- Email: [email protected]
Further studies on the antagonism of stereotyped behaviour induced by amphetamine
EUROPEAN JOURNAL OF PHARMACOLOGY 8 (1969) 73-78. NORTH-HOLLAND PUBLISHING COMPANY, AMSTERDAM
FURTHER OF STEREOTYPED
STUDIES ON THE ANTAGONISM BEHAVIOUR
INDUCED
BY AMPHETAMINE
J. DEL RIO and J. A. FUENTES Department of Medical Chemistry, Institute of Organic Chemistry, C.S.L C., Madrid 6, Spain
Received 30 May 1969
Accepted 26 June 1969
J.DEL RIO, J.A.FUENTES, Further stztdies on the antagonism of stereotyped behaviour induced by amphetamine, European J. Pharmacol. 8 (1969) 73-78. High doses of d-amphetamine cause stereotyped behaviour in rats and an absence of many normal activities such as exploratory motor activity. The antagonism of these two effects of amphetamine was studied by pretreatment with several neuroleptic, adrenolytic and cholinergic drugs. It was found that some neuroleptic drugs antagonized, within a certain dose range, abnormal behaviour with a concomitant increase of exploratory motor activity. The most active drugs in this sense were chlorpromazine and perphenazine. No significant effect was observed with the adrenolytic or cholinergic drugs studied. Some possible implications of the motor reversal by neuroleptic drugs are discussed. d-amphetamine
Stereotyped behaviour
1. INTRODUCTION The amphetamine-induced stereotyped behaviour of the white rat is usually considered as an experimental model of psychotic behaviour and has also been described in man (Randrup et al., 1963; Randrup and Munkvad, 1967). Inhibition of this behaviour is regarded as a very selective test for screening o f neuroleptic activity (Janssen et al., 1965; Janssen et al., 1967). The outstanding features o f this abnormal behaviour are continuous, compulsive and purposeless movements of the mouth and the tongue, i.e. gnawing, licking or biting o f the cage wire netting, the forepaws or the abdominal skin, and an absence o f normal responsiveness towards the experimenter. On the other hand, many normal activities o f the rat, i.e. locomotion, rearing, grooming, are strongly decreased or absent. In a series o f routine experiments which were undertaken in this laboratory in order to examine the antagonism by neuroleptic drugs to this stereotyped behaviour, it was noticed that chlorpromazine was
Neuroleptic drugs
Exploratory motor activity
not only able to antagonize the abnormal behaviour, but also caused a reversal of normal exploratory motor activity which was blocked by the high dose of amphetamine used for inducing stereotypy. Low doses o f perphenazine have been reported to inhibit the abnormal behaviour of the rat while increasing some normal activities such as grooming and locomotion (Randrup and Munkvad, 1965). Since different neuroleptic drugs induce a reversal o f normal motor activities, it was o f interest to examine in more detail some o f the most representative phenothiazines and butyrophenones, as well as some adrenolytic and cholinergic stimulant drugs which are known to a ~ a g o nize certain other effects of amphetamine (see discussion).
2. MATERIAL AND METHODS The subjects were male rats from a Wistar-derived strain, weighing from 170 to 230 g. Experiments were always conducted at the same time o f day, beginning
74
J.DEL RIO and J.A.FUENTES
at I0 a.m. Five animals were placed in a wire cage measuring 35 X 25 X 22 cm and the treatment for each of these animals was assigned at random and was unknown to the observer. Motor activity was measured in an actophotometer similar to that described by Tedeschi et al. (1964)in which the chamber only permits up and down movements of the animals, but not movements from place to place. This actograph registers a very important form of exploratory activity and is specially appropriate for quantifying the effects of psychomotor stimulant drugs. The design of the experiments was as follows. A given dose of a given drug was administered i.p. and was followed 30 rain later by the injection of damphetamine sulphate by the same route. Motor activity was measured 30 min after amphetamine for a period of 15 rain. After being removed from the chamber, the animals were left undisturbed in their cage for 15 min, and after this time, i.e. 1 hr after amphetamine, the degree of stereotyped behaviour was assessed by a trained observer for a period of 5 min. The stereotyped activity was scored by means of a modification of the scoring system of Janssen et al. (1967): Stereotypy score 3: High intensity and continuous sniffing, gnawing, licking or biting. No responsiveness towards the experimenter. Stereotypy score 2: Moderate intensity and continuous sniffing, gnawing, licking or biting. Very slight or no responsiveness towards the experimenter. Stereotypy score 1: Moderate and non continuous sniffing, gnawing, licking or biting. Responsiveness towards the experimenter. Stereotypy score 0: no stereotyped behaviour at all. Each experimental drug was studied at various geometrically spaced dose levels, each dose being given to three or four groups of five rats in three or four different days. The volume injected was always 2.5 ml/kg. The following drugs were used (all doses refer to the salts or to the respective preparations): d-amphetamine sulphate, Barcia, Spain; chlorpromazine hydrochloride, Barcia, Spain; perphenazine dihydrochloride, Trilafon@, Sobering: thioproperazine bis-
methansulphonate, Majeptil®, Specia; thioridazine hydrochloride, Melleril®, Sandoz; promazine hydrochloride, Barcia, Spain; haloperidol® base, Latino; dehydrobenzperidol® base, Latino; triperidol® hydrochloride, Latino; phentolamine methansulphonate, Regitina@, Ciba; phenoxybenzamine hydrochloride, Smith, Kline & French; propranotol hydrochloride, Sumial®, ICI-Farma; pilocarpine hydrochloride, Barcia, Spain; atropine N-methyl bromide, Barcia, Spain; oxotremorine sesquifumarate, Aldrich. Two non-parametric statistical tests were used for calculating the significance of the difference between the rats treated with saline plus amphetamine and the rats treated with a given dose of a given drug plus amphetamine. Each of the treated rats was paired with the control rat of the corresponding experiment. These two tests were the sign test and the Wilcoxon matched-pairs signed-ranks test (Siegel, 1956). The significance was tested at the 0.01 and 0.05 level.
3. RESULTS 3.1. Motor activity and stereotyped behaviour with increasing doses of amphetamine Preliminary work was carried out to assess an adequate dose of d-amphetamine which could induce intense continuous abnormal behaviour in the strain of
S.B. -3
M.A. 180150 -
//'l¢
120906030O-
DOSE
d-AMPHETAMINE
(rng/kg)
Fig. 1. Mean scores of motor activity (c>-----o) and stereotyped behaviour (o------~) with increasing doses of d-amphetamine. M.A. = motor activity, counts per 15-min session. S.B. = stereotyped behaviour one hour after amphetamine. o tB No statistical difference detected between controls and treated groups, om 0.05 > p >0.01. • N p <0.01.
ANTAGONISM OF AMPHETAMINE STEREOTYPED BEHAVIOUR
M.A.
S.B.
M.A.
S.B.
M.A.
60"
60
SO-
50-
50
40-
40-
40
30-
30"
3O
20-
20-
2O
I0-
I0"
I0
O"
0
60-
o_ . . . .
75
O-
0
6 05
i
i
i
~
M.A.
I
0
6 o.iz o,z~o~o DOSE PERPHENAZINE (mg/kg)
S.B.
tr_~.a.....~
S.R.
"3
i~,
DOSE CHLORPROMAZINE (mg/kg)
60.
75
M.A.
-3
60
-2
4O
i
2
DOSE THIOPROPERAZINE ( m g / k g }
S.B.
M.A.
S.B.
-3
o_._. ~____..t~"
5O
50',,
40-
30-
30"
20-
20
IL
JI.
•
\
\
I0'
mo"0
O-
.o
0
6
DOSE
DOSE THIORIDAZINE (mg/kg) M.A.
i
S.B.
60"
-3
40-
40-
30-
30I
le
......
3k e'4
DOSE HALOPERIDOL (mg/kg) S.S. "3
\ "2
20"
-I
I0-
O-
0
6 ohs o.b6 o;,a o;zs o~o ;
2
DOSE DEHYDROBENZPERIDOL (mg/kg)
0
6 om oh6 o~z ~25 o~o i
PROMAZINE ( m g / k g }
M.A. 50
I0"
fi
60"
50-
20"
,i
O-
-0
b o',o3do6o~,2o;25 o,5o i DOSE TRIPERIDOL ( m g / k g )
rat used. Results (fig. 1) showed that a dose of 2.5 mg/kg elicited maximum motility, while a dose of 10 mg/kg induced a high degree of intense stereotyped activity and an extremely low exploratory motor activity. It was decided to use this dose in succesive experiments since the results obtained with 7.5 mg/kg were rather irregular. Throughout the numerous experiments performed, it was also possible to check the innocuity of the highest dose used in normal conditions of temperature ( 1 8 - 25 °C). 3.2. Effect of neuroleptic drugs on stereotyped behaviour Eight neurolepflc drugs were used, five phenothiazines with aliphatic, piperazinic or piperidinic side
i
Fig. 2. Mean scores obtained with different doses of several neuroleptic drugs plus d-amphetamine 10 mg/kg. M.A. = motor activity (o o), counts per 15-rain session. S.B. = stereotyped behaviour (D o) one hour after amphetamine, o D No statistical difference detected between amphetamine controls and treated groups. ®t3 0.05 > p > 0.01. elp
chains and three butyrophenones. The results are given in fig. 2. It may be seen that chlorpromazine ( 2 - 8 mg/kg) and perphenazine ( 0 . 1 2 - 0 . 5 0 mg/kg) reversed very significantly (p <0.01) the exploratory motor activity which was almost completely abolished by the high dose of d-amphetamine in saline-pretreated animals. Psychomotor stimulation was especially prominent with a dose of 2 mg/kg of chlorpromazine. Both drugs decreased concomitantly the intensity of stereotyped behaviour down to a level below stereotypy score 1. Thioproperazine and thioridazine were also active within a narrow dose range (0.25-0.50 and 1 6 - 3 2 mg/kg respectively) but with a lesser degree of significance. Higher doses of these four phenothiazines completely abolished the stereotyped behav-
76
J.DEL RIO and J.A.FUENTES Table l Effect of pretreatment with adrenolytic and cholinergic drugs on rats injected with 10 mg/kg of d-amphetamine. Drug investigated (dose range mg/kg)
Reversal of exploratory motor activity (dose)
Stereotypy score at the highest dose used (mean)
Phentolamine (4-32) Phenoxybenzamine (2-32) Propranolol (4- 32)
None None 32 mg/kg
2.2 2 1.4
Pilocarpine (4-32) Atropine methylbromide (20 mg/kg) plus pilocarpine (4-32) * Oxotremorine (0.06--0.50)
None
2.6
None
2.6
None
2.1
Gross observation of behaviour changes Occasional fighting Stereotypy alternating with quick rotational movements in the cage. High irritability. Tremors. Backward locomotion.
Superimposition of central effects of oxotremorine.
* Atropine was injected 30 minutes before pilocarpine.
iour but did not induce any reversal of motor activity. Finally, promazine did not induce any reversal of motor activity nor did it effectively block the stereotyped behaviour. With respect to butyrophenones, the phenomenon was clearly observed (p < 0.05) with dehydrobenzperidol (0.06-0.5 mg/kg) and haloperidol (0.06-0.25 mg/kg), triperidol (0.12-0.25 mg/kg) being the least effective of the three drugs used in its ability to elicit motor activity reversal. As in the case of phenothiazines, higher doses inhibited abnormal behaviour without reversing exploratory motor activity. 3.3. Effect of other drugs on stereotyped behaviour The results obtained with three adrenolytic drugs (the alpha-blockers phentolamine and phenoxybenzamine, and the beta-blocker propranolol), two centrally-acting cholinergic stimulant drugs (pilocarpine and oxotremorine) and one mixture of quaternary atropine plus pilocarpine, are summarized in table 1.
4. DISCUSSION This work confirms earlier data on the specificity of the antagonism by neuroleptic drugs of amphetamine-induced stereotyped behaviour. Of all the neuroleptic drugs tested, only promazine was not effec-
tive, a fact which is in correlation with the weak actions reported for this phenothiazine in clinical studies (Hollister, 1968). Within certain dose ranges all the neuroleptic drugs tried, with the exception of promazine, reversed the block of exploratory motor activity produced by a high dose of d-amphetamine sulphate. This reversal of motor activity was particularly significant with chlorpromazine and perphenazine, dehydrobenzperidol being the most active butyrophenone tested. There is good evidence that this stereotyped behaviour is caused by a release of endogenous dopamine (Ernst, 1967; Scheel-Kriiger and Randrup, 1967), and its antagonism by neuroleptic drugs is generally understood as a blocking action by the drug at the level of brain dopaminergic receptors (Janssen and Allewijn, 1968). However, many neuroleptic drugs antagonize abnormal behaviour while only some of them seem to elicit also a definite and clearcut reversal of motor activity. The most active neuroleptic drugs in this sense are chlorpromazine and perphenazine, both of them being recognized antidepressants; chlorpromazine is used in the treatment of depressions where agitation and anxiety are prominent (Miller, 1967) while perphenazine is commonly used in combination with amitriptyline (Triavil®, Etrafon®) in the therapy of reactive, anxiety and psychotic depressions. On the other hand, it has been recently reported (Sulser and Dingell, 1968; Borella
ANTAGONISM OF AMPHETAMINESTEREOTYPED BEHAVIOUR et al., 1969) that chlorpromazine, in doses similar to those used in these experiments, causes a marked and prolonged rise in the levels of brain amphetamine which are correlated with an enhancement of motor activity, effects which may be regarded as typical of tricyclic antidepressant drugs (Sulser et al., 1966). Animal models are still relatively inadequate for specific evaluation of the antipsychotic and antidepressant actions of drugs, and this test could perhaps afford some guide to the possible usefulness of new neuroleptic drugs for the treatment of certain forms of depression. The alpha-adrenergic blocking agents used in these tests have been reported to antagonize certain effects of amphetamine, especially toxicity in aggregated mice (Moore, 1964; Mennear and Rudzik, 1965; Boissier et al., 1968), but no effect has been shown against amphetamine-induced abnormal behaviour (Randrup et al., 1963; Herman, 1967). In close agreement with these findings, we were not able to observe any reversal of motor activity or antagonism of abnormal behaviour by phentolamine or phenoxybenzamine. Propranolol, a beta-adrenergic blocking drug, has been reported to antagonize some effects of amphetamine, i.e. hyperthermia, lethality and increased spontaneous activity (Grana and Sossi, 1967; Mantegazza et al., 1968). Propranolol was also reported to diminish the mean duration of abnormal behaviour (Herman, 1967), We estimate that the antagonism found in our experiments at a dose o f 32 mg/kg cannot be taken as a significant result since 64 mg/kg is lethal to a high percentage of animals and doses below 32 mg/kg fail to elicit any effect. The existence in the brain of a dopaminergiccholinergic balance which influences motor activity and stereotypy has been suggested (Arnfred and Randrup, 1968) and the increase in the locomotor activity of mice induced by amphetamine has been found to diminish when the animals are pretreated with a peripheral-acting quaternary atropine plus pilocarpine (Proctor et al., 1967). Mennear (1965) also found a reduction by tremorine in the effect of amphetamine on motor activity. Our experiments have failed to show any antagonism of the stereotyped behaviour by the central cholinergic stimulants pilocarpine, oxotremorine or quaternary atropine plus pilocarpine; a certain superimposition of the effects
77
of these drugs on the amphetamine stereotypy has always been observed, and this makes the observation of the animal more difficult and obscure the results.
ACKNOWLEDGEMENTS The skilful technical assistance of Miss R.M.Caminos, Miss M.L.Toro and Miss M.C.Urdin is gratefully acknowledged. For generous gifts of perphenazine, haloperidol and propranolol, we thank the Schering Corp. USA, the Inst. Farmac. Latino and the ICI-Farma, respectively.
REFERENCES Arnfred, T. and A.Randrup, 1968, Cholinergic mechanism in brain inhibiting amphetamine-induced stereotyped behaviour, Acta Pharmacol. Toxicol. 26,384. Boissier, J.R., P.Simon and J.F.GiudiceUi, 1968, Effets centraux de quelques substances adr6no et/ou sympatholytiques; Ill. Ptosis, catalepsie, antagonisme vis-a-visde l'apomorphine et de l'amph6tamine, Arch. Intern. Pharmacodyn. Ther. 171, 68. Borella, L., F.Herr and A.Wojdan, 1969, Prolongation of certain effects of amphetamine by chlorpromazine, Can. J. Physiol. Pharmacol. 47, 7. Ernst, A.M., 1967, Mode of action of apomorphine and dexamphetamine on gnawing compulsion in rats, Psychopharmacologia 10, 316. Grana, E. and D.Sossi, 1967, Ricerche sull'attivit~ centrale di composti ad attivit~/3-simpatolitica, Farmaco Ed. Sci. 22, 582. Herman, Z.S., 1967, Influence of some psychotropic and adrenergic blocking agents upon amphetamine stereotyped behaviour in white rats, Psychopharmacologia 1l, 136. Hollister, L.E., 1968, Human pharmacology of antipsychotic and antidepressant drugs, Ann. Rev. Pharmacol. 8,491. Janssen, P.A.J., C.J.E.Niemegeers and K.H.L.Schellekens, 1965, Is it possible to predict the clinical effects of neuroleptic drugs (major tranquillizers) from animal data?, Part I, Arzneimittel-Forsch. 15, 104. Janssen, P.A.J., C.J.E.Niemegeers, K.H.L.Schellekens and F.M.Lenaerts, 1967, Is it possible to predict the clinical effects of neuroleptic drugs (major tranquillizers) from animal data?, Part IV, Arzneimittel-Forsch. 17,841. Janssen, P.A.J. and F.T.N.Allewijn, 1968, Pimozide, a chemically novel, highly potent and orally long-acting neuroleptic drug, Part I1, Arzneimittel-Forsch. 18,279. Mantegazza, P., K.B.Naimzada and M.Riva, 1968, Effects of propranolol on some activities of amphetamine, European J. Pharmacol. 4, 25. Mennear, J.H., 1965, Interactions between central cholinergic agents and amphetamine in mice, Psychopharmacologia 7, 107.
78
J.DEL RIO and J.A.FUENTES
Mennear, J.H. and A.D.Rudzik, 1965, The effects of alpha and beta adrenergic blockade on the lethality of amphetamine in aggregated mice, Life Sci. 4, 1425. Miller, H., 1967, DEpression, Brit. Med. J. 1,257. Moore, K.E., 1964, The role of endogenous norepinephrine in the toxicity of d-amphetamine in aggregated mic~, J. Pharmacol. Exp. Ther. 144, 45. Proctor, C.D., J.L.Potts, L.G.Ashley and B.A.Denefield, 1967, Pilocarpine reversal of d-amphetamine induced increase in mouse exploratory locomotor activity, Arch. Int. Pharmacodyn. 167, 61. Randrup, A., l.Munkvad and P.Udsen, 1963, Adrenergic mechanisms and amphetamine induced abnormal behaviour, Acta Pharmacol. Toxicol. 20, 145. Randrup, A. and l.Munkvad, 1965, Special antagonism of amphetamine-induced abnormal behaviour, Psychopharmacologia 7,416.
Randrup, A. and l.Munkvad, 1967, Stereotyped activities produced by amphetamine in several animal species and man, Psychopharmacologia 11,300. ScheeI-Kriiger, J. and A.Randrup, 1967, Stereotype hyperactive behaviour produced by dopamine in the absence of noradrenaline, Life Sci. 6, 1389. Siegel, S., 1956, In: Non parametric statistics for the behavioral sciences (McGraw-Hill, New York) 68-83. Sulser, F., M.L.Owens and J.V.Dingell, 1966, On the mechanism of amphetamine potentiation by desipramine, Life Sci. 5, 2005. Sulser, F. and J.V.Dingell, 1968, Potentiation and blockade of the central actions of amphetamine by chlorpromazine, Biochem. Pharmacol. 17,634. Tedeschi, D.H., P.J.Fowler, W.H.Cromley, J.F.Pauls, R.Z.Eby and E.J.Fellows, 1964, Effects of centrally acting drugs on confinement motor activity, J. Pharm. Sci. 53, 1046.
FURTHER OF STEREOTYPED
STUDIES ON THE ANTAGONISM BEHAVIOUR
INDUCED
BY AMPHETAMINE
J. DEL RIO and J. A. FUENTES Department of Medical Chemistry, Institute of Organic Chemistry, C.S.L C., Madrid 6, Spain
Received 30 May 1969
Accepted 26 June 1969
J.DEL RIO, J.A.FUENTES, Further stztdies on the antagonism of stereotyped behaviour induced by amphetamine, European J. Pharmacol. 8 (1969) 73-78. High doses of d-amphetamine cause stereotyped behaviour in rats and an absence of many normal activities such as exploratory motor activity. The antagonism of these two effects of amphetamine was studied by pretreatment with several neuroleptic, adrenolytic and cholinergic drugs. It was found that some neuroleptic drugs antagonized, within a certain dose range, abnormal behaviour with a concomitant increase of exploratory motor activity. The most active drugs in this sense were chlorpromazine and perphenazine. No significant effect was observed with the adrenolytic or cholinergic drugs studied. Some possible implications of the motor reversal by neuroleptic drugs are discussed. d-amphetamine
Stereotyped behaviour
1. INTRODUCTION The amphetamine-induced stereotyped behaviour of the white rat is usually considered as an experimental model of psychotic behaviour and has also been described in man (Randrup et al., 1963; Randrup and Munkvad, 1967). Inhibition of this behaviour is regarded as a very selective test for screening o f neuroleptic activity (Janssen et al., 1965; Janssen et al., 1967). The outstanding features o f this abnormal behaviour are continuous, compulsive and purposeless movements of the mouth and the tongue, i.e. gnawing, licking or biting o f the cage wire netting, the forepaws or the abdominal skin, and an absence o f normal responsiveness towards the experimenter. On the other hand, many normal activities o f the rat, i.e. locomotion, rearing, grooming, are strongly decreased or absent. In a series o f routine experiments which were undertaken in this laboratory in order to examine the antagonism by neuroleptic drugs to this stereotyped behaviour, it was noticed that chlorpromazine was
Neuroleptic drugs
Exploratory motor activity
not only able to antagonize the abnormal behaviour, but also caused a reversal of normal exploratory motor activity which was blocked by the high dose of amphetamine used for inducing stereotypy. Low doses o f perphenazine have been reported to inhibit the abnormal behaviour of the rat while increasing some normal activities such as grooming and locomotion (Randrup and Munkvad, 1965). Since different neuroleptic drugs induce a reversal o f normal motor activities, it was o f interest to examine in more detail some o f the most representative phenothiazines and butyrophenones, as well as some adrenolytic and cholinergic stimulant drugs which are known to a ~ a g o nize certain other effects of amphetamine (see discussion).
2. MATERIAL AND METHODS The subjects were male rats from a Wistar-derived strain, weighing from 170 to 230 g. Experiments were always conducted at the same time o f day, beginning
74
J.DEL RIO and J.A.FUENTES
at I0 a.m. Five animals were placed in a wire cage measuring 35 X 25 X 22 cm and the treatment for each of these animals was assigned at random and was unknown to the observer. Motor activity was measured in an actophotometer similar to that described by Tedeschi et al. (1964)in which the chamber only permits up and down movements of the animals, but not movements from place to place. This actograph registers a very important form of exploratory activity and is specially appropriate for quantifying the effects of psychomotor stimulant drugs. The design of the experiments was as follows. A given dose of a given drug was administered i.p. and was followed 30 rain later by the injection of damphetamine sulphate by the same route. Motor activity was measured 30 min after amphetamine for a period of 15 rain. After being removed from the chamber, the animals were left undisturbed in their cage for 15 min, and after this time, i.e. 1 hr after amphetamine, the degree of stereotyped behaviour was assessed by a trained observer for a period of 5 min. The stereotyped activity was scored by means of a modification of the scoring system of Janssen et al. (1967): Stereotypy score 3: High intensity and continuous sniffing, gnawing, licking or biting. No responsiveness towards the experimenter. Stereotypy score 2: Moderate intensity and continuous sniffing, gnawing, licking or biting. Very slight or no responsiveness towards the experimenter. Stereotypy score 1: Moderate and non continuous sniffing, gnawing, licking or biting. Responsiveness towards the experimenter. Stereotypy score 0: no stereotyped behaviour at all. Each experimental drug was studied at various geometrically spaced dose levels, each dose being given to three or four groups of five rats in three or four different days. The volume injected was always 2.5 ml/kg. The following drugs were used (all doses refer to the salts or to the respective preparations): d-amphetamine sulphate, Barcia, Spain; chlorpromazine hydrochloride, Barcia, Spain; perphenazine dihydrochloride, Trilafon@, Sobering: thioproperazine bis-
methansulphonate, Majeptil®, Specia; thioridazine hydrochloride, Melleril®, Sandoz; promazine hydrochloride, Barcia, Spain; haloperidol® base, Latino; dehydrobenzperidol® base, Latino; triperidol® hydrochloride, Latino; phentolamine methansulphonate, Regitina@, Ciba; phenoxybenzamine hydrochloride, Smith, Kline & French; propranotol hydrochloride, Sumial®, ICI-Farma; pilocarpine hydrochloride, Barcia, Spain; atropine N-methyl bromide, Barcia, Spain; oxotremorine sesquifumarate, Aldrich. Two non-parametric statistical tests were used for calculating the significance of the difference between the rats treated with saline plus amphetamine and the rats treated with a given dose of a given drug plus amphetamine. Each of the treated rats was paired with the control rat of the corresponding experiment. These two tests were the sign test and the Wilcoxon matched-pairs signed-ranks test (Siegel, 1956). The significance was tested at the 0.01 and 0.05 level.
3. RESULTS 3.1. Motor activity and stereotyped behaviour with increasing doses of amphetamine Preliminary work was carried out to assess an adequate dose of d-amphetamine which could induce intense continuous abnormal behaviour in the strain of
S.B. -3
M.A. 180150 -
//'l¢
120906030O-
DOSE
d-AMPHETAMINE
(rng/kg)
Fig. 1. Mean scores of motor activity (c>-----o) and stereotyped behaviour (o------~) with increasing doses of d-amphetamine. M.A. = motor activity, counts per 15-min session. S.B. = stereotyped behaviour one hour after amphetamine. o tB No statistical difference detected between controls and treated groups, om 0.05 > p >0.01. • N p <0.01.
ANTAGONISM OF AMPHETAMINE STEREOTYPED BEHAVIOUR
M.A.
S.B.
M.A.
S.B.
M.A.
60"
60
SO-
50-
50
40-
40-
40
30-
30"
3O
20-
20-
2O
I0-
I0"
I0
O"
0
60-
o_ . . . .
75
O-
0
6 05
i
i
i
~
M.A.
I
0
6 o.iz o,z~o~o DOSE PERPHENAZINE (mg/kg)
S.B.
tr_~.a.....~
S.R.
"3
i~,
DOSE CHLORPROMAZINE (mg/kg)
60.
75
M.A.
-3
60
-2
4O
i
2
DOSE THIOPROPERAZINE ( m g / k g }
S.B.
M.A.
S.B.
-3
o_._. ~____..t~"
5O
50',,
40-
30-
30"
20-
20
IL
JI.
•
\
\
I0'
mo"0
O-
.o
0
6
DOSE
DOSE THIORIDAZINE (mg/kg) M.A.
i
S.B.
60"
-3
40-
40-
30-
30I
le
......
3k e'4
DOSE HALOPERIDOL (mg/kg) S.S. "3
\ "2
20"
-I
I0-
O-
0
6 ohs o.b6 o;,a o;zs o~o ;
2
DOSE DEHYDROBENZPERIDOL (mg/kg)
0
6 om oh6 o~z ~25 o~o i
PROMAZINE ( m g / k g }
M.A. 50
I0"
fi
60"
50-
20"
,i
O-
-0
b o',o3do6o~,2o;25 o,5o i DOSE TRIPERIDOL ( m g / k g )
rat used. Results (fig. 1) showed that a dose of 2.5 mg/kg elicited maximum motility, while a dose of 10 mg/kg induced a high degree of intense stereotyped activity and an extremely low exploratory motor activity. It was decided to use this dose in succesive experiments since the results obtained with 7.5 mg/kg were rather irregular. Throughout the numerous experiments performed, it was also possible to check the innocuity of the highest dose used in normal conditions of temperature ( 1 8 - 25 °C). 3.2. Effect of neuroleptic drugs on stereotyped behaviour Eight neurolepflc drugs were used, five phenothiazines with aliphatic, piperazinic or piperidinic side
i
Fig. 2. Mean scores obtained with different doses of several neuroleptic drugs plus d-amphetamine 10 mg/kg. M.A. = motor activity (o o), counts per 15-rain session. S.B. = stereotyped behaviour (D o) one hour after amphetamine, o D No statistical difference detected between amphetamine controls and treated groups. ®t3 0.05 > p > 0.01. elp
chains and three butyrophenones. The results are given in fig. 2. It may be seen that chlorpromazine ( 2 - 8 mg/kg) and perphenazine ( 0 . 1 2 - 0 . 5 0 mg/kg) reversed very significantly (p <0.01) the exploratory motor activity which was almost completely abolished by the high dose of d-amphetamine in saline-pretreated animals. Psychomotor stimulation was especially prominent with a dose of 2 mg/kg of chlorpromazine. Both drugs decreased concomitantly the intensity of stereotyped behaviour down to a level below stereotypy score 1. Thioproperazine and thioridazine were also active within a narrow dose range (0.25-0.50 and 1 6 - 3 2 mg/kg respectively) but with a lesser degree of significance. Higher doses of these four phenothiazines completely abolished the stereotyped behav-
76
J.DEL RIO and J.A.FUENTES Table l Effect of pretreatment with adrenolytic and cholinergic drugs on rats injected with 10 mg/kg of d-amphetamine. Drug investigated (dose range mg/kg)
Reversal of exploratory motor activity (dose)
Stereotypy score at the highest dose used (mean)
Phentolamine (4-32) Phenoxybenzamine (2-32) Propranolol (4- 32)
None None 32 mg/kg
2.2 2 1.4
Pilocarpine (4-32) Atropine methylbromide (20 mg/kg) plus pilocarpine (4-32) * Oxotremorine (0.06--0.50)
None
2.6
None
2.6
None
2.1
Gross observation of behaviour changes Occasional fighting Stereotypy alternating with quick rotational movements in the cage. High irritability. Tremors. Backward locomotion.
Superimposition of central effects of oxotremorine.
* Atropine was injected 30 minutes before pilocarpine.
iour but did not induce any reversal of motor activity. Finally, promazine did not induce any reversal of motor activity nor did it effectively block the stereotyped behaviour. With respect to butyrophenones, the phenomenon was clearly observed (p < 0.05) with dehydrobenzperidol (0.06-0.5 mg/kg) and haloperidol (0.06-0.25 mg/kg), triperidol (0.12-0.25 mg/kg) being the least effective of the three drugs used in its ability to elicit motor activity reversal. As in the case of phenothiazines, higher doses inhibited abnormal behaviour without reversing exploratory motor activity. 3.3. Effect of other drugs on stereotyped behaviour The results obtained with three adrenolytic drugs (the alpha-blockers phentolamine and phenoxybenzamine, and the beta-blocker propranolol), two centrally-acting cholinergic stimulant drugs (pilocarpine and oxotremorine) and one mixture of quaternary atropine plus pilocarpine, are summarized in table 1.
4. DISCUSSION This work confirms earlier data on the specificity of the antagonism by neuroleptic drugs of amphetamine-induced stereotyped behaviour. Of all the neuroleptic drugs tested, only promazine was not effec-
tive, a fact which is in correlation with the weak actions reported for this phenothiazine in clinical studies (Hollister, 1968). Within certain dose ranges all the neuroleptic drugs tried, with the exception of promazine, reversed the block of exploratory motor activity produced by a high dose of d-amphetamine sulphate. This reversal of motor activity was particularly significant with chlorpromazine and perphenazine, dehydrobenzperidol being the most active butyrophenone tested. There is good evidence that this stereotyped behaviour is caused by a release of endogenous dopamine (Ernst, 1967; Scheel-Kriiger and Randrup, 1967), and its antagonism by neuroleptic drugs is generally understood as a blocking action by the drug at the level of brain dopaminergic receptors (Janssen and Allewijn, 1968). However, many neuroleptic drugs antagonize abnormal behaviour while only some of them seem to elicit also a definite and clearcut reversal of motor activity. The most active neuroleptic drugs in this sense are chlorpromazine and perphenazine, both of them being recognized antidepressants; chlorpromazine is used in the treatment of depressions where agitation and anxiety are prominent (Miller, 1967) while perphenazine is commonly used in combination with amitriptyline (Triavil®, Etrafon®) in the therapy of reactive, anxiety and psychotic depressions. On the other hand, it has been recently reported (Sulser and Dingell, 1968; Borella
ANTAGONISM OF AMPHETAMINESTEREOTYPED BEHAVIOUR et al., 1969) that chlorpromazine, in doses similar to those used in these experiments, causes a marked and prolonged rise in the levels of brain amphetamine which are correlated with an enhancement of motor activity, effects which may be regarded as typical of tricyclic antidepressant drugs (Sulser et al., 1966). Animal models are still relatively inadequate for specific evaluation of the antipsychotic and antidepressant actions of drugs, and this test could perhaps afford some guide to the possible usefulness of new neuroleptic drugs for the treatment of certain forms of depression. The alpha-adrenergic blocking agents used in these tests have been reported to antagonize certain effects of amphetamine, especially toxicity in aggregated mice (Moore, 1964; Mennear and Rudzik, 1965; Boissier et al., 1968), but no effect has been shown against amphetamine-induced abnormal behaviour (Randrup et al., 1963; Herman, 1967). In close agreement with these findings, we were not able to observe any reversal of motor activity or antagonism of abnormal behaviour by phentolamine or phenoxybenzamine. Propranolol, a beta-adrenergic blocking drug, has been reported to antagonize some effects of amphetamine, i.e. hyperthermia, lethality and increased spontaneous activity (Grana and Sossi, 1967; Mantegazza et al., 1968). Propranolol was also reported to diminish the mean duration of abnormal behaviour (Herman, 1967), We estimate that the antagonism found in our experiments at a dose o f 32 mg/kg cannot be taken as a significant result since 64 mg/kg is lethal to a high percentage of animals and doses below 32 mg/kg fail to elicit any effect. The existence in the brain of a dopaminergiccholinergic balance which influences motor activity and stereotypy has been suggested (Arnfred and Randrup, 1968) and the increase in the locomotor activity of mice induced by amphetamine has been found to diminish when the animals are pretreated with a peripheral-acting quaternary atropine plus pilocarpine (Proctor et al., 1967). Mennear (1965) also found a reduction by tremorine in the effect of amphetamine on motor activity. Our experiments have failed to show any antagonism of the stereotyped behaviour by the central cholinergic stimulants pilocarpine, oxotremorine or quaternary atropine plus pilocarpine; a certain superimposition of the effects
77
of these drugs on the amphetamine stereotypy has always been observed, and this makes the observation of the animal more difficult and obscure the results.
ACKNOWLEDGEMENTS The skilful technical assistance of Miss R.M.Caminos, Miss M.L.Toro and Miss M.C.Urdin is gratefully acknowledged. For generous gifts of perphenazine, haloperidol and propranolol, we thank the Schering Corp. USA, the Inst. Farmac. Latino and the ICI-Farma, respectively.
REFERENCES Arnfred, T. and A.Randrup, 1968, Cholinergic mechanism in brain inhibiting amphetamine-induced stereotyped behaviour, Acta Pharmacol. Toxicol. 26,384. Boissier, J.R., P.Simon and J.F.GiudiceUi, 1968, Effets centraux de quelques substances adr6no et/ou sympatholytiques; Ill. Ptosis, catalepsie, antagonisme vis-a-visde l'apomorphine et de l'amph6tamine, Arch. Intern. Pharmacodyn. Ther. 171, 68. Borella, L., F.Herr and A.Wojdan, 1969, Prolongation of certain effects of amphetamine by chlorpromazine, Can. J. Physiol. Pharmacol. 47, 7. Ernst, A.M., 1967, Mode of action of apomorphine and dexamphetamine on gnawing compulsion in rats, Psychopharmacologia 10, 316. Grana, E. and D.Sossi, 1967, Ricerche sull'attivit~ centrale di composti ad attivit~/3-simpatolitica, Farmaco Ed. Sci. 22, 582. Herman, Z.S., 1967, Influence of some psychotropic and adrenergic blocking agents upon amphetamine stereotyped behaviour in white rats, Psychopharmacologia 1l, 136. Hollister, L.E., 1968, Human pharmacology of antipsychotic and antidepressant drugs, Ann. Rev. Pharmacol. 8,491. Janssen, P.A.J., C.J.E.Niemegeers and K.H.L.Schellekens, 1965, Is it possible to predict the clinical effects of neuroleptic drugs (major tranquillizers) from animal data?, Part I, Arzneimittel-Forsch. 15, 104. Janssen, P.A.J., C.J.E.Niemegeers, K.H.L.Schellekens and F.M.Lenaerts, 1967, Is it possible to predict the clinical effects of neuroleptic drugs (major tranquillizers) from animal data?, Part IV, Arzneimittel-Forsch. 17,841. Janssen, P.A.J. and F.T.N.Allewijn, 1968, Pimozide, a chemically novel, highly potent and orally long-acting neuroleptic drug, Part I1, Arzneimittel-Forsch. 18,279. Mantegazza, P., K.B.Naimzada and M.Riva, 1968, Effects of propranolol on some activities of amphetamine, European J. Pharmacol. 4, 25. Mennear, J.H., 1965, Interactions between central cholinergic agents and amphetamine in mice, Psychopharmacologia 7, 107.
78
J.DEL RIO and J.A.FUENTES
Mennear, J.H. and A.D.Rudzik, 1965, The effects of alpha and beta adrenergic blockade on the lethality of amphetamine in aggregated mice, Life Sci. 4, 1425. Miller, H., 1967, DEpression, Brit. Med. J. 1,257. Moore, K.E., 1964, The role of endogenous norepinephrine in the toxicity of d-amphetamine in aggregated mic~, J. Pharmacol. Exp. Ther. 144, 45. Proctor, C.D., J.L.Potts, L.G.Ashley and B.A.Denefield, 1967, Pilocarpine reversal of d-amphetamine induced increase in mouse exploratory locomotor activity, Arch. Int. Pharmacodyn. 167, 61. Randrup, A., l.Munkvad and P.Udsen, 1963, Adrenergic mechanisms and amphetamine induced abnormal behaviour, Acta Pharmacol. Toxicol. 20, 145. Randrup, A. and l.Munkvad, 1965, Special antagonism of amphetamine-induced abnormal behaviour, Psychopharmacologia 7,416.
Randrup, A. and l.Munkvad, 1967, Stereotyped activities produced by amphetamine in several animal species and man, Psychopharmacologia 11,300. ScheeI-Kriiger, J. and A.Randrup, 1967, Stereotype hyperactive behaviour produced by dopamine in the absence of noradrenaline, Life Sci. 6, 1389. Siegel, S., 1956, In: Non parametric statistics for the behavioral sciences (McGraw-Hill, New York) 68-83. Sulser, F., M.L.Owens and J.V.Dingell, 1966, On the mechanism of amphetamine potentiation by desipramine, Life Sci. 5, 2005. Sulser, F. and J.V.Dingell, 1968, Potentiation and blockade of the central actions of amphetamine by chlorpromazine, Biochem. Pharmacol. 17,634. Tedeschi, D.H., P.J.Fowler, W.H.Cromley, J.F.Pauls, R.Z.Eby and E.J.Fellows, 1964, Effects of centrally acting drugs on confinement motor activity, J. Pharm. Sci. 53, 1046.