- Email: [email protected]
EFFECT OF ANTICONVULSANTS ON THALAMIC AFTERDISCHARGE IN RATS AND CATS
EFFECT
OF
NEUROLEPTIC
BEHAVIOR
DRUGS
AFTER
a-METHYLTYROSINE Makoto
THE
CONDITIONED
PRETREATMENT
OR
OKA, Chiaki
ON
WITH
p-CHLOROPHENYLALANINE*
KAMEI
and Masanao
SHIMIZU
Department of Pharniacologv, Research Laboratories, Dainippon Pharmaceutical Co.. Ltd., Suita ski, Osaka 564, Japan Accepted August 1, 1977
Abstract-Effects of psychotropic drugs on the conditioned behavior after pretreatment with a-rnethyltyrosine(a-MT) or p-chlorophenylalanine(PCPA) were studied using operant conditioning technique in rodents. The operant conditioning procedure used was composed of a concurrent milk-reinforced and avoidance schedule in an operant conditioning chamber with two levers where lever pressing by rats was maintained by milk (VR 6) and electric shock (intertrial interval II min). Chlorpromazine (1 mg/kg i.p.), perphenazine (0.1 mg/kg i.p.), haloperidol (0.05 mg/kg i.p.) and oxypertine (0.25 mg/kg i.p.), at half of the maximum ineffective dose, respectively, caused a significant decrease in both lever pressings after pretreatment with a-MT (20 mg/kg i.p.). Such potentiating effect by a-MT was not observed with diazepam and nortriptyline. PCPA pretreatment (300 mg/kg i.p.) did not influence suppressive effects on responding under both schedules by any drug used. The one-way active avoidance procedure in mice was also employed in this study. On the avoidance response, chlorpromazine, per phenazine, haloperidol, trifluperidol, clozapine and oxypertine showed a suppressive effect, ED50 values of which were considerably lowered by pretreatment with a-MT (25 mg/kg i.p.). No influence was seen with PCPA (200 mg/kg i.p. x 2). Diazepam, phenoxybenzamine, phentolarnine, arecoline, physostigmine and clonidine also had a suppressive effect on the avoidance response, which was not influenced by pretreatment with a-MT. These results suggest that the suppressive effect of neuroleptics on the conditioned behavior is selectively potentiated by (t-MT, and not influenced by PCPA. Ahlenius reinforced
and Engel (1) reported
lever pressing
that
was markedly
the suppressive
potentiated
by pretreatment
(a-MT), a tyrosine hydroxylase inhibitor. Neuroleptics in catecholamine turnover presumably as compensation (2).
This suggests
that
haloperidol-induced
the potentiation
increase
with a-MT
in catecholamine
neurotransmitter
available
such potentiation
with a-MT was also observed
to compete
thioridazine
(3), the cataleptogenic
haloperidol
and pimozide
with a-MT
is specific in neuroleptics
On the other hand, serotonin, has been considered
with a-methyltyrosine
may be due to an inhibition
turnover,
thereby
reducing
for receptor
sites.
behavior
has, however,
(5).
(4) and the suppressive Whether
of
Afterward, and
effect of
or not such potentiation
not been given attention.
amine which exists in various areas of the brain,
to play some role in brain function
relating
to the conditioned
*These data were presented at the 49th General Meeting of the Japanese Pharmacological (March, 1976).
of the
the amount
in the clinical effect of chlorpromazine
effect of haloperidol
another
on food
are known to induce an increase for their receptor blocking activity
with the neuroleptic
on tail-pinch
effect of haloperidol
behavior Society
(6--9).
However,
little work has been done to assess the role of* serotonin
in the action
of
neuroleptics. The present experiment
was designed
ment with (t-MT was observed behavioral
procedures
to determine
generally
in neuroleptics
used were a concurrent
rats (10) and an one-way active avoidance with p-chlorophenylalaniuue
(PUPA),
milk-reinforced
Male Wistar were maintained
a tryptophan
The
HLA strain
operant
and avoidance
Gamalero
(10).
weighing
procedure
hydroxylase
The
schedule
in
The effect of pretreatment
inhibitor,
%vasalso examined.
ill rats
180-220
g at the beginning
of the training,
80 ° of their free feeding weights. used was composed
schedule by a modification
The experimental
and avoidance
AND METHODS
diet at about
conditioning
reinforced
chamber
rats,
by pretreat
and; or only in neuroleptics.
in mice (I 1).
aitd ar'oidcurce procedure
on a restricted
the potentiation
milk-reinforced
schedule
11A !TRIALS Concurrcirt
whether
chamber
of the concurrent
of the method
of Molinengo
was the Grason-Stadler
operant
milk
and Ricci conditioning
for rats which had a milk tray on the center of a side wall and two levers on both
sides of the tray. Food-deprived reinforced
rats were trained
(AC current
of 0.2 mA for I sec).
(VR 6), from a minimum ment after schedule
10 responses, (intertrial
lever during
of I reinforcement
sequence
Although
the minimum
responses.
switched
no drug
was given.
numbers
of responses
on the avoidance
responses.
Drug effects
were compared
calcr,lated from 2 control
lever
were counted
Each session lasted 30 min.
The rats were exposed
periods ss ere separated
the sound
on the avoidance
all the responses
out using a Luroup of 5-6 rats accustomed
and avoidance
avoidance
of a loudspeaker)
and postponed
of responses
The two schedules %yere concurrent.
week and drug administration
(sound
The first response
of the loudspeaker
the number
of 1 reinforce
a discriminated
stimulus
necessary to avoid all shocks,
Drug studies were carried
a variable ratio schedule
to a maximum
procedure,
I min) %~ ith a conditioned
for 1 min.
both milk-reinforced
after 2 responses
by the electric -,hoc].,-. ,vas used.
lever) to be
lever) to avoid an electric shock
procedure,
In the avoidance
the sound presentation
always exceeded as avoidance
In the milk-reinforced
was used.
interval
lasting 30 see, followed
shock
to press the right lever (milk-reinforced
with milk (0.1 nil) and the left lever (avoidance
to 2-3 sessions
by at least 2 control as percentage
well to perform a
sessions in which
change
with the mean
sessions before and after the drug treatment.
Onc-i4'aV active aroidance proc'echire in mice Male STD-ddY
strain mice. sleighing 30-40g,ssere
housed in groups of 5 and maintained
with food and water freely available. The apparatus (I I).
and procedure
used have been extensively
described
In short, the mice were trained in a box of two compartments,
Each trial started
by placing a mouse in the darkened
stimulus was given for 5 sec. 5 sec of the sound stimulus,
compartment
in a previous
darkened
paper
and lightened.
and then a click sound
Unless a mouse moved into the lightened
compartn-rent
an electric shock (5 mA, 50 Hz, 10 m sec) was delivered
vtithin to the
feet for 5 sec.
When the mouse reached the lightened compartment in the first 5 sec (before
the onset of shock), it was counted as an avoidance response. the shock usually escaped within 2 sec.
Mice which did not avoid
The mouse was manually returned to the darkened
compartment and the next trial was started. Most mice were accustomed to performing the avoidance response after 40-50 trials of training.
A group of 10 mice that showed evidence of the avoidance rate (;,, of the
avoidance response in 10 trials) more than 80°C,were used for drug studies.
Drug effects
were evaluated b'; comparing the avoidance rates between drug-treated and control groups. When tests were repeated in the same animals, drugs were administered at intervals of 7 days at a minimum. Drrrlqs Drugs used were chlorpromazine hydrochloride (Wintermin, Shionogi), perphenazine (Triomin, Yarnanouchi), haloperidol (Serenace, Dainippon), trifluperidol (Triperidol, Yoshitomi), clozapine (Leponex, Sandoz-Wander), oxypertine (Font, Daiichi), chlordi azepoxide (Balance, Yamanouchi),
diazepam (Cercine, Takeda),
Fujisawa), nortriptyline
Dainippon),
(Noritren,
phentolamine
imipramine
Geigy), phenoxybenzamine hydrochloride (Tokyo Kasei), arecoline hydrobromide Kasei),
physostigmine sulfate (Merck),
clonidine (Catapres,
tyrosine (Maruwaka) and DL-p-chlorophenylalanine
(Maruwaka).
(Tofranil,
mesylate (Regitine, Ciba Takeda),
(Tokyo
DL-a-methyl-p
All doses were expressed
in terms of total salt. Statistics Differences between groups were evaluated using Student's t-test.
ED50 values were
calculated according to the method of Litchfield and Wilcoxon (12). RFS LT L.TS Concurrent milk-reinforced and avoidance procedure in rats The effects of psychotropic drugs, alone or with a-MT or PCPA, on both milk-reinforced and avoidance responses are shown in Figs. 1-3. In the control sessions, rats pressed the milk-reinforced lever 1000-1600 times and the avoidance lever 23-35 times for 30 min.
Neuroleptics such as chlorpromazine
(3 and
5 mg;'kg i.p.), perphenazine (0.5 and 1 mg/kg i.p.), haloperidol (0.2 and 0.3 mgjkg i.p.) and oxypertine (1 and 2 mg/kg i.p.) caused a dose-related decrease of both milk-reinforced and avoidance lever pressing (Fig. 1).
From the preliminary test, the maximum ineffective
dose of a-MT was found to be 40 mg/kg i.p.
Then, the combined treatment of a-MT and
neuroleptics was examined in doses that were reduced to half of the maximum ineffective dose, respectively.
As shown in Fig. 2, chlorpromazine
1 mg,/kg i.p., perphenazine 0.1 mg/
kg i.p., haloperidol 0.05 mg/kg i.p. and oxypertine 0.25 mg/kg i.p. decreased the numbers of responses under both schedules after pretreatment with a-MT 20 mg/kg i.p. Diazepam (3 and 5 mg/kg i.p.) and nortriptyline (10 and 20 mg/kg i.p.), as did neu roleptics, decreased the numbers of responses under both schedules in a dose-related manner
FIG.
1.
Effects
in
of psychotropic
rats.
and
nortriptyline respectively.
session:
*p ;0.05,
Effects
in
rats
(Fig. 1).
However,
i.p.
of
and
haloperidol,
at 0.5,
Significance
drugs
pretreatment
with
comparison
the milk-reinforced
1, 1,
1, 0.25
difference
in
avoidance
responses
oxypertine, and
0.5
hr
comparison
diazepam before
with
the
the
test
control
**p,,_0.01.
peridol, oxypertine, 0.25, 0.5 and 4 hr in
given
of psychotropic
after
on
perphenazine,
were
session,
FIG. 2.
drugs
Chlorpromazine,
diazepam, before the
with
combined
the
on
the
n-MT.
nortriptyline test session,
control
treatment
kg i.p. with a-MT 20 mg/kg i.p. produced
session:
milk-reinforced
and
Chlorpromazine, and a-MT respectively. *p---~:70.05,
avoidance
responses
perphenazine,
were given i.p. Significance
halo
at 0.5, 1, 1, 1, of difference
**p<0.01.
of diazepam
1 mg/kg i.p. or nortriptyline
no significant
decrease in the numbers
2.5 mg/
of responses
to both levers (Fig. 2). As shown reinforced
in Fig. 3, PCPA at 300 mg/kg i.p. had no significant
and avoidance
did not influence
responses.
the suppressive
mg/kg i.p.), diazepam
In addition,
effects of chlorpromazine
(5 mg/kg i.p.) and nortriptyline
In the drug tests, a difference between rarely observed
with regard
of amine synthesis.
pretreatment
with this same dose of PCPA (5 mg/kg i.p.), haloperidol
(0.2
(20 mg/kg i.p.).
the milk-reinforced
to the effect of psychotropic
effect on both milk
and avoidance
schedules was
drugs with or without
inhibitors
FIG. 3.
Effects
in rats
of psychotropic
after
pretreatment
drugs on the milk-reinforced with
PCPA.
and avoidance
Chlorpromazine,
responses
perphenazine,
halo
peridol, oxypertine, diazepam, nortriptyline and PCPA were given i.p. at 0.5, 1, 1, 1, 0.25, 0.5 and 20 hr before the test session, respectively. Significance of difference in comparison with the control session: *p<0.05, **p<0.01.
These results show that the suppressive effects of neuroleptics on operant behavior in rats are potentiated by a-MT, but not influenced by PCPA. One-way active avoidance procedure in mice An additional experiment was performed using a simple avoidance procedure in mice to confirm the results obtained in the study of operant behavior in rats.
Effects of a variety
of drugs with or without each inhibitor of amine synthesis are shown in Figs. 4-7 and Table 1. Haloperidol caused a dose-related suppression of the avoidance response and the ED50 value was 0.23 mg/kg p.o.
The suppressive effect was markedly potentiated by pretreatment
with half of the maximum ineffective dose of a-MT (25 mg/kg i.p.) and the ED50 value was lowered to 0.043 mg/kg p.o.
On the other hand, pretreatment
with PCPA (200 mg/kg
FIG. 4. Effect of haloperidol on the active avoidance in mice after pretreatment with a-MT or PCPA. Haloperidol was given p.o. after pretreatment with a-MT (25 mg/kg i.p., 0.5 hr) or PCPA (200 mg/kg i.p. X 2, 24 and 48 hr). Vertical bars represent standard errors of means. Significance of difference in comparison with the control group: *p<0.05, **p.0.01.
Fin. 5. Effect of diazepam on the active avoidance in mice after pretreatment with (c-MT or PCPA. Diazepam was given p.o. after pretreatment with n-MT (25 mg/kg i.p., 0.5 hr) or PCPA (200 mg,'kg i.p. 2, 24 and 48 hr). Vertical bars represent standard errors of means. Significance of difference in comparison with the control group: *p<0.05, **p--'0.01.
Fin. 6. Effect of nortriptyline on the active avoidance in mice after pretreatment with n-MT or PCPA. Nortriptyline was given p.o. after pretreatment with n-MT (25 mg~kg i.p., 0.5 hr) or PCPA (200 mgikg i.p. ; 2, 24 and 48 hr). Vertical bars represent standard errors of means. Significance of difference in comparison with the control group: *p--0.05, **p "0.01.
i.p. x 2) had little influence on the suppressive effect of other neuroleptics, oxypertine
Diazepam muscle
i.e. chlorprornazine,
was also potentiated
PCPA (Table
effect of haloperidol
by pretreatment
perphenazine, with a-MT,
(Fig. 4).
The suppressive
trifluperidol,
clozapine
and
whereas it was not affected by
1). suppressed
the avoidance
relaxation
and impaired
did not influence
the suppressive
1).
at 50 and 100 mg/kg p.o. which induced
the escape response. effect of diazepam
kg p.o. had little effect on the avoidance with a-MT or PCPA (Table
response
response
Pretreatment (Fig. 5).
with a-MT
Chlordiazepoxide
which was not affected
or PCPA at 200 mg/
by pretreatment
Nortriptyline
and imipramine
either alone or with a-MT The influence blocked
of pretreatment
the avoidance
at 100 mg/kg p.o. had no effect on the avoidance
response
or PCPA (Fig. 6 and Table 1).
response.
with a-MT was also determined Phentolamine,
phenoxybenzamine,
using other drugs which arecoline,
physostig
Flci. 7. Effect of phenoxybenzamine and arecoline on the active avoidance in mice after pretreatment with (t-NIT. Phenoxybenzamine and arecoline were given i.p. after pretreatment with n-MT (25 mg/kg i.p., 0.5 hr). Vertical bars represent standard errors of means. Significance of difference in comparison with the control group: *p<,0.05, **p<0.01.
TABLE f .
Effect of various
with a-MT
drugs
on the active
avoidance
in mice after
pretreatment
or PCPA
Each drug was administered after pretreatment PCPA (200 mg/kg i.p. 2 at 24 and 48 hr).
with a-MT (25 mg/kg i .p., 0.5 hr) or
mine and clonidine influenced
suppressed
by pretreatment
the avoidance
with or without each inhibitor values
haloperidol,
arecoline, sponse, PCPA
with neuroleptics
clozapine
was potentiated
physostigmine
and clonidine
little change
that the suppressive potentiated
With a-MT,
It means
Diazepam,
little by pretreatment
effect of neuroleptics
that
by c,-MT, but is not influenced
lowering
perphenazine,
the suppressive
effect
phenoxybenzamine,
effect on the avoidance
with a-MT.
of the drugs tested.
on the avoidance
in mice
a considerable
phentolamine,
also had a suppressive
in ED50 values
response
such as chlorpromazine,
and oxypertine.
by a-MT.
but the ED50 values changed produced
drugs tested on the avoidance
of the amine synthesis.
was observed
trifluperidol,
of neuroleptics
but the effect of these drugs was not
with a-MT (Fig. 7 and Table 1).
Table 1 shows ED50 values of various
of ED50
response,
re
Pretreatment These results
response
with suggest
in mice is selectively
by PCPA.
DISCUSSION Neurolptics
induce an increase in catecholamine
for their receptor
blocking
effect of haloperidol treatment hibition
activity (2).
with a-MT
and suggested
be potentiated
effects of the neuroleptics by pretreatment
with a-MT.
in the clinical effect of chlorpromazine haloperidol behavior
(4) and the suppressive (5).
The present
the suppressive concurrent
milk-reinforced
and
the one-way active avoidance In a previous avoidance
response
whereas diazepam response.
showed
paper
blocked
should
pretreatment
activity
of
on the tail-pinch
with a-MT
potentiated
haloperidol
and oxypertine
in the
In addition,
the suppressive
effect
trifluperidol,
clozapine
and oxypertine
by pretreatment
neuroleptics
selectively
on
with a-MT. suppress
the active
in mice at doses lower than those at which the escape response is impaired, and doxepin
have a suppressive
In the present experiment,
differentiated
that
receptor
to
with a-MT was also observed
and pimozide
in mice was also potentiated
the avoidance neuroleptics
response.
effect of neuroleptics
by pretreatment
with a-MT.
The
by such pretreatment.
procedure
in rats,
the potentiation
after
response
with
the avoidance was markedly
with a-MT
milk-reinforced
was observed
neuroleptics.
These results suggest that the effect of neuroleptics
is selectively
potentiated
with a-MT.
a-MT
response. potentiated
the effect of drugs other than neuroleptics
Also in the concurrent
by pretreatment
and clonidine
pretreatment
which suppressed
the escape
(a-adrenergic
(anticholinesterase)
experiment
on the avoidance
However,
influenced
and phenoxybenzamine
physostigmine
from the other drugs
The suppressive
effect at doses which impair
phentolamine
blocking agent), arecoline (cholinomimetic), also
that
schedule.
haloperidol,
(11), we showed
According
(3), the cataleptogenic
perphenazine,
avoidance
perphenazine.
Such potentiation
by pre
might be due to in
turnover.
which block the catecholamine
effect of haloperidol
experiment
as compensation
potentiated
with a-MT
in catecholamine
and thioridazine
effects of chlorpromazine,
of chlorpromazine,
was markedly
the potentiation
increase
presumably
and Engel (1) showed that the suppressive
lever pressing
that
of the haloperidol-induced
this assumption,
Ahlenius
on food-reinforced
turnover,
was not
and avoidance
only in agents classified on the conditioned
as
behavior
Since a-MT inhibits the synthesis of both dopamine and noradrenaline (13, 14) and some neuroleptics act as blocking agents of noradrenaline as well as dopamine (15), the possibility exists that both amines play a role in the potentiation of the effects of neuroleptics by a-MT. However, the suppressive effect of phentolamine and phenoxybenzamine on the avoidance response was not influenced by pretreatment with a-MT 25 mg/kg i.p. in the present experiment. This result suggests that the inhibition of noradrenaline synthesis with this dose of a-MT dose not potentiate an a-blocking action. Therefore, the potentiating effect of a-MT on the action of neuroleptics may be due primarily to an inhibition of dopamine synthesis, as has already been suggested (5, 16). There are reports which concerned the relationship of serotonin to conditioned behavior. Tenen (6) and Brody (7) demonstrated that PCPA caused an increased rate of acquisition of the conditioned avoidance response in rats.
It was shown by Takaori and Tanaka (8) and
Tanaka et al. (9) that PCPA enhanced the avoidance response in Sidman avoidance procedure in rats.
The present experiment showed that PCPA, at doses which were preliminarily
confirmed in biochemical analysis to cause a considerable reduction of serotonin, did not influence the neuroleptic-induced
suppression of the conditioned behavior tested.
Thus,
the suppressive effect of neuroleptics on the conditioned behavior may not be related to the concentration or turnover of serotonin. These results show that, among various drugs which suppress the conditioned behavior in rodents, the suppressive effects of neuroleptics are selectively potentiated by pretreatment with a-MT, but are not influenced by PCPA.
Therefore, the present procedure may be
useful for distinguishing neuroleptics from other types of depressants. REFERENCES 1) AHLENIUS,S. AND ENGEL,J.: Europ. J. Pharmacol. 15, 187 (1971) 2) ANDi N, N.-E., CARLSSON,A. AND HAGGENDAL,J.: A. Rev. Pharmacol. 9, 119 (1969) 3) CARLSSON,A., PERSSON,T., Roos, B.-W. AND WALINDER,J.: J. Neural Transmission 33, 83 (1972) 4) SHORE,P.A. AND DORRIS,R.L.: Europ. J. Pharmacol. 30, 315 (1975) 5) ANTELMAN,S.M., SZECHTMAN,H., CHIN, P. AND FISHER, A.E.: J. Pharm. Pharmacol. 28, 66 (1976) 6) TENEN,S.S.: Psychopharmacologia 10, 204 (1967) 7) BRODY,J.F., JR.: Psychopharmacologia 17, 14 (1970) 8) TAKAORI,S. ANDTANAKA,C.: Japan. J. Pharmacol. 20, 607 (1970) 9) TANAKA,C., YOH, Y.-J. AND TAKAORI,S.: Brain Res. 45, 153 (1972) 10) MOLINENGO,L. AND RICCI-GAMALERO, S.: Psychopharmacologia 24, 247 (1972) 11) OKA, M. AND SHIMIZU,M.: Japan. J. Pharmacol. 25, 121 (1975) 12) LITCHFIELD,J.T., JR. AND WILCOXON,F.: J. Pharmacol. exp. Ther. 96, 99 (1949) 13) SPECTOR,S., SJOERDSMA, A. AND UDENFRIEND,S.: J. Pharmacol. exp. Ther. 147, 86 (1965) 14) RECH, R.H., BORYS,H.K. AND MOORE,K.E.: J. Pharmacol. exp. Ther. 153, 412 (1966) 15) ANDEN, N.-E., BUTCHER, S.G., CORRODI,H., FUXE, K. AND UNGELSTEDT,U.: Europ. J. Pharmacol. 11, 303 (1970) 16) AHLENIUS,S. ANDENGEL, J.: J. Pharm. Pharmacol. 25, 172 (1973)
OF
NEUROLEPTIC
BEHAVIOR
DRUGS
AFTER
a-METHYLTYROSINE Makoto
THE
CONDITIONED
PRETREATMENT
OR
OKA, Chiaki
ON
WITH
p-CHLOROPHENYLALANINE*
KAMEI
and Masanao
SHIMIZU
Department of Pharniacologv, Research Laboratories, Dainippon Pharmaceutical Co.. Ltd., Suita ski, Osaka 564, Japan Accepted August 1, 1977
Abstract-Effects of psychotropic drugs on the conditioned behavior after pretreatment with a-rnethyltyrosine(a-MT) or p-chlorophenylalanine(PCPA) were studied using operant conditioning technique in rodents. The operant conditioning procedure used was composed of a concurrent milk-reinforced and avoidance schedule in an operant conditioning chamber with two levers where lever pressing by rats was maintained by milk (VR 6) and electric shock (intertrial interval II min). Chlorpromazine (1 mg/kg i.p.), perphenazine (0.1 mg/kg i.p.), haloperidol (0.05 mg/kg i.p.) and oxypertine (0.25 mg/kg i.p.), at half of the maximum ineffective dose, respectively, caused a significant decrease in both lever pressings after pretreatment with a-MT (20 mg/kg i.p.). Such potentiating effect by a-MT was not observed with diazepam and nortriptyline. PCPA pretreatment (300 mg/kg i.p.) did not influence suppressive effects on responding under both schedules by any drug used. The one-way active avoidance procedure in mice was also employed in this study. On the avoidance response, chlorpromazine, per phenazine, haloperidol, trifluperidol, clozapine and oxypertine showed a suppressive effect, ED50 values of which were considerably lowered by pretreatment with a-MT (25 mg/kg i.p.). No influence was seen with PCPA (200 mg/kg i.p. x 2). Diazepam, phenoxybenzamine, phentolarnine, arecoline, physostigmine and clonidine also had a suppressive effect on the avoidance response, which was not influenced by pretreatment with a-MT. These results suggest that the suppressive effect of neuroleptics on the conditioned behavior is selectively potentiated by (t-MT, and not influenced by PCPA. Ahlenius reinforced
and Engel (1) reported
lever pressing
that
was markedly
the suppressive
potentiated
by pretreatment
(a-MT), a tyrosine hydroxylase inhibitor. Neuroleptics in catecholamine turnover presumably as compensation (2).
This suggests
that
haloperidol-induced
the potentiation
increase
with a-MT
in catecholamine
neurotransmitter
available
such potentiation
with a-MT was also observed
to compete
thioridazine
(3), the cataleptogenic
haloperidol
and pimozide
with a-MT
is specific in neuroleptics
On the other hand, serotonin, has been considered
with a-methyltyrosine
may be due to an inhibition
turnover,
thereby
reducing
for receptor
sites.
behavior
has, however,
(5).
(4) and the suppressive Whether
of
Afterward, and
effect of
or not such potentiation
not been given attention.
amine which exists in various areas of the brain,
to play some role in brain function
relating
to the conditioned
*These data were presented at the 49th General Meeting of the Japanese Pharmacological (March, 1976).
of the
the amount
in the clinical effect of chlorpromazine
effect of haloperidol
another
on food
are known to induce an increase for their receptor blocking activity
with the neuroleptic
on tail-pinch
effect of haloperidol
behavior Society
(6--9).
However,
little work has been done to assess the role of* serotonin
in the action
of
neuroleptics. The present experiment
was designed
ment with (t-MT was observed behavioral
procedures
to determine
generally
in neuroleptics
used were a concurrent
rats (10) and an one-way active avoidance with p-chlorophenylalaniuue
(PUPA),
milk-reinforced
Male Wistar were maintained
a tryptophan
The
HLA strain
operant
and avoidance
Gamalero
(10).
weighing
procedure
hydroxylase
The
schedule
in
The effect of pretreatment
inhibitor,
%vasalso examined.
ill rats
180-220
g at the beginning
of the training,
80 ° of their free feeding weights. used was composed
schedule by a modification
The experimental
and avoidance
AND METHODS
diet at about
conditioning
reinforced
chamber
rats,
by pretreat
and; or only in neuroleptics.
in mice (I 1).
aitd ar'oidcurce procedure
on a restricted
the potentiation
milk-reinforced
schedule
11A !TRIALS Concurrcirt
whether
chamber
of the concurrent
of the method
of Molinengo
was the Grason-Stadler
operant
milk
and Ricci conditioning
for rats which had a milk tray on the center of a side wall and two levers on both
sides of the tray. Food-deprived reinforced
rats were trained
(AC current
of 0.2 mA for I sec).
(VR 6), from a minimum ment after schedule
10 responses, (intertrial
lever during
of I reinforcement
sequence
Although
the minimum
responses.
switched
no drug
was given.
numbers
of responses
on the avoidance
responses.
Drug effects
were compared
calcr,lated from 2 control
lever
were counted
Each session lasted 30 min.
The rats were exposed
periods ss ere separated
the sound
on the avoidance
all the responses
out using a Luroup of 5-6 rats accustomed
and avoidance
avoidance
of a loudspeaker)
and postponed
of responses
The two schedules %yere concurrent.
week and drug administration
(sound
The first response
of the loudspeaker
the number
of 1 reinforce
a discriminated
stimulus
necessary to avoid all shocks,
Drug studies were carried
a variable ratio schedule
to a maximum
procedure,
I min) %~ ith a conditioned
for 1 min.
both milk-reinforced
after 2 responses
by the electric -,hoc].,-. ,vas used.
lever) to be
lever) to avoid an electric shock
procedure,
In the avoidance
the sound presentation
always exceeded as avoidance
In the milk-reinforced
was used.
interval
lasting 30 see, followed
shock
to press the right lever (milk-reinforced
with milk (0.1 nil) and the left lever (avoidance
to 2-3 sessions
by at least 2 control as percentage
well to perform a
sessions in which
change
with the mean
sessions before and after the drug treatment.
Onc-i4'aV active aroidance proc'echire in mice Male STD-ddY
strain mice. sleighing 30-40g,ssere
housed in groups of 5 and maintained
with food and water freely available. The apparatus (I I).
and procedure
used have been extensively
described
In short, the mice were trained in a box of two compartments,
Each trial started
by placing a mouse in the darkened
stimulus was given for 5 sec. 5 sec of the sound stimulus,
compartment
in a previous
darkened
paper
and lightened.
and then a click sound
Unless a mouse moved into the lightened
compartn-rent
an electric shock (5 mA, 50 Hz, 10 m sec) was delivered
vtithin to the
feet for 5 sec.
When the mouse reached the lightened compartment in the first 5 sec (before
the onset of shock), it was counted as an avoidance response. the shock usually escaped within 2 sec.
Mice which did not avoid
The mouse was manually returned to the darkened
compartment and the next trial was started. Most mice were accustomed to performing the avoidance response after 40-50 trials of training.
A group of 10 mice that showed evidence of the avoidance rate (;,, of the
avoidance response in 10 trials) more than 80°C,were used for drug studies.
Drug effects
were evaluated b'; comparing the avoidance rates between drug-treated and control groups. When tests were repeated in the same animals, drugs were administered at intervals of 7 days at a minimum. Drrrlqs Drugs used were chlorpromazine hydrochloride (Wintermin, Shionogi), perphenazine (Triomin, Yarnanouchi), haloperidol (Serenace, Dainippon), trifluperidol (Triperidol, Yoshitomi), clozapine (Leponex, Sandoz-Wander), oxypertine (Font, Daiichi), chlordi azepoxide (Balance, Yamanouchi),
diazepam (Cercine, Takeda),
Fujisawa), nortriptyline
Dainippon),
(Noritren,
phentolamine
imipramine
Geigy), phenoxybenzamine hydrochloride (Tokyo Kasei), arecoline hydrobromide Kasei),
physostigmine sulfate (Merck),
clonidine (Catapres,
tyrosine (Maruwaka) and DL-p-chlorophenylalanine
(Maruwaka).
(Tofranil,
mesylate (Regitine, Ciba Takeda),
(Tokyo
DL-a-methyl-p
All doses were expressed
in terms of total salt. Statistics Differences between groups were evaluated using Student's t-test.
ED50 values were
calculated according to the method of Litchfield and Wilcoxon (12). RFS LT L.TS Concurrent milk-reinforced and avoidance procedure in rats The effects of psychotropic drugs, alone or with a-MT or PCPA, on both milk-reinforced and avoidance responses are shown in Figs. 1-3. In the control sessions, rats pressed the milk-reinforced lever 1000-1600 times and the avoidance lever 23-35 times for 30 min.
Neuroleptics such as chlorpromazine
(3 and
5 mg;'kg i.p.), perphenazine (0.5 and 1 mg/kg i.p.), haloperidol (0.2 and 0.3 mgjkg i.p.) and oxypertine (1 and 2 mg/kg i.p.) caused a dose-related decrease of both milk-reinforced and avoidance lever pressing (Fig. 1).
From the preliminary test, the maximum ineffective
dose of a-MT was found to be 40 mg/kg i.p.
Then, the combined treatment of a-MT and
neuroleptics was examined in doses that were reduced to half of the maximum ineffective dose, respectively.
As shown in Fig. 2, chlorpromazine
1 mg,/kg i.p., perphenazine 0.1 mg/
kg i.p., haloperidol 0.05 mg/kg i.p. and oxypertine 0.25 mg/kg i.p. decreased the numbers of responses under both schedules after pretreatment with a-MT 20 mg/kg i.p. Diazepam (3 and 5 mg/kg i.p.) and nortriptyline (10 and 20 mg/kg i.p.), as did neu roleptics, decreased the numbers of responses under both schedules in a dose-related manner
FIG.
1.
Effects
in
of psychotropic
rats.
and
nortriptyline respectively.
session:
*p ;0.05,
Effects
in
rats
(Fig. 1).
However,
i.p.
of
and
haloperidol,
at 0.5,
Significance
drugs
pretreatment
with
comparison
the milk-reinforced
1, 1,
1, 0.25
difference
in
avoidance
responses
oxypertine, and
0.5
hr
comparison
diazepam before
with
the
the
test
control
**p,,_0.01.
peridol, oxypertine, 0.25, 0.5 and 4 hr in
given
of psychotropic
after
on
perphenazine,
were
session,
FIG. 2.
drugs
Chlorpromazine,
diazepam, before the
with
combined
the
on
the
n-MT.
nortriptyline test session,
control
treatment
kg i.p. with a-MT 20 mg/kg i.p. produced
session:
milk-reinforced
and
Chlorpromazine, and a-MT respectively. *p---~:70.05,
avoidance
responses
perphenazine,
were given i.p. Significance
halo
at 0.5, 1, 1, 1, of difference
**p<0.01.
of diazepam
1 mg/kg i.p. or nortriptyline
no significant
decrease in the numbers
2.5 mg/
of responses
to both levers (Fig. 2). As shown reinforced
in Fig. 3, PCPA at 300 mg/kg i.p. had no significant
and avoidance
did not influence
responses.
the suppressive
mg/kg i.p.), diazepam
In addition,
effects of chlorpromazine
(5 mg/kg i.p.) and nortriptyline
In the drug tests, a difference between rarely observed
with regard
of amine synthesis.
pretreatment
with this same dose of PCPA (5 mg/kg i.p.), haloperidol
(0.2
(20 mg/kg i.p.).
the milk-reinforced
to the effect of psychotropic
effect on both milk
and avoidance
schedules was
drugs with or without
inhibitors
FIG. 3.
Effects
in rats
of psychotropic
after
pretreatment
drugs on the milk-reinforced with
PCPA.
and avoidance
Chlorpromazine,
responses
perphenazine,
halo
peridol, oxypertine, diazepam, nortriptyline and PCPA were given i.p. at 0.5, 1, 1, 1, 0.25, 0.5 and 20 hr before the test session, respectively. Significance of difference in comparison with the control session: *p<0.05, **p<0.01.
These results show that the suppressive effects of neuroleptics on operant behavior in rats are potentiated by a-MT, but not influenced by PCPA. One-way active avoidance procedure in mice An additional experiment was performed using a simple avoidance procedure in mice to confirm the results obtained in the study of operant behavior in rats.
Effects of a variety
of drugs with or without each inhibitor of amine synthesis are shown in Figs. 4-7 and Table 1. Haloperidol caused a dose-related suppression of the avoidance response and the ED50 value was 0.23 mg/kg p.o.
The suppressive effect was markedly potentiated by pretreatment
with half of the maximum ineffective dose of a-MT (25 mg/kg i.p.) and the ED50 value was lowered to 0.043 mg/kg p.o.
On the other hand, pretreatment
with PCPA (200 mg/kg
FIG. 4. Effect of haloperidol on the active avoidance in mice after pretreatment with a-MT or PCPA. Haloperidol was given p.o. after pretreatment with a-MT (25 mg/kg i.p., 0.5 hr) or PCPA (200 mg/kg i.p. X 2, 24 and 48 hr). Vertical bars represent standard errors of means. Significance of difference in comparison with the control group: *p<0.05, **p.0.01.
Fin. 5. Effect of diazepam on the active avoidance in mice after pretreatment with (c-MT or PCPA. Diazepam was given p.o. after pretreatment with n-MT (25 mg/kg i.p., 0.5 hr) or PCPA (200 mg,'kg i.p. 2, 24 and 48 hr). Vertical bars represent standard errors of means. Significance of difference in comparison with the control group: *p<0.05, **p--'0.01.
Fin. 6. Effect of nortriptyline on the active avoidance in mice after pretreatment with n-MT or PCPA. Nortriptyline was given p.o. after pretreatment with n-MT (25 mg~kg i.p., 0.5 hr) or PCPA (200 mgikg i.p. ; 2, 24 and 48 hr). Vertical bars represent standard errors of means. Significance of difference in comparison with the control group: *p--0.05, **p "0.01.
i.p. x 2) had little influence on the suppressive effect of other neuroleptics, oxypertine
Diazepam muscle
i.e. chlorprornazine,
was also potentiated
PCPA (Table
effect of haloperidol
by pretreatment
perphenazine, with a-MT,
(Fig. 4).
The suppressive
trifluperidol,
clozapine
and
whereas it was not affected by
1). suppressed
the avoidance
relaxation
and impaired
did not influence
the suppressive
1).
at 50 and 100 mg/kg p.o. which induced
the escape response. effect of diazepam
kg p.o. had little effect on the avoidance with a-MT or PCPA (Table
response
response
Pretreatment (Fig. 5).
with a-MT
Chlordiazepoxide
which was not affected
or PCPA at 200 mg/
by pretreatment
Nortriptyline
and imipramine
either alone or with a-MT The influence blocked
of pretreatment
the avoidance
at 100 mg/kg p.o. had no effect on the avoidance
response
or PCPA (Fig. 6 and Table 1).
response.
with a-MT was also determined Phentolamine,
phenoxybenzamine,
using other drugs which arecoline,
physostig
Flci. 7. Effect of phenoxybenzamine and arecoline on the active avoidance in mice after pretreatment with (t-NIT. Phenoxybenzamine and arecoline were given i.p. after pretreatment with n-MT (25 mg/kg i.p., 0.5 hr). Vertical bars represent standard errors of means. Significance of difference in comparison with the control group: *p<,0.05, **p<0.01.
TABLE f .
Effect of various
with a-MT
drugs
on the active
avoidance
in mice after
pretreatment
or PCPA
Each drug was administered after pretreatment PCPA (200 mg/kg i.p. 2 at 24 and 48 hr).
with a-MT (25 mg/kg i .p., 0.5 hr) or
mine and clonidine influenced
suppressed
by pretreatment
the avoidance
with or without each inhibitor values
haloperidol,
arecoline, sponse, PCPA
with neuroleptics
clozapine
was potentiated
physostigmine
and clonidine
little change
that the suppressive potentiated
With a-MT,
It means
Diazepam,
little by pretreatment
effect of neuroleptics
that
by c,-MT, but is not influenced
lowering
perphenazine,
the suppressive
effect
phenoxybenzamine,
effect on the avoidance
with a-MT.
of the drugs tested.
on the avoidance
in mice
a considerable
phentolamine,
also had a suppressive
in ED50 values
response
such as chlorpromazine,
and oxypertine.
by a-MT.
but the ED50 values changed produced
drugs tested on the avoidance
of the amine synthesis.
was observed
trifluperidol,
of neuroleptics
but the effect of these drugs was not
with a-MT (Fig. 7 and Table 1).
Table 1 shows ED50 values of various
of ED50
response,
re
Pretreatment These results
response
with suggest
in mice is selectively
by PCPA.
DISCUSSION Neurolptics
induce an increase in catecholamine
for their receptor
blocking
effect of haloperidol treatment hibition
activity (2).
with a-MT
and suggested
be potentiated
effects of the neuroleptics by pretreatment
with a-MT.
in the clinical effect of chlorpromazine haloperidol behavior
(4) and the suppressive (5).
The present
the suppressive concurrent
milk-reinforced
and
the one-way active avoidance In a previous avoidance
response
whereas diazepam response.
showed
paper
blocked
should
pretreatment
activity
of
on the tail-pinch
with a-MT
potentiated
haloperidol
and oxypertine
in the
In addition,
the suppressive
effect
trifluperidol,
clozapine
and oxypertine
by pretreatment
neuroleptics
selectively
on
with a-MT. suppress
the active
in mice at doses lower than those at which the escape response is impaired, and doxepin
have a suppressive
In the present experiment,
differentiated
that
receptor
to
with a-MT was also observed
and pimozide
in mice was also potentiated
the avoidance neuroleptics
response.
effect of neuroleptics
by pretreatment
with a-MT.
The
by such pretreatment.
procedure
in rats,
the potentiation
after
response
with
the avoidance was markedly
with a-MT
milk-reinforced
was observed
neuroleptics.
These results suggest that the effect of neuroleptics
is selectively
potentiated
with a-MT.
a-MT
response. potentiated
the effect of drugs other than neuroleptics
Also in the concurrent
by pretreatment
and clonidine
pretreatment
which suppressed
the escape
(a-adrenergic
(anticholinesterase)
experiment
on the avoidance
However,
influenced
and phenoxybenzamine
physostigmine
from the other drugs
The suppressive
effect at doses which impair
phentolamine
blocking agent), arecoline (cholinomimetic), also
that
schedule.
haloperidol,
(11), we showed
According
(3), the cataleptogenic
perphenazine,
avoidance
perphenazine.
Such potentiation
by pre
might be due to in
turnover.
which block the catecholamine
effect of haloperidol
experiment
as compensation
potentiated
with a-MT
in catecholamine
and thioridazine
effects of chlorpromazine,
of chlorpromazine,
was markedly
the potentiation
increase
presumably
and Engel (1) showed that the suppressive
lever pressing
that
of the haloperidol-induced
this assumption,
Ahlenius
on food-reinforced
turnover,
was not
and avoidance
only in agents classified on the conditioned
as
behavior
Since a-MT inhibits the synthesis of both dopamine and noradrenaline (13, 14) and some neuroleptics act as blocking agents of noradrenaline as well as dopamine (15), the possibility exists that both amines play a role in the potentiation of the effects of neuroleptics by a-MT. However, the suppressive effect of phentolamine and phenoxybenzamine on the avoidance response was not influenced by pretreatment with a-MT 25 mg/kg i.p. in the present experiment. This result suggests that the inhibition of noradrenaline synthesis with this dose of a-MT dose not potentiate an a-blocking action. Therefore, the potentiating effect of a-MT on the action of neuroleptics may be due primarily to an inhibition of dopamine synthesis, as has already been suggested (5, 16). There are reports which concerned the relationship of serotonin to conditioned behavior. Tenen (6) and Brody (7) demonstrated that PCPA caused an increased rate of acquisition of the conditioned avoidance response in rats.
It was shown by Takaori and Tanaka (8) and
Tanaka et al. (9) that PCPA enhanced the avoidance response in Sidman avoidance procedure in rats.
The present experiment showed that PCPA, at doses which were preliminarily
confirmed in biochemical analysis to cause a considerable reduction of serotonin, did not influence the neuroleptic-induced
suppression of the conditioned behavior tested.
Thus,
the suppressive effect of neuroleptics on the conditioned behavior may not be related to the concentration or turnover of serotonin. These results show that, among various drugs which suppress the conditioned behavior in rodents, the suppressive effects of neuroleptics are selectively potentiated by pretreatment with a-MT, but are not influenced by PCPA.
Therefore, the present procedure may be
useful for distinguishing neuroleptics from other types of depressants. REFERENCES 1) AHLENIUS,S. AND ENGEL,J.: Europ. J. Pharmacol. 15, 187 (1971) 2) ANDi N, N.-E., CARLSSON,A. AND HAGGENDAL,J.: A. Rev. Pharmacol. 9, 119 (1969) 3) CARLSSON,A., PERSSON,T., Roos, B.-W. AND WALINDER,J.: J. Neural Transmission 33, 83 (1972) 4) SHORE,P.A. AND DORRIS,R.L.: Europ. J. Pharmacol. 30, 315 (1975) 5) ANTELMAN,S.M., SZECHTMAN,H., CHIN, P. AND FISHER, A.E.: J. Pharm. Pharmacol. 28, 66 (1976) 6) TENEN,S.S.: Psychopharmacologia 10, 204 (1967) 7) BRODY,J.F., JR.: Psychopharmacologia 17, 14 (1970) 8) TAKAORI,S. ANDTANAKA,C.: Japan. J. Pharmacol. 20, 607 (1970) 9) TANAKA,C., YOH, Y.-J. AND TAKAORI,S.: Brain Res. 45, 153 (1972) 10) MOLINENGO,L. AND RICCI-GAMALERO, S.: Psychopharmacologia 24, 247 (1972) 11) OKA, M. AND SHIMIZU,M.: Japan. J. Pharmacol. 25, 121 (1975) 12) LITCHFIELD,J.T., JR. AND WILCOXON,F.: J. Pharmacol. exp. Ther. 96, 99 (1949) 13) SPECTOR,S., SJOERDSMA, A. AND UDENFRIEND,S.: J. Pharmacol. exp. Ther. 147, 86 (1965) 14) RECH, R.H., BORYS,H.K. AND MOORE,K.E.: J. Pharmacol. exp. Ther. 153, 412 (1966) 15) ANDEN, N.-E., BUTCHER, S.G., CORRODI,H., FUXE, K. AND UNGELSTEDT,U.: Europ. J. Pharmacol. 11, 303 (1970) 16) AHLENIUS,S. ANDENGEL, J.: J. Pharm. Pharmacol. 25, 172 (1973)