Effect of repeated co-treatment with fluoxetine and amantadine on the behavioral reactivity of the central dopamine and serotonin system in rats

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Short communication

Effect of repeated co-treatment with fluoxetine and amantadine on the behavioral reactivity of the central dopamine and serotonin system in rats

Zofia Rogó¿, Gra¿yna Skuza

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:

In the present study we found that repeated co-treatment with fluoxetine and amantadine for 14 days (but not for 7 days) enhanced the hyperactivity induced by amphetamine or quinpirole (a dopamine D_2/3agonist), compared to treatment with either drug alone.

Whereas repeated co-treatment with fluoxetine and amantadine for 7 days more potently inhibited the behavioral syndrome evoked by the 5-hydroxytryptamine (5-HT)_1Areceptor agonist (±)-8-hydroxy-2(di-n-propylamino)-tetralin hydrobromide (8-OH-DPAT), it did not change the action of the 5-HT₂ receptor agonist (±)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride (/±/-DOI). The obtained results support the hypothesis that repeated co-treatment with fluoxetine and amantadine may evoke more effective antidepressant activity than treatment with fluoxetine alone.

Moreover, our results suggest that 5-HT_1Areceptors are useful targets for the development of more rapidly acting and more effective medication.

Key words:

repeated treatment, fluoxetine, amantadine, behavioral test, rats

Introduction

It is estimated that 30–40% of patients suffering from depression do not respond to conventional therapies.

The problem of antidepressant-resistant depression has been the subject of extensive studies, with no ap- parent therapeutic success. Therefore, to improve therapy, a combination of antidepressant drugs (ADs) be- longing to various pharmacological groups, or a combination of an AD and a substance enhancing its effect, is presently used in the clinic. In recent years, much attention has been devoted to the glutamatergic

system in general, and to NMDA receptor antagonists in particular [23, 25]. The antidepressant activities of these compounds have been demonstrated in animal models for competitive NMDA receptor antagonists such as CGP-37849 or AP-1, and for uncompetitive antagonists such as amantadine (AMA), memantine or neramexane [9, 10, 15, 16, 22]. In addition, our earlier studies demonstrated that simultaneous administration of an AD (imipramine, venlafaxine or fluoxetine /FLU/) and an uncompetitive NMDA receptor antagonist (AMA) produces a more potent “antidepressant” effect in the forced swimming test (measured as the shortening of immobility time) than

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does treatment with either of those drugs alone [22].

Positive effects were also observed when the above- mentioned drugs were used jointly in inactive doses.

Moreover, a small clinical trial recently revealed that iv infusion of ketamine produces a short-lasting psy- chotomimetic effect (hours) and long-lasting antidepressive action (days) [1, 29]. Moreover, earlier pre- liminary clinical studies showed that treatment of drug-resistant, unipolarly depressed patients with imipramine and AMA results in significant clinical improvement [18, 19]. A similar result was reported by Stryjer et al. [24], who also observed in a small open- label trial that AMA given with conventional ADs causes a 50% improvement in treatment-resistant patients and decreases the time necessary for a therapeutic improvement in some of those patients. The latter findings suggest that the results obtained with animal models may also be valid in the clinic.

Interestingly, we found previously that repeated (14 days) co-treatment with FLU and AMA induces a substantial increase in BDNF gene expression in the cerebral cortex, and inhibits the behavioral syndrome induced by 5-HT_1Aand 5-HT₂receptor agonists (compared to treatment with either drug given alone) [20].

The aim of the present study was to examine the in- fluence of combined, repeated treatment with FLU and AMA on the central dopamine and serotonin system. First, we studied the effect of repeated treatment (7 and 14 days) with FLU (5 or 10 mg/kg) and AMA (10 mg/kg), given separately or jointly, on the dopamine D_2/3 system in rats. Second, the effects of the two above-mentioned drugs (7-day treatment) on the central serotonin (5-HT_1Aand 5-HT₂) system in rats were also assessed through behavioral measurements.

Materials and Methods

Animals and drug administration

The experiments were carried out on male Wistar rats (obtained from the licensed animal breeder T. Górz- kowska, Warszawa, Poland), all weighing 250–270 g.

The animals had free access to food and water before the experiment, and were kept on a 12–h light/dark cycle (the light on at 07:00) at a constant room tem- perature (22 ± 1°C). FLU in doses of 5 or 10 mg/kg and AMA in a dose of 10 mg/kg were dissolved in dis-

tilled water (at a volume of 2 ml/kg) and administered repeatedly (twice dailypo, for 7 or 14 days), separately or together. The behavioral syndrome evoked by 8-OH-DPAT, a 5-HT_1A receptor agonist, and head twitches induced by (±)-DOI, a 5-HT₂ receptor agonist, as well as amphetamine or quinpirole hyperactivity were measured 24 h after the last dose of FLU and AMA (or after their single administration). Animals were used only once for each experiment. All experiments were conduced in the light phase of the light- dark cycle (between 09:00 and 14:00 h), and were performed in accordance with the European Commu- nities Council Directive of 24 November 1986 (86/609 EEC). All experimental protocols were ap- proved by the Local Bioethics Commission for the Animal Experiments at the Institute of Pharmacology, Polish Academy of Sciences in Kraków.

D-amphetamine- and quinpirole-induced locomotor hyperactivity

Locomotor activity was recorded individually for each animal in Opto-Varimex cages (Columbus In- struments, Columbus, OH, USA), linked to a com- puter, starting from 24 h after the last dose of FLU and AMA (or after their single administration). Each cage (43 × 44 × 25 cm) was surrounded by a 15 × 15 array of photocell beams, located 3 cm from the floor surface as reported previously [28]. Interruption of the photo beams indicated horizontal locomotor activity, defined as the distance traveled expressed in cm. Lo- comotor activity measurements started 30 min after AMP (0.5 mg/kg,sc) or QUI (0.3 mg/kg, sc) admini- stration and lasted for 1 h. Each experimental group consisted of eight rats.

Behavioral syndrome induced by 8-OH-DPAT

8-OH-DPAT (5 mg/kg, ip) was given 24 h after the last dose of FLU or AMA (or after their single administration). Immediately after the injection of 8-OH-DPAT, the animals were separately placed in cages. Observation sessions began 3 min after 8-OH-DPAT injection, and were repeated every 3 min for a period of 15 min. Reciprocal forepaw treading and flat body posture were scored using a ranked in- tensity scale (0 point – absence; 1 point – equivocal; 2 points – present; 3 points – intense). Each score was summed over five observation periods. Each group consisted of six rats.

Repeated co-treatment with fluoxetine and amantadine and behavioral reactivity in rats

Zofia Rogó¿ and Gra¿yna Skuza

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dose of FLU or AMA (or after their single administration). Head twitches were observed immediately after (±)-DOI administration, and recorded for 30 min.

Each group consisted of six rats.

Drugs

Amantadine hydrochloride (AMA; Sigma, USA), D- amphetamine sulfate (AMP; Sigma, USA), fluoxetine hydrochloride (FLU; Farmacom, Kraków, Poland), (±)-8-hydroxy-2(di-n-propylamino)-tetralin hydrobromide (8-OH-DPAT; Research Biochemicals Int., USA), (±)- 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride ((±)-DOI; Research Biochemicals Int., USA) and quinpirole hydrochloride (QUI; Research Bio- chemicals Int., USA) were used for the present study.

Statistical analysis

The data were evaluated by a one-way analysis of vari- ance (ANOVA), followed, when appropriate, by individual comparisons with the control using Dunnett’s test.

D-amphetamine- and quinpirole-induced loco- motor hyperactivity

Both AMP (0.5 mg/kg) and QUI (0.3 mg/kg) increased the locomotor activity of rats compared to vehicle-treated controls (by 205 and 220%, respectively). FLU (5 or 10 mg/kg) and AMA (10 mg/kg), administered as single doses separately or jointly did not affect the locomotor activity of naive rats or the locomotor hyperactivity induced by AMP (0.5 mg/kg) or QUI (0.3 mg/kg) (data not shown).

Repeated treatment with FLU (5 and 10 mg/kg) or AMA (10 mg/kg) insignificantly increased the locomotor hyperactivity induced by AMP (by 17, 19 and 17%, respectively), or the locomotor hyperactivity induced by QUI (by 14, 86 and 124%, respectively).

Co-treatment with FLU (5 or 10 mg/kg) and AMA (10 mg/kg) (twice daily, for 7 days) did not enhance the locomotor hyperactivity evoked by AMP (0.5 mg/kg) or QUI (0.3 mg/kg) (data not shown).

Moreover, repeated co-treatment (twice daily, for 14 days) with a higher dose of FLU (10 mg/kg) and AMA (10 mg/kg) enhanced the locomotor hyperactivity evoked by AMP (0.5 mg/kg), F(5,42) = 13.76, p < 0.001 (Fig. 1) or QUI (0.3 mg/kg), F(5,42) = 7.85, p < 0.001 (Fig. 2).

Fig. 1.The effect of repeated (14 days) co-treatment with fluoxetine (FLU) and amantadine (AMA) on D-amphetamine (AMP)-induced locomotor hyperactivity. The test was carried out 24 h after the last dose of FLU and AMA. AMP (0.5 mg/kg) was injected 30 min before the test. The results are presented as the mean ± SEM of 8 animals/group. The data were statistically evaluated by ANOVA, followed by individual comparisons using Dunnetts test. * p < 0.001 vs.

vehicle group;p < 0.001 vs. appropriate FLU + AMP or AMA + AMP group

Fig. 2.The effect of repeated (14 days) co-treatment with fluoxetine (FLU) and amantadine (AMA) on quinpirole (QUI)-induced locomotor hyperactivity. The test was carried out 24 h after the last dose of FLU and AMA. QUI (0.3 mg/kg) was injected 30 min before the test. The results are presented as the mean ± SEM of 8 animals/group. The data were statistically evaluated by ANOVA, followed by individual comparisons using Dunnetts test. * p < 0.001 vs. vehicle group;

p < 0.001 vs. appropriate FLU + QUI or AMA + OUI group

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Behavioral syndrome induced by 8-OH-DPAT

Neither FLU (5 and 10 mg/kg) nor AMA (10 mg/kg), given in a single dose separately or jointly, changed the behavioral syndrome induced by the 5-HT_1Aagonist 8-OH-DPAT (5 mg/kg) (data not shown). Repeated treatment (twice daily, for 7 days) with FLU (10 mg/kg), but not FLU (5 mg/kg) and AMA (10 mg/kg), inhibited the behavioral syndrome induced by 8-OH-DPAT in a statistically significant manner. Moreover, repeated co-administration of FLU (at either 5 or 10 mg/kg) and AMA (at 10 mg/kg) induced a more potent (statis-

tically significant) inhibition of 8-OH-DPAT action (flat body posture, F(5,30) = 37.69, p < 0.001 (Fig.

3A) and forepaw treading, F(5.30) = 49.45, p < 0.001 (Fig. 3B)) than treatment with FLU or AMA alone.

Head twitches induced by (±)-DOI

When given in a single dose separately or jointly, neither FLU (5 and 10 mg/kg) nor AMA (10 mg/kg) af- fected the head twitch reaction induced by the 5-HT₂ agonist (±)-DOI (2.5 mg/kg) (data not shown). Moreo- ver, repeated administration (twice daily, for 7 days) of FLU (5 and 10 mg/kg) and AMA (10 mg/kg) or co-treatment with FLU (5 or 10 mg/kg) and AMA (10 mg/kg) did not affect head twitches induced by (±)-DOI, F(5,30) = 3.92, ns (data not shown).

Discussion

Earlier studies have shown that ADs administered repeatedly (but not in a single dose) induces upregulation of dopamine D_2/3receptors in the brain, as evaluated by behavioral (potentiation of the hyperactivity induced by dopamine D_2/3 agonists) and bio- chemical experiments [2, 6, 26, 27]. Our present results show that repeated co-treatment with FLU and AMA for 14 days (but not for 7 days) enhances the effects evoked by amphetamine or quinpirole (a dopamine D_2/3agonist) compared to treatment with either drug alone. This finding suggests that AMA enhances the behavioral effects of FLU on the central dopamine D_2/3system in rats.In vivo microdialysis showed that AMA or budipine (NMDA receptor antagonists), given chronically (4, 7, 14 and 21 days), does not significantly alter extracellular DA. Moreover, three ADs (reboxetine, paroxetine or clomipramine) were found to elicit a gradual increase in DA, which be- comes significant after 7 days and tends to reach a plateau thereafter.

When AMA or budipine was co-administered with the above three ADs, two basic differences were observed as compared to the administration of ADs alone. First, the time required for a significant increase in cortical DA was reduced, with elevated lev- els observed after 4 days. Second, the increase in extracellular DA was greater in these rats throughout the experiment [14]. The underlying mechanisms by

FLU AMA 8-OH-DPAT

– 5 10 – 5 10 mg/kg

10

– – – 10 10

+ + + + + +

FLU AMA 8-OH-DPAT

– 5 10 – 5 10 mg/kg

10

– – – 10 10

+ + + + + +

Fig. 3.The effect of repeated (7 days) co-treatment with fluoxetine (FLU) and amantadine (AMA) on the 8-OH-DPAT-induced behavioral syndrome (flat body posture (A) and forepaw treading (B) in rats).

The test was carried out 24 h after the last dose of FLU and AMA.

8-OH-DPAT (5 mg/kg) was injected directly before the test. The results are presented as the mean ± SEM of 6 animals/group. The data were statistically evaluated by ANOVA, followed by individual comparisons using Dunnetts test. * p < 0.001 vs. 8-OH-DPAT group;

p < 0.001 vs. appropriate FLU + 8-OH-DPAT or AMA + 8-OH-DPAT group

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gic transmission or effects on the synthesis of dopamine. It is possible that NMDA receptor antagonists alter glutamatergic tone at NMDA receptors and thus facilitate DA transmission. Additionally, both AMA and budipine have been shown to potentiate the activity of L-DOPA decarboxylase, as well as inhibit monoamine oxidase B [3, 4, 17]. This may lead to an overall increase in DA synthesis, which, if progres- sive, may account for the effects observed after chronic administration of the drugs.

Furthermore, the present study shows that repeated co-treatment (for 7 days) with FLU and AMA more potently inhibits 5-HT_1A(but not 5-HT₂) neurotransmission in behavioral tests than FLU alone, and neither acute nor chronic administration of AMA significantly alters this effect. Moreover, our earlier results indicated that repeated co-treatment (for 14 days) with FLU and AMA also more potently inhibits both 5-HT_1A- and 5-HT₂ neurotransmission in behavioral tests then FLU alone [20]. The latter effect is in line with the previously demonstrated decrease in the den- sity of 5-HT₂receptors observed after repeated treatment with FLU [7] and other ADs [5, 8] in biochemi- cal and behavioral experiments. Using in vivo micro- dialysis, it was previously found that AMA or budipine (NMDA receptor antagonists), given acutely with ADs (reboxetine, paroxetine or clomipramine), elevates extracellular 5-HT concentration in the frontal cortices of freely moving rats. Repeated co- treatment with AMA or budipine and ADs (reboxetine, paroxetine or clomipramine) facilitated and po- tentiated these effects. In addition, neither acute nor chronic (7, 14 and 21 days) administration of AMA or budipine significantly changed extracellular 5-HT concentration in the frontal cortices of freely moving rats [13]. The latter authors suggested that the above- described effect on 5-HT concentration was caused by several factors, including alteration of glutamatergic tone at NMDA receptors, which can facilitate both 5-HT neurotransmission, and the action of budipine and AMA on monoamine metabolism. Moreover, our previous study showed that FLU given repeatedly (for 14 days) increases brain-derived neurotrophic factor (BDNF) gene expression in both brain regions studied (the hippocampus and the cerebral cortex) [20]. The action of FLU in that test was similar to the effect pro- duced by MAOI, tranylcypromine and ECS [11, 12].

administration (for 14 days) compared to either of those drugs alone [20]. In contrast, repeated co- administration (7 days) of FLU and AMA did not change BDNF gene expression in either brain region examined (the hippocampus and the cerebral cortex) [Legutko, personal communication]. The results described in the present paper show that repeated co- treatment (7 days) with FLU and AMA potently inhibits only 5-HT_1Aneurotransmission in behavioral tests.

In conclusion, the results described in the present paper support the hypothesis that repeated joint administration of FLU and AMA evokes more effective antidepressant activity than treatment with FLU alone.

Furthermore, among other mechanisms, 5-HT_1A, 5-HT₂, dopamine D_2/3receptors and the neurotrophic system may also be involved in this action. Thus, 5-HT_1Areceptors appear to be involved in antidepressant efficacy and represent future targets for the development of more effective and rapidly acting medication.

Acknowledgments:

The authors wish to thankFarmacom, Kraków (Poland), for their generous gift of fluoxetine. We would also like to thank Ms. E. Smolak, M.A., for the linguistic supervision of this paper.

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Received:

March 24, 2009; in revised form: October 5, 2009.