Study on the effects of Bacopa monnieri (L.) Wettst extract, a traditional
Vietnamese herb and its potential compounds, bacoside A and – B,on cognitive and emotional deficits in an animal model of dementia,
and molecular mechanism underlying their actions
Name of dispatched researcher（派遣研究者名）

Do Thi Phuong

Instructor（指導教官，研究指導者）

Pham Thi Nguyet Hang, Ph.D.

Affiliation of Instructor（所属）

Department of Pharmacolgy and Biochemistry,

Host Collaborator（受入研究者）

Vietnam National Institute of Medicinal Materials
Kinzo Matsumoto

Abstract:
Bacopa monnieri (L.) Wettst.(BM) is a well-known medicinal plant traditionally used inSouth-east Asian countries as a brain or nerve tonic. This study aimed to clarify the effects of alcoholic extract of BM on cognitive deficits caused in olfactory bulbectomized (OBX) mice and theunderlying molecular mechanisms of its action.
Methods: OBX mice were daily treated with BM (50 – 100 mg/kg per day, p.o) or the reference drug tacrine (2.5 mg/kg per day, i.p.) 1 week before and continuously after 3 days of OBX.
Cognitive performances of the animals was analyzed by the novel object recognition test, modified Y maze test, and fear conditioning test to elucidate non-spatial short term memory, spatial
working memory, and long-term memory, respectively. After completing the behavioral experiments, the animals were decapitated and the brain tissues were dissected for neurochemical andimmunohistochemical studies.
Results: In agreement with our previous study, OBX caused impairment of non-spatial shortterm memory, spatial working memory, and long-term fair memory. BM administration as well
as tacrine treatment significantly ameliorated OBX-induced memory disturbances and the effectof BM in the modified Y-maze test was reversed by scopolamine, a muscarinic receptor antagonist. BM and tacrine enhanced phosphorylation of N-methyl-D-aspartate receptor, calmodulindependent kinase (CaMKII), and cyclic AMP-responsive element binding protein (CREB), neuralsignaling proteins implicated in synaptic plasticity-related-neuronal signaling, and BDNF mRNAin the hippocampi of OBX mice. BM administration also reversed the down-regulated expressionof hippocampal choline acetyltransferase (ChAT), a cholinergic marker protein, inhibited ex vivo
activity of acetylcholinesterase in the cortices, and the decreased number of the septal cholinergic
neurons in the OBX animals.
Conclusion: The present study indicated that that BM treatment ameliorated cognition func-− 82 −

tion of OBX mice via a mechanism implicated in enhancement of synaptic plasticity-related neuronal signaling and BDNF transcription and that protection of cholinergic systems from OBXinduced neuronal damage may in part play an important role in this mechanism.
1. Introduction
Alzheimer's disease (AD) mainly affects elderly individuals, and, because of the ageing of
populations worldwide, this disorder is reaching epidemic proportions, with a large human, social, and economic burden (1). Therefore, new drug discovery and the establishment of new therapeutic treatments for AD are greatly needed.
Bacopa monnieri (L.) Wettst.(BM) is a well-known traditional medicinal plant used in India and other south-east Asian countries as a brain or nerve tonic. Lines of evidence show thatBM enhances learning and memory in neonatal and adult healthy rats (2). Chatterjee et al (3)demonstrated that BM at the dose of 80 mg/kg (po) exhibits an anxiolytic and anti-depressantactivities in mice. Moreover, BM shows neuroprotective effects on epilepsy (4), amnesia (5), andattenuates memory deficits induced by ischemia (6) or intracerebroventricular administration ofcolchicine (7) in rats. In clinical studies, it has been reported that BM can reduce state of anxiety in healthy adults (8, 9) and improve attention, cognitive processing, and working memory inhealthy older persons (10). Accumulated scientific evidence, however, is still needed not only toclarify the mechanism(s) underlying the action of BM but also to promote more possible usages of
BM in dementia therapy.
Olfactory bulbectomy (OBX) in rodent has been widely used as a model of cognitive andemotional dysfunction including AD (11-13). Indeed, evidence indicates that the impairment ofolfactory perceptual acuity not only is present at the early stage of AD (14) but also observed inmild cognitive disorder (MCI) patients (14) as well as in a transgenic AD model of mice over-expressing a mutant form of the human amyloid-β- precursor protein (15). Moreover, OBX induces
elevation of Aβ level in the brain (16) and neurodegeneration of septo-hippocampal cholinergic
innervation in rodents (12, 17). In this study, we elucidated the anti-dementia effects and themechanism underlying the action of BM in an OBX model of mice. Our findings have clearly
demonstrated that BM ameliorates cognitive deficits of OBX mice and enhances neuroplasticityrelated neuronal signaling and BDNF mRNA expression in part via stimulation/protection ofcentral cholinergic systems
2. Materials and Methods
2.1. Animals
The study was conducted according to the experimental protocols as described in Fig. 1.
Male ddY mice (Japan SLC Inc., Shizuoka, Japan) were obtained at the age of 9 weeks old. The
animals were habituated to the laboratory animal room for at least 1 week before surgery. Foodand water were available ad libitum. Housing was thermostatically maintained at 24 ± 1℃ withconstant humidity (65%) and a 12-h light-dark cycle (lights on: 07:00 – 19:00). The behavioral− 83 −

Tap waterBacopa monieri (BM; 50 mg/kg, p.o)Tacrine (THA; 2.5 mg/kg per day, i.p.)

Decapitation
Day -7

Day 0

Neurochemistry
Histochemistry3

Pretreatment

65

17

OBX
Behavioral experiments

Fig. 1 Experimental schedule
experiments were performed during the light phase from 9:00 to 18:00. The present studies wereconducted in accordance with the Guiding Principles (NIH publication #85-23, revised in 1985)
for the Care and Use of Animals and were approved by the Institutional Animal Use and Care
Committee of the University of Toyama.
2.2. Preparation of Plant extract
Bacopa monnieri was collected in Ho Chi Minh city, Vietnam, in 2010. The plant samples
were identified by Dr. Pham Thanh Huyen, a botanist of National Institute of Medicinal Materials (Vietnam). The aerial part was cut into small pieces 1-2 cm in length and dried at 60℃ in a
hot air oven. The herb then was lixiviated in 95% alcohol (1:12 w/v) for 48 h at room temperature.
After filtration, the extract was freeze-dried to yield Bacopa monnieri alcohol extract (BM, 7.2% of moisture) and kept until use. Yield of the extraction from the dried herb was calculated as
11.9% (w/w).
2.3. Surgical operation
OBX of mice was conducted as previously reported (12, 13). Briefly, the mice were anesthetized with sodium pentobarbital (60 mg/kg, i.p.), and fixed on stereotactic instruments (Narishige,
Tokyo, Japan). The skull covering the bulbs was exposed by skin incision, 1% lidocaine solution
was used as a local anesthetic and then a 1 mm burr hole was drilled. The bilateral bulbs wereaspired through a syringe and the cavity of the bulbs was filled with hemostatic gelatin sponge.
After completing the behavioral studies, all the animals were sacrificed and the operated lesion
was verified visually. The data from animals with less than 70% removal or with no intact cortex were excluded from the analysis. Sham operation was performed in a similar way without
removal of the bulb. At the end of the experiments, the olfactory bulbs of sham group mice wereconfirmed to be intact.
2.4. Drug administration
Except in specially stated cases, either vehicle water or test drugs were administered daily
from 3 days after surgery during the experimental period. On a behavioral testing day, admin-− 84 −

istration was conducted 1 h before the testing. Sham group of mice and OBX control group mice
were per orally administered water. Reference standard drugs, tacrine (THA; 9-amino-1,2,3,4tetrahydro-acridine HCl) were dissolved in 0.9% saline and administered once daily at doses of 2.5
mg/kg (i.p.). The BM extract was dissolved in water and given per orally at daily doses of 50 mg/
kg.
2.5. Fear conditioning test
The fear conditioning test was conducted according to the previously described method (12,18) with a slight modification. Briefly, the chamber for fear conditioning consisted of a transparent acrylic chamber (30×30×30 cm) and a stainless-steel grid floor equipped with an electric
shock generator/scrambler SGS-002R, CS Controller CSS-001R, and Cycle TimerCMTR (Muromachi Kikai. Co. Ltd., Tokyo, Japan). The apparatus was placed in a sound-proof observation box(MC-050/CM, Muromachi Kikai, Co. Ltd., Tokyo, Japan) through which the auditory tone (2.9
kHz, 80 dB) (SonalertR, Mallory Sonalert Products Inc., Indianapolis, IN, USA) was delivered tothe animal. In the training sessions performed 9 days after the surgery, the animals were placed
individually into the fear conditioning chamber and allowed to explore freely for 4min. Then,they received an acoustic tone (2.9 kHz, 20 s, 80 dB) co-terminated with electric footshocks (0.8mA, 2 s). The tone-footshock pairing was continuously repeated 5 times with a 1-min interval.
One min after the final footshock delivery, the mice were returned to their home cage. The test
sessions were conducted to measure freezing responses to the context and auditory tone 1 day or
5 days after the training, respectively. For the contextual memory test, 1 day after the training,the mice were placed in the same chamber to provide the contextual stimuli and allowed to movefreely for 6min. Behavioral responses recorded for 5 min from 1 min after the animal was placedin the chamber were analyzed to measure freezing behavior as an index of contextual memory.
Test phase for auditory-dependent fear memory was conducted 5 day after training trial. The
mice were placed in the chamber for 4 min and then received the tone for 2 min. The freezing behavior during the 2-min period was recorded as auditory-dependent fear memory. The animal behavior was video-recorded and analyzed automatically by Smart® system. Freezing was definedas the absence of any movement except for those related to respiration. Footshock-naive mice
were used as controls that likely exhibit pseudo-positive freezing response.
2.6. Neurochemical study
2.6.1. Western blotting
Western blotting was performed as previously described (19, 20). We used the following primary antibodies: anti-NMDAR1 rabbit polyclonal antibody (1:1000 dilution), anti-phospho-NMDAR1 (p-NMDAR1) (pSer896) rabbit polyclonal antibody (1:1000 dilution) (Cell Signaling Technology, USA), anti-GluR1 (AMPA subtype) rabbit polyclonal (1:1000 dilution), anti-phosho-GluR1(pSer 831) rabbit polyclonal antibody (1:1000 dilution) (Sigma–Aldrich, Co., St. Louis, USA),anti-CaMKIIα (A-1: sc-13141) mouse monoclonal antibody (1:1000 dilution), anti-phospho-CaM-− 85 −KII (p-CaMKII) (pThr286) rabbit polyclonal antibody (1:1000 dilution), anti-CREB (48H2) rabbit
monoclonal antibody (1:1000 dilution), anti-phospho-CREB (p-CREB) (pSer133) rabbit monoclonal antibody (1:1000 dilution), anti-PKCα rabbit polyclonal antibody (1:1000 dilution, antiphospho-PKCα/βII (pThr638/641) polyclonal antibody (1:1000) (Cell Signaling Technology, USA),anti-choline acetyltransferase goat polyclonal antibody (1:5000 dilution) (AB-144P, Millipore, CA,
USA) and anti-b actin mouse monoclonal antibody (1:10,000 dilution, Abcam®, Cambridge, UK).
The immune complexes were detected by the enhanced chemiluminescence method (ImmobilonTM
Western Chemiluminescent HRP Substrate) (Millipore, Tumecula, CA, USA) and imaged using
Lumino Image Analyzer LAS-4000R (Fuji Film, Tokyo, Japan). The quantity of immune-reactive
bands was analyzed using ImageQuant TL software (GE Healthcare, Buckinghamshire, UK).
The expression levels of each membrane were re-probed using a Blot Restore Membrane Rejuvenation Kit (Millipore, Tumecula, CA, USA).
2.6.2. Quantitative real-time polymerase chain reaction (PCR)
Quantitative real-time PCR was conducted as previously described (21, 22). Briefly, the animals were decapitated after completing the behavioral experiments. The hippocampus were dissected out and kept at -80℃ until use. Total RNA was extracted from the cortex with Sepazol®
(Nacalai Tesque, Japan) according to the manufacturer’s instructions. First-strand cDNA wassynthesized with oligo (dT) primers and M-MLV reverse transcriptase® (Invitrogen, USA) andwas used as a template for real-time PCR. Quantitative real-time PCR was carried out with FastSYBR Green Master Mix and the StepOne Real-time PCR System® (Applied BioSystem, USA).
The following primer sets of BDNF and b-actin were designed by Perfect Real Time support
system (Takara Bio Inc., Japan): BDNF (NM_007540): 5’-AGCTGAGCTGTGTGACAGT-3’ (forward) and 5’-TCCATAGTAAGGGCCCGAAC-3’ (reverse); β-actin (NM_007393): 5’-CATCCGTAAAGACCTCTATGCCAAC-3’ (forward) and 5’-ATGGAGCCACCGATCC ACA-3’ (reverse). Melting
curve analysis of each gene was performed every time after amplification. In all reactions, b-actinmRNA was used as a control to which the results were normalized. Standard curves of the log
concentration of each gene vs. cycle threshold were plotted to prove inverse linear correlations.
The correlation coefficients for standard curves of target genes were 0.9965 to 0.998.
2.6.3. Ex vivo and in vitro measurements of cholinesterase activity in the brain
Ex vivo measurement: After completing the behavioral experiments, mice were decapitated
and the frontal cortices were dissected out and kept at −80℃ until use. Determination of cholinesterase activity was performed on the basis of the colorimetric method as previously described(12, 21, 23). Briefly, the frozen cortex was weighed and homogenized in 10-times volume of 0.1 M
phosphate buffer (pH 7.4) containing 1% Triton-X-100. After centrifugation at 15,000×g at 4℃ for
20 min, the clear supernatants were collected and served as the enzyme source. Cholinesterase
activity was determined in 10 μl aliquots of homogenates (run as triplicates) in a 96-well flatbottom microplate. The reaction was started by adding 8 μl of 10 mM 5,5´-dithiobis-2-nitrobenzoic− 86 −

acid (DTNB), 16 μl of 7.5 mM acetylthiocholine (ATCI) and 201 μl of 0.1 M sodium phosphatebuffer (pH 8.0). The spectrophotometric absorption at 405 nm during a 3 min incubation period
at 25oC was quantitatively measured using a microplate reader (Sunrise Classic; TECAN Japan,
Kawasaki) and is expressed as nmol ACh hydrolyzed/min/mg tissue.
In vitro measurement: The assay for measuring AChE activity was modified from the assay described by Ellman et al. (23) and Ingkaninan et al. (24). Frontal cortex supernatants wereobtained from naïve ddY mice as the enzyme source. Cholinesterase activity was determined as
described above.
2.7. Data analysis
The data are expressed as the mean ± SEM. The data obtained from the behavioral tests
and neurochemical experiments were analyzed by paired and unpaired Student’s t-test or oneway analysis of variance (ANOVA) followed by a post hoc multiple comparison test (Dunnett’s
method) as appropriate. Differences of P<0.05 were considered as significant. The analysis was
conducted using SigmaStat® ver. 3.5 (SYSTAT Software Inc., Richmond, CA, USA).
3. Results
3.1. BM ameliorates OBX-induced long-term memory deficit in the fear conditioning test
Fear conditioning test was used to clarify the effect of BM treatment on long-term memory
deficit in OBX mice. In this test, mice were fear-conditioned to the context and auditory stimuli
by electrical footshocks as unconditioned stimuli. Hippocampus- dependent memory and amygdala-dependent memory were analysised as freezing responses of each animal group to the contextual and auditory stimuli after 1 day and 5 days training trial, respectively (Fig 4A). Freezing
responses to the context and auditory stimuli of vehicle-treated OBX mice were markedly decreased compared with those of the footshocked sham animals. However, daily treatment of BM
and tacrine significantly increased in the freezing time of OBX mice in both contextual and auditory test, indicating the improvement in hippocampus- and amygdala dependent memory formation in OBX mice (Fig 4B. 4C).
3.2. BM facilitates neuroplasticity-related neuro-signaling pathway in the hippocampus of OBX mice
In order to understand the molecular mechanism(s) underlying BM-induced improvementof cognitive deficits in OBX mice, we examined the effects of BM on neuroplasticity-related
neuro-signaling pathway in the hippocampus of OBX mice. Western blotting data showedthat the total protein expression level of NMDAR1 (NMDAR subunit), GluR1 (AMPAR subunit),
α-CaMKII or CREB have no change under the effect of OBX or drug treatment (Fig. 5). However,vehicle-treated OBX mice showed a significantly reduced in the levels of pSer896-NMDAR1,
pSer831-GluR1 and pThr286-CaMKII compared with sham animals. BM and tacrine treatment
reversed the decrease in levels of pSer896-NMDAR1 and pThr286-CaMKII but these treatments− 87 −

have no influence on the levels of pSer831-GluR1 in OBX mice (Fig. 5 A, B, C, D). No differencein the levels of pSer133-CREB was observed between sham and OBX animals. Interestingly,
pSer133-CREB was over-expressed in BM and tacrine-treated OBX mice (Fig. 5 E).
We also measured the expression levels of BDNF gene transcript which is a functional molecule downstream of the transcriptional activity of CREB, via CREB phosphorylation (Figure 6).
OBX mice had a significantly reduced level of BDNF mRNA in the hippocampus (P<0.05). However, daily administration of BM and tacrine to OBX mice significantly reversed the decreases inthe expression levels of BDNF (P<0.05) (Fig. 6).
Since the activation of PKC is necessary for maintenance of the LTP response, we identified
the the expression levels of PKCα and pThr638-PKCα/pThr641 PKCβin the hippocampus of eachmouse groups. The expression level of PKCα and pThr638-PKCα/pThr641 PKCβ of each animalgroup were found no significant difference.
3.3. The effects of BM on central cholinergic systems
The expression of ChAT, a cholinegic marker in the hippocampus of animals was examinedusing WB. Compared with the sham-operated group, the vehicle-treated OBX group had a significantly reduced expression level of ChAT in the hippocampus (P<0.05). The OBX induced downregulation of ChAT expression was reversed by BM and tacrine (P<0.05) (Fig 8).
In addition, acetylcholinesterase (AChE) activity ex vivo was measured in the cerebral cortex. As shown in Fig. 9A, there was no significant difference in the activity was observed between

Fig. 2
vehicle-treatedsham and OBX groups. However, the activities of cortical AChE in the ATL- andA

B
Contextual memory

1 days

1 min

Auditory memory

5 days

5 min

4 min

Training

2 minTraining

Contextual memory
Auditory memory
OBX

100

Freezing time/ 2 min (%)

Freezing time/ 5 min (%)

100

Sham

##
#

80

60

**
40

20

Sham

OBX

80

#

##

BM

THA

60

**
40

20

g

0

0

Vehicle Vehicle

BM

THA

Vehicle Vehicle

Fig. 2 Effects of BM and THA administration on contextual and auditory (conditional)
fear memory impaired by OBX. Each data column represents the mean ±S.E.M.,
indicated (n=8 -12). **P<0.01 vs. sham-operated group; # P<0.05, ## P<0.01 vs. watertreated OBX group.

− 88 −

Vehicle-treated shamFig. 3
Sham
Vehicle

OBX
VehicleVehicle-treated OBX

BM

THA

BM-treated OBX

A p-NMDAR1

THA-treated OBX
1.5

B

NMDAR1###
Relative density

p-GluR1
GluR1
p-CaMKII
α-CaMKII

1
**

0.5

p-CREBCREB
0NMDAR1
1.5

C

2

D

2.5

p-NMDAR1

E
##

## ##
#

0.5

*
1

Relative density*

1

Relative density

Relative density

2
1.5

1.5
1

0.5
0.5

0

0
GluR1

p-GluR10
CaMKII

p-CaMKII

CREB

p-CREB

Fig. 3 Effects of BM and THA treatment on expression levels of synaptic plasticityrelated neuronal signaling proteins in the hippocampus. Each data column
represents the mean ± S.E.M., indicated (n=5-6). *P<0.05, **P<0.01 vs. sham-operated
group, # P<0.05, ## P<0.01 vs. water-treated OBX group.

tacrine-treated OBX groups were significantly reduced compared with that in the vehicle-treated
OBX mice. On the other hand, treatment of cortical homogenates with tacrine showed a potent
inhibitory effect on the in vitro activity of AChE with IC50 = 21.8 ng/ml, while BM had a negligible effect on the activity with the IC50 value was as great as 739.9 μg/ml (Fig 9B).
3. Discussion
In present study, we demonstrated that BM improves cognition deficits caused by OBX in
mice, and suggested that the effect is at least in part due to facilitation of synaptic plasticityrelated neuron signaling pathway including BDNF transcription and enhancement of cholinergic
systems.
BM improves long-term memory deficits in OBX mice.
In this study, we found that administrations of BM and THA could attenuate OBX-induced
deficits of long-term memory in the fear conditioning test (Fig. 2). It is well known that contextual memory and conditional memory elucidated in this test depend on the hippocampal function
and the hippocampus plus amygdala function, respectively (25), and involve synaptic plasticityrelated signaling and protein synthesis. Moreover, OBX reportedly induces impairment of fear
responses to contextual and conditional auditory stimuli (26) as well as of the cholinergic systems− 89 −

Fig. 4

BDNF mRNA
Sham

OBX

BDNF/β-acttin mRNA ratio

1.8

#

1.6
#
1.4
1.2

*

1
0.8
0.6
0.4
0.2
0
Vehicle Vehicle

BM

THA

Fig. 4 Effects of BM and THA administration on OBX-induced down-regulation of
BDNF mRNA expression in the hippocampus. Each data column represents themean ± S.E.M., indicated (n=5-6). *P<0.05 vs. sham-operated group, # P<0.05vs watertreated OBX group.

in the hippocampus, cortex, and amygdala in rodents (17). Therefore, considering potential involvement of central cholinergic function in the amelioration of short-term memory deficit by BM,
it is also very likely that improvement of OBX-induced long-term fear memory deficits by BM is
at least mediated by the same mechanism(s) as BM-induced amelioration of short-term memory
deficits of OBX animals.
Improvement of neuroplasticity-related signaling by BM administration
To obtain a better understanding, at the molecular level, of BM-induced improvement of cognitive dysfunction in OBX mice, we analyzed alterations of the expression levels of synaptic plasticity-related signaling proteins, a molecular biological feature of learning and memory. Evidence
has demonstrated that the hippocampal glutamatergic neurotransmission is one of the molecularbases underlying learning and memory (27) and that stimulation of glutamate receptors triggers
phosphorylation of some key proteins such as NMDAR, AMPAR, CaMKII and CREB and thereby
is involved in synaptic plasticity in the hippocampus (28). Indeed, stimulation of AMPA- ane
NMDA-type glutamate receptors increases the intracellular Ca2+/Na+ level via the glutamate-gated cation channels on neuronal membranes and thereby activates calmodulin and other Ca2+-dependent enzymes. This signaling elicits the phosphorylation of subunits of NMDAR and AMPAR
at the glutamatergic synapses via the activation of protein kinase C. Moreover, intracellular
Ca2+-dependent activation of calmodulin triggers autophosphorylation of CaMKII and is reportedly implicated in the conversion of short-term memory to long-term memory. CREB, a nuclear
transcription factors that can be activated by protein kinases A and/or C, also plays an importantrole in memory formation in a variety of cognitive tasks involving different brain structures (29).
Its phosphorylated form, p-CREB, is implicated in the transcription of late downstream genes encoding proteins such as neurotrophic/growth factors including BDNF which plays a role in learning and memory (30, 31). In this study, we focused on these factors because of a couple of reasons.

− 90 −

Fig. 5

ShamvehicleOBX
vehicle

BM

THA

ChAT
β-actin

Vehicle-treated sham2

Vehicle-treated OBX#
BM-treated OBX

Relative density#
THA-treated OBX
1.5
*
1

0.5

0

β-actin

ChAT

Fig. 5 Effects of BM and THA treatment on OBX-induced changes in expression levelof cholinergic marker protein, choline acetyltransferase (ChAT) in the hippocampus.
Each data column represents the mean ± S.E.M., indicated (n=5 – 6). *P<0.05 vs.
sham-operated group, # P<0.05 vs. water-treated OBX group.
First, OBX causes deterioration of the septo-hippocampal cholinergic system and thereby impairslearning and memory performance (12, 17). Second, this system can affect hippocampal glutamate receptor function via activation of muscarinic receptors coupled with Gq/11-PKC signaling
systems and thereby modulate glutamatergic neurotransmission.
As shown in Figure 3, this study clearly revealed that OBX reduced levels of p-AMPAR,
p-NMDAR1, p-CaMKII except p-CREB in the hippocampus without affecting basal expression
levels of non-phosphorylated forms of these proteins, indicating that, in agreement with previous
reports, OBX causes down-regulation of synaptic plasticity-related neuronal signaling systems.
The present results that the CREB phosphorylation step analyzed in the hippocampus was apparently insusceptible to OBX agree with Han et al. (32). The reason for this different susceptibility to OBX is unclear but it may be due to a difference in the tissue area used for analysis.
It should be noted that phosphorylation of these neuronal signaling proteins except AMPAR
in OBX animals could be reversed or even elevated in OBX groups treated repeatedly with BM
and THA. These findings prompt us to consider that OBX-induced short-term and long-term
cognitive deficits and their amelioration by THA and BM treatment are at least in part due todysfunction and normalization of synaptic plasticity-related neuronal signaling systems, respectively, in the hippocampus. This hypothesis can be supported by changes in hippocampal expression of BDNF mRNA, one of important downstream factors of the signaling system, by THA andBM treatment of OBX animals, since OBX also down-regulated expression of BDNF mRNA in− 91 −

Fig. 6

B1

Sham

OBX

60
50

70
60
50
40
30
20
10
0

40

*
0

**

B2

30
20
10
0

Vehicle Vehicle BM

THA

10

20

30

40

50

60

THA (ng/ml)

Cholinestrase activity(% inhibition)

AChE activity
g tissue/min)(nmol/mg

80

Cholinestrase activity(% inhibition)

A

70
60
50
40
30
20
10
0
0

200 400 600 800 10001200

BM (µg/ml)

Fig. 6 Effects of BM and THA on ex vivo and in vitro activities of cholinesterase. A:
Cholinesterase activity was measure using cortical tissue homogenates which wereprepared from sham-operated mice or OBX mice treated with test drugs. Each data
column represents the mean ± S.E.M., indicated (n=5 – 6).*P<0.05, ** P<0.01 vs. watertreated OBX group and sham group. B1 and B2: in vitro inhibitory effects of test
drugs were analyzed using the cortical tissue homogenate as an enzyme source.
the hippocampus in a manner reversible by THA and BM treatment (Fig. 4).
Protection/facilitation of septo-hippocampal cholinergic systems by BM and THA
Together with the present behavioral data that THA- and BM-induced amelioration of cognitive deficits caused by OBX was susceptible to a muscarinic receptor antagonist, facilitationof central cholinergic systems by THA and BM is likely to be involved in their ameliorating/enhancing effects on synaptic plasticity-related neuronal signaling system. This hypothesis seems
substantiated by the findings that THA and BM administration attenuated down-regulated expression level of ChAT, a cholinergic nerve maker protein, caused by OBX. Indeed, both OBXinduced decrease in expression level of hippocampal ChAT and its reversal by THA observed inthis study agree with previous reports (12, 13), indicating that OBX causes neurodegenerationof septo-hippocampal cholinergic systems and that THA protects septal cholinergic neurons from
OBX-induced damage via endogenous acetylcholine. Therefore, it is of interest to note that BM
administration could also attenuate OBX-induced down-regulation of ChAT expression in the
hippocampus, suggesting protection of septal cholinergic neurons by BM from OBX damage (Fig.
5).
One of most plausible explanation for the mechanism underlying the effect of BM may bethat BM administration facilitate function of central cholinergic systems by acting like THA,
namely, inhibiting the activity of acetylcholinesterase in the brain. In fact, the present study− 92 −

demonstrated that BM administration, like THA treatment, was capable of reducing ex vivo activity of cholinesterase in the brain tissues and that BM extract itself, like THA, has the potential to inhibit in vitro cholinesterase activity of brain homogenates (Fig. 6). However, the inhibitory effects of BM on ex vivo and in vitro activities of cholinesterase were clearly less potent than
THA even at a dose which exhibited the same extents of behavioral and neurochemical improvements as THA. These findings lead us to suggestion that BM-induced improvement of cognitive
dysfunction in OBX mice is mainly mediated by mechanisms differing from cholinesterase inhibition.
Conclusion
Present study has demonstrated that daily administration of BM ameliorates cognition impairment of OBX mice and that facilitation of LTP-related neuronal signaling and BDNF via
stimulation of cholinergic systems are involved in the action of BM
Acknowledgements
This work was in part supported by a Grant-in-Aid for the 2010 and 2012 Cooperative Research Project II from the Institute of Natural Medicine, University of Toyama (to H.T.N.P. andK.M.). Do Thi Phuong (NIMM, Hanoi, Vietnam) and Le Thi Xoan (NIM, Toyama, Japan) havecontributed to collection and analysis of the data in this collaboration project.
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