Dissociation of Head Twitches and Tremors During the Study of Interactions with 5-Hydroxytryptophan in Mice

PATRICK MARTIN, HENRIETTE FRANCES, AND PIERRE SIMON

lntraperitoneal injection of 5-hydroxytryptophan and S-methoxy-N,N-dimethyl- tryptamine is shown to provoke characteristic behaviors in mice that can be quan- tified. The two principal phenomena described here are head twitches and tremors. Tremors became more frequent when doses of the two substances stud- ied were increased. Head twitches appeared at lower doses, but beyond a certain dose, they decreased and even disappeared. The effects on these movements of agents that modify serotonin function were then studied. The results pose prob- lems of interpretation that are discussed.

Key Words: Head twitch; Tremor; 5-Hydroxytryptophan; S-Methoxy-N,N-di- methyltryptamine; Mice

INTRODUCTION

In previous studies, it was observed that intraperitoneal (i.p.) injection of 5- hydroxytryptophan (5-HTP) in mice was followed by the appearance of a rapid char- acteristic movement called the “head twitch response” (Corne et al., 1963), the “dog head shake” (Matthews and Smith, 1980); or, in rats, the “wet-dog shake” (Yap et al., 1983). The same phenomena were observed after injection of 5-methoxy- N,N-dimethyltryptamine (5-MeODMT) , a serotoninergic agonist (Gessner and Page 1962), and after intracerebral (i.c.) administration of serotonin (Luchowsky et al., 1969).

Numerous studies suggest that the effect of 5-HTP in both mice and rats is me- diated in the central nervous system (Corne et al., 1963; Corne and Pickering, 1967; Nakamura and Fukushima, 1978; Matthews and Smith, 1980). The discordant results obtained in studies of the action of various substances on the serotoninergic system led us to study in greater detail the test as described. Certain substances seem to modify the behaviors induced by serotoninomimetic substances, but the specific results have differed among the various studies. A drug like isoprenaline, for ex- ample, has been shown to both reduce (Corne et al., 1963) and increase (Delini- Stula et al., 1979) 5-HTP-induced head twitches in mice. There seems to be con- sensus, however, with regard to the potentiation of the effects of serotoninomimetic substances by p-adrenergic stimulants (Ortmann et al., 1981; Cowen et al., 1982; Green et al., 1983; Nimgaonkar et al., 1983. Because of the lack of coherence in

From the Faculte de Medecine Piti&SalpCtri&re, DCpartement de Pharmacologic, PARIS, France. Address reprint requests to Dr. P. Simon, Faculte de MCdecine PitiC-Salp@tri&e, DCpartement de

Pharmacologic, 91, 61d de I’H6pital, 75634 Paris Cedex 13, France. Received February, 1984; revised and accepted July, 1984.

193

journal of Pharmacological Methods 13. 193-200 (1985)

0 1985 Elsevier Science Publishmg Co., Inc., 52 Vanderbilt Avenue, New York, NY lW17 01 M)-5402/85/$03.30

194 P. Martin et al.

these past results, and the problems posed by the numerous phenomena that might affect head twitches, we have studied, in depth, both the behavioral test itself as well as the ability of various substances able to modify it.

MATERIALS AND METHODS

Male Swiss NMRI mice (20-24 g, CERJ Breeder, Genest Saint Isle, France, 53940) were used. The animals were housed in plastic boxes, in groups of IO, for one week before the experiments, at a constant temperature 21 t 1°C with free access to food and water.

Soluble substances were dissolved in demineralized water, whereas insoluble substances were suspended in a dilute solution of acacia gum. The substances were administered i.p. in a volume of 0.25 ml/20 g body weight. Controls received de- mineralized water.

Three hours before the test, all the animals (IO per group) received the mono- amine oxidase inhibitor, pargyline, at a dose of 100 mg.kg-’ i.p. The mice were then isolated in transparent plexiglass boxes (20 cm x IO cm x IO cm). Thirty minutes before the test, the animals were given either demineralized water or the substance to be studied. At the beginning of the test (time 0), all the mice received 5-HTP at varying doses. After IO min, the first mouse was observed for 1 min; all of the characteristic head movements (head twitches) were counted, whatever their intensity. Other symptoms, such as abrupt jerks of the head or ear tremors, were disregarded. Tremors were measured in parallel and scored in the following man- ner:

0 = absence of tremor; 1 = intermittent tremor, more or less intense; the mice moved around in their

boxes ; 2 = intermittent tremor; difficulty in moving; possible muscular atony either of

the forepaws or hindpaws; 3 = continuous tremor; no movement; clear atony of all four limbs.

All the mice were observed for 1 min every IO min during a 60-min period. When 5-MeODMT was injected, at various doses, observation of the mice began

immediately after the injection and continued for 7 consecutive min. Other symp- toms, such as treading, stretching of the forepaws, “whip-like movements, raising of the tail, something resembling abdominal cramps, verticalization, stereotypies, were also observed during experiments with 5 HTP or 5-MeODMT. Statistical anal- ysis was performed with Student’s t-test, by use of tables of Fischer and Yates.

The substances used in these studies were obtained from the following sources: L-5-hydroxytryptophan came from Sigma (Coger, France); Methysergide came from laboratoires Sandoz (France); Quipazine came from Miles Laboratories, Inc. (U.S.A.); Pargyline came from Ega-chimie; Clomipramine came from Laboratoires Ciba-Geigy (France), and 5-MeODMT came from Sigma (Coger, France).

5 HTP-induced Head Twitches and Tremor in Mice 195

RESULTS

5-HTP Alone

Figure 1 represents the observed head twitches as a function WTP. The number of twitches increased up to the dose of 12 decreased with further increases of the dose. Tremors appeared

of the dose of 5 mgkg-I; it then at 8 mgkg-’ and

increased progressively with the doses. The evolution of head twitches compared to tremors can be seen in Figure 1; the two phenomena develop in parallel at first, and then dissociate. The same result is observed when the kinetics of the two phenomena as a function of time are considered. The maximai effects are observed 40 and 50 min, respectively, after the start of the test.

of mice

._. Head. twitch

O______~ Tremor

2 4 6 8 lO+2 i 24 32 S_HT P mg-kg-’

-lo- 60- 10_50_P_20_20_80_n_ 30-

FIGURE 1. The effects of increasing doses of 15.HTP in mice. The evolution of head twitches and tremors is shown.

196 P. Martin et al.

5 MeODMT Alone

Figure 2 shows the occurrence of head twitches and tremors as a function of the dose of 5 Me-ODMT. The evolution of the two phenomena is identical to that observed with 5 HTP. Tremors increased regularly with the dose, but head twitches eventually decreased, beginning at a dose of 8 mg.kg-‘.

Methysergide and 5-HTP

The effect of the serotonin antagonist, methysergide was studied under the same experimental conditions, at doses (4, 8, and 16 mg.kg-‘) that do not in themselves induce head twitches or tremors in mice. The effects on head twitches and tremors induced by 5-HTP, at doses of IO and 24 mgekg-I, are summarized in Table 1. Both behaviors are clearly antagonized by methysergide.

Number of mice

._. Head-twitch

o_______~ Tremor

1 2 4 8 16 32 64 5_Me_O T

-lo- 30- 20 - 40 - 10 - P m- mg. P g-1

FIGURE 2. The effects of increasing doses of 5-Me-ODMT in mice. The evolution of head twitches and tremors is shown.

5 HTP-induced Head Twitches and Tremor in Mice 197

TABLE 1 Effects of Methysergide on Head Twitches and Tremors Induced by i.p. injections of 1 5-HTP at doses of 10 and 24 mgkg-‘”

HEAD TWITCHES TREMORS

L 5-HTP METHYSERGIDE (M 2 S.E.M) (M -+ S.E.M.)

(MG.KG-‘) (MG+KG~‘) CONTROL TREATED CONTROL TREATED

4 9.9 + 2.03 3.7 i 1.37b 7.5 2 0.9 7 i 0.84

24 8 9.2 t 1.96 0.5 + 0.22d 9.1 + 0.86 4.9 " 0.67d

16 12.3 2 4.43 0.6 k 0.27' 7.9 + 0.72 4.2 i 0.94c

IO 16 9.6 2 3.28 0.9 k 0.43h 2.2 2 0.99 1.1 i- 0.55

= n = 10. hp < 0.05. c p < 0.01. d/J < 0.001.

Quipazine and 5-HTP

The effect of the serotonin receptor agonist quipazine was also studied under the same experimental conditions, at a dose of 4 rng,kg-‘. Quipazine, when ad- ministered alone at this dose, causes some head twitches (0.4 t 0.16) and moderate tremors (0.8 + 0.2). The modifications of the effects of 5-HTP in the presence of quipazine can be seen in Table 2. In the first part of the curve, head twitches and tremors increased. In the second part of the curve, tremors increased but head twitches decreased compared to controls.

Clomipramine and 5-HTP

Clomipramine, an imipraminelike antidepressant that inhibits the uptake of ser- otonin, was also studied at a dose of 8 mg.kg-’ i.p. The effects on head twitches and tremors are summarized in Table 3. A few head twitches were observed during the 30 min preceding the injection of 5-HTP, but at time 0 and for a period of 40

min thereafter, no head twitches or tremors were observed in the presence of 5- HTP.

TABLE 2 The Effects of Quipazine on Head Twitches and Tremors Induced by i.p. injections of l. 5-HTP at doses of 4,8 and 64 mg-kg-‘”

HEAD TWITCHES TREMORS

QIJIPAZINE L 5-HTP __ (M + 5.E.M) (M + S.E.M.)

_._- (MC.KG-‘) (MG.KC-‘) CONTROL TREATED CONTROL TREATED

_._.

4 1.4 t 0.61 3.1 i; 0.65 0 0

4 8 4.5 z!z 1.13 10.7 t 1.67 1.1 t 0.57 3.6 t 0.67b

64 5.7 -c 1.53 2.7 + l.04b 9.2 rt 1.34 12 + 0.75

rl n = 10.

b p < 0.05.

c p < 0.01.

198 P. Martin et al.

TABLE 3 Effects of Clomipramine on Head Twitches and Tremors Induced by i.p. Injections of 1 5-HTP at doses of 2, 4, 12, and 64 mgkg-‘q”

HEAD TWITCHES TREMORS

CLOMIPRAMINE L5-HTP _ (M ? S.E.M) (M ? S.E.M.)

(MC.KC-- ‘) (MG.KG~.‘) CONTROL TREATED CONTROL TREATED ._~

2 0.4 5 0.27 3.7 + 0.75d 0 0.5 2 0.27

8 4 1.6 " 0.51 3 k 0.67b 0 5.1 k 0.5'

12 8.9 f 2.03 3 _f 0.93b 3.2 ? 0.8 10.5 k 0.65d

64 2.22 2 1.01 0.13 k 0.136 11.89 " 1.02 14.25 2 0.46

a n = IO.

b p < 0.05.

= p < 0.01.

dp -c 0.001

DISCUSSION

The direct precursor of serotonin 5-HTP and the serotonin receptor agonist 5- MeODMT similarly induced head twitches and trembling that can be quantified. These results confirm those of Corne et al. (1963), Matthews and Smith (1980), Ges- sner and Page (19621, and Friedman et al. (1983).

As shown in Figures 1 and 2, head twitches increased as a function of the dose up to 12 mgekg-’ for 5-HTP and 8 mg.kg-’ for 5-MeODMT. At higher doses, the number of head twitches clearly decreased and even disappeared. Tremors ap- peared at 8 mg.kg-’ of 5-HTP and developed in parallel with the head twitches without seeming to affect the evolution of the twitches up to a dose of 12 mg.kg-’ of 5-HTP.

Tremors increased as a direct function of the dose of 5-HTP. At 16 mgakg-’ of 5- HTP, the intensity of the tremors seemed to mask the head twitches; thus explains the decrease in the latter. With 5-MeODMT, a parallel evolution of the two phe- nomena was also observed, followed by a decrease in head-twitches with further increases in tremors.

The same type of curve is observed whether 5-HTP or 5-MeODMT is adminis- tered. Under our experimental conditions, after a certain dose, tremors seem to mask head twitches; this produces an inverted U-curve, that has not yet, to our knowledge, been described in the literature. When the evolution of these behaviors is followed with time, the maximum for both twitches and tremors is almost always obtained 40 to 50 min after injection of 5-HTP (data not shown).

Both behaviors were antagonized by methysergide. The inhibition of head twitches by methysergide was also observed by Corne et al. (1963). At a dose of IO mg.kg-l 5-HTP, both behaviors were clearly inhibited. With 24 mg:kg-’ 5-HTP, however, the head twitches were masked by the tremors only partially.

Both head twitches and tremors seem to be serotoninergic phenomena, but tremors may also involve a cholinergic component. An interaction between sero- tonin and the cholinergic system has been described (Samanin et al., 1978). The

5 HTP-induced Head Twitches and Tremor in Mice YW

watering eyes and salivation that were observed with high doses of 5-HTP are con- sidered to be signs of chofinergic stimulation.

After administration of quipazine, the head twitches and tremors induced by low doses of 5-HTP increased. However, when high doses of 5-HTP were given, tremors increased while head twitches decreased. Thus, it seems likely that the apparent decrease in head twitches resulted from masking by the tremors, as if the two curves in Figures 1 and 2 were displaced towards the left.

When clomipramine was administered, the same results were obtained as with quipazine. Numerous studies have tried to demonstrate that ciomipramine in- creases the concentration of serotonin in the synaptic cleft and, for that reason, potentiates the symptoms studied. However, in the second part of the curve, the decrease in head twitches was greater than in the control mice.

These results demonstrate that it is relatively difficult ta interpret the effects of a substance when its action on the serot~ninergic system is not well established. The results may be interpreted differently depending on the dose administered and the experimental model used. Thus, depending on the dose of 5-HTP used, it is possible to observe, for a given substance, either an increase or a decrease in head twitches. For this reason, it would be more judicious, when studying such effects, to use a range of doses of 5-HTP or 5-MeODMT.

The simultaneous measurement of head twitches and tremors permits a clearer interpretation of the results obtained. It also indicates whether a substance acts on one or the other of the phenomena, or whether it acts on both at the same time.

REFERENCES

Corne S], Pickering RW, Warner BT (1963) A method for assessing the effects of drugs on the central actions of drugs on the central actions of 5-hydro~t~ptamine. Br/ Pharmac 20:106-120.

Come S}, Pickering RW (1967) A possible correfa- tion between drug induced hallucination in man and a behavioral response in mice. fsychophar- macofogia 11: 65-78.

Cowen PJ, Graham+Smith DC, Green AR, Heal DJ (1982) Beta adrenoceptor agonist enhance 5-hy droxyt~ptamine mediated behavioral re- sponses. Br j Pbarmac 76:265-270.

Delini-Stula A, Vassout A, Radeke E, Ortmann R Psychopharmacological profil of beta 2-receptor stimulants. Naunym-Schmiedebergs Forth Phar- mat 307:R65.

Friedman E, Cooper TB, Daliob A (1983) Effects of chronic antidepressant treatment on serotonin receptor activity in mice. Eurj P~armaco~~89:69- 76.

Gessner PK, Page IH (1962) Behavioral effects of 5

Methoxy-N,N dimethyltryptamine, ather tryp- tamines and LSD. Am f fhysioi 203:167.

Green AR, Heal DJ, Johnson P, Laurence BE, Nin- gaonkar VL (1983) Antidepressant treatments: ef- fects in rodents on dose-response CUN~S of 5- hydrox~~ptamine and dopamine behaviours and 5-HT2 receptor number in frontal cortex. Br / Pharmacol in press).

Matthews WD, Smith CD (1980) Pharmacological profile of a model for central serotonin receptor activation. fife Sci 26:1397-1403.

Nakamura M, Fukushima H (1978) Effect of 5,6 dih- ydroxytryptamine on the head twitches induced by 5 HTP, 5 HT, mescaline and fludiazepam in mice. I Pharm Pbarmaco 30:56-S&

Nimgaonkar VL, Green AR, Cowen PI, Heal Dj, Gra- hame-Smith DG, Deakin )FW (19831 Studies on the mecanism by clenbuterol, a beta adrenocep tor agonist, enhances 5HT mediated behavior and increases metabolism of SHT in the brain of the rat. Neuropharmacoiogy 22~739-749.

200 P. Martin et al.

Ortmann R, Martin S, Radeke E, Delini-Stula A (1981) Interaction of beta-adrenoceptor agonists with the serotoninergic system in rat brain. A be- havioral study using the L-5HTP syndrome. Nau- nym-Schmiedeberg’s Arch Pharmac 316:225-230.

Samamin A, Quattrone A, Consolo S, Ladinsky H, Algeri S (1978) Biochemical and pharmacological evidence of the interaction of serotonin with other aminergic systems in the brain. In Inter-

actions Between Putative Neurotransmitters in

the Brain. Eds. S Garattini, JF Pujol, and R Sa- manin. New York: Raven Press, pp. 383-399.

Suchowsky GK, Pegrassi L, Moretti A, Bonsignori A (1969) The effect of 4H-3 methylcarboxamide I-3 benzoxazine 2-one (F16654) on mono ami- noxidase and cerebral 5 HT. Arch Int Pharma-

codyn Ther 182:332-340.

Yap CY, Taylor DA (1983) Involvement of 5 HT2 re- ceptors in the wet-dog shake behavior induced by 5 hydroxytryptophan in the rat. Neurophar-

macology 22 : 801-804.