Biomedicine & Pharmacotherapy 81 (2016) 295–304

Antidepressant effect of pramipexole in mice forced swimming test: Across talk between dopamine receptor and NMDA/nitric oxide/cGMPpathway

Sattar Ostadhadia,b, Muhammad Imran Khanb,d, Abbas Norouzi-Javidana,Ahmad-Reza Dehpoura,b,c,*aBrain and Spinal Cord Injury Research Center, Neuroscience Institute, Tehran University of Medical Sciences, Tehran, IranbDepartment of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iranc Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, IrandDepartment of Pharmacology, School of Medicine, International Campus, Tehran University of Medical Sciences, Tehran, Iran

A R T I C L E I N F O

Article history:Received 20 February 2016Received in revised form 9 April 2016Accepted 11 April 2016

Keywords:DepressionPramipexoleForced swimming testDopamine D2 receptorNMDANitric oxideMice

A B S T R A C T

Pramipexole is a dopamine D2 receptor agonist indicated for treating Parkinson disorder. This study wasaimed to investigate the effect of pramipexole in forced swimming test (FST) in mice and the possibleinvolvement of activation of D2 receptors and inhibition of N-methyl-D-aspartate (NMDA) receptors andnitric oxide-cyclic guanosine monophosphate (NO-cGMP) on this effect. Intraperitoneal administrationof pramipexole (1–3 mg/kg) reduced the immobility time in the FST similar to fluoxetine (20 mg/kg, i.p.).This effect of pramipexole (1 mg/kg, i.p.) was ceased when mice were pretreated with haloperidol(0.15 mg/kg, i.p,) and sulpiride (5 mg/kg, i.p) as D2 receptor antagonists, NMDA (75 mg/kg,i.p.), L-arginine(750 mg/kg, i.p., a substrate for nitric oxide synthase) or sildenafil (5 mg/kg, i.p., a phosphodiesterase5 inhibitor). The administration of MK-801 (0.05 mg/kg, i.p., a NMDA receptor antagonist) L-NG-Nitroarginine methyl ester (L-NAME, 10 mg/kg, i.p., a non-specific nitric oxide synthase (NOS) inhibitor), 7-nitroindazole (30 mg/kg, i.p., a neuronal NOS inhibitor) and methylene blue (10 mg/kg, i.p.), an inhibitorof both NOS and soluble guanylyl cyclase (sGC) in combination with the sub-effective dose ofpramipexole (0.3 mg/kg, i.p.) reduced the immobility. Altogether, our data suggest that theantidepressant-like effect of pramipexole is dependent on the activation of D2 receptor and inhibitionof either NMDA receptors and/or NO-cGMP synthesis. These results contribute to the understanding ofthe mechanisms underlying the antidepressant-like effect of pramipexole and reinforce the role ofD2 receptors, NMDA receptors and L-arginine-NO-GMP pathway in the antidepressant mechanism of thisagent.

ã 2016 Published by Elsevier Masson SAS.

Available online at

ScienceDirectwww.sciencedirect.com

1. Introduction

Depression is one of the debilitating conditions mostlyassociated with the dysregulation of neurotransmitters likenoradrenaline and serotonin (5-HT). The current therapy fordepression is also based on these neurotransmitters like nor-adrenaline and/or 5-HT re-uptake inhibitors [1,2] . Drugs such asselective serotonin re-uptake inhibitors (SSRIs) like as fluoxetine,fluvoxamine or paroxetine are a suitable therapy as it mask thehistaminic and muscarinic receptors affinity, however classical

* Corresponding author at: Department of Pharmacology, School of Medicine,Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran.

E-mail address: Dehpour@sina.tums.ac.ir (A.-R. Dehpour).

http://dx.doi.org/10.1016/j.biopha.2016.04.0260753-3322/ã 2016 Published by Elsevier Masson SAS.

tricyclic antidepressants like imipramine have high affinitytowards these receptors [3].

A large body of research focusing depression have reported thatdepression occurs as a consequence of impairment in dopaminer-gic system [4,5] . Certain classes of drugs that shows antidepres-sant activity both in laboratory animals and human beings arethought to activate dopaminergic (D) receptors [6,7] . Also it isreported that the antidepressant like effects of certain drugs in theFST is mediated by D1/D5 receptors subtypes [8–10], while othersreported that D2/D3 receptor antagonist prevent the antidepres-sant like effect but not the D1/D5 receptor antagonist [11,12].Moreover, the reduction in the immobility time during FST bydopamine reuptake inhibitors has been reported to be initiated bydopamine D1 and D2 receptor [9].

Fig. 1. Effect of i.p administration of pramipexole (PRAM) and fluoxetine (Flu,20 mg/kg, i.p.) on FST (A); and OFT (B) in mice. Values are expressed as mean +SEM(n=8). *P < 0.05 ***P < 0.001 as compared with the vehicle-treated control.

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Pramipexole as a dopamine D2 receptor agonist has beenproved to be effective in treating diseases like Parkinson’s diseaseand restless leg syndrome in patients who has proven antidepres-sant capacity [13,14]. Also It has been reported pramipexoleaugmented the antidepressive effects of other drugs which showedresistance in the treatment of major depressive disorders [15]. Arecent study reported that patient with bipolar disorder, subjectedto antidepressive treatment with additional pramipexole admin-istration, showed significant improvement within 36 weeks oftherapy [16].

Furthermore it is shown that pramipexole decreases the time ofimmobility during force swimming test in mice as well as rats[17–19] through the stimulation of D2 receptors [17]. Despite itsinteraction and known antidepressant effects, still we need to domore in order to uncover the exact mechanism as yet it’s not fullyelucidated.

Dopamine and glutamate are considered as an important andextensively studied neurotransmitter in the brain. Aside from theirmain physiological functions, any variation in the normal harmonyof neurotransmitter release, can lead to various psych- cognitiveabnormalities including depression. Also, it is reported thatD2 receptor activation, decreased NMDA receptor phosphorylationwhich is mediated by protein kinase A (PKA) enzyme [20] andhence blocked the NMDA initiated Ca2+ signaling [21].

It has been suggested that glutamate is involved in pathophysi-ological conditions like depressive disorders [22,23] . Furthermore,it was also affirmed by other research group, which reported that ahigh level of glutamate was found in the frontal cortex of postmortem, who was suffering from major depressive disorder [24].Also pharmacological inhibition of NMDA receptor throughantagonist like ketamine, has an excellent clinical outcome indepressive patients [25,26] . Reports from experimental data arealso in line with the above results, where it shows that drugs whichblock the NMDA receptors have antidepressant like activity[27–30]. Similarly, high doses of antidepressant drugs have intenseeffect on NMDA receptor activity [22,23].

It is well established that NMDA receptor activation can mimicthe activity of nitric oxide synthase (NOS), which in turn catalyzethe synthesis of nitric oxide NO from L-arginine [31,32]. The nitricoxide mediated cGMP synthesis is an essential signaling involvedin pathogenesis of depression [33]. Nitric oxide in brain is alsofound to perform other important functions such as neurotrans-mitter release, pain perception, synaptic plasticity and depression[32]. In recent years, nitric oxide, where decline in the NO level wasfound to have antidepressant, thus supporting that NO isimportant determinant in the instigation of depression [34]. Nitricoxide is powerful modulator of cGMP, which is implicated in themanifestation of depression in experimental animals [35]. Recentliterature on the blockage of NOS activity reported that it canenhance the antidepressant activity of SSRI’s and other agents[36–38].

Taken together these observations, in current study we tried touncover the potential mechanism for the antidepressant-likeactivity of D2 receptor agonist pramipexole, in the mouse FST. Thistest is a reliable model, widely used for the assessment ofantidepressant-like activity of drugs [39]. In order to uncover thisantidepressant-like mechanism, we focused on the followingcomponents as an important determinant of such effect.

- The involvement of dopamine D2 receptor in the antidepres-sant- like effect of pramipexole

- A cross talk with NMDA receptor and Nitrergic system, possiblythrough neural NOS (nNOS) inhibition, in the antidepressant-like effect of pramipexole.

2. Materials and methods

2.1. Animals

Adult male NMRI mice,aged 11–13 weeks, were obtained fromthe Pasteur Institute of Iran, within the range of 20–30 g and. Allanimals were kept under standard conditions of temperature 21�2 �C and light/dark cycle of 12-h each. Freely access to food andwater was given except for the period of experimentation outsidethe cage. Four to five animals were housed in a single cage. Fullefforts were paid to conduct the experiments within 08:00am to16:00 pm. In each group there were 8–10 animals and experimentswere performed by strictly following the institutional guideline ofanimal care and use committee (Department of Pharmacology,School of Medicine, Tehran university of Medical Sciences). Thisstudy was ethically approved by the International Campus researchCommittee of Tehran University of Medical Sciences.

2.2. Open-field test (OFT, locomotor activity test)

The locomotor activity of animals was determined through awell-known test i.e. open-field test, immediately followed by FSTas described previously [27,40]. Briefly the apparatus consist of abox made of wood with dimension of 40 cm � 60 cm � 50 cm. Thebase of the box is divided equally into 12 small squares. At the startof each trial a mouse was placed in the left corner of the field. Thenumber of squares were counted, when crossed with all paws for aperiod of 6-min. After every session for each animal the apparatuswas washed with a 10% ethanol solution.

Fig. 2. The effect of haloperidol (HAL; 0.15 mg/kg, i.p. A) (A), and Sulpiride (SUL,5 mg/kg, i.p. C) pretreatment on antidepressant effect of pramipexole (PRAM, 1 mg/kg) in theFST .The immobility time in FST analyzed for last 4 min of the test. (B and D) the total number of crosses recorded during open-field test. Data are presented as mean � SEM,n = 8 animals/group, and were analyzed using a two-way ANOVA followed by Tukey’s post-test. **P < 0.01 ***P < 0.001 01 compared with saline- treated control; or with thePRAM treated group.

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2.3. Forced swimming test

FST consist of cylindrical jar with dimension of 10 cm diameter,25 cm height and filled with19 cm of water at 24 �1 �C as definedpreviously [41–43]. Each mouse was placed gently and wasallowed to swim freely for 6 min. The mouse was assumed asimmobile when it showed disparity and become motionless in thewater. During the period of immobility they make only thosemoments which were necessary to keep their head outside thewater. The immobility duration was recorded and later on it wasinterpreted for the last 4 min of test.

2.4. Drugs

Drugs used in our study are: pramipexole and fluoxetinepurchased from Abidi Company, Tehran, Iran. haloperidol from Exircompany, Tehran, Iran while other drugs like sulpiride, L-NAME anon-specific NOS inhibitor, 7-NI (7-nitroinidazole), an nNOSspecific inhibitor, L-arginine, NO precursor,methylene blue asinhibitor of both NOS and sGC, NMDA as NMDA receptor agonist,MK-801 as NMDA receptor antagonist were purchased from Sigma,St. Louis, MO, USA. Sildenafil, a phosphodiesterase 5 inhibitor waspurchased from Poursina Company, Tehran, Iran. Drugs solutionwere made in saline (0.9%), except for haloperidol and 7-NI weresolution was prepared by using 5% dimethyl sulfoxide (DMSO) forhaloperidol and 1% twin 80 solution for 7-NI. Intraperitoneal (i.p.)route was followed for all drugs administration in constant volumeof 10 ml/kg of body weight.

2.5. Drugs treatment

For the determination of antidepressant like activity ofpramipexole, various doses (0.1, 0.3, 1 and 3 mg/kg), wereevaluated, given 60 min prior to FST. Fluoxetine (20 mg/kg, i.p.)[44] was also examined as a reference drug for positive control.This agent administrated 30 min before the test. To study theinvolvement of D2/D3 receptor in such effect of pramipexole,animals in each group were treated with a specific drug. Fordopaminergic system involvement in the acute antidepressant likemechanism of pramipexole in the FST, saline and sulpiride (5 mg/kg) as D2 antagonist [9], was concomitantly injected withpramipexole (1 mg/kg). FST and OFT were performed 60 min afteradministration of drugs. Additionally, for the confirmation of theinfluence of dopamine D2/D3 receptor in such effects ofpramipexole, another dopamine D2 receptor antagonist haloperi-dol at sub-effective dose (0.15 mg/kg) [45] was administered30 min prior to pramipexole (1 mg/kg) and FST was than performed60 min later to the injection of pramipexole [17] .

To examine the involvement and modulation of the NMDApathway in the antidepressant-like effect of pramipexole in theforced swimming test (FST), a group of mice (n = 6) was pretreatedwith NMDA, (75 mg/kg i.p.,) 30 min after pramipexole (1 mg/kg)administration. This dose of NMDA do not produce any effectduring FST [30]. Thirty min after injection of NMDA, FST wasperformed.

In order to validate our finding, we tried to uncover theinteraction between pramipexole and NMDA receptors in altering

Fig. 3. Effect of MK-801 (0.05 mg/kg, i.p.) pretreatment on antidepressant effect of pramipexole (PRAM, 0.3 mg/kg, i.p. A) and NMDA (75 mg/kg, i.p) pretreatment onantidepressant effect of pramipexole (PRAM,1 mg/kg, i.p. C) in the FST .The immobility duration in FST for last 4 min of the test. (B, D) Total number of crosses during open-fieldtest. Scores are expressed as the mean � SEM, n = 8 animals/group and were analyzed using two-way ANOVA followed by Tukey’s post-test. **P < 0.01 ***P < 0.001 comparedwith saline- treated control; or with the PRAM treated group.

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the time of immobility in mice; For this, one group of mice wereadministered pramipexole (0.3 mg/kg) sub effective dose and15 min later the sub effective dose of MK-801 (0.05 mg/kg), a dosewhich is incapable to elicit any effect during forced swimming test,was administrated [30]. Forty-four minutes after the administra-tion of MK-801, FST was done.

To study the role of NO on the antidepressant like effect ofpramipexole, L-NAME (10 mg/kg) [17] or 7-NI (30 mg/kg) [11] insub-effective doses co- administered with pramipexole (0.3 mg/kg) sub-effective dose. L-NAME was injected 45 min and 7-NI wasinjected 30 min prior to FST in both pramipexole and saline-treatedanimals. In another set of experimental group, we injected sub-effective dose of L-arginine (750 mg/kg) [18] with an effective doseof pramipexole (1 mg/kg) simultaneously. The control group forthis treatment was only injected with same amount of saline. After60 min of drugs administration, FST was done for both pramipexoleand saline treated animals.

In order to find an association between sGC and antidepressantlike effect of pramipexole, we co- administered subeffective doseof sildenafil (PDE5 inhibitor, 5 mg/kg, i.p.) [19] with pramipexoleeffective dose (1 mg/kg) or with saline. Sixty minutes aftertreatment animals were subjected to FST.

To underpin the cGMP role in the antidepressive like effect ofpramipexole, methylene blue (10 mg/kg, i.p.,) an inhibitor of bothNOS and sGC, were injected into a groups of saline treated orpramipexole (0.3 mg/kg) treated animals [46] .Sixty minutes aftertreatments, FST and OFT were performed for either groups.

2.6. Statistics

One way ANOVA was used to determine the dose effect ofpramipexole on the immobility/motionlessness behavior duringFST while for the determination of locomotor activity in an openfield test one-way analysis of variance (ANOVA), followed byDunnett’s test was used. All other data’s were analyzed using twoway ANOVA followed by Tukey’s test.

3. Results

3.1. Pramipexole effects on the immobility time during FST and onlocomotor activity during OFT

When mice were treated intraperitoneally with different dosesof pramipexole (1–3 mg/kg), a significant reduction in theimmobility time was observed during FST [F (5, 42) = 7.960,p < 0.0001; Fig. 1A]. Also fluoxetine (20 mg/kg, i.p.) led to asignificant reduction in time spent immobile compared to thecontrol group (P < 0.01). Additionally significant reduction in theambulatory activity was also seen during OFT in pramipexole(3 mg/kg) treated group as compare to control one [F (5,42) = 2.908; p < 0.05; Fig. 1B]. These data suggested that prami-pexole tends to decrease locomotor activity in NMRI mice, andtherefore the activity of pramipexole in the mouse FST is not due togeneralized increases in locomotor activity under the conditionsused in the present studies.

Fig. 4. Effect of L-arginine (L-ARG), on pramipexole (PRAM) antidepressant activity.L-ARG (750 mg/kg, i.p.) administrated prior to the administration of either PRAM(1 mg/kg, i.p.) or its vehicle (saline). (A) The duration of immobility in FST for the last4 min of the test. (B) The total number of crosses during open-field test recorded.Scores are presented as the mean � SEM n = 8–10 animals and were analyzed using atwo-way ANOVA followed by Tukey’s post-test. ***P < 0.001, **P < 0.01 comparedwith saline- treated control; or with the PRAM treated group.

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However, pramipexole at dose 3 mg/kg i.p. decreased bothlocomotor activity and immobility time in OFT and FST respec-tively, other doses of pramipexole like 1 mg/kg i.p. (having effectduring FST but do not alter locomotor activity) or a dose of 0.3 mg/kg i.p. which is ineffective during FST and OFT were selected aseffective and sub-effective dose respectively for next experiments

3.2. The involvement of dopamine D2 receptor in the antidepressantlike effect of pramipexole during FST

Fig. 2 shows the effects of sulpiride and haloperidol, a dopamineD2 receptor antagonist on pramipexole (1 mg/kg) anti-immobilityeffects during FST. When haloperidol (0.15 mg/kg) alone wasinjected to mice, it failed to show any effect in mouse forcedswimming test. However, prior administration of haloperidol topramipexole (1 mg/kg) in mice reversed the antidepressant-likeeffect in FST. Similar antagonizing effects were seen with sulpiride(5 mg/kg) on pramipexole (1 mg/kg) activity in FST. Haloperidoland sulpiride failed to show any significant effect on the locomotorbehavior of mice treated with either pramipexole or saline (Fig. 1C,D).

3.3. NMDA receptors involvement in the antidepressant-like effect ofpramipexole during FST and on locomotor activity during OFT

Fig. 3 reveals the effects of co-administration of pramipexoleand MK-801 an NMDA receptor antagonist, on immobility time of

mice during FST (F (3, 28) = 8.090, p < 0.001). Pramipexole (0.3 mg/kg) did not have any effect on the immobility period of mice in FST.Also MK-801 (0.05 mg/kg), when administered as a single drug wasineffective during FST of mice. However, when pramipexole andMK-801 were co-administered in the same doses, they significantlyreduced the immobility time during FST (p < 0.001). Injection ofMK-801 alone or when combined with pramipexole failed to affectthe locomotor activity during OFT (Fig. 3D).

Fig. 3C, shows the effects of co-administration of NMDA andpramipexole on the time lap of mice immobility during FST [F (3,28) = 10.31, p < 0.0001] NMDA (75 mg/kg, i.p.) when given alonehad no substantial effect on the immobility time during FST.However, when the same dose of NMDA was administered beforePRAM, it halted the antidepressant-like effects pramipexole(Fig. 3C). NMDA alone or when combined with effective dosesof pramipexole (1 mg/kg), failed to elicit any effect on ambulatoryactivity of mice during OFT (Fig. 3D).

3.4. Nitric oxide involvement in the antidepressant-like effect ofpramipexole during FST and its effect on locomotor activity during OFT

Fig. 4A illustrates that although L-arginine (750 mg/kg) alonehad no effect on the time of immobility of animals during FST,however, the antidepressant-like activity of pramipexole (1 mg/kg)had significantly (P < 0.01) reversed during FST [F (3, 28) = 9.640,P < 0.001]. L-Arginine when co-administrated with either vehicleor with pramipexole, failed to change the locomotor activity ofanimals during OFT (Fig. 4B).

As shown in Fig. 5A co-administration of per se non-effective l-NAME (10 mg/kg) and pramipexole (0.3 mg/kg) doses havesignificant (P < 0.01) antidepressant-like activity during FST (F(3, 28) = 8.755, P < 0.001). Same effects were also observed whenper se non effective 7-NI (30 mg/kg) and pramipexole (0.3 mg/kg)doses were administered prior to FST (F (3, 28) = 6.472; Fig. 5C).Fig. 5B and D clearly validated that co-injection of pramipexole(0.3 mg/kg) with l-NAME (P > 0.05; Fig. 5 B) or with 7-NI (F (3,28) = 6.472, P > 0.05) failed to bring any significant changes inlocomotor behavior of animals during OFT.

3.5. Involvement of cGMP in the antidepressant-like activity ofpramipexole during FST and effect on locomotor activity during OFT

Methylene blue (10 mg/kg, i.p.,) a direct inhibitor of both NOSand sGC, was unable to affect the immobility time during FST whenused alone. But, when administered with subeffective dose ofpramipexole (0.3 mg/kg, i.p.), we found a significant effect where itconsiderably increased the antidepressant like activity of prami-pexole [F (3, 28) = 9.976, P < 0.001] (Fig. 6A).

Administration of methylene blue alone or in combination withpramipexole did not show any significant affect (p > 0.05) onlocomotor activity during OFT (Fig. 6B).

Fig. 7A displayed the effect of sildenafil (5 mg/kg, i.p., aPDE5 inhibitor) on experimental animals. Pretreatment withsildenafil alone, failed to elicit any effect on the immobility time.However, when the animals were pretreated with sildenafil andpramipexole (1 mg/kg), sildenafil reversed the antidepressanteffect of pramipexole in the FST [F (3, 28) = 10.48, P < 0.001].

Sildenafil administration, alone or in combination withpramipexole did not show any significant affect (p > 0.05) onlocomotor activity during OFT (Fig. 7B).

4. Discussion

In current study, our main concern was to evaluate the possibleantidepressant like effect of pramipexole in Porsolt Forced SwimTest after treatment with acute drugs. Our current results present

Fig. 5. Effect of L-NAME (10 mg/kg, i.p.) pretreatment on antidepressant effect of pramipexole (PRAM, 0.3 mg/kg, i.p. A) And 7-NI (30 mg/kg, i.p) pretreatment onantidepressant effect of pramipexole (PRAM, 0.3 mg/kg, i.p. C) in the FST .The immobility duration in FST for last 4 min of the test. (B, D) Total number of crosses during open-field test. Scores are expressed as the mean � SEM, n = 8 animals/group and were analyzed using two-way ANOVA followed by Tukey’s post-test. **P < 0.01***P < 0.001 compared with saline- treated control; or with the PRAM treated group.

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that agonist at D2 receptor decreases the time of immobility ofmice during forced swimming test. This results are consistent withthe previous report, which have shown that pramipexole has anti-immobility effect in mice subjected to FST [9,17]. Pramipexole itselfcan not be termed as a locomotor behavior enhancer drug. Becausehere we showed that 3 mg/kg dose of pramipexole is effective inFST, while same dose has reduced the locomotor activity. Also, inorder to counteract this ambiguity, we used the most appropriatedose 1 mg/kg of pramipexole which have a significant anti-immobility effect during FST but failed to affect the ambulatorybehavior of mice during OFT.

Obviously, our results also reported that mice treated with3 mg/kg dose of pramipexole have reduced the locomotor function,which was marked during the first hour of injection of drug.

The antidepressant like activity of pramipexole in mouse FSTwas found to be dose-dependent. As acute treatment ofpramipexole (1 and 3 mg/kg) produced antidepressant effect,evident by significantly decreasing the immobility time during FST(Fig. 1). Pramipexole does not have the potential to increase thelocomotor activity, it is clear because dose of pramipexole (1 mg/kg) do not initiate any changes during locomotor activity ofanimals.

Trends from other studies has proposed that dopamine a keyparticipant in the pathophysiology of depression [47,48] and alsomood is found to be regulated by dopamine [49]. A consistentstudy also reported that extracellular concentration of dopaminein the brain regions is reduced when animal is challenged withphysical stress or depression [50]. Recently, dopamine was alsolinked with the activity of antidepressant drugs [51,52] . Forced

swimming test is one of the most prominent tests established [42]to study the antidepressant effects of drugs. The study ofantidepressant potential of drugs, like oxcarbazepine and prami-pexole as dopamine receptor agonists has been found to have anti-immobility effect in the FST [51,52]. Also, dopamine D2 receptoragonists have been reported to have antidepressant effects inindividuals undergoing such therapy [53,54] .

The originality of anti-immobility effect by dopamine lies as afact because, in our study we also found that the anti-immobilityeffect of pramipexole was decreased when the animals werepretreated with dopamine D2 receptor antagonists such assulpiride and haloperidol. This results are in line with the previousstudy, which reported that pramipexole anti-immobility effectscan be unprovoked if pretreated with haloperidol, a D2 receptorantagonist [17]. Hence, it is clear that dopamine D2 receptor have avalid role in anti-immobility potential of pramipexole. Reportsfrom the clinical data also confirms that dopamine D2 receptoragonists are a good candidate to treat patients with depressivedisorders [54]. So far, our results strongly support the hypothesisthat “dopamine D2 receptors activation relieves depression”.

Concomitant administration of pramipexole with haloperidolor with sulpiride did not alter the locomotor activity, showing thatthis effect is not related to changes in the motor activity asconfirmed during OFT. Therefore it is evident, that pramipexolepotential to decrease the time of immobility is attributed bydopamine D2 receptors without affecting the baseline locomotoractivity.

A functional cross talk of dopamine receptors and NMDAreceptors has been reported previously [55]. Based on the above

Fig. 6. Effect of methylene blue (MB), an inhibitor of both nitric oxide synthase andsoluble guanylate cyclase on pramipexole (PRAM) antidepressant activity in the FST.MB (10 mg/kg, i.p.) administered prior to the administration of either PRAM (1 mg/kg, i.p.) or its vehicle (saline). (A) The duration of immobility in FST for the last 4 minof the test. (B) The total number of crosses during open-field test recorded. Scoresare presented as the mean � SEM n = 8–10 animals and were analyzed using a two-way ANOVA followed by Tukey’s post-test. ***P < 0.001 compared with saline-treated control; or with the PRAM treated group.

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approach, various studies have revealed that dopamineD2 receptors activation can trigger the downstream effectors ofNMDA receptors and thereby regulate various functions. Similarfunction was reported during the interaction of neurotensin1Awith D2/NMDA receptor. Dopamine D2 receptor was found to haveinhibitory effect on GABA transmission while on NMDA receptors ithave excitatory effects in brain striatopallidal neurons [56].Dopamine D2 receptor antagonists has the ability to phosphorylateNMDA receptor NR1 [57] and also NR2B subclasses [58].Interestingly, it was found that D2 receptors activation withinCA1 pyramidal neurons decrease the excitatory transmissionmediated by NMDA receptor [59]. Also it is observed thatstimulation of D2 receptors by an agonist halt the calciumsignaling, mediated by NMDA receptor within the brain striatum[21].

Thus, in current study we examined the role of NMDA receptorsand L-arginine-NO-cGMP pathway in the antidepressant activity ofpramipexole.

In our current study, we found that the antidepressant likeactivity of pramipexole in mouse FST was reversed by the NMDA.Also, administration of MK-801with sub-effective dose of prami-pexole boosted the antidepressant like effect of pramipexoleduring FST. Though we did not accomplish the final mechanismfrom our data, through which pramipexole interacted with NMDAreceptor. Yet, it recommends that such effect of PRAM is reliant onthe blockage of NMDA receptor. The result thus obtained iscompatible with our and other previous studies which shows that

prior administration of NMDA reversed the antidepressant likeeffect of certain drugs [28,30,60,61].

Also, in line with the above reports, various clinical andpreclinical findings revealed that NMDA receptor is the mainparticipant in the pathogenesis of depressive disorders. Moreover,those agents which antagonize the effects of NMDA receptor havebeen reported to have antidepressant activity [62,63]. Otherresearchers reported that drugs which attenuate or block theactivity of NMDA receptor have mimicked the effects ofantidepressants including lithium during FST [64–66]. It is worthto mention, that patient who shows resistant to antidepressanttherapy, when undergoes ketamine therapy an NMDA antagonist,respond well and shows long lasting antidepressant effects [25,26].Furthermore, in gene knockout approach, it was found that mousewith NMDA receptor (NR2A) knockout gene possess antidepres-sant like activity in FST [67]

It is true that NMDA receptor activation can leads todownstream signaling, where it can activate nitric oxide synthaseand finally the synthesis of nitric oxide. It is well established thatNMDA receptor activation in the postsynaptic neurons can lead tothe activation of calcium-calmodulin, which in turn activatesneuronal nitric oxide synthase and thus increasing the nitricoxide formation. Similarly, in another study it is reported thatNMDA receptor modulate the activity of NOS through PSD-95 protein and hence accelerates the formation of NO [68,69] .Nevertheless, this strong interplay between NMDA and NO in theantidepressant like effects of certain drugs, led the foundation tostudy the effect of NOS inhibitors in depression [70–72].Undeniably, the effect of cGMP in depression is also wellunderstood, as certain studies have shown that NO and cGMPis involved in the initiation of depression. Drugs which inhibit theL-arginine and NO-cGMP signaling are reported to have antide-pressant effects during FST [61,73–75]. Our results also show thatthe antidepressant like effect of pramipexole in FST can bereversed if the animals are prior treated with L-arginine apotential substrate for NOS.

It is evident from these results that the potential effect ofpramipexole in FST is at least in part, associated with the blockageof nitric oxide formation. There is a good match between ourresults and other studies, where they also reported that theantidepressant like activity of drugs like lithium, imipramine andvenlafaxine can be halted by prior administration of L-arginine[76,77]. Other researchers confirmed that NOS inhibitors elicitantidepressant effect in a dose dependent manner [70–72,78].Furthermore, a decrease in hippocampal nitric oxide level wasfound to attenuate the depressive symptoms, thus confirming themodulation of depression by endogenous high level of hippocam-pus nitric oxide [34].

To further strengthen our hypothesis that pramipexole decreasethe level of nitric oxide and hence shows its antidepressant likeactivity in FST, mice were pretreated with subeffective dose of 7-NI(nNOS inhibitor) or methylene blue (both NOS & sGC inhibitor).They produced a synergistic effect with pramipexole in theantidepressant like activity of pramipexole. Hence taking intoconsideration the effect of NO which activates and thus synthesizecGMP, which performs most of NO function [79], our observationalso proposed that pramipexole antidepressant effect might be dueto decrease production of cGMP due to decrease synthesis of NO.Our results are consistent with other studies, where decrease in theimmobility period during FST was documented with 7-nitro-indazole [72,78] and methylene blue treatment [80]. In last decade,a study reported a synergistic effect of 7-nitroindazole andmethylene blue with antidepressant drugs like venlafaxine inanimals [76]. Similarly, it has been shown that 7-nitroindazolepotentiate the behavior activity of drugs like fluoxetine andimipramine [71] .

Fig. 7. Effect of sildenafil, a PDE5 inhibitor, on pramipexole (PRAM) antidepressantactivity in the FST. Sildenafil (5 mg/kg, i.p.) administered prior to the administrationof either PRAM (1 mg/kg, i.p.) or its vehicle (saline). (A) The duration of immobilityin FST for the last 4 min of the test. (B) The total number of crosses during open-fieldtest recorded. Scores are presented as the mean � SEM n = 8–10 animals and wereanalyzed using a two-way ANOVA followed by Tukey’s post-test.***P < 0.001 compared with saline- treated control; or with the PRAM treatedgroup.

302 S. Ostadhadi et al. / Biomedicine & Pharmacotherapy 81 (2016) 295–304

The contribution of cGMP in the antidepressant like effect ofpramipexole was confirmed by pretreatment of animals withsildenafil. As sildenafil increases the level of cGMP so it reversedthe effect of pramipexole in FST [81]. This outcome highlights andconfirms our statement that pramipexole shows its antidepressantmechanism by decreasing the level of cGMP in FST. This results arein agreement with the previous works which also highlighted thatsildenafil can prevent the anti-immobility effect of venlafaxine [76]and baclofen [60]. Till to date there exist no data in literature,reporting the pharmacokinetic interaction between pramipexoleand NO system.

On the basis of these promising finding, we can say thatpramipexole administered through i.p. route can exerts anantidepressant-like effect in the mouse FST. This antidepressant-like effect of pramipexole was achieved by a cross talk betweenactivated dopamine D2 receptor by pramipexole, which inhibitsthe activated NMDA receptor or NO-cGMP synthesis, thussupporting the idea that these targets are crucial for antidepres-sant like activity of pramipexole.

Hence, summing up the results it can be concluded thatdopamine D2 and NMDA receptors and L-arginine-nitric oxide-cyclic guanosine monophosphate pathway is involved in theantidepressant like mechanism of pramipexole and this mecha-nism is not associated with the locomotor activity.

Conflict of interest

None of the authors of this paper has a financial or personalrelationship with other people or organizations that couldinappropriately influence or bias the content of the paper.

Acknowledgment

This study was supported by Tehran University of MedicalSciences (Grant No. 94-04-30-31245).

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