Short communication

Effect of co-treatment with mirtazapine and

risperidone in animal models of the positive

symptoms of schizophrenia in mice

Zofia Rogó¿

Department of Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12,

PL 31-343 Kraków, Poland

Correspondence: Zofia Rogó¿, e-mail: rogoz@if-pan.krakow.pl

Abstract:

Background: Several clinical reports have suggested that the mirtazapine-induced augmentation of risperidone activity may effec-

tively improve the positive, negative and some cognitive symptoms of schizophrenia.

Methods: The present study was aimed at examining the effect of mirtazapine and risperidone, given separately or jointly in mice,

on the locomotor hyperactivity induced by D-amphetamine or MK-801 as well as a 5-HT�� receptor agonist DOI-induced head

twitches as models for positive symptoms of psychosis.

Results: The obtained results showed that co-treatment with mirtazapine (2.5 or 5 mg/kg) and risperidone (0.01 mg/kg) inhibited the

locomotor hyperactivity induced by D-amphetamine or MK-801. Moreover, co-administration of mirtazapine (1.25 or 2.5 mg/kg)

and risperidone (0.01 mg/kg) reduced the number of head twitches induced by DOI, whereas those drugs given separately changed

neither the locomotor hyperactivity induced by D-amphetamine or MK-801 nor the syndrome induced by DOI.

Conclusion: The obtained results indicated that lower doses of mirtazapine enhanced the antipsychotic-like effect of risperidone in

animal tests of positive symptoms of schizophrenia. Further studies are necessary to elucidate its mechanism of action.

Key words:

mirtazapine, risperidone, animal tests of schizophrenia, mice

Introduction

Schizophrenia is a chronic progressive disease with

a life-time prevalence of approximately 1%. Although

the severity of schizophrenia has been known for

a long time, its etiology and pathophysiology are still

unknown. It is known that the serotonergic system is

potentially an important target for pharmacological

agents. The affinity for 5-HT receptors is one of the

main differences between atypical and conventional

antipsychotic drugs [2, 13, 14]. An atypical antipsy-

chotic agents, e.g., risperidone, whose low doses

block mainly serotonin 5-HT�� receptors and higher

dopamine D� ones, is known to produce minimal ex-

trapyramidal side-effects compared to classic antipsy-

chotics. Several clinical and preclinical studies sug-

gest that some atypical antipsychotic drugs (e.g.,

risperidone, olanzapine, aripiprazole) alleviate not only

positive symptoms of schizophrenia, but also negative

ones (e.g., flat affect, poverty of speech); furthermore,

they bring considerable benefit compared to the first-

generation drugs [4, 9, 15]. Additionally, the role of

antidepressant drugs as adjuncts to the treatment of

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schizophrenic patients with negative symptoms has

been described by several authors [10, 18]. It has also

been ascertained that mirtazapine, a novel antidepres-

sant, enhances noradrenergic and 5-HT��-mediated

serotonergic neurotransmission by antagonizing cen-

tral ��-auto- and hetero-adrenoreceptors, but does not

inhibit the uptake of noradrenaline and 5-HT. It also

blocks 5-HT� and 5-HT� receptors and displays very

low affinity for dopaminergic receptors and high

affinity for histamine H� ones [7]. Several clinical

reports have suggested that the mirtazapine-induced

augmentation of risperidone activity may effectively

improve both negative and some cognitive symptoms

of schizophrenia [1, 5, 20]. On the other hand, Berk et

al. [3] demonstrated that the adjunctive mirtazapine

was not superior to an adjunctive placebo when used

with atypical antipsychotics. Hence, the role of anti-

depressants in general and of mirtazapine in particu-

lar, in the treatment of schizophrenia is still unclear.

To understand the mechanism of the clinical effi-

cacy of a combination of an antidepressant and

risperidone in the therapy of schizophrenia, the pres-

ent study was aimed at examining the effect of mir-

tazapine and risperidone, given separately or jointly

to mice, on the locomotor hyperactivity induced by

D-amphetamine (AMP) or MK-801, as well as on the

head twitches induced by the 5-HT�� receptor agonist

DOI, serving as models of the positive symptoms of

psychosis.

Materials and Methods

Animals

The experiments were carried out on male Albino-

Swiss mice (25 ± 2 g) (Charles River Laboratories,

Sulzfeld, Germany). The animals were housed 10 per

cage (57 × 35 × 20 cm) in a colony room kept at

21 ± 1°C with a 40–50% humidity, on a 12-h light-

dark cycle (the light on at 7 a.m.). The mice had free

access to food and water before the experiments. Each

experimental group consisted of 8–10 animals/dose,

and the animals were used only once for each test. All

the experiments were conduced during the light phase

and were carried out according to the procedures ap-

proved by the Animal Care and Use Committee at the

Institute of Pharmacology, Polish Academy of Sci-

ences in Kraków.

Drugs administration

Mirtazapine and risperidone (Tocris Bioscience, Bris-

tol, UK) were suspended in a 1% aqueous solution of

Tween 80; (+)-MK-801 maleate (MK-801, Tocris

Bioscience, Bristol, UK) or D-amphetamine sulfate

(AMP) and (±)-DOI (Sigma-Aldrich, St. Louis, USA)

were dissolved in a 0.9% NaCl, All the drugs were ad-

ministered in a volume of 10 ml/kg. Risperidone (ip)

and mirtazapine (ip) were injected 30 min before

AMP (5 mg/kg, sc) or MK-801 (0.3 mg/kg, ip) and

60 min before DOI (2.5 mg/kg, ip) treatment.

Locomotor activity test

The locomotor activity was recorded individually for

each animal in OPTO-M3 locomotor activity cages

(Columbus Instruments, Columbus, OH, USA) linked

on-line to a compatible PC. Each cage (13 × 23 × 15 cm)

was surrounded with an array of photocell beams. In-

terruptions of these photobeams resulted in a horizon-

tal activity defined as ambulation scores. Locomotor

activity was measured for 60 (AMP) or 30 min

(MK-801), starting 30 min after treatment with AMP

or MK-801. Each group consisted of 8–10 mice.

Head twitches induced by DOI

The experiment was performed according to

Wieroñska et al. [21]. In order to habituate mice to the

experimental environment, each animal was trans-

ferred to 12 (diameter) × 20 cm (height) glass cylin-

der, linked with sawdust, 30 min before the treatment.

DOI, a 5-HT�� receptor agonist (2.5 mg/kg) was

given 60 min after MIR or risperidone administration.

The number of head twitches of the mice were

counted during a 30-min session, starting immedi-

ately after DOI treatment. Each group consisted of

8 mice.

Statistical analysis

The data were evaluated by a one-way analysis of

variance (ANOVA) followed, when appropriate, by

individual comparisons with the control using Dun-

nett’s test.

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Results and Discussion

In the present study we investigated the effect of mir-

tazapine and risperidone, given separately or jointly,

in animal models of the positive symptoms of schizo-

phrenia. It was used AMP- and MK-801-induced hy-

peractivity, which represents models to test the an-

tipsychotic activity of drugs, mainly with respect to

the positive symptoms of the disease [11], and DOI-

induced head twitches as the model with predictive

validity of psychosis and hallucination [21].

The obtained results showed that mirtazapine in

doses of 2.5 and 5 mg/kg did not change the locomo-

tor activity of mice, while its higher dose (10 mg/kg)

decreased it by ca. 52% [F (3, 36) = 5.96; p < 0.001,

Fig. 1]. Our earlier [16], and present studies indicated

that higher doses (0.3 and 1 mg/kg) of risperidone re-

duced the locomotor activity of mice by ca. 65 or

96%, respectively, while its lower doses of 0.01 or

������������� �� ����� ����� ��� �������� 1569

Effect of co-treatment with mirtazapine and risperidone����� ����

Fig. 1. The effect of mirtazapine (MIR,2.5, 5 and 10 mg/kg, ip) given alone orin combination with risperidone (RIS,0.01 mg/kg, ip) on the amphetamine(AMP, 5 mg/kg, sc)-induced locomotorhyperactivity in mice. The results areshown as the mean ± SEM of 8–10 ani-mals/group. The data were statisticallyevaluated by ANOVA, following by in-dividual comparisons using Dunnett’stest; * p < 0.05; ** p < 0.001 vs.vehicle-treated group, �� p < 0.001 vs.MIR + AMP-treated group

Fig. 2. The effect of mirtazapine (MIR,2.5 and 5 mg/kg, ip) given alone or incombination with risperidone (RIS, 0.01mg/kg, ip) on the MK-801 (0.3 mg/kg,ip)-induced locomotor hyperactivity inmice. The results are shown as themean ± SEM of 8 animals/group. Thedata were statistically evaluated byANOVA, following by individual com-parisons using Dunnett’s test; * p <0.001 vs. vehicle-treated group, � p <0.05; �� p < 0.001 vs. MIR + MK-801-treated group

0.03 mg/kg did not change it [F (5, 54) = 77.60; p <

0.001, data not shown]. In addition, AMP (5 mg/kg)

enhanced the locomotor activity by ca. 200%, while

risperidone in a dose of 0.1 mg/kg completely abol-

ished it. Moreover, risperidone in a lower dose (10

times lower, i.e., 0.01 mg/kg) or mirtazapine in doses

of 2.5 and 5 mg/kg, given separately, did not change

the hyperactivity induced by AMP, while co-treatment

with both those drugs (risperidone, 0.01 mg/kg, and

mirtazapine, 2.5 or 5 mg/kg) reduced that locomotor

hyperactivity [F (5, 42) = 7.69; p < 0.001, Fig. 1].

The glutamatergic therapy of psychosis was based

on the behavioral results indicating psychomimetic

properties of NMDA receptor antagonists (e.g., phen-

cyclidine), which evoked positive, negative, and

cognitive abnormalities in animals, similar to those

observed in patients with psychosis. In addition, the

clinical preliminary studies with the use of ligands

that act on the glycine modulatory site on the NMDA

receptor showed that the compounds improved cogni-

tive and decreased negative syndromes when co-

treated with neuroleptics [6].

The obtained results also showed that MK-801 in

a lower dose (0.1 mg/kg) had no influence on the lo-

comotor activity of mice, but its higher dose (0.3 mg/kg)

enhanced it by ca. 176%, that hyperactivity being com-

pletely abolished by risperidone in a dose of 0.1 mg/kg,

but not in the lower one (0.01 mg/kg) [F (4, 35) =

1570 ������������� �� ����� ����� ��� ��������

Fig. 3. The effect of mirtazapine (MIR, 1.25, 2.5, 5 and 10 mg/kg, ip)(A) or risperidone (RIS, 0.01, 0.03 and 0.1 mg/kg, ip) (B), given aloneor in combination, on the DOI (2.5 mg/kg, ip)-induced head twitches(C) in mice. The results are shown as the mean ± SEM of 8 ani-mals/group. The data were statistically evaluated by ANOVA, follow-ing by individual comparisons using Dunnett’s test; * p < 0.001 vs.DOI-treated group, � p < 0.001 vs. MIR + DOI-treated group

A

B

C

9.17; p < 0.001, Fig. 2]. Moreover, co-treatment with

risperidone in the lower dose (0.01 mg/kg) and mir-

tazapine (2.5 or 5 mg/kg) decreased the MK-801-

induced hyperactivity of mice [F (5, 42) = 8.56; p <

0.001, Fig. 2].

To further investigate the potential antipsychotic-

like action of combined treatment with mirtazapine

and risperidone, we decided to use a behavioral model

of hallucination which is known to be an important

aspect of schizophrenia in humans. Hallucinogenic-

like activity can be achieved via stimulation of seroto-

nergic receptors in both mice and humans [19]. The

5-HT�� receptor agonist DOI induces a characteristic

behavioral effect in mice, consisting of head twitches,

which is attenuated by typical and atypical antipsy-

chotics [8]. The present study showed that mirtazap-

ine [F (4, 35) = 34.46; p < 0.001, Fig. 3A] or risperi-

done [F (3, 28) = 43.85; p < 0.001, Fig. 3B] dose-

dependently inhibited the DOI-induced head twitches

in mice. Moreover, co-administration of risperidone

(0.01 mg/kg) and mirtazapine (1.25 or 2.5 mg/kg) re-

duced the number of head twitches induced by DOI,

but those drugs given separately did not change that

syndrome [F (5, 42) = 36.74; p < 0.001, Fig. 3C].

Since the synergistic antipsychotic-like effect was ob-

served following co-treatment with mirtazapine and

risperidone, an important role of 5-HT�� receptors in

mediating their action has been suggested. In addi-

tion, some earlier studies conduced by other authors

suggested that adjunct treatment with escitalopram,

but not citalopram, may enhance the effect of a sub-

therapeutic dose of risperidone on the positive, nega-

tive, cognitive, and depressive symptoms of schizo-

phrenia [12]. Our earlier data also indicated that mir-

tazapine and other antidepressants may potentiate the

antidepressant like effect of low doses of risperidone

in the forced swimming test in mice [16, 17].

The obtained results indicate that low doses of mir-

tazapine enhance the antipsychotic-like effect of

risperidone in animal tests of positive symptoms of

schizophrenia. Further studies are necessary to eluci-

tate its mechanism of action.

Acknowledgments:

The author wish to thank Ms. El¿bieta Smolak, M.A., for the

linguistic supervision of the paper. This study was financially

supported by statutory funds from the Institute of Pharmacology,

Polish Academy of Sciences, Kraków, Poland.

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Received: July 10, 2012; accepted: September 27, 2012.

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