Title

Fluvoxamine reverses estrogen-dependent decline
in voluntary activities and decreased amygdala
levels of serotonin in ovariectomized rats

Author(s)石橋, 雪子Citation

大阪大学, 2016, 博士論文

Version Type VoR
URL

https://doi.org/10.18910/56164

rights
Note

Osaka University Knowledge Archive : OUKA
https://ir.library.osaka-u.ac.jp/
Osaka University

Fluvoxamine reverses estrogen-dependent decline in voluntary activities
and decreased amygdala levels of serotonin in ovariectomized rats

（フルボキサミンは、卵巣摘出ラットのエストロゲン消失による自発運動量の抑制と扁桃体セロトニン遊離量の減少を回復する）

大阪大学大学院
大阪大学・金沢大学・浜松医科大学・千葉大学・福井大学
連合小児発達学研究科

石橋 雪子

２０１６年２月 博士学位論文

Research and reports

Fluvoxamine reverses estrogen-dependent decline in voluntary activities
and decreased amygdala levels of serotonin in ovariectomized rats

Yukiko Ishibashia,b,†, Nobuo Izumoc,†, Keiko Iwatab,d, Tomomi Morikawae, Toshiki
Kameyamaf, Yasuo Watanabec, Takayuki Manabee,f,g* and Hideo Matsuzakib,d*a

Department of Biochemistry, Yokohama University of Pharmacy, 601

Matano-cho, Totsuka-ku, Yokohama, Kanagawa 246-0066, Japanb

Development of Functional Brain Activities, United Graduate School of Child

Development, Osaka University, Kanazawa University, Hamamatsu University
School of Medicine, Chiba University and University of Fukui, Fukui, Japanc

Genaral Health Medical Center, Yokohama University of Pharmacy, 601

Matano-cho, Totsuka-ku, Yokohama, Kanagawa 246-0066, Japand

Research Center for Child Mental Development, University of Fukui, Fukui,

Japan
e

Laboratory for Neuroanatomy and Neuropharmacology, Research Field of

Professional Basic Medicine, Department of Nursing, Faculty of Nursing,
Chukyogakuin University, 2216 Toki-cho, Mizunami, Gifu 509-6192, Japanf

Division of Gene Expression Mechanism, Institute for Comprehensive Medical

Science, Fujita Health University, 1-98 Dengakugakubo, Kutsukake-cho,
Toyoake, Aichi 470-1192, Japang

Department of Child Development and Molecular Brain Science (Osaka

University), United Graduate School of Child Development, Osaka University,
Kanazawa University, Hamamatsu University School of Medicine, Chiba
University, University of Fukui, 2-2 Yamadaoka, Suita, Osaka, Japan.
†

Contributed equally to this work.

*Corresponding authors:
Name: Hideo MatsuzakiYear of birth: 19
Highest academic degree earned: MD, PhD
Professional titles: Professor
Research direction: Psychiatry
Tel/fax: +81-776-61-8803/
e-mail address matsuzah@u-fukui.ac.jp

Name: Takayuki Manabe
Year of birth: 1974
Highest academic degree earned: PhD
Professional titles: Professor
Research direction: NeurochemistryTel/fax: +81-572-68-4598/+81-572-68-68-4568
e-mail address:

t-manabe@chukyogakuin-u.ac.jp

Abstract
Studies suggest that increased expression of brain-derived neurotrophic factor(BDNF) could mediate the antidepressant effects of drugs. We analyzed theeffects of fluvoxamine on locomotor activities, serotonin levels in the amygdala,and hippocampal expression of BDNF mRNA in ovariectomized (OVX) rats.
Female Wistar rats (8 weeks, 180–200 g) were divided into four groups: sham;
OVX; OVX with estrogen; and OVX with fluvoxamine. Six weeks after
ovariectomy, rats were assessed according to spontaneous locomotor activity,
forced-swimming test (FST), and microdialyses experiments. Body and uterine
weight of OVX rats 6 weeks after surgery were significantly increased anddecreased, respectively, compared with those of the sham group, but thesechanges were returned to sham-group levels upon chronic administration of
estrogen and fluvoxamine. More potent decreases in voluntary activities wereobserved in OVX rats compared with rats in the sham group, but were increased
markedly upon administration of estrogen and fluvoxamine. In the FST,
immobility time and beat counts were increased and decreased significantly by
ovariectomy compared with those of the sham group, respectively, but estrogenand fluvoxamine treatment reversed these changes significantly. More potent

decreases in serotonin release in the amygdala were observed in OVX rats
compared with those of sham rats, but were reversed upon estrogen
replacement. Similar recovery was observed in OVX rats upon fluvoxamine
treatment. These data suggest that, in OVX rats, chronic administration of
fluvoxamine can recover estrogen-dependent changes in behaviors, decreased
serotonin release in the amygdala, and reduced expression of BDNF mRNA.
(245 words)
Keywords: Amygdala, Serotonin, Estrogen, Fluvoxamine, OVX

Abbreviations

BDNF, brain-derived neurotrophic factor; ER, endoplasmic reticulum; OVX,
ovariectmized; RT-PCR, reverse transcription-polymerase chain reaction; SSRI,
selective serotonin reuptake inhibitor; XBP-1, X-box binding protein-1; mRNA,
messenger ribonucleic acid

1. Introduction
The female hormone estrogen has an important role in bone physiology(reviewed in [15, 16]). Moreover, there is a close relationship between estrogenand memory, learning, and emotion in the brain (reviewed in [16]). Also, it has
been reported that replenishment with estrogen can improve recognition,
learning and memory (reviewed in [3, 5, 14]).

We reported that, in female rats 6 weeks after their ovaries had been removed,
depression-like symptoms (as manifested by decreases in spontaneous
locomotor activities and serotonin levels in the amygdala) could be observed [9].

Depression is a complex disorder brought about by genetic and environmental
conditions. Depression involves brain abnormalities as well as dysfunction of theendocrine system, inflammation, altered glucose metabolism and, in some cases,
coronary artery disease [8, 18]. Selective serotonin reuptake inhibitors (SSRIs)
are first-line treatment for depression and depression-like symptoms. However,resistance to the effects of antidepressants has been documented, so
development of new agents with new mechanisms of action is needed [13].

Studies have suggested that brain-derived neurotrophic factor (BDNF) signalingis necessary and sufficient for the action of antidepressant drugs [20]. BDNF
signaling is associated with cyclic adenosine monophosphate responsive
element binding protein, which induces neurogenesis [20]. Moreover, high levelsof BDNF have been observed in post mortem hippocampal samples fromindividuals suffering from depression [20]. Such findings suggest a correlation
between decreased expression of BDNF and the onset of depression.
Furthermore, a recent report demonstrated that the SSRI fluvoxamine reversed
the reduced expression of BDNF messenger ribonucleic acid (mRNA) by chronic
infusion of dexamethasone in mice exhibiting depression-like behaviors [17].
Therefore, the therapeutic effects of antidepressants could be mediated byincreased expression of BDNF.

In the present study, we analyzed the chronic (6-week) effects of theantidepressant fluvoxamine on locomotor activities, serotonin levels in the
amygdala,and expression ofovariectomized (OVX) rats.

BDNF

mRNA in the hippocampusin2. Materials and methods
2.1 Ethical approval of the study protocol
The study protocol was approved by the Ethics Committee of Yokohama Collegeof Pharmacy (Kanagawa, Japan). All experiments using animals were carriedout based on Guidelines for the Care and Use of Laboratory Animals (National
Institutes of Health, Bethesda, MD, USA) as approved by the JapanesePharmacological Society.

2.2 Animals, ovariectomy, and drug administration
Animal care and ovariectomy were as described previously with minor
modification [9]. Briefly, 36 female Wistar rats (8 weeks, 180–200 g) weredivided into four groups of nine. Rats were allowed to acclimatize for ≥1 week totheir surroundings before the start of experimentation (12-h light–dark cycle;
lights on at 7 am and off at 7 pm). After 1 week, OVX and sham operations were
undertaken as described previously (Fukushima et al., 2000). One week after
ovariectomy, fluvoxamine (50 mg/kg body weight; Wako Pure Chemical
Industries, Osaka, Japan) and β-estradiol (50 μg/kg body weight; Sigma–Aldrich,

Saint Louis, MO, USA) were administered (p.o. and i.p., respectively) five times
per week for 6 weeks.

2.3 Voluntary momentum, forced-swim test (FST), and microdialyses
Six weeks after ovariectomy, rats were assessed according to spontaneous
locomotor activity, FST, and microdialyses experiments, as described previously[9, 17].

2.4 Quantitative reverse transcription-polymerase chain reaction (RT-PCR)
RT-PCR was carried out as described previously [17].

2.5 Statistical analyses
Statistical analyses were undertaken as described previously [17].

3. Results
Body weight and uterine weight of OVX rats 6 weeks after surgery wereincreased and decreased significantly, respectively, compared with that of thesham group (Fig. 1a and b; sham and OVX groups). The increased body weightand decreased uterine weight of OVX rats returned to sham-group levels uponchronic administration of estrogen (Fig. 1a and b; sham and estrogen groups). A
tendency for recovery was observed upon chronic administration of fluvoxamine
in terms of increases and decreases of body weight and uterine weight, but thedifferences were not significant (Fig 1a and b; sham and fluvoxamine groups).

Figure 2a shows spontaneous locomotor activity for 12 h in the dark phase (7 pm
to 7 am) for the four groups 6 weeks after ovariectomy. More potent decreases involuntary activities were observed in OVX rats compared with rats in the shamgroup (Fig. 2b). These data were similar to those of our previous report [9].
These decreases in locomotor activities in OVX rats in the dark phase wereincreased markedly upon administration of estrogen and fluvoxamine (Fig. 2b).

Next, we analyzed the immobility time and beat counts on FST of the four rat

groups (Fig. 3). Immobility time and beat counts were increased and decreased
significantly by ovariectomy compared with those of the sham group,
respectively (Fig. 3a and b; sham and OVX groups). These data were consistent
with our previous report [9]. Treatment with estrogen and fluvoxamine reversed
these changes significantly to levels seen in control rats (Figs. 3a and b).

Next, we analyzed serotonin levels in the amygdala under identical conditions(Fig. 4). More potent decreases in serotonin release in the amygdala wereobserved in OVX rats compared with those of sham rats (Fig. 4a). Theseobservations were consistent with our previous report [9]. These decreases inserotonin levels in the amygdala were reversed upon estrogen replacement (Fig.
4a; OVX and estrogen), suggesting that decreases in serotonin level by
ovariectomy were dependent upon the disappearance of estrogen. A similar
recovery was observed in OVX rats upon fluvoxamine treatment (Fig. 4a; OVX
and fluvoxamine). However, this recovery in serotonin release was not detectedin the case of dopamine release in the amygdala (Fig. 4b). This result was
expected because SSRIs have a mechanism of action specific for serotonin.

Next, we analyzed hippocampal BDNF expression of sham rats and OVX rats
using RT-PCR (Fig. 5). No significant changes were observed in samples fromany group obtained from the cerebral cortex or brainstem (Fig. 5; middle andlower panels). More potent decreases in hippocampal BDNF expression wereobserved in the OVX group compared with that in sham groups (Fig. 5; upper
panel). This elevation of BDNF expression was recovered markedly upon
estrogen treatment (Fig. 5; upper panel). Hippocampal BDNF expression in OVX
rats was recovered significantly upon fluvoxamine treatment (Fig. 5; upper
panel). Hippocampal expression of the estrogen receptor-1 gene was notchanged in all groups (data not shown).

4. Discussion
The present study showed that the body weight and uterine weight of OVX rats 6
weeks after ovariectomy were increased and decreased significantly comparedwith that of the sham group, respectively (Fig. 1), and that this phenomenon wasconsistent with previous reports [1,4,9]. These results suggest that we carriedout ovariectomy successfully. Such increases in body weight and decreases inuterine weight in OVX rats were returned to sham-group levels upon chronic
administration of estrogen (Fig. 1), suggesting that the increased weight of OVX
rats was significantly dependent upon disappearance of estrogen. Moreover, thistendency for recovery in increases and decreases in body weight and uterine
weight was also observed upon chronic administration of fluvoxamine exceptthat the differences were not significant (Fig. 1).

The present study demonstrated that BDNF expression was decreased
significantly in the hippocampus, but not the cerebral cortex, of OVX rats, andthese effects were dependent upon estrogen (Fig. 5). Marked recovery ofdecreases in hippocampal BDNF expression was observed in OVX rats upon
fluvoxamine administration, but not in the cerebral cortex. Hippocampal BDNF

and its neurogenic effects are closely related to depression and depression-likebehaviors in animals. Therefore, it is strongly suggested that increased
expression of BDNF causes replacement of depression-like symptoms in OVX
rats. Similar results have been observed in the hippocampi of OVX rats
undergoing exercise [10] and in the whole brains of depressed mice (induced bychronic infusion of dexamethasone) treated with fluvoxamine [17]. Our data
support reports suggesting that the hippocampus is an important component of
the limbic system, is closely related to depression, and a putative area for
estrogens [11,19].

The affinity of fluvoxamine to the sigma-1 receptor (which binds to fluvoxamineand has neuroprotective activities) as a “molecular chaperone” under
endoplasmic reticulum (ER) stress is higher than that observed for otherantidepressants [12]. Unfolded protein responses and ER stress responses arealso associated with depression. These responses are activated and required forneurite outgrowth by BDNF signaling due to increases in expression of X-box
binding protein-1 (XBP-1) [6,7]. However, the depression-like behavior due toovariectomy and recovery upon fluvoxamine administration shown in the present

study may be dependent upon another BDNF signal because the sigma-1
receptor and ER stress-related gene XBP1, in addition to the BDNF signal, havebeen linked to depression-like behavior due to dexamethasone infusion [17].

Our previous study suggested that OVX rats could become models ofdepression because ovariectomy elicits depression-like symptoms, such as adecline in spontaneous movement and decreases in serotonin release in the
amygdala [9]. In the present study, in OVX rats, chronic administration of
fluvoxamine recovered: estrogen-dependent changes in behaviors (Fig. 2 and3); decreased serotonin release in the amygdala (Fig. 4); reduced expression of
BDNF mRNA (Fig. 5). We are the first research team to show such data, though
our experimental setup may be different to that of other scholars [2].
Nevertheless, the observations in the present study strongly support those in ourprevious work [9].

Acknowledgements
T. Manabe and T. Morikawa were supported by Grants-in-Aid for young scientists(B) and for scientific research (C) from the Ministry of Education, Culture, Sports,

Science and Technology of Japan. T. Manabe was supported by a grant for thepromotion of research from the Kanzawa Medical Research Foundation.

K.

Iwata was supported by Grants-in-Aid for scientific research (C) from the Ministryof Education, Culture, Sports, Science and Technology of Japan.

Conflicts of interest
The authors have no conflicts of interest to declare
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Figure legends

Fig. 1. Body weight (a) and uterine weight (b) of rats in sham, OVX, OVX withestrogen, and OVX with fluvoxamine groups. **P<0.01.

Fig. 2. Spontaneous locomotor activities of rats in sham, OVX, OVX withestrogen, and OVX with fluvoxamine groups 6 weeks after ovariectomy. Data are
the number of counts (a) and summed counts over 12 h in the dark phase (7 pm
to 7 am) (b). *P<0.05, **P<0.01.

Fig. 3. Forced-swimming tests in rats of the sham, OVX, OVX with estrogen, andOVX with fluvoxamine groups 6 weeks after ovariectomy. Data are the immobility
time (a) and summed counts of the number of beats (b). *P<0.05, **P<0.01.

Fig. 4. Release of serotonin and dopamine in the amygdala of rats in sham, OVX,OVX with estrogen, and OVX with fluvoxamine groups 6 weeks after
ovariectomy. Data are the summed amounts (pg) of serotonin (a) and dopamine(b) over 12 h in the dark phase (7 pm to 7 am). **P<0.01.

Fig. 5. Quantitative RT-PCR of BDNF mRNA in the hippocampus (upper panel),
cerebral cortex (middle panel) and brainstem (lower panel) of rats in sham, OVX,OVX with estrogen, and OVX with fluvoxamine groups. **P<0.01.