Oxytocin Stimulates Adult Neurogenesis Even Under Conditions ofStress and Elevated Glucocorticoids

Benedetta Leuner, Julia M. Caponiti, and Elizabeth Gould*

ABSTRACT: Oxytocin has been linked to social behavior, includingsocial recognition, pair bonding and parenting, but its potential role inpromoting neuronal growth has not been investigated. We show herethat oxytocin, but not vasopressin, stimulates both cell proliferation andadult neurogenesis in the hippocampus of rats. Oxytocin is also capableof stimulating adult neurogenesis in rats subjected to glucocorticoidadministration or cold water swim stress. These findings suggest that ox-ytocin stimulates neuronal growth and may protect against the suppres-sive effects of stress hormones on hippocampal plasticity. VVC 2011 WileyPeriodicals, Inc.

KEY WORDS: cell proliferation; hippocampus; hypothalamic-pituitaryadrenal (HPA) axis; plasticity; vasopressin

INTRODUCTION

Oxytocin is a neuropeptide, released both peripherally and centrally,that has been linked to social behaviors such as mating and parenting(Young and Wang, 2004; Shelley et al., 2006; Carter et al., 2008; Neu-mann, 2009). Oxytocin has also been shown to buffer against some ofthe negative effects of stress (Windle et al., 2004, 2006; Cohen et al.,2010) - its actions include a reduction in hypothalamic-pituitary adrenal(HPA) axis activity and diminished stress-induced activation of brainregions associated with the HPA axis, including the hippocampus.

Oxytocin enhances hippocampal synaptic plasticity and cognitivefunctions of mother rats (Tomizawa et al., 2003), but its overall influ-ence on this brain region remains unexplored. The hippocampus is asite of structural plasticity, undergoing neurogenesis throughout life—evidence has linked cognitive, anxiety and stress regulation functions ofthe hippocampus to adult neurogenesis (Leuner and Gould, 2010). Oxy-tocin neurons in the hypothalamic paraventricular nucleus project tovarious brain regions, including the hippocampus (Bujis and Swaab,1979; Sofroniew, 1983). Oxytocin receptors are present in the hippo-campus where they are more concentrated in the ventral than dorsalregion and subject to modulation by stress and glucocorticoids (Liberzonand Young, 1997).

Oxytocin has been shown to affect cell proliferationin peripheral systems, exhibiting stimulatory effects onblood cells, osteoblasts, and some types of tumor cells(Petersson, 2008; Maccio et al., 2010) as well asinhibitory effects on prostate and mammary cells(Sapino et al., 1998; Whittington et al., 2007; Peters-son, 2008), but its ability to influence neuronalgrowth in the brain remains unexplored. Since stressand glucocorticoids can be potent regulators of cellproliferation and adult neurogenesis in the hippocam-pus (Mirescu and Gould, 2006; Leuner et al., 2010)and oxytocin is known to attenuate stress responses(Windle et al., 2004, 2006), we investigated whetheroxytocin alters adult neurogenesis and whether thiseffect occurs in the presence of elevated glucocorti-coids. Here we show that peripheral and centraloxytocin, but not the closely related neuropeptidevasopressin, enhances cell proliferation and adult neu-rogenesis. The stimulatory effect of oxytocin on cellproliferation occurred even in rats treated with gluco-corticoids or exposed to a stressor. These findings raisethe possibility that oxytocin acts to protect the hippo-campus from the damaging effects of elevated gluco-corticoids by promoting neuronal growth.

MATERIALS AND METHODS

Animals

Adult (� 60 days of age) male Sprague-Dawley rats(Taconic; Germantown, NY) were provided unlimitedaccess to food and water and maintained on a 12:12light-dark cycle (lights on at 7:00 a.m.). Rats weregroup-housed (2–3/cage) and all rats within a cagewere included in the same experimental group. Proce-dures were conducted in accordance with PrincetonUniversity IACUC and The National Institutes ofHealth Guide for the Care and Use of LaboratoryAnimals.

Peripheral Oxytocin

Rats (n 5 6–11/group) were injected i.p. with oxy-tocin (1 mg/kg or 10 mg/kg; Bachem Americas, Inc.,King of Prussia, PA; doses based on Ring et al., 2006)or an equal volume of saline. Thirty min later, ratswere injected i.p. with the DNA synthesis marker,

Department of Psychology, Neuroscience Institute, Princeton Univer-sity, Princeton, New JerseyBenedetta Leuner is now at Department of Psychology, The Ohio StateUniversity, Columbus, OhioBenedetta Leuner and Julia M. Caponiti contributed equally to this work.Grant sponsor: National Institute of Mental Health; Grant numbers:MH59470, MH84148; Grant sponsor: National Alliance for Research onSchizophrenia and Depression, the Mental Health Research Association*Correspondence to: Elizabeth Gould, Princeton University, Departmentof Psychology, Green Hall, Washington Road, Princeton, NJ 08544,USA. E-mail: goulde@princeton.eduAccepted for publication 17 February 2011DOI 10.1002/hipo.20947Published online 20 June 2011 in Wiley Online Library(wileyonlinelibrary.com).

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VVC 2011 WILEY PERIODICALS, INC.

bromodeoxyuridine (BrdU; 200 mg/kg; Sigma, St. Louis,MO), a dose that labels the maximal number of S phase cellsin the dentate gyrus (DG) (Cameron and McKay, 2001). Ratswere perfused 2 h later, a post-BrdU survival time that is suffi-cient to label cells in S-phase but not to allow the labeled cellsto divide, thus providing a measure of cell proliferation. Addi-tional groups of rats (n 5 10–11/group) were injected i.p. with1 mg/kg oxytocin, 30 min later injected i.p. with BrdU and per-fused after one week or three weeks. At one week, the numberof BrdU-labeled cells is at its maximum and most expressmarkers of immature neurons (Gould et al., 1999). By threeweeks post-BrdU labeling, the majority of new cells in the DGexpress the mature neuronal marker NeuN. A final group of rats(n 5 6–9/group) was injected i.p. with 1 mg/kg oxytocin or anequal volume of saline once daily for seven consecutive days. Onthe last day, rats were injected with BrdU 30 min following theoxytocin or saline injection and perfused three weeks later.

Peripheral Vasopressin

Rats (n 5 4–5/group) were injected i.p. with arginine vaso-pressin (10 lg/kg; Sigma) or an equal volume of saline. Thisdose was chosen based on previous studies of i.p. injections ofvasopressin in rats (Izquierdo et al., 1988). Thirty minuteslater, all rats were injected with BrdU and perfused after 2 h.

Central Oxytocin

Rats (n 5 11) were anesthetized with Nembutal andrestrained in a stereotaxic apparatus. Using a 26 gauge Hamil-ton syringe, rats received hippocampal injections of oxytocin(1 ng/1 ll; Sigma; dose based on Melis et al., 1997; Ringet al., 2006) in one hemisphere and an equal volume of saline(1 ll) in the other hemisphere (stereotaxic coordinates: AP,25.3 from Bregma; LM, 6 4.0; DV, 23.5 from surface ofbrain; Paxinos and Watson, 1998). Prior studies have shownthat injection volumes of �2 ll do not produce degenerationin the granule cell layer (Gould and Tanapat, 1997). The injec-tions in each hemisphere were counter-balanced and the hemi-spheres were analyzed separately. Thirty minutes later, rats wereinjected with BrdU and perfused after a 2 h survival time.

Oxytocin and Corticosterone

Rats (n 5 12) were given an i.p. injection of oxytocin(1 mg/kg; Bachem) and thirty min later were subcutaneouslyinjected with 40 mg/kg corticosterone (Sigma), a dose that hasbeen previously shown to diminish cell proliferation in theDG (Cameron and Gould, 1994). An additional group of rats(n 5 14) was injected with saline followed by corticosterone.One hour later, both groups of rats were injected with BrdUand perfused after a 2 h survival time.

Oxytocin and Cold Swim Stress

Rats (n 5 11) were given i.p. injections of oxytocin (1 mg/kg;Bachem) and then thirty min later exposed to a cold water (2–

68C) swim stress for three min. This stressor has been shown todiminish cell proliferation in the DG (Stranahan et al., 2006).An additional group of rats (n 5 13) was exposed to the coldwater swim stress following treatment with saline. This procedurewas repeated once daily for seven consecutive days. On the lastday, rats were injected with BrdU 1 h following the oxytocin orsaline injection and perfused after a 2 h survival time.

Perfusion

All animals were deeply anesthetized with an overdose ofsodium pentobarbital and transcardially perfused with 4.0%paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brainswere removed and postfixed for at least two days.

Immunohistochemistry

Coronal sections (40 lm thick) throughout the entire ros-trocaudal extent of the DG were cut with a Vibratome into abath of 0.1 M phosphate-buffered saline (PBS). For peroxidasestaining, a 1:12 series of sections were mounted onto slides,dried, heated in 0.1 M citric acid, rinsed in PBS, digested intrypsin, rinsed, denatured in 2N HCl:PBS and rinsed. Slideswere incubated overnight at 48C in mouse monoclonal anti-body against BrdU (1:200; BD Biosciences, San Jose, CA)then rinsed, incubated with biotinylated horse anti-mouse(1:200; Vector, Burlingame, CA), rinsed, incubated with avi-din-biotin complex (Vector), rinsed and reacted in 0.01% dia-minobenzidine (Sigma) with 0.003% H2O2. Slides were coun-terstained with cresyl violet, dehydrated, cleared with Citrisolv(Fisher Scientific) and coverslipped with Permount (FisherScientific).

For assessment of cell phenotype, tissue from rats with athree week post-BrdU survival time was processed for double-labeling immunofluorescence for BrdU and the neuronalmarker NeuN or the astroglial marker GFAP. A 1:12 series offree-floating sections were denatured in 2 N HCl: Tris bufferedsaline (TBS), rinsed in TBS, and incubated with rat anti-BrdU(1:200; Accurate, Westbury, NY) plus mouse anti-NeuN(1:500; Chemicon, Temecula, CA) or guinea pig anti-GFAP(1:1,000; Advanced Immunochemical, Long Beach, CA) fortwo days at 48C. Sections were then rinsed, incubated withbiotinylated anti-rat (Chemicon), rinsed, and incubated withstreptavidin-conjugated Alexa 568 (1:1,000; Molecular Probes,Eugene, OR) for BrdU, and with goat anti-mouse or goat anti-guinea pig Alexa 488 (1:500; Molecular Probes) for NeuN orGFAP. After rinsing, sections were mounted onto slides andcoverslipped using glycerol:TBS (3:1).

Data Analysis

Slides were coded prior to quantitative analysis. For BrdUperoxidase analysis, the number of BrdU-labeled cells in theDG (subgranular zone, granule cell layer and hilus) wascounted on every 12th section of tissue at 100x on an Olym-pus BX-50 light microscope. For experiments with a 2 h sur-

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vival, BrdU-labeled cells were counted throughout the entire ros-trocaudal extent of the DG (2.20-7.20 mm; Paxinos and Watson,1998) as well as in the dorsal and ventral DG separately (Banasret al., 2006). For experiments with one week or three week sur-vival times, BrdU-labeled cells in the dorsal and ventral DG werecounted separately. For experiments involving peripheral injec-tions of oxytocin or vasopressin, half brains were analyzed.Counts were multiplied by 24 to obtain bilateral estimates of thenumber of BrdU-labeled cells per brain. For the central oxytocinstudy, counts were multiplied by 12 because both sides of thebrain (saline and oxytocin) were analyzed separately. Cavalieri’sprinciple (Gundersen et al., 1988) was used for determination ofDG volume (hilus and granule cell layer) from cross-sectionalarea measurements obtained with Image-Pro Plus software(Media Cybernetics, Silver Spring, MD).

For purposes of comparison, the density of BrdU-labeledcells was also determined in the subventricular zone (SVZ).BrdU-labeled cells in the SVZ present on every twelfth coronalsection throughout the DG were counted, the volume of theanalyzed region determined, and the data expressed as densities(number of BrdU-labeled cells/mm3).

For BrdU immunofluorescence, the percentage of BrdU-labeled cells in the granule cell layer and subgranular zoneexpressing NeuN or GFAP was determined using a Zeiss Axio-vert confocal laser scanning microscope (510 LSM; lasers,Argon 458/488 and HeNe 543; Zeiss, Oberkochen, Germany).Dorsal and ventral DG were analyzed separately as describedabove. For each brain, marker, and subregion, 15–25 randomlyselected BrdU-labeled cells were analyzed. Optical stacks of1-lm thick sections were obtained through putative double-

labeled cells. To verify double labeling throughout their extent,cells were examined in orthogonal planes.

Statistical analyses

Unpaired t-test or one-way analysis of variance (ANOVA) fol-lowed by Newman-Keuls post hoc comparisons were used to an-alyze differences in the number of BrdU-labeled cells in the DG(total, dorsal, ventral), BrdU-labeled cells in the subventricularzone, the volume of the DG, and the volume of damage in thegranule cell layer caused by the central injections of oxytocinand saline. When animals were given central injections, pairedt-tests were used because the other side of the brain (saline-injected) served as a control for the oxytocin-injected side.

RESULTS

Acute Peripheral Oxytocin AdministrationEnhances Cell Proliferation in the Ventral DG

To assess the effects of acute oxytocin administration on cellproliferation, animals were given a single peripheral injection ofoxytocin, injected with BrdU 30 min later and perfused after a2 h survival time. A single injection of oxytocin enhanced cellproliferation in the DG (F(2,23) 5 4.49 - P < 0.05). Analysisof dorsal and ventral DG separately revealed that the increasewas evident only in the ventral DG (F(2,23) 5 10.02 - P <0.005; Fig. 1). Rats given 1 mg/kg oxytocin had more BrdU-la-beled cells than rats given saline or a higher dose of oxytocin(10 mg/kg). Groups given saline or 10 mg/kg oxytocin did notdiffer in dorsal, ventral or dorsal-ventral combined (P > 0.05)To determine whether these effects are specific to oxytocin, anadditional group of animals was injected with the closelyrelated neuropeptide vasopressin (AVP), injected with BrdUafter 30 min and perfused 2 h later. Unlike oxytocin, AVP hadno significant effect on the number of proliferating cells in the

FIGURE 1. Peripheral oxytocin administration enhances cellproliferation in the ventral DG. Adult male rats received an i.p.injection of oxytocin (OT) or an equal volume of saline. All ratswere injected with BrdU 30 min later and perfused after 2 h. Ratstreated with 1 mg/kg OT had a greater number of BrdU-labeledcells in the ventral, but not dorsal, DG compared with thosetreated with saline or 10 mg/kg OT. Bars represent mean 6 stand-ard error of mean (SEM), *P < 0.05. Photomicrographs showmore BrdU-labeled cells (brown cells) in the ventral DG of ratstreated with OT. Scale bar, 10 lm. [Color figure can be viewed inthe online issue, which is available at wileyonlinelibrary.com.]

FIGURE 2. Peripheral OT administration does not alter thedensity of BrdU-labeled cells in the SVZ. The density of BrdU-labeled cells in the SVZ of rats treated with 1 mg/kg OT or10 mg/kg OT did not differ from saline-treated rats. Bars representmean 6 SEM. Photomicrographs show BrdU-labeled cells in theSVZ of saline and oxytocin treated rats. Scale bar, 10 lm. [Colorfigure can be viewed in the online issue, which is available atwileyonlinelibrary.com.]

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dorsal (Saline: 1,824 6 249.6, AVP: 2,298 6 181.0 - P > 0.05)or ventral (Saline: 1982 6 256.1, AVP: 2,178 6 418.4 - P >0.05) DG. Furthermore, the effects of oxytocin on cell prolifera-tion were regionally specific since there was no difference in thedensity of BrdU-labeled cells in the SVZ 2 h after BrdU adminis-tration (P > 0.05; Fig. 2). Lastly, no differences in the dorsal,ventral or dorsal-ventral combined volume of the dentate gyruswere detected among the groups (combined dorsal-ventral Saline:10.24 6 0.50, Oxytocin 1 mg/kg: 11.40 6 0.48, Oxytocin 10mg/kg: 10.0 6 0.41- P > 0.05; dorsal Saline: 5.30 6 0.25, Oxy-tocin 1 mg/kg: 5.50 6 0.30, Oxytocin 10 mg/kg: 5.52 6 0.30 -P > 0.05; ventral Saline 4.93 6 0.49, Oxytocin 1 mg/kg: 5.656 0.35, Oxytocin 10 mg/kg: 4.47 6 0.31 - P > 0.05).

Using the same single dose of oxytocin (1 mg/kg) thatenhanced cell proliferation, we found that the number of BrdUlabeled cells was no longer increased at later time points afterBrdU injection. One week and three weeks after BrdU administra-tion, there was no difference in the number of BrdU-labeled cellsin the dorsal, ventral or dorsal-ventral combined DG in groupstreated with saline or 1 mg/kg oxytocin (P-values >0.05).

Repeated Peripheral Oxytocin Increases theNumber of New Neurons

We next examined whether more sustained exposure tooxytocin would produce a lasting enhancement in the number ofBrdU labeled cells by using a repeated oxytocin injection para-digm. Male rats were given seven days of oxytocin injections, aBrdU injection on the last day, and perfused three weeks later.Compared with saline-treated controls, rats treated with oxytocinhad more BrdU-labeled cells in the ventral DG (t(12) 5 2.18 - P< 0.05; Fig. 3a). The number of BrdU-labeled cells in the dorsalDG did not differ between saline and oxytocin treated groups (P> 0.05). Most (�88%) BrdU-labeled cells expressed the markerof mature neurons, NeuN (Fig. 3b). A small percentage (�5%)expressed the astroglial marker, GFAP (Fig. 3c). There was no dif-

ference in the proportion of BrdU-labeled cells expressing thesemarkers across groups or subregions (P-values > 0.05). No differ-ences in the total volume of the dentate gyrus were detected amongthe groups (Saline: 11.78 6 0.24, Oxytocin: 11.87 6 0.49 - P >0.05). No differences were detected in the volume of the dentategyrus at dorsal (Saline: 6.256 0.41, Oxytocin: 5.686 0.22 - P >0.05) or ventral (Saline: 5.53 6 0.28, Oxytocin: 6.19 6 0.30 - P> 0.05) levels. Taken together, these data suggest that repeated ox-ytocin injection enhanced the number of adult-generated cells forat least three weeks, including both neurons and glia.

FIGURE 3. Chronic peripheral oxytocin enhances adult neu-rogenesis. Rats received an i.p. injection of OT (1 mg/kg) or anequal volume of saline once daily for seven consecutive days. Allanimals were injected with BrdU 30 min after the last injectionand perfused after a three-week survival. (a) Chronic OT increasedthe number of BrdU-labeled cells in the ventral, but not dorsal,

DG. Bars represent mean 6 SEM, *P < 0.05. At this time, mostBrdU-labeled cells (red) in the saline and OT groups were co-labeledwith (b) the mature neuronal marker NeuN (green) but not with (c)the astroglial marker GFAP (green). Scale bar, 10 lm. [Color figurecan be viewed in the online issue, which is available atwileyonlinelibrary.com.]

FIGURE 4. Central administration of oxytocin enhances cellproliferation. Adult male rats received hippocampal injections ofOT (1 ng/ll) in one hemisphere or an equal volume of saline intothe other hemisphere. All animals were injected with BrdU 30 minlater and perfused after a 2-h survival. The hemisphere treatedwith OT had a greater number of BrdU-labeled cells in the ventralDG compared to the saline-treated hemisphere. The number ofBrdU-labeled cells in the dorsal DG did not differ between thesaline and OT treated hemispheres. Bars represent mean 6 SEM,*P < 0.05. Photomicrographs show more BrdU-labeled cells(brown cells) in the ventral DG of rats treated with OT. Scale bar,10 lm. [Color figure can be viewed in the online issue, which isavailable at wileyonlinelibrary.com.]

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Hippocampal Oxytocin Enhances CellProliferation

We next evaluated whether oxytocin acts directly within thehippocampus to alter cell proliferation. Rats received hippocam-pal microinfusions of oxytocin in one hemisphere and saline inthe other, allowing the other side of the brain of each animal toserve as its own control. Thirty minutes after the infusion, ratswere injected with BrdU and perfused after a 2 h survival time.We found that the hemisphere treated with oxytocin had a sig-nificantly greater number of BrdU-labeled cells in the ventralDG than the saline-treated hemisphere (t(10) 5 2.69, – P <0.05 Fig. 4). The number of BrdU-labeled cells in the dorsalDG was unaffected by OT (P > 0.0.5). These regional differen-ces were not due to variations in the area of damage in the DGat the injection site which was similar for the saline- and oxyto-cin-treated hemispheres (P > 0.05).

Oxytocin Enhances Cell Proliferation in AnimalsTreated With Corticosterone or Exposed to SwimStress

We next examined whether oxytocin could enhance cell pro-liferation under conditions associated with increased levels ofglucocorticoids. Rats were treated with oxytocin 30 min beforereceiving a single corticosterone injection (OT + CORT) orwith corticosterone alone (CORT). After 1 h, rats were injectedwith BrdU and perfused after a 2 h survival. Corticosteronetreated rats that were also given oxytocin had more BrdU-la-beled cells in total DG than animals that did not receive oxyto-cin (t(24) 5 2.44, – P < 0.05). This difference was concen-trated in the ventral hippocampus which showed a significantincrease in oxytocin treated animals (t(24) 5 3.52, – P <0.005; Table 1). In the dorsal DG, no difference in the numberof BrdU-labeled cells was detected between CORT and OT 6CORT groups (P > 0.05, Table 1). To evaluate the effects ofoxytocin on cell proliferation in animal undergoing repeatedstress, rats were treated daily with oxytocin and 30 min laterexposed to cold water swim stress (OT + Stress) or wereexposed to the stressor without OT (Stress). On the seventhday, rats were injected with BrdU one hr after the stressor andperfused after a 2 h survival. Rats that were treated with oxyto-cin and exposed to cold water swim stress for seven daysshowed an increase in BrdU labeled cells in the total DG

(t(22) 5 2.13 5 2.13 - P < 0.05). However, there was nostatistically significant difference in BrdU cell number in eitherthe dorsal (t(22) 5 1.90 - P 5 0.07) or ventral (t(22) 5 1.87 -P 5 0.08) DG (Table 1).

DISCUSSION

Here we show that acute administration of oxytocin, eithercentrally or peripherally, stimulates cell proliferation in theDG. Although the proliferative effects of acute oxytocin treat-ment did not result in an increase in neurogenesis at one weekor three weeks, more prolonged treatment of oxytocin (i.e., oneweek of daily peripheral oxytocin injections) increased neuro-genesis, an effect that appears to be specific to the ventral por-tion of the DG. In addition, we show that the stimulatoryeffect of oxytocin on cell proliferation occurred even in ratstreated with glucocorticoids or exposed to stress. Takentogether, these results suggest that oxytocin stimulates neuronalgrowth and may protect against the suppressive effects of stresshormones on hippocampal plasticity.

Although we tested two doses of peripheral oxytocin on cellproliferation, only the lower dose was found to stimulate cellproliferation. In this regard it should be noted that effects ofneuropeptides such as oxytocin are often dose-dependent butnot always linear. For example, the effects of peripheral oxyto-cin on social recognition follow an inverted U-shaped doseresponse curve where moderate doses facilitate, and high dosesattenuate social recognition (Popik et al., 1992). Becausewe didn’t examine doses lower than 1 mg/kg or higher than10 mg/kg, the exact dose response relationship between oxyto-cin and cell proliferation remains to be determined.

Only small amounts of oxytocin given peripherally passthrough the blood brain barrier (Ermisch et al., 1985; Engel-mann et al., 1996). Nonetheless, a number of studies haveshown that either subcutaneous or intraperitoneal administra-tion of OT can trigger many of the same responses that occurfollowing the release of, or administration of, OT within theCNS (Arletti et al., 1992; McCarthy et al., 1992; Peterssonet al., 1996). We also found that like peripheral oxytocin, acutecentral administration of oxytocin into the hippocampus simi-larly increased cell proliferation. Taken together, these data sug-gest that oxytocin is acting within the hippocampus to increasethe number of new cells. Oxytocin receptors are known to beexpressed in the hippocampus (Liberzon and Young, 1997;Gimpl and Farrenholz, 2001; Tomozawa et al., 2003) furtherindicating that the hippocampus is a likely target for oxytocin.However, it remains unclear whether the effects of oxytocin oncell proliferation and neurogenesis are mediated via direct acti-vation of oxytocin receptors on precursor cells within the DG.Alternatively, it’s possible that oxytocin modulates these proc-esses indirectly by acting on oxytocin-responsive cells withinbrain regions that provide afferent input to the DG such asthe amygdala (Gimpl and Farrenholz, 2001) or through itsinfluence on other hormonal or neurotransmitter systems thathave been shown to alter cell proliferation.

TABLE 1.

BrdU-Labeled Cells (mean 6 SEM) in the Dorsal and Ventral Dentate

Gyrus of Rats Treated With Oxytocin (OT) Under Conditions of

Elevated Glucocorticoids as a Result of a Single Corticosterone

(CORT) Injection or Seven Days of Cold Water Swim Stress (Stress)

CORT OT 1 CORT Stress OT 1 Stress

Dorsal 4,255 6 228 4,424 6 340 2,245 6 160 2,891 6 319

Ventral 2,755 6 206 4,206 6 376* 2,339 6 219 3,135 6 384

*P < 0.05.

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Oxytocin is known to diminish HPA axis responsiveness andlower levels of circulating glucocorticoids (Windle et al., 2004,2006; Cohen et al., 2010). Another possible interpretation ofour findings demonstrating that oxytocin stimulates cell prolif-eration and neurogenesis is that it does so by lowering levels ofcorticosterone. Numerous studies have shown that elevated cor-ticosterone, applied either exogenously or through exposure toaversive stressors, suppresses cell proliferation and neurogenesis(Cameron and Gould, 1994; Tanapat et al., 2001; Mirescuand Gould, 2006). Conversely, lowering glucocorticoid levelsby adrenalectomy enhances cell proliferation and neurogenesis(Gould et al., 1992). Our results show that oxytocin enhancescell proliferation even after elevating corticosterone levelsacutely by injection. These findings suggest that the stimula-tory effects of oxytocin on cell proliferation and neurogenesisin the DG are unlikely to be mediated only indirectly throughoxytocin-induced reductions in glucocorticoid levels. Thesefindings further suggest the possibility that oxytocin may becapable of overriding some of the negative effects of elevatedglucocorticoids, such as those on neuronal growth inhibition.It should be noted, however, that our stress and oxytocin find-ings were less conclusive than the corticosterone injectionstudy. That is, we observed a statistically significant increase inthe number of BrdU labeled cells in the stress plus oxytocincondition only in the overall DG but not within the dorsal orventral subregions. The lack of a region-specific increase in cellproliferation in oxytocin treated stressed rats suggests that dif-ferent mechanisms may be operating under this conditioncompared with following corticosterone injection. In thisregard, we cannot rule out some potential involvement ofthe HPA axis in mediating these effects without additionalexperimentation.

Nonetheless, it remains possible that oxytocin is responsiblefor promoting neuronal growth under some circumstanceswhere glucocorticoids are naturally elevated. For example, sex-ual experience is rewarding yet elevates corticosterone levels andparadoxically promotes cell proliferation and adult neurogenesis(Leuner et al., 2010). Sexual experience also stimulates oxytocinrelease in the periphery, as well as in the brain (Stonehamet al., 1985; Carmichael et al., 1987; Waldherr and Neumann,2007). Taken together with the present paper, this work sug-gests that oxytocin may buffer the brain from elevated corticos-terone levels during mating by preventing growth inhibitionand stimulating neurogenesis. Similarly, oxytocin is releasedduring physical activity (Michelini, 2001), another rewardingexperience which also paradoxically causes an increase in gluco-corticoids and an increase in adult neurogenesis (van Praaget al., 1999; Stranahan et al., 2006). The release of oxytocinduring these experiences could provide the mechanism bywhich the hippocampus is protected from the rise inglucocorticoids.

Although acute peripheral oxytocin injection rapidlyincreases cell proliferation in the DG, the additional granulecells are lost during the ensuing period of cell death. After dailyinjection with oxytocin, however, a lasting effect on adult neu-rogenesis is observed. This increase is specific to the ventral

portion of the DG. The ventral hippocampus is connectedwith the amygdala, the medial prefrontal cortex and HPA axisstructures (Sahay and Hen, 2007; Fanselow and Dong, 2010)and is also involved in some types of learning and memory(Dalla et al., 2009; Snyder et al., 2009a), though it is moregenerally thought to play a role in anxiety and stress regulation(Herman et al., 1995; Bannerman et al., 2004). Studies haveshown that oxytocin receptors are more abundant in the ventralportion of the hippocampus (Liberzon and Young, 1997).Thus, the effects of oxytocin on neurogenesis in the ventralhippocampus are consistent with the release of oxytocin inresponse to certain stressors and the role of the ventral hippo-campus in stress regulation.

Although the specific role of adult-born neurons is not yetfully understood, there is a wide body of evidence that adult-born cells start to become integrated into the neuronal cir-cuitry shortly after their production (Hastings and Gould,1999; Ide et al., 2008; Snyder et al., 2009b) and that thesecells are important for hippocampal function, especially anxi-ety regulation and learning. A reduction in adult-generatedneurons in the DG produces deficits in hippocampus-depend-ent learning (Shors et al., 2001; Leuner et al., 2006; Snyderet al., 2009b), and learning hippocampus-dependent tasks isimpaired under circumstances in which adult neurogenesis isdiminished, such as after glucocorticoid administration (Luineet al., 1993; Bodnoff et al., 1995) or negative stress paradigms(Luine et al., 1996; Krugers et al., 1997). When the numberof new neurons in the hippocampus is enhanced by experien-ces such as living in an enriched environment (Kempermannet al., 1997; Nilsson et al., 1999) or running (van Praaget al., 1999), performance on these learning tasks is improved.It has also been shown that hippocampal adult neurogenesis isinvolved in anxiety regulation (Revest et al., 2009). Thereforeoxytocin-induced increases in new neurons in the DG couldcontribute to the reductions in anxiety and enhancement inlearning observed with rewarding experiences. These resultstaken together show that oxytocin has the potential for pro-tecting the hippocampus from the adverse effects of elevatedglucocorticoids and that oxytocin released during certain expe-riences may have beneficial effects in the hippocampus.

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