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A possible mechanism of the tyramine-induced head-twitch response
European Journal of Pharmacology, 80 (1982) 163-169 Elsevier Biomedical Press
163
A POSSIBLE MECHANISM OF THE TYRAMINE-INDUCED HEAD-TWITCH RESPONSE s Y u z o ORIKASA and KENSUKE KISARA
Department of Pharmacology, Tohoku College of Pharmacy, 4-4-1, Komatsushima, Sendai 983, Japan Received 8 December 1981, accepted 21 January 1982
S. ORIKASA and K. KISARA, A possible mechanism of the tyramine-inducedhead-twitch response, European J. Pharmacol. 80 (1982) 163-169. The effects of monoaminergic drugs on the head-twitch response (HTR) induced by intracerebroventricular (i.c.v.) injection of tyramine (TyA) in mice pretreated with safrazine, a monoamine oxidase inhibitor, were compared with effects on the response induced by the i.c.v, injection of serotonin (5-HT) in safrazine-pretreated mice. The HTR induced by both TyA and 5-HT were suppressed by i.p. injection of p-chlorophenylalanine. Chlorimipramine enhanced the 5-HT response but not the TyA response. Dimethothiazine, a serotonergic blocker, reduced both the TyA and 5-HT responses. The i.c.v, injection of p-chlorophenylalanine methylester resulted in a reduction of the TyA response but not of the 5-HT response. The i.c.v, injection of 5,6-dihydroxytryptamine (5,6-DHT) 1 day before the test suppressed the TyA response but enhanced the 5-HT response. The i.c.v, injection of noradrenaline reduced both the TyA and 5-HT responses. The i.c.v, injection of 6-hydroxydopamine, and i.p. injection of a-methyl-p-tyrosine and tolazoline accelerated the TyA response. These results suggest that the TyA response may be based on the release of endogenous 5-HT and can be suppressed by the catecholaminergic system. Head-twitch response 6-Hydroxydopamine
Tyramine
Serotonin
p-Chlorophenylalanine methylester
s o m e effects of m o n o a m i n e r g i c drugs on b o t h H T R were e x a m i n e d in the present experiments.
1. Introduction In recent years, t y r a m i n e ( T y A ) has been imp l i c a t e d n o t only in a n u m b e r of neurological a n d p s y c h i a t r i c diseases including P a r k i n s o n ' s disease a n d s c h i z o p h r e n i a (Boulton et al., 1967), b u t also in the effects of a n t i p s y c h o t i c drugs such as chlorp r o m a z i n e a n d h a l o p e r i d o l (Juorio, 1977). F u r thermore, it was recently o b s e r v e d that T y A ind u c e d a h e a d - t w i t c h response ( H T R ) in mice pretreated with monoamine oxidase inhibitors ( S a k u r a d a , 1975). Such evidence has led us to p r o p o s e that the T y A response m a y b e m e d i a t e d via the serotonergic system ( O r i k a s a et al., 1980). I n 1956, K e l l e r a n d U m b r e i t d e m o n s t r a t e d that lysergic acid d i e t h y l a m i d e c o u l d elicit the HT'R in mice, a n d the response was f o u n d to be closely r e l a t e d to the serotonergic system ( C o r n e et al., 1963; M a w s o n a n d W h i t t i n g t o n , 1970). T o characterise the T y A - i n d u c e d H T R in m o r e detail by c o m p a r i n g it with the 5-HT-response, 0014-2999/82/0000-0000/$02.75
5,6-Dihydroxytryptamine
© 1982 Elsevier Biomedical Press
2. Materials and methods 2.1. S u ~ e c ~ M a l e d d Y strain mice ( 2 0 - 2 4 g ) were used t h r o u g h o u t the e x p e r i m e n t s a n d were allowed free access to food a n d water except d u r i n g observation of the H T R . Since T y A a d m i n i s t e r e d i.c.v, is r a p i d l y m e t a b o l i s e d b y m o n o a m i n e oxidase (Breese et al., 1969), safrazine (10 m g / k g ) , a m o n o a m i n e oxidase i n h i b i t o r ( N i s h i n u m a , 1963), was administered i.p. 1 h before T y A . Safrazine was also administered before 5-HT.
2.2. Intracerebroventricular injection T h e i.c.v, injection was d o n e u n d e r very light ether anesthesia. T h e technique used for i.c.v, in-
164
jection was that described by Haley and McCormick (1957) and modified according to Brittain and Handley (1967).
3. Results
3.1. Effects of serotonin and tyramine on head-twitch response
2.3. Observation of head-twitch response
The mice were placed in individual plastic cages (25 X 18 X 13 cm) immediately after the i.c.v, injection of TyA and 5-HT. The number of HTR was counted for 2 min at 10 rain intervals between 10 and 92 min after the i.c.v, injection of 5-HT and TyA. 2. 4. Biochemical assay
The mice were decapitated and the brain removed rapidly and placed on an ice plate. The brains were then weighed and the 5-HT content in the whole brain was measured according to the method of Tadano et al. (1980).
The characteristic HTR was elicited dosedependently by the i.c.v, injection of 5-HT (5.6-24 nmol/mouse) in mice pretreated with safrazine (10 mg/kg i.p.). The i.c.v, administration of TyA (180 -560 nmol/mouse) also caused the HTR in safrazine-pretreated mice (fig. 1). The TyA-induced response was much weaker than that to 5-HT (fig. 2). The supramaximal doses of 5-HT and TyA were 13 and 320 nmol/mouse, respectively, which elicited almost the same total number of HTR (table 1). Therefore, 13 nmol/mouse of 5-HT and 320 nmol/mouse of TyA were used as standard doses in subsequent experiments. 3.2. Effects of serotonergic drugs on head-twitch response
2.5. Drugs
p-Chlorophenylalanine (Tokyo) and cr-methylp-tyrosine (Tokyo) were suspended in distilled water containing 0.5% Tween 80. Chlorimipramine hydrochloride (Ciba-Geigy), dimethothiazine mesylate (Shionogi), tolazoline hydrochloride (Yamanouchi) and safrazine hydrochloride (•no) were dissolved in isotonic saline. Tyramine hydrochloride (Tokyo), serotonin creatinine sulfate complex (Wako), p-chlorophenylalanine methylester (Sigma) and noradrenaline bitartrate (Wako) were dissolved in Ringer solution. 5,6-Dihydroxytryptamine hydrochloride (Sigma) and 6-hydroxydopamine hydrochloride (Sigma) were dissolved in Ringer solution containing 0.1% ascorbic acid. The doses of TyA and 5-HT are given in mol. The doses Of the other drugs refer to the salts. 2.6. Statistics
Student's t-test was used to determine statistical significance.
The i.p. injection of p-chlorophenylalanine (PCPA) reduced the HTR induced by both 5-HT
15
Serotonin
c_ 15
Tyramine
E
e~
%~LQ
10
:., ,,', ,," ~ 0 ",, / ',,
~, IC
~ ~
,"Y ?~/,~--o.,,, ~. ,, 'o q",,
/ .'~,a~.q~
"6
70
10 20 30 40 50 60 ~0 oo so
,0 20 30 40 50 60
1'2 2~2 312 4'2 52 612 7'2 8'2 9J2
1'2 212 3'2 4'2 5'2 6'2 7~2 812 9'2
oo 9o
Time course (rain)
Time course (rain)
Fig. 1. Effects of intracerebroventricular injection of serotonin (left panel), in doses of 7.5 ( • • n = 10), l0 ( t . . . . . . • n = 1 0 ) , 13 ( ~ ~ n=30), 18 ( O O n = 4 0 ) and 24 ( O . . . . . . O n--25) nmol/mouse, and tyramine (right panel), in doses of 180 (O• n -- 15), 240 ( • . . . . . . • n = 20), 320 (~ ~ n=35), 420 ( O O n = 2 0 ) and 560 ( O . . . . - - O n = 15) nmol/mouse, on head-twitch response in nice pretreated with safrazine (10 m g / k g i.p.).
165 4( T
"
6C u
3(
"6
c -
2C
~-
1C
ro_H
Dose (juglrnouse i.c.v.) Control Time betore test 1
10 100 Dose ( nmo( I mouse i.c.v.)
500
500
500
1 day
2 days
3 days
lq
Fig. 2. Dose-response curves of intracerebroventricularly injected serotonin (@ @) and tyramine (O O) in mice pretreated with safrazine (10 m g / k g i.p.). Each point is the mean from 10-40 mice. Vertical bars show the S.E.M.
and TyA. Chlorimipramine enhanced the 5-HT response but not the TyA response. The i.p. injection of dimethothiazine, a serotonergic blocker, suppressed both 5-HT and TyA responses (table 2). The i.c.v, injection of p-chlorophenylalanine
Fig. 3. Effects of intracerebroventricularly injected p-chlorophenylalanine methylester (500 # g / m o u s e ) on head-twitch response induced by serotonin (13 n m o l / m o u s e i.c.v. D) and tyramine (320 n m o l / m o u s e i.c.v. ~ ) in mice pretreated with safrazine (10 m g / k g i.p.). Each value is the mean of 20 mice. Vertical bars show the S.E.M. * P<0.05; when compared with control group.
methylester (PCPA-E), in a dose of 500/~g/mouse, 1 day before the test resulted in a reduction of the TyA response, but failed to produce a significant reduction of the 5-HT response (fig. 3). The i.c.v.
TABLE 1 Effects of intraeerebroventricularly injected serotonin and tyramine on head-twitch response (HTR) in mice pretreated with safrazine (10 m g / k g i.p.). Drug
Vehicle
Serotonin
Tyramine
Dose ( n m o l / m o u s e i.c.v.)
N u m b e r of mice
Incidence ratio a
-
10
0.00
0 . 0 ± 0.0
4.2 5.6 7.5 10 13 18 24
10 10 10 10 30 40 25
0.00 0.20 0.60 0.90 1.00 1.00 1.00
0 . 0 ± 0.0 0 . 5 ± 0.4 6.2--- 2.3 19.0± 8.1 2 8 . 2+- 6.0 41.9 + -- 6.1 62.7--- 10.1
10 15 20 35 20 15
0.00 0.20 0.75 1.00 1.00 1.00
0.01.2-+ 8.0± 26.7± 43.9± 60.7 +
130 180 240 320 420 560
N u m b e r of H T R (mean ± S.E.M.)
0.0 0.8 3.8 3.7 9.2 11.6
a Ratio of the number of animals in which H T R was elicited more than once throughout the observation period of the total number of animals in the group.
166 TABLE 2 Effects of serotonergic drugs on head-twitch response (HTR) induced by serotonin (5-HT, 13 n m o l / m o u s c i.c.v.) and tyraminc (TyA, 320 n m o l / m o u s e i.c.v.) in mice pretreated with safrazine (10 m g / k g i.p.). The number of mice is given in parentheses. Drug
Dose ( m g / k g i.p.)
Time before test
Control 200 2x200/12h 3x200/12h
p-Chlorophenylalanine
Number of HTR (mean ~ S.E.M.) 5-HT-induced
TvA-induccd
-
28.2 ~ 6.0 (3/))
26.7 ~ 3.7 (35)
6 h 6h 6h
22.4± 7.5 (5) 18.8~ 7.7 (5) 10.4-' 3.4 (20)*
17.4 ÷ 11.0 (5) 3.6~ 1.7 (5)* 7.3 ~ 1.3 (20}**
Chlorimipramine
5 10
15 min 15 rain
63.8+-21.8(10)* 113.6-+12.1 (10)**
Dimethothiazine
5 10 20
5min 5 min 5 min
2/).2-+10.4 (5) 3.6z 2.4 (5) 1.4± 0.5 (10)*
20.6 ~ 5.3(1(l) 21.6 ~ 5.6(10) 15.0 ~ 7.0 (5) 8.2: ~ 2.6(10)* 5.6 ~ 1.4 (10)**
* P<0.05, ** P<0.01 when compared with control group.
sponses, a-Methyl-p-tyrosine and tolazoline accelerated the TyA response but almost did not change the 5-HT response (table 3). The i.c.v, injection of 6-hydroxydopamine (6-OHDA), in a dose of 50 fig/mouse, 3 and 7 days before the test markedly enhanced the TyA response but failed to produce a significant increase of the 5-HT-response (fig. 5).
infusion of 5,6-dihydroxytryptamine (5,6-DHT), in a dose of 50 /~g/mouse, 1 day before the test suppressed the TyA response but enhanced the 5-HT response. 5,6-DHT injected 7 days before the test increased the 5-HT response more than the TyA response (fig. 4).
3.3. Effects of catecholaminergic drugs on headtwitch response
3.4. Effects of p-chlorophenylalanine methylester and 5,6-dihydroxytryptamine on serotonin contents
Noradrenaline injected i.c.v, at same time as 5-HT or TyA reduced both 5-HT and TyA re-
The i.c.v, injection of PCPA-E (500/~g/mouse) led to a reduction of the 5-HT contents in mouse
TABLE 3 Effects of catecholaminergic drugs on head-twitch response (HTR) induced by serotonin (5-HT, 13 n m o l / m o u s e i.c.v.) and tyramine (TyA, 320 n m o l / m o u s e i.c.v.) in mice pretreated with safrazine (10 m g / k g i.p.). The number of mice is given in parentheses. Drug
Dose (mg/kg) (/~g/mouse)
Route
Number of HTR (mean -+S.E.M.)
Time before test
5-HT-induced
TyA-induced
2 8 . 2+- 6.0 (30)
26.7 + 3.7 (35)
Control
-
-
-
Noradrenaline
2 #g 5 /~g
i.c.v. i.c.v.
0 min 0 min
100 mg 200mg
i.p. i.p.
6 h 6h
24.0 -+ 6.9 (5) 29.1 + 7.5 (10)
28.9 + 10.2 (10) 58.2 +- 13.4 (10) **
10 mg 20 mg
i.p. i.p.
5 min 5 min
34.9 + 6.8 (10) 36.4 + 13.1 (10)
30.1+ 4.2 (10) 49.3 + 10.6 (|0) *
a-Methyl-p-tyrosine Tolazoline
* P<0.05, ** P<0.01 when compared with control group.
1.7 + 0.7 (10) * 0.8-+ 0.2 (10) *
10.8 -+ 5.3 (5) 4.8+ 1.8 (5)*
167
~u 20
3,(
t
v
"6 10C ~
50
0
Dose OJg/mouse i.c.v.) Control Time before test
_c
L
2(
~.7
I~1 ";' 1/2 1
50
5O
50
1 day
3 days
1 week
Fig. 4. Effects of intracerebroventricularly injected 5,6-dihydroxytryptamine (50 t~g/mouse) on head-twitch response induced by serotonin (13 nmol/mouse i.c.v. Q) and tyramine (320 nmol/mouse i.c.v, la) in *mice pretreated with safrazine (10 mg/kg i.p.). Each value is the mean from 20 mice. Vertical bars. show the S.E.M. ** P<0.01; when compared with control group.
o
,5-.........
o'-'- ..............
0""
2
3
5
7
"0-;;""
Days after injection
Fig. 6. Effects of i.c.v, injected vehicle (0), p-chlorophenylalanine methylester (500 #g/mouse O O) and 5,6-dihydroxytryptamine (50/~g/mouse O . . . . . . O) on serotonin contents in mouse brain. Square shows serotonin contents in normal mice. Each point is the mean from 10 normal, vehicletreated mice and 5 treated mice. Vertical bars show the S.E.M. * P<:0.05, ** P<0.01; when compared with vehicle-treated group.
4. D i s c u s s i o n
Dose Oag/rnouse i.c.v.) Control
Time before test
50
50
50
1 day
3 days
1 week
Fig. 5. Effects of intracerebroventricularly injected 6-hydroxydopamine (50 sg/mouse) on head-twitch response induced by serotonin (13 nmol/mouse i.c.v. []) and tyramine (320 nmol/mouse i.c.v. •) in mice pretreated with safrazine (10 mg/kg i.p.). Each value is the mean from 10 mice. Vertical bars show the S.E.M. ** P<0.01; when compared with control group.
b r a i n I d a y after the injection but, at 2 days, the 5 - H T contents r e t u r n e d to near the control level. T h e r e was a significant r e d u c t i o n of 5 - H T contents 1 d a y after the i.c.v, injection of 5 , 6 - D H T (50 / ~ g / m o u s e ) a n d the 5 - H T contents r e m a i n e d r e d u c e d at 7 d a y s (fig. 6).
It has been r e p o r t e d that several t r e a t m e n t s elicit the characteristic H T R which is closely related to the serotonergic system ( C o r n e et al., 1963; M a w s o n a n d W h i t t i n g t o n , 1970). The results from the present e x p e r i m e n t s indicate that the h e a d - t w i t c h responses i n d u c e d b y 5 - H T a n d T y A have in m a n y aspects a similar m e c h a n i s m of action, b u t there are also some differences between the responses they elicit. W e h a d r e p o r t e d f r o m p r e l i m i n a r y investigations that the i.c.v, injection of 5 - H T increased the T y A - i n d u c e d H T R in safrazine-pretreated mice ( O r i k a s a et al., 1980). It is well k n o w n that P C P A l e a d s to a r e d u c t i o n of 5 - H T contents followed by s u p p r e s s i o n of the function of the serotonergic system ( K o e a n d W e i s s m a n , 1966). A s shown in t a b l e 2 , P C P A r e d u c e d the T y A response. The i.c.v, infusion of P C P A - E , which led to a r e d u c t i o n o f 5 - H T c o n t e n t s in m o u s e b r a i n I d a y after the infusion, also elicited a decrease of the T y A response. Moreover, dimethothiazine, a serotonergic b l o c k e r (Julou et al., 1966), s u p p r e s s e d the T y A response. These results suggest that the T y A response as well as the 5 - H T response is closely r e l a t e d to the serotonergic system.
168
It has been reported that 5,6-DHT produced a relatively selective chemical lesion of 5-HT nerve terminals (Nygren et al., 1974), decreased the concentration of 5-HT and that in addition the 5-HT receptors in mouse brain became 'supersensitive' (Nakamura and Fukushima, 1978). It is highly probable that the potentiation of the 5-HT response by 5,6-DHT was due to the presence of 'supersensitive' 5-HT receptors. There is a report that TyA is able to release 5-HT from striatal synaptosomes (Raiteri et al., 1977). It can be assumed that the TyA response, which differs from the 5-HT response, may be elicited by direct stimulation of 5-HT receptors, leads to the release of endogenous 5-HT. This is because 5,6-DHT injected l day before the test increased the 5-HT respon.se but reduced the TyA response and caused a significant decrease of 5-HT contents. This assumption is supported by the fact that PCPA-E suppressed the TyA response more than the 5-HT response. 5,6-DHT injected 7 days before the test significantly increased the TyA response and did so presumably because the 5-HT receptors were 'supersensitive' and any remaining endogenous 5HT was released by TyA. This suggestion originated from the finding that 5,6D H T injected 7 days before the test enhanced the 5-HT response more strongly than the Ty~, response and decreased the 5-HT contents to about 55%. The i.c.v, injection of noradrenaline resulted in a significant decrease of the 5-HT and TyA response, a-Methyl-p-tyrosine, a tyrosine hydroxylase inhibitor (Nagatsu et al., 1964), and tolazoline, a noradrenergic blocker (King, 1951), increased the TyA response. An increase of the TyA response was also elicited" by the i.c.v, injection of 6-OHDA, a catecholaminergic denervator (Ungerstedt, 1968). These results suggest that the HTR is suppressed by a catecholaminergic system, probably the noradrenergic system, and that TyA itself reduced in part the TyA-induced, HTR because TyA is one of the indirect stimulators of catecholaminergic receptors (Burn and Rand, 1958). However, there is a possibility that other mechanisms and neuronal systems may also be concerned with the TyA-induced HTR, because the effects of monoaminergic drugs on the TyA re-
sponse were quite complicated, in clear contrast with the case of the 5-HT response.
References Boulton, A.A. and R.J. Pollitt, 1967, Identity of a urina~ "pink spot' in schizophrenia and Parkinson's disease, Nature 215, 132. Breese, G.R., T.N. Chase and I.J. Kopin, 1969, Metabolism of tyramine-3H and octopamine-3H by rat brain, Biochem. Pharmacol. 18, 863. Brittain, R.T. and S.L. Handley, 1967, Temperature changes produced by the injection of catecholamines and 5hydroxytryptamine into the cerebral ventricles of the conscious mouse, J. Physiol. 192, 805. Burn, J.H. and M.J. Rand, 1958, The action of sympathomimetic amines in animals treated with reserpine, J. Physiol. 144, 134. Come, S.J., R.W. Pickering and B.T. Warner, 1963, A method for assessing the effects of drugs on the central action of 5-hydroxytryptamine, Br. J. Pharmacol. 20, 106. Haley, T.J. and W.G. McCormick, 1957, Pharmacological effects produced by intracerebral injection of drugs in the conscious mouse, Br. J. Pharmacol. 12, 12. Julou, L., R. Ducrot, M.C. Bardone, J.Y. Detaille, C. Feo, J.C. Guyonnet, G. Loiseau and J. Pasquet, 1966, lktude des propribtbs pharmacologiques de la dim+thylsulfamide-3(dimethylamino-2-propyl)-10-phbnothiazine (8.599 R.P.), Arch. Int. Pharmacodyn. 159, 70. Juorio, A.V., 1977, Effects of D-amphetamine and antipsychotic drug administration on striatal tyramine levels in the mouse, Brain Res. 126, 181. Keller, D.L. and W.W. Umbreit, 1956, 'Permanent' alteration of behavior in mice by chemical and psychological means, Science 124, 723. King, T.O., 1951, Contributions to the mode of action of sympathetic blocking agents, J. Am. Pharm. Assoc. 40, 350. Koe, B.K. and A. Weissman, 1966, p-Chlorophenylalanine: a specific depletor of brain serotonin, J. Pharmacol. Exp. Ther. 154, 499. Mawson, C. and H. Whittington, 1970, Evaluation of the peripheral and central antagonistic activities against 5hydroxytryptamine of some new agents, Br. J. Pharmacol. 39, 223. Nagatsu, T., M. Levitt and S. Udenfriend, 1964, The initial step in norepinephrine biosynthesis, J. Biol. Chem. 239, 2910. Nakamura, M. and H. Fukushima, 1978, Effect of 5,6dihydroxytryptamine on the head-twitches induced by 5HTP, 5-HT, mescaline and fludiazepam in mice, J. Pharm. Pharmacol. 30, 56. Nishinuma, K., 1963, Monoamine oxidase inhibitors as antidepressant, Seishin-Shinkei-Gaku Zasshi 65, 614 (in japanese). Nygren, Lars-G., K. Fuxe, G. Jonsson and L. Olson, 1974, Functional regeneration of 5-hydroxytryptamine nerve terminals in the rat spinal cord following 5,6-dihydroxy-
169 tryptamine induced degeneration, Brain Res. 78, 377. Orikasa, S., S. Sakurada and K. Kisara, 1980, Head-twitch response induced by tyramine, Psychopharmacol. 67, 53. Raiteri, M., R.D. Carmine, A. Bertolline and G. Levi, 1977, Effect of sympathomimetic amines on the synaptosomal transport of noradrenaline, dopamine and 5-hydroxytryptamine, European J. Pharmacol. 41, 133. Sakurada, S., 1975, Central action of fl-phenylethylamine derivatives (6) head-twitches induced by intracerebrally ad-
ministered tyramine in isocarboxazide pretreated mice, Folia Pharmacol. Jap. 71,779. Tadano, T., Y. Endo and K. Kisara, 1980, A simple determination of serotonin, 5-hydroxyindoleacetic acid and 5hydroxytryptophan decarboxylase activity in rat brain areas and parallel correlation among the levels, Jap. J. Pharmacol. 30, 347. Ungerstedt, U., 1968, 6-Hydroxy-dopamine induced degeneration of central monoamine neurons, European J. Pharmacol. 5, 107.
163
A POSSIBLE MECHANISM OF THE TYRAMINE-INDUCED HEAD-TWITCH RESPONSE s Y u z o ORIKASA and KENSUKE KISARA
Department of Pharmacology, Tohoku College of Pharmacy, 4-4-1, Komatsushima, Sendai 983, Japan Received 8 December 1981, accepted 21 January 1982
S. ORIKASA and K. KISARA, A possible mechanism of the tyramine-inducedhead-twitch response, European J. Pharmacol. 80 (1982) 163-169. The effects of monoaminergic drugs on the head-twitch response (HTR) induced by intracerebroventricular (i.c.v.) injection of tyramine (TyA) in mice pretreated with safrazine, a monoamine oxidase inhibitor, were compared with effects on the response induced by the i.c.v, injection of serotonin (5-HT) in safrazine-pretreated mice. The HTR induced by both TyA and 5-HT were suppressed by i.p. injection of p-chlorophenylalanine. Chlorimipramine enhanced the 5-HT response but not the TyA response. Dimethothiazine, a serotonergic blocker, reduced both the TyA and 5-HT responses. The i.c.v, injection of p-chlorophenylalanine methylester resulted in a reduction of the TyA response but not of the 5-HT response. The i.c.v, injection of 5,6-dihydroxytryptamine (5,6-DHT) 1 day before the test suppressed the TyA response but enhanced the 5-HT response. The i.c.v, injection of noradrenaline reduced both the TyA and 5-HT responses. The i.c.v, injection of 6-hydroxydopamine, and i.p. injection of a-methyl-p-tyrosine and tolazoline accelerated the TyA response. These results suggest that the TyA response may be based on the release of endogenous 5-HT and can be suppressed by the catecholaminergic system. Head-twitch response 6-Hydroxydopamine
Tyramine
Serotonin
p-Chlorophenylalanine methylester
s o m e effects of m o n o a m i n e r g i c drugs on b o t h H T R were e x a m i n e d in the present experiments.
1. Introduction In recent years, t y r a m i n e ( T y A ) has been imp l i c a t e d n o t only in a n u m b e r of neurological a n d p s y c h i a t r i c diseases including P a r k i n s o n ' s disease a n d s c h i z o p h r e n i a (Boulton et al., 1967), b u t also in the effects of a n t i p s y c h o t i c drugs such as chlorp r o m a z i n e a n d h a l o p e r i d o l (Juorio, 1977). F u r thermore, it was recently o b s e r v e d that T y A ind u c e d a h e a d - t w i t c h response ( H T R ) in mice pretreated with monoamine oxidase inhibitors ( S a k u r a d a , 1975). Such evidence has led us to p r o p o s e that the T y A response m a y b e m e d i a t e d via the serotonergic system ( O r i k a s a et al., 1980). I n 1956, K e l l e r a n d U m b r e i t d e m o n s t r a t e d that lysergic acid d i e t h y l a m i d e c o u l d elicit the HT'R in mice, a n d the response was f o u n d to be closely r e l a t e d to the serotonergic system ( C o r n e et al., 1963; M a w s o n a n d W h i t t i n g t o n , 1970). T o characterise the T y A - i n d u c e d H T R in m o r e detail by c o m p a r i n g it with the 5-HT-response, 0014-2999/82/0000-0000/$02.75
5,6-Dihydroxytryptamine
© 1982 Elsevier Biomedical Press
2. Materials and methods 2.1. S u ~ e c ~ M a l e d d Y strain mice ( 2 0 - 2 4 g ) were used t h r o u g h o u t the e x p e r i m e n t s a n d were allowed free access to food a n d water except d u r i n g observation of the H T R . Since T y A a d m i n i s t e r e d i.c.v, is r a p i d l y m e t a b o l i s e d b y m o n o a m i n e oxidase (Breese et al., 1969), safrazine (10 m g / k g ) , a m o n o a m i n e oxidase i n h i b i t o r ( N i s h i n u m a , 1963), was administered i.p. 1 h before T y A . Safrazine was also administered before 5-HT.
2.2. Intracerebroventricular injection T h e i.c.v, injection was d o n e u n d e r very light ether anesthesia. T h e technique used for i.c.v, in-
164
jection was that described by Haley and McCormick (1957) and modified according to Brittain and Handley (1967).
3. Results
3.1. Effects of serotonin and tyramine on head-twitch response
2.3. Observation of head-twitch response
The mice were placed in individual plastic cages (25 X 18 X 13 cm) immediately after the i.c.v, injection of TyA and 5-HT. The number of HTR was counted for 2 min at 10 rain intervals between 10 and 92 min after the i.c.v, injection of 5-HT and TyA. 2. 4. Biochemical assay
The mice were decapitated and the brain removed rapidly and placed on an ice plate. The brains were then weighed and the 5-HT content in the whole brain was measured according to the method of Tadano et al. (1980).
The characteristic HTR was elicited dosedependently by the i.c.v, injection of 5-HT (5.6-24 nmol/mouse) in mice pretreated with safrazine (10 mg/kg i.p.). The i.c.v, administration of TyA (180 -560 nmol/mouse) also caused the HTR in safrazine-pretreated mice (fig. 1). The TyA-induced response was much weaker than that to 5-HT (fig. 2). The supramaximal doses of 5-HT and TyA were 13 and 320 nmol/mouse, respectively, which elicited almost the same total number of HTR (table 1). Therefore, 13 nmol/mouse of 5-HT and 320 nmol/mouse of TyA were used as standard doses in subsequent experiments. 3.2. Effects of serotonergic drugs on head-twitch response
2.5. Drugs
p-Chlorophenylalanine (Tokyo) and cr-methylp-tyrosine (Tokyo) were suspended in distilled water containing 0.5% Tween 80. Chlorimipramine hydrochloride (Ciba-Geigy), dimethothiazine mesylate (Shionogi), tolazoline hydrochloride (Yamanouchi) and safrazine hydrochloride (•no) were dissolved in isotonic saline. Tyramine hydrochloride (Tokyo), serotonin creatinine sulfate complex (Wako), p-chlorophenylalanine methylester (Sigma) and noradrenaline bitartrate (Wako) were dissolved in Ringer solution. 5,6-Dihydroxytryptamine hydrochloride (Sigma) and 6-hydroxydopamine hydrochloride (Sigma) were dissolved in Ringer solution containing 0.1% ascorbic acid. The doses of TyA and 5-HT are given in mol. The doses Of the other drugs refer to the salts. 2.6. Statistics
Student's t-test was used to determine statistical significance.
The i.p. injection of p-chlorophenylalanine (PCPA) reduced the HTR induced by both 5-HT
15
Serotonin
c_ 15
Tyramine
E
e~
%~LQ
10
:., ,,', ,," ~ 0 ",, / ',,
~, IC
~ ~
,"Y ?~/,~--o.,,, ~. ,, 'o q",,
/ .'~,a~.q~
"6
70
10 20 30 40 50 60 ~0 oo so
,0 20 30 40 50 60
1'2 2~2 312 4'2 52 612 7'2 8'2 9J2
1'2 212 3'2 4'2 5'2 6'2 7~2 812 9'2
oo 9o
Time course (rain)
Time course (rain)
Fig. 1. Effects of intracerebroventricular injection of serotonin (left panel), in doses of 7.5 ( • • n = 10), l0 ( t . . . . . . • n = 1 0 ) , 13 ( ~ ~ n=30), 18 ( O O n = 4 0 ) and 24 ( O . . . . . . O n--25) nmol/mouse, and tyramine (right panel), in doses of 180 (O• n -- 15), 240 ( • . . . . . . • n = 20), 320 (~ ~ n=35), 420 ( O O n = 2 0 ) and 560 ( O . . . . - - O n = 15) nmol/mouse, on head-twitch response in nice pretreated with safrazine (10 m g / k g i.p.).
165 4( T
"
6C u
3(
"6
c -
2C
~-
1C
ro_H
Dose (juglrnouse i.c.v.) Control Time betore test 1
10 100 Dose ( nmo( I mouse i.c.v.)
500
500
500
1 day
2 days
3 days
lq
Fig. 2. Dose-response curves of intracerebroventricularly injected serotonin (@ @) and tyramine (O O) in mice pretreated with safrazine (10 m g / k g i.p.). Each point is the mean from 10-40 mice. Vertical bars show the S.E.M.
and TyA. Chlorimipramine enhanced the 5-HT response but not the TyA response. The i.p. injection of dimethothiazine, a serotonergic blocker, suppressed both 5-HT and TyA responses (table 2). The i.c.v, injection of p-chlorophenylalanine
Fig. 3. Effects of intracerebroventricularly injected p-chlorophenylalanine methylester (500 # g / m o u s e ) on head-twitch response induced by serotonin (13 n m o l / m o u s e i.c.v. D) and tyramine (320 n m o l / m o u s e i.c.v. ~ ) in mice pretreated with safrazine (10 m g / k g i.p.). Each value is the mean of 20 mice. Vertical bars show the S.E.M. * P<0.05; when compared with control group.
methylester (PCPA-E), in a dose of 500/~g/mouse, 1 day before the test resulted in a reduction of the TyA response, but failed to produce a significant reduction of the 5-HT response (fig. 3). The i.c.v.
TABLE 1 Effects of intraeerebroventricularly injected serotonin and tyramine on head-twitch response (HTR) in mice pretreated with safrazine (10 m g / k g i.p.). Drug
Vehicle
Serotonin
Tyramine
Dose ( n m o l / m o u s e i.c.v.)
N u m b e r of mice
Incidence ratio a
-
10
0.00
0 . 0 ± 0.0
4.2 5.6 7.5 10 13 18 24
10 10 10 10 30 40 25
0.00 0.20 0.60 0.90 1.00 1.00 1.00
0 . 0 ± 0.0 0 . 5 ± 0.4 6.2--- 2.3 19.0± 8.1 2 8 . 2+- 6.0 41.9 + -- 6.1 62.7--- 10.1
10 15 20 35 20 15
0.00 0.20 0.75 1.00 1.00 1.00
0.01.2-+ 8.0± 26.7± 43.9± 60.7 +
130 180 240 320 420 560
N u m b e r of H T R (mean ± S.E.M.)
0.0 0.8 3.8 3.7 9.2 11.6
a Ratio of the number of animals in which H T R was elicited more than once throughout the observation period of the total number of animals in the group.
166 TABLE 2 Effects of serotonergic drugs on head-twitch response (HTR) induced by serotonin (5-HT, 13 n m o l / m o u s c i.c.v.) and tyraminc (TyA, 320 n m o l / m o u s e i.c.v.) in mice pretreated with safrazine (10 m g / k g i.p.). The number of mice is given in parentheses. Drug
Dose ( m g / k g i.p.)
Time before test
Control 200 2x200/12h 3x200/12h
p-Chlorophenylalanine
Number of HTR (mean ~ S.E.M.) 5-HT-induced
TvA-induccd
-
28.2 ~ 6.0 (3/))
26.7 ~ 3.7 (35)
6 h 6h 6h
22.4± 7.5 (5) 18.8~ 7.7 (5) 10.4-' 3.4 (20)*
17.4 ÷ 11.0 (5) 3.6~ 1.7 (5)* 7.3 ~ 1.3 (20}**
Chlorimipramine
5 10
15 min 15 rain
63.8+-21.8(10)* 113.6-+12.1 (10)**
Dimethothiazine
5 10 20
5min 5 min 5 min
2/).2-+10.4 (5) 3.6z 2.4 (5) 1.4± 0.5 (10)*
20.6 ~ 5.3(1(l) 21.6 ~ 5.6(10) 15.0 ~ 7.0 (5) 8.2: ~ 2.6(10)* 5.6 ~ 1.4 (10)**
* P<0.05, ** P<0.01 when compared with control group.
sponses, a-Methyl-p-tyrosine and tolazoline accelerated the TyA response but almost did not change the 5-HT response (table 3). The i.c.v, injection of 6-hydroxydopamine (6-OHDA), in a dose of 50 fig/mouse, 3 and 7 days before the test markedly enhanced the TyA response but failed to produce a significant increase of the 5-HT-response (fig. 5).
infusion of 5,6-dihydroxytryptamine (5,6-DHT), in a dose of 50 /~g/mouse, 1 day before the test suppressed the TyA response but enhanced the 5-HT response. 5,6-DHT injected 7 days before the test increased the 5-HT response more than the TyA response (fig. 4).
3.3. Effects of catecholaminergic drugs on headtwitch response
3.4. Effects of p-chlorophenylalanine methylester and 5,6-dihydroxytryptamine on serotonin contents
Noradrenaline injected i.c.v, at same time as 5-HT or TyA reduced both 5-HT and TyA re-
The i.c.v, injection of PCPA-E (500/~g/mouse) led to a reduction of the 5-HT contents in mouse
TABLE 3 Effects of catecholaminergic drugs on head-twitch response (HTR) induced by serotonin (5-HT, 13 n m o l / m o u s e i.c.v.) and tyramine (TyA, 320 n m o l / m o u s e i.c.v.) in mice pretreated with safrazine (10 m g / k g i.p.). The number of mice is given in parentheses. Drug
Dose (mg/kg) (/~g/mouse)
Route
Number of HTR (mean -+S.E.M.)
Time before test
5-HT-induced
TyA-induced
2 8 . 2+- 6.0 (30)
26.7 + 3.7 (35)
Control
-
-
-
Noradrenaline
2 #g 5 /~g
i.c.v. i.c.v.
0 min 0 min
100 mg 200mg
i.p. i.p.
6 h 6h
24.0 -+ 6.9 (5) 29.1 + 7.5 (10)
28.9 + 10.2 (10) 58.2 +- 13.4 (10) **
10 mg 20 mg
i.p. i.p.
5 min 5 min
34.9 + 6.8 (10) 36.4 + 13.1 (10)
30.1+ 4.2 (10) 49.3 + 10.6 (|0) *
a-Methyl-p-tyrosine Tolazoline
* P<0.05, ** P<0.01 when compared with control group.
1.7 + 0.7 (10) * 0.8-+ 0.2 (10) *
10.8 -+ 5.3 (5) 4.8+ 1.8 (5)*
167
~u 20
3,(
t
v
"6 10C ~
50
0
Dose OJg/mouse i.c.v.) Control Time before test
_c
L
2(
~.7
I~1 ";' 1/2 1
50
5O
50
1 day
3 days
1 week
Fig. 4. Effects of intracerebroventricularly injected 5,6-dihydroxytryptamine (50 t~g/mouse) on head-twitch response induced by serotonin (13 nmol/mouse i.c.v. Q) and tyramine (320 nmol/mouse i.c.v, la) in *mice pretreated with safrazine (10 mg/kg i.p.). Each value is the mean from 20 mice. Vertical bars. show the S.E.M. ** P<0.01; when compared with control group.
o
,5-.........
o'-'- ..............
0""
2
3
5
7
"0-;;""
Days after injection
Fig. 6. Effects of i.c.v, injected vehicle (0), p-chlorophenylalanine methylester (500 #g/mouse O O) and 5,6-dihydroxytryptamine (50/~g/mouse O . . . . . . O) on serotonin contents in mouse brain. Square shows serotonin contents in normal mice. Each point is the mean from 10 normal, vehicletreated mice and 5 treated mice. Vertical bars show the S.E.M. * P<:0.05, ** P<0.01; when compared with vehicle-treated group.
4. D i s c u s s i o n
Dose Oag/rnouse i.c.v.) Control
Time before test
50
50
50
1 day
3 days
1 week
Fig. 5. Effects of intracerebroventricularly injected 6-hydroxydopamine (50 sg/mouse) on head-twitch response induced by serotonin (13 nmol/mouse i.c.v. []) and tyramine (320 nmol/mouse i.c.v. •) in mice pretreated with safrazine (10 mg/kg i.p.). Each value is the mean from 10 mice. Vertical bars show the S.E.M. ** P<0.01; when compared with control group.
b r a i n I d a y after the injection but, at 2 days, the 5 - H T contents r e t u r n e d to near the control level. T h e r e was a significant r e d u c t i o n of 5 - H T contents 1 d a y after the i.c.v, injection of 5 , 6 - D H T (50 / ~ g / m o u s e ) a n d the 5 - H T contents r e m a i n e d r e d u c e d at 7 d a y s (fig. 6).
It has been r e p o r t e d that several t r e a t m e n t s elicit the characteristic H T R which is closely related to the serotonergic system ( C o r n e et al., 1963; M a w s o n a n d W h i t t i n g t o n , 1970). The results from the present e x p e r i m e n t s indicate that the h e a d - t w i t c h responses i n d u c e d b y 5 - H T a n d T y A have in m a n y aspects a similar m e c h a n i s m of action, b u t there are also some differences between the responses they elicit. W e h a d r e p o r t e d f r o m p r e l i m i n a r y investigations that the i.c.v, injection of 5 - H T increased the T y A - i n d u c e d H T R in safrazine-pretreated mice ( O r i k a s a et al., 1980). It is well k n o w n that P C P A l e a d s to a r e d u c t i o n of 5 - H T contents followed by s u p p r e s s i o n of the function of the serotonergic system ( K o e a n d W e i s s m a n , 1966). A s shown in t a b l e 2 , P C P A r e d u c e d the T y A response. The i.c.v, infusion of P C P A - E , which led to a r e d u c t i o n o f 5 - H T c o n t e n t s in m o u s e b r a i n I d a y after the infusion, also elicited a decrease of the T y A response. Moreover, dimethothiazine, a serotonergic b l o c k e r (Julou et al., 1966), s u p p r e s s e d the T y A response. These results suggest that the T y A response as well as the 5 - H T response is closely r e l a t e d to the serotonergic system.
168
It has been reported that 5,6-DHT produced a relatively selective chemical lesion of 5-HT nerve terminals (Nygren et al., 1974), decreased the concentration of 5-HT and that in addition the 5-HT receptors in mouse brain became 'supersensitive' (Nakamura and Fukushima, 1978). It is highly probable that the potentiation of the 5-HT response by 5,6-DHT was due to the presence of 'supersensitive' 5-HT receptors. There is a report that TyA is able to release 5-HT from striatal synaptosomes (Raiteri et al., 1977). It can be assumed that the TyA response, which differs from the 5-HT response, may be elicited by direct stimulation of 5-HT receptors, leads to the release of endogenous 5-HT. This is because 5,6-DHT injected l day before the test increased the 5-HT respon.se but reduced the TyA response and caused a significant decrease of 5-HT contents. This assumption is supported by the fact that PCPA-E suppressed the TyA response more than the 5-HT response. 5,6-DHT injected 7 days before the test significantly increased the TyA response and did so presumably because the 5-HT receptors were 'supersensitive' and any remaining endogenous 5HT was released by TyA. This suggestion originated from the finding that 5,6D H T injected 7 days before the test enhanced the 5-HT response more strongly than the Ty~, response and decreased the 5-HT contents to about 55%. The i.c.v, injection of noradrenaline resulted in a significant decrease of the 5-HT and TyA response, a-Methyl-p-tyrosine, a tyrosine hydroxylase inhibitor (Nagatsu et al., 1964), and tolazoline, a noradrenergic blocker (King, 1951), increased the TyA response. An increase of the TyA response was also elicited" by the i.c.v, injection of 6-OHDA, a catecholaminergic denervator (Ungerstedt, 1968). These results suggest that the HTR is suppressed by a catecholaminergic system, probably the noradrenergic system, and that TyA itself reduced in part the TyA-induced, HTR because TyA is one of the indirect stimulators of catecholaminergic receptors (Burn and Rand, 1958). However, there is a possibility that other mechanisms and neuronal systems may also be concerned with the TyA-induced HTR, because the effects of monoaminergic drugs on the TyA re-
sponse were quite complicated, in clear contrast with the case of the 5-HT response.
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