J Acupunct Meridian Stud 2017;10(1):26e32

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Journal of Acupuncture and Meridian Studies

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RESEARCH ARTICLE

Effect of Manual Acupuncture Stimulation at

“Bai-Hui” (GV 20) or “Yintáng” (Ex-HN3) on
Depressed Rats
Ken Takagi 1,2, Nobuyuki Tanahashi 1, Nozomi Amagasu 3,
Kaito Mizuno 4, Jun Kawanokuchi 1, Guo Yi 2, Torao Ishida 1,2,*

1
Institute of Traditional Chinese Medicine, Suzuka University of Medical Science,
1001-1 Kishioka, Suzuka, Mie 510-0293, Japan
2
Acupuncture and Moxibustion College, Tianjin University of Traditional Chinese Medicine,
Tianjin 300193, China
3
Graduate School of Health Science, Suzuka University of Medical Science, 1001-1 Kishioka,
Suzuka, Mie 510-293, Japan
4
Department of Acupuncture and Moxibustion, Suzuka University of Medical Science,
1001-1 Kishioka, Suzuka, Mie 510-0293, Japan
Available online 9 December 2016

Received: Feb 29, 2016 Abstract

Revised: Nov 26, 2016 A previous study on rats showed that simultaneous acupuncture stimulation at the “Bai-
Accepted: Nov 30, 2016 Hui” (GV 20) and the “Yintáng” (Ex-HN3) acupoints alleviated the state of depression to
an extent similar to that achieved by pharmacotherapy. This study investigated whether
KEYWORDS the alleviation of the depressed state required simultaneous acupuncture at these two
acupuncture; acupuncture points. For the purposes of testing the effect of acupuncture on depressive
anxiety; symptoms, we treated a depression model rat, where depression had been induced by us-
Bai-Hui; ing a mild water-immersion stress technique, with either acupuncture stimulation at only
depression; one acupuncture point (GV 20 or Ex-HN3) or an antidepressant, and we measured the
immobilization stress; immobile time for evaluating the state of depression. Anxiety, as a symptom commonly
water-immersion stress; associated with depression, was also evaluated by measuring the number of head dips.
Yintáng Neither the immobile time nor the number of head dips decreased upon acupuncture
stimulation. From this study, single acupuncture stimulation at either “GV 20” or “Ex-

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any
medium, provided the original work is properly cited.
* Corresponding author. Institute of Traditional Chinese Medicine, Suzuka University of Medical Science, 1001-1, Kishiokacho, Suzukashi,
Mieken 510-0293, Japan.
E-mail: ishida-t@suzuka-u.ac.jp (T. Ishida).
pISSN 2005-2901 eISSN 2093-8152
http://dx.doi.org/10.1016/j.jams.2016.11.006
Copyright ª 2016, Medical Association of Pharmacopuncture Institute. Publishing services by Elsevier B.V.
Acupuncture at GV 20 or Ex-HN 3 on Depressed Rats
HN3” alleviated neither the state of depression nor the anxiety. The water-immersion
stress used to make the depression model rats was shown not to induce anxiety; however,
the stress induced by immobilizing the rats for acupuncture stimulation did lead to
anxiety.
1. Introduction
Depression is a mood disorder characterized by depressed
mood, anxiety, impaired mental activity, reduced appetite,
and insomnia [1]. Pharmacotherapy and electroconvulsive
therapy are considered the main effective options against
depression, and the rate of recurrence of depression is
reportedly decreased when multiple approaches are used
at the same time [2]. The clinical condition of depression is
not yet clear, and depression may be attributable to many
possible mechanisms. Monoamine metabolic dysfunction is
thought to be one of the mechanisms via which depression
can occur as a result of disorders of monoamine metabolism
in the brain because of stress and decreased monoamine
levels [3,4]. This may suggest that the hypothal-
amicepituitaryeadrenal (HPA) axis adapts to stimulation
from an external environment. However, reports in the
literature suggest that the reverse, in which prolonged
exposure to stressors alters the HPA axis, which, in turn,
leads in time to the development of depressive symptoms,
is often the case. Monoamine concentrations are restored
to normal levels several hours after antidepressant admin-
istration, but treatment for several weeks is necessary to
improve the state of depression. A theory that has gained
attention in recent years is the neural plasticity hypothesis
[5e7]. This suggests that the HPA axis adapts to stimulation
from an external environment. Accordingly, depression
causes an abnormality of this system, resulting in increased
blood glucocorticoid concentrations, leading to hippocam-
pal atrophy [3].
Acupuncture or electroacupuncture stimulation is used
in China to treat patients suffering from depression [8,9].
One of the treatments is simultaneous electroacupuncture
stimulation at the GV 20 and the Ex-HN3 acupoints [8]. Few
animal experiments have examined the treatment of
depression with acupuncture. In acupuncture research
using rat models of depression created by exposure to
chronic stress, electroacupuncture at the “Bai-Hui” (GV 20)
and the “Yintáng” (Ex-HN3) or the GV 20 and the “An-Mian”
(EX17) acupoints showed remedial effects against depres-
sion [10]. For the purposes of testing the effect of
acupuncture on depressive symptoms, our own research
found that a state of mild depression could be induced in
rats by using water-immersion stress at night. Also, when
these mildly depressed rats were stimulated with simulta-
neous acupuncture without any electrical stimulation at GV
20 in combination with Ex-HN3, the depressive state was
alleviated to an extent similar to that achieved with anti-
depressants [11].
The present study used behavioral assessments to
analyze whether depression induced by water-immersion
stress would be alleviated by using nonelectroacupuncture
stimulation at only one acupuncture point, either the GV 20
or the Ex-HN3 acupoint, or whether simultaneous
27
nonelectroacupuncture at the GV 20 and the Ex-HN3 acu-
points, as shown in our previous paper, is necessary [11].
Part of this research was presented at the World Federation
of AcupunctureeMoxibustion Societies’ 8th World Confer-
ence on Acupuncture [12].
2. Materials and methods
We purchased 4-week-old male Wister rats (body weight,
100e120 g) from CLEA Japan, Inc. (Tokyo, Japan). We
maintained the animals in an environment at room tem-
perature (22
3 C) with 60% humidity and a 12-hour light/
dark cycle. The rats were given free access to animal feed
(CE-2; CLEA Japan, Inc.) and water. Rats were acclimatized
to handling for 1 week after purchase. Thirty-five rats were
randomly divided into two groups with 10 rats in the non-
depression group and 25 rats in the mild depression group.
The rats in the nondepression group were maintained under
the conditions described above. For the rats in the mild
depression groups, mild depression was created using
water-immersion stress according to Tanahashi et al [11].
The night water-immersion stress was continued from 22:00
to 10:00 of the next day for 1 week. The floor of the cage
was flooded with water at 25
2 C to a depth equivalent to
ankle height on the rat. The degree of mild depression
created in the rats by using water-immersion stress was
evaluated by using the forced swimming test described by
Porsolt et al [13].
To avoid disruptions in the rats’ sleep cycle, which may
produce or contribute to the depressive symptomology, and
disruptions in the rats’ circadian rhythm, which are asso-
ciated with stress, anxiety, and depression, we performed
water immersion during the time described above because
rats are nocturnal and are usually awake and active at
night. The weight of each rat and its food intake were
measured every week.
The 10 rats in the nondepression group were randomly
divided into two groups with five rats in each group: Group
1 [water-immersion stress () þ immobilization ()] and
Group 2 [water-immersion stress () þ immobilization (þ)].
In addition, the 25 mildly depressed rats were randomly
divided in five groups with five rats in each group: Group 3
[water-immersion stress (þ) þ immobilization ()], Group 4
[water-immersion stress (þ) þ immobilization (þ)], Group 5
[water-immersion stress (þ) þ acupuncture at “Yintáng”
(Ex-HN3)], Group 6 [water-immersion stress (þ) þ
acupuncture at “Bai-Hui” (GV 20)], and Group 7 [water-
immersion stress (þ) þ imipramine] (Table 1). No significant
differences in body weights among the groups were noted
at the time of grouping. Experiments were then started.
Approval from the Suzuka University of Medical Science’s
Animal Experiment Ethical Review Board was obtained for
the experiments conducted in this research.
28
Table 1 List of the processes applied to the experimental
groups.
Name of Water-immersion Intervention
group stress
Group 1  d looked into a hole.
Group 2  Immobilization
Group 3 þ d mean and were analyzed at first by using Excel Statistics
Group 4 þ Immobilization
Group 5 þ Acupuncture at Ex-HN3
Group 6 þ Acupuncture at GV 20
Group 7 þ Imipramine
Mild depression was induced in the rats by using water
immersion (from 22:00 to 10:00 of the next day) for 1 week
[11]. Thereafter, rats exhibiting symptoms suggestive of
mild depression underwent either stimulation by acupunc-
ture or administration of imipramine as an antidepressant
for 3 weeks. Body weights and food intakes were measured
every week. To clarify the effects of acupuncture stimu-
lation and pharmacotherapy on rats exhibiting symptoms
suggestive of mild depression, we randomly divided the rats
into seven groups with five rats per groups, as has already
been described (Table 1).
Acupuncture stimulation was continued for 20 minutes
by inserting a stainless-steel needle (diameter, 0.25 mm;
length, 15 mm; Acupuncture Needle D-Type; SEIRIN Co.
Ltd., Shizuoka, Japan), without any spinning, flicking,
moving up and down, or application of electrical current, to
a depth of 5 mm into the GV 20 or the Ex-HN3 acupoint. This
procedure was performed 5 days a week for 3 weeks [11].
Groups that did not receive acupuncture stimulation,
except Group 1 and Group 3, were immobilized for a similar
length of time in a plastic bag in which a hole had been
made to allow the rats to breathe. The rats in Group 1 and
Group 3 were not immobilized for comparison with other
groups to study the effects of the immobilization.
The antidepressant imipramine (10 mg/kg body weight)
was dissolved in saline and administered intraperitoneally
5 days a week for 3 weeks [14,15]. Groups not receiving
antidepressants, including Group 1 [water-immersion () þ
immobilization ()], were intraperitoneally administered
with saline, instead of antidepressants, without
immobilization.
The forced swimming test was performed using the
method described by Porsolt et al [13]. Rats were placed in
an open cylindrical container with a diameter of 25 cm and
a height of 50 cm containing fresh water at 25
2 C to a
depth of body height þ 5 cm for 13 minutes. The actions of
the rats were recorded with a video camera. The immobile
time for the 10 minutes after the first 3 minutes of the 13-
minute recording had been excluded was measured, the
first 3 minutes being regarded as the acclimation time. The
immobile state was defined as the state in which the rat
stopped most movements and only performed the small-
limb movements necessary to maintain balance. The
immobile time was defined as the time during which that
state continued.
A hole board apparatus comprising a gray wooden box
(50 cm  50 cm  50 cm) with four holes of 3.8 cm in
K. Takagi et al.
diameter spaced equally across the floor [16,17] was used
in this experiment. The rat was placed in the center of the
apparatus and allowed to freely explore the apparatus for 5
minutes. The anxiety state was evaluated by measuring the
number of head dips and the number of times the rat
Data are presented as means
standard errors of the
2012 (Society Information Service) and were then rean-
alyzed using IBM SPSS Statistics 22. The presence of sig-
nificant differences was assessed using the one-way
analysis of variance and a multiple comparison assay
(TukeyeKramer), except for the analysis for Figure 1. The
analysis for Figure 1 was done using a single comparison
assay (ManneWhitney). A p value < 0.05 were considered
statistically significant.
3. Results
Our previous paper showed that water-immersion stress at
night led to the induction of symptoms akin to mild
depression and that simultaneous nonelectroacupuncture
at both the GV 20 and the Ex-HN3 acupoints alleviated
those symptoms to an extent similar to that for an anti-
depressant [11].
This time, we studied whether acupuncture stimulation
at only one acupoint, either GV 20 or Ex-HN3, would alle-
viate the state of depression in mildly depressed rats.
The weight changes in the rats from the seven study
groups over time are shown in Figure 2. For more than 1
week after immobilization began, the rate of increase in
the body weights of the rats in Group 2 [water-immersion
stress () þ immobilization (þ)) (,) began to decrease.
By contrast, the weights of the rats in Group 3 (water-im-
mersion stress (þ) þ immobilization ()] (O), Group 4
[water-immersion stress (þ) þ immobilization (þ)] (B),
Group 5 [water-immersion stress (þ) þ acupuncture at Ex-
HN3] (<), Group 6 [water-immersion stress (þ) þ
Figure 1 Comparison between the numbers of head dips in
Group 1 and Group 3 at 4 weeks. Group 1 and Group 3 were the
nonwater-immersion and the water-immersion stress groups,
respectively. The p value of Group 1 versus Group 3 is p > 0.99.
The treatments for Groups 1 and 2 were the same as those in
Figure 2.
Acupuncture at GV 20 or Ex-HN 3 on Depressed Rats
400
300
Body Weight ( g )
200
100
0
0 1 2 3 4 5
pre-stress
Figure 2 Changes in body weight in depressed and nonde-
pressed rats for each treatment. The treatments for experi-
mental groups 1 to 7 are as follows: Group 1 (), water-
immersion stress () þ immobilization (); Group 2 (,),
water-immersion stress () þ immobilization (þ); Group 3
(O), water-immersion stress (þ) þ immobilization (); Group
4 (B), water-immersion stress (þ) þ immobilization (þ);
Group 5 (<), water-immersion stress (þ) þ acupuncture at Ex-
HN3; Group 6 (;), water-immersion stress (þ) þ acupuncture
at GV 20; Group7 (-), water-immersion stress (þ) þ
imipramine.
acupuncture at GV 20] (;), and Group 7 [water-immersion
stress (þ) þ imipramine] (-) did not increase during the 1
week with water-immersion stress, but started to increase
after water-immersion stress had been stopped, but the
weights did not attain the level of the nonstress group.
Food intake was also found to have decreased for the week
of the water-immersion stress. However, the food intakes in
Groups 5, 6, and 7 increased after the water-immersion
stress had been stopped. Furthermore, the food intake in
Group 2 tended to decrease slightly, and those in Groups 3
to 7 tended to be lower compared with Group 1 [water-
Figure 3 Food intake of each group in each week for 4
weeks: 0e1, 1e2, 2e3, and 3e4. Stress started in Week 0. The
treatments for experimental groups 1 to 7 are the same as
those in Figure 2.
29
immersion stress () þ immobilization ()] (Figure 3).
However, these differences among the groups did not attain
statistical significance.
The immobile times for the seven groups in the forced
swimming test are shown in Figure 4. In Group 1 () and
Group 2 (,), the immobile times remained relatively
constant over the 4-week period of the experiment. The
immobile times for Group 3 (O) and Group 4 (B)
increased during the water-immersion stress for 1 week,
continued to increase during 1 week after the end of water-
immersion stress, and then decreased during the remaining
2 weeks of the experiment. The degree of mild depression
of rats created by using water-immersion stress was
confirmed for the immobility time of the mild depression
week rats to be 99
50 seconds compared with 37.5
7 seconds
of the nondepression rats’ immobility time. The differences
in the immobile times between Group 1 () and Group 4
(B) were statistically significant not only after 1 week but
also after 2 weeks (p < 0.01).
Furthermore, the immobile time for Group 7 (-)
decreased significantly during the week after the cessation
of water-immersion stress (p < 0.01). A previous paper
reported that the immobile time for a water-immersion
stress (þ) þ simultaneous acupuncture at the GV 20 and
the Ex-HN3 acupoint group decreased significantly during
the week after cessation of water-immersion stress (p <
0.01). In addition, in the present study, the immobile times
for Group 5 (<) and Group 6 (;) continued to increase in
the week after cessation of water-immersion stress in the
same manner as Group 4 (B).
Rats perform exploratory behaviors, including head dips
to looking into holes, when placed in an apparatus under a
new environment [16,17]. However, fear and anxiety
regarding the holes occurs, and the number of head dips
decreases when the rat is under stress. We, therefore,
analyzed the anxiety in the five groups of depressed rats
and the two groups of nondepressed rats (Figure 5). No
Figure 4 Immobile time in the forced swimming test in
depressed and nondepressed rats for each treatment, with the
treatments being the same as those in Figure 2. ##p < 0.01:
Group 1 versus Group 4 after 1 week or Group 1 versus Group 4
after 2 weeks; þþp < 0.01: Group 4 versus Group 5, Group 6, or
Group 7 after 1 week; **p < 0.01: Group 4 versus Group 5,
Group 6, or Group 7 after 2 weeks.
30 K. Takagi et al.

30

25

Head Dip (counts/5 min)

20

15

++ + +
+ +
10

0
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7

Figure 5 Number of head dips in the hole-board test for depressed and nondepressed rats for each treatment, with the
treatments being the same as those in Figure 2: ,, 0 week; , 1 week; , 2 weeks; , 3 weeks; -, 4 weeks. þp < 0.05: 0 week
versus each week. þþp < 0.01: 0 weeks versus each week.

large changes were seen in the numbers of head dips in state of depression induced by chronic stress [15]. Animals
Groups 1 and 3. The numbers of head dips in Group 2 in a depressive state have been stimulated with electro-
significantly decreased over the time of the experiment acupuncture at the “Bai-Hui” (GV 20) and the “Yintáng”
compared to the numbers during the pre-stress period (p < (Ex-HN3) acupoints, at the GV 20 and “Sanyinjiao” (Sp-6)
0.01). The numbers of head dips in Groups 4 to 7 were acupoints, or at the GV 20 and “An-Mian” (EX17) acupoints
significantly decreased compared to the numbers during [10,18,19]. A previous paper found that rats with depres-
the pre-stress period (p < 0.05). sion induced by water-immersion stress showed improve-
To demonstrate both that the water-immersion stress ment with antidepressant pharmacotherapy [11]. Reports
did not induce anxiety but rather the stress induced by have indicated that the pause in weight gain with 1 week of
immobilization did and that the stress was not suppressed water-immersion stress is thought to depend on decreased
by acupuncture stimulation at only one acupoint, Ex-HN3 or food consumption and functional declines in digestion
GV 20, we compared and analyzed the numbers of head
dips for Groups 1 and 3 at the end of the experiment, and
we simultaneously compared and analyzed the numbers of
head dips for the non-immobilization groups (Groups 1 and
3), the immobilization groups (Groups 2 and 4), and the
acupuncture groups (Groups 5 and 6). No statistically sig-
nificant difference was seen between Groups 1 and 3 (p >
0.99; Figure 1). This result showed that water-immersion
stress did not induce anxiety. However, a statistically sig-
nificant difference was seen not only between the non-
immobilization groups (Groups 1 and 3) and the
immobilization groups (Groups 2 and 4) (p < 0.05), but also
between the nonimmobilization groups (Groups 1 and 3)
and the acupuncture groups (Groups 5 and 6) (p < 0.01)
(Figure 6). Again, no statistically significant difference was
seen between the immobilization groups (Groups 2 and 4)
and the acupuncture groups (Groups 5 and 6; p > 0.99;
Figure 6). These results show that immobilization induced
the anxiety and that the anxiety was not suppressed with Figure 6 Simultaneous comparisons between the numbers of
acupuncture at only one acupuncture point (GV 20 or Ex- head dips in the nonimmobilization groups (Group 1 þ Group
HN3). 3), the immobilization groups (Group 2 þ Group 4), and the
acupuncture groups (Group 5 þ Group 6) at 4 weeks. *p < 0.05:
nonimmobilization groups (Group 1 þ Group 3) versus immo-
4. Discussion bilization groups (Group 2 þ Group 4) at 4 weeks. **p < 0.01:
nonimmobilization groups (Group 1 þ Group 3) versus
Many animal studies of depression have been reported, and acupuncture groups (Group 5 þ Group 6) at 4 weeks. The
a large proportion has been concerned with animals in a treatments for Groups 1e6 are the same as those in Figure 2.
Acupuncture at GV 20 or Ex-HN 3 on Depressed Rats
[11,20]. When the stress ended, the weight increased
again. This may be the reason for the increase in food
consumption and the slight recoveries of the digestion and
the absorption functions. In the rats exposed to stress caused
by immobilization in this study, body weights still increased,
but not to the same extent, as in control rats. This was
attributed to an effect similar to that of water-immersion
stress, but the detailed causes remain unclear. In addition,
acupuncture stimulation at the GV 20 or the Ex-HN3 acupoint
did not have an effect for immobilization stress.
The stimulation of rats in a depressive state with
acupuncture at the GV 20 and the Ex-HN3 acupoints at the
same time has been reported to decrease the immobile
time after exposure to water-immersion stress [11]. How-
ever, in this study, stimulation of depressed rats with
acupuncture at a single acupoint, either GV 20 or Ex-HN3,
did not decrease the immobile time. Thus, stimulation at
both acupoints simultaneously seems to be necessary to
alleviate depression. Moreover, the stress of immobilization
was not found to exacerbate the depressive state.
An evaluation of anxiety based on behavior clarified
three issues. First, water-immersion stress does not induce
a state of anxiety because the decrease in the number of
head dips was not statistically different in Group 3, whose
rats were subjected to water-immersion stress. Second,
stress caused by immobilization leads to a state of anxiety
as evidenced by the observations that the decreases in the
number of head dips were statistically significant in the
immobilization groups (Groups 2 and 4) and in the
acupuncture groups (Groups 5 and 6) after 4 weeks of
immobilization, but not in the nonimmobilization groups
(Groups 1 and 3) (see Figures 1 and 6). Third, a report
indicated that a decrease in the number of head dips was
induced with anxiogenic drugs and suppressed with anti-
anxiety drugs, but not with antidepressants [16]. There-
fore, one can conclude that immobilization stress causes
anxiety. One can also conclude that in this study, anxiety
and depression in the rats may have been caused by
different stresses.
The mood disorder of depression has been proposed to
result from decreased levels of monoamine in the brain
[4,14]. However, monoamine concentrations have been
found to increase because of acupuncture stimulation at
the “Zusanli” (ST36) and the “Shangjuxu” (ST37) acupoints
[21] or at the “Baihui” (GV 20) and the “Dazhui” (GV 14)
acupoints [22].
Discussions of the results using animal models have a
serious limitation. In rats experiencing trauma in the form
of water-immersion stress, immobilization, and intraperi-
toneal injection, as well as acupuncture, the normal stress-
reducing and pain-attenuating mechanisms activated in all
of these animals in response to their environment may have
been sufficiently large to override the ability to detect
differences in the effect of acupuncture among the groups.
This needs to be addressed in future research.
Disclosure statement
The authors declare that they have no conflicts of interest
and no financial interests related to the material of this
manuscript.
31
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