17?-ESTRADIOL ATTENUATES HIPPOCAMPAL NEURONAL LOSS
AND COGNITIVE DYSFUNCTION INDUCED BY CHRONIC RESTRAINT
STRESS IN OVARIECTOMIZED RATS
K. TAKUMA,aA. MATSUO,aY. HIMENO,aY. HOSHINA,a
Y. OHNO,aY. FUNATSU,aS. ARAI,aH. KAMEI,a
H. MIZOGUCHI,aT. NAGAI,aK. KOIKE,bM. INOUEb
AND K. YAMADAa*
aLaboratory of Neuropsychopharmacology, Graduate School of Natu-
ral Science and Technology, Kanazawa University, Kakuma-machi,
Kanazawa 920-1192, Japan
bDepartment of Obstetrics and Gynecology, Kanazawa University
Graduate School of Medical Science, 13-1 Takara-machi, Kanazawa
Abstract—Several lines of evidence suggest that hormonal
changes after menopause may play an important role in the
incidence of cognitive dysfunction, and also in the develop-
ment of Alzheimer’s disease. In this study, we investigated
the effect of estrogen on cognitive function in rats under
different stress environment. Female rats were divided into
four groups: two groups were ovariectomized (OVX) and two
were sham-operated. One group each of OVX and sham rats
was kept in a normal environment, and the other groups were
assigned to a daily restraint stress (6 h/day) for 21 days from
2 months after the operation. Following the stress period,
subjects were tested for performance in novel object recog-
nition test and then used for morphological and neurochem-
ical analyses. The OVX plus stress (OVX/stress) group
showed a significant impairment of recognition of novel ob-
jects, compared with the other groups. The OVX/stress group
also showed a marked decrease in the number of pyramidal
cells of the CA3 region and levels of brain-derived neurotro-
phic factor mRNA in the hippocampus. We further examined
the effect of estrogen against cognitive dysfunction and hip-
pocampal changes of OVX/stress rats. Vehicle or 17?-estra-
diol (E2) at 20 ?g/day was s.c. administered to OVX/stress
rats from 2 days before the stress period to the end of be-
havioral analysis through an implantable osmotic pump.
Chronic E2 treatment decreased stress response and im-
proved the cognitive and morphological impairments relative
to vehicle group. These data have important implications for
cognition enhancing effect of estrogen treatment in post-
menopausal women. © 2007 IBRO. Published by Elsevier Ltd.
All rights reserved.
Key words: 17?-estradiol, ovariectomy, cognition, hippocam-
Estrogens are known as potent regulators of neuronal
function, including neuronal proliferation, survival and plas-
ticity (McEwen, 2001; Behl, 2002), and have been shown
to alter the density of pyramidal cells in the hippocampus
(Gould et al., 1990; Woolley et al., 1990; Woolley and
McEwen, 1992; McEwen and Woolley, 1994; Leranth et
al., 2000; Leranth and Shanabrough, 2001) via the activa-
tion of two intracellular receptors for estrogen, ER? and
ER?, especially expressed in the CA3 region of hippocam-
pus (Mehra et al., 2005). Consistent with these morpho-
logical changes, enhancement of working memory in the
Morris water maze task was observed within the time
frame of estrogen-induced increases in hippocampal pyra-
midal cell density (Sandstrom and Williams, 2001, 2004).
These results suggest that estrogen-dependent changes
in hippocampal neuronal networks are, at least in part,
responsible for the hormonal effects on cognitive functions.
Furthermore, estrogen is known to exert neurotrophic ef-
fect by the expression of the neurotrophic factors, such as
brain-derived neurotrophic factor (BDNF) (Lu and Chow,
1999; Thoenen, 2000).
Clinical studies have reported that memory decline is a
very common complaint during the menopausal transition
and early postmenopause (Woods et al., 2000). Meno-
pause is associated with decreased ovarian hormones,
such as estrogens (Zapantis and Santoro, 2003). Estrogen
replacement therapy may improve cognitive function in
postmenopausal women (Hogervorst et al., 2000; Mulnard
et al., 2000; LeBlanc et al., 2001; Genazzani et al., 2002),
although the Women’s Health Initiative Memory Study
(WHIMS) demonstrated that estrogen or hormone replace-
ment therapy does not improve or even had an adverse
effect on cognition in postmenopausal woman (Rapp et al.,
2003; Espeland et al., 2004). Experimentally, ovariecto-
mized (OVX) animals are used as models to investigate
the roles of ovarian hormones in the pathogenesis of mem-
ory deficits. Although there is growing evidence to support
the hypothesis that ovarian hormone loss plays a role in
cognition of menopausal women (Gibbs and Aggarwal,
1998; Luine et al., 1998), postmenopausal women do not
always suffer from the memory decline. These findings
suggest that other additional factors besides the ovarian
hormone loss are required for the development of post-
menopausal memory deficits.
In addition to ovarian hormones, environmental stress
is known to cause morphological changes in the hip-
pocampus. Stress activates the hypothalamic–pituitary–
adrenocortical axis, leading to glucocorticoid secretions
from the adrenal. Corticosterone, the adrenal hormone of
rodents, can enter the brain and bind to the glucocorticoid
receptors (GR) or the mineralocorticoid receptors (MR)
*Corresponding author. Tel: ?81-76-234-4465; fax: ?81-76-234-4416.
E-mail address: firstname.lastname@example.org (K. Yamada).
Abbreviations: BDNF, brain-derived neurotrophic factor; CS, chronic
stress; E2, 17?-estradiol; GR, glucocorticoid receptor; MR, mineralo-
corticoid receptor; NS, no stress; OVX, ovariectomized; PBS, phos-
phate-buffered saline; RT-PCR, reverse transcription polymerase
Neuroscience 146 (2007) 60–68
0306-4522/07$30.00?0.00 © 2007 IBRO. Published by Elsevier Ltd. All rights reserved.
with a 10-fold higher affinity (Reul and de Kloet, 1985).
Neurons in the CA1 region and dentate gyrus of the hip-
pocampus express high levels of both GR and MR. In
contrast, CA3 pyramidal neurons contain much lower
amounts of GR but high levels of MR (McEwen et al.,
1986). Exposure of animals to chronic stress (CS), which
leads to an increase in corticosterone levels, causes an
atrophy of CA3 pyramidal cells (Magariños and McEwen
1995). In fact, it is reported that CS impairs cognitive
function (Luine et al., 1994; McEwen and Sapolsky, 1995;
Sandi, 2004). Accordingly, synergistic effects of a dramatic
decrease in circulating estrogen with the chronic daily
stress during menopausal transition and early postmeno-
pause may eventually cause cognitive dysfunction in post-
menopausal women. Furthermore, stress or glucocorticoid
administration is shown to cause a dramatic decrease in
BDNF expression in male rodents (Smith et al., 1995;
Vaidya et al., 1997, 1999). This stress-induced decrease in
BDNF levels may be critical, because BDNF inhibits glu-
cocorticoid-induced cell death (Nitta et al., 1999). How-
ever, little is known about the role of interaction of ovarian
hormone loss and stress response on the pathogenesis of
the memory deficits in menopause.
In this study, to clarify the involvement of environmen-
tal stress on the pathogenesis of postmenopausal cogni-
tive impairment and to develop a reliable animal model of
the disease, we examined the combined effects of ovari-
ectomy and chronic restraint stress on cognitive function
and neurochemical changes in female rats with or without
chronic estrogen treatment. After the chronic restraint
stress, subjects were tested for recognition performance,
and then, neuronal cell density and BDNF mRNA levels in
the hippocampus were evaluated.
Animals and treatment
Female Fisher 344 rats (Charles River Japan, Yokohama, Japan)
weighing 160–200 g at the beginning of the experiments were
used. They were housed two or three per cage under standard
12-h light/dark conditions (12-h light cycle starting at 8:45 a.m.) at
a constant temperature of 23?1 °C, and had free access to food
and water. All animal experiments were carried out in accordance
with the guidelines established by the Institutional Animal Care
and Use Committee of Kanazawa University, the Guiding Princi-
ples for the Care and Use of Laboratory Animals approved by the
Japanese Pharmacological Society, and the United States Na-
tional Institutes of Health Guide for the Care and Use of Labora-
tory Animals. All efforts were made to minimize the number of
animals used and their suffering or distress.
One week after arrival, all experimental animals were bilater-
ally OVX or sham-operated under pentobarbital (50 mg/kg) anes-
thesia. After a recovery period of 2 months, some animals were
anesthetized again with pentobarbital (50 mg/kg) and implanted
s.c. with a 28-day Alzet miniosmotic pump (Model 2ML4, Durect
Co., Cupertino, CA, USA), which was filled with either 17?-estra-
diol (E2; 0.33 mg/ml) or vehicle (97% 1,2-polypropylene glycol and
3% ethanol). Since the pump releases the contents at a rate of
2.5 ?l/h, the dose of E2 was estimated to be 20 ?g/day/body,
which is reported to attenuate chronic hypoxia-induced pulmonary
hypertension and erythropoietin gene expression (Mukundan et
al., 2002; Resta et al., 2001).
It has already been reported that the 21 consecutive days of
chronic immobilization stress (6 h/day) affects the rat performance
on a radial arm maze in related to hippocampal function (Luine et
al., 1994; Bowman et al., 2001, 2002). Thus, similar immobiliza-
tion stress was selected and began 2 days after the start of E2
treatment and was repeated every day for 21 days. The stress
was performed with a stainless mesh (Magariños et al., 1997) that
allowed for a close fit to rats for 6 h (between 9:00 a.m. and
3:00 p.m.) in their home cages. Some animals were not subjected
to stress (no stress (NS) group), but were handled at 9:00 a.m. for
a few seconds. Following the repeated restraint stress for 21 days,
behavioral analysis was performed according to the following
procedure. And then, the animals were randomly subjected to the
histological or biochemical analyses.
Blood sampling and determination of serum
estradiol and corticosterone levels
Rats were anesthetized with pentobarbital (40 mg/kg) on the day
before blood sampling, and a venous cannula of SILASCON®
brand silicon tube (internal diameter 0.5 mm, external diameter
1.0 mm; Kaneka Medix Corporation, Osaka, Japan) was placed in
the right atrium via the right jugular vein. The end of the cannula
was exteriorized through a s.c. tunnel to the back of the neck and
was filled with a stainless-steel plug to readily access for the
sampling. Blood sampling (500 ?l at each time point) was made
immediately before and 0.5, and 6 h after commencing immobili-
zation stress on day 1 of the 21-day stress period. As for the
sampling after 21-day stress period, trunk blood was collected
after decapitation. The blood samples were centrifuged at 800?g
for 10 min, the serum was separated and stored at ?80 °C until
assayed. Serum concentrations of estradiol and corticosterone
were quantified using the enzyme immunoassay (EIA) kits (Estra-
diol EIA Kit and Corticosterone EIA Kit; Cayman Chemical, Ann
Arbor, MI, USA). Data are expressed as pg of estradiol and ng of
corticosterone per 1 ml of serum, respectively.
For the novel object recognition test, a rat was habituated to a gray
empty open field (35 cm high?60 cm diameter) for 3 days (10
min/day). In the training session, two novel objects were placed in
the field, and the rat was allowed to explore freely for 10 min. Time
spent exploring each object was recorded manually. In the reten-
tion session at 24 h after the training session, the rat was placed
back in the same field, in which one of the familiar objects used in
the training was replaced by a novel object, and the rat was
allowed to explore for 5 min. The objects, which varied in shape
and color and were made of glass and plastic, were fixed to the
floor and cleaned thoroughly between trials to ensure the absence
of olfactory cues. The objects had been previously tested with
naive rats to ensure an equivalent level of spontaneous prefer-
ence. Locomotor activity in the training and retention sessions was
measured using infrared counters (NS-AS01; NeuroScience Idea,
Osaka, Japan). Exploratory preference, a ratio of time spent ex-
ploring any one of the two objects (training) or the novel one
(retention) over the total time spent exploring both objects, was
used to measure recognition memory (Kamei et al., 2006).
Morphological data analysis
After completion of behavioral analyses, rats were deeply anes-
thetized with pentobarbital and perfused intracardially with 4%
paraformaldehyde in phosphate-buffered saline (PBS). The brains
were removed, post-fixed with the same fixative and cryoprotected
with 30% sucrose-containing PBS. Sections (20 ?m) containing
hippocampus were obtained using a rotary microtome (HM505E;
Microm International GmbH, Walldorf, Germany), mounted on
slides, and stored at ?80 °C until use. Nissl staining was done
K. Takuma et al. / Neuroscience 146 (2007) 60–6861
according to standard procedures (Yamada et al., 2005). A fluo-
rescence microscope with a cooled CCD digital camera system
(Axio Imager A1/AxioCam MRc5; Carl Zeiss, Jene, Germany) was
used to scan the Nissl-stained sections. Nissl-positive neuronal
cell numbers were manually and rigidly counted within the hip-
pocampal pyramidal cell layer (CA1 and CA3 regions) and the
dentate gyrus of the scanned digital images. The total cell counts
were averaged from at least three sections per animal. Nissl-
positive neuronal cell size within the CA1, CA3 regions and the
dentate gyrus of the hippocampus were calculated from the digi-
tized images using an image analyzing software Win ROOF (ver.
5.6, Mitani Co., Fukui, Japan).
Reverse transcription polymerase chain reaction
(RT-PCR) and determination of BDNF mRNA levels
The levels of BDNF mRNA in brain tissues were determined by a
method previously described (Mizuno et al., 2000). In brief, total
RNA was extracted from brain tissues and converted into cDNA
using SuperScript™First-Strand Synthesis System for RT-PCR
(Invitrogen, Carlsbad, CA, USA). The mRNA for ?-actin was used
for an internal control. PCR was performed using one twentieth of
the RT-reaction mixture, 0.5 ?M of each (forward and reverse)
primer, and 0.5 units of TaKaRa Taq™(Takara Bio, Tokyo, Japan)
in a total reaction volume of 20 ?l. The primers used were as
follows: BDNF: 5=-CGTGATCGAGGAGCTGTTGG-3= (forward)
and 5=-CTGCTTCAGTTGGCCTTTCG-3= (reverse), and ?-actin:
5=-TGCTCGACAACGGCTCCGGCATGT-3= (forward) and 5=-
CCAGCCAGGTCCAGACGCAGGAT-3= (reverse). In a prelimi-
nary experiment, the number of PCR cycles and denaturation
temperature were tested to ascertain a linear working range for all
PCR products. The experimental amplification protocol consisted
of a first round at 94 °C for 3 min and then 30 cycles of denatur-
ation for 30 s at 94 °C, annealing for 1 min at 58 °C, and extension
for 1 min at 72 °C on a programmable thermal cycler (GeneAmp®
PCR System 9700; Applied Biosystems, Foster, CA, USA). The
PCR products were visualized using a SYBR®Gold nucleic acid
gel stain (Molecular Probes, Eugene, OR, USA) under UV light
after electrophoresis on a 1.5% agarose gel, and quantified with
an Atto Lane Analyzer 2.2 (Atto, Tokyo, Japan). The levels of
BDNF mRNA were expressed as percentage of control after nor-
malization with the levels of ?-actin mRNA.
Determination of BDNF protein level
The levels of BDNF protein in the hippocampus were determined
using a sensitive two-site ELISA kit (BDNF Emax®ImmunoAssay
System; Promega Corporation, Madison, MI, USA) according to
the manufacture’s instructions. In brief, the tissues were homog-
enized in five volumes of lysis buffer (137 mM NaCl, 20 mM
Tris–HCl, pH 8.0, 1% NP40, 10% glycerol, 1 mM PMSF, 10 ?g/ml
aprotinin, 1 ?g/ml leupeptin, and 0.5 mM sodium vanadate) and
removed any particulates by centrifugation at 1500?g for 15 min.
The resulting supernatants were assayed for BDNF determina-
tion. The 96-well, flat-bottomed ELISA plates (cat. # 3590; Corning
Inc., Acton, MA, USA) were coated with anti-BDNF polyclonal
antibody. The plates containing samples and standards were
incubated at room temperature for 6 h on a plate shaker. BDNF
standards, ranging from 7.8–500 pg/ml, were prepared using
recombinant human BDNF. The captured NGF was reacted first
with rat anti-NGF monoclonal antibody, and then with horseradish
peroxidase-conjugated anti-rat IgY antibody (1:500). The protein
content of each sample was determined by a BioRad DC protein
assay (BioRad Laboratories, Hercules, CA, USA). Data are ex-
pressed as pg of BDNF per mg protein.
Statistical analysis of the experimental data were carried out using
StatView 5.0 Windows. The significance of differences was deter-
mined by Student’s t-test for two-group comparison, and by a
one-way ANOVA, followed by the Student-Newman-Keuls test for
multigroup comparisons, respectively. The criterion for statistical
significance was P?0.05.
Effects of ovariectomy and estrogen treatment on
serum estradiol levels and corticosterone responses
against restraint stress in female rats
Ovariectomy significantly decreased the serum estradiol
levels (15.2?0.96 pg/ml, n?10; P?0.05 by Student’s
t-test), compared with sham-operated control group includ-
ing all estrous cycle rats (25.7?3.7 pg/ml, n?12). After the
21-day restraint stress period, serum estrogen levels in E2
(20 ?g/day)-treated rats reached 402?33.2 pg/ml (n?3),
which was significantly higher than the levels in other two
groups (F(2,22)?495.6, P?0.0001 by one-way ANOVA,
P?0.01 by post hoc).
Ovariectomy did not affect the basal serum corticoste-
rone levels (568?86.2 ng/ml, n?12; P?0.3546 by Stu-
dent’s t-test) compared with sham-operated control
(730?147 ng/ml, n?10). Ovariectomy also failed to affect
the corticosterone levels in the NS group or the corticoste-
rone response 30 min after the beginning of restraint stress
on day 1 of the 21-day stress period, compared sham-
operated control (Fig. 1). In contrast, 2-day treatment of E2
(20 ?g/day) slightly decreased the stress-induced increase
in serum corticosterone (Fig. 1), although the E2 treatment
Fig. 1. Effects of ovariectomy, restraint stress and estrogen treatment
on serum corticosterone levels in female rats. Serum samples at 30
min after first restraint stress were determined by an ELISA system.
Results are shown as means?S.E. (sham-operated control group
(NS/sham): n?3, OVX group (NS/OVX): n?5, sham-operated stress
group (CS/sham): n?5, OVX plus stress group (CS/OVX): n?5, E2-
treated OVX plus stress group (CS/OVX/E2): n?3). ** P?0.01, vs.
Keuls test). F(4,16)?8.657, P?0.0006.
#P?0.05, vs. CS/OVX (ANOVA and Student-Newman-
K. Takuma et al. / Neuroscience 146 (2007) 60–6862
had no effect on the basal serum corticosterone levels
(576?306 ng/ml, n?3, F(2,20)?0.456, P?0.6405), com-
pared with sham-operated and OVX groups. The effects of
restraint stress and E2 on changes in serum corticosterone
levels were still observed 6 h after the beginning of re-
straint stress (F(4,16)?7.353, P?0.0015).
Combination effects of ovariectomy, CS and E2
treatment on cognitive function of rats
Ovariectomy, repeated daily restraint stress for 21 days
and chronic E2 treatment did not affect the locomotor
activity (F(4,58)?1.033, P?0.391), total exploratory time
(F(4,58)?1.943, P?0.1154) and exploratory preference to
two objects (F(4,58)?0.671, P?0.615, Fig. 2A) in female
rats during the training session of the novel object recog-
In the retention session at 24 h after the training ses-
sion, a decrease in exploratory preference to a novel ob-
ject was observed in CS/OVX rats, compared with NS/
sham, NS/OVXand CS/sham
P?0.01 by one-way ANOVA, P?0.05 by post hoc, Fig.
2B), although there was no difference in total exploratory
time between these groups (F(4,58)?2.198, P?0.0804).
Chronic treatment of E2 (20 ?g/day) markedly attenuated
CS-induced cognitive dysfunction of OVX rats, as ob-
served in the novel object recognition test (P?0.05 by post
hoc, Fig. 2B).
Effects of CS and E2 treatment on neuronal cell
numbers and BDNF levels in the hippocampus of
After behavioral analysis, size and density of neuronal
cells and BDNF levels in the hippocampus were evaluated.
Fig. 3 shows typical microscopic images of the hippocam-
pal CA3 region after Nissl staining. Between five groups,
the significant changes in size of the stained cells were not
detected the CA1 (NS/sham: 53.5?3.6 ?m2; F(4,16)?
0.0698; P?0.990), CA3 (NS/sham: 101.3?7.1 ?m2;
F(4,16)?0.0879; P?0.985) regions and dentate gyrus
(NS/sham: 49.7?2.8 ?m2; F(4,16)?1.109; P?0.386) of
the hippocampus. In contrast, density of the stained cells in
CS/OVX rats was lower than that in the other groups.
Table 1 numerically expresses Nissl-positive cells in the
CA1, CA3 regions and dentate gyrus of the hippocampus.
Ovariectomy or chronic restraint stress alone slightly, but
significantly, decreased the Nissl-positive cells, compared
with NS/sham controls, in the hippocampal CA3 region
(F(4,16)?21.020, P?0.0001 by one-way ANOVA, P?0.05
P?0.0001 by one-way ANOVA, P?0.05 by post hoc), but
Fig. 2. Effects of ovariectomy, chronic restraint stress and estrogen treatment on recognition memory in female rats. Rats were exposed to two objects
in the training session (A) and recognition memory was tested 24 h later with a familiar object replaced by a novel object (B). Results are expressed
as percentage time spent exploring one object (A) or the novel object (B) over the total time of object exploration, and means?S.E. (NS/sham: n?13,
NS/OVX: n?10, CS/sham: n?11, CS/OVX: n?17, CS/OVX/E2: n?12). All animals were exposed to chronic restraint stress for 21 days. E2
(20 ?g/day) was administered from 2 days before the stress exposure to the end of behavioral analyses. The horizontal dashed line represents equal
exploration of the novel and familiar objects. * P?0.05, vs. NS/sham,#P?0.05, vs. NS/OVX or CS/sham,†P?0.05, vs. the indicated group (ANOVA
and Student-Newman-Keuls test). (A) F(4,58)?0.671, P?0.615; B: F(4,58)?3.803, P?0.0082.
K. Takuma et al. / Neuroscience 146 (2007) 60–6863
not in the CA1 region (F(4,16)?2.561, P?0.0787), respec-
tively. Moreover, their combination caused a marked de-
crease in cell number of CA3 region (P?0.01 by post hoc,
Table 1), compared with NS/sham controls. Similar effects
were observed in the levels of the hippocampal BDNF
mRNA (F(4,27)?12.593; P?0.0001, Fig. 4), although the
significant changes in the protein levels were not detected
(F(4,21)?0.822; P?0.522). Similar to the behavioral ef-
fect, chronic treatment of E2 (20 ?g/day) attenuated the
CS-induced decrease in neuronal cell density in CA3 re-
gion of the hippocampus (P?0.05 by post hoc, Fig. 3) and
hippocampal BDNF mRNA levels (P?0.05 by post hoc,
Fig. 4) in the OVX rats.
The present study aimed to clarify how depletion of female
sex hormones and environmental stress influence the cog-
nitive function of female animals. We observed that com-
bination of ovariectomy and chronic restraint stress caused
recognition impairment and hippocampal neuronal loss in
female rats, compared with the other groups. We also
found that E2 treatment decreased stress response and
attenuated the behavioral deficits and neuronal loss of
OVX rats induced by the chronic restraint stress. These
behavioral and morphological examinations suggest the
neuroprotective role of estrogen in stress-induced behav-
ioral and neuronal deficits.
The results of this study demonstrated that combina-
tion of ovariectomy and chronic restraint stress decreased
the numbers of hippocampal CA3 pyramidal neurons in
female rats, which was accompanied by recognition im-
pairment. The CA3 pyramidal cells that receive excitatory
inputs from three regions, the dentate gyrus, entorhinal
cortex, and the CA3 themselves (Ishizuka et al., 1990; Lee
Fig. 3. Effects of ovariectomy, chronic restraint stress and estrogen treatment on histological changes in hippocampal CA3 region of female rats.
Nissl-positive cells were visualized by Cresyl Violet staining. Animals were OVX (B, D and E) or sham-operated (A and C) and then exposed to chronic
restraint stress for 21 days (C, D and E). E2 (20 ?g/day) was administered from 2 days before the stress exposure (E). A typical result of three
independent experiments is shown. Scale bar?100 ?m.
Table 1. Effects of ovariectomy, chronic restraint stress and estrogen
treatment on numbers of Nissl-positive cells in hippocampal CA1, CA3
regions and dentate gyrus of female rats
Treatment Nissl-positive cells (number/0.01 mm2)
CA1 CA3 Dentate gyrus
Results are shown as means?S.E. (sham/no stress: n?5, OVX/no
stress: n?4, sham/stress: n?3, OVX/stress: n?5, E2/OVX/stress:
P?0.0001; dentate gyrus: F(4,16)?12.050, P?0.0001.
** P?0.01, vs. no stress/sham.
#P?0.05, vs. no stress/OVX.
††P?0.05, vs. chronic stress/OVX (ANOVA and Student-Newman-
K. Takuma et al. / Neuroscience 146 (2007) 60–6864
et al., 2004), subserve different computational functions,
such as spatial and temporal pattern separation, pattern
association, novelty detection, short-term memory and in-
termediate-term memory (Kesner et al., 2000, 2004; He et
al., 2002). Similar morphological alterations of the hip-
pocampus have been observed in various animal models
leading to cognitive deterioration (Kadar et al., 1994, 1998;
Luine et al., 1994). Taken together, the present results
suggest that the neuronal loss in the CA3 subfield of the
hippocampus plays a fundamental role in the pathogenesis
of memory deficit in menopausal women.
The present study showed that restraint stress ele-
vated the levels of serum corticosterone in both sham-
operated and OVX rats, compared with NS controls, and
that estrogen might attenuate the stress-induced cortico-
sterone secretion. In this regard, it has been shown that
the changes in corticosteroid levels depend on estrous
cycle (Carey et al., 1995). In the brain, corticosteroids are
critical for processes involved in neuronal regeneration
and death and for behaviors involved in learning, memory,
and adaptation (Bohn et al., 1994), through the activation
of two types of corticosteroid receptors, GR and MR (de
Kloet, 2003; Rashid and Lewis, 2005). It has been pro-
posed that the physiological levels of corticosteroids main-
tain a stable activation of MR and the higher levels of
corticosteroids following stress activate GR to normalize
the perturbations of hippocampal activity (Joëls, 1997). It is
notable that both GR and MR are highly expressed in the
hippocampus and subiculum (Arriza et al., 1988; Herman,
1993). A recent study demonstrated that both GR and MR
were colocalized in the CA1 and CA2 pyramidal neurons
and granule cells of the dentate gyrus, while MR was
expressed only in the CA3 pyramidal neurons (Han et al.,
2005). These findings indicate that CA3 pyramidal neurons
may be more vulnerable to stress than other hippocampal
regions. In addition, female rats exposed to stress showed
an increase in MR immunoreactivity in the CA3 subfield of
the hippocampus (Kitraki et al., 2004). Taken together with
our findings, it is suggested that estrogen regulates the
stress responses and plays a neuroprotective role in cor-
ticosteroid-mediated neuronal vulnerability, which may be
especially important in the CS-induced neuronal loss in the
CA3 subfield of the hippocampus. Our findings also sug-
gest that environmental stress and the stress-induced cor-
ticosterone secretion play a crucial role synergistically with
the depletion of estradiol levels in the postmenopausal
cognitive impairment although further studies are required
to address the issue.
It is well documented that estrogen can modulate the
morphology and physiology of the hippocampus, enhance
cognitive function, and protect neurons from a variety of
insults (McEwen and Alves, 1999; Garcia-Segura et al.,
2001). The present study demonstrated that ovariectomy
decreased neuronal cell numbers in both hippocampal
CA3 region and dentate gyrus of female rats and that E2
rescued the neuronal cell decline in both regions.
A recent study has revealed that estradiol enhances
cell proliferation in the dentate gyrus of adult female ro-
dents (Mazzucco et al., 2006). In adult hippocampal neu-
rogenesis, it has been proposed that the newly generated
cells in dentate gyrus mature within 4 weeks and extend
axons to the CA3 region to form mossy fibers, which take
part in synaptic plasticity (Lie et al., 2004; Nicoll and
Schmitz, 2005). These findings suggest that a possible
perturbation of neurogenesis and axonal formation in-
duced by estradiol depletion would be involved in the
development of postmenopausal memory deficits.
Several studies also have shown that, in specific neu-
ronal populations, estrogen regulates the expression of
neurotrophins such as BDNF (Solum and Handa, 2002),
which affects neuronal survival, differentiation, and synap-
tic plasticity (Lu and Chow, 1999; Thoenen, 2000; Yamada
and Nabeshima, 2003). Estrogen can modulate BDNF
expression through an estrogen response element that
has been identified in the BDNF gene in rats (Sohrabji et
al., 1995). The present study also demonstrated that OVX
decreased hippocampal BDNF mRNA levels synergisti-
cally with CS and that E2 treatment attenuated the OVX-
induced changes in BDNF mRNA levels. However, we
could not detect the OVX and/or stress-induced changes in
BDNF protein levels. Regulation of BDNF expression is
complex, because it is synthesized as a precursor pro-
BDNF and processed to its mature form by prohormone
convertases (Seidah et al., 1996). For instance, it has been
reported that estrogen increases BDNF mRNA levels in
the frontal cortex, but decreased cortical BDNF protein
contents (Singh et al., 1995). Thus, in regard to these
paradoxical results, we suppose that estrogen may regu-
late the BDNF expression by translational and post-trans-
Fig. 4. Effects of ovariectomy, chronic restraint stress and estrogen
treatment on BDNF mRNA levels in the hippocampus of female rats.
Results are shown as means?S.E. (NS/sham: n?8, NS/OVX: n?6,
CS/sham: n?5, CS/OVX: n?8, CS/OVX/E2: n?5). ** P?0.01, vs.
NS/sham,#P?0.05, vs. NS/OVX or CS/sham,†P?0.05, vs. the indi-
catedgroup (ANOVA and
K. Takuma et al. / Neuroscience 146 (2007) 60–68 65
lational modifications in addition to transcription. Consis-
tent with our findings, a recent study has revealed that
decrease in pro-BDNF levels is more drastic than those in
mature BDNF in patients of mild cognitive impairment that
is considered a pre-clinical stage of Alzheimer’s disease
(Peng et al., 2005). It is also reported that pro-BDNF can
activate both of Trk B receptors (Mowla et al., 2001) and
p75NTR(Teng et al., 2005; Woo et al., 2005), leading to cell
survival and apoptosis, respectively. Therefore, further
studies are required to address whether pro-BDNF is in-
volved in the estrogen and stress-mediated memory
On the other hand, several in vitro studies have shown
that estrogen rapidly activates cell growth and survival
signal pathways, the mitogen-activated protein kinase cas-
cade (Carrer et al., 2005) and the phosphoinositol-3 kinase
cascade (Yu et al., 2004). Estrogen is thought to exert
neuroprotective effects by several mechanisms, acting
through two classical nuclear ERs, ER? and ER?, which
are highly expressed in CA3 pyramidal layer in normal
adult rats (Mehra et al., 2005; Shughrue and Merchentha-
ler, 2000), and through membrane-associated ER, which
activates G-protein-associated intracellular signaling cas-
cade (Revankar et al., 2005), leading to enhanced mito-
chondrial function, increased calcium load tolerance, and
promotion of antioxidant defense mechanisms (Morrison et
al., 2006). Accordingly, although the exact mechanisms of
the region-specific neuronal cell death and E2 action in the
CA3 subfield of the hippocampus are still unclear in our
animal model, we assume that E2 may mediate its neuro-
protective effects and improve the stress-induced cognitive
impairment through BDNF expression and activation of
ERs-mediated cell survival signaling.
Luine and co-workers (Wallace et al., 2006) have re-
cently reported that ovariectomy alone decreases recog-
nition memory (1 week after OVX) and spine density in the
hippocampal CA1 region and prefrontal cortex (7 weeks
after OVX) of female Sprague–Dawley rats, although these
findings differ from our observation in Fisher 344 rats
(11–13 weeks after OVX). Luine et al. (2003) also showed
that acute treatment of estradiol enhanced visual and
place memory in OVX rats. Consistently, E2 (20 ?g/day)
treatment in the present study, which was referred to pre-
vious studies (Mukundan et al., 2002; Resta et al., 2001;
Zhou et al., 2005), prevented the stress-induced memory
impairment in CS/OVX rats, but the serum estradiol levels
reached abnormally higher levels than sham-operated
control rats. Therefore, to elucidate the molecular basis of
estrogen-mediated memory function, further studies are
required with careful consideration to the recovery period
after ovariectomy, strains and species of animals, and
duration and doses of drug treatments.
In conclusion, our study demonstrates that combination of
ovariectomy and chronic restraint stress affects the mor-
phology and function of the hippocampal CA3 region and
induces recognition impairment in female rats. We propose
that the loss of CA3 pyramidal neurons in the hippocampus
of OVX rats may be associated with the diminution of
neuroprotective mechanism of estradiol such as BDNF
expression. Estrogen replacement therapy could thus offer
an interesting approach to prevent cognitive disorders in
postmenopausal women. Further, the present study sug-
gests that the CS/OVX rats are useful in the capacity of an
animal model of postmenopausal memory deficits.
Acknowledgments—This study was supported in part by a grant
for the 21st Century COE Program from the Ministry of Education,
Culture, Sports, Science and Technology of Japan, a Grant-in-Aid
for Scientific Research from the Japan Society for the Promotion
of Science, and a grant from Kanzawa Medical Research Foun-
Arriza JL, Simerly RB, Swanson LW, Evans RM (1988) The neuronal
mineralocorticoid receptor as a mediator of glucocorticoid re-
sponse. Neuron 1:887–900.
Behl C (2002) Oestrogen as a neuroprotective hormone. Nat Rev
Bohn MC, O’Banion MK, Young DA, Giuliano R, Hussain S, Dean DO,
Cunningham LA (1994) In vitro studies of glucocorticoid effects on
neurons and astrocytes. Ann N Y Acad Sci 746:243–258.
Bowman RE, Ferguson D, Luine VN (2002) Effects of chronic restraint
stress and estradiol on open field activity spatial memory and
monoaminergic neurotransmitters in ovariectomized rats. Neuro-
Bowman RE, Zrull MC, Luine VN (2001) Chronic restraint stress
enhances radial arm maze performance in female rats. Brain Res
Carey MP, Deterd CH, de Koning J, Helmerhorst F, de Kloet ER (1995)
The influence of ovarian steroids on hypothalamic-pituitary-adrenal
regulation in the female rat. J Endocrinol 144:311–321.
Carrer HF, Cambiasso MJ, Gorosito S (2005) Effects of estrogen on
neuronal growth and differentiation. J Steroid Biochem Mol Biol
de Kloet ER (2003) Hormones brain and stress. Endocr Regul
Espeland MA, Rapp SR, Shumaker SA, Brunner R, Manson JE,
Sherwin BB, Hsia J, Margolis KL, Hogan PE, Wallace R, Dailey M,
Freeman R, Hays J; Women’s Health Initiative Memory Study
(2004) Conjugated equine estrogens and global cognitive function
in postmenopausal women: Women’s Health Initiative Memory
Study. JAMA 291:2959–2968.
Garcia-Segura LM, Azcoitia I, DonCarlos LL (2001) Neuroprotection
by estradiol. Prog Neurobiol 63:29–60.
Genazzani AR, Monteleone P, Gambacciani M (2002) Hormonal in-
fluence on the central nervous system. Maturitas 43:S11–S17.
Gibbs RB, Aggarwal P (1998) Estrogen and basal forebrain cholinergic
neurons: implications for brain aging and Alzheimer’s disease-
related cognitive decline. Horm Behav 34:98–111.
Gould E, Woolley CS, Frankfurt M, McEwen BS (1990) Gonadal
steroids regulate dendritic spine density in hippocampal pyramidal
cells in adulthood. J Neurosci 10:1286–1291.
Han F, Ozawa H, Matsuda K, Nishi M, Kawata M (2005) Colocalization
of mineralocorticoid receptor and glucocorticoid receptor in the
hippocampus and hypothalamus. Neurosci Res 51:371–381.
He J, Yamada K, Nabeshima T (2002) A role of Fos expression in the
CA3 region of the hippocampus in spatial memory formation in
rats. Neuropsychopharmacology 26:259–268.
Herman JP (1993) Regulation of adrenocorticosteroid receptor mRNA
expression in the central nervous system. Cell Mol Neurobiol
K. Takuma et al. / Neuroscience 146 (2007) 60–68 66
Hogervorst E, Williams J, Budge M, Riedel W, Jolles J (2000) The
nature of the effect of female gonadal hormone replacement ther-
apy on cognitive function in post-menopausal women: a meta-
analysis. Neuroscience 101:485–512.
Ishizuka N, Weber J, Amaral DG (1990) Organization of intrahip-
pocampal projections originating from CA3 pyramidal cells in the
rat. J Comp Neurol 295:580–623.
Joëls M (1997) Steroid hormones and excitability in the mammalian
brain. Front Neuroendocrinol 18:2–48.
Kadar T, Arbel I, Silbermann M, Levy A (1994) Morphological hip-
pocampal changes during normal aging and their relation to cog-
nitive deterioration. J Neural Transm Suppl 44:133–143.
Kadar T, Dachir S, Shukitt-Hale B, Levy A (1998) Sub-regional hip-
pocampal vulnerability in various animal models leading to cogni-
tive dysfunction. J Neural Transm 105:987–1004.
Kamei H, Nagai T, Nakano H, Togan Y, Takayanagi M, Takahashi K,
Kobayashi K, Yoshida S, Maeda K, Takuma K, Nabeshima T,
Yamada K (2006) Repeated methamphetamine treatment impairs
recognition memory through a failure of novelty-induced ERK1/2
activation in the prefrontal cortex of mice. Biol Psychiatry 59:
Kesner RP, Gilbert PE, Wallenstein GV (2000) Testing neural network
models of memory with behavioral experiments. Curr Opin Neuro-
Kesner RP, Lee I, Gilbert PE (2004) A behavioral assessment of
hippocampal function based on a subregional analysis. Rev Neu-
Kitraki E, Kremmyda O, Youlatos D, Alexis MN, Kittas C (2004) Gen-
der-dependent alterations in corticosteroid receptor status and
spatial performance following 21 days of restraint stress. Neuro-
LeBlanc ES, Janowsky J, Chan BK, Nelson HD (2001) Hormone
replacement therapy and cognition: systematic review and meta-
analysis. JAMA 285:1489–1499.
Lee I, Rao G, Knierim JJ (2004) A double dissociation between hip-
pocampal subfields: differential time course of CA3 and CA1 place
cells for processing changed environments. Neuron 42:803–815.
Leranth C, Shanabrough M (2001) Supramammillary area mediates
subcortical estrogenic action on hippocampal synaptic plasticity.
Exp Neurol 167:445–450.
Leranth C, Shanabrough M, Horvath TL (2000) Hormonal regulation of
hippocampal spine synapse density involves subcortical media-
tion. Neuroscience 101:349–356.
Lie DC, Song H, Colamarino SA, Ming GL, Gage FH (2004) Neuro-
genesis in the adult brain: new strategies for central nervous
system diseases. Annu Rev Pharmacol Toxicol 44:399–421.
Lu B, Chow A (1999) Neurotrophins and hippocampal synaptic trans-
mission and plasticity. J Neurosci Res 58:76–87.
Luine VN, Jacome LF, Maclusky NJ (2003) Rapid enhancement of
visual and place memory by estrogens in rats. Endocrinology
Luine VN, Richards ST, Wu VY, Beck KD (1998) Estradiol enhances
learning and memory in a spatial memory task and effects levels of
monoaminergic neurotransmitters. Horm Behav 34:149–162.
Luine V, Villegas M, Martinez C, McEwen BS (1994) Repeated stress
causes reversible impairments of spatial memory performance.
Brain Res 639:167–170.
Magariños AM, Verdugo JM, McEwen BS (1997) Chronic stress alters
synaptic terminal structure in hippocampus. Proc Natl Acad Sci
U S A 94:14002–14008.
Magariños AM, McEwen BS (1995) Stress-induced atrophy of apical
dendrites of hippocampal CA3c neurons: involvement of glucocor-
ticoid secretion and excitatory amino acid receptors. Neuroscience
Mazzucco CA, Lieblich SE, Bingham BI, Williamson MA, Viau V, Galea
LA (2006) Both estrogen receptor a and estrogen receptor b ago-
nists enhance cell proliferation in the dentate gyrus of adult female
rats. Neuroscience 141:1793–1800.
McEwen BS (2001) Estrogens effects on the brain: multiple sites and
molecular mechanisms. J Appl Physiol 91:2785–2801.
McEwen BS, Alves SE (1999) Estrogen actions in the central nervous
system. Endocr Rev 20:279–307.
McEwen BS, Woolley CS (1994) Estradiol and progesterone regulate
neuronal structure and synaptic connectivity in adult as well as
developing brain. Exp Gerontol 29:431–436.
McEwen BS, Sapolsky RM (1995) Stress and cognitive function. Curr
Opin Neurobiol 5:205–216.
McEwen BS, De Kloet ER, Rostene W (1986) Adrenal steroid recep-
tors and actions in the nervous system. Physiol Rev 66:
Mehra RD, Sharma K, Nyakas C, Vij U (2005) Estrogen receptor a and
b immunoreactive neurons in normal adult and aged female rat
hippocampus: a qualitative and quantitative study. Brain Res
Mizuno M, Yamada K, Olariu A, Nawa H, Nabeshima T (2000) Involve-
ment of brain-derived neurotrophic factor in spatial memory forma-
tion and maintenance in a radial arm maze test in rats. J Neurosci
Morrison JH, Brinton RD, Schmidt PJ, Gore AC (2006) Estrogen,
menopause, and the aging brain: how basic neuroscience can
inform hormone therapy in women. J Neurosci 26:10332–10348.
Mowla SJ, Farhadi HF, Pareek S, Atwal JK, Morris SJ, Seidah NG,
Murphy RA (2001) Biosynthesis and post-translational processing
of the precursor to brain-derived neurotrophic factor. J Biol Chem
Mukundan H, Resta TC, Kanagy NL (2002) 17b-Estradiol decreases
hypoxic induction of erythropoietin gene expression. Am J Physiol
Regul Integr Comp Physiol 283:R496–R504.
Mulnard RA, Cotman CW, Kawas C, van Dyck CH, Sano M, Doody R,
Koss E, Pfeiffer E, Jin S, Gamst A, Grundman M, Thomas R, Thal
LJ (2000) Estrogen replacement therapy for treatment of mild to
moderate Alzheimer disease: a randomized controlled trial. JAMA
Nicoll RA, Schmitz D (2005) Synaptic plasticity at hippocampal mossy
fibre synapses. Nat Rev Neurosci 6:863–876.
Nitta A, Ohmiya M, Sometani A, Itoh M, Nomoto H, Furukawa Y,
Furukawa S (1999) Brain-derived neurotrophic factor prevents
neuronal cell death induced by corticosterone. J Neurosci Res
Peng S, Wuu J, Mufson EJ, Fahnestock M (2005) Precursor form of
brain-derived neurotrophic factor and mature brain-derived neuro-
trophic factor are decreased in the pre-clinical stages of Alzhei-
mer’s disease. J Neurochem 93:1412–1421.
Rapp SR, Espeland MA, Shumaker SA, Henderson VW, Brunner RL,
Manson JE, Gass ML, Stefanick ML, Lane DS, Hays J, Johnson
KC, Coker LH, Dailey M, Bowen D; WHIMS Investigators (2003)
Effect of estrogen plus progestin on global cognitive function in
postmenopausal women: the Women’s Health Initiative Memory
Study: a randomized controlled trial. JAMA 289:2663–2672.
Rashid S, Lewis GF (2005) The mechanisms of differential glucocor-
ticoid and mineralocorticoid action in the brain and peripheral
tissues. Clin Biochem 38:401–409.
Resta TC, Kanagy NL, Walker BR (2001) Estradiol-induced attenua-
tion of pulmonary hypertension is not associated with altered
eNOS expression. Am J Physiol Lung Cell Mol Physiol 280:
Reul JM, de Kloet ER (1985) Two receptor systems for corticosterone
in rat brain: microdistribution and differential occupation. Endocri-
Revankar CM, Cimino DF, Sklar LA, Arterburn JB, Prossnitz ER
(2005) A transmembrane intracellular estrogen receptor mediates
rapid cell signaling. Science 307:1625–1630.
Sandi C (2004) Stress, cognitive impairment and cell adhesion mole-
cules. Nat Rev Neurosci 5:917–930.
K. Takuma et al. / Neuroscience 146 (2007) 60–6867
Sandstrom NJ, Williams CL (2001) Memory retention is modulated by Download full-text
acute estradiol and progesterone replacement. Behav Neurosci
Sandstrom NJ, Williams CL (2004) Spatial memory retention is en-
hanced by acute and continuous estradiol replacement. Horm
Seidah NG, Benjannet S, Pareek S, Chretien M, Murphy RA (1996)
Cellular processing of the neurotrophin precursors of NT3 and
BDNF by the mammalian proprotein convertases. FEBS Lett
Shughrue PJ, Merchenthaler I (2000) Evidence for novel estrogen
binding sites in the rat hippocampus. Neuroscience 99:605–
Singh M, Meyer EM, Simpkins JW (1995) The effect of ovariectomy
and estradiol replacement on brain-derived neurotrophic factor
messenger ribonucleic acid expression in cortical and hippocampal
brain regions of female Sprague-Dawley rats. Endocrinology
Smith MA, Makino S, Kvetnansky R, Post RM (1995) Effects of stress
on neurotrophic factor expression in the rat brain. Ann N Y Acad
Sohrabji F, Miranda RC, Toran-Allerand CD (1995) Identification of a
putative estrogen response element in the gene encoding brain-
derived neurotrophic factor. Proc Natl Acad Sci U S A 92:
Solum DT, Handa RJ (2002) Estrogen regulates the development of
brain-derived neurotrophic factor mRNA and protein in the rat
hippocampus. J Neurosci 22:2650–2659.
Teng HK, Teng KK, Lee R, Wright S, Tevar S, Almeida RD, Kermani
P, Torkin R, Chen ZY, Lee FS, Kraemer RT, Nykjaer A, Hempstead
BL (2005) ProBDNF induces neuronal apoptosis via activation of a
receptor complex of p75NTR and sortilin. J Neurosci 25:
Thoenen H (2000) Neurotrophins and activity-dependent plasticity.
Prog Brain Res 128:183–191.
Vaidya VA, Marek GJ, Aghajanian GK, Duman RS (1997) 5-HT2A
receptor-mediated regulation of brain-derived neurotrophic factor
mRNA in the hippocampus and the neocortex. J Neurosci
Vaidya VA, Terwilliger RM, Duman RS (1999) Role of 5-HT2Arecep-
tors in the stress-induced down-regulation of brain-derived neuro-
trophic factor expression in rat hippocampus. Neurosci Lett
Wallace M, Luine V, Arellanos A, Frankfurt M (2006) Ovariectomized
rats show decreased recognition memory and spine density in the
hippocampus and prefrontal cortex. Brain Res 1126:176–182.
Woo NH, Teng HK, Siao CJ, Chiaruttini C, Pang PT, Milner TA,
Hempstead BL, Lu B (2005) Activation of p75NTRby proBDNF
facilitates hippocampal long-term depression. Nat Neurosci 8:
Woods NF, Mitchell ES, Adams C (2000) Memory functioning among
midlife women: observations from the Seattle Midlife Women’s
Health Study. Menopause 7:257–265.
Woolley CS, Gould E, Frankfurt M, McEwen BS (1990) Naturally
occurring fluctuation in dendritic spine density on adult hippocam-
pal pyramidal neurons. J Neurosci 10:4035–4039.
Woolley CS, McEwen BS (1992) Estradiol mediates fluctuation in
hippocampal synapse density during the estrous cycle in the adult
rat. J Neurosci 12:2549–2554.
Yamada K, Nabeshima T (2003) Brain-derived neurotrophic factor/TrkB
signaling in memory processes. J Pharmacol Sci 91:267–270.
Yamada K, Takayanagi M, Kamei H, Nagai T, Dohniwa M, Kobayashi
K, Yoshida S, Ohhara T, Takuma K, Nabeshima T (2005) Effects
of memantine and donepezil on amyloid ?-induced memory im-
pairment in a delayed-matching to position task in rats. Behav
Brain Res 162:191–199.
Yu X, Rajala RV, McGinnis JF, Li F, Anderson RE, Yan X, Li S, Elias
RV, Knapp RR, Zhou X, Cao W (2004) Involvement of insulin/
phosphoinositide 3-kinase/Akt signal pathway in 17?-estradiol-me-
diated neuroprotection. J Biol Chem 279:13086–13094.
Zapantis G, Santoro N (2003) The menopausal transition: character-
istics and management. Best Pract Res Clin Endocrinol Metab
Zhou J, Zhang H, Cohen RS, Pandey SC (2005) Effects of estrogen
treatment on expression of brain-derived neurotrophic factor
and cAMP response element-binding protein expression and
phosphorylation in rat amygdaloid and hippocampal structures.
(Accepted 13 January 2007)
(Available online 22 February 2007)
K. Takuma et al. / Neuroscience 146 (2007) 60–6868