Gender differences in the long-term effects of chronic prenatal stress on the HPA axis and hypothalamic structure in rats.
ABSTRACT Stress during pregnancy can impair biological and behavioral responses in the adult offspring and some of these effects are associated with structural changes in specific brain regions. Furthermore, these outcomes can vary according to strain, gender, and type and duration of the maternal stress. Indeed, early stress can induce sexually dimorphic long-term effects on diverse endocrine axes, including subsequent responses to stress. However, whether hypothalamic structural modifications are associated with these endocrine disruptions has not been reported. Thus, we examined the gender differences in the long-term effects of prenatal and adult immobilization stress on the hypothalamic-pituitary-adrenocortical (HPA) axis and the associated changes in hypothalamic structural proteins. Pregnant Wistar rats were subjected to immobilization stress three times daily (45 min each) during the last week of gestation. One half of the offspring were subjected to the same regimen of stress on 10 consecutive days starting at postnatal day (PND) 90. At sacrifice (PND 180), serum corticosterone levels were significantly higher in females compared to males and increased significantly in females subjected to both stresses with no change in males. Prenatal stress increased pituitary ACTH content in males, with no effect in females. Hypothalamic CRH mRNA levels were significantly increased by prenatal stress in females, but decreased in male rats. In females neither stress affected hypothalamic cell death, as determined by cytoplasmic histone-associated DNA fragment levels or proliferation, determined by proliferating cell nuclear antigen levels (PCNA); however, in males there was a significant decrease in cell death in response to prenatal stress and a decrease in PCNA levels with both prenatal and adult stress. In all groups BrdU immunoreactivity colocalized in glial fibrillary acidic protein (GFAP) positive cells, with few BrdU/NeuN labelled cells found. Furthermore, in males the astrocyte marker S100β increased with prenatal stress and decreased with adult stress, suggesting affectation of astrocytes. Synapsin-1 levels were increased by adult stress in females and by prenatal stress in males, while, PSD95 levels were increased in females and decreased in males by both prenatal and adult stress. In conclusion, hypothalamic structural rearrangement appears to be involved in the long-term endocrine outcomes observed after both chronic prenatal and adult stresses. Furthermore, many of these changes are not only different between males and females, but opposite, which could underlie the gender differences in the long-term sequelae of chronic stress, including subsequent responses to stress.
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Gender differences in the long-term effects of chronic
prenatal stress on the HPA axis and hypothalamic
structure in rats
Cristina Garcı ´a-Ca ´ceresa, Natalia Lagunasb, Isabel Calmarza-Fontb,
In ˜igo Azcoitiac, Yolanda Diz-Chavesb, Luis M. Garcı ´a-Segurab,
Eva Baquedanoa, Laura M. Fragoa, Jesu ´s Argentea, Julie A. Chowena,*
aHospital Infantil Universitario Nin ˜o Jesu ´s, Universidad Auto ´noma de Madrid, CIBER Fisiopatologı ´a de Obesidad y Nutricio ´n
(CIBERobn), Instituto de Salud Carlos III, Madrid 28009, Spain
bInstituto Cajal, CSIC. Avenida Dr. Arce 37, Madrid 28002, Spain
cDepartamento de Biologı ´a Celular, Facultad de Biologı ´a, Universidad Complutense de Madrid, Madrid 28040, Spain
Received 22 December 2009; received in revised form 19 April 2010; accepted 20 May 2010
Psychoneuroendocrinology (2010) 35, 1525—1535
offspring and some of these effects are associated with structural changes in specific brain
of the maternal stress. Indeed, early stress can induce sexually dimorphic long-term effects on
diverse endocrine axes, including subsequent responses to stress. However, whether hypotha-
lamic structural modifications are associated with these endocrine disruptions has not been
reported. Thus, we examined the gender differences in the long-term effects of prenatal and
adult immobilization stress on the hypothalamic—pituitary—adrenocortical (HPA) axis and the
associated changes in hypothalamic structural proteins. Pregnant Wistar rats were subjected to
immobilization stress three times daily (45 min each) during the last week of gestation. One half
of the offspring were subjected to the same regimen of stress on 10 consecutive days starting at
postnatal day (PND) 90. At sacrifice (PND 180), serum corticosterone levels were significantly
higher in females compared to males and increased significantly in females subjected to both
stresses with no change in males. Prenatal stress increased pituitary ACTH content in males, with
no effect in females. Hypothalamic CRH mRNA levels were significantly increased by prenatal
stressinfemales, butdecreased inmalerats.Infemales neitherstressaffected hypothalamic cell
death, as determined by cytoplasmic histone-associated DNA fragment levels or proliferation,
determined by proliferating cell nuclear antigen levels (PCNA); however, in males there was a
significant decrease in cell death in response toprenatal stressand a decrease in PCNA levels with
Stress during pregnancy can impair biological and behavioral responses in the adult
* Corresponding author at: Servicio de Endocrinologı ´a, Hospital Infantil Universitario Nin ˜o Jesu ´s, Avenida Mene ´ndez Pelayo 65, Madrid 28009,
Spain. Tel.: +34 91 503 5939; fax: +34 91 503 5939.
E-mail address: email@example.com (J.A. Chowen).
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/psyneuen
0306-4530/$ — see front matter # 2010 Elsevier Ltd. All rights reserved.
Author's personal copy
Exposure of pregnant humans or animals to chronic stress
during critical periods of fetal brain development may raise
the risk of depression, attention and learning deficits, hormo-
nal imbalances and metabolic disorders in the offspring, some
of which have been associated with structural alterations in
the brain (Schneider, 1992; Van Os and Selten, 1998; Geddes,
1999; Rhees et al., 1999a; Weinstock, 2001; Wadhwa et al.,
2001; Linnet et al., 2003; Maccari et al., 2003; Walker, 2005;
Abe et al., 2007; Yaka et al., 2007; Bogoch et al., 2007).
Indeed, behavioral disturbances produced by prenatal stress
offspring (Lemaireetal.,2000;Koo etal.,2003;VandenHove
long-term behavioral alterations caused by prenatal stress, as
well as the structural alterations in brain areas controlling
behavior, are gender specific (Reznikov et al., 1999; Rhees
et al., 1999a; Bowman et al., 2004; Tobe et al., 2005; Wein-
stock, 2007; Mueller and Bale, 2008; Zuena et al., 2008).
Prenatal stress also has long-term effects on distinct
endocrine axes (Kofman, 2002; Mairesse et al., 2008; Mueller
and Bale, 2008), but whether modifications in hypothalamic
structures are involved in this process remains largely
unknown. Moreover, some of these endocrine alterations in
response to early stress are sexually dimorphic (Horst et al.,
2009; Garcı ´a-Ca ´ceres et al., 2010). As normal hypothalamic
development differs between males and females, resulting in
sexually dimorphic hypothalamic structures and functions
(Ward, 1972; Rhees et al., 1999a,b), it follows that environ-
mental challenges or changes during this critical period may
differentially affect each gender. Hence, it is conceivable
that maternal and early stresses not only induce structural
changes in the hypothalamus, but that these effects are
sexually dimorphic and this could contribute to the gender
differences in endocrine outcomes.
the possible structural alterations in the hypothalamus in
response to chronic stress or whether these changes are
the effect of maternal stress on hypothalamic cell turnover
and synaptic density in the adult offspring, (2) determine
whether these structural changes are different between
males and females, (3) examine whether exposure to a
second chronic stress during adulthood results in long-lasting
hormonal and hypothalamic alterations and if exposure to
chronic prenatal stress modulates this response and (4)
determine whether the long-term response of the hypotha-
lamic—pituitary—adrenal axis to maternal stress, which is
different between males andfemales, can becorrelated with
structural changes in the hypothalamus.
2. Materials and methods
All chemicals and reagents were purchased from Sigma Che-
mical Co. (St. Louis, MO) or Merck (Barcelona, Spain) unless
All experiments were designed according to the European
Union laws foranimal care and thestudy wasapproved by the
local institutional Ethical Committee. Young adult pregnant
Wistar rats were housed individually under alternate light
(12 h)—dark (12 h) periods and allowed free access to rat
chow and tap water.
2.3. Experimental design
Prenatal restraint stress was performed daily in pregnant rats
during the last week of gestation (gestational days 14—21) by
placing them in transparent plastic cylinders (7 cm inner
diameter, 19 cm long) along with bright light exposition,
for 45 min, three times a day, as previously described (Ward
and Weisz, 1980). Female rats from the control group
remained undisturbed in their home cage.
At birth pups were housed with their mother with no
handling of either the pups or the mothers until postnatal
day 21 (P21) at which time they were weaned. Only litters of
9—14 pups were employed in the study. At P21, pups were
distributed (four/cage) according to origin from control or
stressed dams, with males and females being housed sepa-
rately. At approximately P90, 9—10 animals from each of the
two groups (control; C or prenatally stressed; PnS) for both
sexes were subjected to adult stress (AS). Female rats were
by daily vaginal swab). Adult stress was performed by using a
similar protocol tothat describedforprenatalstress, butover
10 days. Either 2 or 14 days before sacrifice rats received i.p.
injections of 5-bromo-20-deoxyuridine (BrdU; Sigma—Aldrich,
at a concentration of 10 mg/ml in sterile saline).
both prenatal and adult stress. In all groups BrdU immunoreactivity colocalized in glial fibrillary
acidic protein (GFAP) positive cells, with few BrdU/NeuN labelled cells found. Furthermore, in males
the astrocyte marker S100b increased with prenatal stress and decreased with adult stress,
suggesting affectation of astrocytes. Synapsin-1 levels were increased by adult stress in females
and by prenatal stress in males, while, PSD95 levels were increased in females and decreased in
males by both prenatal and adult stress. In conclusion, hypothalamic structural rearrangement
appears to be involved in the long-term endocrine outcomes observed after both chronic prenatal
and adult stresses. Furthermore, many of these changes are not only different between males and
females, but opposite, which could underlie the gender differences in the long-term sequale of
chronic stress, including subsequent responses to stress.
# 2010 Elsevier Ltd. All rights reserved.
1526C. Garcı ´a-Ca ´ceres et al.
Author's personal copy
All rats were sacrificed at 180 days of age between 1900 h
and 2200 h. Thus, females were at random stages of the
estrous cycle. The brain and pituitary were removed and
rapidly frozen in dry ice and stored at ?70 8C until processed.
Adrenal glands were removed and weighed and trunk blood
was collected, allowed to clot and the serum separated and
stored at ?70 8C until processed.
This experimental design resulted in the following four
groups (n = 9—10/group): C non-stressed (C), prenatally
stressed (PnS), adult stressed (AS), and prenatally and adult
2.4. Tissue preparation
The hypothalami were isolated on ice by using the following
boundaries: an anterior cut was made at the level of the
optic chiasm, a posterior coronal section anterior to the
mammilary bodies, two sagittal cuts parallel to the lateral
ventricles, and a dorsal horizontal cut at the level of the
anterior commissure. Each hypothalamus was then cut into
two halves by following the sagittal axis. One half was
in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4)
for 48 h at 4 8C and then rinsed with phosphate buffer (PB;
0.1 M, pH 7.4). Hypothalamic sections (30 mm) were
obtained using a vibratome (VT 1200S, Leica Microsystems,
Wetzlar, Germany) and stored at ?20 8C in cryoprotector
solution (30% sucrose, 30% etylene glycol in PB). The second
half was stored at ?70 8C until processed for Western blot-
ting or ELISA.
The pituitaries were weighed and transferred to a tube
and stored at ?70 8C until processed for Western blotting.
2.5. Protein extraction
Tissue for ELISA was homogenized in lysis buffer provided by
the manufacturer of the commercial kit (Roche Diagnostics,
Mannheim, Germany). Tissue for Western blotting was homo-
genized on ice in 500 ml of radioimmunoprecipitation assay
lysis (RIPA) buffer with an EDTA-free protease inhibitor cock-
tail (Roche Diagnostics, Mannheim, Germany). After homo-
genization for Western blotting, samples were centrifuged at
14,000 rpm for 20 min at 4 8C. Supernatants were transferred
to a new tube and total protein concentration was deter-
mined by the method of Bradford (Protein Assay; Bio-Rad
Laboratories, Hercules, CA, USA).
2.6. Cell death detection ELISA
This photometric enzyme immunoassay for the quantitative
determination of cytoplasmic histone-associated DNA frag-
ments (mono- and oligo-nucleosomes) that are produced
after induced cell death was carried out according to the
manufacturer’s instructions (Roche Diagnostics). Each sam-
ple was measured in duplicate in each assay. Background
measurements were made and this value was subtracted
from the mean value of each sample. This assay has a
detection limit of approximately 50 dead cells/well and
results were normalized to protein levels in each sample.
The inter- and intra-assay coefficients of variation were 8.5%
and 4.3%, respectively.
2.7. Western blotting
Depending on the specific protein to be detected 10, 20 or
60 mg of protein were resolved on a 12% SDS-polyacrylamide
gel under denaturing conditions. The proteins were then
electro-transferred to poly-vinylidene difluoride membranes
(Bio-Rad). Membranes were blocked in TBS (20 mM) contain-
ing 5% nonfat dried milk or 5% bovine serum albumin (BSA)
and 0.1% Tween 20 for 2 h. Primary antibodies, used at a
concentration of 1:1000, were as follows: anti-proliferating
cell nuclear antigen (PCNA) was purchased from Signet
Laboratories (Dedham, MA, USA), the antibody towards the
astrocyte protein S100b from Abcam (Cambridge, UK) and
anti-neuron-specific beta-III tubulin (Tuj-1) from RD Systems
(Minneapolis, MN, USA). The antibodies for the presynaptic
protein synapsin-1 and the post-synaptic protein PSD95 were
purchased from Calbiochem (Madison, WI, USA). Antibodies
for the glial cell markers vimentin and glial fibrillary acidic
protein (GFAP) were from Sigma—Aldrich (St. Louis, MO,
USA). Anti-b-actin was from Santa Cruz Biotechnology (Santa
was from Bachem (Torrance, CA, USA). Membranes were
incubated with primary antibodies overnight at 4 8C under
agitation. The membranes were then washed and incubated
with the corresponding secondary antibody conjugated with
peroxidase (Pierce, Rockford, IL, USA) at a dilution of 1:2000.
Bound peroxidase activity was visualized by chemiluminis-
cence (PerkinElmer Life Science, Boston, MA, USA) and
quantified by densitometry by using a Kodak Gel Logic
1500 Image Analysis system and Molecular Imaging Software,
version 4.0(Rochester,NY,USA). Allresults werefirst normal-
ized to actin levels in each lane and then to control values on
Both single and double immunohistochemistry were carried
out in free-floating sections under moderate shaking. After
washing in phosphate buffer (PB, 0.1 M, pH 7.4), endogenous
peroxidase was inhibited with a 50% methanol, 3% H2O2
solution in PB for 15 min. After washing in PB, the sections
wereincubatedin2NHClat37 8Cfor30 minandthenblocked
for 90 min at room temperature in PB with 1% Triton X-100
and 3% BSA (blocking buffer). For single immunohistochem-
istry of BrdU sections were incubated for 48 h at 4 8C with
anti-BrdU containing 5% normal goat serum (1:5000). The
BrdU monoclonal antibody was developed by Stephen J.
Kaufman under the auspices of the NICHD and maintained
byThe University ofIowa,Department of BiologicalSciences,
Iowa City, IA and obtained from the Developmental Studies
Hybridoma Bank. Sections were then washed three times in
PB with 0.3% bovine serum albumin (BSA) and 0.3% Triton X-
100 (PBT, 0.1 M pH 7.4) and incubated for 90 min at room
temperature with goat anti-mouse IgG biotin (1:1000). The
sections were washed and then incubated for 45 min in
avidin—biotin—peroxidase complex (ABC peroxidase staining
kit, Pierce Rockford, IL). After three washes with PB, per-
oxidase was then detected by using 3,30-diaminobenzidine
tetrahydrochloride (DAB; Sigma) as a chromogen (0.05% DAB,
0.02% H2O2in PB). The sections were washed with PB and
Sexual dimorphism in stress effects on hypothalamic structural proteins1527
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mounted on gelatin-coated slides, stained with methyl green
and dehydrated through a series of graded alcohols, cleared
in xylene, and covered with a coverslip in Depex.
For double immunohistochemistry of GFAPand BrdU, after
the blocking, the sections were incubated for 24 h at 4 8C
anti-GFAP (Sigma) at a dilution of 1:500 in blocking buffer
containing 5% normal goat serum. Sections were then rinsed
three times in buffer and incubated for 2 h at room tem-
perature with biotinylated secondary anti-rabbit IgG anti-
body (1:1000), followed by incubation for 45 min in ABC
complex and detected by using DAB as described above.
The sections were washed three times at RT and treated
with 2N HCl for 30 min at 37 8C. Afterwards, they were rinsed
in PB and incubated with blocking buffer containing anti-
BrdU overnight at 4 8C as described above. After incubation,
the sections were washed and incubated with a biotinylated
secondary anti-mouse IgG antibody (1:1000) for 2 h, rinsed
and incubated for 45 min in ABC, followed by DAB with 1%
cobalt chloride and 1% nickel sulphate in sterile water.
Finally, the sections were washed and mounted as described
Double-fluorescent immunohistochemistry for BrdU and
NeuN was carried out in free-floating sections, after washed
PB containing 0.3% bovine serum albumin (BSA) and 0.3%
Triton X-100 (PBT, 0,1 M, pH 7.4). This same buffer was used
90 min at RT in PB with 1% Triton X-100 and 3% BSA and
incubated for 48 h at 4 8C with anti-BrdU (1:750; Abcam) and
NeuN (1:500, Chemicon International, Billerica, MA). Sec-
tions were then washed three times in buffer and incubated
for 90 min at RT with goat anti-mouse IgG biotin (1:1000,
Pierce Biotechnology). Afterwards sections were washed and
incubated under dark conditions with streptavidin, Alexa
Fluor 488 (1:2000, Molecular Probes, Leiden, The Nether-
lands) and Alexa Fluor 633 anti-sheep IgG (1:1000, Molecular
Probes) for 90 min at RTand washed three times with buffer.
Finally, sections were mounted and cover-slipped with Clear
Mount (Electronic Microscopy Sciences, Hatfield, PA, USA).
Preliminary assays were performed to determine the
concentration of antibodies to be used. For every experi-
ment, sections for all groups were incubated in parallel. In
each assay control slides consisted of omission of primary
antibodies and verification that immunostaining was absent.
Peroxidase-stained sections were examined with a light
microscope (Zeiss Axioplan, Go ¨ttingen, Germany). Images
were captured using a digital camera and processed using
Image-Pro Plus software (version 5.0 for Windows; Media
Cybernetics Inc., Silver Spring, MD). Immunofluorescence
was visualized by using a confocal microscope (Leica model
DMIRB; Leica, Wetzlar, Germany).
2.9. RNA extraction and real-time PCR
Total RNA was extracted from the hypothalami according to
Tri-Reagent protocol (Chomczynski, 1993). Briefly, quantita-
tive real-time PCR was performed on cDNA prepared from
1 mg of total RNA isolated from hypothalami by reverse
transcription (High capacity cDNARTkit,Applied Biosystems,
Foster City, CA, USA). Predesigned primers for corticotro-
phin-releasing hormone (CRH) and b-actin and assay-on-
demand kits using TaqMan Universal PCR Master Mix (Applied
Biosystems) were used according to the manufacturer’s pro-
tocol, and analyzed by using the ABI PRISM 7000HT Sequence
Detection System (Applied Biosystems). Values were normal-
ized to the reference gene actin. According to manufac-
turer’s guidelines, the DDCTmethod was used for relative
quantification. Statistics were performed using DDCTvalues.
2.10. Determination of serum corticosterone
levels by RIA
Levels of corticosterone were measured by radioimmunoas-
say following the manufacturer’s instructions (MP Biomedi-
7.7 ng/ml and the intra- and inter-assay variations were 7.3%
and 6.9%, respectively.
2.11. Statistical analysis
To determine if there was an interaction between sex and
stress on a parameter, 2-way ANOVAs were performed. One-
way ANOVAs were performed when appropriate and post hoc
comparisons were made using the Scheffe f-test. All data are
presented as mean ? SEM. The values were considered sig-
nificantly different when the p value was lower than 0.05.
Statistics were performed using the statistics program Stat-
View (version 4.0).
3.1. Circulating corticosterone concentrations
Serum corticosterone levels were found to be significantly
affected by both sex (F: 108.827; p < 0.0001) and stress (F:
3.022; p < 0.05), with a significant interaction between
these two factors (F: 3.579; p < 0.02). Control female rats
had significantly higher corticosterone levels than control
males. In males, there was no difference in corticosterone
levels regardless of their previous stress experience. In con-
trast, corticosterone levels were significantly higher in
of prenatal and adult stress compared to control females or
females that were only prenatally stressed, even though the
last immobilization stress was performed 90 days before
sacrifice (Fig. 1A).
3.2. Adrenal gland weight
There was no effect of prenatal or adult stress on adrenal
gland weight in either males or females, but there was a
significant effect of sex with females having significantly
0.232 ? 0.010 g, PnS females: 0.235 ? 0.015 g, AS females:
0.206 ? 0.013 g,APSfemales:0.235 ? 0.016 g,malecontrol:
0.118 ? 0.010 g, PnS males: 0.108 ? 0.003 g, AS males:
0.108 ? 0.005 g, APS males: 0.108 ? 0.010 g; F: 233.347;
p < 0.0001, n = 9—10/group).
3.3. Pituitary ACTH content
In female rats, there was no significant effect of prenatal or
adult stress on pituitary ACTH content (Fig. 1B). In males,
1528C. Garcı ´a-Ca ´ceres et al.
Author's personal copy
prenatal stress increased ACTH levels (F: 18.120; p < 0.002),
with no effect of adult stress (Fig. 1C).
3.4. Hypothalamic CRH mRNA
In female rats PnS induced a significant increase in hypotha-
lamicCRHmRNAlevels(F:13.093;p < 0.01)withnoeffectof
AS (Fig. 1D). In males, there was a significant effect of
prenatal stress on CRH mRNA levels (F: 10.353, p < 0.005)
by 2-way ANOVA with no significant interaction between the
two stresses. Males exposed to prenatal stress had a decline
in CRH mRNA levels with this reaching significance when
exposed to both stresses (Fig. 1E; p < 0.02).
3.5. Hypothalamic cell turnover
Infemalerats,stress hadnosignificanteffect oncelldeathas
represented by cytoplasmic histone-associated DNA frag-
ment levels (Fig. 2A). In contrast, in male rats PnS and the
combination of both stresses significantly reduced this cell
death marker in the hypothalamus compared to controls
(Fig. 2B; F: 5.700, p < 0.04).
adrenocorticotropic hormone (ACTH) levels in the pituitary of female (B) and male (C) rats in response to stress (n = 4—5/group).
Effect of prenatal and adult stress on hypothalamic corticotrophin-releasing hormone (CRH) mRNA levels in females (D) and males
(E), n = 4—5/group. C, control; PnS, prenatal stress; AS, adult stress; PAS, prenatal and adult stress. NS, non-significant. *, ANOVA
p < 0.05.
Circulating levels of corticosterone in female and male rats in response to stress, n = 9—10/group (A). Changes in
Sexual dimorphism in stress effects on hypothalamic structural proteins1529
Author's personal copy
Stress had no effect on hypothalamic cell proliferation, as
represented by PCNA levels, in the hypothalamus of female
rats (Fig. 2C). However, in males there was an interaction
between the two stresses (F: 9.140; p < 0.01) with both
prenatal and adult stress significantly reducing PCNA levels
and the combination of stresses returning them to control
levels (Fig. 2D).
Thedistribution ofBrdUpositive cellsinthehypothalamus
was similar between all experimental groups, with sparse
cells being found in the lateral hypothalamus, dorsomedial
hypothalamus, anterior hypothalamus and in the white mat-
ter. Slightly higher densities of BrdU positive cells were found
lining and close to the 3rd ventricle and in the arcuate,
supraoptic and paraventricular nuclei.
Cells immunolabeled for both BrdU and GFAP were found
(Fig. 3A), with very few cells found double labelled for BrdU
and NeuN in any of the experimental groups.
3.6. Glial and neuronal markers in the
We found no change in neuron-specific beta-III tubulin (Tuj-1)
levels in response to either stress in males or females (data
not shown). However, the effect of chronic stress on astro-
cyte marker levels differed according to sex. In females,
there was an interaction between prenatal and adult stress
on GFAP levels with the combination of stresses inducing a
significant decrease (C: 100 ? 8.8, PnS: 128.0 ? 12.6, AS:
122.7 ? 8.3, PAS: 77.9 ? 14.9; F: 10.536; p < 0.01; n = 4—5/
group). In contrast, in males GFAP levels were unchanged in
response to stress (C: 100 ? 21.5, PnS: 111.9 ? 26.5, AS:
94.2 ? 17.1, PAS: 88.4 ? 14.2; n = 4—5/group). Hypothala-
mic vimentin levels were highly variable and no significant
differences were found between any of the experimental
groups (males C:100 ? 17.8,
88.8 ? 26.8, PAS: 53.4 ?18.1; females C: 100 ? 20.8, PnS:
172.6 ? 92.7, AS: 163.7 ? 62.8, PAS: 92.5 ? 33.7; n = 4—5/
There was no effect of prenatal or adult stress on S100b
levels in females (Fig. 3B). In contrast, both prenatal and
adult stress significantly modulated S100b levels, a marker of
mature astrocyte, in males (PnS; F: 18.289; p < 0.002 and
AS; F: 27.793; p < 0.001). Male rats exposed to prenatal
stress had significantly higher and those exposed to adult
stress had significantly lower S100b levels compared to con-
trols. In male rats subjected to both stresses S100b returned
to control levels (Fig. 3C; p < 0.002).
PnS: 95.7 ? 23.5, AS:
nuclear antigen (PCNA) levels in the hypothalamus of females (C) and males (D) in response to stress. C, control; PnS, prenatal stress;
AS, adult stress; PAS, prenatal and adult stress. NS, non-significant. *, ANOVA p < 0.05; n = 4—5/group.
Effect of prenatal and adult stress on hypothalamic cell death in female (A) and male (B) rats. Changes in proliferating cell
1530C. Garcı ´a-Ca ´ceres et al.
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3.7. Determination of synaptic proteins levels in
The effect of stress on hypothalamic synaptic proteins was
also sexually dimorphic. In females, adult stress increased
synapsin-1 levels (F: 18.702; p < 0.002), with prenatal stress
having no effect (Fig. 4A). In contrast, in males prenatal
stress increased synapsin-1 levels, with adult stress having no
effect (Fig. 4B; p < 0.005).
The effect of stress on the relative levels of the post-
synaptic protein PSD95 was also sexually dimorphic. In
of these two stresses all increased PSD95 levels (Fig. 4C;
p < 0.03). In contrast, there was a decrease in PSD95 in male
rats in response to prenatal and adult stress (Fig. 4D;
p < 0.006), while the combination of these stresses returned
to control values.
During development gonadal steroids play a key role in the
(Patchev et al., 1995, 1999; Weinstock, 2007; Zuena
et al., 2008; Koehl et al., 2009) and this underlies, at least
in part, the differences found between males and females in
neuroendocrine functions and behavior. As brain develop-
ment is different between the sexes, it follows that an insult
during development may have variable outcomes. Indeed,
long-term responses to different types of stress have been
shown to be sexually dimorphic (Rhees et al., 1999a; Wein-
stock, 2007; Zuena et al., 2008; Andreano and Cahill, 2009;
Bowman et al., 2009), although the mechanisms underlying
this phenomenon remain largely unknown. Here we report
that not only is the hypothalamic—pituitary—adrenal axis
modulated differently in male and female rats, but that both
prenatal and adult stress inducechanges in hypothalamic cell
turnover and synaptic protein concentrations, with these
modifications differing dramatically between the sexes.
Hence, these results suggest that structural reorganization
of the hypothalamus underlies not only the affectation of
endocrine axes by chronic early stress, but that it could also
participate in the sexually dimorphic long-term endocrine
The stress axis differs between males and females not
only at baseline, but also in response to different stresses
(Rhees et al., 1999a; Drossopoulou et al., 2004; Weinstock,
2007; Zuena et al., 2008; Andreano and Cahill, 2009; Bow-
man et al., 2009), which may be due at least in part to
differences in gonadal steroid levels (Lund et al., 2004).
Indeed, we found corticosterone levels to be higher in
control females compared to control males, indicating that
hypothalamus of male rats (A). Effect of prenatal and adult stress on hypothalamic S100b levels of female (B) and male (C) rats. C,
control; PnS, prenatal stress; AS, adult stress; PAS, prenatal and adult stress. NS, non-significant. **, ANOVA p < 0.005.
Immunolabeling of BrdU (black) was found to colocalize with glial fibrillary acidic protein (GFAP; brown) in cells of the
Sexual dimorphism in stress effects on hypothalamic structural proteins1531
Author's personal copy
the basal situation of this system is different between the
sexes and could participate in the differential responses to
stress in the adult animal. Furthermore, in female rats
corticosterone levels were increased in response to the
combination of stresses, with hypothalamic CRH mRNA
levels increasing in response to prenatal stress. The lack
of statistical significanceinresponse to adultstress could be
due to the fact that females were sacrificed at random
moments of the estrous cycle, which could underlie the high
variability seen in CRH mRNA levels in female rats, as well as
many of the studied variables. Increased CRH mRNA levels in
response to early stress is in agreement with a previous
report showing an increase in the paraventricular nucleus
of adult female rats (Bosch et al., 2007) and suggests that
this axis is up-regulated in females. In contrast, pituitary
ACTH levels increased and hypothalamic CRH mRNA levels
decreased in prenatally stressed males, suggesting a possi-
ble decrease in ACTH liberation from the anterior pituitary
and a down-regulation of this axis in males. These gender
specific long-term changes in the stress axis in response to
early stress, as well as adult stress, could help to explain the
differential responses to later stress, where females may be
Modifications in hippocampal cell turnover and synaptic
density have been associated with stress induced modifica-
tions in behavior (Koo et al., 2003; Bogoch et al., 2007;
Darnaude ´ry and Maccari, 2008). Hence, we hypothesized
lamus that could be associated with the endocrine outcomes.
Indeed, we found that markers of cell turnover and synaptic
density were affected long-term by both prenatal and adult
stress. This hypothesis is further supported by the fact that
the majority of changes in structural proteins were gender
specific, as are many of the reported long-term endocrine
responses to stress (Reznikov and Nosenko, 2000; Matthews,
2002; Luine, 2002; Welberg et al., 2006; Darnaude ´ry and
Maccari, 2008; Mueller and Bale, 2008; Garcı ´a-Ca ´ceres et al.,
Prenatal stress significantly decreased both cell death
and proliferation markers in the hypothalamus of male
rats, suggesting a decrease in basal cell turnover. In con-
trast, no effect of either type of stress was observed in
females. Decreased neurogenesis in the hippocampus of
adult rats as a result of early life experiences has been
suggested to inhibit the structural plasticity and diminish
the ability of the hippocampus to respond to stress
in adulthood (Mirescu et al., 2004). However, how the
adult stress; NS, non-significant. *, ANOVA p < 0.05; n = 4—5/group.
Effect of prenatal and adult stress on synapsin-1 and post-synaptic density protein (PSD) 95 levels in the hypothalamus of
1532C. Garcı ´a-Ca ´ceres et al.
Author's personal copy
functioning of the hypothalamus and its response to further
stresses is affected by this change in cell turnover is
unknown, but could also indicate a modification in the
ability to respond to future challenges. It is interesting
to note that cell proliferation, as indicated by PCNA levels,
was decreased by both prenatal and adult stresses, while
the combination of these two stresses normalized this
parameter. The normalization of hypothalamic cell turn-
over could possibly participate in the habituation of some
endocrine parameters to immobilization stress (Girotti
et al., 2006; Garcı ´a-Ca ´ceres et al., 2010).
In the adult hypothalamus the majority of cells under-
going turnover have been reported to be glial cells (Garcia-
Segura and McCarthy, 2003) and we found BrdU to be
incorporated into GFAP positive astrocytes. Gliosis, as
indicated by increased expression of GFAP and vimentin,
in the cortex and hippocampus after chronic immobiliza-
tion stress (Barros et al., 2006; Jang et al., 2008) is
suggested to indicate neurodegeneration (Jang et al.,
2008). However, in the hypothalamus there was no change
in vimentin levels and GFAP levels actually decreased in
females in response to both prenatal and adult stress, with
no effect in males. Thus, gliosis does not appear to occur in
the hypothalamus in response to immobilization stress.
Previous studies have demonstrated that S100b, an astro-
cyte-derived protein, can also be increased in response to
neurodegeneration, inflammation and in psychiatric dis-
eases (Rothermundt et al., 2003; Mrak and Griffin, 2005;
Craft et al., 2005; Ralay et al., 2006) and prenatal stress
resulted in increased hypothalamic S100b levels in males.
Therefore, increased S100b levels in response to maternal
stress could represent increased susceptibility to patholo-
gic processes in males. However, adult stress decreased
S100b levels and the combination of the two stresses
returned them to control levels. The inverse changes in
hypothalamic S100b in response to prenatal and adult
stress could be due to the age of the animals at the
moment of stress, as S100b has also been reported
to be a regulator of neurite outgrowth and to enhance
survival of neurons only during development (Rothermundt
et al., 2003). Thus, the changes in this glial marker could
be related to other astrocytic functions as astrocytes
are involved in many aspects of neuronal function
(Pellerin etal., 2007; Brown
Milligan and Watkins, 2009) and also participate in the
regulation of synaptic changes in the hypothalamus (Theo-
dosis et al., 2006; Prevot et al., 2007; Garcia-Segura et al.,
2008; Ojeda et al., 2008). Hence, changes in glial cells
could be associated with the changes in synaptic protein
Synapsin-1, a presynaptic protein related to the number
of synaptic inputs, was significantly increased by prenatal
stress in males and by adult stress in females. Inverse
effects in males and females were also seen with PSD-
95, with both prenatal and adult stress increasing this
protein in females and decreasing it in males. It is clear
that synaptic protein density is affected and that this
occurs in a sexually dimorphic manner even though the
hypothalamic neuronal systems where these synaptic
changes are occurring cannot be determined as these
parameters were analyzed in whole hypothalamic homo-
genates. However, the modifications in PSD95 levels indi-
cate that at least some of the synapses involved are
glutamatergic and that these stimulatory inputs may be
increased in females and decreased in males in response to
both prenatal and adult stress. Indeed, prenatal stress
alters synaptic inputs in the frontal cortex, striatum and
hippocampus (Barros et al., 2006; Michelsen et al., 2007),
with the responsiveness of the glutamatergic system in the
prefrontal cortex known to be affected (Fumagalli et al.,
2009). Whether these changes in hypothalamic excitatory
inputs are related to the increase in the activation of the
stress axis, including the increase in CRH mRNA levels in
females and the decrease in males deserves further inves-
Early brain development is vulnerable to high levels of
circulating corticosterone, which in rats has been shown to
cross the placental barrier especially during the last week
of gestation leading to disturbances in the formation of
neural circuits (Weinstock, 2007; Mabandla et al., 2008).
Indeed, chronic corticosterone exposure modulates cell
morphology and cell death in various brain areas, including
the hypothalamus (Skynner et al., 2006). Thus, the changes
in hypothalamic synaptic proteins and cell turnover
markers could be due to high levels of maternal glucocor-
ticoids in response to prenatal stress (Barros et al., 2006)
and this could underlie the neuroendocrine alterations
that persist into adulthood. Early events have previously
been show to induce long-term alterations in central CRH
and corticosterone levels that affect the responsiveness of
the HPA axis in the adult animal (Weaver et al., 2004;
McGill et al., 2006; Mabandla et al., 2008; Mueller and
Bale, 2008). Here we show that both prenatal and adult
immobilization stress induce long-term changes in the HPA
axis, as well as modifications in synaptic and structural
protein levels in the hypothalamus. Furthermore, these
changes are gender specific, which could help to explain
some of the differences between the sexes in the physio-
logical manifestations in response to stress and repeated
stresses, as well as the pathological responses to chronic
Role of the funding sources
Funding for this study was provided by grants from Fondo de
Investigacio ´n Sanitaria (PI070182), Ministerio de Ciencia e
Innovacio ´n (BFU2008-02950 C03-1, C03-2 and C03-3), CIBER
de Fisiopatologı ´a de Obesidad y Nutricio ´n (CIBEROBN) Insti-
tuto de Salud Carlos II and Fundacio ´n de Endocrinologı ´a y
the Ministerio de Educacio ´n y Ciencı ´a (FPU AP2006/02761)
of the Consejerı ´a de Sanidad y Consumo de la Comunidad de
None of the funding institution had any further role in the
study design, the collection, analysis and interpretation of
data, writing of the report or in the decision to submit the
paper for publication. None of the authors have anything to
Conflict of interest
Sexual dimorphism in stress effects on hypothalamic structural proteins 1533
Author's personal copy
The authors would like to thank Dr. Vicente Barrios for advice
in performing the hormone assays and Francisca Dı ´az and
Sandra Canelles for the excellent technical support.
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