Kamishoyosan (a Japanese traditional herbal formula), which effectively
reduces the aggressive biting behavior of male and female mice, and
potential regulation through increase of Tph1, Tph2, and Esr2 mRNA levels
Kento Igarashi a
, Toshiko Kuchiiwa b,c
, Satoshi Kuchiiwa c
, Haruki Iwai d
, Kazuo Tomita a
Tomoaki Sato a,*
a Department of Applied Pharmacology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan b Department of Clinical Psychology, Graduate School of Human Science, Kagoshima Immaculate Herat University, 2365 Amatatsu-Cho, Satsuma-Sendai 895-0011,
Japan
c Department of Morphological Science, Field of Neurology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima
890-8544, Japan d Department of Oral Anatomy and Cell Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544,
Japan
ARTICLE INFO
Keywords:
Kamishoyosan
Traditional herbal medicine
Social isolation
Pathological aggression
Serotonin
Estrogen receptor
ABSTRACT
Kamishoyosan (KSS), a Japanese traditional herbal formula, is used to treat symptoms related to the autonomic
nervous system in men and women; it is especially known for improving the symptoms of irritability (e.g., bad
temper and persistent anger). Although clinical and ethological studies of KSS have been conducted, its efficacy
in reducing irritability remains to be validated. In the present study, male and female ddY-strain mice were
isolation-reared for 8 weeks (from the third postnatal week) to induce pathologically aggressive biting behavior
(ABB), which was used as an indicator of irritability. The ABB of mice toward metal rods was measured using the
Aggressive Response Meter. An intraperitoneal administration of KSS (100 mg/kg) effectively reduced ABB in
male and female mice at 2 h after the administration; however, this effect was canceled by prior administration of
WAY-100635 [a 5-hydroxytryptoamine (5-HT)-1A receptor antagonist; 0.5 mg/kg] and bicuculline (a type-A
gamma-aminobutyric acid receptor antagonist; 1.0 mg/kg). Additionally, tamoxifen, ICI-182780, and G-15 (all
estrogen receptor antagonists) inhibited the action of KSS in a dose-dependent manner. Furthermore, gene
expression of tryptophan hydroxylase (Tph) 1 and Tph2 were increased and 5-HT immunofluorescence was slightly
increased in the dorsal raphe nucleus (DRN) of isolation-reared mice administered with KSS. Collectively, these
results indicate that KSS effectively reduces ABB in isolation-reared male and female mice through stimulation of
5-HT production in the DRN. Our findings also suggest that gene expression of estrogen receptor (Esr) 2 increased
in the DRN might be associated with the reduction of ABB.
1. Introduction
Kamishoyosan (KSS; Kami-shoyo-san or Jia-Wey Shiau-Yau San in
Chinese) is a traditional herbal formula in Japan that is used to treat
symptoms related to autonomic nervous system symptoms, such as
palpitations, sweating, and hot flashes, as well as psychiatric-related
symptoms, such as irritability (e.g., bad temper or persistent anger),
insomnia, and general malaise. KSS is considered an effective supple￾ment to improve these symptoms especially for men with some frailty
and women (regardless of their constitution). Several clinical pilot
studies have reported that KSS is an effective treatment for menstrual￾related mood disorders, including premenstrual syndrome, premen￾strual dysphoric disorder, and perimenopausal depression with climac￾teric symptoms (Chen et al., 2003; Hidaka et al., 2013; Yamada and
Abbreviations: ABB, aggressive biting behavior; ANOVA, analysis of variance; ARM, Aggressive Response Meter; DRN, dorsal raphe nucleus; ER, estrogen receptor;
E2, estradiol; GABA, gamma-aminobutyric acid; i.p., intraperitoneal; KSS, Kamishoyosan; 5-HT, 5-hydroxytryptamine.
* Corresponding author at: Department of Applied Pharmacology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka,
Kagoshima 890-8544, Japan.
E-mail address: [email protected] (T. Sato).
Contents lists available at ScienceDirect
Brain Research
journal homepage: www.elsevier.com/locate/brainres

https://doi.org/10.1016/j.brainres.2021.147580

Received 1 February 2021; Received in revised form 23 June 2021; Accepted 6 July 2021
Brain Research 1768 (2021) 147580
Kanba, 2007). Case studies have reported that KSS is effective for
treating female patient of panic disorder (Mantani et al., 2002; Sato￾Nishimori et al., 2008). Recently, it was also reported that KSS im￾proves women’s excitability and irritability by a randomized controlled
study (Takamatsu et al., 2021). In addition to above studies, it was also
reported that KSS is effective for psychiatric symptoms of men. A case
study was reported that KSS improved the symptoms of male patients
with autonomic nervous system dysfunctions complaining of easy fati￾gability, palpitation, and so on (Matsuda, 1975). Another clinical study
of Free and Easy Wanderer Plus (FEWP), a Chinese prescription similar
to KSS, suggested that FEWP monotherapy effectively improved
depressive symptoms in bipolar patients of both sexes (Zhang et al.,
2007). These previous studies suggest that KSS exerts psychiatric action
on both man and women, although the exact mechanistic action of KSS
and any difference between sexes remains unclear. Animal (typically
rodent) models have been used to investigate its anxiolytic action or
promotion of prosocial behaviors; such studies help to understand the
psychiatric action of KSS (Guo et al., 2019a, b; Mizowaki et al., 2001).
Based on these findings, we hypothesized that KSS would also be an
effective treatment for irritability, which can trigger impulsive and/or
pathological aggression. To date, however, the efficacy of KSS on
pathologically aggressive behavior has yet to be studied in detail. Thus,
in the present study, we used an experimental mouse model to examine
the efficacy of KSS against irritability or pathologically aggressive
behavior.
A variety of psychiatric disorders include symptoms involving
persistent anger and/or aggressive behavior (Matson and Jang, 2014;
Patel and Barzman, 2013; Topiwala and Fazel, 2011; Tsiouris et al.,
2011). Both genetic background and environmental factors, which can
include mistreatment, harsh parenting, and impaired interpersonal re￾lationships during development, may be associated with a tendency
toward pathologically aggressive behavior (Waltes et al., 2016). Ani￾mals maintained under stress-rearing conditions can be used as experi￾mental models to evaluate the causal relationships between
environmental factors and aggressive behaviors. In particular, the
maternal separation (MS) model and postweaning social isolation
(PWSI) model are commonly applied to induce pathologically aggressive
behavior in rodents (Haller et al., 2014). In representative MS experi￾ments, pups are typically separated from both the dam and their litter￾mates for 3–4 h per day to model adverse childhood experiences such as
emotional neglect and social deprivation (Furukawa et al., 2017; Vee￾nema et al., 2006). Numerous effects of MS have been described; they
include not only excessive aggression but also anxiety-related behavior
and impaired cognitive function (Cao et al., 2014; Wigger and Neumann,
1999). In representative PWSI experiments, male rats are usually reared
in isolation after weaning (postnatal days 21–80) (Toth ´ et al., 2008).
PWSI is considered a useful model for studying the developmental as￾pects of human reactive aggression, which often results from early social
neglect.
Within the last decade, development of the Aggressive Response
Meter (ARM) has improved measurements of aggressive behavior in
male and female mice (Kuchiiwa and Kuchiiwa, 2014, 2016). In previ￾ous studies, the ARM was used to measure increasing aggressive biting
behavior (ABB) of ddY-strain mice that were isolation-reared from 3 to
10 weeks after birth; ABB toward two metal rods was measured by a load
sensor in the ARM (Kuchiiwa and Kuchiiwa, 2014, 2016). In these
studies, healthy (group-housed) mice showed little or no aggressive
behavior, whereas isolation-reared mice showed increasing ABB, which
was considered a measurement of pathological aggression. In addition to
measuring aggressive behavior in mice, the ARM device was intended to
evaluate the effect of psychotropic drugs such as buspirone, which is a 5-
hydroxytryptamine (5-HT)-1A receptor agonist that can function as an
antidepressant.
In the present study, we examined the intensity and frequency of ABB
in male and female mice, and we evaluated the effect of KSS on the ir￾ritability of these mice using the ARM. We first focused that the effect of
KSS was due to its action on 5-HT1A receptors, since agonistic action of
buspirone on these receptors effectively reduced ABB in a previous study
(Kuchiiwa and Kuchiiwa, 2014). In addition, it recently has been re￾ported that KSS exerts antidepressive effects through 5-HT1A receptor
(Shimizu et al., 2019). To test this, we examined whether a 5-HT1A re￾ceptor antagonist (WAY-100635) interfered with the effect of KSS. In
addition, we hypothesized that estrogen receptors (ERs) were associated
with the psychiatric action of KSS, since KSS is reported to act on ERβ
(Kumagai et al., 2005; Watanabe et al., 2006). To test this, we applied
ER antagonists (i.e., tamoxifen, ICI-182,780, and G-15) to determine
whether this action on ERs was related to reduced ABB, and we inves￾tigated the amelioration of ABB by 17α- and 17β-estradiol (E2).
Furthermore, we evaluated mRNA expression of the tryptophan hydrox￾ylase gene (Tph; the tryptophan hydroxylase enzyme being rate-limiting
within 5-HT synthesis) in brain tissue fragments of the dorsal raphe
nucleus (DRN) and assessed 5-HT immunofluorescence in the DRN by
quantitative real-time polymerase chain reaction (qRT-PCR) and
immunofluorescence staining, respectively. Finally, we examined es￾trogen receptor 2 (Esr2) mRNA and ERβ protein expression.
2. Results
2.1. KSS-treated mice show reduced aggressive biting behavior
In the present study, male and female mice were reared in socially
isolated conditions (i.e., lacking physical contact with other mice) from
3 weeks after their birth. Socially isolated mice were tested within the
period during which they had received 6–8 weeks of isolation rearing (i.
e., postnatal week 9–11). At postnatal week 10–11, the subject mouse is
placed in the cylinder on the top of ARM and measured ABB. As in a
previous study (Kuchiiwa and Kuchiiwa, 2014), metal rods with a dome￾shape head are automatically controlled to make an up-and-down mo￾tion 30 times and the load sensor connected to the rods measures ABB
intensity (average intensity of biting) and ABB frequency (total number
of bitings within 30 presentations of the rods; see Supplementary Fig. 1).
ABB intensity is expressed in numerical values as an integral of biting
force within one-second presentation of the rods (milliNewton × second:
mNs). In a previous study, isolation-reared mice showing a certain range
of ABB intensity and intraperitoneal (i.p.) administration were used to
examine the effect of buspirone on the aggressive behavior of isolation￾reared mice (Kuchiiwa and Kuchiiwa, 2014). In the present study, we
prescreened isolation-reared mice for their ABB intensity within 7–15
mNs, and investigated whether i.p. administration of KSS attenuated the
ABB of isolation-reared mice (Fig. 1B–E). The dose of KSS was set at 100
mg/kg, which is not higher than the dose for human in a day. One-way
analysis of variance (ANOVA) showed that the intensity and frequency
of ABB in mice administered with saline via i.p. injection were similar to
those of mice tested before saline administration (F(2,27) = 0.76, p = 0.47
in ABB intensity of saline-treated males, F(2,27) = 0.48, p = 0.63 in ABB
frequency of saline-treated males, F(2,23) = 0.12, p = 0.89 in ABB in￾tensity of saline-treated females, and F(2,23) = 0.45, p = 0.64 in ABB
frequency of saline-treated females). In contrast, one-way ANOVA
showed statistically significant change in the intensity of ABB in both
male and female mice intraperitoneally administered KSS (F(2,25) =
11.34, p = 0.0003 in ABB intensity of KSS-treated males, F(2,25) = 5.67, p
= 0.009 in ABB frequency of KSS-treated males, F(2,21) = 14.18, p =
0.0001 in ABB intensity of saline-treated females, and F(2,21) = 10.74, p
= 0.0006 in ABB frequency of KSS-treated females). Post hoc analysis
revealed that ABB intensity significantly decreased at 1 and 2 h after
injection when compared with the ABB of mice before administration (p
= 0.002 at 1 h and p = 0.001 at 2 h in male mice; p = 0.0002 at 1 h and p
= 0.002 in female mice). Similarly, the frequency of ABB in mice
significantly decreased at 1 and 2 h after i.p. administration of KSS (p =
0.02 at 1 h and p = 0.02 at 2 h in male mice; p = 0.0008 at 1 h and p =
0.07 in female mice). These results indicated that i.p. administration of
KSS effectively reduces the ABB of both male and female isolation￾K. Igarashi et al.
Brain Research 1768 (2021) 147580
reared mice. In isolation-reared female mice, we also confirmed that the
effect of i.p. KSS administration on ABB was not dependent on their
estrous cycle (see Supplementary Fig. 2). Thus, we did not examine
estrous cycle of female mice in the following experiments.
We then examined whether a single oral administration of KSS (100,
200, and 400 mg/kg) through a plastic sonde attenuated the ABB of
isolation-reared male and female mice (Fig. 2), since KSS is orally
administered for human patients. One-way ANOVA showed no statisti￾cal significance within saline treatment group (F(2,9) = 0.51, p = 0.62 in
saline-treated males and F(2,12) = 0.86, p = 0.45 in saline-treated fe￾males). In contrast, a statistical significance was found within male mice
orally treated with 400 mg/kg of KSS (F(2,6) = 12.12, p = 0.0078). Post
hoc analysis showed a significant decrease in ABB intensity at 1 and 2 h
after oral administration (p = 0.014 at 1 h and p = 0.011 at 2 h). In
addition, there was statistically significant change found in female mice
orally treated with 200 mg/kg KSS and those with 400 mg/kg KSS (F(2,9)
= 10.19, p = 0.0049 in 200 mg/kg, and F(2,12) = 4.80, p = 0.029 in 400
mg/kg). Post hoc analysis revealed a significant decrease in biting in￾tensity at 1 and 2 h after oral administration (p = 0.032 at 1 h and p =
0.0045 at 2 h in 200 mg/kg KSS treated female; p = 0.11 at 1 h and p =
0.028 at 2 h in 400 mg/kg KSS treated female). However, no significant
effect was observed in ABB frequency at any of the three doses in both
male and female mice. From these observations, we considered i.p.
administration is more sensitive application to measure the effect of KSS
and applied it in the following experiments.
2.2. Action of KSS was less effective in group-housed mice
We administrated KSS to group-housed mice to compare its psychi￾atric action in isolation-reared and group-housed treatments (Fig. 3).
Group-housed mice showed smaller ABB intensity and frequency,
compared with those used as isolation-reared mice. The intensity and
frequency of ABB in group-housed mice did not show a statistically
significant difference between before and after KSS (100 mg/kg) i.p.
administration (F(2,15) = 0.66, p = 0.53 in ABB intensity of KSS-treated
males; F(2,15) = 1.27, p = 0.31 in ABB frequency of KSS-treated males;
F(2,6) = 3.26, p = 0.11 in ABB intensity of KSS-treated females; F(2,6) =
0.96, p = 0.43 in ABB frequency of KSS-treated females), suggesting that
KSS was less effective in subjects without pathological aggression.
2.3. KSS action was blocked by a 5-HT1A receptor antagonist in female
and in males by combinatory use of a 5-HT1A receptor antagonist and a
GABAA receptor antagonist
To test our hypothesis that 5-HT1A receptors play some role in KSS￾mediated amelioration of ABB, mice were pretreated with WAY-
100635, a 5-HT1A receptor antagonist (Fig. 4A). One-way ANOVA
showed a statistical significance in the effect on ABB intensity (F(2,15) =
6.78, p = 0.008) and on ABB frequency (F(2,15) = 11.30, p = 0.001) after
WAY-100635 and KSS administration on male mice (Fig. 4B and C). Post
hoc analysis revealed that ABB intensity of male mice after KSS treat￾ment was significantly decreased (p = 0.02 vs. before administration,
Fig. 1. Reduction of biting behavior in isolation-reared mice administered with Kamishoyosan (KSS). (A) Scheme of the experimental procedure. After 6–8 weeks of
social isolation, the biting intensity of each mouse was measured to screen for mice with a biting intensity of 7–15 mNs. These mice were intraperitoneally
administered with KSS extract or saline. At 1 and 2 h after administration, ABB intensity and ABB frequency (number of biting behaviors in measurement session)
were measured. (B–E) The effect of KSS on the biting behavior of male (B, C) and female (D, E) mice. The intensity and frequency of ABB in these measurement
sessions were respectively averaged within each experimental group; thus, data represent means ± standard errors. Numbers in brackets represent numbers of mice
used in each experimental group. * p < 0.05 (vs. preadministration; one-way ANOVA followed by Tukey–Kramer test).
K. Igarashi et al.
Brain Research 1768 (2021) 147580
and p = 0.01 vs. after WAY-100635 treatment), and that ABB frequency
of male mice after KSS treatment was significantly decreased (p = 0.003
vs. before administration, and p = 0.003 vs. after WAY-100635 treat￾ment). Thus, it was suggested that the action of KSS on ABB was not
canceled by inhibition of 5-HT1A receptors in male mice. Since it was
previously shown that the action of KSS on social behavior involves the
type-A gamma-aminobutyric acid (GABAA) receptor (Guo et al., 2019a,
2019b), we also tested pretreatment of male mice with bicuculline, a
GABAA receptor antagonist (Fig. 4B and C). The intensity and frequency
of ABB in male mice treated with KSS following bicuculline significantly
decreased after administration (F(2,15) = 6.81, p = 0.0079 in ABB in￾tensity of male mice, and F(2,15) = 19.93, p = 0.00006 in ABB frequency
of male mice), suggesting that the action of KSS on ABB was not canceled
by inhibition of the GABAA receptor in male mice. In addition, we tested
a treatment of KSS following WAY-100635 and bicuculline in male mice
(Fig. 4B and C). We found that the intensity of ABB in these mice did not
significantly decrease after administration (F(2,21) = 2.47, p = 0.11) and
that the frequency of ABB in male mice was not significantly affected by
KSS following WAY-100635 and bicuculline pretreatment (F(2,21) =
0.59, p = 0.56). These results suggest that the action of KSS on ABB was
partly canceled by concomitant inhibition of the 5-HT1A and GABAA
receptors in male mice.
In contrast, statistically significant change was not shown in ABB
intensity or frequency of female mice treated with KSS following WAY-
100635 by one-way ANOVA (F(2,20) = 1.94, p = 0.17 in ABB intensity of
male mice and F(2,20) = 1.32, p = 0.29 in ABB frequency of female mice;
Fig. 4D and E), suggesting that the action of KSS on ABB was canceled by
inhibition of 5-HT1A receptors in female mice. We also examined the
effect of bicuculline preadministration and following KSS administra￾tion, and statistically significant change was shown by one-way ANOVA
Fig. 2. Reduced aggressive biting behavior in mice orally administered with Kamishoyosan (KSS). (A) Scheme of the experimental procedure. ABB intensity of male
(B) and female (D) mice. ABB frequency (Number of biting behavior in measurement session) of male (C) and female (E) mice. Error bars represent standard errors.
Numbers in brackets represent numbers of mice used in each experimental group. * p < 0.05 (one-way ANOVA followed by Tukey’s HSD test).
K. Igarashi et al.
Brain Research 1768 (2021) 147580
(F(2,15) = 26.37, p = 0.00001 in ABB intensity, and F(2,15) = 19.98, p =
0.00006 in ABB frequency), suggesting that the action of KSS on ABB
was not canceled by inhibition of the GABAA receptor in female mice.
We further examined whether the combinatory preadministration of
WAY-100635 and bicuculline affects ABB of female mice, and no sta￾tistically significant change was shown in both ABB intensity and fre￾quency by one-way ANOVA (F(2,6) = 0.36, p = 0.71 in ABB intensity, and
F(2,6) = 3.08, p = 0.12 in ABB frequency).
2.4. KSS action is blocked by estrogen receptor antagonists
Since KSS is used to mitigate the symptoms of menopausal syn￾dromes, such as hot flashes and dysphoria, in combination with hor￾mone replacement therapy (Hidaka et al., 2013), we hypothesized
whether KSS action on estrogen receptors (ER) has a certain association
with reducing ABB. In order to investigate the hypothesis, we used
tamoxifen and ICI-182,780 as antagonists for classical ERs (ERα and
ERβ), and G-15 as an antagonist for GPR30 (a 7-transmembrane G
protein-coupled ER) (Prossnitz et al., 2008; Shanle and Xu, 2010).
Isolation-reared mice were preadministered with ER antagonists
(tamoxifen, ICI-182,780, or G-15) by i.p. injection 30 min before
administration of 100 mg/kg KSS by i.p. injection. Statistically signifi￾cant changes within the ABB intensity of male and female mice pre￾treated with vehicle was shown by one-way ANOVA (F(2,6) = 10.65, p =
0.01 in males and F(2,6) = 9.62, p = 0.01 in females). Post hoc analysis
revealed reduction of ABB intensity after administration of KSS by about
56% in males (p = 0.02 vs. before administration, and p = 0.01 vs. after
vehicle treatment) and 63% in females (p = 0.02 vs. before adminis￾tration, and p = 0.02 vs. after vehicle); however, the ABB intensity of
male and female mice that were pretreated with tamoxifen (5.0 mg/kg),
ICI-182,780 (5.0 mg/kg), or G-15 (0.35 mg/kg) before KSS adminis￾tration did not show statistically significant change (F(2,9) = 0.07, p =
0.94 in tamoxifen (5.0 mg/kg)-treated male, F(2,6) = 1.05, p = 0.41 in
tamoxifen (5.0 mg/kg)-treated female, F(2,9) = 0.97, p = 0.41 in ICI-
182,780 (5.0 mg/kg)-treated male, F(2,6) = 0.84, p = 0.48 in ICI-
182,780 (5 mg/kg)-treated female, F(2,9) = 2.60, p = 0.13 in G-15
(0.35 mg/kg)-treated male, F(2,9) = 0.09, p = 0.92 in G-15 (0.35 mg/kg)-
treated female; Fig. 5B and D). One-way ANOVA analysis also showed
statistical significant changes in the frequency of ABB in male and fe￾male mice pretreated with vehicle (F(2,6) = 7, p = 0.027 in male and
F(2,6) = 6.19, p = 0.035 in female), and post hoc analysis revealed
reduction of ABB intensity after administration of KSS in males (p = 0.04
vs. before administration, and p = 0.04 vs. after vehicle treatment) and
in females (p = 0.04 vs. before administration, and p = 0.04 vs. after
vehicle). On the other hand, statistically significant changes were not
found within the frequency of ABB in male and female mice pretreated
with tamoxifen, ICI-182,780, or G-15 (F(2,9) = 1.19, p = 0.34 in
tamoxifen (5.0 mg/kg)-treated male, F(2,6) = 0.016, p = 0.98 in
tamoxifen (5.0 mg/kg)-treated female, F(2,9) = 0.80, p = 0.48 in ICI-
182,780 (5.0 mg/kg)-treated male, F(2,6) = 0.50, p = 0.63 in ICI-
182,780 (5.0 mg/kg)-treated female, F(2,9) = 0.33, p = 0.72 in G-15
(0.35 mg/kg)-treated male, F(2,9) = 0.13, p = 0.88 in G-15 (0.35 mg/kg)-
treated female; Fig. 5C and E). These results suggest that KSS acted in
some manner on ER to reduce ABB in both male and female mice.
Fig. 3. Kamishoyosan (KSS) had no significant effect on aggressive biting behavior in group-housed mice. (A) Scheme of the experimental procedure. ABB intensity
of group-housed male (B) and female (D) mice. ABB frequency of group-housed male (C) and female (E) mice. Error bars represent standard errors. Numbers in
brackets represent numbers of mice used in each experimental group. Data were analyzed using one-way ANOVA and statistical significance at p < 0.05 was
not detected.
K. Igarashi et al.
Brain Research 1768 (2021) 147580
2.5. ABB is reduced in mice administered with 17β-E2
Since it has been reported that 1 g of KSS exerts estrogen-like activity
equivalent to 13.3 pg of 17β-E2 in a luciferase reporter assay (Kumagai
et al., 2005), we examined whether the equivalent amount of 17α- or
17β-E2 would reduce ABB in isolation-reared mice at 1 h after the
administration, as KSS exerted the ABB-reducing effect at 1 h after the
administration. The ABB intensity of mice administered with 17β-E2 was
reduced after treatment by about 46% in males (p = 0.02) and about
29% in females (p = 0.006), whereas the ABB intensity of mice of both
sexes administered with 17α-E2 did not differ significantly before and
after treatment (p = 0.21 in males and p = 0.08 in females; Fig. 6B and
D). The frequency of ABB in male mice administered with 17β-E2 was
also significantly reduced after treatment, but it did not differ before and
after treatment in females (p = 0.049 in males and p = 0.52 in females;
Fig. 6C and E). Collectively, these results support the hypothesis that ERs
are partly involved in the ABB-reducing action of KSS.
2.6. Tph1 and Tph2 mRNA increases in DRN of KSS-treated mice
A large proportion of serotonin in the brain is produced by a major
group of serotonergic neurons in the DRN of the midbrain (Dahlstrom ¨
and Fuxe, 1964; Hensler, 2006). To investigate gene expression of Tph,
which encodes a rate-limiting enzyme during 5-HT synthesis, we per￾formed qRT-PCR analysis on DRN fragments sampled from isolation￾reared mice 2 h after administration of KSS (Fig. 7B and C). Since Tph
has two isoforms, Tph1 and Tph2, we examined respective mRNA levels
of each isoform. Two-way ANOVA showed a significant effect of the
treatment on Tph1 mRNA level (F(1,8) = 40.80, p = 0.0002) and of the
sex (F(1,8) = 40.72, p = 0.0002) with no interaction effect (F(1,8) =
0.0036, p = 0.95). Post hoc analysis revealed increases of Tph1 mRNA
level in KSS-treated male mice (p = 0.0022) and KSS-treated female mice
(p = 0.0020). In addition, a statistically significant difference was found
between saline-treated male and saline-treated female mice (p =
0.0020). Two-way ANOVA analysis was also showed a significant effect
of the treatment on Tph2 mRNA level (F(1,8) = 89.75, p = 0.00001) and
of the sex (F(1,8) = 136.74, p = 2.6 × 10− 6
) with significant interaction
effect (F(1,8) = 23.06, p = 0.0014). Post hoc analysis revealed increases of
Tph2 mRNA level in KSS-treated male mice (p = 0.0002) and KSS-treated
female mice (p = 0.011). In addition, a statistically significant difference
was found between saline-treated male and saline-treated female mice
(p = 0.0002).
To examine 5-HT production in the DRN of mice brains following
administration of KSS, we also performed immunofluorescence staining
using an anti-5-HT antibody (Fig. 7D and E). A characteristic pattern was
confirmed, which has been reported in a previous study (VanderHorst
et al., 2005). Two-way ANOVA analysis showed a significant effect of
the treatment on immunofluorescence intensity (F(1,8) = 6.52, p =
0.034) and of the sex (F(1,8) = 28.92, p = 0.0007) with no interaction
effect (F(1,8) = 0.21, p = 0.66). Post hoc analysis revealed a statistically
significant difference was found between saline-treated male and saline￾treated female mice (p = 0.0085). However, no significant increase was
found in KSS-treated male mice (p = 0.18) or in KSS-treated female mice
(p = 0.066).
It has been previously reported that prefrontal cortex (PFC) regulates
intermale aggressive behavior and that PFC is one of the common brain
regions innervated by the serotonergic projection from the middle part
of the DRN, which we have examined here (Fernandez et al., 2016;
Takahashi et al., 2014; Waselus et al., 2011). In order to further examine
the efficacy of KSS administration, we thus examined c-fos gene
Fig. 4. Preadministration of the 5-HT1A serotonin receptor inhibitor WAY-100635 abolished the Kamishoyosan (KSS)-induced reduction in biting behavior in
isolation-reared female mice, while in males this blocking effect was only observed when the GABAA receptor antagonist bicuculline was also present. (A) Scheme of
the experimental procedure. After 6–8 weeks of social isolation, the biting intensity of each mouse was measured to screen for mice with a biting intensity of 7–15
mNs. These mice were intraperitoneally administered with the 5-HT1A serotonin receptor inhibitor WAY-100635 (WAY; 0.5 mg/kg) and/or the GABAA receptor
inhibitor bicuculline (Bic; 1 mg/kg). Thirty minutes after administration of inhibitors, ABB intensity and ABB frequency were measured, and then mice were
intraperitoneally administered with KSS (100 mg/kg). At 1 h after KSS administration, the intensity and frequency were measured. (B–E) The biting behaviors of
male (B, C) and female (D, E) mice. The intensity and frequency of ABB in these measurement sessions were respectively averaged within each experimental group;
thus, data represent means ± standard errors. Numbers in brackets represent numbers of mice used in each experimental group. * p < 0.05 (one-way ANOVA
followed by Tukey’s HSD test).
K. Igarashi et al.
Brain Research 1768 (2021) 147580
expression in DRN and PFC after KSS administration (Fig. 7F and G).
Two-way ANOVA of c-fos gene expression in DRN showed a significant
effect of the treatment on c-fos mRNA level (F(1,8) = 506.8, p = 1.6 ×
10− 8
) and of the sex (F(1,8) = 1839, p = 9.6 × 10− 11) with significant
interaction effect (F(1,8) = 161.4, p = 1.4 × 10− 6
). Post hoc analysis
revealed increases of c-fos mRNA level in KSS-treated male mice (p =
0.0002) and KSS-treated female mice (p = 0.0003). These results suggest
that the level of neuroactivation in the DRN was decreased in the male
and female mice that were administered with KSS. In addition, a sta￾tistically significant difference was found between saline-treated male
and saline-treated female mice (p = 0.0002). Two-way ANOVA of c-fos
gene expression in PFC showed no significant effect of the treatment on
c-fos mRNA level (F(1,8) = 1.03, p = 0.34) and of the sex (F(1,8) = 42.76, p
= 0.0002) with significant interaction effect (F(1,8) = 74.64, p =
0.00003). Post hoc analysis revealed increase of c-fos mRNA level in KSS￾treated male mice (p = 0.0008) and decrease of c-fos mRNA level in KSS￾treated female mice (p = 0.0003). These results suggest that the level of
neuroactivation in the PFC was not decreased in the male, while it was
decreased in female mice. Statistically significant difference was not
found between saline-treated male and saline-treated female mice (p =
0.18).
2.7. Estrogen receptors are associated with Tph2 mRNA induction in the
DRN of male mice that were administrated with KSS
From above results, we surmised that ERs are associated with the
action of Tph1 and Tph2 mRNA induction. We examined Tph1 and Tph2
mRNA expression in the DRN of the mice that were administrated with
tamoxifen (5.0 mg/kg) or ICI-182,780 (5.0 mg/kg), followed by KSS
treatment (Fig. 8). Statistically significant changes were not found
Fig. 5. Preadministration of estrogen receptor inhibitors abolished the Kamishoyosan (KSS)-mediated reduction in biting behaviors in isolation-reared mice. (A)
Scheme of the experimental procedure. After 6–8 weeks of social isolation, the biting intensity of each mouse was measured to screen for mice with a biting intensity
of 7–15 mNs. These mice were intraperitoneally administered with estrogen receptor inhibitor tamoxifen (Tam), ICI-182,780 (ICI), G-15, or vehicle. Thirty minutes
after administration of inhibitors, ABB intensity and ABB frequency were measured, and then mice were intraperitoneally administered with KSS. At 1 h after KSS
administration, ABB intensity and frequency were measured. (B–E) The biting behaviors of male (B, C) and female (D, E) mice. The intensity and frequency of biting
behaviors in these measurement sessions were respectively averaged within each experimental group; thus, data represent means ± standard errors. Numbers in
brackets represent numbers of mice used in each experimental group. * p < 0.05 (one-way ANOVA followed by Tukey’s HSD test).
K. Igarashi et al.
Brain Research 1768 (2021) 147580
within Tph1 mRNA expression in the DRN of male and female mice
(F(2,6) = 0.34, p = 0.73 in males; F(2,6) = 2.20, p = 0.19 in females). In
contrast, statistically significant changes were found within Tph2 mRNA
expression in the DRN of male and female mice (F(2,6) = 81.2, p =
0.00005 in males; F(2,6) = 36.8, p = 0.0004 in females). Post hoc analysis
revealed decrease of Tph2 mRNA expression in male mice administered
with tamoxifen or ICI-182,780 (p = 0.0002 in comparison between
tamoxifen and vehicle, p = 0.0002 in comparison between ICI and
182,780 and vehicle), suggesting that ERs are associated with Tph2
mRNA induction in the DRN of male mice administered with KSS. It was
also revealed that Tph2 mRNA expression increases in female mice
administered with tamoxifen or ICI-182,780 (p = 0.0008 in comparison
between tamoxifen and vehicle, p = 0.0009 in comparison between ICI
and 182,780 and vehicle).
2.8. Esr2 (ERβ) mRNA levels increases in brain tissue fractions of the
DRN and ERβ are involved with the ABB-reducing action of KSS
From the above results, it was surmised that ERs are associated with
the action of Tph2 mRNA induction in the DRN of mice administered
with KSS. Since it was previously reported that ERβ regulates Tph2
mRNA expression in the DRN (Donner and Handa, 2009), we focused on
Esr2 (which encodes ERβ) mRNA expression, and performed qRT-PCR
analysis on brain tissue fractions of the DRN sampled at 2 h after the
administration of KSS or saline (Fig. 9B). Two-way ANOVA analysis was
also showed a significant effect of the treatment on Esr2 mRNA level
(F(1,8) = 34.06, p = 0.0004) but no significant effect of the sex (F(1,8) =
4.35, p = 0.07) with no interaction effect (F(1,8) = 0.0023, p = 0.96). Post
hoc analysis revealed increases of Esr2 mRNA level in KSS-treated male
mice (p = 0.0033) and KSS-treated female mice (p = 0.036).
In addition, we used immunoblot analysis to investigate ERβ protein
expression in isolation-reared mice 2 h after an i.p. administration of
KSS or saline. Male mice administered with KSS showed a statistically
significant increase (p = 0.04) in ERβ protein levels compared with male
mice administered with saline (Fig. 9C). Female mice administered with
KSS showed an increase in ERβ protein levels compared with female
mice administered with saline, although a statistically significant in￾crease was not detected (p = 0.07; Fig. 9D).
In order to validate whether ERβ are associated with the ABB￾reducing action of KSS, isolation-reared mice were preadministered
with an ERβ-selective antagonist (PHTPP) by i.p. injection 30 min before
administration of 100 mg/kg KSS by i.p. injection (Fig. 9E–I). Statisti￾cally significant changes were not found within the intensity of ABB in
male and female mice pretreated with PHTPP (F(2,6) = 1.71, p = 0.25 in
PHTPP (0.5 mg/kg)-treated male; F(2,6) = 0.02, p = 0.98 in PHTPP (1.0
mg/kg)-treated male; F(2,6) = 2.37, p = 0.17 in PHTPP (0.5 mg/kg)-
treated female; F(2,6) = 0.13, p = 0.88 in PHTPP (1.0 mg/kg)-treated
female). No statistically significant change was found either within the
frequency of ABB in male and female mice pretreated with PHTPP (F(2,6)
= 2.40, p = 0.17 in PHTPP (0.5 mg/kg)-treated male; F(2,6) = 2.13, p =
0.20 in PHTPP (1.0 mg/kg)-treated male; F(2,6) = 0.96, p = 0.43 in
PHTPP (0.5 mg/kg)-treated female; F(2,6) = 0.29, p = 0.77 in PHTPP
(1.0 mg/kg)-treated female). These results suggest that KSS acted in
some manner on ERβ to reduce ABB in both male and female mice.
3. Discussion
In the present study, we used an ARM device to evaluate the irrita￾bility of isolation-reared mice based on their propensity to aggressively
bite metal rods. Our results indicate that administration of KSS effec￾tively reduces the ABB of both male and female mice. Although KSS is
conventionally used as an effective treatment for menopause-related
psychological disorders and is also considered an effective supplement
for men with autonomic nervous system dysfunctions (easy fatigability,
palpitation and so on), the molecular basis for the action of KSS has yet
to be fully elucidated (Hidaka et al., 2013; Matsuda, 1975). It is likely
Fig. 6. The effects of 17α-E2 and 17β-E2 on the biting behavior of isolation-reared female and male mice. (A) Scheme of the experimental procedure. After 6–8 weeks
of social isolation, the biting intensity of each mouse was measured to screen for mice with a biting intensity of 7–15 mNs. These mice were intraperitoneally
administered with 17α- or 17β-E2 (1.33 pg/kg). One hour after administration of 17α- or 17β-E2, ABB intensity and ABB frequency were measured. (B–E) Biting
behaviors in male (B, C) and female (D, E) mice. The intensity and frequency of biting behaviors in these measurement sessions were respectively averaged within
each experimental group; thus, data represent means ± standard errors. Numbers in brackets represent numbers of mice used in each experimental group. * p < 0.05
(Student’s t test).
K. Igarashi et al.
Brain Research 1768 (2021) 147580
Fig. 7. Tph1 and Tph2 mRNA expression and 5-HT immunofluorescence in brain tissue fractions of the dorsal raphe nucleus (DRN). (A) Scheme of the experimental
procedure. Isolation-reared mice were intraperitoneally administered with Kamishoyosan (KSS; 100 mg/kg) or saline, and brain tissue fractions of the DRN were
resected at 2 h after the administration. (B, C) Expression levels of Tph1 and Tph2 according to qRT-PCR; data were normalized to Actb mRNA expression and shown
as relative values compared with expression levels in male mice administered with saline. (D) Representative images of immunofluorescence staining using anti-5-HT
antibody. (E) Fluorescence intensity ere measured in each mouse. (F, G) Expression levels of c-fos in DRN (F) and in prefrontal cortex (G) according to qRT-PCR; data
were normalized to Actb mRNA expression and shown as relative values compared with expression levels in male mice administered with saline. Error bars represent
standard deviations. Numbers in brackets represent numbers of mice used in each experimental group. * p < 0.05 (two-way ANOVA followed by Tukey’s HSD test).
K. Igarashi et al.
Brain Research 1768 (2021) 147580
10
that multiple components are acting synergistically during KSS treat￾ment, since traditional herbal formulas are complex and contain various
crude ingredients. Our results indicated that KSS acts on 5-HT1A re￾ceptors, leading to reduced aggressiveness in socially isolated male and
female mice. In addition, KSS action on both 5-HT1A receptors, and
GABAA receptors seemed to be involved in reduced ABB in male mice.
We also found that Tph1 and Tph2 induction after KSS treatment in the
DRN. Our findings also suggest that increase of ERs in the DRN might be
associated with reduction of ABB.
We showed that KSS reduced aggressive behavior in isolation-reared
mice; previously, buspirone, a psychotic drug acting on 5-HT1A re￾ceptors, was also shown to reduce aggressive behavior in mice reared in
isolation (Kuchiiwa and Kuchiiwa, 2014). Our study included exami￾nation of the DRN, which is a major source of 5-HT in the brain and is
functionally associated with emotional behaviors such as aggression
(Azmitia et al., 2011; Dahlstrom ¨ and Fuxe, 1964; Faccidomo et al., 2008;
Sijbesma et al., 1991; Vergnes et al., 1986). Specifically, we observed an
increase in Tph1 and Tph2 gene expression in the DRN of mice admin￾istered with KSS. We also observed that Tph1 and Tph2 mRNA levels in
females were higher compared with those in males. In a previous study,
they performed restraint stress on pregnant rats and found that Tph2
gene expression in the DRN of male offspring rats was decreased
compared with female offspring rats (Huang et al., 2017). However, in
the human study, it was reported that the mean rate of 5-HT synthesis
was higher in normal males than in normal females (Nishizawa et al.,
1997). Further study is required to verify whether expression levels of
these genes are physiologically different or resulted from stress treat￾ments. Our immunofluorescence staining results did not denied that 5-
HT production was promoted in the DRN after KSS treatment. Seroto￾nergic neurons in the DRN are reported to project to distinct brain areas,
such as PFC and nucleus accumbens (Waselus et al., 2011). Given these
investigations, we postulate one hypothesis that KSS administration
induces Tph1 and Tph2 gene expression in the DRN, and consequently
reduces ABB. Further research is needed to examine the relation be￾tween 5-HT increase in the DRN and other brain areas regulating
aggressive behaviors. It would be helpful to distinguish newly activated
serotonergic neuron, for example, by coimmunostaining with anti-5-HT
antibody and anti-c-Fos antibody.
As many as seven subfamilies of 5-HT receptors exist, each of which
induces distinct responses (Zmudzka ˙ et al., 2018). For example, 5-HT2C
receptors in the bed nucleus of the stria terminalis have been implicated
in fear-promoting action of mice (Marcinkiewcz et al., 2016). Addi￾tionally, a 5-HT4 receptor agonist has been shown to acutely reduce
immobility time of rats in a forced swim test (Lucas et al., 2007).
However, based on the results of our experiment, in which we used a 5-
HT1A receptor antagonist, we propose that the reduction of ABB was
achieved through activation of 5-HT1A receptors. Although the exact
mechanism underlying the induced selective action on 5-HT1A receptors
remains to be determined, a previous study reported that shogaol
(composed of ginger) acts as a partial agonist on 5-HT1A receptors
(Nievergelt et al., 2010). Further study will be required to ascertain the
concerted mechanism by which KSS components selectively act on 5-
HT1A receptors. The activity of 5-HT1A receptors usually results in
reduction of neuronal activity (Huang et al., 2017). However, the 5-
HT1A receptors have dual role: a first role as postsynaptic receptors to
reduce different neuronal cells and a second role as autoreceptors to
regulate serotonergic cell activity. Indeed, we found that c-fos gene
expression is decreased in the DRN of both male and female mice that
were administered with KSS, suggesting that 5-HT1A autoreceptors in
the DRN were activated in both male and female mice that were
administered with KSS. It was also found that c-fos gene expression in
the PFC of male mice that were administered with KSS was increased,
Fig. 8. Tph1 and Tph2 mRNA expression in the dorsal raphe nucleus (DRN) of the mice that were administrated with tamoxifen (5.0 mg/kg) or ICI-182,780 (5.0 mg/
kg), and following KSS (100 mg/kg) treatment. Isolation-reared mice were intraperitoneally administered with tamoxifen (5.0 mg/kg), ICI-182,780 (5.0 mg/kg), or
vehicle. Thirty minutes after the administration of these ER inhibitors, the mice were intraperitoneally administered with Kamishoyosan (KSS; 100 mg/kg), and brain
tissue fractions of the DRN were resected at 1 h after the KSS administration. (B–E) Expression levels of Tph1 (B, D) and Tph2 (C, E) according to qRT-PCR; data were
normalized to Actb mRNA expression and shown as relative values compared with expression levels in male (B, C) or female (D, E) mice administered with vehicle.
Error bars represent standard deviations. Numbers in brackets represent numbers of mice used in each experimental group. * p < 0.05 (one-way ANOVA followed by
Tukey’s HSD test).
K. Igarashi et al.
Brain Research 1768 (2021) 147580
Fig. 9. The effect of Kamishoyosan (KSS) administration on Esr2 mRNA expression and ERβ protein expression in the DRN of mouse brains. (A) Scheme of the
experimental procedure for expression analysis. Brain tissue fractions from the DRN were resected from mice administered with KSS (100 mg/kg) or saline at 2 h after
administration. (B) Expression levels of Esr2 (ERβ) according to qRT-PCR; data were normalized to Actb mRNA expression and shown as relative values compared
with expression levels in male mice administered with saline (n = 3 animals in each group). Error bars represent standard deviations. * p < 0.05 (two-way ANOVA
followed by Tukey’s HSD test). (C, D) Expression levels of ERβ according to western blot; data were normalized to Actin protein expression and shown as relative
values compared with expression levels in male (C) or female (D) mice administrated with saline (n = 3 in each group). Error bars represent standard deviations. * p
< 0.05 (Student’s t test). (E) Scheme of the experiment procedure for inhibitor analysis. (F–I) The biting behaviors of male (F, G) and female (H, I) mice. The intensity
and frequency of biting behaviors in these measurement sessions were respectively averaged within each experimental group; thus, data represent means ± standard
errors. Numbers in brackets represent numbers of mice used in each experimental group. Data were analyzed using one-way ANOVA and statistical significance at p
< 0.05 was not detected.
K. Igarashi et al.
Brain Research 1768 (2021) 147580
while c-fos gene expression in the PFC of female mice that were
administered with KSS was decreased. In the previous study, it was re￾ported that male rats show higher performance in context-mediated
renewal test and Fos-positive neurons in the PFC compared with fe￾males and that the males’ performance was lowered by silencing the
neuroactivation in the PFC, suggesting that a certain sex-specific phys￾iological machinery activates or suppresses Fos induction in the PFC
(Anderson and Petrovich, 2018). Further study is needed to elucidate
how KSS works on distinct 5-HT1A receptors and related neuronal
circuits.
As we have discussed, our results suggest that activation of seroto￾nergic neurons in the DRN may be involved in the efficacy of KSS against
ABB. On the other hand, our results also indicate that KSS reduces ABB
in male mice through combinatory action on the GABAergic and sero￾tonergic systems. In addition, it was previously suggested that neural
crosstalk between the GABAergic and serotonergic neuron (Huang et al.,
2017). Thus, it is possible that KSS reduces male mouse ABB partly by
sedative action that occurs via the GABAergic system. Additional studies
are required to elucidate the synergistical action mechanism of KSS and
sex differences in the neural circuits that govern pathological
aggression.
We found that KSS-mediated reduction in mouse ABB was inhibited
by preadministration of ER antagonists, suggesting that potential roles
of ERα, ERβ, and GPR30 on the ABB-reducing action of KSS. It has been
reported that ERα and ERβ have different distributions in the mouse
brain (Mitra et al., 2003; Sheng et al., 2004; VanderHorst et al., 2005). In
the present study, we focused the expression of ERs in the DRN, since
increases of Tph1 and Tph2 mRNA expression were observed in the DRN,
which is a major source of brain serotonin. Indeed, we found that Tph2
mRNA induction in the DRN of male mice was abolished by pre￾administration of ER antagonists. It was previously reported that around
the midbrain, ERβ is predominantly distributed in the DRN, whereas
ERα is predominantly distributed in the periaqueductal gray (Mitra
et al., 2003; Nomura et al., 2005). ER, when bound to agonists, dimerize
and interact with regulatory DNA sequences to modulate gene tran￾scription (Klinge, 2001; Powell and Xu, 2008; Yas¸ar et al., 2017).
Interestingly, the colocalization of ERβ with TPH in the DRN has been
reported as ~ 96% (Nomura et al., 2005; Sheng et al., 2004). In addition,
Gundlah et al. (2005) reported that the Tph mRNA signal detected by in
situ hybridization in ERβ-deficient mice was lower than that detected in
wild-type mice. We found that Esr2 mRNA expression and ERβ protein
expression are increased by about 30% in the DRN of male and female
mice. We also found that an ERβ-selective antagonist inhibits ABB￾reducing action of KSS, supporting the notion that the action of KSS
on ERβ might be involved in Tph induction and reduced aggressiveness.
However, it has been reported that ERs are distributed not only in the
DRN but also in a variety of brain regions and other neurotransmitter
systems, whose contribution to the ABB-reducing effect of KSS have yet
to be examined (Cui et al., 2013; Isgor et al., 2003).
Kumagai et al. (2005) showed some activity of KSS on ERs using a
reporter assay; therefore, we compared the action of 17α- and 17β-E2 on
ABB. It has also been reported that KSS and liquiritigenin, the estrogenic
compound from the root of Glycyrrhizae uralensis, act agonistically on
ERβ (Mersereau et al., 2009; Watanabe et al., 2006). Taken together, the
results of the present and previous studies suggest that a certain ingre￾dient of KSS might act on ERβ in the DRN neurons to facilitate Tph1 gene
expression and 5-HT production. A 3D-HPLC chart of KSS, which has
previously been described in Wang et al. (2018), would be useful for
determining the active pharmaceutical ingredients of KSS in relation to
reduced ABB. Interestingly, a trace amount of E2 was capable of
reducing ABB, although it can be hypothesized that mice had sufficient
flavonoid levels, since mice were fed with an ordinary diet, not depleted
in phytoestrogens such as genistein and daidzein, ingredients of soy￾beans (Lian et al., 2001; Thigpen et al., 1999). Further study is required
to explore and characterize the estrogen-like ingredients in KSS.
Finally, our results indicate that the action on 5-HT1A receptor is
associated with the reduction of ABB and the action of KSS on ERs might
stimulate 5-HT production. However, since KSS is composed of 10 herbs,
work remains to be done to elucidate the action of each herb and how
their combination concertedly exert the complex action of KSS.
4. Conclusion
KSS is widely used as a traditional herbal formula in Japan to
improve psychiatric symptoms, such as general malaise, in menopausal
women. However, limited ethological validation of KSS action has been
reported until recently. In the present study, we showed that KSS
effectively reduces ABB in isolation-reared male and female mice pre￾sumably through stimulation of 5-HT production in the DRN. In male
mice, the combinatory effect of KSS on 5-HT1A receptors and GABAA
receptors seems to be involved in reducing ABB. In female mice, how￾ever, the effect of KSS on 5-HT1A receptors seems to be the primary
element in ABB reduction. Our findings also suggest that increased ERs
are associated with the action of KSS. Further research building on the
results provided here should focus on the individual components of KSS;
such studies will provide a better understanding of the synergistic effect
of KSS and its ability to improve psychiatric symptoms.
5. Materials and methods
5.1. Animals
All experiments were performed using ddY-strain mice, which were
purchased from Japan SLC (Slc: ddY; Japan SLC, Inc., Shizuoka, Japan).
Normal male and female mice were housed together for mating to obtain
offspring. For the isolation-rearing experiments, male and female pups
were separated from the dam at 3-weeks-old and then isolation-reared in
individual cages (16 × 23 × 12 cm) until 11 weeks after birth. For the
group-housing experiments, pups were separated from the dam at 3
weeks and housed in groups of 2–3 per cage (32 × 23 × 12 cm) until 11
weeks after birth. Measurement of ABB was performed within the period
during which they had received 6–8 weeks of isolation rearing (i.e.,
postnatal week 9–11; see Supplementary Fig. 1). To assess the effects of
a single oral administration of KSS, it was administered to isolation￾reared mice using a plastic sonde (Fuchigami Kikai, Kyoto, Japan). For
i.p. administration of drugs, each mouse was placed on wire netting,
picked up by its tail, and the drug was intraperitoneally administrated
using a Terumo 29 × 1/2′′ gauge syringe (Terumo Co., Tokyo, Japan), as
previously described (Kuchiiwa and Kuchiiwa, 2014, 2016). Monitoring
of vaginal smear images was performed following the method of McLean
et al. (2012). All animals were housed under controlled conditions
(temperature: 22 ◦C ± 2 ◦C; lighting: lights on at 7:00, lights off at
19:00), with food and water administered ad libitum. Cage exchange was
performed every 10 days. All animal procedures were approved by the
Committee of Animal Experimentation, Kagoshima University, and
performed in accordance with the guidelines of the Japanese Pharma￾cological Society.
5.2. Drugs and reagents
KSS (TJ-24; Tsumura Co. Ltd., Tokyo, Japan) was purchased as a
freeze-dried powdered extract. A daily dose of powder (7.5 g) of this
medicine contains 4.0 g of KSS extract obtained from the following 10
herbs: Bupleurum falcatum (3.0 g), Paeonia lactiflora (3.0 g), Atractylodes
lancea (3.0 g), Angelica acutiloba (3.0 g), Paria cocos (3.0 g), Gardenia
jasminoides (2.0 g), Paeonia suffruticosa (2.0 g), Glycyrrhizae uralensis
(1.5 g), Zingiber officinale (1.0 g), and Menthae arvensis (1.0 g) (Yamada
and Kanba, 2007). Magnesium stearate and lactose hydrate are added as
inactive ingredients for granulation by the manufacturer. At each
experimental day, KSS suspension was prepared by vortex. The sus￾pension of KSS was intraperitoneally injected to the subject mouse at the
experimental day within postnatal week 9–11 at 100 mg/kg or orally
K. Igarashi et al.
Brain Research 1768 (2021) 147580
administered at 100, 200, or 400 mg/kg.
N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyr￾idinylcyclohexanecarboxamide (WAY-100635) maleate (ab120550;
Abcam, Cambridge, UK) and [S-(R*,S*)]-5-(6,8-Dihydro-8-oxofuro[3,4-
e]-1,2-benzodioxol-6-yl)5,6,7,8-tetrahydro-6,6-dimethyl-1,3-dimethyl-
1,3-dioxolo[4,5-g]isoquinolinium iodide ((-)-bicuculline) methiodide
(ab120108; Abcam) were dissolved in distilled water and intraperito￾neally administered to each mouse (0.5 mg/kg and 1 mg/kg, respec￾tively). The doses of WAY-100635 and (-)-bicuculline were determined
according to the previous studies (Guo et al., 2019a; Harada et al., 2018;
Kanno et al., 2009; Magen et al., 2010; Mizowaki et al., 2001).
Tamoxifen (2-[4-[(1Z)-1,2-diphenyl-1-buten-1-yl]phenoxy]-N,N￾dimethyl-ethanamine, item no. 13258; Cayman Chemical Company, MI,
USA) and PHTPP (4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo [1,5-a]
pyrimidin-3-yl]phenol, Selleck Biotech, Tokyo, Japan) were suspended
in corn oil containing 10% ethanol. ICI-182,780 ((7R,9S,13S,14S,17S)-
7-(9-(4,4,5,5,5-Pentafluoropentylsulfinyl)nonyl)-
7,8,9,11,12,13,14,15,16,17-decahydro-13-methyl-6H-cyclopenta[a]
phenanthrene-3,17-diol, ab120131; Abcam) and G-15 ((3aS,4R,9bR)-4-
(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]
quinoline, item no. 14673; Cayman Chemical Company) were respec￾tively suspended in corn oil containing 5% ethanol. 17α- and 17β-E2
were purchased from Nacalai Tesque Inc. (Kyoto, Japan; code: 145–48
and 145-41, respectively) and dissolved in corn oil containing 1%
ethanol. The doses of tamoxifen, PHTPP, ICI-182780, and G-15 were
determined according to previous studies using mice (Alfinito et al.,
2008; Kim et al., 2010; Lai et al., 2017; Li et al., 2016; Liu et al., 2010;
Shooshtari et al., 2021; Zhao et al., 2017). The doses of 17α- and 17β-E2
were determined based on assumption of the previous study (Kumagai
et al., 2005).
5.3. ABB measurement
The intensity and frequency of ABB toward an inanimate object was
measured using an ARM device (Muromachi Kikai Co. Ltd., Tokyo,
Japan), as previously described (Kuchiiwa and Kuchiiwa, 2014, 2016).
In brief, the subject mouse is placed in the cylinder on the top of ARM
and measured ABB (see Supplementary Fig. 1). Measurement of ABB is
consisted of two sessions; first irritation session and second measure￾ment session. Metal rods with a dome-shape head are automatically
controlled to make an up-and-down motion 30 times to touch the hind
limb of the subject mouse in the first session and to propose the rods in
front of the subject mouse in the second session. The load sensor con￾nected to the rods measures ABB intensity (average intensity of one
biting) and ABB frequency (total number of bitings within 30 pre￾sentations of the rods). ABB intensity is expressed in numerical values as
an integral of biting force within one-second presentation of the rods
(milliNewton × second: mNs).
5.4. Quantitative RT-PCR
To surgically resect brain tissue, mice were first deeply anesthetized
with pentobarbital (100 mg/kg; Kyoritsu Seiyaku, Tokyo, Japan) and
then transcardially perfused with phosphate-buffered saline (PBS). To
obtain the tissue of the DRN, we referred to the Franklin and Paxinos’
mouse brain atlas (Franklin and Paxinos, 2007). Briefly, the brain was
first coronally sliced on ice at the inferior colliculi. After that, a triangle
tissue fraction, extending about 0.8–1 mm from the aqueduct to whitish
area and about 0.5 mm width from the aqueduct to each bilateral
whitish area was resected. This triangle tissue fraction was used as DRN.
Each fraction was homogenized in Isogen (Nippongene, Tokyo, Japan)
and stored at − 80 ◦C until total RNA was prepared; at this point, the
fractions of three mice per group were mixed. After using a standard
extraction procedure using Isogen, following DNase I (QIAGEN) treat￾ment, and purification using NucleoSpin RNA Clean-up kit (Macherey
Nagel GmbH & Co. KG), 600 ng of total RNA was used for reverse
transcription with ReverTra Ace (Toyobo, Osaka, Japan). First-strand
cDNA was stored at − 20 ◦C until use. Relative gene expression was
analyzed via the relative standard curve method with THUNDERBIRD
SYBR qPCR Mix (Toyobo) on an ABI 7300 real-time PCR system (Applied
Biosystems, Thermo, MA, USA). The forward and reverse primer se￾quences used were 5′
-ACA TGC CAA AGT CAA GCC CT-3′ and CCG CAA
CTC ATT CAT GGC AC for Tph1 (ENSMUST00000049298); 5′
-AGA TGC
TCT CAC CGA GTC CT-3′ and 5′
-GCA AGC ATG AGT CGG GTA GA-3′ for
Tph2 (ENSMUST00000006949); 5′
-CTG TCC AGC CAC GAA TCA GT-3′
and 5′
-GGC TTT GTT CAG GCA ATG CA-3′ for Esr2
(ENSMUST00000076634); 5′
- TGT TCT CGG GTT TCA ACG CC-3′ and
5′
-CTG GTG GAG ATG GCT GTC AC-3′ for c-fos
(ENSMUST00000021674); 5′
-GAG GCC CAG AGC AAG AGA GG-3′ and
5′
-GCT ACG TAC ATG GCT GGG GT-3′ for Actb
(ENSMUST00000100497). The primer sequence was designed using
Primer3Plus software (Untergasser et al., 2012).
5.5. Immunohistochemistry
Mice were deeply anesthetized with pentobarbital (100 mg/kg) and
transcardially perfused with PBS (pH 7.4) followed by 4% formalde￾hyde. After post fixation, brains were equilibrated in 30% sucrose
overnight at 4 ◦C. Brain tissues were frozen in O.C.T. compound (Sakura
Finetek Japan Co., Ltd., Tokyo, Japan) at − 80 ◦C overnight, after which
they were coronally sectioned to a thickness of 40 μm on a freezing
microtome (CryoStar NX70 Cryostat; Thermo) and stored in PBS sup￾plemented with 0.1% sodium azide at 4 ◦C until use.
For immunostaining, sections were blocked with 1% BSA in PBS
supplemented with 0.2% Triton X-100 at room temperature for 1 h.
Sections were then incubated with anti-5-HT antibody (S5545; Sigma￾Aldrich, Merck KGaA, Darmstadt, Germany; 1:1,000) as the primary
antibody at room temperature overnight. The sections were subse￾quently incubated with Alexa488-conjugated anti-rabbit IgG antibody
(A-11008; Thermo; 1:1,000) as the secondary antibody at room tem￾perature for 3 h. Each section was counterstained with 4′
6-diamidino-2-
phenylindole (i.e., DAPI). Images were captured on a confocal micro￾scope system (Leica TCS SP8; Leica Microsystems, Tokyo, Japan).
For measuring the mean immunofluorescence intensity of DRN in
each mouse, three sections (approximately around bregma − 4.72) were
used per an individual by referring to the standard mouse brain atlas
(Franklin and Paxinos, 2007). Immunofluorescence intensity of each
image was quantified by using ImageJ (NIH).
5.6. Immunoblot analysis
For immunoblot analysis, each fraction was mixed in cell lysis buffer
(containing 50 mM Tris, 150 mM NaCl, 1% NP-40, 0.1% sodium deox￾ycholate, 1 mM NaF, and 1 mM sodium orthovanadate) supplemented
with protease inhibitor cocktail (Nacalai Tesque); after centrifugation of
this mixture, the supernatant was collected as protein extract. Following
reduction with 100 mM dithiothreitol under thermal denaturing con￾ditions (95 ◦C for 10 min.), 10 μg of each protein extract was electro￾phoresed on polyacrylamide gel (10% acrylamide, 29:1) and blotted on
a polyvinylidene difluoride (PVDF) membrane (Cytiva, UK). Blocking
was performed with 2% bovine serum albumin (BSA) in Tris-buffered
saline supplemented with 0.1% Tween-20 for 30 min. Anti-β-actin
antibody (no. 4970; Cell Signaling Technology, MA, USA; 3,000) and
anti-ERβ antibody (PA1-310B; Thermo; 1:1,000) were used as primary
antibodies, whereas anti-Rabbit IgG, HRP-linked antibody (no. 7074;
Cell Signaling Technology; 3,000) was used as the secondary antibody.
After activation with ImmunoStar Zeta (Fujifilm-Wako, Osaka, Japan),
chemical luminescence was captured on a ChemiDoc XRS + system
(BioRad, CA, USA).
K. Igarashi et al.
Brain Research 1768 (2021) 147580
5.7. Statistical analysis
Tukey’s HSD test or Tukey–Kramer test after one-way or two-way
ANOVA was used for analyses. Student’s t test was used for analysis of
ABB after 17α- or 17β-E2 treatment. Significance was set at p < 0.05.
Statistical analyses were performed using StatPlus software (AnalystSoft
Inc., CA, USA).
Funding
This work was in part supported by Japanese Society for the Pro￾motion of Science (JSPS) KAKENHI Grant Number 19 K19170 and a
Grant from the Kodama Memorial Fund for Medical Research Grant
Number 311.
CRediT authorship contribution statement
Kento Igarashi: Conceptualization, Methodology, Data curation,
Formal analysis, Writing – original draft. Toshiko Kuchiiwa: Concep￾tualization, Writing – review & editing. Satoshi Kuchiiwa: Conceptu￾alization, Writing – review & editing. Haruki Iwai: Methodology,
Writing – review & editing. Kazuo Tomita: Data curation, Writing -
review & editing. Tomoaki Sato: Conceptualization, Methodology,
Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors are thankful to Dr. Kohjitani and Dr. Ohno for kindly
assisting our use of the ABI-7300 real-time PCR system.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.brainres.2021.147580.
References
Alfinito, P.D., Chen, X., Atherton, J., Cosmi, S., Deecher, D.C., 2008. ICI 182,780
penetrates brain and hypothalamic tissue and has functional effects in the brain after
systemic dosing. Endocrinology 149, 5219–5226. https://doi.org/10.1210/en.2008-
0532.
Anderson, L.C., Petrovich, G.D., 2018. Ventromedial prefrontal cortex mediates sex
differences in persistent cognitive drive for food. Sci. Rep. 8, 1–9. https://doi.org/
10.1038/s41598-018-20553-4.
Azmitia, E.C., Singh, J.S., Whitaker-Azmitia, P.M., 2011. Neuropharmacology Increased
serotonin axons (immunoreactive to 5-HT transporter) in postmortem brains from
young autism donors. Neuropharmacology 60 (7-8), 1347–1354. https://doi.org/
10.1016/j.neuropharm.2011.02.002.
Cao, X., Huang, S., Cao, J., Chen, T., Zhu, P., Zhu, R., Su, P., Ruan, D., 2014. The timing
of maternal separation affects morris water maze performance and long-term
potentiation in male rats. Dev. Psychobiol. 56 (5), 1102–1109. https://doi.org/
10.1002/dev.v56.510.1002/dev.21130.
Chen, L.-C., Tsao, Y.-T., Yen, K.-Y., Chen, Y.-F., Chou, M.-H., Lin, M.-F., 2003. A pilot
study comparing the clinical effects of Jia-Wey Shiau-Yau San, a traditional Chinese
herbal prescription, and a continuous combined hormone replacement therapy in
postmenopausal women with climacteric symptoms. Maturitas 44 (1), 55–62.

https://doi.org/10.1016/S0378-5122(02)00314-6.

Cui, J., Shen, Y., Li, R., 2013. Estrogen synthesis and signaling pathways during aging:
From periphery to brain. Trends Mol. Med. 19 (3), 197–209. https://doi.org/
10.1016/j.molmed.2012.12.007.
Dahlstrom, ¨ A., Fuxe, K., 1964. Localization of monoamines in the lower brain stem.
Experientia 20 (7), 398–399. https://doi.org/10.1007/BF02147990.
Donner, N., Handa, R.J., 2009. Estrogen receptor beta regulates the expression of
tryptophan-hydroxylase 2 mRNA within serotonergic neurons of the rat dorsal raphe
nuclei. Neuroscience 163 (2), 705–718. https://doi.org/10.1016/j.neuroscience:
2009.06.046.
Faccidomo, S., Bannai, M., Miczek, K.A., 2008. Escalated aggression after alcohol
drinking in male mice: Dorsal raph´e and prefrontal cortex serotonin and 5-HT1B
receptors. Neuropsychopharmacology 33 (12), 2888–2899. https://doi.org/
10.1038/npp.2008.7.
Fernandez, S.P., Cauli, B., Cabezas, C., Muzerelle, A., Poncer, J.-C., Gaspar, P., 2016.
Multiscale single-cell analysis reveals unique phenotypes of raphe 5-HT neurons
projecting to the forebrain. Brain Struct. Funct. 221 (8), 4007–4025. https://doi.org/
10.1007/s00429-015-1142-4.
Franklin., K.B., Paxinos, G., 2007. In: Paxinos, G. (Ed.), The Mouse Brain in Stereotaxic
Coordinates, Third ed. Academic Press, New York.
Furukawa, M, Tsukahara, T, Tomita, K, Iwai, H, Sonomura, T, Miyawaki, S, Sato, T,
2017. Neonatal maternal separation delays the GABA excitatory-to-inhibitory
functional switch by inhibiting KCC2 expression. Biochem. Biophys. Res. Commun.
493 (3), 1243–1249. https://doi.org/10.1016/j.bbrc.2017.09.143.
Guo, Q., Ebihara, K., Fujiwara, H., Toume, K., Awale, S., Araki, R., Yabe, T., Dong, E.,
Matsumoto, K., 2019a. Kami-shoyo-san ameliorates sociability deficits in
ovariectomized mice, a putative female model of autism spectrum disorder, via
facilitating dopamine D1 and GABAA receptor functions. J. Ethnopharmacol. 236,
231–239. https://doi.org/10.1016/j.jep.2019.03.010.
Guo, Qing-Yun, Ebihara, Ken, Shimodaira, Takafumi, Fujiwara, Hironori,
Toume, Kazufumi, Dibwe, Dya Fita, Awale, Suresh, Araki, Ryota, Yabe, Takeshi,
Matsumoto, Kinzo, Lee, Joohyung, 2019b. Kami-shoyo-san improves ASD-like
behaviors caused by decreasing allopregnanolone biosynthesis in an SKF mouse
model of autism. PLoS One 14 (1), e0211266. https://doi.org/10.1371/journal.
pone.0211266.
Haller, J., Harold, G., Sandi, C., Neumann, I.D., 2014. Effects of adverse early-life events
on aggression and anti-social behaviours in animals and humans.
J. Neuroendocrinol. 26 (10), 724–738. https://doi.org/10.1111/jne.12182.
Harada, Y., Yamaguchi, T., Hu, A., Otani, S., Han, C., Kurihara, Y., Kobayashi, H.,
Arai, H., 2018. Effect of hangekobokuto for amelioration of aggressiveness and social
behavior in socially isolated mice. Tradit. Kampo Med. 5 (2), 75–82. https://doi.org/
10.1002/tkm2.2018.5.issue-210.1002/tkm2.1095.
Hensler, Julie G., 2006. Serotonergic modulation of the limbic system. Neurosci.
Biobehav. Rev. 30 (2), 203–214. https://doi.org/10.1016/j.neubiorev.2005.06.007.
Hidaka, T., Yonezawa, R., Saito, S., 2013. Kami-shoyo-san, Kampo (Japanese traditional
medicine), is effective for climacteric syndrome, especially in hormone-replacement￾therapy-resistant patients who strongly complain of psychological symptoms WAY-100635.
J. Obstet. Gynaecol. Res. 39, 223–228. https://doi.org/10.1111/j.1447-
0756.2012.01936.x.
Huang, X., Yang, J., Yang, S., Cao, S., Qin, D., Zhou, Y., Li, X., Ye, Y., Wu, J., 2017. Role
of tandospirone, a 5-HT1A receptor partial agonist, in the treatment of central
nervous system disorders and the underlying mechanisms. Oncotarget 8,
102705–102720. https://doi.org/10.18632/oncotarget.22170.
Isgor, C., Cecchi, M., Kabbaj, M., Akil, H., Watson, S.J., 2003. Estrogen receptor β in the
paraventricular nucleus of hypothalamus regulates the neuroendocrine response to
stress and is regulated by corticosterone. Neuroscience 121 (4), 837–845. https://
doi.org/10.1016/S0306-4522(03)00561-X.
Kanno, H., Sekiguchi, K., Yamaguchi, T., Terawaki, K., Yuzurihara, M., Kase, Y.,
Ikarashi, Y., 2009. Effect of yokukansan, a traditional Japanese medicine, on social
and aggressive behaviour of para-chloroamphetamine-injected rats. J. Pharm.
Pharmacol. 61, 1249–1256. https://doi.org/10.1211/jpp/61.09.0016.
Kim, D.H., Jung, H.A., Park, S.J., Kim, J.M., Lee, S., Choi, J.S., Cheong, J.H., Ko, K.H.,
Ryu, J.H., 2010. The effects of daidzin and its aglycon, daidzein, on the scopolamine￾induced memory impairment in male mice. Arch. Pharm. Res. 33 (10), 1685–1690.

https://doi.org/10.1007/s12272-010-1019-2.

Klinge, C.M., 2001. Estrogen receptor interaction with estrogen response elements.
Nucleic Acids Res. 29, 2905–2919. https://doi.org/10.1093/nar/29.14.2905.
Kuchiiwa, S., Kuchiiwa, T., 2014. A novel semi-automated apparatus for measurement of
aggressive biting behavior in mice. J. Neurosci. Methods 228, 27–34. https://doi.
org/10.1016/j.jneumeth.2014.02.017.
Kuchiiwa, T., Kuchiiwa, S., 2016. Evaluation of aggressiveness of female mice using a
semi-automated apparatus for measurement of aggressive biting behavior toward an
inanimate object. J. Neurosci. Methods 257, 179–184. https://doi.org/10.1016/j.
jneumeth.2015.10.005.
Kumagai, Y., Hyuga, S., Hyuga, M., Watanabe, K., Kawanishi, T., Hanawa, T., 2005.
Estrogen-like activity in Kampo medicines used for menopausal symptoms and
gynecological diseases. J. Tradit. Med. 22, 228–236. https://doi.org/10.11339/j
tm.22.228.
Lai, Y.-J., Liu, L., Hu, X.-T., He, L., Chen, G.J., 2017. Estrogen Modulates ubc9 Expression
and Synaptic Redistribution in the Brain of APP/PS1 Mice and Cortical Neurons.
J. Mol. Neurosci. 61 (3), 436–448. https://doi.org/10.1007/s12031-017-0884-2.
Li, Y., Xu, J., Jiang, F., Jiang, Z., Liu, C., Li, L., Luo, Y., Lu, R., Mu, Y., Liu, Y., Xue, B.,
2016. G protein-coupled estrogen receptor is involved in modulating colonic motor
function via nitric oxide release in C57BL/6 female mice. Neurogastroenterol. Motil.
28 (3), 432–442. https://doi.org/10.1111/nmo.12743.
Lian, Z., Niwa, K., Tagami, K., Hashimoto, M., Gao, J., Yokoyama, Y., Mori, H.,
Tamaya, T., 2001. Preventive effects of isoflavones, genistein and daidzein, on
estradiol-17β-related endometrial carcinogenesis in mice. Jpn. J. Cancer Res. 92,
726–734. https://doi.org/10.1111/j.1349-7006.2001.tb01154.x.
Liu, J.L., Tian, D.S., Li, Z.W., Qu, W.S., Zhan, Y., Xie, M.J., Yu, Z.Y., Wang, W., Wu, G.,
2010. Tamoxifen alleviates irradiation-induced brain injury by attenuating
microglial inflammatory response in vitro and in vivo. Brain Res. 1316, 101–111.

https://doi.org/10.1016/j.brainres.2009.12.055.

Lucas, G., Rymar, V.V., Du, J., Mnie-Filali, O., Bisgaard, C., Manta, S., Lambas-Senas, L.,
Wiborg, O., Haddjeri, N., Pineyro, ˜ G., Sadikot, A.F., Debonnel, G., 2007. Serotonin4
K. Igarashi et al.
Brain Research 1768 (2021) 147580
(5-HT4) receptor agonists are putative antidepressants with a rapid onset of action.
Neuron 55 (5), 712–725. https://doi.org/10.1016/j.neuron.2007.07.041.
Magen, I., Avraham, Y., Ackerman, Z., Vorobiev, L., Mechoulam, R., Berry, E.M., 2010.
Cannabidiol ameliorates cognitive and motor impairments in bile-duct ligated mice
via 5-HT1A receptor activation. Br. J. Pharmacol. 159, 950–957. https://doi.org/
10.1111/j.1476-5381.2009.00589.x.
Mantani, N., Hisanaga, A., Kogure, T., Kita, T., Shimada, Y., Terasawa, K., 2002. Four
cases of panic disorder successfully treated with Kampo (Japanese herbal)
medicines: Kami-shoyo-san and Hange-koboku-to. Psychiatry Clin. Neurosci. 56,
617–620. https://doi.org/10.1046/j.1440-1819.2002.01064.x.
Marcinkiewcz, C.A., Mazzone, C.M., D’Agostino, G., Halladay, L.R., Andrew
Hardaway, J., DiBerto, J.F., Navarro, M., Burnham, N., Cristiano, C., Dorrier, C.E.,
Tipton, G.J., Ramakrishnan, C., Kozicz, T., Deisseroth, K., Thiele, T.E., McElligott, Z.
A., Holmes, A., Heisler, L.K., Kash, T.L., 2016. Serotonin engages an anxiety and fear￾promoting circuit in the extended amygdala. Nature 537 (7618), 97–101. https://
doi.org/10.1038/nature19318.
Matson, J.L., Jang, J., 2014. Treating aggression in persons with autism spectrum
disorders: a review. Res. Dev. Disabil. 35 (12), 3386–3391. https://doi.org/10.1016/
j.ridd.2014.08.025.
Matsuda, K., 1975. The effect of KAMISHOYOSAN on two male cases of vegetative
stigmata. J. Japan Soc. Orient. Med. 26, 158–160. https://doi.org/10.14868/
kampomed1950.26.158.
McLean, A.C., Valenzuela, N., Fai, S., Bennett, S.A.L., 2012. Performing vaginal lavage,
crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle
staging identification. J. Vis. Exp. 4–9 https://doi.org/10.3791/4389.
Mersereau, J.E., Levy, N., Staub, R.E., Baggett, S., Zogric, T., Chow, S., Ricke, W.A.,
Tagliaferri, M., Cohen, I., Bjeldanes, L.F., Leitman, D.C., 2009. Liquiritigenin is a
plant-derived highly selective estrogen receptor β agonist. Mol. Cell. Endocrinol. Cel
Endocrinol 283, 49–57.
Mitra, S.W., Hoskin, E., Yudkovitz, J., Pear, L., Wilkinson, H.A., Hayashi, S., Pfaff, D.W.,
Ogawa, S., Rohrer, S.P., Schaeffer, J.M., McEwen, B.S., Alves, S.E., 2003.
Immunolocalization of estrogen receptor β in the mouse brain: Comparison with
estrogen receptor α. Endocrinology 144, 2055–2067. https://doi.org/10.1210/
en.2002-221069.
Mizowaki, M., Toriizuka, K., Hanawa, T., 2001. Anxiolytic effect of Kami-Shoyo-San (TJ-
24) in mice possible mediation of neurosteroid synthesis. Life Sci. 69 (18),
2167–2177. https://doi.org/10.1016/S0024-3205(01)01290-5.
Nievergelt, A., Huonker, P., Schoop, R., Altmann, K.H., Gertsch, J., 2010. Identification
of serotonin 5-HT1A receptor partial agonists in ginger. Bioorganic Med. Chem. 18
(9), 3345–3351. https://doi.org/10.1016/j.bmc.2010.02.062.
Nishizawa, S., Benkelfat, C., Young, S.N., Leyton, M., Mzengeza, S., de Montigny, C.,
Blier, P., Diksic, M., 1997. Differences between males and females in rates of
serotonin synthesis in human brain. Proc. Natl. Acad. Sci. U.S.A. 94 (10),
5308–5313. https://doi.org/10.1073/pnas.94.10.5308.
Nomura, M., Akama, K.T., Alves, S.E., Korach, K.S., Gustafsson, J.-Å., Pfaff, D.W.,
Ogawa, S., 2005. Differential distribution of estrogen receptor (ER)-α and ER-β in the
midbrain raphe nuclei and periaqueductal gray in male mouse: predominant role of
ER-β in midbrain serotonergic systems. Neuroscience 130 (2), 445–456. https://doi.
org/10.1016/j.neuroscience:2004.09.028.
Patel, B.D., Barzman, D.H., 2013. Pharmacology and pharmacogenetics of pediatric
ADHD with associated aggression: a review. Psychiatr. Q. 84 (4), 407–415. https://
doi.org/10.1007/s11126-013-9253-7.
Powell, E., Xu, W., 2008. Intermolecular interactions identify ligand-selective activity of
estrogen receptor α/β dimers. Proc. Natl. Acad. Sci. U.S.A. 105, 19012–19017.

https://doi.org/10.1073/pnas.0807274105.

Prossnitz, E.R., Sklar, L.A., Oprea, T.I., Arterburn, J.B., 2008. GPR30: a novel therapeutic
target in estrogen-related disease. Trends Pharmacol. Sci. 29 (3), 116–123. https://
doi.org/10.1016/j.tips.2008.01.001.
Sato-Nishimori, F., Matsukawa, Y., Matsuda, K., Kida, M., Saito, T., Fujiwara, H., 2008.
Three cases of panic disorder successfully treated with kampo formulae. Kampo Med.
59, 721–726. https://doi.org/10.3937/kampomed.59.721.
Shanle, Erin K., Xu, Wei, 2010. Selectively targeting estrogen receptors for cancer
treatment. Adv. Drug Deliv. Rev. 62 (13), 1265–1276. https://doi.org/10.1016/j.
addr.2010.08.001.
Sheng, Z., Kawano, J., Yanai, A., Fujinaga, R., Tanaka, M., Watanabe, Y., Shinoda, K.,
2004. Expression of estrogen receptors (α, β) and androgen receptor in serotonin
neurons of the rat and mouse dorsal raphe nuclei; sex and species differences.
Neurosci. Res. 49 (2), 185–196. https://doi.org/10.1016/j.neures.2004.02.011.
Shimizu, S., Ishino, Y., Takeda, T., Tohyama, M., Miyata, S., 2019. Antidepressive effects
of kamishoyosan through 5-HT1AReceptor and PKA-CREB-BDNF signaling in the
hippocampus in postmenopausal depression-model mice. Evidence-based
Complement. Altern. Med. 2019, 1–11. https://doi.org/10.1155/2019/9475384.
Khombi Shooshtari, M., Farbood, Y., Mansouri, S.M.T., Badavi, M., Khorsandi, L.S.,
Ghasemi Dehcheshmeh, M., Sarkaki, A.R., 2021. Neuroprotective effects of chrysin
mediated by estrogenic receptors following cerebral ischemia and reperfusion in
male rats. Basic Clin. Neurosci. 12 (1), 149–162.
Sijbesma, H., Schipper, J., de Kloet, E.R., Mos, J., van Aken, H., Olivier, B., 1991.
Postsynaptic 5-HT1 receptors and offensive aggression in rats: A combined
behavioural and autoradiographic study with eltoprazine. Pharmacol. Biochem.
Behav. 38 (2), 447–458. https://doi.org/10.1016/0091-3057(91)90305-L.
Takahashi, A., Nagayasu, K., Nishitani, N., Kaneko, S., Koide, T., Siegel, A., 2014. Control
of intermale aggression by medial prefrontal cortex activation in the mouse. PLoS
One 9 (4), e94657. https://doi.org/10.1371/journal.pone.0094657.
Takamatsu, K., Ogawa, M., Obayashi, S., Takeda, T., Terauchi, M., Higuchi, T., Kato, K.,
Kubota, T., Ferraro, M.G., 2021. A multicenter, randomized, double-blind, placebo￾controlled trial to investigate the effects of Kamishoyosan, a traditional Japanese
medicine, on menopausal symptoms: The KOSMOS study. Evid. Based Complement.
Altern. Med. 2021, 1–9. https://doi.org/10.1155/2021/8856149.
Thigpen, J.E., Setchell, K.D.R., Ahlmark, K.B., Locklear, J., Spahr, T., Caviness, G.F.,
Goelz, M.F., Haseman, J.K., Newbold, R.R., Forsythe, D.B., 1999. Phytoestrogen
content of purified, open- and closed-formula laboratory animal diets. Lab. Anim.
Sci. 49, 530–536.
Toth, ´ M., Hal´
asz, J., Mikics, E., ´ Barsy, B., Haller, J., 2008. Early social deprivation
induces disturbed social communication and violent aggression in adulthood. Behav.
Neurosci. https://doi.org/10.1037/0735-7044.122.4.849.
Topiwala, A., Fazel, S., 2011. The pharmacological management of violence in
schizophrenia: a structured review. Expert Rev. Neurother. 11 (1), 53–63. https://
doi.org/10.1586/ern.10.180.
Tsiouris, J.A., Kim, S.Y., Brown, W.T., Cohen, I.L., 2011. Association of aggressive
behaviours with psychiatric disorders, age, sex and degree of intellectual disability: a
large-scale survey. J. Intellect. Disabil. Res. 55, 636–649. https://doi.org/10.1111/
j.1365-2788.2011.01418.x.
Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B.C., Remm, M., Rozen, S.
G., 2012. Primer3-new capabilities and interfaces. Nucleic Acids Res. 40, 1–12.

https://doi.org/10.1093/nar/gks596.

VanderHorst, V.G.J.M., Gustafsson, J.-Å., Ulfhake, B., 2005. Estrogen receptor-α and -β
immunoreactive neurons in the brainstem and spinal cord of male and female mice:
Relationships to monoaminergic, cholinergic, and spinal projection systems.
J. Comp. Neurol. 488 (2), 152–179. https://doi.org/10.1002/cne.v488:210.1002/
cne.20569.
Veenema, A.H., Blume, A., Niederle, D., Buwalda, B., Neumann, I.D., 2006. Effects of
early life stress on adult male aggression and hypothalamic vasopressin and
serotonin. Eur. J. Neurosci. 24, 1711–1720. https://doi.org/10.1111/j.1460-
9568.2006.05045.x.
Vergnes, M., Depaulis, A., Boehrer, A., 1986. Parachlorophenylalanine-induced serotonin
depletion increases offensive but not defensive aggression in male rats. Physiol.
Behav. 36 (4), 653–658. https://doi.org/10.1016/0031-9384(86)90349-5.
Waltes, R., Chiocchetti, A.G., Freitag, C.M., 2016. The neurobiological basis of human
aggression: A review on genetic and epigenetic mechanisms. Am. J. Med. Genet. Part
B Neuropsychiatr. Genet. https://doi.org/10.1002/ajmg.b.32388.
Wang, Z., Kanda, S., Shimono, T., Enkh-Undraa, D., Nishiyama, T., 2018. The in vitro
estrogenic activity of the crude drugs found in Japanese herbal medicines prescribed
for menopausal syndrome was enhanced by combining them. BMC Complement.
Altern. Med. 18, 1–12. https://doi.org/10.1186/s12906-018-2170-4.
Waselus, M., Valentino, R.J., Van Bockstaele, E.J., 2011. Collateralized dorsal raphe
nucleus projections: a mechanism for the integration of diverse functions during
stress. J. Chem. Neuroanat. 41 (4), 266–280. https://doi.org/10.1016/j.
jchemneu.2011.05.011.
Watanabe, K., Hyuga, S., Masashi, H., Kawanishi, T., Hanawa, T., 2006. Agonistic or
antagonistic action of Kampo medicines used for menopausal symptoms on estrogen
receptor subtypes, ERα and ERβ. J. Tradit. Med. 23, 203–207. https://doi.org/
10.11339/jtm.23.203.
Wigger, A., Neumann, I.D., 1999. Periodic maternal deprivation induces gender￾dependent alterations in behavioral and neuroendocrine responses to emotional
stress in adult rats. Physiol. Behav. 66 (2), 293–302. https://doi.org/10.1016/
S0031-9384(98)00300-X.
Yamada, K., Kanba, S., 2007. Effectiveness of kamishoyosan for premenstrual dysphoric
disorder: open-labeled pilot study. Psychiatry Clin. Neurosci. 61 (3), 323–325.

https://doi.org/10.1111/pcn.2007.61.issue-310.1111/j.1440-1819.2007.01649.x.

Yas¸ar, P., Ayaz, G., User, S.D., Güpür, G., Muyan, M., 2017. Molecular mechanism of
estrogen–estrogen receptor signaling. Reprod. Med. Biol. 16 (1), 4–20. https://doi.
org/10.1002/rmb2.12006.
Zhang, Z.-J., Kang, W.-H., Li, Q., Tan, Q.-R., 2007. The beneficial effects of the herbal
medicine Free and Easy Wanderer Plus (FEWP) for mood disorders: double-blind,
placebo-controlled studies. J. Psychiatr. Res. 41 (10), 828–836. https://doi.org/
10.1016/j.jpsychires.2006.08.002.
Zhao, Y., He, L., Zhang, Y., Zhao, J., Liu, Z., Xing, F., Liu, M., Feng, Z., Li, W., Zhang, J.,
2017. Estrogen receptor alpha and beta regulate actin polymerization and spatial
memory through an SRC-1/mTORC2-dependent pathway in the hippocampus of
female mice. J. Steroid Biochem. Mol. Biol. 174, 96–113. https://doi.org/10.1016/j.
jsbmb.2017.08.003.
Zmudzka, ˙ E., Sałaciak, K., Sapa, J., Pytka, K., 2018. Serotonin receptors in depression
and anxiety: Insights from animal studies. Life Sci. 210, 106–124. https://doi.org/
10.1016/j.lfs.2018.08.050.
K. Igarashi et al.