Hormones and Behavior 63 (2013) 361–369

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Hormones and Behavior

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Estradiol reduces depressive-like behavior through inhibiting nitric oxide/cyclic GMPpathway in ovariectomized mice

Pouria Heydarpour a,b, Mohammad Salehi-Sadaghiani a, Mehrak Javadi-Paydar a,b, Reza Rahimian a,b,Gohar Fakhfouri c, Mohsen Khosravi a, Shayan Khoshkish a, Mohammad Hadi Gharedaghi a,b,Mehdi Ghasemi a,d, Ahmad Reza Dehpour a,e,⁎a Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iranb Brain and Spinal Injury Repair Research Center, Tehran University of Medical Sciences, Tehran, Iranc Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, P.O. Box: 19615-1178, Tehran, Irand Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USAe Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran

Abbreviations: OVX, Ovariectomy; NO, Nitric oxideNω-nitro-L-arginine methyl ester; 7-NI, 7-nitroindazoForced swimming test.⁎ Corresponding author at: Department of Pharmacolo

University of Medical Sciences, P.O. Box: 13145-784, TehraE-mail address: dehpour@yahoo.com (A.R. Dehpour

0018-506X/$ – see front matter © 2012 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.yhbeh.2012.12.005

a b s t r a c t

a r t i c l e i n f o

Article history:Received 26 June 2012Revised 5 December 2012Accepted 7 December 2012Available online 19 December 2012

Keywords:DepressionOvariectomyEstradiolNitric oxide

Estradiol decline has been associatedwith depressive-like behavior in femalemice andNO has been suggested toplay a major role in the pathogenesis of major depression. This study was conducted to investigate theantidepressant-like effects of acute estradiol administration in female ovariectomized (OVX) mice and the pos-sible role of nitric oxide (NO)/cyclic GMP (cGMP) pathway. To this end, bilateral ovariectomy was performed infemalemice and different doses of estradiolwere injected alone or in combinationwith non-specificNO synthase(NOS) inhibitor (L-NAME), selective neural NOS (nNOS) inhibitor (7-NI), an NO precursor (L-arginine) or selec-tive phosphodiesterase type 5 inhibitor (sildenafil). The duration of immobilitywas recorded in the forced swim-ming test (FST) to assess the depressive behavior. Moreover, hippocampal levels of NOwere determined in selectgroups. 10 days following the procedure, OVXmice showed significantly prolonged immobility time in comparisonwith the shamgroup. Estradiol (3, 10, and 30 μg/kg, s.c.), when injected 1 h prior to FST, exerted antidepressant-likeeffects in OVX mice. Both L-NAME (30 mg/kg, i.p.), and 7-NI (50 mg/kg, i.p.) significantly reduced the immobilitytimes of OVX mice. Administration of a sub-effective dose of L-NAME (10 mg/kg), 15 min after a sub-effectivedose of estradiol (1 μg/kg, s.c.) had a robust antidepressant-like effect in OVX mice. Also a sub-effective dose of7-NI (25 mg/kg), 30 min after a sub-effective dose of estradiol (1 μg/kg, s.c.) showed antidepressant-like effect inOVX mice. Both the NO precursor L-arginine (750 mg/kg, i.p.) and the cGMP-specific phosphodiesterase type 5 in-hibitor sildenafil (5 mg/kg, i.p.), 30 min before estradiol treatment, prevented the antidepressant-like effect of a po-tent dose of estradiol (10 μg/kg, s.c.) in OVXmice. The presentfindings suggest that suppression of theNO synthase/NO/cGMP pathway may be involved in the antidepressant-like effects of estradiol in OVX mice.

© 2012 Elsevier Inc. All rights reserved.

Introduction

Alterations in hormonal milieu during menopausal transition maycontribute to episodes of lowered mood, reduction of energy and de-crease in activity in many women as they age (Avis et al., 1994; Cohenet al., 2006; Freeman, 2010; Freeman et al., 2004). Pre-clinical studieson bilaterally ovariectomized mice point to the role of ovarian sex

; cGMP, Cyclic GMP; L-NAME,leand; L-Arg, L-arginine; FST,

gy, School of Medicine, Tehrann, Iran. Fax: +98 21 6640 2569.).

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hormones in menopausal depressive-like state (Bekku et al., 2006;de Chaves et al., 2009; Lagunas et al., 2010). Aged female mice receiv-ing estradiol have demonstrated anti-anxiety and antidepressant effects(Stoffel and Craft, 2004;Walf and Frye, 2010). Estradiol reversed the an-xiolytic and depressive-like behavior in OVX mice in various studies(Bernardi et al., 1989; Estrada-Camarena et al., 2003; Kandi andHayslett, 2011; Morgan and Pfaff, 2002; Rachman et al., 1998). Estradiolreduced the latency to the onset of action for some antidepressants inthe forced swimming test (Estrada-Camarena et al., 2008). Althoughthe mechanism for its effect is not well-understood, estradiol mayexert its effects partly via modulation of dopaminergic and serotonergicreceptors (Dhir and Kulkarni, 2008).

Nitric oxide (NO), an important neurotransmitter involved in manyneurological disorders is produced from L-arginine by NO synthase(NOS) enzymes (Knowles et al., 1989). NO may have a central role in

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the neurobiology of depression by its modulatory effects on serotonin(Harkin et al., 2004), dopamine (Dhir and Kulkarni, 2007b) and norepi-nephrine (Dhir and Kulkarni, 2007a). Inhibition of neuronal or inducibleNOS in the rat hippocampus exerts antidepressant-like effects (Joca andGuimaraes, 2006; Wang et al., 2008). NO acts as an endogenous activa-tor of guanylyl cyclase, thereby increasing the level of cyclic guanosinemonophosphate (cGMP) (Snyder and Bredt, 1991). Given that cGMP isa downstream effector of NO, phosphodiesterase type 5 (PDE5) inhibi-tors via enhancement of cGMP bioavailability, augment the biological ef-fectsmediated by NO (Puzzo et al., 2008). It has been also suggested thatthe nNOS isoform is crucial to the effects of PDE5 inhibitors; for instance,sildenafil failed to enhance the diminished erectile responses in micelacking the nNOS (Cashen et al., 2002). NOS inhibitors have beensuggested as a novel class of therapeutics for major depressive disordersby reducing cyclic guanosine monophosphate (cGMP) and NO levels(Harkin et al., 1999; Heiberg et al., 2002).

Evidence from clinical and biological studies indicates that thehippocampus plays an important role in major depression disorder(MacQueen and Frodl, 2011). Rapid changes occurring in nitrergicsystem of female hippocampus during ovarian cycle may proposethis system as a target for estrogen actions (Gotti et al., 2009).Lower basal NO level in female hippocampus than inmale counterparts;diminished nNOS expression and NO shortage in female hippocampusin mice subjected to a chronic stress model for depression and impor-tantly increased immobility in the FST after receiving an NO donor intofemale hippocampus may suggest the role of hippocampal nitric oxidein sex differences of affective behaviors (Hu et al., 2012).

Regarding the protective effects of estradiol on affective disordersand the complex role of NO in this context, the present study wasconducted to investigate the antidepressant-like effects of acute es-tradiol administration in female ovariectomized (OVX) mice in FSTand the possible role of nitric oxide (NO)/cyclic GMP (cGMP) path-way. We hypothesized that estrogens may partly exert their behav-ioral effects through the L-arginine/NO/cGMP signaling pathway.

Materials and methods

Animals

Female NMRI mice (Pasteur Institute) weighing 25–35 g were usedthroughout the study. The animals were housed in a temperature-controlled room (24±1 °C) on a 12-h light/dark cycle. Animals hadfree access to food and water except for brief time in which they wereremoved from their cages at the time of ovariectomy and the behavioraltest. Animals were divided into 3 main groups: unoperated control,sham-operated and ovariectomized. Each animal was used only onceand the number of animals in each groupwas 6–8. All behavioral exper-iments were conducted between 12:00 and 18:00 h. All animal experi-ments were performed according to the institutional guidelines foranimal care and use.

Ovariectomy

Anesthesia was induced by intraperitoneal injection of 50 mg/kgKetamine and 5 mg/kg Xylazine (Alfasan, Woerden, Holland). Afterthe onset of anesthesia, the lumbar dorsum was shaved, and theexposed skin prepared for aseptic surgery (a 10% povidene-iodinescrub followed by a sterile saline wipe). Surgery was performed aspreviously described (Kalbasi Anaraki et al., 2008; Sadeghi et al.,2009). In brief, skin was opened with a 1- to 2-cm incision in the mid-line on the lumbar vertebral line. About 1 cm to each flank,parovarian fatty tissue was identified and pulled out through asmall incision. The exposed ovary and associated oviduct were re-moved. Then the skin incision was sutured (4-0 nonabsorbable). Inthe sham-operated animals, the parovarian fatty tissues and ovaries

were just retracted and replaced. All behavioral tests were initiatedafter a recovery period of 10 days (Mirbaha et al., 2009).

17β-estradiol assay

Cardiac puncture under anesthesia with Ketamine (50 mg/kg) andXylazine (5 mg/kg) was performed to obtain approximately 1 mlblood from each mouse (this was a terminal procedure and all themice in these experimental groups were sacrificed with CO2 afterthe procedure), whole blood samples were allowed to clot at roomtemperature for 30 min, and then centrifuged at 10,000 rpm for10 min to isolate serum and were stored at –80 ° C until processedfor 17β-estradiol (E2). Serum E2 level was determined by direct andcompetitive chemiluminescence immunoassay (CLIA) detection usingLIASION Estradiol (310400) kit and according tomanufacturer's instruc-tions (Diasorin Inc, USA). Inter- and intra-assay coefficients of variationswere below 5 and 9.3%, respectively. Sensitivity level of thismethodwas12–1100 pg/ml. Each experimental group consisted of 4–6 animals(This Experiment was performed in NOOR laboratory) (Lv and Shi da,2010; Malekinejad Hassan et al., 2011).

Drugs

The following compounds were used in the study: estradiol valer-ate (E2V) (Aboureihan Pharmaceutical Company, Tehran, Iran);Nω-nitro-L-arginine methyl ester (L-NAME), a non-specific NOS in-hibitor; 7-nitroindazole (7-NI), a neuronal NOS inhibitor; L-arginine,a NO precursor and sildefanil, a selective phosphodiesterase (PDE)-5inhibitor (Sigma, St. Louis, MO, USA). All drugs were freshly dilutedin physiological saline except for estradiol valerate, which wasdissolved in sesame oil and 7-nitroindazole that was dissolved in sa-line with few drops of Tween80. All compounds were administeredi.p. in a constant volume of 10 ml/kg body weight except for estradiolvalerate which was administered subcutaneously (s.c.).

Open-field locomotor activity

To ensure that alterations in the duration of immobility were not dueto changes in motor activity, the ambulatory behavior of mice wasassessed in an open-field test immediately before the FST (Kaster et al.,2005; Salehi-Sadaghiani et al., 2012). The apparatus consisted of a wood-en boxmeasuring 40×60×50 cm. Thefloor of the arenawas divided into12 equal squares. The animalswere gently placed in the center of thefieldand the number of squares crossed with all paws (crossing) was countedin a 6-min session. After each test, the apparatus was cleaned with 10%ethanol solution to hide animal clues. The light inside the apparatus wasmaintained at minimum to avoid anxiety behavior.

Forced swimming test (FST)

Mice were individually placed in an open cylindrical containercontaining 19 cmofwater at 25±1 °C as previously described (diameter10 cm, height 25 cm).Micewere allowed to swim for 6 min. Eachmousewas judged to be immobilewhen it ceased struggling and remainedfloat-ing motionless in water, making only those movements necessary tokeep its head abovewater. The duration of immobility was recorded dur-ing the last 4 min of the test (Ghasemi et al., 2009; Porsolt et al., 1977).

Experiments

To assess the effects of estradiol (1, 3, 10, and 30 μg/kg, s.c.) in ovari-ectomized (OVX)mice, the drug was administered 1 h before the FST indifferent experimental groups (respectively: n=6, 7, 8, and 6). To ruleout the effect of surgery, the immobility time was compared with anunoperated control group (n=7) and a sham-operated group (n=7)in which sesame oil was injected as the vehicle. Estradiol doses were

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chosen based on a pilot study and in accordance with previous studies(Sadeghi et al., 2009; Walf and Frye, 2010).

Possible involvementof theNOsystem in the antidepressant-like effectof estradiol was evaluated. Different doses of L-NAME (10, 30 mg/kg, i.p.)were injected 45 min before behavioral tests and 15 min after injectionof sesame oil (n=6, 6). The immobility time was compared with sesameoil/saline group and the non-effective dose of L-NAMEwas determined.Then the non-effective dose of L-NAME was administered 15 min afteradministration of the non-effective dose of estradiol (n=6). L-NAMEdoses were chosen based on previous studies (Ghasemi et al., 2008;Sadaghiani et al., 2011).

To further evaluated the role of NO system, 7-nitroindazole (25,50 mg/kg, i.p., a neuronal NOS inhibitor) was injected 30 min beforebehavioral tests and 30 min after injection of sesame oil (n=6, 6).The immobility time was compared with sesame oil/saline groupand the non-effective dose of 7-NI was determined. Then thenon-effective dose of 7-NI was administered 30 min after administra-tion of the non-effective dose of estradiol (n=6) (Ludka et al., 2012;Moretti et al., 2011).

We further evaluated the effect of the NO precursor, L-arginine750 mg/kg on the antidepressant-like property of a potent dose of es-tradiol in the FST. In this regard, L-arginine was injected 30 min be-fore the administration of estradiol (n=8). Sixty minutes afterestadiol administration, animals were assessed in the FST. To evaluatethe role of cGMP pathway, the specific cGMP inhibitor sildenafil5 mg/kg was administered 30 min before a potent dose of estradiolin the FST in the ovariectomized mice (n=7) (Ghasemi et al., 2008;Payandemehr et al., 2012; Tahsili-Fahadan et al., 2006).

Hippocampal nitrite assay

Nitric oxide (NO) levels in the hippocampus of mice was deter-mined in the form of nitrite, a major stable product of NO, using theGriess reagent as described previously (Coker and Laurent, 1998).Briefly, hippocampal homogenates were centrifuged and aliquots ofsupernatants reacted with the same volume of Griess reagent (1% sul-fanilamide, 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride,2.5% H3PO4) (each hippocampus of experimental mice was lysed sep-arately). Nitrite concentrations were quantified spectrophotomericallyat 540 nm with regard to a standard curve plotted for known con-centrations of sodium nitrite. Each experimental group consisted of6–8 hippocampi.

Statistical analysis

All data were expressed as mean±S.E.M. and analyzed using SPSSstatistical software package (version 13.0). Differences within mainexperimental groups in immobility time and locomotor activitywere analyzed by one-way analysis of variance (ANOVA), whereasinter-group differences were analyzed by two-way ANOVA and bothwere followed by Tukey's post test. Pb0.05 was considered statistical-ly significant in all experiments.

Results

17β-estradiol assay

Serum E2 measurement was conducted by chemiluminescenceimmunoassay in various groups. As shown in Table 1, the E2 level in

Table 117β-estradiol level measurements performed by chemiluminescence immunoassay detectioanalyzed using a one-way ANOVA followed by Tukey's post test. # Pb0.05 compared to sha

Sham OVX

Serum E2 level (pg/ml) 60.45±8.63 28.67±4.1

OVX group (28.67±4.12 pg/ml) was significantly lower (Pb0.05)than the Sham group (60.45±8.63 pg/ml). The E2 level in ovariecto-mized mice receiving 10 μg/kg of estradiol valerate 1 h beforeobtaining blood samples was significantly increased (Pb0.01) to71.99±11.98 pg/ml. The E2 level in ovariectomized mice receiving1 μg/kg of estradiol valerate was also moderately increased to49.41±2.46 pg/ml.

Effect of ovariectomy on forced swimming test in mice

As shown in Fig. 1, ovariectomy significantly increased theimmobility time in FST in comparison with sham-operated group(F (1, 13)=1.162, Pb0.05; Fig. 1a). Ovariectomy had no effecton locomotor activity when the crosses of this group were com-pared with the sham-operated group locomotion (F (1, 13)=0.656, P>0.05; Fig. 1b).

Effect of estradiol on ovariectomized mice

The results depicted in Fig. 1 show that administration of estradioldecreased the immobility time of the ovariectomized mice in the FST.ANOVA revealed a significant effect of estradiol (F (4, 31)=5.213,Pb0.01). Estradiol at the doses of 3, 10 and 30 μg/kg (Pb0.01) signif-icantly decreased the immobility time in the FST. Different doses ofestradiol had no significant effect on the locomotor activity of micein the open-field test (F (4, 31)=0.537, P>0.05)

Effect of L-NAME on anti-immobility effect of estradiol

As shown in Fig. 2, the administration of different doses of L-NAMEhad anti-immobility effects on mice in the FST (F (2, 18)=10.175,Pb0.001; Fig. 2a). L-NAME at the dose of 30 mg/kg (Pb0.001) signifi-cantly decreased the immobility time in the FST, whereas at the doseof 10 mg/kg produced no significant anti-immobility effect. In addition,different doses of L-NAME had no significant effect on the locomotor ac-tivity of mice in the open-field test (F (2, 18)=3.035, P>0.05; Fig. 2b).

Fig. 3a shows that combination of a per se non-effective dose ofL-NAME (10 mg/kg) and estradiol (1 μg/kg) demonstrated a signifi-cant antidepressant-like effect in the mouse FST in comparison withestradiol-treated (F (1, 10)=73.054, Pb0.001); but not L-NAME-treated mice (F (1, 11)=2.757, P>0.05). Concurrent administrationof estradiol with L-NAME did not alter the locomotor activity ofmice in the open-field test in comparison with estradiol-treated andalso L-NAME-treated mice (F (3, 23)=1.821, P>0.05; Fig. 3b).

Effect of 7-NI on anti-immobility effect of estradiol

In order to elucidate the NOS isotypemediating the effects of estradiolon NO pathway, we also evaluated the effect of 7-NI, a neuronal nitricoxide inhibitor on the forced swimming test in ovariectomized mice. Asshown in Fig. 4 administration of 7-NI (50 mg/kg, i.p.) 30 min beforethe behavioral tests had a significant antidepressant effect, while the25 mg/kg dose did not reduce immobility time (F (2, 18)=11.906,Pb0.001; Fig. 4a). Different doses of 7-NI had no significant effect on thelocomotor activity of mice in the open-field test (F (2, 18)=0.738,P>0.05; Fig. 4b).

A sub-effective dose of 7-NI (25 mg/kg) administered in combina-tion with sub-effective estradiol (1 μg/kg) demonstrated a significantantidepressant-like effect in the mouse FST in comparison with

n in mice sera. Values are expressed as the mean±S.E.M. from 4 to 6 animals and werem-operated group; **Pb0.01 compared to ovarectomized group.

OVX+EST 1 μg/kg OVX+EST 10 μg/kg

2# 49.41±2.46 71.99±11.98⁎⁎

Fig. 1. Effect of acute administration of estradiol (1, 3, 10, and 30 μg/kg, s.c.) administered 1 h before FST. A) The duration of immobility in FST during the last 4 min of the test andB) the total number of crosses in an open-field test is recorded. Values are expressed as the mean±S.E.M. from 6 to 8 animals and were analyzed using a one-way ANOVA followedby Tukey's post test. # Pb0.05 compared to sham-operated group; *Pb0.05, **Pb0.01 compared to ovarectomized group.

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estradiol-treated (F (1, 10)=40.034, Pb0.001; Fig. 5a), but not with7-NI-treated mice (F (1, 11)=4.541, P>0.05; Fig. 5a). Concurrent ad-ministration of estradiol with 7-NI did not alter the locomotor activityof mice in the open-field test (F (3, 23)=1.067, P>0.05; Fig. 5b).

Effect of L-arginine pretreatment on anti-immobility effect of estradiol

Fig. 6a shows that although L-arginine (750 mg/kg, i.p.) individuallydid not alter the immobility time of ovariectomizedmice in the FST (F (1,12)=5.175, P>0.05), it significantly reversed antidepressant-likeeffects of 10 μg/kg estradiol in the FST (F (1, 15)=14.095, Pb0.01).L-Arginine concurrentwith sesame oil or estradiol did not alter the loco-motor activity of mice in the open-field test in comparison withestradiol-treated and also L-NAME-treated mice (F (3, 28)=0.952,P>0.05; Fig. 6b).

Effect of sildenafil pretreatment on anti-immobility effect of estradiol

Fig. 7a shows that sildenafil (5 mg/kg) individually did not alter theimmobility time of ovariectomized mice in the FST (F (1, 13)=0.080,P>0.05), while it significantly reversed the antidepressant-like effectof 10 μg/kg estradiol in the FST (F (1, 14)=42.852, Pb0.001). Sildenafilconcurrentwith sesame oil or estradiol did not alter the locomotor activ-ity of mice in the open-field test (F (3, 28)=1.269, P>0.05; Fig. 7b).

Hippocampal nitrite assay

To further investigate the involvement of NO system in the antide-pressant effect of estradiol, as shown in Fig. 8 we determined the

content of NO as nitrite in the hippocampus of experimental mice.Ovariectomy induced a significant rise in the hippocampal NO levelscompared to shamoperation (pb0.01). Administration of either estradi-ol (1 μg/kg) or 7-NI (25 mg/kg), that did not show any antidepressanteffect in FST, failed to alter theNO content in ovariectomizedmice. How-ever, exertion of the antidepressant effect by 10 μg/kg estradiol onovariectomized mice was associated with 1.81-fold reduction in nitriteconcentration (pb0.01). Co-administration of sub-effective doses of es-tradiol and 7-NI (1 μg/kg and 25 mg/kg respectively) also brought abouta 2.32-fold decrease in nitrite levels compared with the values of ovari-ectomized group receiving no further treatment (pb0.001).

Discussion

Our present study suggests that estradiol (3, 10, and 30 μg/kg, s.c.),1 h after injection, exerted antidepressant-like effects in OVX micein the FST. The non-selective NO synthase inhibitor, L-NAME(30 mg/kg, i.p.), also significantly reduced the immobility times ofOVX mice in the FST. Administration of a sub-effective dose ofL-NAME (10 mg/kg), 15 min after a sub-effective dose of estradiol(1 μg/kg) had a robust antidepressant-like effect in OVXmice, show-ing the additive effects of these two compounds and implying thepossibility of involvement of NO signaling in the antidepressant ef-fects of estradiol. The observation that L-arginine, the enhancer of NOproduction, per se did not alter the depressive-like behavior of ovariec-tomizedmicewhile it reversed the beneficial effect of 10 μg/kg estradiol,suggests that the antidepressant-like action of estradiol is mediated bysuppression of the NO pathway. In addition, to directly show whetherestradiol can alter the NO content in the hippocampus to exert its

Fig. 2. Effect of administration of L-NAME (0, 10, 30 mg/kg, i.p.) administered 45 minbefore FST. A) The duration of immobility in FST during the last 4 min of the test andB) the total number of crosses in an open-field test is recorded. Values are expressedas the mean±S.E.M. from 6 to 8 animals and were analyzed using a one-way ANOVAfollowed by Tukey's post test. *** Pb0.001 compared to ovarectomized group.

Fig. 3. Effect of co-administration of non-effective doses of L-NAME on effect of es-tradiol, administered 1 h prior the test on the FST. L-NAME (10 mg/kg, i.p.) was ad-ministered 45 min before FST in either vehicle or estradiol- (1 μg/kg, s.c.) treatedgroups. A) The duration of immobility in FST during the last 4 min of the test andB) the total number of crosses in an open-field test is recorded. Values areexpressed as the mean±S.E.M. from 6 to 8 animals and were analyzed using atwo-way ANOVA followed by Tukey's post test. +++ Pb0.001 compared toestradiol-treated ovarectomized group.

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antidepressant properties, we measured nitrite level, a major stableproduct of NO in hippocampal homogenates in various groups in anadditional experiment. Analysis of the data revealed that estradiol, atthe same dose and time that elicited antidepressant-like effects, signifi-cantly reduced the hippocampal NO concentration. Interestingly, a doseof estradiol which failed to produce any effect on the immobility behav-ior of ovariectomizedmice in FST (1 μg/kg), did not alter the hippocam-pal NO content as well. These findings strongly suggest the involvementof NO in antidepressant effects of estradiol observed in this setting. Wealso observed that administration of a sub-effective estradiol (1 μg/kg)and a per se noneffective neuronal nitric oxide inhibitor, 7-NI(25 mg/kg) had a robust antidepressant effect. This finding wasfurther strengthened by the observations that both the NO precur-sor L-arginine (750 mg/kg, i.p.) and the cGMP-specific phosphodi-esterase type 5 inhibitor sildenafil (5 mg/kg, i.p.), 30 min beforeestradiol treatment, prevented the antidepressant-like effect of apotent dose of estradiol (10 μg/kg) in OVX mice.

Several parameters have been proposed to influence the meno-pausal depressive-like state in rodents following ovariectomy: strains(Bekku et al., 2006), age at ovariectomy (Kiss et al., 2012) and timeafter ovariectomy (Estrada-Camarena et al., 2011). Behavioral bene-fits of estrogens in ovariectomized rodents have also been associatedwith type of estrogen (Estrada-Camarena et al., 2003) and treatmentdose and duration (Walf and Frye, 2005). Our female NMRI mice wereovariectomized at 8 weeks and the efficacy of the procedure and es-tradiol treatment in study groups was assessed by serum E2 concen-tration 10 days after ovariectomy procedure. E2 level in the OVXgroup dropped in 10 days in comparison with the Sham group, Kimet al also found E2 levels of 19.8±2.9 pg/ml two weeks after

ovariectomy, significantly lower in comparison with their shamgroup E2 level of 38.8±7.6 pg/ml (Kim et al., 2010) Depressive-likebehavior was abolished when estradiol was injected at doses of 3,10 and 30 μg/kg. According to serum E2 measurements, it appearsthat estradiol demonstrates its antidepressant properties even at thedose that produces concentrations similar to its physiological levelin intact mice. Estradiol treatment reduces forebrain c-fos expressionin ovariectomized rats subjected to FST, suggesting a similar effect tochronic antidepressant treatment (Rachman et al., 1998).

The antidepressant-like actions of estrogens could involve es-trogen receptor (ERs), serotonergic and noradrenergic receptors(Estrada-Camarena et al., 2010). Estrogens act via the nuclear re-ceptors ERα and ERβ, the latter being predominantly expressed inhippocampal formation (Ostlund et al., 2003). Absence of estradiolinduced antidepressant-like effects in the FST in ERβ knockoutmice implies their necessary role for estradiol's actions on affectivebehavior (Rocha et al., 2005). E2 has been suggested to modulatenNOS expression via activating ERβ in the cultured hippocampal neu-rons incubatedwith a nonselective ER antagonist, an ERα-selective ago-nist or diarylpropionitrile (DPN) (an ERβ-selective agonist)(Hu et al.,2012). In a genetic model of depression, altered 5HT receptor mRNAlevel was reversedwith estradiol treatment (Ostlund et al., 2003). In ad-dition to mediating antidepressant-like effects of estrogens, serotoninhas been suggested to suppress nNOS expression in hippocampus via5-HT1A receptors and modulate anxiety-related behavior (Zhang et al.,2010).

NO has been suggested to play a major role in the pathogenesis ofmajor depression (Dhir andKulkarni, 2011). Hippocampal sections in pa-tients suffering frommajor depression or bipolar disorder demonstrated

Fig. 4. Effect of administration of 7-NI (0, 25, and 50 mg/kg, i.p.) administered 30 minbefore FST. A) The duration of immobility in FST during the last 4 min of the test andB) the total number of crosses in an open-field test is recorded. Values are expressedas the mean±S.E.M. from 6 to 8 animals and were analyzed using a one-way ANOVAfollowed by Tukey's post test. *** Pb0.001 compared to ovarectomized group.

Fig. 5. Effect of co-administration of non-effective doses of 7-NI on effect of estra-diol, administered 1 h prior the test on the FST. 7-NI (25 mg/kg, i.p.) was admin-istered 30 min before FST in either vehicle or estradiol (1 μg/kg, s.c.) treatedgroups. A) The duration of immobility in FST during the last 4 min of the testand B) the total number of crosses in an open-field test is recorded. Values areexpressed as the mean±S.E.M. from 6 to 8 animals and were analyzed using atwo-way ANOVA followed by Tukey's post test. ***Pb0.001 compared to estradiol-treated ovarectomized group.

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increased nNOS expressing neurons (Oliveira et al., 2008). Furthermore,8 weeks of antidepressant therapy has been associatedwith a significantdecrease in xanthine oxidase activity and plasmanitrite levels (Herken etal., 2007). In this study, spectrophotomectrical inspection of the hippo-campi from OVX mice, 10 days following gonadectomy, also revealed asignificant elevation of NO contents. The non-selective NOS inhibitor,L-NAME (30 mg/kg, i.p.) showed an antidepressant-like effect in OVXmice in the FST. Several studies have shown the antidepressant propertyof NOS inhibition in FST (Harkin et al., 1999, 2004; Yildiz et al., 2000). Assuggested in our study, the role of neuronal NOS in the behavioral effectsof NO is noteworthy; genetic deletion or pharmacological inhibition ofnNOS has been suggested to prevent the behavioral despair in micesubjected to chronic mild stress by reversing hippocampal neurogenesis(Zhou et al., 2007). Indeed, calcium excess during insults could lead toovert NO generation which contributes to hippocampal neurotoxicityin major depression (Sapolsky, 2000).

Both NO excess and shortage result in depression-like behaviorsthrough affecting cAMP response element binding protein (CREB) ac-tivation. Conflicting evidence also exists regarding the interaction ofovarian hormones with NO. Ovarian hormones could induce NOS ex-pression in several brain regions in rodents (Panzica et al., 2006). Huet al. have suggested that estrogen-mediated nNOS expression in thehippocampus is critical for sex difference in affective behaviors.OVX-induced decrease in hippocampal nNOS was reversed by E2 mi-croinjection which was ascribed to ERβ receptors (Hu et al., 2012).Supraestrus levels of E2 through local inhibition of nNOS expressionincreased urethral tone, whereas physiological levels of E2 had no ef-fect (Game et al., 2008). It has been suggested that inhibitory effect ofE2 on NO release could be caused by a reduction in iNOS protein ex-pression in a murine macrophage cell line or a chondrocyte model

(Hayashi et al., 1998; Richette et al., 2007). Estradiol facilitates cou-pling of glutamatergic fluxes for NO production in primary neuronalcultures (d'Anglemont de Tassigny et al., 2009). Estradiol stimulatesNO release through activation of a cell-surface estrogen receptorthat is coupled to increases in intracellular calcium in human endo-thelial cells (Stefano et al., 2000). It was observed that estradiolmodulates endothelial and neuronal nitric oxide synthase expressionvia an estrogen receptor beta-dependent mechanism in hypothalamicslice cultures. Further studies have suggested that activation of ERβincreases levels of phosphorylated nNOS and NO production (Gingerichand Krukoff, 2005, 2008). In our hands, administration of estradiol atthe same dose and time that produced an antidepressant-like effect inthe FST, induced a substantial reduction of the NO content in the hippo-campus. Also the intrahippocampal NO donor significantly increased im-mobility in the TST and FST both in males and females (Hu et al., 2012).

In the current study, administration of a per se ineffective dose ofL-NAME (10 mg/kg) or 7-NI (25 mg/kg), respectively 15 min or30 min after a sub-effective dose of estradiol (1 μg/kg) also reducedthe immobility time in OVX mice. These findings suggest that theantidepressant-like effect of estradiol might be mediated through itsaptitude to inhibit NOS, especially nNOS, and NO/cGMP signalingpathway. This implication is powered by our results that the NO pre-cursor L-arginine (750 mg/kg, i.p.), though not influencing the immo-bility time experienced by ovariectomized mice in FST, prevented theantidepressant-like effect of a potent dose of estradiol (10 μg/kg) inOVX mice. Corroborating these findings, dehydroepiandrosterone(DHEA) reportedly inhibits NO production to confer neuroprotectionagainst NMDA induced neurotoxicity in primary cultured hippocampalneurons (Kurata et al., 2004). L-Arg but not D-Arg, given 10 min before7-Nitroindazole (7-NI), reversed its decreasing effect on immobility

Fig. 7. Effect of pretreatment with specific cGMP inhibitor, sildenafil on antidepressanteffect of estradiol, administered 1 h prior the test on the FST. Sildenafil (5 mg/kg, i.p.)was administered 30 min before administration of either estradiol (10 μg/kg, s.c.) or itsvehicle (sesame oil). A) The duration of immobility in FST during the last 4 min of thetest and B) the total number of crosses in an open-field test is recorded. Values areexpressed as the mean±S.E.M. from 6 to 8 animals and were analyzed using atwo-way ANOVA followed by Tukey's post test ***Pb0.001 compared to ovarectomizedgroup +++ Pb0.001 compared to estradiol-treated ovarectomized group.

Fig. 8. Hippocampal nitrite assay. To further investigate the involvement of NO systemin the antidepressant effect of estradiol, we determined the content of NO as nitrite inthe hippocampus of experimental mice. Values are expressed as the mean±S.E.M.from 6 to 8 hippocampi and were analyzed using a two-way ANOVA followed byTukey's post test. **Pb0.01 as compared with the sham group. ## Pb0.01, ###Pb0.001 as compared with the values of ovariectomized group receiving no furthertreatment.

Fig. 6. Effect of pretreatment with NO precursor, L-arginine on antidepressant effect ofestradiol, administered 1 h prior the test on the FST. L-Arginine (750 mg/kg, i.p.) wasadministered 30 min before administration of either estradiol (10 μg/kg, s.c.) or its ve-hicle (sesame oil). A) The duration of immobility in FST during the last 4 min of the testand B) the total number of crosses in an open-field test is recorded. Values areexpressed as the mean±S.E.M. from 6 to 8 animals and were analyzed using atwo-way ANOVA followed by Tukey's post test. ***Pb0.001 compared to ovarectomizedgroup ++Pb0.01compared to estradiol-treated ovarectomized group.

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time in rats, suggesting that NOmight be an importantmodulator of de-pression (Yildiz et al., 2000).

The observation that under normal condition, female rodents bearlower hippocampal NO contents than males shows that this moleculeis somehow implicated in pathogenesis of depression. However, consis-tent with its dual role in the periphery, NO displays disparate effects indepression that may arise from its complex interactions with multiplemediators and signaling pathways, among which are K+ channelsand serotoninergic system (Joca and Guimaraes, 2006). In our hands,sex hormone depletion (ovariectomy) led to the elevation of NO levelsin the hippocampus, which was associated with the appearance ofdepressive-like behaviors in FST while nonselective NOS and selectivenNOS inhibitors had an antidepressant impact. In accordance with ourresults favoring the pro-depressive aspect of NO, Joca and Guimarãesreported that inhibition of nNOS in the rat hippocampus inducedantidepressant-like effects (Joca and Guimaraes, 2006). In addition, anumber of studies show a bimodal effect for NO in FST. Da Silva et al.demonstrated that NO has a dual role in antidepressant-like behaviorin FST and the tail suspension test. They reported that both L-arginine,an NO precursor, and NG-nitro-L-arginine, a NOS inhibitor, exhibitantidepressant-like effects at a specific dose range, while higher andlower doses of these compounds had no significant effect (da Silva etal., 2000). Moreover, Inan et al. suggested that NOmay exert its dual ef-fects on FST by modifying K+ channels, they observed that moderatedose of L-arginine (100 mg/kg ip, ineffective in the FST) reversedantidepressant-like effects of potassium channel blockers, but high con-centration of L-arginine (500 mg/kg ip) potentiated the antidepressant-like effects of K+ channel blockers. They also observed dose dependentdual effects of NO on the mechanisms of depression and serotonin re-lease, low doses of L-arginine reversed and high doses of L-arginine

potentiated the antidepressant-like effects of sertraline. Cyprohepta-dine (a serotonin antagonist) antagonizing the antidepressant-like ef-fects of L-arginine may confirm the involvement of serotonin in theNO-mediated changes (Inan et al., 2004).

The NO-mediated actions are mostly due to cGMP formation(Zhou et al., 2011). Cellular cGMP concentrations are regulated by

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both soluble guanylyl cyclase (sGC) and phosphodiesterase (PDE),the latter catalyzing the hydrolysis of cAMP and cGMP (Beavo,1995). It has been previously reported that estrogen and progester-one administration does not alter the expression of soluble guanylylcyclase in vivo while it enhances the NO-dependent cGMP synthesisin the preoptic area and hypothalamus (Chu et al., 2004; Reyna-Neyra et al., 2007). Here, we found that the phosphodiesterase type5 inhibitor sildenafil (5 mg/kg, i.p.) that increases the cGMP level intarget tissues, 30 min before estradiol treatment, prevented theantidepressant-like effect of a potent dose of estradiol (10 μg/kg) inOVX mice. This suggests that the cGMP pathway may be partly medi-ating the antidepressant-like effect of estradiol.

In summary, the present study suggested that estradiol as well asinhibitors of nitrergic system exerted a significant antidepressant-likeeffect in OVX mice in the FST. We also showed that L-arginine/NO/cGMP pathway may play a role in the antidepressant-like effects ofestradiol in OVX mice in the same behavioral test.

Conclusion

The contribution of nitrergic system to the acute antidepressant-like properties of estradiol might be of value in clinical setting, espe-cially in mood changes due to the depletion of estrogens. Our findingswarrant further investigation to delineate whether estradiol affectsthe expression and/or activity of NOS isoenzymes. It also remains tobe explored whether the observed acute antidepressant-like effectsof estradiol in the present study are mediated through its known nu-clear receptors or else, other receptors are involved.

Acknowledgments

Authors wish to express their gratitude to Dr. Parvin Pasalar(Biochemistry department) who generously provided us with L-Arg.This study was supported by Grant number 13876 from TUMS.

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