Pharmacology & Pharmacy, 2011, 2, 10-16
doi:10.4236/pp.2011.21002 Published Online January 2011 (http://www.SciRP.org/journal/pp)
Restraint-Induced Expression of Endoplasmic
Reticulum Stress-Related Genes in the Mouse
Brain
Mitsue Ishisaka1, Takashi Kudo2, Masamitsu Shimazawa1, Kenichi Kakefuda1, Atsushi Oyagi1,
Kana Hyakkoku1, Kazuhiro Tsuruma1, Hideaki Hara1
1Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University; 2Department of Psychiatry,
Osaka University Graduate School of Medicine.
Received October 26th, 2010; revised November 23rd, 2010; accepted November 30th, 2010.
ABSTRACT
Depression is a significant public health concern but its pathology remains unclear. Previously, increases in an endo-
plasmic reticulum (ER) stress-related protein were reported in the temporal cortex of subjects with major depressive
disorder who had died by suicide. This finding suggests an association between depression and ER stress. The present
study was designed to investigate whether acute stress co uld affect th e ER stress response. Mice were immobilized fo r a
period of 6 hr and then expression of ER stress response-related genes was measured by real-time PCR. We also used
enzyme-linked immunosorbent assay for concomitant measurement of the plasma corticosterone levels in the mice. The
effect of corticosterone on ER stress proteins was fu rther investigated by treatin g mice with corticosterone for 2 weeks
and then measuring ER protein expression by Western blotting. After a 6 hr restraint stress, mRNA levels of ER
stress-related genes, such as the 78-kilodalton glucose regulated protein (GRP78), the 94-kilodalton glucose regulated
protein (GRP94), and calreticulin, were increased in the cortex, hippocampus, and striatum of mouse brain. Blood
plasma corticosterone level was also increased. In the corticosterone-treated mouse model, the expression of GRP78
and GRP94 was significan tly increased in the hippo campus. These re sults suggest tha t acute stress may affect ER func-
tion and that ER stress may be involve d in the pathogenesis of restraint stress, including the development of depression.
Keywords: Corticosteron e, Depression, Endoplasmic Reticulum Stress, Restraint Stress
1. Introduction
Major depression, along with bipolar disorder, has be-
come a common psychiatric disorder in modern society.
About 1% of the population is estimated to be affected
by major depression one or more times during their life-
time [1]. Even though extensive studies have led to a
variety of hypotheses regarding the molecular mecha-
nism underlying depression, the pathogenesis of this dis-
order remains to be fully elucidated.
The endoplasmic reticulum (ER) is the cell organelle
where secretory and membrane proteins are synthesized
and folded. It also functions as a Ca2+ store and resource
of calcium signals. The disturbance of ER functions
through events such as disruption of Ca2+ homeostasis,
inhibition of protein glycosylation or disulfide bond for-
mation, hypoxia and viral or bacterial infection, can re-
sult in the accumulation of unfolded or misfolded pro-
teins and may trigger stress responses in the cell (ER
stress). To overcome ER stress, an unfolded protein re-
sponse (UPR) is invoked by the activation of several
signaling pathways; this UPR promotes an adaptive re-
sponse to ER stress and reestablishes homeostasis in the
ER [2,3]. Molecular chaperones such as the 78-kilodalton
glucose regulated protein (GRP78) and the 94-kilodalton
glucose regulated protein are induced and promote cor-
rect protein folding. If the damage is too severe to repair,
C/EBP-homologous protein (CHOP) and other factors
are activated and induce cell apoptosis [4]. On the other
hand, if misfolded protein aggregates into insoluble
higher-order structures, it can give rise to various dis-
eases. For example, rhodopsin misfolding causes auto-
somal dominant retinitis pigmentosa [5], while the ac-
cumulation of amyloid β-peptide is associated with Alz-
heimer’s disease [6].

Restraint-Induced Expression of Endoplasmic Reticulum Stress-Related Genes in the Mouse Brain 11
Some reports have also suggested a relationship be-
tween mental disorder and ER stress. In bipolar disorder
patients, DNA microarray analysis of cell derived from
twins discordant with respect to the disease revealed a
down-regulated expression of genes related to ER stress
responses such as x-box binding protein 1 (XBP1) and
GRP78 [7]. In schizophrenia patients, a similar abnor-
mality of these genes was found [8]. In addition,
mood-stabilizing drugs such as valproate and lithium
have been reported to increase the expression of GRP78,
GRP94, and calreticulin [9]. Similarly, olanzapine, one
of the second-generation “atypical” anti-psychotic drugs,
appears to potentiates neuronal survival and neural stem
cell differentiation by regulation of ER stress response
proteins [10].
A recent study reported that significantly increased
levels of GRP78, GRP94, and calreticulin were found in
the temporal cortex of subjects with major depressive
disorder who had died by suicide compared with control
subjects who had died of other causes [11]. In addition,
hippocampal atrophy [12] and reduction of glial density
in the subgenual prefrontal cortex [13] were found in
patients with major depression. Stress, a risk factor for
depression, has been shown to induce atrophy of the api-
cal dendrites of the hippocampal neurons [14], and to
promote neuronal apoptosis in the cerebral cortex [15] in
animal depression models. These findings suggest that a
stressful situation, which may increase the risk for sui-
cide, serves as an ER stressor. To clarify the relationship
between exogenous stress and ER stress, in the present
study, we investigated the expression of ER stress-related
genes after restraint stress. We also focused on the eleva-
tion of corticosterone in the plasma and used a corticos-
terone-treated depression model to clarify the relation-
ship between chronic corticosterone elevation and ER
stress.
2. Materials and Methods
2.1. Animals
Male 9-week-old ddY mice and male 6-week-old ICR
mice (Japan SLC, Hamamatsu, Japan) were used for all
experiments. Mice were housed at 24 ± 2˚C under a 12 hr
light-dark cycle (lights on from 8:00 to 20:00) and had ad
libitum access to food and water when not under restraint.
Animals were acclimatized to laboratory conditions be-
fore the experiment. All procedures relating to animal
care and treatment conformed to the animal care guide-
lines of the Animal Experiment Committee of Gifu
Pharmaceutical University. All efforts were made to
minimize both suffering and the number of animal used.
2.2. Restraint Stress
Male 9-week-old ddY mice (Japan SLC) weighing 30-40
g were used for real-time PCR studies. Mice were placed
into 50-mL perforated plastic tubes, which prevented
them from turning in any direction. Each mouse was
maintained in the tube for 6 hr without any access to food
or water.
2.3. Sampling
After this restraint stress, a blood sample was collected
from the tail and the mouse was decapitated. The brain
was quickly removed from the skull, briefly washed in
ice-cold saline, and laid on a cooled (4˚C) metal plate.
The brain was rapidly dissected to separate the hippo-
campus, striatum, and cortex and stored at -80°C until
use.
2.4. RNA Isolation
Total RNA was isolated from frozen brain using High
Pure RNA Isolation Kit (Roche, Tokyo, Japan). RNA
concentrations were determined spectrophotometrically
at 260 nm. First-standed cDNA was synthesized in a
20-μl reaction volum using a random primer (Takara,
Shiga, Japan) and Moloney murine leukemia virus re-
verse transcriptase (Invitrogen, Carlsbad, CA, USA).
2.5. Reai-Time PCR
Real-time PCR (TaqMan; Applied Biosystems, Foster
City, CA, USA) was performed as described previously
[16]. Single-standard cDNA was synthesized from total
RNA using a high capacity cDNA archive kit (Applied
Biosystems). Quantitative real-time PCR was performed
using a sequence detection system (ABI PRISM 7900HT;
Applied Biosystems) with a PCR master mix (TaqMan
Universal PCR Master Mix; Applied Biosystems), ac-
cording to the manufacturer’s protocol. A gene expression
product (Assays-on-Demand Gene Expression Product;
Applied Biosystems) was used for measurements of
mRNA expression by real-time PCR. The primers used
for amplification were as follows: GRP78: 5’-GTTTG
CTGAGGAAGACAAAAAGCTC-3’ and 5’-CACTTCC
ATAGAGTTTGCTGATAATTG-3’; CHOP: 5’-GGAG
CTGGAAGCCTGGTATGAGG-3’ and 5’-TCCCTGGT
CAGGCGCTCGATTTCC-3’; GRP94: 5’-CTCACCATT
TGGATCCTGTGTG-3’ and 5’-CACATGACAAGATT
TTACATCAAGA-3’; calreticulin: 5’-GCCAAGGACG
AGCTGTAGAGAG-3’ and 5’-GGTGAGGGCTGAAG
GAGAATC-3’; ERdj4: 5’-TCTAGAATGGCTACTCCC
CAGTCAATTTTC-3’ and 5’-TCTAGACTACTGTCCT
GAACAGTCAGTG-3’; EDEM: 5’-TGGGTTGGAAAG
CAGAGTGGC-3’ and 5’-TCCATTCCTACATGGAGG
TAG-3’; p58IPK 5’-GAGGTTTGTGTTTGGGATGCAG-
3’ and 5’-GCTCTTCAGCTGACTCAATCAG-3’; ASNS:
5’-AGGTTGATGATGCAATGATGG-3’ and 5’-TCCC
CTATCTACCCACAGTCC-3’; β-actin: 5’-TCCTCCCT

Restraint-Induced Expression of Endoplasmic Reticulum Stress-Related Genes in the Mouse Brain 12
GGAGAAGAGCTAC-3’ and 5’-TCCTGCTTGCTGAT
CCACAT-3’ The thermal cycler conditions were as fol-
lows: 2 min at 50˚C and then 10 min at 95˚C, followed
by two-step PCR for 50 cycles consisting of 95˚C for 15s
followed by 60˚C for 1 min. For each PCR measurement,
we checked the slope value, R2 value, and linear range of
a standard curve of serial dilutions. All reactions were
performed in duplicate. The results were expressed rela-
tive to a β-actin internal control.
2.6. Measurement of Plasma Corticosterone
Plasma was obtained as described previously [17] and
the concentration of corticosterone was determined via a
corticosterone EIA kit (Assay Designs, Inc., Ann Arbor,
MI, USA) according to the manufacturer’s protocol.
2.7. Chronic Corticosterone Treatment
Male 6-week-old ICR mice (Japan SLC) weighing 20-25
g were used for chronic oral corticosterone exposure as
described in a previous report [18]. Briefly, corticoster-
one (25 μg/mL free base; 4-pregnen-11β 21-DIOL-3
20-DIONE 21-hemisuccinate; Steraloids, Inc., RI, USA)
was add to tap water and the pH was brought to 12-13
with 10 N NaOH (Kishidai Chemical, Osaka, Japan),
followed by stirring at 4˚C until dissolved (3 to 7 hr).
Following dissolution, the pH was brought to 7.0-7.4
with 10 N HCl (Wako, Osaka, Japan). Group-housed
ICR mice were presented with this corticosterone solu-
tion in place of normal drinking water for 14 days, re-
sulting in a dose of approximately 8.7 mg/kg/day (p.o).
Animals were weaned with 3 days of 12.5 μg/mL, and
then 3 days with 6.25 μg/mL, to allow for gradual recov-
ery of endogenous corticosterone secretion.
2.8. Western Blot Analysis
At 35 days, each mouse was decapitated and its brain
was quickly removed from the skull, briefly washed in
ice-cold saline, and laid on a cooled (4˚C) metal plate.
The brain was rapidly dissected to separate the hippo-
campus and stored at -80˚C until use. Brain samples were
homogenized in 10 mL/g tissue ice-cold lysis buffer [50
mM Tris-HCl (pH 8.0) containing 159 mM NaCl, 50 mM
EDTA, 1% Triton X-100, and protease/phosphatase in-
hibitor mixture] using a homogenizer (Physcotron; Mi-
crotec Co. Ltd., Chiba, Japan). Lysates were centrifuged
at 12,000×g for 15 min at 4˚C. Supernatants were col-
lected and boiled for 5 min in SDS sample buffer (Wako).
Equal amounts of protein were subjected to 10% SDS-
PAGE gradient gel and then transferred to poly (vi-
nylidene difluoride) membranes (Immobilon-P; Millipore,
MA, USA). After blocking with Block Ace (Snow Brand
Milk Products Co. Ltd., Tokyo, Japan) for 30 min, the
membranes were incubated with primary antibody. The
primary antibodies used were as follows: mouse anti-BiP
antibody (BD Bioscience, CA, USA) for GRP78, mouse
anti-KDEL antibody (Stressgen Bioreagents Limited
Partnership, B.C., Canada) for GRP94, and mouse anti-
actin antibody (Sigma-Aldrich, St. Louis, MO, USA).
Subsequently, the membrane was incubated with the
secondary antibody [goat anti-mouse (Pierce Biotech-
nology, IL, USA)]. The immunoreactive bands were
visualized using Super Signal West Femto Maximum
Sensitivity Substrate (Pierce Biotechnology) and then
measured using LAS-4000 mini (Fujifilm, Tokyo, Ja-
pan).
2.9. Statistical Analysis
Statistical comparisons were made by Student’s t-test
using Statview version 5.0 (SAS Institute Inc., NC, USA),
with p < 0.05 being considered statistically significant.
3. Results and Discussion
Real-time PCR was carried out to investigate whether the
expression of ER stress response-related genes in the
brain was changed by 6-hr restraint stress. In this study,
we investigated the expression of GRP94, carleticulin,
ER degradation-enhancing α-mannosidase-like protein
(EDEM), protein kinase inhibitor of 58 kDa (p58IPK),
asparagines synthetase (ASNS), GRP78, ER-localized
DnaJ 4 (ERdj4), and C/EBP homologous protein (CHOP).
The expression of GRP78, GRP94, and calreticulin
mRNA was significantly increased in the hippocampus,
striatum, and cortex (Figure 1). In addition, there was
significantly increased expression of p58IPK mRNA in the
cortex, but not in the hippocampus or striatum.
We next investigated whether restraint stress affected
the plasma concentrations of corticosterone, as previ-
ously reported. Immediately following the 6-hr restraint
stress, significantly higher plasma corticosterone concen-
trations were found in stressed mice compared to un-
stressed mice. Seven days after the restraint stress, the
plasma corticosterone recovered to the normal control
level (Figure 2).
To clarify the mechanism of ER stress-related mRNA
elevation, we artificially elevated the plasma concentra-
tions of corticosterone in mice for 2 weeks and then
measured the levels of ER stress-related proteins. In the
corticosterone-treated animal model, the expression of
GRP78 and GRP94 in the hippocampus was significantly
increased compared to control levels (Figure 3).
Restraint stress is used widely to induce stress re-
sponses in animals, and it is known that a number of
stresses, including restraint stress, can cause depression
in animals. In the present study, we found that several
ER stress-related genes were increased in the mouse
hippocampus, striatum, and cortex after restraint stress.

Restraint-Induced Expression of Endoplasmic Reticulum Stress-Related Genes in the Mouse Brain 13
(a)
(b)
(c)
Figure 1. The expression mRNA of ER stress-related factors
in the mouse brain after 6 hr restraint-stress. Mice were
immobilized for 6 hr in a 50-mL perforated plastic tube.
White and black bars represent the control group and the
restraint group, respectively. Immediately after restraint,
mice were killed and real-time PCR was performed on
brain tissues from the (a) hippocampus, (b) striatum, and (c)
cortex. Data represent means and S.E.M., n = 3 to 5. *p <
0.05, **p < 0.01 vs. control group. GRP94: the 94-kilodalton
glucose regulated protein, EDEM: ER degradation-enhancing
α-mannosidase-like protein, p58IPK: protein kinase inhibi-
tor of 58 kilodalton, ASNS: asparagines synthetase, GRP78:
the 78-kilodalton glucose regulated protein, ERdj4: ER-
localized DnaJ 4, CHOP: C/EBP-homologous protein.
The significant increases in expression of GRP78,
GRP94, and calreticulin agreed with the findings of a
previous report of changes in the temporal cortex of sub-
jects with major depression who died by suicide [11].
However, no study has yet specifically investigated ex-
pression changes of these genes in the hippocampus or
the striatum in subjects with depression.
Figure 2. The effect of 6 hr restraint stress on the concen-
tration of corticosterone in mouse plasma. Mice were im-
mobilized for 6 hr. Immediately after restraint and 7 days
later, blood samples were collected and concentration of
plasma corticosterone was measured by ELISA. Restraint
stress significantly increased the concentration of corticos-
terone in plasma. The corticosterone levels decreased to the
normal control levels 7 days after restraint stress. Data
represent means and S.E.M., n = 7. *p < 0.05 vs. control
group.
GRP78, otherwise known as BiP, is one of the best-
characterized ER chaperone proteins and is regarded as a
classical marker of UPR activation. Overexpression of
GRP78 has been reported to inhibit the upregulation of
CHOP, which plays a key role in regulating cell growth
and which has been implicated in apoptosis [19,20].
GRP94 and calreticulin are also ER chaperone proteins
and show protective effects against ER stress [21]. The
increase in these chaperones after restraint stress (Figure
1) may represent an attempt to oppose the toxic effect of
prolonged stress and the high concentrations of glucocor-
ticoid, such as corticosterone, on the brain. Dysregulation
of the hypothalamic-pituitary-adrenal (HPA) axis, which
controls glucocorticoid levels, has been reported in most
depression patients and glucocorticoid level of depres-
sion patients was higher than those of normal ones
[22-24]. In the mice in the present study, 6-hr restraint
stress elevated the concentration of corticosterone in
plasma, suggesting that restraint stress induced a re-
sponse similar to depression.
Recently, corticosterone has been reported to exert
immunostimulatory effects on macrophages via induction
of ER stress [25]. Following corticosterone treatment, the
glucocorticoid receptor (GR) binds onto B-cell lym-
phoma 2 (Bcl-2), a protein that affects cytochrome C and
calcium release from mitochondria. Subsequently, this
GR/Bcl-2 complex moves into mitochondria and regu-
lates mitochondrial functions in an inverted “U”-shaped
manner–i.e., a high dose treatment with corticosterone
decreased levels of GRs and Bcl-2 in mitochondria and
intracellular calcium was increased [26,27]. Substances

Restraint-Induced Expression of Endoplasmic Reticulum Stress-Related Genes in the Mouse Brain 14
(a)
(b)
(c)
Figure 3. The expression of GRP78 and GRP94 in the hip-
pocampus in a mouse model of chronic corticosterone in-
duced depression. (a) Representative band images show
immunoreactivities against GRP94, GRP78, and β-actin. (b)
GRP78 expression was significantly increased by corticos-
terone exposure. (c) GRP94 expression was also increased
by corticosterone exposure. Data represent means and
S.E.M., n = 5 or 6. *p < 0.05 vs. control group.
that deplete the ER Ca2+ stores, such as thapsigargin, are
widely used an ER stressors. Therefore, elevation of Ca2+
via GR may be sufficient for control of ER stress re-
sponses. In the present study, the restraint stress induced
the expressions of only GRP78, GRP94, and calreticulin,
but not other ER proteins. GRP78, GRP94, and cal-
reticulin function as Ca2+ binding proteins [28]. Under
the high concentration of corticosterone, the intracellular
Ca2+ level might be higher, therefore, the expressions of
GRP78, GRP94, and calreticulin might be increased.
Intracerebroventricular administration of thapsigargin
has been reported to produce a depressant-like behavior
[29]. A 14-days corticosterone treatment has also shown
to induce depression symptoms in mice [18]. We used
this animal model to investigate the effect of chronic
elevation of corticosterone on ER stress responses in
brain. As expected, significant increases in GRP78 and
GRP94 proteins were observed in the hippocampus (Fig-
ure 3). The increase of GRP78 was consistent with the
result of a previous report [30]. On the other hand, no
change in these proteins was observed in the cortex (data
not shown). Mineralocorticoid receptor (MR) and GR,
which are the targets of corticosterone, are known to be
well expressed in the hippocampus [31,32]. These reports,
together with our findings, indicate that the hippocampus
may be more sensitive to corticosterone exposure than
are other brain regions. Many reports have referred to
hippocampal atrophy in patients with depression [12,14].
In the cortex, it had been reported that chronic stress in-
creased the caspase-3 positive neurons, in other words,
exogenous stress was contributing to the cell apoptosis
[15]. In our study, corticosterone exposure was per-
formed for 2 weeks, but, in fact, long-term cortisol eleva-
tion has been observed in most depression patients. More
extended corticosterone treatment may affect the expres-
sion of ER stress proteins in the cortex.
Recently, many experiments have focused on the rela-
tionship between depression and neurogenesis. Interest-
ingly, ER stress also affects adult neurogenesis in the
brain [33]. Brain-derived neurotrophic factor (BDNF),
which promotes neurogenesis, is also known to inhibit
neuronal cell death induced by ER stress [34]. These
reports may also point to an involvement of ER stress in
depression.
4. Conclusions
Restraint stress, which may contribute to depression in
mice, may up-regulate the ER stress response via corti-
costerone elevation. This suggests the possibility of an
ER stress involvement in the pathogenesis of stress-related
depression disorders.
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