17-ESTRADIOL ATTENUATES HIPPOCAMPAL NEURONAL LOSS

AND COGNITIVE DYSFUNCTION INDUCED BY CHRONIC RESTRAINT

STRESS IN OVARIECTOMIZED RATS
K. TAKUMA,
a
A. MATSUO,
a
Y. HIMENO,
a
Y. HOSHINA,
a
Y. OHNO,
a
Y. FUNATSU,
a
S. ARAI,
a
H. KAMEI,
a
H. MIZOGUCHI,
a
T. NAGAI,
a
K. KOIKE,
b
M. INOUE
b
AND K. YAMADA
a
*
a
Laboratory of Neuropsychopharmacology, Graduate School of Natu-
ral Science and Technology, Kanazawa University, Kakuma-machi,
Kanazawa 920-1192, Japan
b
Department of Obstetrics and Gynecology, Kanazawa University
Graduate School of Medical Science, 13-1 Takara-machi, Kanazawa
920-8641, Japan
AbstractSeveral 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 Alzheimers 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 signicant 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-
pus.
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 Womens 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 decits. 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 ndings
suggest that other additional factors besides the ovarian
hormone loss are required for the development of post-
menopausal memory decits.
In addition to ovarian hormones, environmental stress
is known to cause morphological changes in the hip-
pocampus. Stress activates the hypothalamicpituitary
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: kyamada@p.kanazawa-u.ac.jp (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
chain reaction.
Neuroscience 146 (2007) 6068
0306-4522/07$30.000.00 2007 IBRO. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuroscience.2007.01.017
60
with a 10-fold higher afnity (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 (Magarios 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 decits 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.
EXPERIMENTAL PROCEDURES
Animals and treatment
Female Fisher 344 rats (Charles River Japan, Yokohama, Japan)
weighing 160200 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 231 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 lled 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 (Magarios et al., 1997) that
allowed for a close t 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 lled 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 800g
for 10 min, the serum was separated and stored at 80 C until
assayed. Serum concentrations of estradiol and corticosterone
were quantied 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.
Behavioral analysis
For the novel object recognition test, a rat was habituated to a gray
empty open eld (35 cm high60 cm diameter) for 3 days (10
min/day). In the training session, two novel objects were placed in
the eld, 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 eld, 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 xed to the
oor 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-xed with the same xative 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) 6068 61
according to standard procedures (Yamada et al., 2005). A uo-
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 amplication protocol consisted
of a rst 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 quantied 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 manufactures instructions. In brief, the tissues were homog-
enized in ve volumes of lysis buffer (137 mM NaCl, 20 mM
TrisHCl, 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 1500g for 15 min.
The resulting supernatants were assayed for BDNF determina-
tion. The 96-well, at-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 7.8500 pg/ml, were prepared using
recombinant human BDNF. The captured NGF was reacted rst
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.
Statistics
Statistical analysis of the experimental data were carried out using
StatView 5.0 Windows. The signicance of differences was deter-
mined by Students 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
signicance was P0.05.
RESULTS
Effects of ovariectomy and estrogen treatment on
serum estradiol levels and corticosterone responses
against restraint stress in female rats
Ovariectomy signicantly decreased the serum estradiol
levels (15.20.96 pg/ml, n10; P0.05 by Students
t-test), compared with sham-operated control group includ-
ing all estrous cycle rats (25.73.7 pg/ml, n12). After the
21-day restraint stress period, serum estrogen levels in E2
(20 g/day)-treated rats reached 40233.2 pg/ml (n3),
which was signicantly higher than the levels in other two
groups (F(2,22)495.6, P0.0001 by one-way ANOVA,
P0.01 by post hoc).
Ovariectomy did not affect the basal serum corticoste-
rone levels (56886.2 ng/ml, n12; P0.3546 by Stu-
dents t-test) compared with sham-operated control
(730147 ng/ml, n10). 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 rst restraint stress were determined by an ELISA system.
Results are shown as meansS.E. (sham-operated control group
(NS/sham): n3, OVX group (NS/OVX): n5, sham-operated stress
group (CS/sham): n5, OVX plus stress group (CS/OVX): n5, E2-
treated OVX plus stress group (CS/OVX/E2): n3). ** P0.01, vs.
NS/sham,
#
P0.05, vs. CS/OVX (ANOVA and Student-Newman-
Keuls test). F(4,16)8.657, P0.0006.
K. Takuma et al. / Neuroscience 146 (2007) 6068 62
had no effect on the basal serum corticosterone levels
(576306 ng/ml, n3, F(2,20)0.456, P0.6405), com-
pared with sham-operated and OVX groups. The effects of
restraint stress and E2 on changes in serumcorticosterone
levels were still observed 6 h after the beginning of re-
straint stress (F(4,16)7.353, P0.0015).
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, P0.391), total exploratory time
(F(4,58)1.943, P0.1154) and exploratory preference to
two objects (F(4,58)0.671, P0.615, Fig. 2A) in female
rats during the training session of the novel object recog-
nition test.
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/OVX and CS/sham rats (F(4,58)3.803,
P0.01 by one-way ANOVA, P0.05 by post hoc, Fig.
2B), although there was no difference in total exploratory
time between these groups (F(4,58)2.198, P0.0804).
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 (P0.05 by post
hoc, Fig. 2B).
Effects of CS and E2 treatment on neuronal cell
numbers and BDNF levels in the hippocampus of
female rats
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 ve groups,
the signicant changes in size of the stained cells were not
detected the CA1 (NS/sham: 53.53.6 m
2
; F(4,16)
0.0698; P0.990), CA3 (NS/sham: 101.37.1 m
2
;
F(4,16)0.0879; P0.985) regions and dentate gyrus
(NS/sham: 49.72.8 m
2
; F(4,16)1.109; P0.386) 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
signicantly, decreased the Nissl-positive cells, compared
with NS/sham controls, in the hippocampal CA3 region
(F(4,16)21.020, P0.0001 by one-way ANOVA, P0.05
by post hoc) and dentate gyrus (F(4,16)12.050,
P0.0001 by one-way ANOVA, P0.05 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 meansS.E. (NS/sham: n13,
NS/OVX: n10, CS/sham: n11, CS/OVX: n17, CS/OVX/E2: n12). 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. * P0.05, vs. NS/sham,
#
P0.05, vs. NS/OVX or CS/sham,

P0.05, vs. the indicated group (ANOVA
and Student-Newman-Keuls test). (A) F(4,58)0.671, P0.615; B: F(4,58)3.803, P0.0082.
K. Takuma et al. / Neuroscience 146 (2007) 6068 63
not in the CA1 region (F(4,16)2.561, P0.0787), respec-
tively. Moreover, their combination caused a marked de-
crease in cell number of CA3 region (P0.01 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; P0.0001, Fig. 4), although the
signicant changes in the protein levels were not detected
(F(4,21)0.822; P0.522). 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 (P0.05 by post hoc, Fig. 3) and
hippocampal BDNF mRNA levels (P0.05 by post hoc,
Fig. 4) in the OVX rats.
DISCUSSION
The present study aimed to clarify how depletion of female
sex hormones and environmental stress inuence 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 decits 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 decits.
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 bar100 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 mm
2
)
CA1 CA3 Dentate gyrus
No stress
Sham 40.61.6 53.12.1 124.84.1
OVX 36.81.3 44.81.2** 87.96.9**
Chronic stress
Sham 37.41.4 42.20.3** 99.49.3
OVX 35.11.1 37.20.5**
,#
75.73.5**
OVXE2 37.61.2 49.51.4

101.46.3

Results are shown as meansS.E. (sham/no stress: n5, OVX/no

stress: n4, sham/stress: n3, OVX/stress: n5, E2/OVX/stress:
n4). CA1: F(4,16)2.561, P0.0787; CA3: F(4,16)21.020,
P0.0001; dentate gyrus: F(4,16)12.050, P0.0001.
** P0.01, vs. no stress/sham.
#
P0.05, vs. no stress/OVX.

P0.05.

P0.05, vs. chronic stress/OVX (ANOVA and Student-Newman-

Keuls test).
K. Takuma et al. / Neuroscience 146 (2007) 6068 64
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 subeld of the
hippocampus plays a fundamental role in the pathogenesis
of memory decit 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 (Jols, 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 ndings 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 subeld of
the hippocampus (Kitraki et al., 2004). Taken together with
our ndings, 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 subeld of the hippocampus. Our ndings 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 bers, which take
part in synaptic plasticity (Lie et al., 2004; Nicoll and
Schmitz, 2005). These ndings suggest that a possible
perturbation of neurogenesis and axonal formation in-
duced by estradiol depletion would be involved in the
development of postmenopausal memory decits.
Several studies also have shown that, in specic 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 identied 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 meansS.E. (NS/sham: n8, NS/OVX: n6,
CS/sham: n5, CS/OVX: n8, CS/OVX/E2: n5). ** P0.01, vs.
NS/sham,
#
P0.05, vs. NS/OVX or CS/sham,

P0.05, vs. the indi-
cated group (ANOVA and Student-Newman-Keuls test).
F(4,27)12.593, P0.0001.
K. Takuma et al. / Neuroscience 146 (2007) 6068 65
lational modications in addition to transcription. Consis-
tent with our ndings, 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 Alzheimers disease
(Peng et al., 2005). It is also reported that pro-BDNF can
activate both of Trk B receptors (Mowla et al., 2001) and
p75
NTR
(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
function.
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-specic neuronal cell death and E2 action in the
CA3 subeld 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 SpragueDawley rats, although these
ndings differ from our observation in Fisher 344 rats
(1113 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.
CONCLUSION
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 decits.
AcknowledgmentsThis 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 Scientic Research from the Japan Society for the Promotion
of Science, and a grant from Kanzawa Medical Research Foun-
dation.
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(Accepted 13 January 2007)
(Available online 22 February 2007)
K. Takuma et al. / Neuroscience 146 (2007) 6068 68