Progesterone promotes the survival of newborn neurons in the dentate gyrus of adult male mice

Progesterone Promotes the Survival of Newborn Neuronsin the Dentate Gyrus of Adult Male Mice

Zhuo Zhang,1,2 Rong Yang,2 Rong Zhou,2 Liang Li,2

Masahiro Sokabe,3,4,5 and Ling Chen1,2,3*

ABSTRACT: This study investigated the effects of progesterone (P4)on the production and survival of neurons in the hippocampal dentategyrus of adult male mice. The administration of P4 (4 mg/kg) for 3 con-secutive days beginning on the 0–2nd day after the first BrdU-injection(BrdU-D0–2) produced an approximately twofold increase in the numberof 28- and 56-day-old BrdU1 cells in comparison to the controls,whereas it did not alter the number of 24/48-h-old BrdU1 cells. P4 pref-erentially promoted the survival of newborn neurons when administeredat BrdU-D5–7, but not at BrdU-D10–12 and BrdU-D15–17. Androstene-dione (Ad), testosterone (TE), or estradiol (E2) at the same-dose of P4,when administered at BrdU-D0–2, could not replicate the effect of P4,while the inhibition of 5a-reductase by finasteride did not affect the P4-action, indicating that the P4-effect is exerted by P4 itself but not by itsmetabolites. On the other hand, the P4R antagonist RU486 partiallysuppressed the P4-effect, while inhibitors for Src, MEK, or PI3K totallysuppressed the P4-effect. Finally, the P4-enhanced survival of newbornneurons was accompanied by a potentiation of spatial learning andmemory, which was P4R-dependent. These findings suggest that P4enhances the survival of newborn neurons through P4R and/or the Src-ERK and PI3K pathways independent of its influence on cell prolifera-tion, which is well correlated with the potentiated spatial cognitivefunction of P4-treated animals. VVC 2009 Wiley-Liss, Inc.

KEY WORDS: progesterone (P4); dentate gyrus (DG); proliferation;survival; R4 receptor

INTRODUCTION

Recent studies have demonstrated that progenitor cells in the hippo-campal dentate gyrus (DG) retain the ability to proliferate and that asignificant number of the daughter cells develop into neurons in adult

mammalian species (Eriksson et al., 1998; Christieand Cameron, 2006). Newly formed neurons, similarto established ones, are electrically active and makeconnections to the hippocampal CA3 field, thus sug-gesting that they are functional (Markakis and Gage,1999; Shors et al., 2001). This is reinforced by evi-dence that increased survival of newborn neurons isstrongly correlated with hippocampus-dependentmemory, while the inhibition of neurogenesis hasadverse effects on hippocampus-dependent behaviors(for review, see Leuner et al., 2006; Winocur et al.,2006), thus implying that hippocampal neurogenesiscontributes, at least in part, to learning and memory.However, little is known about the factors that regu-late the processes of neurogenesis including the prolif-eration of neural progenitor cells and the survival ofnewborn cells.

Past studies have revealed sex difference in the cellproliferation in the DG only when females are in ahigh estradiol (E2) state in comparison to that ofmales (Galea, 2008). The neurogenesis in the subven-tricular zone and the subgranular zone (SGZ) of theDG significantly increases during pregnancy (Banasret al., 2001; Shingo et al., 2003; Furuta and Bridges,2005), and then declines immediately after parturition(Darnaudery et al., 2007; Leuner et al., 2007; Paw-luski and Galea, 2007). Pregnancy, parturition, andpostpartum coincide with dramatic fluctuations in thelevels of steroid hormones. The serum levels of E2 inrats increase during late pregnancy and decrease dur-ing parturition, while those of progesterone (P4)increase steadily during pregnancy, peak prior to par-turition and decline thereafter until after parturition(Rosenblatt et al., 1988). Evidence indicates that infemale adult rodents acute exposure to E2 initiallyenhances cell proliferation (within 4 h) and subse-quently suppresses cell proliferation after 48 h(Ormerod and Galea, 2001; Ormerod et al., 2003),whereas in male meadow voles E2 enhances the sur-vival of young neurons when administered during their‘‘axon extension’’ phase (Ormerod et al., 2004). Intri-guingly, P4 is naturally present at a similar level in thebrains of men and women (Stein, 2008). Recent stud-ies confirmed that the P4’s metabolites influence theprocess of neurogenesis. For example, allopregnanolone(ALL) is a potent and highly efficacious proliferative

1 Laboratory of Reproductive Medicine, Nanjing Medical University,Jiangsu, China; 2Department of Physiology, Nanjing Medical University,Jiangsu, China; 3Department of Physiology, Nagoya University GraduateSchool of Medicine, Nagoya, Japan; 4 ICORP/SORST Cell Mechanosens-ing, JST, Nagoya, Japan; 5Department of Molecular Physiology NationalInstitute for Physiological Sciences, Okazaki, JapanGrant sponsor: PCSIRT; Grant number: IRT0631; Grant sponsor: 973;Grant number: 2009CB941701; Grant sponsor: NSFC; Grant number:30872725; Grant sponsor: JSPBHG; Grant number: H200505; Grantsponsor: Scientific Research on Priority Areas; Grant number: 15086270;Grant sponsor: Creative Scientific Research; Grant number: 16GS0308.*Correspondence to: Ling Chen, Laboratory of Reproductive Medicine,Department of Physiology, Nanjing Medical University, Hanzhong Road140, Jiangsu, China. E-mail: [email protected] for publication 2 April 2009DOI 10.1002/hipo.20642Published online 27 May 2009 in Wiley InterScience (www.interscience.wiley.com).

HIPPOCAMPUS 20:402–412 (2010)

VVC 2009 WILEY-LISS, INC.

agent in vitro and in vivo in both rodent and human neuralstem cells (Wang et al., 2005; Brinton and Wang, 2006). Testos-terone (TE) upregulates hippocampal neurogenesis (via cell sur-vival), but not cell proliferation, in adult male rats (Spritzer andGalea, 2007). Glucocorticoid suppresses neurogenesis in adultmale rats (Karishma and Herbert, 2002). To date, however, thespecific effect of P4 on adult hippocampal neurogenesis has notyet been fully elucidated.

In adult animals, P4 affects neuronal function by modulatinggene transcription and cellular activity through classical intra-cellular P4 receptors (P4R) abundant in the CNS (Brintonet al., 2007) and splicing variants of both the seven trans-mem-brane domain 7TMPRb (Peluso et al., 2006) and the mem-brane-associated P4 receptor component 1 (PGRMC1) (Krebset al., 2000). The classical nuclear P4R regulates gene expres-sion and triggers a signal pathway leading to the activation ofextracellular signal-regulated kinase (ERK) (Boonyaratana-kornkit et al., 2001; Faivre et al., 2005; Faivre and Lange,2007). A recent study (Cai et al., 2008) demonstrated that asingle administration of P4 triggers a persistent phosphorylationof ERK1/2 lasting for 48 h, while also enhancing the transloca-tion of phosphorylated ERK2 to the nucleus. The activation ofERK1/2 promotes cell survival (Singh, 2005, 2006). Further-more, multiple laboratories have demonstrated that P4 pro-motes cell meiosis by initiating a cascade that stimulates ERKactivation (Fisher et al., 1999; Karaiskou et al., 2001). Activa-tion of MAP kinase by P4 leads to the formation of M-phasepromoting protein complex (CDC2 and cyclin B), which pro-motes the G2 to M phase transition (Masui, 2001). In addi-tion, rapid effects of P4 such as increasing the level of phos-phorylated Akt in neuronal cells may be mediated byPGRMC1 (Singh, 2001). P4 or its metabolite ALL regulatesthe GABAA receptor (Smith and Gong, 2005) and the sigmareceptor (Monnet and Maurice, 2006).

This study investigated whether P4 influences cell prolifera-tion and the survival of newborn neurons in the adult mouseDG, and if so, whether the P4-induced change in neurogenesisis associated with hippocampus-dependent cognitive behavior.The results showed that the short-term administration of P4promotes the survival of newborn neurons in the DG of adultmale mice, which is strongly correlated with hippocampus-dependent learning and memory.

MATERIALS AND METHODS

Experimental Animals

All procedures were conducted in accordance with the guide-lines of the Institute for Laboratory Animal Research of theNanjing Medical University. Adult male mice weighing 25–30 gwere maintained under constant environmental conditions[temperature (22 6 2)8C, humidity 55% 6 5%, and 12:12 hlight/dark cycle] in the Animal Research Center of NanjingUniversity. They had free access to food and water before and

after all procedures. We avoided using female mice, because P4significantly alters the influence of E2 on adult neurogenesis infemale animals (Tanapat et al., 2005).

Drug Administration

Bromodeoxyuridine (BrdU, Sigma-Aldrich, St. Louis, MO),a thymidine analog that is incorporated into the DNA of divid-ing cells during S phase, was used for mitotic labeling. BrdUwas dissolved in freshly prepared 0.9% saline to a concentra-tion of 10 mg/ml just prior to injection, and was given by in-traperitoneal injection (i.p.). At least four injections of BrdU(50 mg/kg) are necessary (Mayo et al., 2005) to label the new-born cells in 2- to 18-month-old male rats. Therefore, the drug(50 mg/kg) was injected twice a day at 12-h intervals for 2consecutive days. P4 was dissolved in dimethyl sulfoxide(DMSO), then in sesame oil to a final concentration of 2%DMSO (Gibson and Murphy, 2004) following the confirma-tion that DMSO alone did not affect the neurogenesis in theadult mice DG. 2-Hydroxypropyl-b-cyclodextrin, a more pop-ular vehicle for P4, was not used since the drug has beenreported to enhance the production of androstadienedione, aprecursor of a variety of steroid hormones (Manosroi et al.,2008), which might influence the neurogenesis in the DG. P4and vehicle (2% DMSO/0.2 ml/mouse) were injected subcuta-neously (s.c.) once a day for 3 consecutive days. All of theexperiments used 4 mg/kg of P4, because P4 at this dosagesignificantly increases the phosphorylation of ERK1/2 in thehippocampus of adult male rats (Cai et al., 2008), thereby pro-ducing a maximal increase in the number of newborn neurons(see the result in Fig. 2A). To investigate the injection timingdependency of P4-effect, the drug was injected at 0–2nd day(BrdU-D0–2), 5–7th day (BrdU-D5–7), 10–12th day (BrdU-D10–12), or 15–17th day (BrdU-D15–17) after the first BrdU-injection (see the time chart of experimental procedure in Fig.1B). This timing roughly corresponds to that of mitosis of theprogenitor cells (24 h), neuronal migration and the expressionof differentiation markers (day 4–10), and mature neuronalmarker expression (week 2–3), respectively, in rats and mice(Galea, 2008). Other steroids including Ad, TE, and E2 wereprepared and injected following the same way as that of P4.Finasteride and RU486 were given by intraperitoneal injection(i.p.). U0126, PP2, PP3, and LY294002 were infused into thecereboventricle (i.c.v.). The mice were anesthetized with keta-mine (80 mg/kg i.p.) for i.c.v. implantation. A guide cannula(4-mm length, 23 gauge) aimed above the left lateral ventriclewas implanted and anchored to the skull with four stainlesssteel screws and dental cement. The guide cannula wasimplanted above the rostral ventricle to avoid damaging thehippocampus as described previously (Mayo et al., 2005). Theinhibitors or vehicle were injected with a stepper-motorizedmicro-syringe (Stoelting, Wood Dale, IL) at a rate of 0.5 ll/min. The inhibitors were prepared freshly on the day of theexperiment (final volume 5 3 ll/mouse). Control mice weregiven an equal volume of vehicle.

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BrdU Staining

The animals were treated with P4 or vehicle at BrdU-D0–2

and were perfused with BrdU-staining agents at 48 h, 7th day,28th day, and 56th day after the first injection of BrdU to inves-tigate the effects of P4 on cell proliferation and the survivalof newborn neurons (see the time chart in Fig. 1A). The ani-mals received the treatment with P4 or vehicle at BrdU-D0–2,BrdU-D5–7, BrdU-D10–12, or BrdU-D15–17, and were perfused

with BrdU-staining agents at 28th day after the first BrdU-injec-

tion to determine the administration time-dependency of

P4-effect on the survival of newborn neurons (see the time chart

of experimental procedure in Fig. 1B). To examine the effects

of P4 on the maturation of newborn neurons, animals were

treated with P4 or vehicle at BrdU-D0–2, and were perfused

with BrdU/NeuN-staining agents at 28th day after the first

injection of BrdU (see the time chart in Fig. 1C). All mice were

FIGURE 1. P4 promotes the survival of newborn neurons inthe dentate gyrus of adult male mice. A: Effects of P4 on cell pro-liferation and survival. BrdU1 cells were stained at various timepoints after injection of BrdU as indicated in the upper panel.Open bars depict the cell density from the control data at 48 h,7th day, 28th day, or 56th day after the first BrdU-injection,hatched bars those from the mice treated with P4 (4 mg/kg) atBrdU-D0–2. B: The time-dependency of the P4-effect on the sur-vival of newborn neurons. P4 was injected at various times as indi-cated in the upper panel. Bars indicate density of 28-day-oldBrdU1 cells from mice treated with vehicle (control) or P4 at

BrdU-D0–2, BrdU-D5–7, BrdU-D10–12, or BrdU-D15–17, respec-tively. C: The effect of P4 on mature of newborn neurons. BrdUand NeuN were double stained at 28th day after injection of BrdUas indicated by the upper panel. The middle panel shows represen-tative fluorescence images of DG immunolabeled with BrdU (red)and NeuN (green) antibodies in control and P4-treated mice.BrdU1/NeuN1 cells show yellow color (asterisk). Lower panel:The calculated density of 28-day-old BrdU1/NeuN1 cells fromP4-treated mice is ca. twofold greater than the control. Scale bars,100 lm. *P < 0.05 and **P < 0.01 vs. the corresponding to dataobtained from control mice.

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transcardially perfused with ice-cold phosphate-buffered saline(PBS) followed by 4% ice-cold phosphate-buffered paraformal-dehyde (PFA). Thereafter, their brains were removed, fixed over-night in 4% PFA, and consecutive 40-lm-thick coronal sectionswere cut using a vibrating microtome (Microslicer DTK 1,500,Dousaka EM Co, Kyoto, Japan).

Peroxidase immunohistochemistry was performed asdescribed previously to estimate the mean number of BrdU-labeled cells (BrdU1 cells) (Karishma and Herbert, 2002).Free-floating sections were rinsed repeatedly in PBS (pH 7.4)between steps. The sections were prepared with 50% formam-ide-23SSC, at 658C for 2 h, then washed three times (10 mineach) in PBS at room temperature (RT), and denatured by

incubating in 2 mol/l hydrochloric acid (30 min at RT) beforeBrdU staining. They were next washed for 30 s with boricacid, pH 8.5, and then three more times (5 min each at RT)in PBS. Nonspecific binding sites were blocked for 1 h inblocking solution (PBS in 3% bovine serum albumin) contain-ing 1% Triton X-100. The sections were then incubated at 48Cfor 24 h in PBS blocking solution containing 0.5% Triton X-100 in the presence of primary mouse monoclonal antibodyagainst BrdU (1:200; Chemicon International). The next day,the tissue was rinsed repeatedly and then incubated in mousesecondary antisera (Vector Laboratories) in PBS for 4 h andthen in avidin-biotin horseradish peroxidase complex (ABCElite; Vector Laboratories) for 1 h. Two controls were used tocheck that the staining was specific: some animals received onlysaline injections instead of BrdU; in others the primary anti-body was omitted from the immunohistochemistry procedurein the BrdU-treated rats.

Immunofluorescent Staining

Immunofluorescent labeling was done to identify the pheno-type of BrdU- and NeuN (a mature neuronal marker)-labeledcells as described previously (Ormerod et al., 2004). The sec-tions were double-stained for BrdU and NeuN. Free-floatingcoronal sections (40 lm) were prepared with 50% formamide-23SSC, at 658C for 2 h, then washed three times (10 mineach) in PBS at RT, and denatured by incubation in 2 mol/lhydrochloric acid (30 min at RT) before BrdU staining. Theywere washed for 30 s with boric acid, pH 8.5, and then threemore times (5 min each at RT) in PBS. The nonspecific bind-ing sites were blocked for 1 h in blocking solution (PBS in 3%bovine serum albumin) containing 1% Triton X-100. The sec-tions were then incubated at 48C for 24 h in PBS blocking so-lution containing 0.5% Triton X-100 in the presence of pri-mary rat monoclonal antibody against BrdU (1:200; AccurateChemical Scientific Corporation) and mouse monoclonal anti-neuronal nuclei (NeuN) antibody (1:500; Chemicon Interna-tional). After three more 5-min washes in PBS, the sectionswere labeled for 2 h with Cy3-labeled antirat secondary anti-body (1:200; Chemicon International) and fluorescein-labeledantimouse secondary antibody (1:50; Chemicon International).After several washes with PBS, the sections were mounted onglass slides, and then coverslipped with the antifading agentdiazobicyclooctane.

Counting Labeled Cells

All the slide preparations were randomly coded so thatobservers were blinded to the experimental conditions. BrdU1

cells in the entire DG were counted by eye on every 5th sectionthrough the granule cell layer and SGZ on peroxidase-treatedtissues using a conventional light microscope (PD70) with a1003 objective. Areas of the granule cell layer were measuredwith the software Image J (NIH) and multiplied by the thick-ness of sections (40 lm) to calculate the volumes of granule celllayer in each section and the total DG. Fluorescent-stainedpreparations (BrdU and NeuN) underwent essentially the same

FIGURE 2. Dose-dependency and specificity of P4-action inthe survival of newborn neurons. A: Dose–response curve of P4-enhanced survival of newborn neurons. The mice were treatedwith various concentrations P4 (0.1, 1, 4, or 8 mg/kg) at BrdU-D0–2 and BrdU1 cells were stained at 28 days after BrdU injection.Note that the P4-enhanced survival of newborn neurons shows aU-shaped dose-dependent curve. B: Effects of Ad, TE, and E2(4 mg/kg) on the survival of newborn neurons. Open bars repre-sent the density of 28-day-old BrdU1 cells at respective conditionindicated. C: Effect of the 5a-reductase inhibitor finasteride on theP4-enhanced survival of newborn neurons. Finasteride was admin-istered 30 min before the injection of P4 at BrdU-D0–2. **P <0.01 vs. the corresponding control mice treated with vehicle.


Hippocampus

procedure, but the NeuN1 region was measured to calculatethe volume of the granule cell layer. The densities of BrdU1

cells and BrdU1/NeuN1 cells in the DG were calculated bydividing the number of cells by the volume of the granule celllayer (106 lm3) as described elsewhere (Tashiro et al., 2007).Each group data contained five mice.

Morris Water Maze Training

All animals were trained in the standard Morris water mazetask (Morris, 1984). For the Morris water maze task, a pool(90-cm diameter, 45-cm height) made of black-colored plasticwas prepared, and was filled with water [(20 6 1)8C] renderedopaque with nontoxic blue-paint. The swimming paths wereanalyzed by a computer system with a video camera (AXIS-90Target/2; Neuroscience). In the hidden-platform test, the plat-form (7 cm in diameter) was submerged 1 cm below the watersurface. The first day of training began with a free swim, inwhich the swim speed was assessed in absence of the platform,and then immediately after the free swim the mice were givenfour training trials per day over 5 days, in which they weregiven 90 s to swim to the hidden platform. If a mouse did notfind the platform in the allotted time, the mouse was placedon the platform for 15 s. On day 6 of the Morris water mazetest, a probe trial was administered in which the platform wasremoved from the maze, and the percent time spent in eachquadrant was assessed. The behavior in the water maze was vid-eotaped. The average swim speed (cm/s) and latency (s) toreach the platform were scored on all trials and analyzed. Datawere obtained from groups of 10 mice throughout theexperiments.

Statistical Analysis

Data were retrieved and processed using the Micro cal Ori-gin 6.1 software program. The group data were expressed asthe means 6 standard error. Dependent variables (density ofBrdU1) were analyzed using analysis of variance (ANOVA)with conditions (time of P4-injection, time of BrdU-staining,treatment with inhibitors). The density of BrdU1/NeuN1 cellsbetween the control and P4-treated mice was analyzed by theunpaired t-test. Dependent variables (latency and path-lengthto reach the platform, swim speed) were analyzed usingrepeated-measures ANOVAs followed by the Bonferroni’s post-hoc comparisons as the between-subjects factor. Dependent var-iables (time in each quadrant) were analyzed using two-wayANOVA followed by Bonferroni posthoc. The statistical analy-sis was performed using the State7 software program (StateCorporation, USA). A probability of 0.05 was considered to besignificant.

Chemicals

Progesterone (P4), androstenedione (Ad), testosterone (TE),estradiol (E2), U0126 (4-diamino-2,3-dicyano-1–4-bis [2-ami-nophynylthio] butadiene), LY294002 (2-(4-Morpholinyl)-8-phenyl-4 H-1-benzopyran-4-one), finasteride, and RU486

(mifepristone) were from Sigma (St. Louis, MO). PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine)and PP3 (4-amino-7-phenylpyrazolo[3,4-d]pyrimidine) werepurchased from Wako (Calbiochem, Darmstadt, Germany).Other chemicals of special grade were obtained from Wako(Osaka, Japan).

RESULTS

P4 Promotes the Survival of Newborn Neuronsin the DG of Adult Male Mice

P4 (4 mg/kg) was administered at BrdU-D0–2, and BrdU1

cells were counted at 48 h, 7th day, 28th day, and 56th dayafter the first BrdU-injection to investigate the effect of P4 oncell proliferation and the survival of newborn neurons in theadult mouse DG. As seen in Figure 1A, the P4-treatmentcaused an �1.4-fold increase in the number of 7-day-oldBrdU1 cells in comparison to that in mice treated with vehicle(DMSO) alone (P < 0.05), whereas the numbers of 24/48-h-old BrdU1 cells in the P4-treated mice were not significantlydifferent from the controls (P > 0.05). Although the numberof BrdU1 cells decreased with time, the P4-treatment increasedthe rate of the number of BrdU1 cells in comparison to thecontrols, approximately twofold larger at 28th day and 56thday after the first BrdU-injection (28-day-old: P < 0.01; 56-day-old: P < 0.01). These results suggest that the short-termtreatment with P4 enhances the survival of newborn neurons inthe adult mouse DG.

Next, P4 was administered at BrdU-D5–7, BrdU-D10–12, orBrdU-D15–17, spanning the period when progenitor cells in ratsand mice completed a single mitotic division (Cameron andMcKay, 2001; Hayes and Nowakowski, 2002) to examine thetime-dependency of the P4-effect on the survival of newbornneurons. As shown in Figure 1B, treatment with P4 at BrdU-D0–2 and BrdU-D5–7 significantly increased the number of 28-day-old BrdU1 cells (F(4, 49) 5 7.8, P < 0.01) in comparisonto that in the mice treated with vehicle alone. However, theP4-treatment at BrdU-D10–12 and BrdU-D15–17 failed to pro-mote the survival of newborn neurons (P > 0.05). Theseresults suggest that P4 enhances the survival of newborn neu-rons independent of the influence on cell proliferation, and theP4-effcet is more efficient in younger neurons.

To examine the effect of P4 on the maturation of newbornneurons, preparations were stained with NeuN, a marker formature neurons, along with BrdU. The density of 28-day-oldBrdU1/NeuN1 cells was measured in the mice treated with P4or vehicle at BrdU-D0–2. As shown in Figure 1C, the densityof 28-day-old BrdU1/NeuN1 cells in P4-treated mice was sig-nificantly larger by more than twofold in comparison to thecontrols (P 5 0.011). This number (ca. twofold) is roughly thesame as that of 28-day-old BrdU1 cells in comparison to eachcontrol as presented in Figure 1A, thus suggesting that P4 pro-motes the maturation of surviving newborn neurons. The pro-

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portion of BrdU1/NeuN1 cells in the total BrdU1 populationwas �57.8% in P4-treated mice, which was roughly consistentwith the control results in mice treated with vehicle (�55.9%;Fig. 1C) and in adult male meadow voles (�60%, Ormerodet al., 2004). In addition, �18.6% 28-day-old BrdU1 cellswere found to express the glial marker GFAP (data not shown).The percentage of BrdU1/NeuN1 (56%) is lower in compari-son to that reported by other laboratories (�75%, Tashiroet al., 2007). This may be due to differences in the efficiencyof fluorescent-labeling and/or the method of fluorescence imag-ing. Generally higher numbers of BrdU1 cells are found byconfocal microscopy than by classical fluorescence microscopy(Tashiro et al., 2007). When BrdU signals cover only a smallportion of the nucleus, the resulting weak signals could not besufficiently visualized by conventional light microscopy.

Dose-Dependency and Specificity of P4-Actionin the Survival of Newborn Neurons

P4 was administered at various concentrations (0, 0.1, 1, 4,or 8 mg/kg) at BrdU-D0–2 to examine the dose-dependency ofthe P4-effect on the survival of newborn neurons. Interestingly,P4 at the dose of 1–4 mg/kg markedly increased the numberof 28-day-old BrdU1 cells (F(4, 49) 5 5.15, P 5 0.002; 1 mg/kg: P < 0.05; 4 mg/kg: P < 0.01; Fig. 2A). In contrast, theeffect of P4 diminished at both lower (0.1 mg/kg: P > 0.05)and higher (8 mg/kg: P > 0.05) doses in comparison to thattreated with vehicle, thus resulting a U-shaped dose-dependentcurve.

Neurosteroidogenic enzymes convert progesterone into othersteroids including Ad, TE, and E2 in the hippocampus. Amongthem, E2 (Ormerod et al., 2004) and TE (Spritzer and Galea,2007) are known to promote hippocampal neurogenesis bypromoting the cell survival in the DG of adult male rats andvoles. Mice were treated with Ad, TE, or E2 at 4 mg/kg (s.c.)instead of P4 to determine whether the P4-effect on the sur-vival of newborn neurons is specifically caused by P4 itself orits metabolites. As shown in Figure 2B, the administration ofAd, TE, and E2 at BrdU-D0–2 caused no detectable change inthe density of 28-day-old BrdU1 cells in comparison to cellstreated with vehicle (F(4, 49) 5 8.95, P < 0.001; P4: P <0.01; Ad: P > 0.05; TE: P > 0.05; E2: P > 0.05).

The P4-metabolite ALL promotes the proliferation of neuro-nal progenitor cells in rodent and human (Wang et al., 2005).The 5a-reductase inhibitor finasteride was used to examinewhether P4 enhances the survival of newborn neurons throughits metabolite ALL. The result in Figure 2C indicates that theinhibition of 5a-reductase by finasteride (20 mg/kg i.p.) beforeP4-administration at BrdU-D0–2 did not affect the P4-inducedincrease in the density of 28-day-old BrdU1 cells in compari-son to that of mice treated with P4 alone (F(3, 39) 5 11.6, P <0.001; P4 vs. P4/1finasteride: P > 0.05). Furthermore, treat-ment with finasteride alone at BrdU-D0–2 did not alter thenumber of 28-day-old BrdU1 cells in comparison to the con-trol mice (P > 0.05).

Involvement of P4R-Signaling in P4-EnhancedSurvival of Newborn Neurons

A single injection of 4 mg/kg P4 triggers P4R-mediatedERK phosphorylation and enhances the translocation of phos-phorylated ERK2 into the nucleus (Cai et al., 2008). The P4Rantagonist RU486 (3 mg/kg, i.p.) was used to examine the pos-sible involvement of P4R-mediated signaling in the P4-enhanced survival of newborn neurons. The results are shownin Figure 3 (F(8, 89) 5 8.28, P < 0.001), and clearly indicatethat pretreatment with RU486 significantly attenuated the P4-induced increase in the number of 28-day-old BrdU1 cells(lane-3 vs. lane-2 P4-treated mice, P < 0.05). Treatment withRU486 alone at BrdU0–2 did not significantly affect the basalnumber of newborn neurons (lane-4 vs. lane-1 control mice,P > 0.05).

Agonist binding of P4R reportedly activates the Src-Ras-ERK1/2 signal pathway during cell growth (Migliaccio et al.,2002). Thereupon, inhibitors for ERK kinase (MEK) and Srcfamily kinase were used to determine the involvement of thesignal pathway in the P4-enhanced survival of newborn neu-rons. The MEK inhibitor U0126 (100 lM/3 ll) or the Src in-hibitor PP2 (100 lM/3 ll) was microinjected (i.c.v.) at 30 minbefore P4-administration for 3 days. As expected, followingthe pretreatment with U0126 or PP2, P4 failed to increasethe number of 28-day-old BrdU1 cells (lane-5 U0126/P4-treated mice vs. lane-2 P4-treated mice, P < 0.01; lane-6

FIGURE 3. An analysis of the signal pathway underlying theP4-enhanced survival of newborn neurons. Bars indicate the den-sities of 28-day-old BrdU1 cells in the presence of various inhibi-tors (P4R blocker RU486, MEK inhibitor U0126, Src inhibitorPP2, PP2 inactive analog PP3, PI3K inhibitor LY294002) with(hatched bars) and without (open bars) P4-treatment. The P4-effect seems to partially depend on the classical nuclear P4R, whiletotally on Src ERK, and PI3K (see text). All drugs were adminis-tered 30 min before the injection of P4 or vehicle at BrdU-D0–2.**P < 0.01 vs. the corresponding mice treated with P4 alone.


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PP2/P4-treated mice vs. lane-2 P4-treated mice, P < 0.01),while the pretreatment with PP3, an inactive analog of PP2,did not alter the P4-action in the survival rate of newborn neu-rons (lane-7 vs. lane-2 P4-treated mice, P > 0.05). The activa-tion of Akt by P4 in cortical slice cultures promotes neuronalsurvival (Singh, 2001), thus its involvement in the P4-enhancedsurvival of newborn neurons was examined using LY294002 aninhibitor for PI3 kinase (PI3K) that is an upstream regulator ofAkt. Pretreatment with LY294002 (100 lM//3 ll, i.c.v.) largelyattenuated the P4-induced increase in the number of 28-day-old BrdU1 cells (lane-8 vs. lane-2 P4-treated mice, P < 0.01).This result indicates that the effect of P4 on the survival ofnewborn neurons is mediated through not only the Src-ERKpathway, but also the PI3K pathway. Collectively, these datasuggested that the P4-effects on the survival of newborn neu-rons are exerted by a P4R and unidentified P4 receptor(s)insensitive to RU486 followed by the Src-ERK or Akt signalpathway.

Role of P4 in Spatial Memory

The Morris water maze test was used to determine whetherthe P4-induced increase in the number of newborn neurons isrelated to hippocampus-dependent cognitive behavior in mice

from the 28th day after the first BrdU-injection. No significantdifference was observed in the swim speed between control(12.83 6 2.51 cm/s) and P4-treated mice (14.54 6 3.11 cm/s,P > 0.05) in the baseline trial in which the platform wasabsent from the pool (Fig. 4A). Notably, on the 3rd day and4th day after the training the mice treated with P4 at BrdU-D0–2 showed a decrease in the escape-latency to the hidden-platform in comparison to the control mice (3rd day: P <0.01; 4th day: P < 0.05, n 5 10). On the 6th day after theMorris water maze test, the platform was removed from thepool, and the probe trial performance was measured to evaluatethe percent of time spent for swimming in each quadrant(hidden-platform, opposite and adjacent quadrants), which pos-itively correlated with the spatial memory function. As shownin Figure 4B, the P4-treated mice spent more swimming timein the quadrant (43.6% 6 3.2%) that previously housed theplatform during the training trials than the control mice did(32.07% 6 2.69%, P < 0.05, n 5 10).

The P4R antagonist RU486 was used to confirm whetherthe improvement of spatial memory function was correlatedwith the increased survival levels of newborn neurons caused byP4. As expected, pretreatment with RU486 completely blockedthe effects of P4 on the decrease in the escape-latency to reachthe hidden-platform (vs. control mice, 3rd day: P > 0.05; 4th

FIGURE 4. P4 improves hippocampus-dependent cognitivebehavior. In the graphs, the open circles or bars depict the per-formance of mice treated with vehicle, and the black circles orhatched bars depict the data recorded in mice treated with P4 atBrdU-D0–2. A: Effects of P4 on water maze acquisition trials dur-ing BrdU-D28–32. The standard training following the scheduleindicated by the upper panel. B: Effects of P4 on the water maze

probe trial at BrdU-D33. Note that mice treated with P4 spendmore swimming time in the quadrant previously housing the plat-form during training trials than that in control mice. C and D:Role of the P4R antagonist RU486 in P4-potentiated spatial mem-ory. *P < 0.05 and **P < 0.01 vs. *corresponding control micetreated with vehicle.

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day: P > 0.05, n 5 10; Fig. 4C) and increased the swimmingtime in the quadrant that previously housed the platform (vs.control mice, P > 0.05, n 5 10; Fig. 4D), thus indicating thatthe increased BrdU1 cells by P4 is positively correlated withthe potentiation of spatial learning and memory of the animals.

DISCUSSION

The present study provides evidence that P4 promotes thesurvival of newborn neurons in the hippocampal DG of adultmale mice. This finding was supported by three major results.(1) Treatment with P4 for 3 days produced an approximatelytwofold increase in the relative numbers of 28- and 56-day-oldBrdU1 cells in comparison to each control without affectingthe number of 24/48-h-old BrdU1 cells. (2) P4 by itself, butnot its metabolites, enhanced the survival of newborn neurons.(3) The P4-effect was inhibited by the P4R antagonist RU486and inhibitors for Src, MEK, or PI3K. The functional contri-bution of the P4-enhanced survival of newborn neurons wasdemonstrated by a positive correlation with the potentiated spa-tial memory in P4-treated animals.

P4 Promotes the Survival of Newborn Neurons

This study has clearly demonstrated that treatment with P4produces an approximately twofold increase in the relativenumber of 28- and 56-day-old BrdU1 cells. Neurogenesis is aprocess that includes both cell proliferation (production of newcells) and cell survival (newborn cells to maturity). Therefore,the number of mature newborn neurons increased not only byincreased cell proliferation, but also by the enhanced survival ofnewborn neurons (Kempermann et al., 1997; van Praag et al.,1999). Recent studies have revealed that ALL promotes theproliferation of rodent and human neural progenitor cellsthrough the regulation of cell-cycle genes (Wang et al., 2005;Brinton et al., 2007), while P4 reduces the E2-inducedenhancement of cell proliferation (Tanapat et al., 2005). ALLenhances the proliferation of neuroprogenitor cells throughGABAA receptor-activated voltage-gated L-type calcium chan-nels (L-VGCC) (Wang et al., 2005), while P4 does not directlyregulate GABAA receptor activity (Ardeshiri et al., 2006). Thecurrent results revealed that P4 increased the relative numbersof 7-, 28-, and 56-day-old BrdU1 cells in comparison to therespective controls, but did not alter the number of 24/48-h-old BrdU1 cells. In addition, the administration of P4 atBrdU-D5–7 but not at BrdU-D10–12 and BrdU-D15–17

enhanced the survival of newborn neurons, suggesting that theP4-effect arises mainly from action on relatively young new-born cells. However, these data including the total numbers of24- and 48-h-old BrdU1 cells could not determine whether P4affected cell proliferation; because progenitor cells in rats andmice complete one mitotic division within 24 h (Hayes andNowakowski, 2002). Therefore, further studies examining the

effects of P4 on the cell proliferation within one mitotic divi-sion are required to make any definitive conclusions.

Recently, an increasing number of papers report the acuteand chronic effects of estrogens and androgens on hippocampalneurogenesis in adult rodents (Galea, 2008). Estradiol at0.2 mg/kg enhances cell survival only when administered dur-ing the ‘‘axon extension phase’’ of cell maturation (BrdU-D6–10) in male meadow voles (Ormerod et al., 2004). TE(3 mg/kg) when administered continuously for 30 daysthroughout the period of cell survival, promotes the survival ofnewborn neurons in the DG of castrated adult male rats(Spritzer and Galea, 2007), while short-term (3 days) TE expo-sure enhances only cell proliferation (Fowler et al., 2003). Incontrast, the present study revealed that an estrogen (E2) orandrogens (TE and Ad) at either 4 mg/kg or 0.5 mg/kg (datanot shown) at BrdU-D0–2 did not increase the relative numberof 28-day-old BrdU1 cells. Considering the known positiveeffects of E2 and TE on the neurogenesis in the DG (Ormerodet al., 2004; Spritzer and Galea, 2007), the current results maybe due to differences in the experimental conditions includingthe administration-timing and -duration of these steroids. Inaddition, the effect of P4 on the survival of newborn neuronswas not suppressed by the inhibition of 5a-reductase. This sug-gests that P4 regulates the survival of newborn neurons byitself, rather than via its metabolites.

Possible Mechanisms UnderlyingP4-Enhanced Cell Survival

Another principal observation in the present study is that theeffect of P4 on the survival of newborn cells depends upon itsbinding to the classical P4R and following Src-ERK1/2 andPI3K signaling. P4R, a dual functioning steroid hormone re-ceptor, directly regulates Src-related intracellular signaling andtranscription processes (Ballare et al., 2003). A specific polypro-line motif in the amino-terminal domain of P4R interacts withSH3 domains of cytoplasmic c-Src kinase (Boonyaratanakornkitet al., 2001; Faivre et al., 2005; Faivre and Lange, 2007). P4triggers a Src-ERK1/2 signal cascade (Migliaccio et al., 2002)that enhances the translocation of ERK2 into the nucleus (Caiet al., 2008), which is necessary for the activation of cAMPresponse element binding protein (CREB). ERK1/2-CREB sig-naling is thought to play a critical role in neuronal cell survival(Singh, 2006) through increasing the expression of antiapop-totic molecules, such as Bcl-2 and Bcl-XL (Yao et al., 2005),and decreasing the expression of proapoptotic molecules, suchas Bax, Bad, and caspase-3 (Djebaili et al., 2004). In addition,at the transcriptional level, P4R increases the expression of c-Src kinase and ERK (Boonyaratanakornkit et al., 2001) andEGF (Gerstenberg et al., 1999) that may stimulate the neuro-genesis in adult and aged rodent brains (Cheng et al., 2001;Jin et al., 2003). More recently, Kaur et al. (2007) reportedthat P4 increases the expression of brain-derived neurotrophicfactor and protects against glutamate toxicity in an ERK- andPI3K-dependent manner.


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Interestingly, the inhibition of P4R by RU486 could notcompletely abolish the P4-effect on the survival of newbornneurons (see lane-2 and lane-3 in Fig. 3), while the RU486-insensitive P4-effect was blocked by the inhibitors for Src-ERKand PI3K (lane-5, lane-6, and lane-8), thus suggesting the exis-tence of a membrane-mediated P4-action independent of theclassical nuclear P4R. Recently, much attention has been paidto the rapid action of P4 on membrane-associated P4-bindingproteins (Meffre et al., 2005). For example, P4 binding toPGRMC1 increases the level of phosphorylated Akt in neuro-nal cells (Singh, 2001) and induces subsequent PI3K-mediatedphosphorylation of ERK1/2 (Swiatek-De et al., 2007). Pelusoet al. (2008) reported that PGRMC1 mediates the antiapop-totic actions of P4, which raises the possibility that the R4R-in-dependent P4-effect may work through the PGRMC1-medi-ated PI3K-Akt or ERK signaling pathway. On the other hand,RU486 has a potent antagonistic effect on glucocorticosteroidreceptors (GR), which prevents the suppression of newbornneurons by corticosterone (Wong and Herbert, 2005). In addi-tion, some neuroprotective effects of RU486 have been demon-strated to be independent of P4R and GR (Zhu et al., 2004).RU486 is known to promote the survival of Purkinje cells via aP4R-independent way (Ghoumari et al., 2003). These observa-tions raise the possibility that inhibition of GR by RU486, orRU486 itself enhances cell proliferation and survival of new-born neurons. However, the current results do not support thishypothesis, because RU486 when solely administered at BrdU-D0–2 failed to increase the number of 28-day-old BrdU1 cells(lane-4 in Fig. 3) in comparison to control (lane-1).

P4-Enhanced Cell Survival Is Well CorrelatedWith Spatial Memory

Notably, the number of the mature newborn neurons in theDG were increased in P4-treated mice (Fig. 1C) associatedwith their improved cognitive performances such as shorter la-tency to reach the platform (Fig. 4A) and longer swimmingtime in the quadrant where the platform had been housed(Fig. 4B). Although this result does not immediately mean thatthe increased newborn neurons in the DG are responsible forthe improved cognitive performance, accumulated knowledgehas demonstrated that neurogenesis in the DG plays an impor-tant role in spatial learning of adult rodents. Spatial learningincreases the proportion of multiple synapse boutons in theDG but not in the CA1, thus indicating that the DG is moreinvolved in spatial learning than the CA1 (Thuret et al., 2009).In the Morris water maze test, the aged-rats with a well-pre-served capability of spatial memory exhibited a higher rate ofcell proliferation and a higher number of newborn neurons inthe DG in comparison to the aged-rats with spatial memoryimpairments (Drapeau et al., 2003), thus suggesting a strongcorrelation between the newborn neurons in the DG and thecapability of spatial learning. Further support can be seen inthe recent findings indicating that the newborn neurons areintegrated into the hippocampal circuitry only after 4–8 weeksof their maturation, during which their dendritic spines contact

perforant path synapses and their axons are projected in a CA3area (Zhao et al., 2006; Toni et al., 2007, 2008). Theimproved connectivity resulting from a prolonged maturationof newborn neurons may contribute to the formation of place-neurons in the CA3 (Nakazawa et al., 2004). Interestingly, thenewborn neurons have lower activation thresholds and higherresting potentials, thus leading to the facilitation of long-termpotentiation induction in comparison with mature neurons(van Praag et al., 2002; Schmidt-Hieber et al., 2004). In addi-tion, unlike mature granule cells, the newborn granule cells areinsensitive to GABAergic inhibition (Schmidt-Hieber et al.,2004), which may provide a better environment for quicklearning. Taken together with these observations, it is highlylikely that the increased newborn neurons in the DG inducedby P4 may therefore significantly contribute to the improvedperformance of the spatial learning in the P4-treated mice.

Dose-Dependency of P4-Effect

The present study demonstrates that the effect of P4 on thesurvival of newborn cells shows a U-shaped dose-dependentcurve. This is consistent with previous studies showing that theneuroprotective dose of P4 is in the range of 1–4 mg/kg,whereas at a higher dose (8 mg/kg) it loses its neuroprotectiveeffect in ovariectomized rats (Hoffman et al., 2003; Cirizaet al., 2006). P4 easily penetrates the blood–brain barrier, thusthe concentration of P4 in the brain is thought to followclosely the circulating serum levels, and thus P4 is naturallypresent in the brains of men and women at comparable levels(Brinton et al., 2008). In rats, the plasma level of P4 fluctuatesranging from 18–70 ng/ml at the basal level (Nequin et al.,1979; Ciriza et al., 2006) to 160–180 ng/ml in pregnancy (Kaiet al., 1997). Absorption of 4 mg/kg (i.p.) results in plasma P4levels of P4 between 42 and 70 ng/ml (Ciriza et al., 2006;Gibson et al., 2008), indicating that the administration of P4at 4 mg/kg does not dramatically affect the gonadal-origin hor-mone levels. The present study contributes to the understand-ing of P4 as a modulator promoting neurogenesis in adultmale mice, which may help to clarify the influence and impor-tance of P4 in hippocampus-dependent learning and memory.Further studies are currently underway to determine the neuro-genic potential of P4 for cognitive deterioration in the agingpopulation, neurodegenerative disease, and cerebral injury.

Acknowledgments

The authors declare that there is no competing financialassociation that could be construed as influencing the results orinterpretation of the reported study.

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Progesterone promotes the survival of newborn neurons in the dentate gyrus of adult male mice