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Endoplasmic Reticulum Stress Proteins in the Rat

Cerebral Cortex and Hippocampus

Biao Chen, Jun Feng Wang, and L. Trevor Young

Background: Sodium valproate is a highly effective

treat-ment for bipolar disorder, but its mechanism of action

remains poorly understood. We recently found with

differ-ential display polymerase chain reaction that valproate

regulates the expression of the endoplasmic reticulum

stress protein GRP78 in the rat cerebral cortex. In our

study, we investigated the effect of this drug on the other

members of the endoplasmic reticulum stress protein

family, GRP94 and calreticulin, and we studied the brain

regional distribution of GRP78, GRP94, and calreticulin.

Methods: Immunohistochemistry was used to measure

protein levels of GRP78, GRP94, and calreticulin after

treatment with sodium valproate (300 mg/kg,

intraperito-neal) in specific rat brain regions.

Results: We report here that chronic treatment with

valproate also increased expression of other members of

the endoplasmic reticulum stress protein family, such as

GRP94 and calreticulin. The brain regional distribution of

these changes was similar for all three proteins, with

marked increase detected in the frontal cortex, parietal

cortex, and CA1 region of the hippocampus.

Conclusions: Because GRP78, GRP94, and calreticulin

possess molecular chaperone activity and bind Ca



the endoplasmic reticulum, the pharmacologic action of

valproate may involve one or more of these processes.

Biol Psychiatry 2000;48:658 – 664 © 2000 Society of

Biological Psychiatry

Key Words: GRP78, GRP94, calreticulin, mood

stabiliz-ers, valproate, anticonvulsants



odium valproate (VPA) is a first-line treatment for

bipolar disorder (BD), with a broad spectrum of

efficacy in this disorder (Davis et al 2000; Keck et al 1998;

Tohen and Grundy 1999). Despite extensive investigation,

however, the mechanism of its therapeutic action is still

poorly understood. Earlier studies found that chronic

treatment with VPA regulated the expression of several

genes such as Na


channel subunit (Yamamoto et al 1997)







(MARCKS; Lenox et al 1996). Using differential display

polymerase chain reaction, we recently reported that

chronic treatment with VPA increased mRNA and protein

expression of 78-kilodalton glucose-regulated protein

(GRP78) in rat brain (Wang et al 1999). This suggests a

novel and potentially clinically relevant mechanism

through which this drug may work.

GRP78, along with GRP94 and calreticulin, are gene

products induced as part of the stress response in

eukary-otic cells. They play an important role in the cellular

response to stress and are involved in endoplasmic

retic-ulum (ER) glycoprotein trafficking, molecular chaperone

activity, and ER Ca


binding (Brostrom et al 1998; Kim

et al 1987; Little et al 1994; Wooden et al 1991). Because

these proteins act in concert, it was of interest to study the

effects of VPA on other members of the ER protein

family. Moreover, understanding the brain regional

distri-bution of any changes in expression is critical to clarifying

the functional significance of these drug effects. In the

present study, we used immunohistochemistry to study

GRP78, GRP94, and calreticulin expression in rat brain

after chronic treatment with VPA and report here

brain-specific changes in all three members of the ER stress

protein family.

Methods and Materials

Animals and Drug Treatment

Male Sprague–Dawley rats (150 –170 g) were housed two per cage and maintained on a 12-hour light/dark cycle with food and water freely available. Rats were divided into 2 groups: control animals (n5 6) and VPA-treated animals (1 day [1d], 7 days [7d], and 14 days [14d], n56 for each time point). Rats were injected intraperitoneally (IP) daily with saline or VPA (300 mg/kg).

After treatment, rats were anesthetized with sodium

pentobar-From the Department of Psychiatry and Behavioral Neuroscience, McMaster University, Hamilton, Canada.

Address reprint requests to L. Trevor Young, M.D., Ph.D., McMaster University, 1200 Main Street, West, 4N77A, Hamilton Ontario L8N 3Z5, Canada. Received November 25, 1999; revised February 29, 2000; accepted March 6, 2000.

© 2000 Society of Biological Psychiatry 0006-3223/00/$20.00


bital (60 mg/kg) and perfused transcardially with 250 mL of 100 mmol/L phosphate buffer (pH 7.5) at room temperature. Brains were removed, rapidly frozen in280°C and serial 20mm coronal sections (from Bregma 22.30 mm to Bregma 24.80 mm) prepared at218°C. The sections were thaw-mounted on poly-L-lysine-coated glass slides. Each slide contained corresponding sections from one animal in each treatment groups (control, 1d, 7d, and 14d) to allow comparisons across groups and minimize differences in background.

Immunohistochemical Staining

Immunohistochemistry was carried out using the streptavidin-peroxidase method (Elias et al 1989; Ozaki et al 1997). Briefly, sections were treated with 0.3% Triton X-100/phosphate-buff-ered saline (PBS) and incubated in 3% H2O2/methanol to block

endogenous peroxidase. The sections were preincubated with normal serum and then incubated at 4°C for 48 hours with specific primary antisera that recognize GRP78 (Stressgen Bio-technologies, Victoria, Canada), GRP94 (Stressgen Biotecholo-gies), and calreticulin (Upstate Biotechnology, Lake Placid, NY). These primary antisera were used at dilutions of 1:800, 1:800, and 1:1000 for GRP78, GRP94, and calreticulin, respectively. The sections were then incubated for 30 min with biotinylated secondary antibody (Zymed, South San Francisco, CA), and finally with streptavidin-horseradish peroxidase (HRP) conjugate for 30 min at room temperature. The peroxidase reaction was carried out with a DAB Kit (Zymed) according to the manufac-turer’s protocol. The specificity of immunoreactivity was tested by incubating rat brain sections with no primary antibody or after the addition of nonimmune rabbit serum in which no immuno-staining was observed.

Quantitative Image Analysis

Immunohistochemically stained sections were examined at 503 magnification by creating a digitized image with a microcom-puter imaging device (MCID) image analysis system (Brock University, St. Catherines, Canada) attached to a light micro-scope (Zeiss Axioskop, Oberkochen, Germany) with a high-resolution charge-coupled device (CCD) camera (MTI CCD 72). The density of immunoreactive positive neurons in frontal cortex, parietal cortex, dentate gyrus, and CA1 and CA3 of hippocampus were measured using standard landmarks. Back-ground values were obtained at a ten small boxes cursor in the molecular layer of cortex. The density of immunostaining in different regions was expressed as relative optical density (ROD), which was the density of each region divided by the background value of the same section. Six sections from each animal per each antibody through each region of interest were counted, and the same regions were compared as closely as possible between animals.

Statistical Analysis

Data were presented as the mean6SEM. from six independent experiments. Statistical analysis of data was performed with one way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test.


GRP78, GRP94, and calreticulin immunoreactivity were

restricted to the cytoplasm of neurons with substantial

overlap across the expression pattern for each protein

(Figure 1). In saline-treated rats, immunoreactive positive

neurons of GRP78, GRP94, and calreticulin were

distrib-uted throughout the entire brain. The pyramidal cells in the

parietal cortex showed particularly strong

immunoreactiv-ity to GRP78, GRP94, and calreticulin when compared

with other cells (Figures 2, 3, and 4). The

immunoreac-tivity of GRP94 and calreticulin was relatively weaker in

Figure 1. Immunohistochemical staining of GRP78, GRP94, and calreticulin of pyramidal cell laminae of the rat parietal cortex treated with saline. Magnification 3363.

Valproate Increases ER Stress Proteins BIOL PSYCHIATRY 659


dentate gyrus compared with CA1 and CA3 regions of

hippocampus (Figures 3 and 4).

GRP78 immunoreactivity was significantly increased

(Figure 5A) by intraperitoneal injection of VPA for 1, 7,

and 14 days in both frontal and parietal cortex (p



Sodium valproate increased GRP78 expression by 30%,

38%, and 28% in the frontal cortex and 21%, 21%, and

17% in the parietal cortex. Although VPA treatment for 1,

7, and 14 days had no effect on GRP78 expression in CA3

region of hippocampus, VPA significantly increased

GRP78 expression in CA1 and dentate gyrus (p



VPA increased GRP78 expression by 38%, 27%, and 20%

at 1, 7, and 14 days in CA1; by 46% and 43% at 1 and 7

days in dentate gyrus (p




Similarly, calreticulin immunoreactivity was

signifi-cantly increased (Figure 5B) after intraperitoneal injection

of VPA for 1, 7, and 14 days in frontal cortex, parietal

cortex, CA1, and dentate gyrus, but not in the CA3 region.

VPA increased calreticulin expression by 22%, 38%, and

5% at 1, 7, and 14 days in frontal cortex (p


.01); by

18%, 29%, and 16% at 1, 7, and 14 days in parietal cortex



.01); by 39%, 38%, and 25% at 1, 7, and 14 days in

CA1 (p


.05); and by 30% and 28% at 1 and 7 days in

dentate gyrus (p



Finally, intraperitoneal injection of VPA for 1, 7, and 14

days significantly increased GRP94 immunoreactivity in

all five measured regions including frontal cortex, parietal

cortex, CA1, CA3, and dentate gyrus. VPA increased

GRP94 expression by 38%, 29%, and 27% at 1, 7, and 14

days in frontal cortex (p


.05); by 24%, 28%, and 35%

Figure 3. GRP94 immunoreactivity in different brain regions of the rat treated with sodium valproate (VPA). Rats were treated with saline (a, e, and i) and VPA (300 mg/kg, intraperitoneal) for 1 day (b, f, and j), 7 days (c, g, and k), and 14 days (d, h, and l). Arrowheads point to pyramidal cells. Magnification 1003. DG, dentate gyrus.

Valproate Increases ER Stress Proteins BIOL PSYCHIATRY 661


at 1, 7, and 14 days in parietal cortex (p


.05); by 53%,

49%, and 55% at 1, 7, and 14 days in CA1 (p


.05); and

32%, 35%, and 41% at 1, 7, and 14 days in dentate gyrus.


We previously demonstrated that VPA increased GRP78,

one of the ER stress proteins in rat cerebral cortex (Wang et


regulate the cellular response to stress to bring about its

therapeutic effects.

ER stress proteins function in ER glycoprotein

traffick-ing, have molecular chaperone activity, and protect cells

from the deleterious effects of damaged proteins by

binding and disposing of malfolded protein (Kim et al

1987; Little et al 1994; Wooden et al 1991). ER stress

proteins are also Ca


binding proteins and play an

important role in maintaining ER Ca



(Lie`vremont et al 1997). Because VPA appears to increase

expression of ER stress proteins that perform these

partic-ular processes, our findings suggest that VPA treatment

may target one or more of these processes. Although the

mechanism of the regulation of ER stress proteins by VPA

is not yet known, it is possible that this regulation is

important in the pharmacologic action of VPA.

The effect of VPA on ER stress protein expression may

also be relevant to the pathophysiology of BD. It has been

known that intracellular free Ca


in peripheral blood

cells was increased in BD subjects (Dubovsky et al 1992).

Increasing evidence suggests that altered

polyphosphoino-sitide (PI)– generated second messenger signaling occurs

in blood cells and brain tissue obtained from BD patients,

which may relate to these increased intracellular Ca


levels (Jope et al 1996; Wang et al 1997). Under these

conditions, an increase in ER stress proteins might be

expected to bind ER Ca


and prevent depletion of ER

stores due to increased PI signaling and in turn lower

intracellular Ca


levels. Some recent evidence suggests

that VPA inhibits 5-hydroxytryptamine-induced Ca


in-flux in C6 glioma cells (Yamaji et al 1996), consistent

with this hypothesis.

VPA-induced increases in ER stress proteins may have

a neuroprotective role in specific brain regions in which

cellular damage may occur in BD. Although there is no

direct evidence to support this hypothesis, there is

increas-ing interest in the role of ER stress proteins in central

nervous system (CNS) disease and injury. GRP78

expres-sion is induced after CNS injuries (Lowenstein et al 1994),

such as seizures, global ischemia, and acute trauma, and in

neurodegenerative diseases like Alzheimer’s (Hamos et al

1991). ER stress proteins induction by chronic VPA

treatment may play a neuroprotective role by clearing

malfolded proteins.

In conclusion, chronic treatment with the mood

stabi-lizer VPA upregulates expression of ER stress proteins

GRP78, GRP94, and calreticulin in rat cerebral cortex and

hippocampus, which may have particular relevance for the

prophylactic effect of VPA in the long term management

of patients with BD. The molecular chaperone and Ca


binding activities of ER stress proteins may be involved in

the pharmacologic action of VPA.

This work is supported by grants from the Ontario Mental Health Foundation and the Stanley Foundation. B.C. is supported by a Wyeth Ayerst Fellowship. LTY is a Career Scientist of the Ontario Ministry of Health.


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Figure 5. Expression of GRP78, GRP94, and calreticulin in different regions of the rat treated with sodium valproate (300 mg/kg, intraperitoneal). Immunoreactive granule density in the frontal cortex (Fr-Cx), parietal cortex (Par-Cx), hippocampus CA1, hippocampus CA3, and dentate gyrus (DG) regions is expressed as relative optical density. The data are means 6 SEMs (n56). *p,.05 and **p,.01, when compared with control animals.

Valproate Increases ER Stress Proteins BIOL PSYCHIATRY 663


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Figure 1. Immunohistochemical staining of GRP78, GRP94, andcalreticulin of pyramidal cell laminae of the rat parietal cortextreated with saline
Figure 2. GRP78 immunoreactivity in different brain regions of the rat treated with sodium valproate (VPA)
Figure 3. GRP94 immunoreactivity in different brain regions of the rat treated with sodium valproate (VPA)
Figure 4. Calreticulin immunoreactivity in different brain regions of the rat treated with sodium valproate (VPA)


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