f l e x c o n t r a c t i o n s s h o w i n g t h a t i t s s i t e of a c t i o n i s e n t i r e l y w i t h i n t h e b r a i n . A t r o p i n e (0.5 m g / k g ) p r e v e n t e d t h e e f f e c t s of a s u b s e q u e n t i n j e c t i o n of t r e m o r i n e o n t h e r e f l e x c o n t r a c t i o n s (fig. 2), b u t did not r e d u c e t h e e f f e c t s of h a r m i n e (fig. 3).
3.2. Conscious chicks In t h e e x p e r i m e n t s on c o n s c i o u s c h i c k s , a l l injections were given intraperitoneally unless otherwise stated. 3.2.1. T r e m o r In c h i c k s a g e d 1-3 d a y s , t r e m o r i n e , in d o s e s of 4 m g / k g a n d a b o v e , p r o d u c e d s e d a t i o n a n d t h e w i n g s t e n d e d to d r o o p . T h e c h i c k s r e m a i n e d standing but spontaneous movement was curtailed. T r e m o r w a s p r o d u c e d b y d o s e s of 10 m g / k g a n d a b o v e a f t e r a l a t e n t p e r i o d of 1 - 1 5 m i n , b u t u s u a l l y a f t e r a b o u t 10 m i n , a n d w a s e v i d e n t in the head, neck, body and drooping wings. A similar latent period occurred when the same doses w e r e i n j e c t e d i n t r a v e n o u s l y . A f t e r a d o s e of 20 m g / k g , t h e m a x i m u m e f f e c t in c h i c k s a ~ e d 1 d a y occurred 20-30 min after injection and at this t i m e t h e r e c o r d e d f r e q u e n c y of t h e t r e m o r w a s 1 5 - 2 0 / s e c . T o w a r d s t h e e n d of t h e f i r s t h o u r a f t e r i n j e c t i o n , b r i e f p e r i o d s of q u i e s c e n c e of 1 - 3 min duration interrupted the tremor. The quiescent periods then became more freqeunt and longer lasting. Tremor could be restarted during a q u i e s c e n t p e r i o d , f o r up to 3 h o u r s a f t e r i n -
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1 5 - 2 5 m i n a f t e r i n j e c t i o n . W h e n d o s e s of 1 0 - 4 0 mg/kg were given a second time, 2-4 hrs after the first dose, the tremor count recorded was greater than the initial count by 5-10°. The tremor effect produced by tremorine was f o u n d to d i m i n i s h w i t h i n c r e a s i n g age. In a s s e s s i n g t h e e f f e c t of a g e , d i f f e r e n t g r o u p s of 5 c h i c k s w e r e t a k e n d a i l y , f r o m t h e s e c o n d to t h e e i g h t h d a y of a g e , f r o m a c a g e o r i g i n a l l y c o n t a i n i n g 100 c h i c k s a g e d 1 day. E a c h 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 t r e m o r i n e a n d t h e t r e m o r produced was counted for 1 hour after injection. T h e r e s u l t s a r e e x p r e s s e d g r a p h i c a l l y in fig. 5 ( u p p e r c u r v e ) . T r e m o r w a s g r e a t e s t in c h i c k s a g e d 3 d a y s a n d d i m i n i s h e d to l e s s t h a n o n e t h i r d of t h i s m a x i m u m by t h e t i m e t h e c h i c k s w e r e 8 d a y s old. In c h i c k s a g e d 10 d a y s o r m o r e t r e m o r w a s s l i g h t o r a b s e n t a f t e r d o s e s of 20 mg/kg tremorine. Adult rabbits have also been f o u n d to b e i n s e n s i t i v e to t h e t r e m o r p r o d u c i n g e f f e c t of t r e m o r i n e ( F a r q u h a r s o n a n d J o h n s t o n , 1959; F r i e d m a n , 1964), b u t it i s n o t k n o w n whether new-born rabbits respond. When tremorine was injected into the same c h i c k on s u c c e s s i v e d a y s , t h e r e s u l t i n g t r e m o r response a p p e a r e d to b e c o m e smaller at a g r e a t e r r a t e t h a n c o u l d b e a c c o u n t e d f o r by t h e f a l l - o f f of r e s p o n s e w i t h age. In a s s e s s i n g t h e
Fig. 3. Chick aged 3 days and weighing 48 g. Recording as in fig. 2. At H, 0.2 mg h a r m i n e before (above) and a f t e r (below) 50 /ig atropine. All injections i n t r a venously. Atropine did not reduce the effects of h a r mine. jection, by environmental stimuli such as a sound or movement. Such stimuli evoked gross m o v e m e n t s of t h e s e d a t e d c h i c k s a n d t h i s t r i g g e r e d a b o u t of t r e m o r . Consequently in the q u a n t i t a t i v e a s s e s s m e n t of t r e m o r i t w a s i m p o r t a n t to c o n t r o l t h e e n v i r o n m e n t a l conditions closely. All quantitative measurements were therefore made in a quiet, artificially-lighted r o o m w i t h a t e m p e r a t u r e of 1 9 - 2 1 o c . Fig. 4 i n c l u d e s a d o s e / r e s p o n s e c u r v e p l o t t e d for tremorine using chicks aged 3 days. Each p o i n t w a s o b t a i n e d f r o m r e s u l t s o n 10 c h i c k s . Each chick was studied individually under identical environmental conditions, the total count during the first hour after injection being rec o r d e d . D o s e s g r e a t e r t h a n 40 m g / k g p r o d u c e d pronounced parasympathetic symptoms, occasional convulsions and sometimes death within
2c
.l
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20 Dose n~/kg
30
4O
Fig. 4. D o s e / r e s p o n s e c u r v e s for t r e m o r i n e and h a r mine. The dose in m g / k g i n t r a p e r i t o n e a l l y is plotted against the total t r e m o r count produced in 1 hr. The c u r v e s c r o s s because h a r m i n e , though effective in s m a l l e r doses than t r e m o r i n e , produced a t r e m o r of lower frequency and s h o r t e r duration than that produced by t r e m o r i n e . Each point is the mean of r e s u l t s on 10 chicks. The v e r t i c a l lines are the standard errors.
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extent of tachyphylaxis, a group of 75 chicks w e r e each i n j e c t e d with 20 m g / k g t r e m o r i n e on the second day a f t e r hatching. F i v e of t h e s e c h i c k s w e r e then p l a c e d singly in the r e c o r d i n g a p p a r a t u s and the t r e m o r count r e c o r d e d f o r 1 hr a f t e r injection. T h e s e chicks w e r e then d i s carded. On the next day, the r e m a i n i n g 70 c h i c k s w e r e again i n j e c t e d with the s a m e dose and the r e s p o n s e r e c o r d e d f r o m 5 of them. T h i s p r o c e d u r e was continued up to and including the 8th day a f t e r hatching. Using this p r o c e d u r e , any one chick was p l a c e d in the r e c o r d i n g a p p a r a t u s only once, so r e m o v i n g the p o s s i b i l i t y that any additional f a l l - o f f in r e s p o n s e o v e r that due to age was the r e s u l t of the chick b e c o m i n g a c c u s t o m e d to the e n v i r o n m e n t of the r e c o r d i n g apparatus. The r e s u l t s obtained a r e a l s o e x p r e s s e d in fig. 5 (lower curve). They indicate that the t o l e r a n c e which d e v e l o p s to r e p e a t e d daily dosage is g r e a t e r than the t o l e r a n c e s i m ply due to age. Thus, a f t e r 7 days of r e p e a t e d dosage, the t r e m o r r e s p o n s e was l e s s than one eighth of its m a x i m u m , w h e r e a s the f a l l - o f f due to age alone was only to just o v e r o n e - t h i r d of the m a x i m u m . T o l e r a n c e to the e f f e c ts of r e peated dosage of t r e m o r i n e has a l s o been noted in m i c e (Baker and T r i o l o , 1960; D e s c i , V a r s z e g i and Mehes, 1961). The chicks i n j e c t e d daily up to 8 days of age showed a l o s s of weight when c o m p a r e d with unt r e a t e d c o n t r o l s and of the 60 c h i c k s i n j e c t e d on 4 or m o r e days, 18 had died by the end of the 8th day of age. H a r m i n e in d o s e s of 2.5-40 m g / k g p r o d u c e d t r e m o r which was m o r e powerful and c o a r s e r than that p r o d u c e d by s i m i l a r d o s e s of t r e m -
i
u
Fig. 5. Graphs showing diminishing tremor response to tremorine (20 mg/kg) with age (upper curve) and diminishing tremor response resulting from age and repeated dosage (lower curve). Each point, except the first, is the mean of results on 5 chicks. The first point is the mean of results with the first dose in both experiments and is, therefore, from 10 chicks. For further explanation see text.
orine. The latent p e r i o d b e t w e e n i n j ect i o n and o n set of t r e m o r w as s h o r t e r than that a f t e r t r e m o r i n e , being u s u a l l y about 2 min but o c c a sionally as long a s 5 min. A f t e r i n j ect i o n of 20 m g / k g h a r m i n e , the m a x i m u m d e g r e e of t r e m o r o c c u r r e d in 15-25 m i n and was r e c o r d e d as a f r e q u e n c y of 1 0 - 1 5 / s e c . A f t e r 45-65 min, the ef fect had d i s a p p e a r e d . A d o s e / r e s p o n s e c u r v e in c h i c k s aged 3 days w as c o m p i l e d as f o r t r e m o r i n e (fig. 4). D o s e s of h a r m i n e of 50 m g / k g and above p r o d u c e d convulsions. When 20 m g / k g h a r m i n e was i n j e c t e d into c h i c k s of d i f f e r e n t a g e s (2-8 days) as in the ex p e r i m e n t s with t r e m o r i n e , t h e r e was no e v i d e n c e of a d i m i n i s h i n g e f f e c t with age. When the s a m e chicks w e r e i n j e c t e d daily up to the 8th day of age with 20 m g / k g h a r m i n e , only slight or no tachyphylaxis was evident. Chicks which had b e c o m e r e s i s t a n t to the t r e m o r - p r o d u c i n g effect of t r e m o r i n e , r e s p o n d e d n o r m a l l y to h a r m i n e . 3.2.2. A k i n e s i a T r e m o r i n e p r o d u c e d a k i n e s i a and r i g i d i t y of the l i m b s in d o s e s of 10 m g / k g and above. Its o n set u su al l y p r e c e d e d that of t r e m o r and it p e r s i s t e d a f t e r t r e m o r had c e a s e d . With a dose of 20-30 m g / k g , a k i n e s i a was m o s t pronounced 3070 m i n a f t e r i n j e c t i o n and was o c c a s i o n a l l y s t i l l evident as long a s 8 h r a f t e r injection. N o r m a l u n t r e a t e d c h i c k s p l a c e d on a bench m o v ed spontaneously and quickly changed t h e i r position when pushed gently with a s m a l l rod. When the legs w e r e f o r c i b l y s p r e a d apart, they w e r e quickly r e t u r n e d to the n o r m a l position. With a mild d e g r e e of a k i n e s i a (after about 10 mg/kg tremorine) spontaneous movements c e a s e d and the c h i c k s r e m a i n e d standing in an u p r i g h t position. When gently pushed they changed t h e i r p o s i t i o n with a h i g h - s t e p p i n g gait. A g r e a t e r d e g r e e of a k i n e s i a (after 20 m g / k g ) was e v i d e n c e d by swaying head m o v e m e n t s and difficulty in r e s t o r i n g the l e g s to t h e i r n o r m a l p o s i t i o n when s p r e a d apart. S e v e r e a k i n e s i a (after 30 m g / k g and above) was e v i d e n c e d by inability to r e s t o r e the legs to t h e i r n o r m a l p o s i tion when s p r e a d apart. H a r m i n e p r o d u c e d a k i n e s i a and rigidity in s o m e chicks but the e f f e c t was m i l d in c o m p a r i son with that p r o d u c e d by an e q u a l l y - s i z e d dose of t r e m o r i n e . S m a l l e r d o s e s of h a r m i n e (2-5 m g / k g ) have been shown to p r o d u c e a k i n e s i a in m o n k ey s (Chen and Chen, 1939) and p i g eo n s (Hara, 1953). 3.2.3. Vocal e f f e c t s The f r e q u e n c y of c h i r p i n g was d e c r e a s e d in
TREMORINE AND HARMINE IN THE CHICK c h i c k s about 20 m i n a f t e r i n j e c t i o n of t r e m o r i n e (20-30 m g / k g ) ; about 30 m in a f t e r i n j e c ti o n they b e c a m e c o m p l e t e l y s i l e n t even in the p r e s e n c e of u n t r e a t e d chicks. A d e p r e s s a n t e f f e c t of t r e m o r i n e on evoked s q u e a l s of adult g u i n e a - p i g s has a l s o been r e p o r t e d by F r i e d m a n and Ringal (1965). In c o n t r a s t , h a r m i n e (5-20 mg/kg) s t i m u l a t e d c h i r p i n g in chicks, an effect r e s e m b l i n g that p r o d u c e d by a m p h e t a m i n e as r e c o r d e d by Z a i m i s (1960b). 3.2.4. H y p o t h e r m i a Body t e m p e r a t u r e c o n t r o l is d e f e c t i v e in the chick during the f i r s t few days a f t e r hatching and, p o s s i b l y for this r e a s o n , the h y p o t h e r m i c effect of t r e m o r i n e in young chicks was p r o nounced. In d o s es of 20-30 m g / k g , t r e m o r i n e produced a fall in body t e m p e r a t u r e of 2 - 1 3 o c below the n o r m a l r a n g e of 3 9 - 4 2 o c in chicks aged f r o m 1-10 days, and kept in an e n v i r o n m e n t a l t e m p e r a t u r e of 1 9 - 2 1 o c . The fall in t e m p e r a t u r e was m a x i m a l about 1 hour a f t e r i n j e c tion and p e r s i s t e d for 2-5 hours. A s m a l l e r hyp o t h e r m i c effect of t r e m o r i n e has been r e c o r d e d in m i c e but in this s p e c i e s it p e r s i s t s for up to 18 hr ( E v e r e t t , 1964). The t r e m o r - p r o d u c i n g effect of t r e m o r i n e did not a p p e a r to be a c o n s e q u e n c e of its h y p o t h e r mic effect. Thus, the onset of the fall in t e m p e r a t u r e p r e c e d e d and o u t la s te d the t r e m o r r e sponse. Chicks aged ]0 days exhibited only v e r y slight t r e m o r but the fall in body t e m p e r a t u r e r e s e m b l e d that in y o u n g e r chicks. In u n t r e a t e d chicks, a fall in body t e m p e r a t u r e induced by a cold e n v i r o n m e n t induced negligible t r e m o r - l i k e a c t i v i t y when c o m p a r e d with the e f f e c ts of t r e m orine. Finally, the in te n s i ty of t r e m o r produced by t r e m o r i n e was not r e l a t e d to the magnitude of the fall in t e m p e r a t u r e . M a r k e d t r e m o r was s o m e t i m e s a c c o m p a n i e d by only a s m a l l hypot h e r m i c effect and v i c e v e r s a . Other w o r k e r s using d i f f e r e n t s p e c i e s have a l s o concluded that the t r e m o r and h y p o t h e r m i c e f f e c ts a r e independent r e s p o n s e s (Blockus and E v e r e t t , 1957; P a t ten et al., 1964; S p e n c e r , 1965). N e v e r t h e l e s s , the d e g r e e of t r e m o r p r o d u c e d by t r e m o r i n e was influenced by e n v i r o n m e n t a l t e m p e r a t u r e , being b r i e f e r in duration at high t e m p e r a t u r e s (e.g. the chick incubator at 32°C) and m o r e p r o lo n g e d at low t e m p e r a t u r e s . A k i n e s i a was also i n t e n s if i e d in a cold e n v i r o n m e n t . At an e n v i r o n m e n t a l t e m p e r a t u r e of 0°C, t r e m o r p r o d u c e d by t r e m o r i n e was prolonged, but was eventually inhibited as the body t e m p e r a t u r e fell to about 20oc. On r e moving the chicks f r o m the cold e n v i r o n m e n t ,
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t r e m o r r e c o m m e n c e d when the body t e m p e r a t u r e r o s e to 2 2 - 2 5 o c . P r o l o n g e d t r e m o r in a cold e n v i r o n m e n t may be the r e s u l t of s h i v e r i n g s u p e r i m p o s e d on the t r e m o r . In chicks aged 20 days or m o r e , t r e m o r i n e (20 mg/kg) did not p r o d u c e a h y p o t h e r m i c effect. In fact, it often p r o d u c e d a s m a l l and s h o r t l ast i n g r i s e in body t e m p e r a t u r e . Cho, F a i r hurst, H a s l e t t and J e n d e n (1964) r e p o r t e d that o x o t r e m o r i n e , the a c t i v e m e t a b o l i t e of t r e m urine, produced a s m a l l r i s e in body t e m p e r a t u r e in adult cats. When p l a c e d t o g e t h e r in a cage, young chicks tend to a g g r e g a t e and this tendency was inc r e a s e d a f t e r injection of t r e m o r i n e p o s s i b l y as an a t t e m p t to r e d u c e heat loss during the hypot h e r m i c effect of the drug. H a r m i n e in d o s e s of 20 m g / k g a l s o p r o d u c e d a fall in body t e m p e r a t u r e in young chicks, but the effect was s m a l l e r (1-3oc) than that p r o duced by t r e m o r i n e . In c o n t r a s t to the effect of t r e m o r i n e , h a r m i n e antagonised the tendency of chicks to a g g r e g a t e when placed t o g e t h e r in a cage. 3.2.5. P a r a s y m p a t h e t i c e f f e c t s T r e m o r i n e , in d o s e s of 20-30 m g / k g p r o d u c e d i n t en se d i a r r h o e a in chicks aged 3 days or m o r e and this was the p r e d o m i n a n t p a r a s y m p a t h e t i c effect. Salivation was a l s o produced but only in o l d e r chicks (6 days and over) at this dose level. In I or 2-day old chicks, n e i t h e r d i a r r h o e a nor s a l i v a t i o n was produced, p o s s i b l y b e c a u s e of i m m a t u r i t y of the s e c r e t o r y glands at this stage. P a r a s y m p a t h e t i c ef f ect s i n c r e a s e d in intensity with age up to about 8 days a f t e r which they w e r e r e p r o d u c i b l e at a s i m i l a r level. Thus in chicks aged 8 days or m o r e , p a r a s y m p a t h e t i c act i o n s w e r e the p r e d o m i n a n t e f f e c t s of t r e m o r i n e , the b i r d s having b e c o m e r e s i s t a n t to the t r e m o r p r o d u ci n g action of the drug at this time. I s o l a t e d s e g m e n t s of g u i n e a - p i g i l eu m t r e a t e d with dyflos c o n t r a c t e d in r e s p o n s e to 1 n g / m l of t r e m o r i n e . T a c h y p h y l a x i s to this effect d e v e l oped rapidly when s u c c e s s i v e d o ses w e r e added to the p r e p a r a t i o n . L a r g e c o n c e n t r a t i o n s of t r e m o r i n e (1 /zg/ml) p o s s e s s e d a t r o p i n e - l i k e action, s e l e c t i v e l y blocking the r e s p o n s e s of the g u i n e a - p i g i l e u m to a c e t y l c h o l i n e . H a r m i n e did not p r o d u ce d i a r r h o e a in chicks. O c c a s i o n a l l y slight s a l i v a t i o n was p r o d u c e d by d o s e s of 30 m g / k g and a b o v e 3.3. B i o c h e m i c a l c h a n g e s in b r a i n The c o n c e n t r a t i o n s of n o r a d r e n a l i n e , dopamine, t y r a m i n e , 5-HT, a c e t y l c h o l i n e and e r g o -
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W. C. BOWMAN and G. OSUIDE Table 1 E f f e c t s of t r e m o r i n e (30 m g / k g i n t r a p e r i t o n e a l l y ) on l e v e l s of n a t u r a l l y - o c c u r r i n g s u b s t a n c e s in brain. Substance e s t i m a t e d Noradrenaline * 5-Hydroxytryptamine Acetylcholine * Ergothioneine Tyramine Dopamine
Concentration in control b r a i n s (/lg/g ± S.E.) 0.26 0.34 1.9 0.445 0.326 0.207
± ± ± ± ± ±
0.041 0.032 0.18 0.039 0.08 0.05
Concentration a f t e r t r e m o r i n e (/lg/g ± S.E.) 0.14 0.402 2.56 0.45 0.322 0.20
± ± ± ± ± ±
0.03 0.034 0.21 0.05 0.08 0.05
% change 46% d e c r e a s e 18% i n c r e a s e 35% i n c r e a s e
none none none
* F o r both n o r a d r e n a l i n e and acetylcholine the t value calculated f r o m the d i f f e r e n c e s between the m e a n s is a p p r o x imately 2.3 and with 22 d e g r e e s of f r e e d o m , the value significant at the 5% level is 2.08. This s u g g e s t s that the change for t h e s e two s u b s t a n c e s is significant. Table 2 Effects of h a r m i n e (30 m g / k g intraperitoneally) on levels of n a t u r a l l y - o c c u r r i n g s u b s t a n c e s in brain. Substance e s t i m a t e d Noradrenaline * 5-Hydroxytryptamine Acetylcholine E rgothioneine Tyramine Dopamine
Concentration in control b r a i n s ( # g / g ± S.E.) 0.21 0.32 1.8 0.5 0.34 0.207
± 0.02 i0.035 ± 0.19 ± 0.06 ± 0.03 i 0.011
Concentration after h a r m i n e ( # g / g ± S.E.) 0.28 0.41 2.0 0.51 0.36 0.227
~- 0.024 4- 0.038 ± 0.16 ± 0.05 ± 0.04 ± 0.01"/
% change 33% i n c r e a s e 28% i n c r e a s e none none none none
* The t value for n o r a d r e n a l i n e calculated f r o m the difference between the m e a n s is 2.24 and with 22 d e g r e e s of f r e e d o m , the value significant at the 5% level is 2.08, s u g g e s t i n g that the change for this a m i n e is significant. t h i o n e i n e in w h o l e b r a i n s of c o n t r o l c h i c k s a n d in c h i c k s p r e - t r e a t e d w i t h 30 m g / k g of t r e m o r i n e o r 30 m g / k g h a r m i n e 40 r a i n p r e v i o u s l y a r e p r e s e n t e d in t a b l e s 1 and 2 r e s p e c t i v e l y . Tremorine produced a significant decrease in
the brain level of noradrenaline and a significant increase in the level of acetylcholine. There was also a small increase in the level of 5-HT, but this effect was not significant. No change was detected in the brain levels of dopamine, tyramine or ergothioneine. Others (Friedman, 1963, 1964; Friedman, Aylesworth and Friedman, 1963) have recorded an increase in 5-HT levels and a decrease in noradrenaline levels in mice, and Holmstedt (1964) found an increase in acetylcholine levels in rats. Harmine produced a small but significant increase in the level of noradrenaline, but did not change the levels of acetylcholine, ergothioneine or tyramine. There may also have been a small increase in the levels of 5-HT and dopamine but these changes were not significant at the P=0.95 level. Holzer and Hornykiewicz (1959) noted an increase in brain dopamine levels in rats after injections of harmine.
4. DISCUSSION T h e r e s u l t s s h o w e d t h a t t h e e f f e c t s of t r e m o r i n e a n d h a r m i n e in y o u n g c h i c k s a r e e s s e n t i a l ly s i m i l a r to t h o s e p r o d u c e d in m a m m a l i a n s p e c i e s w h i c h r e s p o n d to the d r u g . T h e a d v a n t a g e s of u s i n g y o u n g c h i c k s f o r s t u d y i n g t r e m o r i n e e f fects include their availability and cheapness, and the ease with which tremor and akinesia may b e d e t e c t e d . O b s e r v a t i o n of t r e m o r i s f a c i l i t a t e d by the feathers which amplify the fine movements of t h e u n d e r l y i n g m u s c l e s a n d s k i n , a n d a k i n e s i a and r i g i d i t y a r e m a d e e s p e c i a l l y o b v i o u s by the fact that the chick is a biped with long legs. The chief d i s a d v a n t a g e in u s i n g c h i c k s i s that they r e s p o n d w e l l to t r e m o r i n e o n l y d u r i n g t h e f i r s t 4 o r 5 d a y s a f t e r h a t c h i n g . It i s p o s s i b l e t h a t t h e f a l l - o f f of r e s p o n s e to t r e m o r i n e w i t h a g e i s a r e f l e c t i o n of t h e g r a d u a l d e v e l o p m e n t of t h e b l o o d - b r a i n b a r r i e r . H o w e v e r , it s e e m s u n l i k e l y that this is the chief factor responsible, as inc r e a s i n g a g e h a d l i t t l e e f f e c t on t h e t r e m o r r e s p o n s e t o h a r m i n e a n d t h e r e i s no e v i d e n c e i n other species that the blood-brain barrier diff e r e n t i a t e s b e t w e e n t h e s e two d r u g s . F u r t h e r m o r e , t r e m o r i n e i s effective in p r o d u c i n g t r e m o r i n a d u l t m i c e , c a t s a n d d o g s a n d t h e r e i s no
TREMORINE AND HARMINE IN THE CHICK e v i d e n c e that the b l o o d - b r a i n b a r r i e r in the adult f o w l i s a n y m o r e r e s i s t a n t to p e n e t r a t i o n by d r u g s t h a n t h a t of o t h e r s p e c i e s . A l t h o u g h n o t d i r e c t l y r e l a t e d , it i s p o s s i b l e t h a t t h e r e s i s t a n c e to t r e m o r i n e w h i c h d e v e l o p s w i t h a g e , i s s i m i l a r to t h e t a c h y p h y l a x i s w h i c h i s p r o d u c e d b y r e p e a t e d d o s a g e . R e p e a t e d a d m i n i s t r a t i o n of s e v e r a l d r u g s i s k n o w n to s t i m u l a t e t h e f o r m a t i o n of l i v e r m i c r o s o m a l e n z y m e s w h i c h d e t o x i f y t h e m ( s e e , f o r e x a m p l e , B r o d i e , 1964). S u c h a n e f f e c t m i g h t e x p l a i n t a c h y p h y l a x i s to t r e m o r i n e , a n d if t h e f o r m a t i o n of t h e s a m e o r s i m i l a r e n z y m e s i s s t i m u l a t e d b y c e r t a i n c o n s t i t u e n t s of d i e t , w h i c h a r e n o t p r e s e n t i n t h e y o l k s a c in o v u m , t h e l e s s p r o n o u n c e d d i m i n u t i o n of r e s p o n s i v e n e s s with age would be a c c o u n t e d for. The interaction between tremorine and harm i n e and o t h e r d r u g s in the c h i c k will be the s u b j e c t of a s e p a r a t e r e p o r t .
ACKNOWLEDGEMENTS T h i s work was supported by a grant made to W.C.B. by the B r i t i s h Egg M a r k e t i n g Board. G.O. is indebted to the I n t e r - U n i v e r s i t y Council for Higher Education O v e r s e a s for a studentship and to the B r i t i s h C o m m o n wealth S c h o l a r s h i p C o m m i s s i o n for a s c h o l a r s h i p d u r ing the t e n u r e s of which this work was undertaken. We a r e grateful to Dr. J. C r o s s l a n d of the University of Nottingham for d e m o n s t r a t i n g to u s the method of e x t r a c t i o n and e s t i m a t i o n of ergothioneine.
REFERENCES A r i e n s - K a p p e r s , C . U . , 1929, The evolution of the n e r v o u s s y s t e m in i n v e r t e b r a t e s , v e r t e b r a t e s and m a n (De E r v e n F. Boh, H a a r l e m ) p. 126. Baker, W.W. and A . J . Triolo, 1960, Additional s t u d i e s on t r e m o r i n e induced involuntary m o v e m e n t s , P h a r m a c o l o g i s t 2, 87. Beani, L. and C. Bianchi, 1963, The e x t r a c t i o n of a c e tylcholine in s m a l l s a m p l e s of c e r e b r a l t i s s u e , J. P h a r m . P h a r m a c . 15, 281. Blaber, L . C . and A.W. Cuthbert, 1961, A s e n s i t i v e method for the a s s a y of acetylcholine, J. P h a r m . P h a r m a c . 13, 445. Bloekus, L . E . and G . M . E v e r e t t , 1957, T r e m o r p r o ducing d r u g 1 , 4 - d i p y r r o l i d i n o - 2 - b u t y n e ( T r e m orine), Fedn. P r o c . 16, 283. Bowman, W.C., B.A. C a l l i n g h a m and G. Osuide, 1964, E f f e c t s of t y r a m i n e on a spinal reflex 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. Brodie, B . B . , 1964, Distribution and fate of d r u g s ; t h e r a p e u t i c i m p l i c a t i o n s . In: Absorption and d i s t r i bution of d r u g s , eds. T . B . Binns and C. Dodds (Livingstone, Edinburgh) p. 199. Biilbring, E., 1955, C o r r e l a t i o n between m e m b r a n e potential, spike d i s c h a r g e and tension in smooth m u s c l e , J. P h y s i o l . (London) 128, 200.
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C a l l i n g h a m , B . A . and R. C a s s , 1963, A modification of the b u t a n o l e x t r a c t i o n method for the f l u o r i m e t r i c a s s a y of c a t e c h o l a m i n e s in biological m a t e r i a l s , J. P h a r m . P h a r m a e . 15, 699. C a r l s s o n , A., 1959, Detection and a s s a y of dopamine, P h a r m a c . Rev. 11, 300. C a r l s s o n , A. and B. Waldeck, 1958, A f l u o r o m e t r i c method for the d e t e r m i n a t i o n of dopamine (3-hyd r o x y t y r a m i n e ) , Acta Physiol. Scand. 44, 293. C a s s , R. and T . L . B . Spriggs, 1961, T i s s u e a m i n e levels and s y m p a t h e t i c blockade a f t e r guanethidine and b r e t y l i u m , Brit. J. P h a r m a c . C h c m o t h e r . 17, 442. Chen, A . L . and K . K . Chen, 1939, H a r m i n e the alkaloid of Caapi, Quart. J. P h a r m . P h a r m a c o l . 12, 30. Cho, A.K., A.S. F a i r h u r s t , W. L. t l a s l e t t and D. J. J e n den, 1964, The m e t a b o l i s m of t r e m o r i n e , P r o c e e d i n g s of the 2nd International P h a r m a c o l o g i c a l C o n g r e s s , Vol. 2 ( P e r g a m o n P r e s s , Oxford) p. 75. Cho, A.K., W . L . Haslctt and D . J . Jenden, 1961, The identification of an active mctabolite of t r e m o r i n e , Biochem. Biophys. R e s . C o m m u n . 5, 276. C r o s s l a n d , J., J. F. Mitchell and G. N. Woodruff, 1964, Identity of the c e r e b e l l a r factor, Nature (London) 203, 1388. C r o s s l a n d , J., J. F. Mitchell and G. N. Woodruff, 1966, The p r e s e n c e of ergothioneine in the c e n t r a l n e r vous s y s t e m and its probable identity with the c e r e b e l l a r factor, J. Physiol. (London) 182, 427. Desei, L., M. V a r s z e g i and J. Mehes, 1961, T o l e r a n c e to t r e m o r i n e , J. P h a r m . P h a r m a c . 13, 127. E v e r e t t , G.M., 1956, T r e m o r produced by d r u g s , Nature (London) 177, 1238. E v e r e t t , G.M., 1964, P h a r m a c o l o g i c a l s t u d i e s on t r e m o r i n e , P r o c e e d i n g s of the 2nd International Pharmacological Congress, Vol. 2 ( P e r g a m o n P r e s s , Oxford) p. 69. E v e r e t t , G.M., L . E . Blockus and I. M. Shepperd, 1956, T r e m o r i n e induced t r e m o r and its a n t a g o n i s m by a n t i - P a r k i n s o n d r u g s , Science 13, 79. F a r q u h a r s o n , M . E . and R.G. Johnston, 1959, Antagon i s m of the effects of t r e m o r i n e by atropine d e r i v a tives, Brit. J. P h a r m a c . C h e m o t h e r . 14, 559. F r i e d m a n , A.H., 1963, Norepinephrine depletion in the b r a i n s t e m by t r e m o r i n e , Fedn. Proc. 22, 272. F r i e d m a n , A.H., 1964, P r o c e e d i n g s of the 2nd I n t e r national P h a r m a c o l o g i c a l C o n g r e s s , Vol. 2 ( P e r gamon P r e s s , Oxford) p. 130. F r i e d m a n , 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 : Effects 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 , Science 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 logical a s p e c t s of P a r k i n s o n i s m , Advances in P h a r macology 3, e d s . S. Garattini and P. A. Shore (Academic P r e s s , New York) p. 83. F r i e d m a n , A.H. and R . L . RingeI, 1965, S p e c t r o graphic a n a l y s i s of evoked vocalisation a s a method of evaluating d r u g action in v i v o , Fedn. P r o c . 24, 675. Hara, S., 1953, P h a r m a c o l o g i c a l s t u d i e s on the functions of the e x t r a p y r a m i d a l s y s t e m , Japan. J. P h a r m a c . 2, 127. Hara, S. and K. K a w a m o r i , 1954, P h a r m a c o l o g i c a l s t u d i e s on the functions of the e x t r a p y r a m i d a l s y s t e m , Report No. 2, M e c h a n i s m of the a p p e a r a n c e of t r e m o r due to e x t r a p y r a m i d a l poisons, Japan. J. P h a r m a e . 3, 149.
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Holmstedt, B., 1964, P r o c e e d i n g s of the 2nd International P h a r m a c o l o g i c a l C o n g r e s s (Pergamon P r e s s , Oxford) p. 128. Holzer, G. and O. Hornykiewiez, 1959, Ueber den Dopamin (Hydroxytyramin) Stoffwechsel im Gehirn der Ratte, Arch. Exp. Path. Pharmak. 237, 27. Key, B . J . and E . M a r l e y , 1962, The effect of s y m pathomimetie amines on behaviour and e l e e t r o c o r tical activity of chickens, Electron. Neurophysiol. 14, 90. Kocsis, J . J . and R.M. Welch, 1960, Conversion of t r e m o r i n e (1,4-dipyrollidino-2-butyne) to an active metabolite, P h a r m a c o l o g i s t 2, 87. Melville, D.B. and W . H . H o r n e r , 1953, Blood e r g o thioneine in germ free chickens, J. Biol. Chem. 202, 187. Melville, D.B., W.H. Horner and R. Lubsehez, 1954, Tissue ergothioneine, J. Biol. Chem. 206, 221. Pale~ek, F., 1963, A simple r e s p i r a t o r for laboratory use, J. Appl. Physiol. 18, 443. Patten, B.M., A. Sakamoto, M.H. Van Woert, P.S. P a pavasiliou and G. C. Cotzias, 1964, T r e m o r i n e induced t r e m o r v e r s u s extrapyramidal d i s e a s e , Nature (London) 201, 929. Shore, P . A . and J . S . Olin, 1958, Identification and chemical a s s a y of norepinephrine in brain and other t i s s u e s , J. P h a r m a c . Exp. Ther. 122, 295. Spector, S., K. Melmon, W. Lovenberg and A. S j o e r d s ma, 1962, P r e s e n c e of tyramine in the spinal cord and brain, P h a r m a c o l o g i s t 4, 148.
Spector, S., K. Melmon, W. Lovenberg and A. S j o e r d s ma, 1963, The p r e s e n c e and distribution of t y r amine in mammalian t i s s u e s , J. P h a r m a c . Exp. Ther. 140, 229. Spencer, P. S. J:, 1965, Activity of centrally acting and other drugs against t r e m o r and hypothermia induced in mice by t r e m o r i n e , Brit. J. P h a r m a c . Chemother. 25, 442. Stern, P., N. Radovic and S. Buljubasic, 1965, P h a r macolog~¢ of experimental t r e m o r , Nature {London} 206, 1261. Udenfriend, S., H. Weissbach and C. T. Clark, 1955, The estimation of 5-hydroxytryptamine (serotonin} in biological t i s s u e s , J. Biol. Chem. 215, 337. V e r n i e r , U.G., 1964, Anti-Parkinson agents: Evaluation of drug activities: P h a r m a c o m e t r i c s , eds. D.R. Laurence and A . L . Bacharach {Academic P r e s s , New York and London} p. 301. Waelsch, H., 1955, The turnover of components of the developing brain; the blood brain b a r r i e r , Biochemi s t r y of the developing nervous s y s t e m {Academic P r e s s , New York} p. 187. Zaimis, E., 1960a, Ciba Foundation Symposium on Adrenergic Mechanisms {Churchill, London} p. 562. Zaimis, E., 1960b, Simultaneous recording of activity and rate of chirping in the young chick, J. Physiol. {London} 152, l l P . Zetler, G., 1957, Der H a r m i n - T r e m o r und seine Antagonisten, Arch. Exp. Path. Pharmak. 231, 34.
EFFECTS OF T R E M O R I N E A N D H A R M I N E I N THE CHICK W. C. BOWMAN and G. OSUIDE *
Department of Pharmacology, School of Pharmacy, University of London and University of Strathclyde, Glasgow, C. 1, U.K. Accepted 14 November 1966
W. C. BOWMAN and G. OSUIDE, Effects oftremorine and harmine in the chick, European J. Pharmacol. 1 (1967) 71-80. T r e m o r i n e produced pronounced t r e m o r , akinesia, hypothermia and parasympathetic effects in young chicks. T r e m o r was r e c o r d e d quantitatively and 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 was found to diminish with increasing age and with repeated dosage. The optimal age of the chicks for obtaining a t r e m o r r e s p o n s e to t r e m o r i n e was 3 to 4 days. Harmine also produced t r e m o r , and some akinesia and hypothermia. The harmine effect did not diminish with age or with repeated dosage. T r e m o r i n e produced a significant i n c r e a s e in the brain levels of acetylcholine, and a d e c r e a s e in the brain levels of noradrenaline. It did not affect the levels of tyramine, dopamine or ergothioneine. Harmine produced an i n c r e a s e in the brain levels of noradrenaline but did not affect the levels of a c e tylcholine, tyramine or ergothioneine.
Acetylcholine effect of harmine on chick brain levels effect of t r e m o r i n e on chick brain levels
Dopamine effect of harmine on chick brain levels effect of t r e m o r i n e on chick brain levels
Ergothioneine effect of harmine on chick brain levels effect of t r e m o r i n e on chick brain levels
Harmine akinesia in the chick produced by hypothermia in the chick produced by t r e m o r in the chick produced by
5- Hydroxy trypta mine effect of harmine on chick brain levels effect of t r e m o r i n e on chick brain levels
Noradrenaline effect of harmine on chick brain levels effect of t r e m o r i n e on chick brain levels
Tremorine akinesia in the chick produced by hypothermia in the chick produced by parasympathetic effects in the chick produced by t r e m o r in the chick produced by
Tyramine effect of harmine on chick brain levels effect of t r e m o r i n e on chick brain levels
I. INTRODUCTION
H a r m i n e is an alkaloid obtained f r o m Pegahum harmala a n d f r o m Banisteria caapi. It too
T r e m o r i n e p r o d u c e s t r e m o r a n d a k i n e s i a in m i c e , a n d in s o m e o t h e r m a m m a l s , by a n a c t i o n b e l i e v e d to b e e x e r t e d in t h e b r a i n s t e m r e t i c u l a r f o r m a t i o n ( E v e r e t t , 1956; E v e r e t t , B l o c k u s a n d S h e p p e r d , 1956; B l o c k u s a n d E v e r e t t , 1957; S t e r n , R a d o v i c a n d B u l j u b a s i c , 1965). T r e m o r i n e o w e s i t s a c t i v i t y to a m e t a b o l i t e , o x o t r e m o r i n e , f o r m e d in t h e l i v e r m i c r o s o m e s (Kocsis and W e l c h , 1960; Cho, H a s l e t t a n d J e n d e n , 1961). T h e s y m p t o m s of t r e a t m e n t w i t h t r e m o r i n e in animals superficially resemble s o m e of t h e s y m p t o m s of P a r k i n s o n ' s d i s e a s e a n d m a n y a n t i P a r k i n s o n d r u g s a r e e f f e c t i v e in a n t a g o n i s i n g t h e a c t i o n s of t r e m o r i n e .
p r o d u c e s t r e m o r in s o m e a n i m a l s p e c i e s by a n a c t i o n in t h e e x t r a - p y r a m i d a l s y s t e m ( H a r a , 1953; H a r a a n d K a w a m o r i , 1954). H o w e v e r , a n t i P a r k i n s o n d r u g s u s u a l l y do not a n t a g o n i s e the t r e m o r r e s p o n s e p r o d u c e d by h a r m i n e ( E v e r e t t , 1956; Z e t l e r , ]957; 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). A c c o r d i n g to A r i e n s - K a p p e r s (1929) and to Patten, Sakamoto, Van Woert, P a p a v a s i l i o u and C o t z i a s (1964), t h e c o r p u s s t r i a t u m of b i r d s (and r e p t i l e s ) i s w e l l d e v e l o p e d a n d i s the h i g h e s t c e n t r e controlling m o t o r activity. During the f i r s t few d a y s a f t e r h a t c h i n g , t h e b l o o d - b a r r i e r of t h e c h i c k i s d e f e c t i v e , at l e a s t w i t h r e s p e c t to s o m e s u b s t a n c e s ( W a e l s c h , 1955; Z a i m i s , 1960a; K e y a n d M a r l e y , 1962). T h e s e f a c t s s u g g e s t e d that young chicks might p r o v i d e useful t e s t ani-
* P r e s e n t a d d r e s s : Department of Pharmacology, University of Ibadan, Nigeria
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m a l s for studying the actions of d r u g s acting on the e x t r a - p y r a m i d a l s y s t e m , including antiP a r k i n s o n drugs. T h i s p a p e r d e s c r i b e s e x p e r i me nts in which the e f f e c ts of t r e m o r i n e and h a r m i n e w e r e studied as a p r e l i m i n a r y to i n v e s tigating the i n t e r a c t i o n of a n t i - P a r k i n s o n drugs with them.
2. METHODS The 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 d o m e s tic fowl chicks (Silver link) aged f r o m 1-25 days a f t e r hatching.
2.1. Spinal reflexes Crossed extensor reflexes were recorded f r o m one limb of chicks lightly a n a e s t h e t i s e d with c h l o r a l o s e (30-60 m g / k g ) i n j e c t e d i n t r a p e r i t o n e a l l y . The method used was that des c r i b e d by Bowman, Callingham and Osuide (1964). Artificial v e n t il a t io n was applied throughout by the method d e s c r i b e d by P a le ~ e k (1963), and drugs w e r e i n j e c t e d through a cannula in a j u g u l ar vein. 2.2. Recording of tremor Chicks w e r e p l a c e d individually in a jiggle cage made f r o m w i r e m e s h with a r u b b e r base.
A t h r e a d fixed to the r u b b e r b a s e was attached to a s t r a i n gauge made f r o m a RCA 5734 m e c h a n o e l e c t r i c t r a n s d u c e r v a l v e (Btllbring, 1955), the output of which was co n n ect ed to a T e k t r o n i x (type 502) double b e a m o s c i l l o s c o p e through a b ack i n g - o f f p o t e n t i o m e t e r used to find the null point of the s t r a i n gauge. The input of a C o s s e r p r e - a m p l i f i e r (model 1440) was connected in p a r a l l e l to the input of the o s c i l l o s c o p e . A m p l i fied signals w e r e fed into a T e k t r o n i x (type 161) pulse g e n e r a t o r . The p u l s e s p r o d u c e d during t r e m o r of the chick w e r e counted by a Panax Counter (type SA 102) connected to the pulse g e n e r a t o r . Fig. 1 i l l u s t r a t e s a t r e m o r g r a m r e c o r d e d on the o s c i l l o s c o p e f r o m a chick t r e a t e d with t r e m o r i n e . Chicks u su al l y do not exhibit e x p l o r a t o r y m o v e m e n t s and they usually r e mained s t a t i o n a r y in the middle of the jiggle cage. This was e s p e c i a l l y so a f t e r t r e a t m e n t with t r e m o r i n e which e x e r t s a s e d a t i v e effect. Consequently, g r o s s m o v e m e n t a r t i f a c t s did not usually c o m p l i c a t e the t r e m o r count and the few chicks that did exhibit spontaneous m o v e m e n t s w e r e d i s c a r d e d . E a c h chick was p l a c e d in the jiggle cage and r e m a i n e d t h e r e f o r 1 hr b e f o r e drug t r e a t m e n t . During this p e r i o d the s e n s i t i v i ty of the s t r a i n gauge was adjusted to avoid counting the c h i c k ' s r e s p i r a t o r y m o v e m e n t s and h e a r t beat. Under t h e s e conditions, the total count during 1 hr did not e x c e e d 1% of the total
1 HR 45 MIN
|
0.5 sec
I
Fig. 1. Tremorgram recorded on a cathode ray oscilloscope from a chick injected with 20 mg/kg tremorine intraperitoneally. These signals were electronically counted as described in the test. The times denote the times after injection.
TREMORINE AND HARMINE IN THE CHICK
73
count produced in 1 hr by the s m a l l e s t dose of t r e m o r i n e used.
w e r e pooled to make each sample, and 12 s a m ples w e r e used for the d e t e r m i n a t i o n s .
2.3. Body temperature The body t e m p e r a t u r e of chicks was r e c o r d e d by m e a n s of a s m a l l t h e r m i s t o r (Grant I n s t r u m e n t s Ltd., Cambridge) i n s e r t e d into the cloaca.
2.6. Acetylcholine assay I m m e d i a t e l y a f t e r r e m o v a l , b r a i n s from 12 control chicks and f r o m 12 chicks t r e a t e d either with t r e m o r i n e or with h a r m i n e were placed s e p a r a t e l y in McIlwan's buffer at pH4 and homogeni'sed by grinding with sand in a glass m o r tar. The buffer i n a c t i v a t e s the c h o l i n e s t e r a s e and acetylcholine is stable at this pH. The ext r a c t i o n method was that d e s c r i b e d by Beani and Bianchi (1963). I m m e d i a t e l y before a s s a y the test solutions were n e u t r a l i s e d to pH 7.0 by the dropwise addition of HC1 and then a s s a y e d against s t a n d a r d acetylcholine solutions on the s e n s i t i s e d guinea-pig ileum (Blaber and Cuthb e r t , 1961). Tyrode solution containing m e p y r a m i n e 10 -7 was used to bathe the ileum, and before the a s s a y was c o m m e n c e d the i l e u m was r e n d e r e d i n s e n s i t i v e to the s t i m u l a n t action of t r e m o r i n e by applying repeated s m a l l doses of t r e m o r i n e until complete tachyphylaxis was p r o duced. The substance p r e s e n t was identified as an a c e t y l c h o l i n e - l i k e substance by the facts that its action was completely blocked by atropine (10 n g / m l ) and it r e q u i r e d s e n s i t i s a t i o n of the i l e u m with dyflos before its p r e s e n c e could be demonstrated.
2.4. Spectrophotofluorornetric assays Chicks aged 3 days w e r e killed by cutting through the thorax into the h e a r t and allowing them to bleed to death. The b r a i n s were r e moved, d r i e d with blotting paper to remove any blood and kept at - 1 0 o c until the a s s a y was b e gun. Whole b r a i n s from control groups of chicks and from groups t r e a t e d e i t h e r with t r e m o r i n e or with h a r m i n e w e r e a s s a y e d for n o r a d r e n a l i n e , dopamine, t y r a m i n e and 5 - h y d r o x y t r y p t a m i n e , u s i n g an A m i n c o - B o w m a n s p e c t r o p h o t o f l u o r i m eter. In the a s s a y s of the a m i n e s , each sample c o m p r i s e d a single b r a i n , and 12 s a m p l e s were used for each d e t e r m i n a t i o n except in the case of 5 - h y d r o x y t r y p t a m i n e w h e r e 24 s a m p l e s were used. Noradrenaline was e x t r a c t e d and a s s a y e d by the method of Shore and Olin (1958) as modified by Cass and Spriggs (1961) and Callingham and Cass (1963). Doparnine was e x t r a c t e d by the method of Shore and Olin (1958) as modified by Callingham and Cass (1963). The s a m p l e s w e r e a s s a y e d a c c o r d ing to the method of C a r l s s o n and Waldeck (1958) and C a r l s s o n (1959). Tyrarnine was e x t r a c t e d and a s s a y e d according to the method of Spector, Melmon, Lovenberg and S j o e r d s m a (1962, 1963) except that the p o s s i bility of loss of a m i n e due to heat generated d u r ing the p r e p a r a t i o n of the t i s s u e s for e x t r a c t i o n was avoided by u s i n g liquid n i t r o g e n (Callingham and Cass, 1963). 5-Hydroxytryptarnine was extracted and a s s a y e d a c c o r d i n g to the method of Udenfriend, W e i s s bach and Clark (1955) with the exception that the t i s s u e was p r e p a r e d for e x t r a c t i o n using liquid n i t r o g e n (Callingham and Cass, 1963). 2.5. Ergothioneine assay The b r a i n s of chicks aged 3 days were r e moved as d e s c r i b e d above. Ergothioneine was a s s a y e d in c o n t r o l b r a i n s and in b r a i n s from chicks t r e a t e d with t r e m o r i n e or h a r m i n e a c c o r d i n g to the method of C r o s s l a n d , Mitchell and Woodruff (1964, 1966), which is s i m i l a r to the methods of Melville and H o r n e r (1953) and Melville, H o r n e r and Lubschez (1954), using a Hilger H810 Biochem A b s o r p t i o m e t e r . T h r e e b r a i n s
2.7. Drugs The drugs used were t r e m o r i n e hydrochloride (Abbott's l a b o r a t o r i e s ) , h a r m i n e hydrochloride (L. Light and Co.), acetylcholine chloride (Roche), b r o m o l y s e r g i c acid diethylamide (Sandoz), atropine sulphate (British Drug Houses), m e p y r a m i n e m a l e a t e (May and Baker Ltd.) and dyflos (DFP, Boots).
3. RESULTS
3.1. Anaesthetised chicks. Crossed extensor r e flex In m i n i m a l effective doses (20-25 mg/kg) i n jected i n t r a v e n o u s l y , t r e m o r i n e slightly augm e n t e d t h e reflex c o n t r a c t i o n s and converted the single r e s p o n s e to each s t i m u l u s into a double or t r i p l e c o n t r a c t i o n (fig. 2). Occasional c o n t r a c tions u n r e l a t e d to the e l e c t r i c a l s t i m u l a t i o n also o c c u r r e d u n d e r the influence of t r e m o r i n e . The effects lasted 15-45 min and after the f i r s t injection t h e r e was always a latent period of 10-15 rain before the onset of the effect. With s u b s e quent doses the latent period b e c a m e s h o r t e r . After s e v e r a l doses of t r e m o r i n e , the effect
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Fig. 2. Chick aged 3 days and weighing 50 g. C r o s s e d extensor reflex elicited e v e r y 15 sec; contractions downwards. At T, 1.5 mg t r e m o r i n e intravenously before (above) and 15 min a f t e r (below) the intravenous injection of 50 ttg atropine. These injections were the 3rd and 4th injections of the same dose of t r e m o r i n e in this e x p e r i m e n t so that the latent period between injection and effect was brief. The double contractions to which t r e m o r i n e gives r i s e can be distinguished by the thickening of the contraction r e c o r d s in the upper panel. Atropine blocked the effects of tremorine.
c h a n g e d to a d e p r e s s i o n of t h e e v o k e d c o n t r a c tions, but the double or triple responses and the c o n t r a c t i o n s not e v o k e d b y e l e c t r i c a l s t i m u l a t i o n persisted. After sectioning the spinal chord at t h e l e v e l of t h e 10th c e r v i c a l v e r t e b r a ( s e e B o w m a n et al., 1964) a s e c o n d d o s e of t r e m o r i n e p r o d u c e d a n e f f e c t s i m i l a r to, b u t m u c h w e a k e r than, the effect produced by the first dose given to t h e i n t a c t c h i c k , i n d i c a t i n g t h a t t h e s i t e of a c t i o n of t r e m o r i n e i s m a i n l y , t h o u g h n o t e n t i r e l y , w i t h i n the b r a i n . In d o s e s of 2 . 5 - 1 0 m g / k g i n t r a v e n o u s l y , h a r mine slightly potentiated the reflex contractions in c h i c k s w i t h i n t a c t c e n t r a l n e r v o u s s y s t e m a n d produced vigorous muscular activity which was u n r e l a t e d to t h e e l e c t r i c a l s t i m u l a t i o n (fig. 3). The second and succeeding doses were more effective than the first. After sectioning the spinal c o r d at t h e l e v e l of C10, h a r m i n e w a s w i t h o u t e f f e c t on the r e f l e x c o n t r a c t i o n s s h o w i n g t h a t i t s s i t e of a c t i o n i s e n t i r e l y w i t h i n t h e b r a i n . A t r o p i n e (0.5 m g / k g ) p r e v e n t e d t h e e f f e c t s of a s u b s e q u e n t i n j e c t i o n of t r e m o r i n e o n t h e r e f l e x c o n t r a c t i o n s (fig. 2), b u t did not r e d u c e t h e e f f e c t s of h a r m i n e (fig. 3).
3.2. Conscious chicks In t h e e x p e r i m e n t s on c o n s c i o u s c h i c k s , a l l injections were given intraperitoneally unless otherwise stated. 3.2.1. T r e m o r In c h i c k s a g e d 1-3 d a y s , t r e m o r i n e , in d o s e s of 4 m g / k g a n d a b o v e , p r o d u c e d s e d a t i o n a n d t h e w i n g s t e n d e d to d r o o p . T h e c h i c k s r e m a i n e d standing but spontaneous movement was curtailed. T r e m o r w a s p r o d u c e d b y d o s e s of 10 m g / k g a n d a b o v e a f t e r a l a t e n t p e r i o d of 1 - 1 5 m i n , b u t u s u a l l y a f t e r a b o u t 10 m i n , a n d w a s e v i d e n t in the head, neck, body and drooping wings. A similar latent period occurred when the same doses w e r e i n j e c t e d i n t r a v e n o u s l y . A f t e r a d o s e of 20 m g / k g , t h e m a x i m u m e f f e c t in c h i c k s a ~ e d 1 d a y occurred 20-30 min after injection and at this t i m e t h e r e c o r d e d f r e q u e n c y of t h e t r e m o r w a s 1 5 - 2 0 / s e c . T o w a r d s t h e e n d of t h e f i r s t h o u r a f t e r i n j e c t i o n , b r i e f p e r i o d s of q u i e s c e n c e of 1 - 3 min duration interrupted the tremor. The quiescent periods then became more freqeunt and longer lasting. Tremor could be restarted during a q u i e s c e n t p e r i o d , f o r up to 3 h o u r s a f t e r i n -
TREMORINE AND HARMINE IN THE CHICK
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1 5 - 2 5 m i n a f t e r i n j e c t i o n . W h e n d o s e s of 1 0 - 4 0 mg/kg were given a second time, 2-4 hrs after the first dose, the tremor count recorded was greater than the initial count by 5-10°. The tremor effect produced by tremorine was f o u n d to d i m i n i s h w i t h i n c r e a s i n g age. In a s s e s s i n g t h e e f f e c t of a g e , d i f f e r e n t g r o u p s of 5 c h i c k s w e r e t a k e n d a i l y , f r o m t h e s e c o n d to t h e e i g h t h d a y of a g e , f r o m a c a g e o r i g i n a l l y c o n t a i n i n g 100 c h i c k s a g e d 1 day. E a c h 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 t r e m o r i n e a n d t h e t r e m o r produced was counted for 1 hour after injection. T h e r e s u l t s a r e e x p r e s s e d g r a p h i c a l l y in fig. 5 ( u p p e r c u r v e ) . T r e m o r w a s g r e a t e s t in c h i c k s a g e d 3 d a y s a n d d i m i n i s h e d to l e s s t h a n o n e t h i r d of t h i s m a x i m u m by t h e t i m e t h e c h i c k s w e r e 8 d a y s old. In c h i c k s a g e d 10 d a y s o r m o r e t r e m o r w a s s l i g h t o r a b s e n t a f t e r d o s e s of 20 mg/kg tremorine. Adult rabbits have also been f o u n d to b e i n s e n s i t i v e to t h e t r e m o r p r o d u c i n g e f f e c t of t r e m o r i n e ( F a r q u h a r s o n a n d J o h n s t o n , 1959; F r i e d m a n , 1964), b u t it i s n o t k n o w n whether new-born rabbits respond. When tremorine was injected into the same c h i c k on s u c c e s s i v e d a y s , t h e r e s u l t i n g t r e m o r response a p p e a r e d to b e c o m e smaller at a g r e a t e r r a t e t h a n c o u l d b e a c c o u n t e d f o r by t h e f a l l - o f f of r e s p o n s e w i t h age. In a s s e s s i n g t h e
Fig. 3. Chick aged 3 days and weighing 48 g. Recording as in fig. 2. At H, 0.2 mg h a r m i n e before (above) and a f t e r (below) 50 /ig atropine. All injections i n t r a venously. Atropine did not reduce the effects of h a r mine. jection, by environmental stimuli such as a sound or movement. Such stimuli evoked gross m o v e m e n t s of t h e s e d a t e d c h i c k s a n d t h i s t r i g g e r e d a b o u t of t r e m o r . Consequently in the q u a n t i t a t i v e a s s e s s m e n t of t r e m o r i t w a s i m p o r t a n t to c o n t r o l t h e e n v i r o n m e n t a l conditions closely. All quantitative measurements were therefore made in a quiet, artificially-lighted r o o m w i t h a t e m p e r a t u r e of 1 9 - 2 1 o c . Fig. 4 i n c l u d e s a d o s e / r e s p o n s e c u r v e p l o t t e d for tremorine using chicks aged 3 days. Each p o i n t w a s o b t a i n e d f r o m r e s u l t s o n 10 c h i c k s . Each chick was studied individually under identical environmental conditions, the total count during the first hour after injection being rec o r d e d . D o s e s g r e a t e r t h a n 40 m g / k g p r o d u c e d pronounced parasympathetic symptoms, occasional convulsions and sometimes death within
2c
.l
i~)
20 Dose n~/kg
30
4O
Fig. 4. D o s e / r e s p o n s e c u r v e s for t r e m o r i n e and h a r mine. The dose in m g / k g i n t r a p e r i t o n e a l l y is plotted against the total t r e m o r count produced in 1 hr. The c u r v e s c r o s s because h a r m i n e , though effective in s m a l l e r doses than t r e m o r i n e , produced a t r e m o r of lower frequency and s h o r t e r duration than that produced by t r e m o r i n e . Each point is the mean of r e s u l t s on 10 chicks. The v e r t i c a l lines are the standard errors.
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extent of tachyphylaxis, a group of 75 chicks w e r e each i n j e c t e d with 20 m g / k g t r e m o r i n e on the second day a f t e r hatching. F i v e of t h e s e c h i c k s w e r e then p l a c e d singly in the r e c o r d i n g a p p a r a t u s and the t r e m o r count r e c o r d e d f o r 1 hr a f t e r injection. T h e s e chicks w e r e then d i s carded. On the next day, the r e m a i n i n g 70 c h i c k s w e r e again i n j e c t e d with the s a m e dose and the r e s p o n s e r e c o r d e d f r o m 5 of them. T h i s p r o c e d u r e was continued up to and including the 8th day a f t e r hatching. Using this p r o c e d u r e , any one chick was p l a c e d in the r e c o r d i n g a p p a r a t u s only once, so r e m o v i n g the p o s s i b i l i t y that any additional f a l l - o f f in r e s p o n s e o v e r that due to age was the r e s u l t of the chick b e c o m i n g a c c u s t o m e d to the e n v i r o n m e n t of the r e c o r d i n g apparatus. The r e s u l t s obtained a r e a l s o e x p r e s s e d in fig. 5 (lower curve). They indicate that the t o l e r a n c e which d e v e l o p s to r e p e a t e d daily dosage is g r e a t e r than the t o l e r a n c e s i m ply due to age. Thus, a f t e r 7 days of r e p e a t e d dosage, the t r e m o r r e s p o n s e was l e s s than one eighth of its m a x i m u m , w h e r e a s the f a l l - o f f due to age alone was only to just o v e r o n e - t h i r d of the m a x i m u m . T o l e r a n c e to the e f f e c ts of r e peated dosage of t r e m o r i n e has a l s o been noted in m i c e (Baker and T r i o l o , 1960; D e s c i , V a r s z e g i and Mehes, 1961). The chicks i n j e c t e d daily up to 8 days of age showed a l o s s of weight when c o m p a r e d with unt r e a t e d c o n t r o l s and of the 60 c h i c k s i n j e c t e d on 4 or m o r e days, 18 had died by the end of the 8th day of age. H a r m i n e in d o s e s of 2.5-40 m g / k g p r o d u c e d t r e m o r which was m o r e powerful and c o a r s e r than that p r o d u c e d by s i m i l a r d o s e s of t r e m -
i
u
Fig. 5. Graphs showing diminishing tremor response to tremorine (20 mg/kg) with age (upper curve) and diminishing tremor response resulting from age and repeated dosage (lower curve). Each point, except the first, is the mean of results on 5 chicks. The first point is the mean of results with the first dose in both experiments and is, therefore, from 10 chicks. For further explanation see text.
orine. The latent p e r i o d b e t w e e n i n j ect i o n and o n set of t r e m o r w as s h o r t e r than that a f t e r t r e m o r i n e , being u s u a l l y about 2 min but o c c a sionally as long a s 5 min. A f t e r i n j ect i o n of 20 m g / k g h a r m i n e , the m a x i m u m d e g r e e of t r e m o r o c c u r r e d in 15-25 m i n and was r e c o r d e d as a f r e q u e n c y of 1 0 - 1 5 / s e c . A f t e r 45-65 min, the ef fect had d i s a p p e a r e d . A d o s e / r e s p o n s e c u r v e in c h i c k s aged 3 days w as c o m p i l e d as f o r t r e m o r i n e (fig. 4). D o s e s of h a r m i n e of 50 m g / k g and above p r o d u c e d convulsions. When 20 m g / k g h a r m i n e was i n j e c t e d into c h i c k s of d i f f e r e n t a g e s (2-8 days) as in the ex p e r i m e n t s with t r e m o r i n e , t h e r e was no e v i d e n c e of a d i m i n i s h i n g e f f e c t with age. When the s a m e chicks w e r e i n j e c t e d daily up to the 8th day of age with 20 m g / k g h a r m i n e , only slight or no tachyphylaxis was evident. Chicks which had b e c o m e r e s i s t a n t to the t r e m o r - p r o d u c i n g effect of t r e m o r i n e , r e s p o n d e d n o r m a l l y to h a r m i n e . 3.2.2. A k i n e s i a T r e m o r i n e p r o d u c e d a k i n e s i a and r i g i d i t y of the l i m b s in d o s e s of 10 m g / k g and above. Its o n set u su al l y p r e c e d e d that of t r e m o r and it p e r s i s t e d a f t e r t r e m o r had c e a s e d . With a dose of 20-30 m g / k g , a k i n e s i a was m o s t pronounced 3070 m i n a f t e r i n j e c t i o n and was o c c a s i o n a l l y s t i l l evident as long a s 8 h r a f t e r injection. N o r m a l u n t r e a t e d c h i c k s p l a c e d on a bench m o v ed spontaneously and quickly changed t h e i r position when pushed gently with a s m a l l rod. When the legs w e r e f o r c i b l y s p r e a d apart, they w e r e quickly r e t u r n e d to the n o r m a l position. With a mild d e g r e e of a k i n e s i a (after about 10 mg/kg tremorine) spontaneous movements c e a s e d and the c h i c k s r e m a i n e d standing in an u p r i g h t position. When gently pushed they changed t h e i r p o s i t i o n with a h i g h - s t e p p i n g gait. A g r e a t e r d e g r e e of a k i n e s i a (after 20 m g / k g ) was e v i d e n c e d by swaying head m o v e m e n t s and difficulty in r e s t o r i n g the l e g s to t h e i r n o r m a l p o s i t i o n when s p r e a d apart. S e v e r e a k i n e s i a (after 30 m g / k g and above) was e v i d e n c e d by inability to r e s t o r e the legs to t h e i r n o r m a l p o s i tion when s p r e a d apart. H a r m i n e p r o d u c e d a k i n e s i a and rigidity in s o m e chicks but the e f f e c t was m i l d in c o m p a r i son with that p r o d u c e d by an e q u a l l y - s i z e d dose of t r e m o r i n e . S m a l l e r d o s e s of h a r m i n e (2-5 m g / k g ) have been shown to p r o d u c e a k i n e s i a in m o n k ey s (Chen and Chen, 1939) and p i g eo n s (Hara, 1953). 3.2.3. Vocal e f f e c t s The f r e q u e n c y of c h i r p i n g was d e c r e a s e d in
TREMORINE AND HARMINE IN THE CHICK c h i c k s about 20 m i n a f t e r i n j e c t i o n of t r e m o r i n e (20-30 m g / k g ) ; about 30 m in a f t e r i n j e c ti o n they b e c a m e c o m p l e t e l y s i l e n t even in the p r e s e n c e of u n t r e a t e d chicks. A d e p r e s s a n t e f f e c t of t r e m o r i n e on evoked s q u e a l s of adult g u i n e a - p i g s has a l s o been r e p o r t e d by F r i e d m a n and Ringal (1965). In c o n t r a s t , h a r m i n e (5-20 mg/kg) s t i m u l a t e d c h i r p i n g in chicks, an effect r e s e m b l i n g that p r o d u c e d by a m p h e t a m i n e as r e c o r d e d by Z a i m i s (1960b). 3.2.4. H y p o t h e r m i a Body t e m p e r a t u r e c o n t r o l is d e f e c t i v e in the chick during the f i r s t few days a f t e r hatching and, p o s s i b l y for this r e a s o n , the h y p o t h e r m i c effect of t r e m o r i n e in young chicks was p r o nounced. In d o s es of 20-30 m g / k g , t r e m o r i n e produced a fall in body t e m p e r a t u r e of 2 - 1 3 o c below the n o r m a l r a n g e of 3 9 - 4 2 o c in chicks aged f r o m 1-10 days, and kept in an e n v i r o n m e n t a l t e m p e r a t u r e of 1 9 - 2 1 o c . The fall in t e m p e r a t u r e was m a x i m a l about 1 hour a f t e r i n j e c tion and p e r s i s t e d for 2-5 hours. A s m a l l e r hyp o t h e r m i c effect of t r e m o r i n e has been r e c o r d e d in m i c e but in this s p e c i e s it p e r s i s t s for up to 18 hr ( E v e r e t t , 1964). The t r e m o r - p r o d u c i n g effect of t r e m o r i n e did not a p p e a r to be a c o n s e q u e n c e of its h y p o t h e r mic effect. Thus, the onset of the fall in t e m p e r a t u r e p r e c e d e d and o u t la s te d the t r e m o r r e sponse. Chicks aged ]0 days exhibited only v e r y slight t r e m o r but the fall in body t e m p e r a t u r e r e s e m b l e d that in y o u n g e r chicks. In u n t r e a t e d chicks, a fall in body t e m p e r a t u r e induced by a cold e n v i r o n m e n t induced negligible t r e m o r - l i k e a c t i v i t y when c o m p a r e d with the e f f e c ts of t r e m orine. Finally, the in te n s i ty of t r e m o r produced by t r e m o r i n e was not r e l a t e d to the magnitude of the fall in t e m p e r a t u r e . M a r k e d t r e m o r was s o m e t i m e s a c c o m p a n i e d by only a s m a l l hypot h e r m i c effect and v i c e v e r s a . Other w o r k e r s using d i f f e r e n t s p e c i e s have a l s o concluded that the t r e m o r and h y p o t h e r m i c e f f e c ts a r e independent r e s p o n s e s (Blockus and E v e r e t t , 1957; P a t ten et al., 1964; S p e n c e r , 1965). N e v e r t h e l e s s , the d e g r e e of t r e m o r p r o d u c e d by t r e m o r i n e was influenced by e n v i r o n m e n t a l t e m p e r a t u r e , being b r i e f e r in duration at high t e m p e r a t u r e s (e.g. the chick incubator at 32°C) and m o r e p r o lo n g e d at low t e m p e r a t u r e s . A k i n e s i a was also i n t e n s if i e d in a cold e n v i r o n m e n t . At an e n v i r o n m e n t a l t e m p e r a t u r e of 0°C, t r e m o r p r o d u c e d by t r e m o r i n e was prolonged, but was eventually inhibited as the body t e m p e r a t u r e fell to about 20oc. On r e moving the chicks f r o m the cold e n v i r o n m e n t ,
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t r e m o r r e c o m m e n c e d when the body t e m p e r a t u r e r o s e to 2 2 - 2 5 o c . P r o l o n g e d t r e m o r in a cold e n v i r o n m e n t may be the r e s u l t of s h i v e r i n g s u p e r i m p o s e d on the t r e m o r . In chicks aged 20 days or m o r e , t r e m o r i n e (20 mg/kg) did not p r o d u c e a h y p o t h e r m i c effect. In fact, it often p r o d u c e d a s m a l l and s h o r t l ast i n g r i s e in body t e m p e r a t u r e . Cho, F a i r hurst, H a s l e t t and J e n d e n (1964) r e p o r t e d that o x o t r e m o r i n e , the a c t i v e m e t a b o l i t e of t r e m urine, produced a s m a l l r i s e in body t e m p e r a t u r e in adult cats. When p l a c e d t o g e t h e r in a cage, young chicks tend to a g g r e g a t e and this tendency was inc r e a s e d a f t e r injection of t r e m o r i n e p o s s i b l y as an a t t e m p t to r e d u c e heat loss during the hypot h e r m i c effect of the drug. H a r m i n e in d o s e s of 20 m g / k g a l s o p r o d u c e d a fall in body t e m p e r a t u r e in young chicks, but the effect was s m a l l e r (1-3oc) than that p r o duced by t r e m o r i n e . In c o n t r a s t to the effect of t r e m o r i n e , h a r m i n e antagonised the tendency of chicks to a g g r e g a t e when placed t o g e t h e r in a cage. 3.2.5. P a r a s y m p a t h e t i c e f f e c t s T r e m o r i n e , in d o s e s of 20-30 m g / k g p r o d u c e d i n t en se d i a r r h o e a in chicks aged 3 days or m o r e and this was the p r e d o m i n a n t p a r a s y m p a t h e t i c effect. Salivation was a l s o produced but only in o l d e r chicks (6 days and over) at this dose level. In I or 2-day old chicks, n e i t h e r d i a r r h o e a nor s a l i v a t i o n was produced, p o s s i b l y b e c a u s e of i m m a t u r i t y of the s e c r e t o r y glands at this stage. P a r a s y m p a t h e t i c ef f ect s i n c r e a s e d in intensity with age up to about 8 days a f t e r which they w e r e r e p r o d u c i b l e at a s i m i l a r level. Thus in chicks aged 8 days or m o r e , p a r a s y m p a t h e t i c act i o n s w e r e the p r e d o m i n a n t e f f e c t s of t r e m o r i n e , the b i r d s having b e c o m e r e s i s t a n t to the t r e m o r p r o d u ci n g action of the drug at this time. I s o l a t e d s e g m e n t s of g u i n e a - p i g i l eu m t r e a t e d with dyflos c o n t r a c t e d in r e s p o n s e to 1 n g / m l of t r e m o r i n e . T a c h y p h y l a x i s to this effect d e v e l oped rapidly when s u c c e s s i v e d o ses w e r e added to the p r e p a r a t i o n . L a r g e c o n c e n t r a t i o n s of t r e m o r i n e (1 /zg/ml) p o s s e s s e d a t r o p i n e - l i k e action, s e l e c t i v e l y blocking the r e s p o n s e s of the g u i n e a - p i g i l e u m to a c e t y l c h o l i n e . H a r m i n e did not p r o d u ce d i a r r h o e a in chicks. O c c a s i o n a l l y slight s a l i v a t i o n was p r o d u c e d by d o s e s of 30 m g / k g and a b o v e 3.3. B i o c h e m i c a l c h a n g e s in b r a i n The c o n c e n t r a t i o n s of n o r a d r e n a l i n e , dopamine, t y r a m i n e , 5-HT, a c e t y l c h o l i n e and e r g o -
78
W. C. BOWMAN and G. OSUIDE Table 1 E f f e c t s of t r e m o r i n e (30 m g / k g i n t r a p e r i t o n e a l l y ) on l e v e l s of n a t u r a l l y - o c c u r r i n g s u b s t a n c e s in brain. Substance e s t i m a t e d Noradrenaline * 5-Hydroxytryptamine Acetylcholine * Ergothioneine Tyramine Dopamine
Concentration in control b r a i n s (/lg/g ± S.E.) 0.26 0.34 1.9 0.445 0.326 0.207
± ± ± ± ± ±
0.041 0.032 0.18 0.039 0.08 0.05
Concentration a f t e r t r e m o r i n e (/lg/g ± S.E.) 0.14 0.402 2.56 0.45 0.322 0.20
± ± ± ± ± ±
0.03 0.034 0.21 0.05 0.08 0.05
% change 46% d e c r e a s e 18% i n c r e a s e 35% i n c r e a s e
none none none
* F o r both n o r a d r e n a l i n e and acetylcholine the t value calculated f r o m the d i f f e r e n c e s between the m e a n s is a p p r o x imately 2.3 and with 22 d e g r e e s of f r e e d o m , the value significant at the 5% level is 2.08. This s u g g e s t s that the change for t h e s e two s u b s t a n c e s is significant. Table 2 Effects of h a r m i n e (30 m g / k g intraperitoneally) on levels of n a t u r a l l y - o c c u r r i n g s u b s t a n c e s in brain. Substance e s t i m a t e d Noradrenaline * 5-Hydroxytryptamine Acetylcholine E rgothioneine Tyramine Dopamine
Concentration in control b r a i n s ( # g / g ± S.E.) 0.21 0.32 1.8 0.5 0.34 0.207
± 0.02 i0.035 ± 0.19 ± 0.06 ± 0.03 i 0.011
Concentration after h a r m i n e ( # g / g ± S.E.) 0.28 0.41 2.0 0.51 0.36 0.227
~- 0.024 4- 0.038 ± 0.16 ± 0.05 ± 0.04 ± 0.01"/
% change 33% i n c r e a s e 28% i n c r e a s e none none none none
* The t value for n o r a d r e n a l i n e calculated f r o m the difference between the m e a n s is 2.24 and with 22 d e g r e e s of f r e e d o m , the value significant at the 5% level is 2.08, s u g g e s t i n g that the change for this a m i n e is significant. t h i o n e i n e in w h o l e b r a i n s of c o n t r o l c h i c k s a n d in c h i c k s p r e - t r e a t e d w i t h 30 m g / k g of t r e m o r i n e o r 30 m g / k g h a r m i n e 40 r a i n p r e v i o u s l y a r e p r e s e n t e d in t a b l e s 1 and 2 r e s p e c t i v e l y . Tremorine produced a significant decrease in
the brain level of noradrenaline and a significant increase in the level of acetylcholine. There was also a small increase in the level of 5-HT, but this effect was not significant. No change was detected in the brain levels of dopamine, tyramine or ergothioneine. Others (Friedman, 1963, 1964; Friedman, Aylesworth and Friedman, 1963) have recorded an increase in 5-HT levels and a decrease in noradrenaline levels in mice, and Holmstedt (1964) found an increase in acetylcholine levels in rats. Harmine produced a small but significant increase in the level of noradrenaline, but did not change the levels of acetylcholine, ergothioneine or tyramine. There may also have been a small increase in the levels of 5-HT and dopamine but these changes were not significant at the P=0.95 level. Holzer and Hornykiewicz (1959) noted an increase in brain dopamine levels in rats after injections of harmine.
4. DISCUSSION T h e r e s u l t s s h o w e d t h a t t h e e f f e c t s of t r e m o r i n e a n d h a r m i n e in y o u n g c h i c k s a r e e s s e n t i a l ly s i m i l a r to t h o s e p r o d u c e d in m a m m a l i a n s p e c i e s w h i c h r e s p o n d to the d r u g . T h e a d v a n t a g e s of u s i n g y o u n g c h i c k s f o r s t u d y i n g t r e m o r i n e e f fects include their availability and cheapness, and the ease with which tremor and akinesia may b e d e t e c t e d . O b s e r v a t i o n of t r e m o r i s f a c i l i t a t e d by the feathers which amplify the fine movements of t h e u n d e r l y i n g m u s c l e s a n d s k i n , a n d a k i n e s i a and r i g i d i t y a r e m a d e e s p e c i a l l y o b v i o u s by the fact that the chick is a biped with long legs. The chief d i s a d v a n t a g e in u s i n g c h i c k s i s that they r e s p o n d w e l l to t r e m o r i n e o n l y d u r i n g t h e f i r s t 4 o r 5 d a y s a f t e r h a t c h i n g . It i s p o s s i b l e t h a t t h e f a l l - o f f of r e s p o n s e to t r e m o r i n e w i t h a g e i s a r e f l e c t i o n of t h e g r a d u a l d e v e l o p m e n t of t h e b l o o d - b r a i n b a r r i e r . H o w e v e r , it s e e m s u n l i k e l y that this is the chief factor responsible, as inc r e a s i n g a g e h a d l i t t l e e f f e c t on t h e t r e m o r r e s p o n s e t o h a r m i n e a n d t h e r e i s no e v i d e n c e i n other species that the blood-brain barrier diff e r e n t i a t e s b e t w e e n t h e s e two d r u g s . F u r t h e r m o r e , t r e m o r i n e i s effective in p r o d u c i n g t r e m o r i n a d u l t m i c e , c a t s a n d d o g s a n d t h e r e i s no
TREMORINE AND HARMINE IN THE CHICK e v i d e n c e that the b l o o d - b r a i n b a r r i e r in the adult f o w l i s a n y m o r e r e s i s t a n t to p e n e t r a t i o n by d r u g s t h a n t h a t of o t h e r s p e c i e s . A l t h o u g h n o t d i r e c t l y r e l a t e d , it i s p o s s i b l e t h a t t h e r e s i s t a n c e to t r e m o r i n e w h i c h d e v e l o p s w i t h a g e , i s s i m i l a r to t h e t a c h y p h y l a x i s w h i c h i s p r o d u c e d b y r e p e a t e d d o s a g e . R e p e a t e d a d m i n i s t r a t i o n of s e v e r a l d r u g s i s k n o w n to s t i m u l a t e t h e f o r m a t i o n of l i v e r m i c r o s o m a l e n z y m e s w h i c h d e t o x i f y t h e m ( s e e , f o r e x a m p l e , B r o d i e , 1964). S u c h a n e f f e c t m i g h t e x p l a i n t a c h y p h y l a x i s to t r e m o r i n e , a n d if t h e f o r m a t i o n of t h e s a m e o r s i m i l a r e n z y m e s i s s t i m u l a t e d b y c e r t a i n c o n s t i t u e n t s of d i e t , w h i c h a r e n o t p r e s e n t i n t h e y o l k s a c in o v u m , t h e l e s s p r o n o u n c e d d i m i n u t i o n of r e s p o n s i v e n e s s with age would be a c c o u n t e d for. The interaction between tremorine and harm i n e and o t h e r d r u g s in the c h i c k will be the s u b j e c t of a s e p a r a t e r e p o r t .
ACKNOWLEDGEMENTS T h i s work was supported by a grant made to W.C.B. by the B r i t i s h Egg M a r k e t i n g Board. G.O. is indebted to the I n t e r - U n i v e r s i t y Council for Higher Education O v e r s e a s for a studentship and to the B r i t i s h C o m m o n wealth S c h o l a r s h i p C o m m i s s i o n for a s c h o l a r s h i p d u r ing the t e n u r e s of which this work was undertaken. We a r e grateful to Dr. J. C r o s s l a n d of the University of Nottingham for d e m o n s t r a t i n g to u s the method of e x t r a c t i o n and e s t i m a t i o n of ergothioneine.
REFERENCES A r i e n s - K a p p e r s , C . U . , 1929, The evolution of the n e r v o u s s y s t e m in i n v e r t e b r a t e s , v e r t e b r a t e s and m a n (De E r v e n F. Boh, H a a r l e m ) p. 126. Baker, W.W. and A . J . Triolo, 1960, Additional s t u d i e s on t r e m o r i n e induced involuntary m o v e m e n t s , P h a r m a c o l o g i s t 2, 87. Beani, L. and C. Bianchi, 1963, The e x t r a c t i o n of a c e tylcholine in s m a l l s a m p l e s of c e r e b r a l t i s s u e , J. P h a r m . P h a r m a c . 15, 281. Blaber, L . C . and A.W. Cuthbert, 1961, A s e n s i t i v e method for the a s s a y of acetylcholine, J. P h a r m . P h a r m a c . 13, 445. Bloekus, L . E . and G . M . E v e r e t t , 1957, T r e m o r p r o ducing d r u g 1 , 4 - d i p y r r o l i d i n o - 2 - b u t y n e ( T r e m orine), Fedn. P r o c . 16, 283. Bowman, W.C., B.A. C a l l i n g h a m and G. Osuide, 1964, E f f e c t s of t y r a m i n e on a spinal reflex 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. Brodie, B . B . , 1964, Distribution and fate of d r u g s ; t h e r a p e u t i c i m p l i c a t i o n s . In: Absorption and d i s t r i bution of d r u g s , eds. T . B . Binns and C. Dodds (Livingstone, Edinburgh) p. 199. Biilbring, E., 1955, C o r r e l a t i o n between m e m b r a n e potential, spike d i s c h a r g e and tension in smooth m u s c l e , J. P h y s i o l . (London) 128, 200.
79
C a l l i n g h a m , B . A . and R. C a s s , 1963, A modification of the b u t a n o l e x t r a c t i o n method for the f l u o r i m e t r i c a s s a y of c a t e c h o l a m i n e s in biological m a t e r i a l s , J. P h a r m . P h a r m a e . 15, 699. C a r l s s o n , A., 1959, Detection and a s s a y of dopamine, P h a r m a c . Rev. 11, 300. C a r l s s o n , A. and B. Waldeck, 1958, A f l u o r o m e t r i c method for the d e t e r m i n a t i o n of dopamine (3-hyd r o x y t y r a m i n e ) , Acta Physiol. Scand. 44, 293. C a s s , R. and T . L . B . Spriggs, 1961, T i s s u e a m i n e levels and s y m p a t h e t i c blockade a f t e r guanethidine and b r e t y l i u m , Brit. J. P h a r m a c . C h c m o t h e r . 17, 442. Chen, A . L . and K . K . Chen, 1939, H a r m i n e the alkaloid of Caapi, Quart. J. P h a r m . P h a r m a c o l . 12, 30. Cho, A.K., A.S. F a i r h u r s t , W. L. t l a s l e t t and D. J. J e n den, 1964, The m e t a b o l i s m of t r e m o r i n e , P r o c e e d i n g s of the 2nd International P h a r m a c o l o g i c a l C o n g r e s s , Vol. 2 ( P e r g a m o n P r e s s , Oxford) p. 75. Cho, A.K., W . L . Haslctt and D . J . Jenden, 1961, The identification of an active mctabolite of t r e m o r i n e , Biochem. Biophys. R e s . C o m m u n . 5, 276. C r o s s l a n d , J., J. F. Mitchell and G. N. Woodruff, 1964, Identity of the c e r e b e l l a r factor, Nature (London) 203, 1388. C r o s s l a n d , J., J. F. Mitchell and G. N. Woodruff, 1966, The p r e s e n c e of ergothioneine in the c e n t r a l n e r vous s y s t e m and its probable identity with the c e r e b e l l a r factor, J. Physiol. (London) 182, 427. Desei, L., M. V a r s z e g i and J. Mehes, 1961, T o l e r a n c e to t r e m o r i n e , J. P h a r m . P h a r m a c . 13, 127. E v e r e t t , G.M., 1956, T r e m o r produced by d r u g s , Nature (London) 177, 1238. E v e r e t t , G.M., 1964, P h a r m a c o l o g i c a l s t u d i e s on t r e m o r i n e , P r o c e e d i n g s of the 2nd International Pharmacological Congress, Vol. 2 ( P e r g a m o n P r e s s , Oxford) p. 69. E v e r e t t , G.M., L . E . Blockus and I. M. Shepperd, 1956, T r e m o r i n e induced t r e m o r and its a n t a g o n i s m by a n t i - P a r k i n s o n d r u g s , Science 13, 79. F a r q u h a r s o n , M . E . and R.G. Johnston, 1959, Antagon i s m of the effects of t r e m o r i n e by atropine d e r i v a tives, Brit. J. P h a r m a c . C h e m o t h e r . 14, 559. F r i e d m a n , A.H., 1963, Norepinephrine depletion in the b r a i n s t e m by t r e m o r i n e , Fedn. Proc. 22, 272. F r i e d m a n , A.H., 1964, P r o c e e d i n g s of the 2nd I n t e r national P h a r m a c o l o g i c a l C o n g r e s s , Vol. 2 ( P e r gamon P r e s s , Oxford) p. 130. F r i e d m a n , 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 : Effects 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 , Science 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 logical a s p e c t s of P a r k i n s o n i s m , Advances in P h a r macology 3, e d s . S. Garattini and P. A. Shore (Academic P r e s s , New York) p. 83. F r i e d m a n , A.H. and R . L . RingeI, 1965, S p e c t r o graphic a n a l y s i s of evoked vocalisation a s a method of evaluating d r u g action in v i v o , Fedn. P r o c . 24, 675. Hara, S., 1953, P h a r m a c o l o g i c a l s t u d i e s on the functions of the e x t r a p y r a m i d a l s y s t e m , Japan. J. P h a r m a c . 2, 127. Hara, S. and K. K a w a m o r i , 1954, P h a r m a c o l o g i c a l s t u d i e s on the functions of the e x t r a p y r a m i d a l s y s t e m , Report No. 2, M e c h a n i s m of the a p p e a r a n c e of t r e m o r due to e x t r a p y r a m i d a l poisons, Japan. J. P h a r m a e . 3, 149.
80
W.C. BOWMAN and G. OSUIDE
Holmstedt, B., 1964, P r o c e e d i n g s of the 2nd International P h a r m a c o l o g i c a l C o n g r e s s (Pergamon P r e s s , Oxford) p. 128. Holzer, G. and O. Hornykiewiez, 1959, Ueber den Dopamin (Hydroxytyramin) Stoffwechsel im Gehirn der Ratte, Arch. Exp. Path. Pharmak. 237, 27. Key, B . J . and E . M a r l e y , 1962, The effect of s y m pathomimetie amines on behaviour and e l e e t r o c o r tical activity of chickens, Electron. Neurophysiol. 14, 90. Kocsis, J . J . and R.M. Welch, 1960, Conversion of t r e m o r i n e (1,4-dipyrollidino-2-butyne) to an active metabolite, P h a r m a c o l o g i s t 2, 87. Melville, D.B. and W . H . H o r n e r , 1953, Blood e r g o thioneine in germ free chickens, J. Biol. Chem. 202, 187. Melville, D.B., W.H. Horner and R. Lubsehez, 1954, Tissue ergothioneine, J. Biol. Chem. 206, 221. Pale~ek, F., 1963, A simple r e s p i r a t o r for laboratory use, J. Appl. Physiol. 18, 443. Patten, B.M., A. Sakamoto, M.H. Van Woert, P.S. P a pavasiliou and G. C. Cotzias, 1964, T r e m o r i n e induced t r e m o r v e r s u s extrapyramidal d i s e a s e , Nature (London) 201, 929. Shore, P . A . and J . S . Olin, 1958, Identification and chemical a s s a y of norepinephrine in brain and other t i s s u e s , J. P h a r m a c . Exp. Ther. 122, 295. Spector, S., K. Melmon, W. Lovenberg and A. S j o e r d s ma, 1962, P r e s e n c e of tyramine in the spinal cord and brain, P h a r m a c o l o g i s t 4, 148.
Spector, S., K. Melmon, W. Lovenberg and A. S j o e r d s ma, 1963, The p r e s e n c e and distribution of t y r amine in mammalian t i s s u e s , J. P h a r m a c . Exp. Ther. 140, 229. Spencer, P. S. J:, 1965, Activity of centrally acting and other drugs against t r e m o r and hypothermia induced in mice by t r e m o r i n e , Brit. J. P h a r m a c . Chemother. 25, 442. Stern, P., N. Radovic and S. Buljubasic, 1965, P h a r macolog~¢ of experimental t r e m o r , Nature {London} 206, 1261. Udenfriend, S., H. Weissbach and C. T. Clark, 1955, The estimation of 5-hydroxytryptamine (serotonin} in biological t i s s u e s , J. Biol. Chem. 215, 337. V e r n i e r , U.G., 1964, Anti-Parkinson agents: Evaluation of drug activities: P h a r m a c o m e t r i c s , eds. D.R. Laurence and A . L . Bacharach {Academic P r e s s , New York and London} p. 301. Waelsch, H., 1955, The turnover of components of the developing brain; the blood brain b a r r i e r , Biochemi s t r y of the developing nervous s y s t e m {Academic P r e s s , New York} p. 187. Zaimis, E., 1960a, Ciba Foundation Symposium on Adrenergic Mechanisms {Churchill, London} p. 562. Zaimis, E., 1960b, Simultaneous recording of activity and rate of chirping in the young chick, J. Physiol. {London} 152, l l P . Zetler, G., 1957, Der H a r m i n - T r e m o r und seine Antagonisten, Arch. Exp. Path. Pharmak. 231, 34.