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Pharmacological profile of a model for central serotonin receptor activation
Life Sciences, Vol. 26, pp. 1397-1403 Printed in the U.S.A.
Pergamon Press
PHARMACOLOGICAL PROFILE OF A MODEL FOR CENTRAL SEROTONIN RECEPTORACTIVATION William D. Matthews and Cynthia D. Smith Department of Biological Research Smith Kline and French Laboratories Philadelphia, Pa. 19101, U.S.A. (Received in final form February 22, 1980)
Summary The head shake response in rats after systemic administration of the serotonin (5HT) precursor 5-hydroxytryptophan (5HTP) was pharmacologically characterized and shown to be a useful animal model to quantify brain 5HT receptor activation. The behavior occurred in a dose-dependent manner after injection of 5HTP and the 5HT agonist quipazine. Head shakes were also observed after injection of L-tryptophan, 5-methoxydimethyltryptamine and fenfluramine. The 5HT antagonists cyproheptadine and metergoline were potent blockers of the response. Xylamidine, a peripheral 5HT antagonist, had no effect on head shaking. Inhibition of 5HT uptake with fluoxetine potentiated the head shake response after 5HTP. Manipulation of central chclinergic or GA~Aergic mechanisms did not a l t e r 5HTP-induced shakes. Alpha-noradrenergic receptor blockade had no s i g n i f i c a n t effect on head shakes. However, desmethylimipramine was equipotent with methyser(lide as an antagonist of the behavior. Beta-noradrenergic receptor blockade had no specific effect on 5HTP head shakes. Concomitant dopamine receptor activation with SK&F 38393 did not affect head shakes but the neuroleptics chlorpromazine and pimozide reduced the number of head shakes after 5HTP. The Hl receptor antagonist pyrilamine had no effect on head shakes. I t is concluded that 5HTP-induced head shakes in rats is a quantitative model of brain 5HT receptor activation which is p a r t i c u l a r l y sensitive to 5HT antagonists. Systemic administration of serotonin (5HT) precursors or agonists leads to pharmacological activation of 5HT receptors in the rodent central nervous system (1). Several stereotyped behaviors have been described following central 5HT receptor stimulation includir/l a complex motor syndrome in rats (2,3,4), myoclonus in guinea piqs (5) and shaking behavior in mice and rats (6,7). Because of the postulated role of 5HT neurotransmission in mood, sleep and motor a c t i v i t y (g) interest in an intact animal model which allows qu'antitative assessment of central serotonin a c t i v i t y has increased. The shaking response is the most easily quantified of these behaviors and has been described as head twitches in mice (6) and wet dog shakes in the rat (7). Preliminary experiments in our laboratory revealed that rats displayed vigorous shakin~ of the head. when treated with .~-hydroxytryptophan (5HTP) in the presence of an i n h i b i t o r of peripheral l-aromatic amino acid decarboxylase. This report describes the pharmacological characterizaticn cf the 5HTP-induced head shake in the rat. The results of our studies dem,onstrate the s e n s i t i v i t y oo24-32o5/8o/171397-o7so2.oo/o Copyright
(c) 1980 Pergamon Press Ltd
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of the response to i n t e r r u p t i o n or a c t i v a t i o n of 5HT neurotransmission and establish 5-HTP-induced head shakes as a r e l i a b l e q u a n t i t a t i v e ~odel for study of brain 5HT receptors in the whole animal. Methods Animal s F1ale Sprague-Dawley rats (250-330 g) were used in all experiments. Animals were housed in constant temperature, humidity and l i g h t cycles. Food and vlater were available ad libitum. Head Shake Behavior Compounds which induced head shakes were qiven i . p . or s.c. in a dose volume cf 2 ml/kg. Animals were observed for two hours a f t e r i n j e c t i o n and the total number of head shakes was counted. Rats were injected s.c. with doses of 5HTP (25-200 mg/kg, 4 ml/kg) 30 minutes a f t e r pretreatment with s-methyl DOPA hydrazine (carbidopa, 25 mg/kg i . p . ) . The number of head shakes of each animal was counted for two hours. The greatest frequency of shakes occurred 90-120 minutes a f t e r 5HTP i n j e c t i o n . A head shake is a rapid twitch of the head similar to a pinna r e f l e x . Usin~ carbidopa plus 200 ~g/kg 5HTP to e l i c i t a standard response, the e f f e c t on head shakes of drugs of d i f f e r e n t pharmacological classes was investigated. Ninety minutes a f t e r 5HTP administration the number of head shakes in a 30minute interval (90-120 minutes post 5HTP) was counted for each rat. Compounds tested were given i . p . at a suitable time p r i o r to the counting i n t e r val. Control animals received s t e r i l e water i n j e c t i o n s . The amine concentration of perchloric acid extracts of whole rat brain (less cerebellum) was determined by a modification of the procedure described by Allenmark and Hed~an (9). Statistical
Analysis
The mean head shakes of animals in each dose group (n = 5-10) of test compound was compared to the mean number of head shakes in the control group by a 2t a i l e d Student's t t e s t . S i g n i f i c a n t deviation from control had a p r o b a b i l i t y less than 0.05. The ED75 (dose of drug necessary to i n h i b i t 75% of the mean number of head shakes of the control group) with 95% confidence l i m i t s was determined by a log I0 transformation regression l i n e analysis ( I 0 ) . Dru9s Carbidopa (Merck, Sharp and Dohme), 5-hydroxy-DL-tryptophan (Sigma), cyproheptadine (Merck, Sharp and Dohme), quipazine, 5-methoxy-N, N-dimethyltryptamine (Sigma) and L-tryptophan (Sigma) were all dissolved in d i l u t e HCI. Methysergide (Sandoz), l-phenyl-7,8-dihydroxy-2,3,4,5-tetrahydro-IH-3-benzazepine (SK&F 38393), phenoxybenzamine, scopolamine (K+K Labs), 2-(2,6-dimethoxyphenoxyethyl) amino methyl-l,4-benzodioxane (WB4101, Ward Blinkinsop L t d . ) , chlorpromazine, viloxazine ( S t u a r t ) , t o l a z o l i n e , fenfluramine, muscimol, l proprano!ol, desmethylimipramine, f l u o x e t i n e ( L i l l y ) and pyrilamine (Merck, Sharp and Dohme) were all dissolved in s t e r i l e water. Yohimbine (Penick), metergoline (Farmitalia) and pimozide (McNeil) were administered in d i l u t e t a r t a r i c acid. Xylamidine (Burroughs Wellcome) was dissolved in d i l u t e dimethylformamide. All doses are expressed as the base and administered in a volume of 2 ml/kg.
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Resul ts 5HTP-Induced Head Shakes Systemic administration of carbidopa (25 mg/kg) plus 5HTP (25-200 mg/kg) produced an increasing number of head shakes in a dose-related manner. The behavior was c h a r a c t e r i s t i c a l l y a rapid head movement with l i t t l e or no body involvement. The shake behavior resembles the pinna r e f l e x evoked by t a c t i l e stimulation of the auditory canal in rodents. Preliminary experiments revealed that 200 mg/kg 5HTP produced the maximum number of head shakes with no other stereotyped behavior. Moreover the shake frequency was greatest in the 30-minute i n t e r v a l from 90 to 120 minutes post-5HTP. Thus, the standard of 200 mg/kg 5HTP and a 30-minute i n t e r v a l (90-120 minutes post-5HTP) was chosen f o r a l l antagonism experiments. A large population of animals so treated has been accumulated. The mean ± SE of this control population is 51 ± 1 shakes/ 30 minutes (n = 407). The 200 mg/kg dose of 5HTP produced a greater than t h r e e - f o l d increase in 5HT concentration in whole brain measured 90 minutes a f t e r 5HTP i n j e c t i o n (Table I ) , while dopamine and norepinephrine l e v e l s decreased by 26% and 32% r e s p e c t i v e l y . TABLE I
Treatment
Amine Concentration (ng/g whole brain) Serotonin
Dopamine Norepinephrine
H20
n = 6
280 ± 13
526 ± 27
235 ± 26
Carbidopa (25 mg/kg)
n = 6
300 ± 94
529 ± 52
247 ± 33
392 ± 138"
169 ± 55*
Carbidopa + 5HTP (200 mg/kg) n = 6
1018 ± 44*
Table values are mean ± SD * p < 0.05 by Student's ~ t e s t vs. Carbidopa alone Effects of L-Tryptophan and 5HT Agonists Quipazine produced head shakes at doses of I - I 0 mg/kg. The maximum frequency of shakes was observed at 5 mg/kg (~ = 26 ± 2 (SE)/30 min, n = 8). L-Tryptophan (200 mg/kg) plus pargyline (25 mg/kg, 30-min pretreatment) also caused head shakes (15 ± 5/60 min, n = 3). Fenfluramine was tested at 5, I0 and 20 mg/kg. The maximum shaking response occurred at I0 mq/kg (9 ± 3/60 min, n = 3); higher doses of fenfluramine evoked the stereotyped motor syndrome (3). 5-Methoxydimethyltryptamine (5-HeODMT) produced some head shakes (5 ± 3/30 min, n = 4) at 0.5 mn/kq. In general, the putative serotonin agonists were not as potent as 5HTP in producinn head shakes because high doses of the agonists caused a behavioral syndrome which seemed to preclude the head shake response.
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A~ents A f f e c t i n 9 Cholinergic and Dopaminer~cic Mechanisms Scopolamine did not a f f e c t 5HTP-induced head shakes at any dose tested. Chlorpromazine (ED7s = l . l mg/ko) and pimozide (ED7s = 1.5 mq/kg) depressed head shakes in a dose-related manner. The novel dopamine agonist SK&F 38393 had no e f f e c t on the shaking behavior. Effects of 5HT Antagonists Methysergide (ED7s = 2.2 mg/kg) was the weakest of the c l a s s i c a l indoleamine antagonists tested against head shakes (Table I I ) . Cyproheptadine (ED7s = 0.15 mg/kg) and metergoline (ED7s = 0.07 mg/kg) were potent i n h i b i t o r s of 5HTP-induced head shakes. The antagonist of peripheral 5HT responses, x y l a midine, was i n e f f e c t i v e at lO mn/kg at blocking the behavior. I n h i b i t o r s of Amine Uptake Pretreatment with f l u o x e t i n e (lO mg/kg) markedly increased the number of head shakes observed a f t e r a submaximal dose of 511TP (IgO mg/kg). Control animals e x h i b i t e d 18 ± 3 shakes/30 min, n = lO, whereas 35 + 7 shakes/30 min, n = lO, were counted in the f l u o x e t i n e - t r e a t e d group. In contrast desmethylimipramine caused dose-related decreases in 5HTP head shakes (ED7s = 1.8 mg/kg). V i l o x azine also reduced head shakes by 42% at 20 mg/kg. GABA Agonist and Histarline Antagonist The GABA agonist muscimol had no s i g n i f i c a n t action on head shakes at doses between 0.I and 4 mg/kg. Pyrilamine, an HI antagonist, f a i l e d to i n h i b i t head shakes at any dose tested. TABLE I I Compounds I n h i b i t i n g 511TP-Induced Head Twitch Pretreatment Time (Min)
ED7s (mg/kg)
Metergoline
60
0.07 (0.02 - 0.27)*
Cyproheptadine
90
0.15 (0.09 - 0.30)
Chlorpromazine
60
l.l
Compound
Pimozide
(0.2-
If.9)
120
1.5 (0.4 - I f . 3 )
Desmethylimipramine
60
1.8 (0.2 - 34.7)
Methysergide
20
2.2 (0.8 - 7.0)
l-Propranolol
40
24.0 (12.1 - 57.8)
* 95% Confidence l i m i t s
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TABLE I I I Compounds Having L i t t l e or No Effect on 5HTP-Induced Head Twitch
Compound WB4101
Pretreatment Time (Min)
Maximum Dose Given (mg/kg)
Effect on Head Twitch
60
1,0
150
16.0
+51% *
Tolazoline
30
I0.0
N.A.
Yohimbine
30
5.0
+56% *
Scopolamine
30
2.0
N.A.
SK&F 38393
30
25.0
N.A.
Xylamidine
60
I0.0
N.A.
Fluoxetine
150
I0.0
÷100% *
Viloxazine
60
20.0
+42% *
Muscimol
30
4.0
N.A.
Pyrilamine
30
I0.0
N.A.
Phenoxybenzamine
N.A.
N.A. - not active * - s i g n i f i c a n t (p < 0.05) change from control DISCUSSION The results of the experiments described in t h i s report demonstrate a r e l a tionship between head shakin~ in rats and stimulation of brain 5HT receptors. Systemic administration of the serotonin precursor 5HTP, with peripheral decarboxylase blockade, c o n s i s t e n t l y induced head shakes in a dose-dependent manner. The 5HT agonist quipazine ( I I ) also produced dose-related head shakes and the response was induced by another 5HT agonist 5-methoxydimethyltryptamine (2), the 5HT precursor L-tryptophan and fenfluramine, a putative 5HT releasing agent (12). During the period of maximum shaking behavior brain 5HT concentration was elevated more than t h r e e - f o l d while dopamine and norepinephrine concentrations were reduced. The large increase in brain 5HT ref l e c t s the uptake and subsequent decarboxylation of 5HTP to 5HT w i t h i n neurons. Chan~es in the fluorescence pattern of central dopaminergic and noradrenergic neurons i n d i c a t i v e of catecholamine loss have been reported a f t e r peripheral decarboxylase i n h i b i t i o n and 5HTP administration (13). 5-Hydroxytryptophan also enhances the release of 3H-dopamine from s l i c e s of r a t brain (14). Moreover, reductions in brain catecholamine concentrations occurred in rats which exhibited the complex motor syndrome a f t e r i n j e c t i o n of 5HTP alone (4). Taken together, these observations suggest that a portion of exogenously administered 5HTP may enter catecholaminerqic terminals and undergo decarboxyl a t i o n to 5HT with r e s u l t a n t displacement of the endogenous catecholamine. The head shake behavior, however, appears dependent upon the a c t i v a t i o n of 5HT receptors since pi~ozide or phenoxybenzamine only attenuated the response at
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Head Shakes
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r e l a t i v e l y high doses while metergoline and cyproheptadine were potent antagonists of head shaking. Our observations confirm the appearance of shaking behavior described in mice (6) and rats (7) after 5HT precursors or agonists. However, several differences do exist between the head shake response observed in this study and wet dog shakes (7) previously reported in rats. The head shake consists of a rapid movement or twitch of the head with l i t t l e involvement of the trunk musculature. The head shake resembles a pinna r e f l e x and appears the same behavior as described by Corne et al (6) in mice. The present experiments are the f i r s t comprehensive pharmacological characterization of the head shake response in the rat.
5HTP-induced head shaking is a very sensitive model for detection of 5HT antagonists. Metergoline and cyproheptadine, well-known 5HT antagonists, are e f f e c t i v e i n h i b i t o r s of head shakes in the ug/kg range when given i n t r a p e r i toneally (Table I I ) . Methysergide also blocked head shake behavior but was 15 times less potent than cyproheptadine. Xylamidine, an i n h i b i t o r of peripheral 5|IT responses (15), did not antagonize 5HTP-induced head shakes suggesting shakes are due to central 5HT receptor a c t i v a t i o n . Blockade of alpha-adrenergic receptors with a variety of m~ and ~e antagonists produced no potent i n h i b i t i o n of head shakes. Phenoxybenzamine antagonized the behavior but the doses necessary evoked non-specific side e f f e c t s . Likewise, l - p r o p r a n o l o l , which is reported to displace 3H-5HT from binding sites (16) and antagonize head twitches in mice (17), i n h i b i t e d 5HTP-induced head shakes in rats only at r e l a t i v e l y high doses. Further evidence for mediation of the head twitch response by 5HT comes from the present results with f l u o x e t i n e , a highly selective i n h i b i t o r of 5HT uptake in vivo (18). A marked potentiation of head twitches evoked by submaximal doses of 5HTP was observed in animals treated with f l u o x e t i n e (Table I l l ) . The a b i l i t y of f l u o x e t i n e to enhance head twitches produced by 5HTP strongly suggests the behavior is mediated by activation of 5HT receptors. Of the other amine uptake blockers studied only desmethylimipramine (DMI) prevented head shakes at low doses. Recent experiments have revealed a central 5HT antagonist a c t i v i t y of other t r i c y c l i c antidepressants as well (19,20). The a b i l i t y of DMI to antagonize 5HTP-induced head shakes may also suggest a modulation of this 5HT behavior by noradrenergic mechanisms. Cholinergic blockade with scopolamine and activation of dopamine receptors with SK&F 38393 (21) did not a l t e r head shake behavior. The neuroleptics chlorpromazine and pimozide reduced the head shake response. This may be a r e s u l t of antiserotonin properties of tile neuroleptics which have recently come to l i g h t (22,23). In this behavioral model these neuroleptics were j u s t as potent as methyserqide at antagonizing 5HT-induced behavior. GABAergic mechanisms do not appear to modulate the 5HTP head shakes since muscimol had l i t t l e e f f e c t on shaking behavior. Furthermore, Hl receptors are also not involved for pyrilamine had no e f f e c t on 5HTP-induced head shake. The head shake model has several d i s t i n c t advantages over other 5HT-related behaviors. Use of the i n t a c t animal eliminates the surgery necessary in the f l e x o r r e f l e x of the spinal rat model (24). The head shake response is readily q u a n t i f i a b l e without recording equipment and a graded response can be used to assess data. Horeover, the head shake model is extremely sensitive for detection of 5HT antagonists. The 5HT motor syndromes in guinea pig (5) and rat (3) allow only quantal responses to be studied and require much higher doses of 5HT antagonists to block these behaviors.
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In summary, systemic administration of the serotonin precursor, 5HTP, in the presence of an i n h i b i t o r of peripheral L-aromatic amino acid decarboxylase, produces vigorous head shaking behavior in the rat. The head shake response is evoked or inhibited by a~ents which activate or interrupt 5HT neurotrans~ission, respectively, and potentiated by blockade of 5HT uptake mechanisms. The head shake behavior is a reliable quantitative model for study of brain 5HT receptors and is especially useful for evaluating 5HT antagonist properties of compounds. ACKNOWLEDGE~.IENT The authors g r a t e f u l l y acknowledge I~r. Elwood Stack for neurochemical of amine concentrations.
analysis
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 18. 17. 18. 19. 20. 21. 22. 23. 24.
D. G. GRAIIA;~E-SHITH, J. Neurochem. 18:1053-1066 (1971) D. G. GRAHAr,~,E-SHITH, Br. J. PharmacoT. t~3:856-864 (1971) B. L. JACOBS, Life Sci. 19:777-786 (1976-) R. S. SLOVlTER, E. G. DR~T and J. C. CONNOR, J. Pharmacol. Exp. Ther. 206:339-347 (1978) W. WEINER, C. GOETZ, P. NAUSIEDA and H. KLAWANS, Eur. J. Pharmacol. 46: 21-24 (1977) S. CORNE, R. PICKERING and B. WARNER, Br. J. Pharmacol. 20:106-120 (1963) P. BEDARD and C. J. PYCOCK, Neuropharmacology 16:663-670 (1977) A. R. GREENand D. G. GP~HAME-SHITH, Handbook ~ Psychopharmacology Vol. 3, pp 169-245, Plenum Press, New York (1975) S. ALLENMARKand L. HEDI,IAN, J. Liquid Chromatography 2:277-286 (1979) D. J. FINNEY, S t a t i s t i c a l Hethod in Biological Assay,-Hafner Publishing Company (1952) E. HONG, L. SANClLIO, R. VARGAS and E. PARDO, Eur. J. Pharmacol. 6: 274280 (1969) J. DUHAULT and C. VERDAVAINNE, Arch. Int. Pharmacodyn. 170:276-286 (1967) K. FUXE, L. L. BUTCHERand J. ENGEL, J. Pharm. Pharmac. 23': 420-424 (1971) L. K. Y. NG, T. N. CHASE, R. W. COLBURNand I. J. KOPIN, B---rain Res. 45: 499-505 (1972) C. MAWSONand H. WHITTINGTO,N, Br. J. Pharmacol. 39: 223P (1970) D. MIDDLEMISS, L. BLAKEBOROUGHand S. LEATHER, N~ure 267:289-290 (1979) M. WEINSTOCK, C. WEISS and S. GITTER, Neuropharmacology 16:273-276 (1977) R. W. FULLER and K. W. PERRY, J. Pharm. Pharmacol. 2 9 : 7 ~ - 7 1 1 (1977) K. FUXE, S. O. OGREN, L. AGNATI, J. K. GUSTAFSSONa ~ G. JONSSON, Neurosci. Letters 6:339-344 (1977) J. MAJ, A. LEWANDO~TSKAand A. RAWTON, Pharmakopsychiat. 12:281-285 (1979) P. E. SETLER, H. M. SARAU, C. L. ZIRKLE and H. L. SAUNDE~, Eur. J. Pharmacol. 50:419-430 (1978) J. LEYSEN, C__ NIEMEGEERS, J. TOLLENAERE and P. LADURON, Nature 272: 168171 (1978) J. MAJ, L. BARAN, I. BIGAJSKS, Z. ROGOZand G. SKUZA, Pol. J. Pharmacol. 30:431-440 (1978) J. MAJ, W. PALIDER and L. BARAN, J. Neural Transmission 38:131-147 (1976)
Pergamon Press
PHARMACOLOGICAL PROFILE OF A MODEL FOR CENTRAL SEROTONIN RECEPTORACTIVATION William D. Matthews and Cynthia D. Smith Department of Biological Research Smith Kline and French Laboratories Philadelphia, Pa. 19101, U.S.A. (Received in final form February 22, 1980)
Summary The head shake response in rats after systemic administration of the serotonin (5HT) precursor 5-hydroxytryptophan (5HTP) was pharmacologically characterized and shown to be a useful animal model to quantify brain 5HT receptor activation. The behavior occurred in a dose-dependent manner after injection of 5HTP and the 5HT agonist quipazine. Head shakes were also observed after injection of L-tryptophan, 5-methoxydimethyltryptamine and fenfluramine. The 5HT antagonists cyproheptadine and metergoline were potent blockers of the response. Xylamidine, a peripheral 5HT antagonist, had no effect on head shaking. Inhibition of 5HT uptake with fluoxetine potentiated the head shake response after 5HTP. Manipulation of central chclinergic or GA~Aergic mechanisms did not a l t e r 5HTP-induced shakes. Alpha-noradrenergic receptor blockade had no s i g n i f i c a n t effect on head shakes. However, desmethylimipramine was equipotent with methyser(lide as an antagonist of the behavior. Beta-noradrenergic receptor blockade had no specific effect on 5HTP head shakes. Concomitant dopamine receptor activation with SK&F 38393 did not affect head shakes but the neuroleptics chlorpromazine and pimozide reduced the number of head shakes after 5HTP. The Hl receptor antagonist pyrilamine had no effect on head shakes. I t is concluded that 5HTP-induced head shakes in rats is a quantitative model of brain 5HT receptor activation which is p a r t i c u l a r l y sensitive to 5HT antagonists. Systemic administration of serotonin (5HT) precursors or agonists leads to pharmacological activation of 5HT receptors in the rodent central nervous system (1). Several stereotyped behaviors have been described following central 5HT receptor stimulation includir/l a complex motor syndrome in rats (2,3,4), myoclonus in guinea piqs (5) and shaking behavior in mice and rats (6,7). Because of the postulated role of 5HT neurotransmission in mood, sleep and motor a c t i v i t y (g) interest in an intact animal model which allows qu'antitative assessment of central serotonin a c t i v i t y has increased. The shaking response is the most easily quantified of these behaviors and has been described as head twitches in mice (6) and wet dog shakes in the rat (7). Preliminary experiments in our laboratory revealed that rats displayed vigorous shakin~ of the head. when treated with .~-hydroxytryptophan (5HTP) in the presence of an i n h i b i t o r of peripheral l-aromatic amino acid decarboxylase. This report describes the pharmacological characterizaticn cf the 5HTP-induced head shake in the rat. The results of our studies dem,onstrate the s e n s i t i v i t y oo24-32o5/8o/171397-o7so2.oo/o Copyright
(c) 1980 Pergamon Press Ltd
1398
5-Hydroxy-DL-tryptophan-lnduced Head Shakes
Vol. 26, No. 17, 1980
of the response to i n t e r r u p t i o n or a c t i v a t i o n of 5HT neurotransmission and establish 5-HTP-induced head shakes as a r e l i a b l e q u a n t i t a t i v e ~odel for study of brain 5HT receptors in the whole animal. Methods Animal s F1ale Sprague-Dawley rats (250-330 g) were used in all experiments. Animals were housed in constant temperature, humidity and l i g h t cycles. Food and vlater were available ad libitum. Head Shake Behavior Compounds which induced head shakes were qiven i . p . or s.c. in a dose volume cf 2 ml/kg. Animals were observed for two hours a f t e r i n j e c t i o n and the total number of head shakes was counted. Rats were injected s.c. with doses of 5HTP (25-200 mg/kg, 4 ml/kg) 30 minutes a f t e r pretreatment with s-methyl DOPA hydrazine (carbidopa, 25 mg/kg i . p . ) . The number of head shakes of each animal was counted for two hours. The greatest frequency of shakes occurred 90-120 minutes a f t e r 5HTP i n j e c t i o n . A head shake is a rapid twitch of the head similar to a pinna r e f l e x . Usin~ carbidopa plus 200 ~g/kg 5HTP to e l i c i t a standard response, the e f f e c t on head shakes of drugs of d i f f e r e n t pharmacological classes was investigated. Ninety minutes a f t e r 5HTP administration the number of head shakes in a 30minute interval (90-120 minutes post 5HTP) was counted for each rat. Compounds tested were given i . p . at a suitable time p r i o r to the counting i n t e r val. Control animals received s t e r i l e water i n j e c t i o n s . The amine concentration of perchloric acid extracts of whole rat brain (less cerebellum) was determined by a modification of the procedure described by Allenmark and Hed~an (9). Statistical
Analysis
The mean head shakes of animals in each dose group (n = 5-10) of test compound was compared to the mean number of head shakes in the control group by a 2t a i l e d Student's t t e s t . S i g n i f i c a n t deviation from control had a p r o b a b i l i t y less than 0.05. The ED75 (dose of drug necessary to i n h i b i t 75% of the mean number of head shakes of the control group) with 95% confidence l i m i t s was determined by a log I0 transformation regression l i n e analysis ( I 0 ) . Dru9s Carbidopa (Merck, Sharp and Dohme), 5-hydroxy-DL-tryptophan (Sigma), cyproheptadine (Merck, Sharp and Dohme), quipazine, 5-methoxy-N, N-dimethyltryptamine (Sigma) and L-tryptophan (Sigma) were all dissolved in d i l u t e HCI. Methysergide (Sandoz), l-phenyl-7,8-dihydroxy-2,3,4,5-tetrahydro-IH-3-benzazepine (SK&F 38393), phenoxybenzamine, scopolamine (K+K Labs), 2-(2,6-dimethoxyphenoxyethyl) amino methyl-l,4-benzodioxane (WB4101, Ward Blinkinsop L t d . ) , chlorpromazine, viloxazine ( S t u a r t ) , t o l a z o l i n e , fenfluramine, muscimol, l proprano!ol, desmethylimipramine, f l u o x e t i n e ( L i l l y ) and pyrilamine (Merck, Sharp and Dohme) were all dissolved in s t e r i l e water. Yohimbine (Penick), metergoline (Farmitalia) and pimozide (McNeil) were administered in d i l u t e t a r t a r i c acid. Xylamidine (Burroughs Wellcome) was dissolved in d i l u t e dimethylformamide. All doses are expressed as the base and administered in a volume of 2 ml/kg.
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Resul ts 5HTP-Induced Head Shakes Systemic administration of carbidopa (25 mg/kg) plus 5HTP (25-200 mg/kg) produced an increasing number of head shakes in a dose-related manner. The behavior was c h a r a c t e r i s t i c a l l y a rapid head movement with l i t t l e or no body involvement. The shake behavior resembles the pinna r e f l e x evoked by t a c t i l e stimulation of the auditory canal in rodents. Preliminary experiments revealed that 200 mg/kg 5HTP produced the maximum number of head shakes with no other stereotyped behavior. Moreover the shake frequency was greatest in the 30-minute i n t e r v a l from 90 to 120 minutes post-5HTP. Thus, the standard of 200 mg/kg 5HTP and a 30-minute i n t e r v a l (90-120 minutes post-5HTP) was chosen f o r a l l antagonism experiments. A large population of animals so treated has been accumulated. The mean ± SE of this control population is 51 ± 1 shakes/ 30 minutes (n = 407). The 200 mg/kg dose of 5HTP produced a greater than t h r e e - f o l d increase in 5HT concentration in whole brain measured 90 minutes a f t e r 5HTP i n j e c t i o n (Table I ) , while dopamine and norepinephrine l e v e l s decreased by 26% and 32% r e s p e c t i v e l y . TABLE I
Treatment
Amine Concentration (ng/g whole brain) Serotonin
Dopamine Norepinephrine
H20
n = 6
280 ± 13
526 ± 27
235 ± 26
Carbidopa (25 mg/kg)
n = 6
300 ± 94
529 ± 52
247 ± 33
392 ± 138"
169 ± 55*
Carbidopa + 5HTP (200 mg/kg) n = 6
1018 ± 44*
Table values are mean ± SD * p < 0.05 by Student's ~ t e s t vs. Carbidopa alone Effects of L-Tryptophan and 5HT Agonists Quipazine produced head shakes at doses of I - I 0 mg/kg. The maximum frequency of shakes was observed at 5 mg/kg (~ = 26 ± 2 (SE)/30 min, n = 8). L-Tryptophan (200 mg/kg) plus pargyline (25 mg/kg, 30-min pretreatment) also caused head shakes (15 ± 5/60 min, n = 3). Fenfluramine was tested at 5, I0 and 20 mg/kg. The maximum shaking response occurred at I0 mq/kg (9 ± 3/60 min, n = 3); higher doses of fenfluramine evoked the stereotyped motor syndrome (3). 5-Methoxydimethyltryptamine (5-HeODMT) produced some head shakes (5 ± 3/30 min, n = 4) at 0.5 mn/kq. In general, the putative serotonin agonists were not as potent as 5HTP in producinn head shakes because high doses of the agonists caused a behavioral syndrome which seemed to preclude the head shake response.
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5-Hydroxy-DL-tryptophan-lnduced Head Shakes
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A~ents A f f e c t i n 9 Cholinergic and Dopaminer~cic Mechanisms Scopolamine did not a f f e c t 5HTP-induced head shakes at any dose tested. Chlorpromazine (ED7s = l . l mg/ko) and pimozide (ED7s = 1.5 mq/kg) depressed head shakes in a dose-related manner. The novel dopamine agonist SK&F 38393 had no e f f e c t on the shaking behavior. Effects of 5HT Antagonists Methysergide (ED7s = 2.2 mg/kg) was the weakest of the c l a s s i c a l indoleamine antagonists tested against head shakes (Table I I ) . Cyproheptadine (ED7s = 0.15 mg/kg) and metergoline (ED7s = 0.07 mg/kg) were potent i n h i b i t o r s of 5HTP-induced head shakes. The antagonist of peripheral 5HT responses, x y l a midine, was i n e f f e c t i v e at lO mn/kg at blocking the behavior. I n h i b i t o r s of Amine Uptake Pretreatment with f l u o x e t i n e (lO mg/kg) markedly increased the number of head shakes observed a f t e r a submaximal dose of 511TP (IgO mg/kg). Control animals e x h i b i t e d 18 ± 3 shakes/30 min, n = lO, whereas 35 + 7 shakes/30 min, n = lO, were counted in the f l u o x e t i n e - t r e a t e d group. In contrast desmethylimipramine caused dose-related decreases in 5HTP head shakes (ED7s = 1.8 mg/kg). V i l o x azine also reduced head shakes by 42% at 20 mg/kg. GABA Agonist and Histarline Antagonist The GABA agonist muscimol had no s i g n i f i c a n t action on head shakes at doses between 0.I and 4 mg/kg. Pyrilamine, an HI antagonist, f a i l e d to i n h i b i t head shakes at any dose tested. TABLE I I Compounds I n h i b i t i n g 511TP-Induced Head Twitch Pretreatment Time (Min)
ED7s (mg/kg)
Metergoline
60
0.07 (0.02 - 0.27)*
Cyproheptadine
90
0.15 (0.09 - 0.30)
Chlorpromazine
60
l.l
Compound
Pimozide
(0.2-
If.9)
120
1.5 (0.4 - I f . 3 )
Desmethylimipramine
60
1.8 (0.2 - 34.7)
Methysergide
20
2.2 (0.8 - 7.0)
l-Propranolol
40
24.0 (12.1 - 57.8)
* 95% Confidence l i m i t s
Vol. 26, No. 17, 1980
5-Hydroxy-DL-tryptophan-lnduced Head Shakes
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TABLE I I I Compounds Having L i t t l e or No Effect on 5HTP-Induced Head Twitch
Compound WB4101
Pretreatment Time (Min)
Maximum Dose Given (mg/kg)
Effect on Head Twitch
60
1,0
150
16.0
+51% *
Tolazoline
30
I0.0
N.A.
Yohimbine
30
5.0
+56% *
Scopolamine
30
2.0
N.A.
SK&F 38393
30
25.0
N.A.
Xylamidine
60
I0.0
N.A.
Fluoxetine
150
I0.0
÷100% *
Viloxazine
60
20.0
+42% *
Muscimol
30
4.0
N.A.
Pyrilamine
30
I0.0
N.A.
Phenoxybenzamine
N.A.
N.A. - not active * - s i g n i f i c a n t (p < 0.05) change from control DISCUSSION The results of the experiments described in t h i s report demonstrate a r e l a tionship between head shakin~ in rats and stimulation of brain 5HT receptors. Systemic administration of the serotonin precursor 5HTP, with peripheral decarboxylase blockade, c o n s i s t e n t l y induced head shakes in a dose-dependent manner. The 5HT agonist quipazine ( I I ) also produced dose-related head shakes and the response was induced by another 5HT agonist 5-methoxydimethyltryptamine (2), the 5HT precursor L-tryptophan and fenfluramine, a putative 5HT releasing agent (12). During the period of maximum shaking behavior brain 5HT concentration was elevated more than t h r e e - f o l d while dopamine and norepinephrine concentrations were reduced. The large increase in brain 5HT ref l e c t s the uptake and subsequent decarboxylation of 5HTP to 5HT w i t h i n neurons. Chan~es in the fluorescence pattern of central dopaminergic and noradrenergic neurons i n d i c a t i v e of catecholamine loss have been reported a f t e r peripheral decarboxylase i n h i b i t i o n and 5HTP administration (13). 5-Hydroxytryptophan also enhances the release of 3H-dopamine from s l i c e s of r a t brain (14). Moreover, reductions in brain catecholamine concentrations occurred in rats which exhibited the complex motor syndrome a f t e r i n j e c t i o n of 5HTP alone (4). Taken together, these observations suggest that a portion of exogenously administered 5HTP may enter catecholaminerqic terminals and undergo decarboxyl a t i o n to 5HT with r e s u l t a n t displacement of the endogenous catecholamine. The head shake behavior, however, appears dependent upon the a c t i v a t i o n of 5HT receptors since pi~ozide or phenoxybenzamine only attenuated the response at
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5-Hydroxy-DL-tryptophan-lnduced
Head Shakes
Vol. 26, No. 17, 1980
r e l a t i v e l y high doses while metergoline and cyproheptadine were potent antagonists of head shaking. Our observations confirm the appearance of shaking behavior described in mice (6) and rats (7) after 5HT precursors or agonists. However, several differences do exist between the head shake response observed in this study and wet dog shakes (7) previously reported in rats. The head shake consists of a rapid movement or twitch of the head with l i t t l e involvement of the trunk musculature. The head shake resembles a pinna r e f l e x and appears the same behavior as described by Corne et al (6) in mice. The present experiments are the f i r s t comprehensive pharmacological characterization of the head shake response in the rat.
5HTP-induced head shaking is a very sensitive model for detection of 5HT antagonists. Metergoline and cyproheptadine, well-known 5HT antagonists, are e f f e c t i v e i n h i b i t o r s of head shakes in the ug/kg range when given i n t r a p e r i toneally (Table I I ) . Methysergide also blocked head shake behavior but was 15 times less potent than cyproheptadine. Xylamidine, an i n h i b i t o r of peripheral 5|IT responses (15), did not antagonize 5HTP-induced head shakes suggesting shakes are due to central 5HT receptor a c t i v a t i o n . Blockade of alpha-adrenergic receptors with a variety of m~ and ~e antagonists produced no potent i n h i b i t i o n of head shakes. Phenoxybenzamine antagonized the behavior but the doses necessary evoked non-specific side e f f e c t s . Likewise, l - p r o p r a n o l o l , which is reported to displace 3H-5HT from binding sites (16) and antagonize head twitches in mice (17), i n h i b i t e d 5HTP-induced head shakes in rats only at r e l a t i v e l y high doses. Further evidence for mediation of the head twitch response by 5HT comes from the present results with f l u o x e t i n e , a highly selective i n h i b i t o r of 5HT uptake in vivo (18). A marked potentiation of head twitches evoked by submaximal doses of 5HTP was observed in animals treated with f l u o x e t i n e (Table I l l ) . The a b i l i t y of f l u o x e t i n e to enhance head twitches produced by 5HTP strongly suggests the behavior is mediated by activation of 5HT receptors. Of the other amine uptake blockers studied only desmethylimipramine (DMI) prevented head shakes at low doses. Recent experiments have revealed a central 5HT antagonist a c t i v i t y of other t r i c y c l i c antidepressants as well (19,20). The a b i l i t y of DMI to antagonize 5HTP-induced head shakes may also suggest a modulation of this 5HT behavior by noradrenergic mechanisms. Cholinergic blockade with scopolamine and activation of dopamine receptors with SK&F 38393 (21) did not a l t e r head shake behavior. The neuroleptics chlorpromazine and pimozide reduced the head shake response. This may be a r e s u l t of antiserotonin properties of tile neuroleptics which have recently come to l i g h t (22,23). In this behavioral model these neuroleptics were j u s t as potent as methyserqide at antagonizing 5HT-induced behavior. GABAergic mechanisms do not appear to modulate the 5HTP head shakes since muscimol had l i t t l e e f f e c t on shaking behavior. Furthermore, Hl receptors are also not involved for pyrilamine had no e f f e c t on 5HTP-induced head shake. The head shake model has several d i s t i n c t advantages over other 5HT-related behaviors. Use of the i n t a c t animal eliminates the surgery necessary in the f l e x o r r e f l e x of the spinal rat model (24). The head shake response is readily q u a n t i f i a b l e without recording equipment and a graded response can be used to assess data. Horeover, the head shake model is extremely sensitive for detection of 5HT antagonists. The 5HT motor syndromes in guinea pig (5) and rat (3) allow only quantal responses to be studied and require much higher doses of 5HT antagonists to block these behaviors.
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5-Hydroxy-DL-tryptophan-Induced Head Shakes
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In summary, systemic administration of the serotonin precursor, 5HTP, in the presence of an i n h i b i t o r of peripheral L-aromatic amino acid decarboxylase, produces vigorous head shaking behavior in the rat. The head shake response is evoked or inhibited by a~ents which activate or interrupt 5HT neurotrans~ission, respectively, and potentiated by blockade of 5HT uptake mechanisms. The head shake behavior is a reliable quantitative model for study of brain 5HT receptors and is especially useful for evaluating 5HT antagonist properties of compounds. ACKNOWLEDGE~.IENT The authors g r a t e f u l l y acknowledge I~r. Elwood Stack for neurochemical of amine concentrations.
analysis
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