Lactation-Induced Reduction in Hippocampal Neurogenesis isReversed by Repeated Stress Exposure

K.M. Hillerer,1,2 I.D. Neumann,1 S. Couillard-Despres,3 L. Aigner,4* and D.A. Slattery1

ABSTRACT: The peripartum period is a time of high susceptibility formood and anxiety disorders, some of which have recently been associatedwith alterations in hippocampal neurogenesis. Several factors includingstress, aging, and, perhaps unexpectedly, lactation have been shown todecrease hippocampal neurogenesis. Intriguingly, lactation is also a time ofreduced stress responsivity suggesting that the effect of stress on neuro-genic processes may differ during this period. Therefore, the aim of thepresent study was to assess the effect of repeated stress during lactation[2 h restraint stress from lactation day (LD) 2 to LD13] on brain weight,hippocampal volume, cell proliferation and survival, and on neuronal andastroglial differentiation. In addition to confirming the known lactation-associated decrease in cell proliferation and survival, we could reveal thatstress reversed the lactation-induced decrease in cell proliferation, while itdid not affect survival of newly born cells, nor the number of mature neu-rons , nor did it alter immature neuron production or the number of astro-glial cells in lactation. Stress exposure increased relative brain weight andhippocampal volume mirroring the observed changes in neurogenesis.Interestingly, hippocampal volume and relative brain weight were lower inlactation as compared to nulliparous females under nonstressed conditions.This study assessed the effect of stress during lactation on hippocampalneurogenesis and indicates that stress interferes with important peripartumadaptations at the level of the hippocampus. VC 2014 Wiley Periodicals, Inc.

KEY WORDS: lactation; hippocampal volume; neurons; differentia-tion; corticosterone

INTRODUCTION

Throughout all mammalian species, motherhood is characterized bynumerous neuroendocrine and behavioral adaptations. Among these, the

most obvious are lactogenesis and maternal behavior,including maternal care and aggression (Walker et al.,1995; Neumann, 2001; Russell et al., 2001; Hillereret al., 2012). However, in addition to changes directlyassociated with reproductive functioning, a host ofimportant behavioral and physiological alterationsoccur (Walker et al., 1995; Carter et al., 2001; Neu-mann, 2001, 2003), with decreased anxiety (Altemuset al., 1995; Carter et al., 2001; Heinrichs et al.,2001) [and see (Slattery and Neumann, 2008) forreview] and increased basal cortisol/corticosterone(CORT) levels amongst the most prominent. The lat-ter alteration is related to the presence of nursingpups (Stern et al., 1973; Walker et al., 1995) and hasbeen directly correlated with the concomitant decreasein hippocampal neurogenesis observed during the firstpostpartum weeks (Leuner et al., 2007).

In addition to the peripartum-associated hypercorti-cism/hypercortisolism, there are numerous alterationsthat act in concert to decrease the response of theHPA axis to external stressors (Altemus et al., 1995;Heinrichs et al., 2001; Brunton et al., 2008). Thisraises the intriguing possibility that stress during theperipartum period may affect neurogenesis in a differ-ent fashion to that observed in male and female (nul-liparous) rodents. Thus, it has repeatedly beendemonstrated that exposure to a variety of stressorsdecreases hippocampal cell proliferation and survivalin males, and cell survival in females (Gould et al.,1997, 1998; Lucassen et al., 2001; Pham et al., 2003;Hillerer et al., 2013). Such stress-related alterations inneurogenesis have been posited to play a crucial rolein the etiology of anxiety and depression (Fuchs,2007; Snyder et al., 2011). Moreover, there is a grow-ing body of evidence suggesting that antidepressantsmediate their effects, at least in part, by increasinghippocampal neurogenesis (Hanson et al., 2011;Snyder et al., 2011). In addition, numerous sex differ-ences in various aspects of hippocampal neurogenesis,under both basal and stress conditions, have beenreported (Galea et al., 2008; Hillerer et al., 2013).The peripartum period is a time of high susceptibilityfor mood and anxiety disorders (Robertson et al.,2004; Beck, 2006; Lonstein, 2007; Bridges, 2008)and stress during the peripartum period is one of themost prominent risk factors to develop postpartummood disorders (Robertson et al., 2004). Therefore, itmay be hypothesized that alterations in hippocampal

1 Department of Behavioural and Molecular Neurobiology, University ofRegensburg, Regensburg, Germany; 2 Department of Obstetrics andGynaecology, Salzburger Landeskrankenhaus (SALK), Paracelsus MedicalUniversity, Salzburg, Austria; 3 Institute of Experimental Neuroregenera-tion, Spinal Cord Injury and Tissue Regeneration Center Salzburg, Para-celsus Medical University, Salzburg, Austria; 4 Institute of MolecularRegenerative Medicine, Spinal Cord Injury and Tissue RegenerationCenter Salzburg, Paracelsus Medical University, Salzburg, AustriaGrant sponsor: the Deutsche Forschungsgemeinschaft; Grant number:DFG SL141/4-1; Grant sponsor: the European Union’s Seventh Frame-work Programme HEALTH-F2-2011-278850 (INMiND); Grant number:FP7/2007-2013; Grant sponsors: Bavarian State Ministry of Sciences,Research and the Arts, the state of Salzburg.*Correspondence to: Ludwig Aigner, Institute of Molecular RegenerativeMedicine, Spinal Cord Injury and Tissue Regeneration Center Salzburg,Paracelsus Medical University, Salzburg, Austria, Strubergasse 21, 5020Salzburg, Austria. E-mail: ludwig.aigner@pmu.ac.atAccepted for publication 31 January 2014.DOI 10.1002/hipo.22258Published online 00 Month 2014 in Wiley Online Library(wileyonlinelibrary.com).

VC 2014 WILEY PERIODICALS, INC.

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neurogenesis underlie some of the observed disturbances inpostpartum mood. However, there is a lack of evidence for theimpact of stress during lactation on postpartum hippocampalneurogenesis to date.

Therefore, the aim of our study was to determine the effectsof repeated restraint stress (RS) during the lactation period ondifferent aspects of hippocampal neurogenesis within the den-tate gyrus (DG). We evaluated the number of proliferating andsurviving cells, as well as neuronal and astroglial differentiationpatterns of the proliferating cells. Moreover, as lactation hasbeen shown to affect brain size in humans (Oatridge et al.,2002), we also evaluated absolute and relative brain weight, aswell as hippocampal volume. Finally, we assessed the effect ofstress on basal and acute-stress induced CORT levels across thestress period.

MATERIALS AND METHODS

Animals

Female Wistar rats (200–250 g; Charles River, Sulzfeld,Germany), housed in groups of four in standard polycarbon-ate rat cages under standard laboratory conditions (12 hlight/dark cycle, lights on at 06:00 h, 22 6 1�C, 60 6 5%humidity; free access to food and water) and allowed tohabituate for at least seven days were used in these studies.All experimental procedures were performed between 08:00and 12:00, approved by the Committee on Animal Healthand Care of the local government of the Oberpfalz, andcomplied with international guidelines on ethical use ofanimals.

Experimental Cohorts

A total of three different experimental cohorts were used forthe present studies. One cohort of animals (nulliparous n 5

18; lactating n 5 16) was used to analyze relative brain weight,hippocampal volume, hippocampal cell proliferation, andimmature neurons (Fig. 1A), one cohort (nulliparous n 5 15;lactating n 5 14) was used to analyze hippocampal cell survivaland cell fate, (Fig. 1B) and a final cohort of animals (lactatingnonstressed n 5 13; lactating stressed n 5 13) was used to assessplasma CORT levels.

Mating Procedure and Confirmation ofPregnancy

After habituation, all female rats were mated (two to threefemales to one male), and pregnancy was verified by vaginalsmears (designated pregnancy day (PD) 0). Nonpregnant ratswere assumed to be nulliparous. All animals were returned togroup cages with 4 females housed together (cage size 55 3

35 3 20 cm) until PD16 (or equivalent in nulliparous ani-mals), when they were single-housed. To rule out the possi-bility of pseudopregnancy in nulliparous females and to

confirm that these animals were normally cycling after mat-ing, vaginal smears were taken on days 1 and 3 after the endof the mating procedure. Females that were in proestrus onthe day of perfusion were excluded from the study to ruleout the possibility of estradiol-mediated changes in cell prolif-eration (Tanapat et al., 1999). On the day of birth, i.e., onlactation day (LD) 1, the number of pups in the litter andthe average birth weight were determined, and then all litterswere culled to 8 pups to ensure comparable conditions acrossall dams.

Stress Procedure

Animals of the stressed groups were subjected to 2 h RS for12 consecutive days (LD2–LD13, or equivalent in nulliparousanimals) between 10:00 and 12:00 A.M. RS has been repeat-edly shown to be an effective stressor in female rats (Darnau-dery et al., 2004; Hillerer et al., 2011, 2012) and to affectproliferation and survival rate of new hippocampal cells(Pham et al., 2003; Luo et al., 2005; Rosenbrock et al., 2005;Hillerer et al., 2013). Each rat was placed in a plexiglass col-umn with ventilation holes (12 cm diameter). Nonstressedcontrols were single-housed and left undisturbed in theirhome cages in the same room. Dams that were separated fromtheir pups, but not exposed to stress, were also included torule out an effect of pup separation on the parametersassessed. However, this group did not differ from nonstressedcontrols, which remained with their pups, in any parameter

FIGURE 1. Schematic representation of the temporal designsused in the present studies to assess (A) cell proliferation, imma-ture neuron production (DCX positive cells), brain weight, andhippocampal volume, and (B) cell survival and neuronal/astroglialdifferentiation. After 1 week of habituation, all females weremated. After birth, animals were exposed to 2 h of restraint stress(RS) for 12 consecutive days from (LD2 to LD13, or equivalent innulliparous animals). They received either (A) a single BrdU injec-tion (50 mg/kg; i.p.) on the last day of stress (LD13 or equivalentin nulliparous animals) or (B) four BrdU injections (50 mg/kg;i.p.) on four consecutive days (LD2-LD5 or equivalent in nullipar-ous animals/day 1–day 4 of stress exposure). Animals were intra-cardiacally perfused either (A) 24 h after (LD14 or equivalent innulliparous animals) or (B) 16 days after (LD21 or equivalent innulliparous animals) the last BrdU injection.

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(data not shown). The body weight of each animal wasrecorded daily from the beginning of the stress procedure untilthe day of sacrifice.

Nulliparous Females

To determine the effects of the reproductive status and stresson hippocampal neurogenesis in lactating females, the presentstudy was carried out in parallel with our recent study (Hillereret al., 2013). Therefore, the data on cell proliferation, cell sur-vival, and differentiation patterns from the nulliparous group isreplicated here in order to determine, whether the observedeffects were specific to the peripartum period or to females ingeneral.

Experiments 1 and 2: Assessmentof Hippocampal Cell Proliferation,Cell Survival, Brain Weightand Hippocampal Volume inNulliparous and Lactating Females

BrdU labeling

Proliferation: To examine proliferation of precursor cells, ratswere injected with 5-bromo-20-desoxyuridine (BrdU; 50 mg/kg, i.p; 0.9% NaCl solution; Sigma/Aldrich) once on the lastday of stress, immediately after removal from the restrainttubes, i.e., 12:00. After 24 h the animals were deeply anesthe-tized with ketamine/xylazine (90–120 mg/kg ketamine; 6–8mg /kg xylazin in 0.9% NaCl), intracardiacally perfused with4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4. Thistime-point has been reported to be sufficient for newborn cellsto complete one cell cycle (Takahashi et al., 1992).

Survival: To trace the survival and fate of recently born cells,rats received daily BrdU injections (50 mg/kg, i.p.) during thefirst four days of stress exposure (LD2–LD5; or equivalent innulliparous animals) as described above. Sixteen days after thelast BrdU injection (LD21, or equivalent day in nulliparousanimals) rats were anesthetized and perfused as describedabove. BrdU was freshly prepared in 0.9% NaCl solution to adilution of 20 mg/ml on each injection day (for a schematicrepresentation of the study design see Fig. 1).

Histological procedures

Immediately after perfusion, brains were weighed to deter-mine absolute and relative brain weights, postfixed in 4% para-formaldehyde at 4�C overnight, before transfer to 30%sucrose, 0.1M sodium phosphate solution (pH 7.4) in sterilewater for at least one week. Sagittal brain sections (40 mm)were prepared using a sliding microtome on dry ice, and subse-quently stored at 4�C in a cryoprotection solution [glycerol,ethylene glycol, and 0.1M phosphate buffer, pH 7.4, at a ratioof 1:1:2 by volume; (Kandasamy et al., 2010)]. Cresyl violetstaining was performed on free-floating sections to analyze hip-pocampal volume. Immunostaining of BrdU-labeled cells wasperformed on free-floating sections using the DAB peroxiodasemethod. Briefly, brain sections were treated with 0.6% H2O2

in Tris-buffered saline (TBS: 0.15M NaCl, 0.1M Tris-HCl, pH7.5) for 30 min. For DNA denaturation, sections were incu-bated for 2 h in 50% formaldehyde/23 saline-sodium citrate(SSC) (0.3M NaCl, 0.03M sodium citrate) at 65�C, rinsed for5 min in 23 SSC, incubated in 2M HCl for 30 min at 37�Cand washed for 10 min in 0.1M boric acid, pH 8.5. There-after, sections were incubated in FSGB for 1 h, followed byincubation with the primary rat a-BrdU antibody (1:500,Oxford Biotechnology, Oxford, UK) in FSGB overnight at4�C. The next day, the sections were incubated with biotinyl-ated secondary donkey a-rat antibody (1:500, MolecularProbes), followed by the avidin-biotin-peroxidase complex reac-tion [1 h; Vectastain elite ABC kit; Vector Laboratories, Burlin-game, CA (Kandasamy et al., 2010)]. Thereafter, the signalwas visualized using DAB (25 mg/ml in water with 0.01%H2O2, 0.04 NiCl2). Stained slides were mounted on micro-scopic slides, washed with NeoClear (Merck) and cover-slippedwith NeoMount (Merck). Immunostaining of DCX-labeledcells was performed on free-floating sections as described earlier(antibodies used: 1� AB goat a DCX, 1:250 in FSGB [SantaCruz Biotechology; Santa Cruz, CA); 2� AB biotinylated don-key a goat (Molecular Probes); (Kandasamy et al., 2010)].Triple-immunfluorescence for BrdU/GFAP/NeuN was per-formed using a standardized protocol (Kandasamy et al.,2010). Briefly, free floating brain sections were incubated in50% formaldehyde/23 saline-sodium citrate (SSC) (0.3MNaCl, 0.03M sodium citrate) at 65�C for 1 h, rinsed for 5min in 23 SSC, incubated in 2M HCl for 30 min at 37�Cand washed for 10 min in 0.1M boric acid, pH 8.5. There-after, sections were washed four times in TBS for 5 min, beforethey were incubated with FSGB for 30 min, followed by incu-bation with the antibody mix (BrdU a rat, 1:500,Oxford Bio-technology, Oxford, UK; GFAP a guinea pig, 1:500, Progen;NeuN a mouse, 1:500, Chemicon) in FSGB for 48 h at 4�C.Forty-eight hours later, the sections were incubated overnightwith the secondary antibody mix (donkey a rat conjugatedwith rhodamine red, 1.500; donkey a guinea pig conjugatedwith IgG Cy5, 1:500; donkey a mouse conjugated with AlexaFluor 488, 1.500) in FSGB, shaking in the dark. Stained slideswere mounted on microscopic slides and cover-slipped withProlong-Antifade (Molecular Probes).

Stereology

To analyze hippocampal volume, pictures were taken at a103 magnification and volume was assessed using polygonselection measurement in ImageJ. To determine the number ofBrdU-positive cells, every sixth section (240 mm interval) ofthe right hemisphere was examined for BrdU-positive cellsthroughout the rostral-caudal extent of the granule cell layerand the adjacent SGZ. Cells were counted regardless of shapeor size under 1003 magnification. A semiautomatic stereologysystem (Stereoinvestigator, MicroBrightField) and a 53 objec-tive to trace a defined area of the DG/SGZ were employed.The defined area was used to calculate the number of BrdU-positive cells per 100 mm2 of the DG.

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Confocal analysis

All morphological analyses were performed by an experi-menter blind to the group. To determine the frequency of neuro-nal differentiation of newborn cells, a series was examined usinga grid confocal laser microscope (Olympus XI81) using everysixth section (240 mm interval). Z-stacks were built using theVolocity Software (Perkin Elmer). 50 BrdU-positive labeled cellsper animal were analyzed for neuronal differentiation. BrdU-positive cells were counted as solely BrdU-positive (newborncells), BrdU/ NeuN (newborn neurons) double-positive cells,and BrdU/GFAP (newborn astrocytes) double-positive cells.

Experiment 3: Effect of RS Exposure on PlasmaCORT Levels

As CORT levels have been shown to be an important regulatorof neurogenesis (Magarinos and McEwen, 1995; Galea et al.,2008; Barha et al., 2011), we measured plasma CORT levelsunder basal and stress conditions. Therefore, a jugular vein surgerywas performed on PD19—as previously described (Neumannet al., 1998; Hillerer et al., 2013). Briefly, the jugular vein wasexposed by blunt dissection, then a catheter consisting of silicontubing (Dow Corning, Midland, MI) and PE-50 polyethylenetubing was inserted �3 cm into the vessel through an incision in acardiac direction and exteriorized at the neck of the animal. Thecatheter was filled with sterile saline containing gentamicin(30,000 IU/ml; Centravet, Bad Bentheim, Germany). Five daysafter surgery, on the first day of stress procedure (LD2), and onLD4 and LD6, at 07:30, the catheter was attached to an extensiontube connected to a 1 ml plastic syringe filled with sterilized hep-arinized 0.9% saline (30 IU/ml, Heparin-Natrium, Ratiopharm,Ulm, Germany). Each rat was then left undisturbed for 2 h. Twobasal samples (basal sample one: 0.6 ml and basal sample two: 0.2ml) were taken 30 min apart and were used to calculate the meanbasal concentrations for CORT. Immediately after the basal sam-pling, rats were placed into the restraint tubes and additionalblood samples were taken 30 and 90 min after start of the stressprocedure. After 2 h, rats were removed from the restraint tubesand returned to their home-cage. All blood samples were immedi-

ately replaced with the same volume of intravenous sterile 0.9%saline. All blood samples were collected on ice in EDTA-tubescontaining aprotinin (Trasylol, Bayer AG, Leverkusen, Germany)and analyzed for CORT using a commercially available ELISA kit(DRG Instruments GmbH, Marburg, Germany).

Statistical Analysis

All data are expressed as mean 6 SEM and were statisticallyanalyzed using either an unpaired Student’s t-test, a Mann Whit-ney U test, or a one- or two- way analysis of variance (ANOVA)with or without repeated measures, as appropriate. ANOVAs werecalculated separately for number of cells in the DG with group(nulliparous/lactating and control/stress) as between-subject fac-tors. Any statistical differences, which were set at P < 0.05, werefurther analyzed using a Fisher’s post-hoc test. Statistical analyseswere performed using SPSS for Windows (version 16; SPSS, Chi-cago, IL); overall statistics are shown in Table 1. Because of techni-cal issues some sections could not be analyzed (proliferation andimmature neurons: one nulliparous nonstressed animal, one lactat-ing nonstressed, and three lactating stressed animals; survival: twolactating stressed animals; neuronal/astroglial differentiation: twonulliparous nonstressed animals).

RESULTS

Stress Reverses Lactation-AssociatedHippocampus Volume Shrinkage (Experiment 1)

To assess the effects of the reproductive status and stress on rela-tive brain weight and on hippocampal volume animals were sacri-ficed after 12 days of RS on LD13 (or equivalent in nulliparousanimals; see Fig. 1A). Lactating animals showed decreased absolute(data not shown) and relative brain weight (Fig. 2A) under bothbasal and RS conditions when compared with nulliparous animals(Fig. 2A; P< 0.01). However, RS animals, either nulliparous or lac-tating, showed an increase in relative brain weight compared withnonstressed rats (Fig. 2A; P< 0.05). An even more pronouncedeffect was present at the level of the hippocampus volume.

TABLE 1.

Overall statistical effects for Figures 2, 3, 4, and 5

Status effect Stress effect Status 3 Stress effect

Absolute brain weight F(1,28) 5 10.81, P< 0.001*

Relative brain weight (Fig. 2A) F(1,28) 5 84.83, P< 0.0001* F(1,28) 5 5.31, P< 0.01*

Hippocampal volume (Fig. 2B) F(1,27)5 29.72, P <0.0001*

Cell proliferation (Fig. 2D) F(1,23) 5 26.45, P< 0.0001* F(1,23) 5 7.40, P< 0.05*

Immature neurons (Fig. 3A) F(1,24) 5 0.46, P 5 0.505 F(1,24) 5 0.13, P 5 0.723

Cell survival (Fig. 3B) F(1,23) 5 4.47, P< 0.05*

Neuronal differentiation (Fig. 4A) F(1,23) 5 0.001, P 5 0.972 F(1,23) 5 11.54, P< 0.01*

Astroglial differentiation (Fig. 4B) F(1,23) 5 2.32, P 5 0.142 F(1,23) 5 2.32, P 5 0.142

Corticosterone levels (Fig. 5) F(1,28) 5 9.04, P< 0.01*

One- or two-way analysis of variance (ANOVA) with or without repeated measures was applied as appropriate followed by Fisher’s post hoc test. *P < 0.05.

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Compared to nulliparous animals, lactating animals showed adecrease in hippocampal volume under basal conditions (Figs.2B,C; P< 0.001). Whereas repeated stress led to a decrease in hip-pocampal volume in nulliparous animals (Fig. 2B; P< 0.01), it ledto an increase in the lactating group (Fig. 2B; P< 0.001), resultingin comparable hippocampus volumes between nulliparous non-stressed animals and lactating stressed dams. Figure 2C shows a rep-resentative image of a cresyl violet staining [lactating stressed animal(a) and lactating nonstressed animal (b)].

Repeated RS Exposure Elevates CellProliferation in the DG SGZ During Lactation,But Does Not Affect the Number ofDoublecortin Expressing Cells (Experiment 1)

To assess the effects of the reproductive status and stress oncell proliferation animals were given a single BrdU injection

after 12 days of RS (LD13 or equivalent in nulliparous ani-mals) and were perfused 24 h later (Fig. 1A). Lactating animalsshowed a decreased number of BrdU-positive cells both underbasal and repeated stress conditions (Fig. 3A; P< 0.05). How-ever, stressed lactating animals showed an increased prolifera-tion rate as compared to nonstressed dams (Fig. 3A; P< 0.01).

Next we questioned if the stress-associated effects on hippo-campal cell proliferation in lactating animals translated into dif-ferent numbers of immature neurons by assessing the number ofDCX positive cells in the DG. The analysis revealed no signifi-cant differences in the number of DCX positive cells (Fig. 3B).However, the morphology of DCX cells in the stressed groupappeared to be less mature as compared to nonstressed animals,as indicated by a less elaborated dendrite arborisation. Figure 3Cshows a representative image of a DAB-immunostaining (lactat-ing nonstressed animal (I) and lactating stressed animal (II))against DCX.

FIGURE 2. Effect of reproductive status and stress on relativebrain weight and hippocampal volume. The relative brain weight(A) (experimental timeline see Fig. 1A) and the hippocampal vol-ume (B) (experimental timeline see Fig. 1A) was assessed in theDG of the hippocampus in nulliparous and lactating femalesunder basal (gray bars) or repeated stress (black bars) conditions.(A) Lactating animals showed a lower relative brain weight underbasal conditions; stress during lactation led to an increase in rela-tive brain weight, when compared with the respective nonstressedgroup. (B) Analysis of hippocampal volume revealed a reducedhippocampal volume in lactating animals under basal conditions;stress led to a reduced volume in nulliparous animals, whereas it

had the opposite effect in lactating animals. (C) Representativeimage of the Cresyl violet staining for the analysis of hippocampalvolume (a) nulliparous nonstressed animal; black dottedarrow 5 242 mm, white dotted arrow 5 466 mm, (b) lactating non-stressed animals; black dotted arrow 5 230 mm, white dottedarrow 5 420 mm). Data represent mean 6 SEM with the numbersin parentheses representing the group sizes. *P < 0.05 vs. respectivenonstressed group; **P < 0.01 vs. respective nonstressed group;***P < 0.001 vs. respective nonstressed group; ##P < 0.01 vs.respective nulliparous group; ###P < 0.001 vs. respective nullipar-ous group; RS, restraint stress. [Color figure can be viewed in theonline issue, which is available at wileyonlinelibrary.com.]

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Reproductive Status and Repeated RS Modulatethe Fate of Newly Born Cells in the DG(Experiment 2)

We next assessed the effect of reproductive status and stresson the fate of newly born cells in the DG. Therefore, animalsreceived BrdU injections on the first 4 days of stress (LD2–LD5 or equivalent in nulliparous and nonstressed groups) andwere perfused 16 days later on LD21 (or equivalent in nulli-parous animals; see Fig. 1B). The quantitative analysis revealeda reduction in the number of BrdU-positive cells in nonstressedand stressed lactating animals and in stressed nulliparous ani-mals as compared to the nonstressed nulliparous animals (Fig.4A; P< 0.01) (Fig. 4A; P> 0.05).

To evaluate if nulliparous and lactating animals vary in theirastroglial and neuronal differentiation patterns under basal and RS

conditions the hippocampus was analyzed for cells double-positivefor BrdU and NeuN for neuronal differentiation, or BrdU andGFAP for astroglial differentiation. Although there was no effectof reproductive status on astroglial or neuronal differentiation,nulliparous, and lactating rats that were exposed to RS showed areduction in neuronal differentiation (Fig. 4B; P< 0.05 vs. respec-tive non-stress groups), astroglial differentiation patterns wereunchanged by RS exposure (Fig. 4C). Figures 4D–F shows repre-sentative images of BrdU/NeuN/GFAP triple labeling of the DG(4D: BrdU; 4E: NeuN; 4F: GFAP; 4G: merge).

Repeated RS Leads to an Increase in BasalCORT Levels on LD6 (Experiment 3)

To assess the effect of stress during lactation on CORT lev-els, basal blood samples were taken immediately prior to stress

FIGURE 3. Effect of reproductive status and stress on cell pro-liferation and immature neuron production in the DG. The totalnumber of proliferating cells (A) (experimental timeline see Fig.1A) and the number of immature neurons (DCX1 cells). (B)(experimental timeline see Fig. 1A) was assessed in the DG of thehippocampus in nulliparous and lactating females under basal(gray bars) and stress (black bars) conditions. (A) Lactating ani-mals showed a lower number of proliferating cells, both underbasal and repeated stress conditions; stress during lactation led to

an increase in cell proliferation when compared with the respectivenonstressed group; (B) There was no effect of reproductive statusor stress on immature neuron production; (C) Representativeimage of a DAB-immunostaining against DCX (lactating non-stressed animal (I) and lactating stressed animal (II)), scalebar 5 50 mm. Data represent mean 6 SEM with the numbers inparentheses representing the group sizes. **P < 0.01 vs. respectivenonstressed group; #P < 0.05 vs. respective nulliparous group;###P < 0.001 vs. respective nulliparous group; RS, restraint stress.

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exposure on LD2, LD4, and LD6 in separate groups of lactat-ing rats. Two-way repeated-measures ANOVA revealed a signif-icant interaction effect of day and stress (F1,28 5 9.042;P 5 0.006). Post hoc analysis revealed higher basal CORT levelsin RS dams on LD6 as compared with nonstressed dams(P< 0.01; Fig. 5). Acute stress-induced CORT levels did notsignificantly differ in between groups (data not shown).

DISCUSSION

This study provides the first evidence that hippocampal mor-phology and distinct stages of adult hippocampal neurogenesisare affected by stress in lactation. Lactating rats showed a

decrease in absolute and relative brain weight during mid-lactation, which reflects previously published MR studies inhumans revealing reduced brain size during the peripartumperiod (Oatridge et al., 2002). The observed lactation-associated effect on brain weight was reversed repeated stressexposure. Changes in brain weight were also reflected in hippo-campal volume and hippocampal cell proliferation. As previ-ously reported, lactating dams showed a decrease in cellproliferation in the DG of the hippocampus as compared withnulliparous females (Darnaudery et al., 2007; Leuner et al.,2007), which was reversed by stress exposure. Stressed damsdisplayed a lower number of dividing cells during the first daysof lactation and survived for approximately two weeks, as wellas a reduced neuronal differentiation, whereas immature neu-ron production and astroglial differentiation were unaffected

FIGURE 4. Effect of reproductive status and stress on cell sur-vival and differentiation patterns in the DG. The number of sur-viving cells (A) (experimental timeline see Fig. 1B), the percentageof cells differentiating into neurons (BrdU positive, GFAP nega-tive, and NeuN positive cells) (B) (experimental timeline see Fig.1B) and astroglial cells (BrdU positive, GFAP positive, NeuN neg-ative cells) (C) (experimental timeline see Fig. 1B) was assessed inthe DG of the hippocampus in nulliparous and lactating femalesunder basal (gray bars) or stress (black bars) conditions. (A) Lac-tating animals showed a reduction in cell survival under nonstressconditions; stress led to a reduction in cell survival in nulliparous,but not lactating animals. (B) Repeated stress reduced neuronal

differentiation in nulliparous and lactating females, no basal dif-ferences were observed between nulliparous and lactating animals.(C) There was no effect of the reproductive status or stress onastroglial differentiation patterns. (D–G) Representative images ofthe triple-immunfluorescent image (lactating nonstressed animal)with antibodies against BrdU (4D) (red), GFAP (4E) (violet),NeuN (4F) (green), and merge (4G), scale bar 5 50 mm. Data rep-resent mean 6 SEM with the numbers in parentheses representingthe group sizes. *P < 0.01 vs. respective nonstressed group;##P < 0.01 vs. respective nulliparous group. RS, restraint stress.[Color figure can be viewed in the online issue, which is availableat wileyonlinelibrary.com.]

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by RS in lactation. Moreover, the results of the present studyreveal that basal CORT levels are only partly involved in theregulation of adult hippocampal neurogenesis under stress con-ditions during lactation, which further suggests that fluctua-tions in other hormones that occur classically during theperipartum period may play a major role, such as prolactin(Torner et al., 2009). Collectively, these findings highlight thatrepeated stress during lactation has detrimental effects on phys-iologically occurring postpartum-associated adaptations at thelevel of the hippocampus.

Repeated RS During Lactation Prevents theLactation-Associated Decrease in HippocampalVolume and Cell Proliferation and Leads to aDecrease in Cell Survival

The brain, and more specifically hippocampal morphology,undergo a postpartum reorganization (Galea et al., 2000;Oatridge et al., 2002), which includes dendritic pruning ofCA3 and CA1 pyramidal neurons of the hippocampus at thetime of weaning, as well as an increase in spine density in theseregions during pregnancy and lactation (Pawluski and Galea,2006; Kinsley et al., 2006). Interestingly, we were able to showa reduction in hippocampal volume during lactation, whichmirrors the decrease in hippocampal cell proliferation and cellsurvival seen in our study; as well as in other previous findings(Darnaudery et al., 2007; Leuner et al., 2007; Pawluski andGalea, 2007). The aforementioned changes in hippocampalneurogenesis have been shown to be related to the lactationperiod and are associated with the hypercorticism observedduring that time (Leuner et al., 2007). Interestingly, we couldshow that the lactation-associated decrease in hippocampal vol-

ume and cell proliferation was prevented by stress exposure.Despite these reversals, repeated RS actually led to a decreasein cell survival, as well as an increase in basal CORT levels oneday after the last BrdU injection. These results may initiallyseem paradoxical, as psychosocial and physical stress are com-monly thought to impair adult hippocampal neurogenesis(Gould et al., 1997; Czeh et al., 2002; Pham et al., 2003;Heine et al., 2005), mainly due to an effect of increased basalCORT levels (Gould et al., 1992; Cameron and Gould, 1994;Tanapat et al., 2001). However, these studies have been per-formed in males, whereas the general consensus of studies infemales is that stress reduces cell survival (Kuipers et al., 2006;Hillerer et al., 2013) either without affecting the number ofproliferating cells (Falconer and Galea, 2003; Westenbroeket al., 2004; Hillerer et al., 2013), or increasing the number ofproliferating cells in virgin or pregnant females (Pawluski et al.,2011). Thus, it appears that males and females react differen-tially to elevated CORT levels and stress situations in general.Additionally, it has been shown that the DG proliferation ratemay habituate to stress exposure and, thus, display a reducedsensitivity to HPA axis hormones (Czeh et al., 2002). Despitethe literature supporting a link between a reduction in adulthippocampal neurogenesis and high basal CORT levels duringstress and lactation, only a small percentage of precursor cellsexpress CORT receptors (Cameron et al., 1993a). In addition,a decrease in hippocamapal [3H]-Dexamethasone bindingcapacity (Meaney et al., 1989), as well as reduced coriticoste-roid binding globulin levels (Pawluski et al., 2009), during lac-tation might play a regulatory role in this context. Anotherconsideration is that there might be a shift from CORTtowards other regulatory mechanisms playing a predominantrole during lactation stress. Given the fact that repeated stressduring lactation led to an increase in active (i.e., arched backnursing) behavior (data not shown) in the 2 h after stress andpup exposure has been shown to increase hippocampal cellproliferation in nulliparous rats (Pawluski and Galea, 2007) itis possible that maternal behavior itself might regulate hippo-campal cell proliferation in stressed dams. Further, the genera-tion and removal of cells is delicately balanced andincorporation of newborn cells above a critical set-point mightalso be maladaptive as observed following epileptic seizures(Varodayan et al., 2009). It is tempting to speculate that a sim-ilar situation may apply to the lactation period, as the normaldecrease in cell proliferation may be involved in the reducedacute stress responsiveness observed at this time (Neumann,2001; Lonstein, 2007). Therefore, increased cell proliferationas seen in stressed lactating animals may have long-term conse-quences on HPA axis activity.

In addition, closer inspection of hippocampal cell prolifera-tion and survival in both lactation groups revealed that ahigher percentage of cells died in stressed dams over the dis-tinct time course assessed as compared with nonstressed dams.This suggests that the lactation period, under normal circum-stances, is associated with a greater efficiency in neurogenicprocesses, as fewer proliferating cells are required to result inthe same net cell survival as occurs in nulliparous females/

FIGURE 5. Effect of reproductive status and stress on plasmaCORT levels. Plasma CORT levels on LD2, LD4, and LD6 wereassessed immediately prior to stress exposure. Stressed damsshowed higher basal CORT levels on LD6 when compared withthe nonstressed dams. Data represent mean 6 SEM with the num-bers in parentheses representing the group sizes. **P < 0.01 vs.respective nonstressed group. NS, nonstressed; S, stressed.

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males, which is in accordance with previous studies (Cameronet al., 1993b; Tanapat et al., 2001). In contrast, increased celldeath occurred in nulliparous animals despite no changes incell proliferation. Thus, increased efficiency of neurogenesisduring the lactation period might be an evolutionary adapta-tion, as the generation of new cells is a highly metabolic activ-ity and the dam requires high metabolic resources tosuccessfully raise her offspring. Furthermore, the observedresults indicate that either apoptotic processes of progenitorcells and/or cell cycle length differs between the two lactatinggroups. The discrepancy between cell proliferation and survivalin the stressed dams could indicate increased apoptosis ofnewly generated cells and, thereby, suggest a compensatoryincrease in cell proliferation as a result of an increased celldeath of older cells after stress exposure (Gould et al., 1991;Lucassen et al., 2001; Abrous et al., 2005).

Another possible explanation for the differences observedbetween stressed and nonstressed dams is an alteration in cellcycle length, as speculated above. Interestingly, increased cellproliferation in epilepsy is not only associated with an increasein apoptotic rate, but, moreover, with a shortened cell cyclelength (Varodayan et al., 2009). Therefore, it can be hypothe-sized that the turnover of the DG is accelerated by an increaseddivision rate in stressed dams. In this regard, oestrogen couldplay an important role, as it decreases in lactation from highlevels throughout pregnancy and stimulates a population ofDG granule cells to divide faster, by regulation of the G1/S-phase transmission of the cell cycle (Geum et al., 1997; Honget al., 1998). Therefore, repeated stress may maintain high oes-trogen levels in the dams, which, at least in part, would explainthe observed changes in hippocampal cell proliferation and sur-vival. Future studies will, therefore, assess apoptotic rates andcell cycle length by use of specific markers (i.e., Ki-67), as wellas oestrogen levels, to gain better insight in factors regulatingcell proliferation and survival under stress conditions duringlactation.

Repeated RS During Lactation Does Not AffectImmature Neuron Production, But Leads to anAlteration in Astroglial and NeuronalDifferentiation Patterns

In addition to proliferation and survival, selection, differen-tiation, and integration of adult generated cells into the cir-cuitry are important neurogenic components (Lledo et al.,2006). Disruption of these processes by environmental changesor stress exposure may cause a lack of cell populations withspecific, temporally-necessary, properties in hippocampal func-tion (Abrous et al., 2005). Although immature neuron produc-tion during lactation was found to be decreased in a previousstudy (Leuner et al., 2007), we could not replicate this in ourstudy. However, the discrepancy may be explained by the useof different immature neuronal lineage markers (TuJ1 in theformer and DCX in ours). In agreement with previous findingsin male and nulliparous rats (Pham et al., 2003; Hillerer et al.,2013), stress during lactation did not affect neuronal progeni-

tor cells. Again, these results may initially appear to differ fromthe cell proliferation and survival results. However, upon closerinspection, despite not affecting the number of immature neu-rons, repeated stress appears to affect dendritic morphology ofimmature neurons; particularly in stressed dams (Figs. 3C/Iand II). In more detail, we observed distraction of dendriticprocesses, including a lack of orientation towards the hilus ofthe DG. This is an interesting observation, as dendritic mor-phology and spine density has already been shown to beincreased during the peripartum period in the prefrontal cortexand hippocampus (Kinsley et al., 2006; Leuner and Gould,2010) and given the fact that immature neurons play animportant role as pattern integrators and pattern separation[for review see (Aimone et al., 2010, #70)]. Therefore, futurestudies should include a more detailed analysis of immatureneurons in the DG of the hippocampus. Aside from theobserved changes in dendritic morphology of immature neu-rons, we also observed a trend towards a decrease in immatureneuron production, which might facilitate the interpretation ofour results. With respect to the hypothesized stress-inducedincrease in apoptosis, it seems feasible that the unchangedimmature neuron production in stressed dams mirrors the shiftfrom the increased proliferation towards decreased survival, asalso occurred in nulliparous females. This becomes even moreevident when considering the neuronal and astroglial differen-tiation patterns. Although neuronal differentiation wasunchanged during the lactation period, stress exposure led to areduction in mature neurons with a concurrent trend towardsan increase in mature astroglial cells. These results reveal thatDG cells that are engaged in apoptotic processes in the post-partum stress group are solely neurons. The addition of newneurons that reach maturity is thought to be of importance inrelation to motherhood, especially in the context of maternalbonding and care (Gandelmann et al., 1971; Santarelli et al.,2003; Leuner and Shors, 2006) and vice versa (Furuta andBridges, 2009). Therefore, alterations in the number of neu-rons within the hippocampus might both directly and indi-rectly via DG pathways to other brain regions involved inmaternal behavior contribute to changes in maternal care andmaternal anxiety, as seen after stress exposure during the peri-partum period (Maestripieri and D’Amato, 1991; Hillereret al., 2011; Purcell et al., 2011).

CONCLUSIONS

The results of the present study reveal important consequen-ces of repeated stress exposure during lactation on maternalbrain adaptations and particularly on different stages of adulthippocampal neurogenesis in the DG. The observed changessuggest that stress prevents or reverses the physiologicallyreduced proliferation and increased efficiency of neurogenesisin the peripartum period. These stress-induced changes in neu-rogenesis are likely to be mediated, at least in part, via

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increased apoptosis or a shortened cell cycle length, given thereduced neuronal differentiation observed in the stressed group.They may further play a role in the behavioral and neuroendo-crine adaptations observed following lactation stress exposure.However, further research is required to elucidate specific regu-latory mechanisms of cell proliferation, selection, differentia-tion, and integration of newly generated cells, which are alteredby repeated stress. This study reveals that repeated postpartumstress exerts a variety of negative effects on the maternal brainand particularly in specific stages of hippocampal neurogenesisthat might underlie the increased risk for depressive disordersduring that susceptible time.

Acknowledgments

The authors thank Dr. Mahesh Kandasamy for support ofstaining procedures, and Rodrigue Maloumby, Andrea Havasi,and Gabriele Schindler (University of Regensburg) for technicalassistance.

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