J Pharmacol Sci 124, 000 – 000 (2014) Journal of Pharmacological Sciences

© The Japanese Pharmacological Society

Full Paper

Butea superba–Induced Amelioration of Cognitive and Emotional

Deficits in Olfactory Bulbectomized Mice and Putative Mechanisms
Underlying Its Actions
Daishu Mizuki1, Zhao Qi1, Ken Tanaka1, Hironori Fujiwara1, Tsutomu Ishikawa2, Yoshihiro Higuchi3,
and Kinzo Matsumoto1,*
1
Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama-shi, Toyama 930-0194, Japan
2
Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan
3
Material Development Laboratories, Shiratori Pharmaceutical Co., Ltd., 6-11-24 Tsudanuma, Narashino,
Chiba 275-0016, Japan

Received November 28, 2013; Accepted February 4, 2014

Abstract.  This study investigated the effects of alcoholic extract of Butea superba (BS) on
cognitive deficits and depression-related behavior using olfactory bulbectomized (OBX) mice and
the underlying molecular mechanisms of its actions. OBX mice were treated daily with BS (100
and 300 mg/kg, p.o.) or reference drugs, tacrine (2.5 mg/kg, i.p.) and imipramine (10 mg/kg, i.p.)
from day 3 after OBX. OBX impaired non-spatial and spatial cognitive performances, which
were elucidated by the novel object recognition test and modified Y maze test, respectively. These
deficits were attenuated by tacrine and BS but not imipramine. OBX animals exhibited depression-
like behavior in the tail suspension test in a manner reversible by imipramine and BS but not
tacrine. OBX down-regulated phosphorylation of synaptic plasticity–related signaling proteins:
NMDA receptor, AMPA receptor, calmodulin-dependent kinase II, and cyclic AMP-responsive
element-binding protein. OBX also reduced choline acetyltransferase in the hippocampus. BS
and tacrine reversed these neurochemical alterations. Moreover, BS inhibited ex vivo activity of
acetylcholinesterase in the brain. These results indicate that BS ameliorates not only cognition
dysfunction via normalizing synaptic plasticity–related signaling and facilitating central cholinergic
systems but also depression-like behavior via a mechanism differing from that implicated in BS
amelioration of cognitive function in OBX animals.

Keywords: Butea superba, olfactory bulbectomy, cognitive and emotional deficit,

synaptic plasticity–related signaling, cholinergic system

Introduction blind clinical trial (3) showed that oral administration of

powdered tubers of this plant to patients with erectile
Butea superba (BS) (Red Kwao Kua in Thai) is an dysfunction (ED), aged 30 to 70 years, caused noticeable
herb in the family Papilionaceae (Leguminosae) which improvement without apparent toxicity. In fact, this
is abundantly distributed in Thai deciduous forests. The clinical finding was supported by pharmacological (2)
tuberous roots of this plant have long been taken as a folk and chemical studies of this plant (4), demonstrating
medicine not only to increase physical and mental that BS enhances penile erection in rats and that some
strength but also to prevent aging-related symptoms isoflavonolignans such as butespurerins A and B are
such as impaired sexual performance in middle-aged or chemical constituents with inhibitory activities against
elderly males (1, 2). Evidence from a randomized double- phosphodiesterase type 3A and type 5, enzymes targeted
for ED therapy. Its ethno-medical uses and chemical
*Corresponding author.  [email protected] constituents suggest that BS may have potential for the
Published online in J-STAGE treatment of neurocognitive and/or neuropsychiatric
doi: 10.1254/jphs.13252FP symptoms.

1
2 D Mizuki et al

Alzheimer’s disease (AD) is a progressive neuro­ induced dysfunction of neuroplasticity-related neuronal

degenerative disorder that is characterized by memory signaling and cholinergic systems. Our findings sug-
dysfunction and behavioral and psychological symptoms gested that BS is useful for the treatment of AD patients
of dementia (BPSD), including depression. An increas- with depressive symptoms.
ing population of AD patients is a serious social and
economic problem in super-aging societies worldwide Materials and Methods
(5). Current approved treatment with anti-dementia drugs
such as donepezil, an acetylcholinesterase inhibitors, and Animals
memantine, an N-methyl-d-aspartate receptor (NMDA) This study was conducted according to the experi­
antagonist, has provided marginal therapeutic benefits mental protocols as described in Fig. 1. A total of 85
without affecting the progression of the disease (6). male ddY mice were obtained at the age of 8 weeks
Therefore, new drug discovery and the establishment (Japan SLC, Inc., Shizuoka). The animals were habituated
of new therapeutic methods are greatly needed. to the laboratory animal room for at least 1 week before
Olfactory bulbectomy (OBX) in rodents has been surgery. Food and water were available ad libitum.
widely used as an animal model to investigate emotional Housing was thermostatically maintained at 24°C ± 1°C
dysfunction such as depression (7 – 10). Indeed, OBX with constant humidity (65%) and a 12-h light-dark cycle
causes depression-like behavioral and neurochemical (lights on: 07:00 – 19:00). The behavioral experiments
alterations in rodents that are susceptible to antidepres- were performed during the light phase from 9:00 to
sant treatment (11 – 13). Moreover, OBX has been used 18:00. The present studies were conducted in accordance
as one of the AD models since impairment of olfactory with the Guiding Principles (NIH publication #85-23,
perceptual acuity is observed not only at the early stage revised in 1985) for the Care and Use of Animals and
of AD (14, 15) but also in a transgenic AD model of mice were approved by the Institutional Animal Use and
over-expressing a mutant form of the human amyloid-b- Care Committee of the University of Toyama.
precursor protein (16). In addition, OBX causes elevation
of the Ab level in the brain (17) and the degeneration of Preparation of plant extract
septo-hippocampal cholinergic system in rodents (8, 9, The ethanol extract of BS was prepared as previously
18), and thereby induces cognitive dysfunction (8 – 10). described. Briefly, the root of BS was collected in
In this study, we elucidated the anti-dementia and anti- Lampang Province in Thailand in 2011 and identified by
depressant drug-like effects of BS and the mechanism Dr. Chaiyo Chaichantipyuth (Department of Pharmacog-
underlying its action using an OBX model mouse to nosy, Faculty of Pharmaceutical Sciences, Chulalongkorn
obtain a better understanding of the potential availability University, Thailand). The dried powder of the root
of BS for the treatment of cognitive and emotional dys- (52 g) was extracted with 200 ml of 95% ethanol at 75°C
function. The present findings clearly demonstrated that for 2 h and filtrated. This step was repeated 3 times; and
BS ameliorates cognitive and depression-like emotional the filtrated samples were combined, concentrated under
deficits of OBX mice and that the effects on the cognitive reduced pressure at 40°C, and then dried in vacuo. The
deficits are mediated in part by normalization of OBX- yield of the extraction from the dried root was calculated

Fig. 1.  Schematic drawing of experimental protocol. ddY mice received daily administration of Butea superba extract
(BS; 100 – 300 mg/kg, p.o.), tacrine (THA; 2.5 mg/kg per day, i.p.), imipramine (IMP; 10 mg/kg per day, i.p.) or tap water from
day 3 after the operation. The administration was repeated during the experimental period. Each experiment was conducted at
1 – 2-week interval as described in the text. After completing the behavioral experiments, the mice were decapitated under
pentobarbital anesthesia. The brain tissues were used for neurochemical studies.
 Anti-dementia Effects of Butea superba 3

as 8.9% (w/w). The BS (voucher specimen No. TMPW- saline and administered once daily at doses of 2.5 and
27997) and its extract (voucher specimen no INM-531) 10 mg/kg (i.p.), respectively. BS extract was suspended
used in this study were deposited at our institute. in water and given perorally at daily doses equivalent to
LC–MS analyses were also performed with a Shi- 100 and 300 mg (dried herb weight)/kg. The daily doses
madzu LC-IT-TOF mass spectrometer equipped with of BS used in the present animal experiments were
an ESI interface. The ESI parameters were as follows: almost 10 – 30 times more than that used for human ED
source voltage, +4.5 kV; capillary temperature, 200°C; therapy (3).
and nebulizer gas, 1.5 l/min. The mass spectrometer
was operated in positive ion mode scanning from m/z Behavioral study
200 to 2000. A Waters Atlantis T3 column (2.1 mm The following behavioral experiments were started
i.d. × 150 mm) was used and the column temperature from 2 weeks after surgery.
was maintained at 40°C. The mobile phase was a binary
eluent of A) 5 mM ammonium acetate solution and Novel object recognition test (ORT)
B) acetonitrile under the following gradient conditions: ORT was conducted at days 17 – 20. This test is based
0 – 30 min, linear gradient from 10% to 100% B and on the tendency of mice to discriminate a familiar from
30 – 40 min, isocratic at 100% B. The flow rate was a new object. This test was conducted as previously
0.2 ml/min. Mass spectrometry data obtained from the described (8, 9, 20) with minor modification. Briefly,
extract have been listed in the MassBank database (19) mice were individually habituated to an observation
and stored together with the pharmacological informa- box (50 × 50 × 40 cm) for 10 min on the day before the
tion on the extract in the Wakan-Yaku DataBase system experiment. The ORT consisted of a sample phase trial
(http://wakandb.u-toyama.ac.jp/wiki/LCMS:Butea_ and a test phase trial. In the sample phase trial, each
INM-531), Institute of Natural Medicine, University of mouse was placed in the observation box where two
Toyama. identical objects, object 1 and object 2, were placed
separately, and allowed to explore the area freely for
Surgical operation 5 min. The total time that the mouse spent exploring
OBX of mice was conducted on day 0 as previously each of the two objects was measured and then the mouse
described (8 – 10). The mice were anesthetized with was returned to the home cage. In the test phase trial
sodium pentobarbital (60 mg/kg, i.p.) and fixed on performed 30 min after the sample phase trial, one of the
stereotactic instruments (Narishige, Tokyo). With 1% two objects was replaced by an identical copy (familiar
lidocaine solution used as a local anesthetic, the skull object) and the other by a novel object. Performance of
covering the bulbs was exposed by skin incision, and the animals in this test was video-recorded for later
then a 1-mm burr hole was drilled. The bilateral bulbs analysis. In these trials, the exploration of an object
were aspired through a syringe and the cavity of the was defined as directing the nose to the object at a
bulbs was filled with a hemostatic gelatin sponge. After distance of < 2 cm according to previous reports (8, 9,
completing the behavioral studies, all the animals were 20) and the time spent exploring each of the two objects
sacrificed and the operated lesion was verified visually. was analyzed using SMARTW ver. 2.5 (PanLab, S.L.,
The data from animals with less than 70% removal Barcelona, Spain) with a tri-wise module to detect the
or with no intact cortex were excluded from the data head, center mass, and base tail.
analysis. The success rate of surgical operation was 98%
(49/50). Sham operation was performed in a similar Modified Y-maze test
way without removal of the bulb. At the end of the A modified version of the Y-maze test was conducted
experiments, the olfactory bulbs of the sham-group mice at days 24 – 27 according to Yamada et al. (9). The
were also confirmed to be intact. apparatus used for this test consists of black polypro­
pylene walls with 3 arms, each 40-cm-long, 12-cm-wide
Drug administration at the top, 3-cm-wide at the bottom, and 18-cm-high.
Either vehicle water or test drugs were administered This test was a two-trial task with a sample phase trial
daily from day 3 after surgery during the experimental and a test phase trial that were separated by an inter-trial
period. On the behavioral testing day, administration was interval. In the sample phase trial, each mouse was
conducted 1 h before the testing. Sham-group mice and individually placed in the maze with one of the 3 arms
OBX-control group mice were perorally administered closed. The animal was allowed to explore the other
water. Tacrine (9-amino-1,2,3,4-tetrahydro-acridine HCl) 2 arms freely for 5 min. Thirty minutes after the sample
and imipramine HCl (Sigma-Aldrich Co., St. Louis, MO, phase trial, the animal was again placed in the maze with
USA), reference standard drugs, were dissolved in 0.9% all 3 arms opened, and allowed to explore the arms
4 D Mizuki et al

freely. The previously closed arm that was opened in the Analyzer LAS-4000R (Fuji Film, Tokyo). The quantity
test phase trial was defined as the new arm. The animal of immune-reactive bands was analyzed using
behavior was video-recorded for later analysis. Percent ImageQuant TL software (GE Healthcare, Buckingham-
time spent in the new arm was analyzed using SMART® shire, UK). The quantity of immunoreactive bands was
system ver. 2.5 (PanLab). normalized by comparison with their expression levels
in treatment-naïve control mice. The expression levels of
Tail suspension test (TST) each target protein were reprobed using a Blot Restore
We employed TST to assess the antidepressant effects Membrane Rejuvenation Kit (Millipore).
of the test drugs (21). This test was conducted at days
38 – 40 as previously described (10). Briefly, mice were Ex vivo measurements of acetylcholinesterase (AChE)
subjected to the short-term inescapable stress of being activity in the brain
suspended by the tail, which leads to the development Ex vivo AChE activity in the cerebral cortex was
of an immobile posture. The animals were separately determined by the colorimetric method as previously
suspended 50 cm above the floor in a chamber by described (8 – 10, 24). Briefly, 9-week-old male mice
adhesive tape placed approximately 2 cm from the tip of received daily administration of 300 mg/kg BM for 14
the tail. The animal behavior in the test was video- days. The animals were decapitated 1 h after the last
recorded for later analysis. Immobility was defined as administration and the cerebral cortices were dissected
a state with movement speed no more than 0.05 cm2/s out from the brain. The frozen cortex was weighed and
using SMART® system ver. 2.5; immobility time was homogenized in 10 volumes of 0.1 M phosphate buffer
recorded for 8 min and the performance during the last (pH 7.4) containing 1% Triton-X-100. After centrifuga-
6-min period was analyzed. tion at 15,000 × g and 4°C for 20 min, the clear superna-
tants were collected and served as the enzyme source.
Neurochemical study Cholinesterase activity was determined in 50 ml aliquots
One week after completing the behavioral experiments, of homogenates (run as duplicates) in a 96-well flat-
mice were decapitated under pentobarbital (50 mg/kg, bottomed microplate. The reaction was started by
i.p.) anesthesia. The hippocampal tissues were quickly adding 20 ml of 10 mM 5,5′-dithiobis-2-nitrobenzoic
dissected out and stored at −80°C until use. acid (DTNB), 20 ml of 7.5 mM acetylthiocholine (ATCI),
and 160 ml of 0.1 M sodium phosphate buffer (pH 8.0).
Western blotting The spectrophotometric absorption at 405 nm during
Expression of synaptic plasticity–related proteins in a 5-min incubation period at 25°C was quantitatively
the hippocampus was analyzed using western blotting as measured using a microplate reader (Sunrise Classic;
previously described (8, 22, 23). The following primary TECAN Japan, Kawasaki) and is expressed as nmol
antibodies were used: anti-NMDAR1 rabbit polyclonal ACh hydrolyzed/min per mg tissue.
antibody (1:1000 dilution), anti-phospho-NMDAR1
(p-NMDAR1) (pSer896) rabbit polyclonal antibody Data analyses
(1:1000 dilution) (Cell Signaling Technology, Danvers, The data are expressed as the mean ± S.E.M. The data
MA, USA); anti-glutamate receptor 1 (GluR1) rabbit obtained from the object recognition test were analyzed
polyclonal antibody (1:1000 dilution), anti-phosho- by the paired Student’s t-test and the data obtained from
GluR1 (p-GluR1) (pSer 831) rabbit polyclonal antibody other behavioral and neurochemical experiments were
(1:1000 dilution) (Sigma-Aldrich); anti-CaMKIIa (A-1: analyzed by one-way analysis of variance (ANOVA)
sc-13141) mouse monoclonal antibody (1:1000 dilution), followed by a post hoc multiple comparison test (Student-
anti-phospho-CaMKII (p-CaMKII) (pThr286) rabbit Newman-Keuls method) as appropriate. Differences of
polyclonal antibody (1:1000 dilution), anti-CREB (48H2) P < 0.05 were considered significant. The analysis was
rabbit monoclonal antibody (1:1000 dilution), anti- conducted using SigmaStat® ver. 3.5 (SYSTAT Software
phospho-CREB (p-CREB) (pSer133) rabbit monoclonal Inc., Richmond, CA, USA).
antibody (1:1000 dilution), anti-choline acetyltransferase
(ChAT) goat polyclonal antibody (1:5000 dilution) Results
(AB-144P; Millipore, Temecula, CA, USA); and anti-b-
actin mouse monoclonal antibody (1:10,000 dilution, BS attenuates OBX-induced non spatial short-term
Abcam®, Cambridge, UK). The immune complexes were memory deficits in the object recognition test
detected by the enhanced chemiluminescence method The object recognition test was used to evaluate the
(ImmobilonTM Western Chemiluminescent HRP Sub- effects of BS on the non-spatial cognitive memory of
strate) (Millipore) and imaged using Lumino Image OBX mice (25). In the sample phase trials, none of the
 Anti-dementia Effects of Butea superba 5

Fig. 2.  Treatment of BS (300 mg/kg) and tacrine (THA) improves object recognition memory deficit in OBX mice. A: Experi-
mental protocols depicting the novel object recognition test (ORT). B and C: The sample phase (B) and the test phase trials (C)
were conducted at a 30-min interval. Each data column represents the mean ± S.E.M. (n = 10). *P < 0.05 and ***P < 0.001 vs.
time spent exploring a familiar object (paired t-test).

animal groups showed significant differences in time

spent exploring each identical object. In the test phase
trial, the sham group spent a significantly longer time
exploring the novel object than exploring the familiar
one (t = −10.500, df = 18, P < 0.001), while the vehicle-
treated OBX group showed no preference for the novel
object (t = 1.521, df = 18, P = 0.163). BS (300 mg/kg per
day)- and tacrine-treated OBX mice, as well as the
sham group, spent a significantly longer time exploring
the novel object than the familiar one. (BS-treated OBX
group: t = −2.843, df = 18, P = 0.019; tacrine-treated
OBX group: t = −5.525, df = 18, P < 0.001) (Fig. 2). In
contrast, BS (100 mg/kg per day)- and imipramine-
treated OBX mice did not show a preference for the
novel object.

BS treatment reverses spatial short-term memory deficits

of OBX mice in the modified Y-maze test
We also examined the effect of BS treatment on time Fig. 3.  Effects of BS and tacrine (THA) on OBX-induced spatial
visiting the novel arm in the modified Y-maze test. As working memory deficit. A: Experimental protocols depicting the
modified Y-maze test. The maze was surrounded by different spatial
shown in Fig. 3, the ratio of the time that the sham group cues. The sample and test trials were conducted for 5 min at a 30-min
spent visiting the novel arm to the time spent visiting the interval. In the sample trial, each mouse was individually placed in
other two familiar arms was higher than the chance level the maze with one of the 3 arms closed. The previously closed arm
that was opened in the test trial was defined as the novel arm. Results
of 33.3%, indicating a preference for the novel arm over are expressed as % time animals spent exploring the novel arm in the
the familiar arms. The vehicle-treated OBX mice spent a test trial. B: Spatial short-term working memory performance of sham
significantly shorter time exploring the novel arm than and OBX groups in the test trials conducted after 30 min. Each data
column represents the mean ± S.E.M. (n = 9 – 12). ***P < 0.001 vs.
the sham mice [one-way ANOVA: F(5,50) = 8.420, vehicle-treated OBX group (post hoc Student-Newman-Keuls test).
6 D Mizuki et al

P < 0.001; post hoc test: sham vs. vehicle-treated OBX, BS reversed OBX-induced downregulation of glutama-
P < 0.001]. BS (100 – 300 mg/kg per day)- and tacrine- tergic neural plasticity in the hippocampus
treated OBX mice spent a significantly longer time To understand the molecular mechanisms underlying
exploring the novel arm than the vehicle-treated OBX BS-induced improvement of cognitive deficits in OBX
group [post hoc test: vehicle-treated OBX vs. BS mice, we examined the effects of BS on the synaptic
(100 mg/kg per day)-treated OBX, P < 0.001; vehicle- plasticity–related neuro-signaling pathway in the hippo-
treated OBX vs. BS (300 mg/kg per day)-reated OBX, campus of OBX mice by western blotting. No differences
P < 0.001; vehicle-treated OBX vs. tacrine-treated OBX, in the hippocampal NMDAR1 (NMDAR subunit),
P < 0.001]. On the other hand, imipramine administra- GluR1 (AMPAR subunit), CaMKIIa, and CREB levels
tion had no effect on spatial working memory deficits in were found among OBX, BS, or reference drugs (Fig. 5).
OBX mice [post hoc test: vehicle-treated OBX vs. The expression levels of phosphorylated NMDAR1,
imipramine-treated OBX, P = 0.096]. GluR1, CaMKIIa, and CREB in the OBX mice were
significantly decreased compared with the levels in the
BS ameliorates depression-like behavior of OBX mice in sham mice. However compared with vehicle-treated
the TST sham mice, vehicle-treated OBX mice showed a signifi-
This test was used to evaluate OBX-induced depres- cant decrease in the levels of pSer896-NMDAR1 [one-
sive-like behavior. The vehicle-treated OBX mice way ANOVA: F(4,15) = 3.505, P = 0.033; post hoc
showed a significantly longer immobility time than the test: sham vs. vehicle-treated OBX group, P = 0.039],
sham group [one-way ANOVA: F(5,54) = 4.303, P =  pSer831-GluR1 [one-way ANOVA: F(4,15) = 5.863,
0.002; post hoc test: sham vs. vehicle-treated OBX, P = 0.005; post hoc test: sham vs. vehicle-treated OBX
P = 0.006]. BS (300 mg/kg per day)- and imipramine- group, P = 0.041], pThr286-CaMKII [one-way ANOVA:
treated OBX mice showed a significantly shorter F(4,15) = 4.725, P = 0.011; post hoc test: sham vs.
immobility time than the vehicle-treated OBX mice [post vehicle-treated OBX group, P = 0.010], and pSer133-
hoc test: vehicle-treated OBX vs. BS (300 mg/kg per CREB [one-way ANOVA: F(4,15) = 4.270, P = 0.017;
day)-treated OBX, P = 0.023; vehicle-treated OBX vs. post hoc test: sham vs. vehicle-treated OBX group,
imipramine-treated OBX, P = 0.005], whereas BS (100 P =  0.032]. BS and tacrine treatment ameliorated
mg/kg per day) and tacrine treatment did not significantly OBX-induced downregulation of pSer896-NMDAR1
reduce immobility time [post hoc test: vehicle-treated [post hoc test: vehicle-treated OBX group vs. BS-treated
OBX vs. BS (100 mg/kg per day)-treated OBX, OBX group, P = 0.042; vehicle-treated OBX group vs.
P = 0.069; vehicle-treated OBX vs. tacrine-treated OBX, tacrine-treated OBX group, P = 0.036] and pSer831-
P = 0.204] (Fig. 4). GluR1 [post hoc test: vehicle-treated OBX vs. BS (300
mg/kg per day)-treated OBX, P = 0.046; vehicle-treated
OBX vs. tacrine-treated OBX, P = 0.021] and pSer133-
CREB [post hoc test: vehicle-treated OBX group vs.
BS (300 mg/kg per day)-treated OBX group, P = 0.025;
vehicle-treated OBX group vs. tacrine-treated OBX
group, P = 0.011] and pThr286-CaMKII [post hoc test:
vehicle-treated OBX vs. BS (300 mg/kg per day)-treated
OBX, P = 0.017; vehicle-treated OBX vs. tacrine-treated
OBX, P = 0.012] (Fig. 5: B – E). Imipramine treatment
significantly reversed OBX-induced down-regulation
of pThr286-CaMKII [post hoc test: vehicle-treated OBX
vs. imipramine-treated OBX, P = 0.028] and pSer133-
CREB [post hoc test: vehicle-treated OBX vs. imipra-
mine-treated OBX, P = 0.043], but it had no effect on
pSer896-NMDAR1 [post hoc test: vehicle-treated OBX
vs. imipramine-treated OBX, P = 0.103], pSer831-
GluR1 [post hoc test: vehicle-treated OBX vs. imipra-
mine-treated OBX, P = 0.502].
Fig. 4.  Effects of BS, imipramine (IMP) and tacrine (THA) on
OBX-induced depression–related behavior in the tail suspension test.
BS facilitated central cholinergic systems in the brain of
Each data column represents the mean ± S.E.M. (n = 9 – 10).
*P < 0.05, **P < 0.01, and ***P < 0.001 vs. vehicle-treated OBX OBX mice
group (post hoc Student-Newman-Keuls test). The expression of ChAT, a cholinergic marker in the
 Anti-dementia Effects of Butea superba 7

Fig. 5.  Effects of BS and tacrine (THA) on synaptic plasticity–related signaling pathway in the hippocampus of OBX mice.
A: Typical photos indicate expression levels of NMDAR1, p-NMDAR1 (p-Ser 896), GluR1, p-GluR1 (p-Ser831), CaMKIIa,
p-CaMKII (p-Thr286), CREB, and p-CREB (pSer 133) in the hippocampi of sham and vehicle-, BS-, and THA-treated OBX
animals. B – E: Quantitative comparisons of expression levels of p-NMDAR1/NMDAR1, p-GluR1 (p-Ser831)/GluR1, p-CaMKII/
CaMKIIa, and p-CREB (p-Ser133)/CREB. Each data column represents the mean ± S.E.M. (n = 4). *P < 0.05, **P < 0.01, and
***P < 0.001 vs. vehicle-treated OBX group (post hoc Student-Newman-Keuls test).

hippocampus, was examined by western blotting. Com- shown in Fig. 7, importantly, the activity of cortical
pared with that in the sham-operated group, the vehicle- AChE in the mice treated with BS and tacrine for 2
treated OBX group exhibited a reduced expression weeks was significantly lower than that in the vehicle-
level of ChAT in the hippocampus [one-way ANOVA: treated mice [one-way ANOVA: F(2,19)  = 
8.129,
F(4,15) = 11.080, P < 0.001; post hoc test: sham vs. P = 0.003; post hoc test: vehicle-treated group vs.
vehicle-treated OBX, P = 0.007]. OBX-induced down- BS-treated group, P = 0.016; vehicle-treated group vs.
regulation of ChAT expression was reversed by BS and tacrine-treated group, P = 0.002].
tacrine but not by imipramine [post hoc test: vehicle-
treated OBX group vs. BS (300 mg/kg per day)-treated Discussion
OBX group, P = 0.005; vehicle-treated OBX group vs.
tacrine-treated OBX group, P < 0.001; vehicle-treated In this study, we investigated the effects of BS on
OBX group vs. imipramine-treated OBX group, P =  OBX-induced cognitive deficits and depression-like
0.087] (Fig. 6). behavior. The present findings demonstrated that BS
administration ameliorates not only cognitive deficits
The effect of tacrine and BS on ex vivo AChE activity in but also depression-like behavior in OBX animals and
the brain suggested that the anti-dementia effect is at least in part
We analyzed the effect of BS on ex vivo activities of due to restoring synaptic plasticity–related signaling
AChE in the brain to examine the possible involvement and enhancement of cholinergic systems via inhibiting
of endogenous acetylcholine in the action of BS. As the activity of AChE in the brain.
8 D Mizuki et al

Fig. 6.  BS and tacrine (THA) treatment reverses

OBX-induced down-regulation of ChAT expression
in the hippocampus. A: Typical photos indicating
expression levels of ChAT. B: Quantitative compari-
sons of expression levels of ChAT. Each data column
represents the mean ± S.E.M. (n = 4), **P < 0.01 and
***P < 0.001 vs. vehicle-treated OBX group (post
hoc Student-Newman-Keuls test).

In the test trial, sham-operated mice spent more time

exploring a novel object, while vehicle-treated OBX
mice failed to discriminate between familiar and novel
objects, indicating impairment of non-spatial working
memory. The result that OBX-induced impairment of
object recognition performance was reversed by the
AChE inhibitor tacrine agrees with our previous studies
(8, 10) and supports the idea that OBX-induced impair-
ment of object recognition performance involves central
cholinergic dysfunction (8, 18).
Interestingly, BS treatment as well as tacrine could
significantly ameliorate OBX-induced deficit of novel
object recognition. The ameliorative effect of BS on the
working memory deficit was further supported by the
Fig. 7.  Effects of BS and tacrine (THA) on the ex vivo acetylcholin-
data obtained from the modified version of the Y-
esterase (AChE) activity in the cerebral cortices of mice. Nine-week-
old male mice received daily administration of 300 mg/kg BM for 14 maze test. Previous studies demonstrated that central
days. The animals were decapitated 1 h after the last administration cholinergic and glutamatergic systems are involved in
and the cerebral cortices were dissected out from the brain. The activ- short-term spatial working memory performance eluci-
ity of AChE was analyzed as described in the text. Each data column dated by the modified Y-maze test since the muscarinic-
represents the mean ± S.E.M. (n = 7 – 8), *P < 0.05 and **P < 0.01 receptor antagonist scopolamine and the anti-NMDA–
vs. vehicle-treated group (post hoc Student-Newman-Keuls test).
receptor antagonist MK-801 interfere with the perfor-
mance in a manner reversible by AChE inhibitors (9, 28).
We first employed an object recognition test to In this study, we found that, consistent with previous
elucidate short-term non-spatial working memory (26). reports (8 – 10), OBX mice exhibited significantly
Working memory is one of the types of short-term impaired spatial working memory performance in the
memory that have been reported to be impaired at an modified Y-maze test, and that the impairment was
early stage in patients with AD (27). In the sample trial reversed by tacrine and BS but not by imipramine. These
of this test, no significant difference in total time spent findings suggest that BS treatment can exhibit an anti-
exploring two identical objects was observed between dementia effect like tacrine in OBX mice. Taken together
sham and OBX groups, indicating virtually no differ- with the data obtained using an object recognition test,
ences in the ability to recognize objects between animals. it is likely that BS has the potential to improve both
 Anti-dementia Effects of Butea superba 9

non-spatial and spatial working memory deficits. regulation of CaMKII phosphorylation in the hippo­
We analyzed the expression levels of synaptic plastic- campus. Considering the data that imipramine failed to
ity–related signaling proteins, a molecular biological affect the cognitive dysfunction of OBX animals, it is
feature of learning and memory (8, 29, 30), to understand likely that an increase in CaMKII autophosphorylation
the mechanism underlying the effects of BS on the may be insufficient to induce neuroplasticity related to
impaired cognitive performance of OBX animals. Pre­ cognitive function.
vious studies have reported that glutamatergic systems, The present study revealed that administration of
such as NMDAR1 and GluR1, are one of the molecular tacrine and BS attenuated the OBX-induced down-
biological bases underlying learning and memory, as regulated expression level of ChAT in the hippocampus.
well as depression, and that glutamate receptor stimula- OBX-induced decrease in the expression level of ChAT
tion–triggered phosphorylation of some key protein and its reversal by tacrine are indeed consistent with our
such as NMDAR, AMPAR, CaMKII, and CREB is a previous reports (8, 10). Daily tacrine administration–
molecular mechanism underlying neuroplasticity in the induced protection of septal cholinergic neurons has
hippocampus (31 – 33). Indeed, stimulation of AMPA- also been observed in an animal model of type 2 diabetes,
and NMDA-type glutamate receptors increases the the cognitive dysfunction of which was ameliorated
intracellular Ca2+/Na+ level via the glutamate-gated by tacrine (23, 37, 38), suggesting that elevation of
cation channels on neuronal membranes, leading to the endogenous acetylcholine protects cholinergic neuron
activation of calmodulin and other Ca2+-dependent from degeneration under pathological conditions. Inter-
enzymes, and thereby elicits phosphorylation of subunits estingly, in this study, we found that systemic admin­
of NMDAR and AMPAR at glutamatergic synapses via istration of BS, as well as of tacrine, inhibited the ex vivo
the activation of protein kinases A and/or C, CaMKII activity of AChE in brain tissue. Therefore, a possible
(32). Moreover, autophosphorylation of CaMKII trig- explanation for the effect of BS on ChAT expression in
gered by Ca2+-dependent activation of calmodulin plays OBX animals is that BS may elevate the endogenous
an important role in the conversion of short-term memory acetylcholine level via its tacrine-like action and thereby
to long-term memory (18). A phosphorylated form of protect the septo-hippocampal cholinergic systems from
CREB is also known to play a role in the transcription OBX-induced neurodegenerative damage in the brain.
of late downstream genes encoding proteins such as This mechanism is also very likely to be involved in
neurotrophic/growth factors which have been implicated ameliorating/enhancing the effects of BS on the synaptic
in memory formation (33) and depression (34). plasticity–related neuronal signaling system and cogni-
In this study, we focused on these factors for the fol- tive performance in OBX animals, since evidence indi-
lowing reasons: First, previous reports from this labora- cates that endogenous acetylcholine exhibits a facilitatory
tory and others (9, 18, 27) demonstrated that OBX dete- role in the NMDA-receptor function via M1 muscarinic
riorates the septo-hippocampal cholinergic system and receptors in the brain (36) and that the M1 receptor–
thereby induces learning and memory performance mediated cognitive behavior is mediated by neuro-
deficits. Secondly, cholinergic systems affect glutamate signaling pathways including CREB phosphorylation
receptor function via the activation of muscarinic recep- and BDNF expression and secondary messenger cascades,
tors coupled with Gq/11-PKC signaling systems and like Gq/11-PKC signaling, and intracellular Ca2+ mobiliza-
thereby modulate glutamatergic neurotransmission (9, tion (36).
35, 36). As shown in Fig. 5, OBX significantly reduced The present study also investigated using the TST
the levels of p-GluR1, p-NMDAR1, p-CaMKII, and as a model to detect depression-like behavior (39),
p-CREB in the hippocampus without affecting the whether BS treatment affects emotional deficits that
basal expression levels of non-phosphorylated forms of are distinctively observed in OBX animals (10, 40).
these proteins. These findings agree with our previous The vehicle-treated OBX mice showed significantly
reports (8) and indicate that cognitive deficits observed prolonged immobility time in a manner reversible by
in OBX animals are at least partly due to OBX-induced imipramine treatment in the TST. These data agree with
dysfunction of the synaptic plasticity–related neuronal previous studies (10, 21) and indicate that increased
signaling system in the hippocampus. This idea is sup- immobility of OBX animals in this test can be used as
ported by the fact that OBX-induced dysfunction of the an index of a behavioral symptom relevant to depression.
aforementioned neuronal signaling systems was signifi- It should be noted that the depression-like behavior
cantly ameliorated in the OBX groups, the cognitive deficits caused by OBX were ameliorated in the BS-
performance of which was improved by BS and tacrine treated OBX group, as well as in the imipramine-treated
treatment. Moreover, it is of interest to note that imipra- OBX animals. These findings raise the possibility that
mine treatment attenuated only OBX-induced down- BS has an antidepressant-like effect. This idea seemed to
10 D Mizuki et al

be supported by our recent observations that BS admin- Acknowledgment

istration exhibited imipramine-like effects in an animal
KM reports a grant and Butea superba from Shiratori Pharmaceuti-
model of chronic mild stress (Mizuki et al., unpublished
cal Co., Ltd.
data).
The mechanism(s) by which BS exhibits the anti­
Conflicts of Interest
depressant-like effect is unclear. The present study
suggested involvement of synaptic plasticity–related The authors declare that they have no competing interests.
signaling and central cholinergic systems in the amelio-
rative effects of BS and tacrine on OBX-induced memory References
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