Volume 70 · Supplement 1 · 2020

Volume 70 · Supplement 1 · 2020
The 97th Annual Meeting of the Physiological Society of Japan
Plenary Lectures 
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Plenary Lectures

Special Lectures 
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The Sunao Tawara Memorial Lecture
The Makoto Arita Memorial Lecture
The Susumu Hagiwara Memorial Lecture

Planned Symposia 
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Japan-Canada Joint Symposium Cutting-edge approaches to the functioning mechanisms and pathophysiology of ion channels
Neuro-immune communication and homeostatic responses
Integration of sensory and metabolic signals by neuropeptide in hypothalamus (navigates metabolic state.)
The metabolic modulation by sensory input from peripheral organs
Brain function for physical therapy and its physiological mechanisms
Recent Advancement of the Multidisciplinary Approaches to the Pathophysiology Underlying Arrhythmia
The emerging roles of pallidal nuclei in the basal ganglia circuitry
Systems biology of hearing: from the inner ear to the brain
Scientific basis of the oriental medicine: mechanisms improving muscle blood flow at rest
Japan-China Joint Symposium Cutting edge of cell fate determination and migration in the developing cerebral cortex
Women scientists in the optogenetic field – the new fact has come to light using optical approaches
New frontiers of locomotion research using zebrafish as a model system
Combined approaches of cutting-edge techniques and structural information to reveal the elaborate dynamics of membrane proteins
Past, present and future of JPS
Leading-edge approach for regulated exocytosis in neural system
New Insights into Vascular Aging Mechanisms and Therapeutic Targets
Cardiac mechano-physiology: physiology and pathophysiology of the mechano-electrical coupling
Inter-organ communication: molecular mechanism and pathophysiology induced by its disruption
Progress and perspectives of neuroscience with data-driven intelligence
Neural Mechanism of Psychological Stress: Molecules, Circuits and Disorders
The ethics, laws, and guidelines for human and animal researches
Challenge for the unsolved mechanism of arrhythmias; from the view of physiologists and cardiologists
Angiology evolving into new research fields
What is ‘data-driven’ science
Mechanisms of experience- or metabolism-dependent behavioral changes
New insights of sensory and brain functions affecting feeding behaviors: circadian, swallowing, taste and pain
Pathophysiology of ion channels and transporters in tumor growth
Basic and applied researches to progress the knowledge and therapy for heat stroke
Recent advances in muscle physiology

Symposia 
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Deeper insights into smooth muscle physiology using natural products
Warm-blooded cool animals: hibernation and torpor physiology
Frontiers of biophotonics in physiology: Ion environments and molecule behaviors in the nano-space detected by Raman spectrum
Neural mechanisms unveiled by combined in vitro and in silico approaches
Mitochondrial function and its roles in cellular physiology/pathophysiology
Crosstalk in modifiable and non-modifiable risk factors for cardiovascular disease
Molecular and Neural basis for appetite and food preference
Physiological importance of environmental temperatures in ectotherms
Reverse engineering the brain functions for the control of adaptive behaviors
A new world of physiology developed by peptide hormones
AMED support for medical R&D: From basics to practiceControl of endogenous regulatory responses in glial cells in neuroinflammation - From embryonic development to pathogenesis of brain diseases
New strategies for understanding functions and mechanisms of ion channels/transporters/pumps in the upcoming post-structure era
The new sunrise of zinc physiology and zinc pathophysiology
Aging-related changes in physiological functions induced by the space environmentState-of-the-art physiology in urinary continence
Current advance in endocrine disruptor research
History and Up-to-date stories on the mechanism of smooth muscle contraction in health and disease

Educational Programs 
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Educational Program

Continued insede back cover

Volume 70 · Supplement 1 · 2020 · pp 1–000

Memorial Lectures Proceedings of the 97th Annual Meeting of the Physiological Society of Japan
March 17–19, 2020, Beppu, Oita, Japan

Continued from back cover

Lunchtime Sessions 
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Luncheon Seminars 
S61Award Presentations ( Formerly The Japanese Journal of Physiology )Official Journal of The Physiological Society of Japan

Aims and Scope:
The Journal of Physiological Sciences
publishes peer-reviewed original papers,
reviews, short communications, technicalnotes, and letters to the editor, based on
the principles and theories of modern
physiology and addressed to theinternational scientific community.
All fields of physiology are covered,encompassing molecular, cellular andsystems physiology. The emphasis is on
human and vertebrate physiology, but
comparative papers are also considered.
The process of obtaining results must be
ethically sound.
Fields covered:·	Adaptation and environment·	Autonomic nervous function·	Biophysics·	Cell sensors and signaling·	Central nervous system and brain
sciences·	Endocrinology and metabolism·	Excitable membranes and neural cell
physiology·	Exercise physiology·	Gastrointestinal and kidney physiology·	Heart and circulatory physiology·	Molecular and cellular physiology·	Muscle physiology·	Physiome/systems biology·	Respiration physiology·	Senses

Luncheon Seminar

Editorial Board of The Journal of Physiological Sciences
Chief Editor

Associate Editors

Harumi Hotta, Tokyo
Hironobu Morita, Gifu
Makoto Tominaga, Okazaki

Review Editor

Yasunobu Okada, Okazaki
Yasuo Sakuma, Tokyo
Satomi Adachi-Akahane, Tokyo
Wataru Aoi, Kyoto
Hsiao Chang Chan, Hong Kong
Ying-Shing Chan, Hong Kong
Douglas C. Eaton, Atlanta
Yong-Jian Geng, Houston
Katsumasa Goto, Toyohashi
Yukiko Hayashi, Tokyo
Akihiro Hazama, Fukushima
Masaki Kameyama, Kagoshima
Masanobu Kano, Tokyo
Katsumasa Kawahara, Sagamihara
Toru Kawata, Suita
Kyungjin Kim, Seoul
Yoshihiro Kubo, Okazaki
Manabu Kubokawa, Morioka
Yoshihisa Kurachi, Suita
Tomoyuki Kuwaki, Kagoshima
Weimin Li, Shanghai
Yoshinori Marunaka, Kyoto
Satoshi Matsuoka, Kyoto
Kenju Miki, Nara
Yasuhiko Minokoshi, Okazaki
Shohei Mitani, Tokyo
Kei Nagashima, Tokorozawa
Mitsuyuki Nakao, Sendai
Yasuhiro Nishida, Tokorozawa
Kazunori Nosaka, Joondalup

Hiroshi Nose, Matsumoto
Shigehiko Ogoh, Kawagoe
Yasushi Okamura, Suita
Satoshi Okumura, Yokohama
Yoshitaka Oku, Nishinomiya
Brian Oldfield, Clayton
Lawrence G. Palmer, New York
Mathieu Piché, Trois-Rivières
Eduardo Rios, Chicago
Hideki Sakai, Toyama
Ichiro Sakuma, Tokyo
Motohiko Sato, NagakuteGeert Schmid-Schönbein, San Diego
Manabu Shibasaki, Nara
Yasutake Shimizu, Gifu
Masahiro Sokabe, Nagoya
Hideaki Soya, TsukubaMark Stewart, Brooklyn
Eiji Takahashi, Saga
Miyako Takaki, Kashihara
Makoto Takano, Kurume
Shigeru Takemori, Tokyo
Akiyuki Taruno, Kyoto
Yoichi Ueta, Kitakyushu
Marcel A.G. van der Heyden, Utrecht
Paul A. Welling, Baltimore
Kazuhiro Yagita, Kyoto
Jianqun Yan, Xi’an

Organizers of the 97th Annual Meeting in Beppu
Katsushige Ono, Yufu
Reiko Hanada, Yufu
Osamu Tokumaru, Oita
Tatsuki Kurokawa, Yufu
Shinichiro Kume, Yufu

21st Promotion Award of the Physiological Society of Japan for Young Scientists
10th Hiroshi and Aya Irisawa Memorial Promotion Award for Young Physiologists
10th Aya Irisawa Memorial Promotion Award for Excellence by Women Physiologists
10th Hiroshi and Aya Irisawa Memorial Award for Excellent Papers in The Journal of Physiological Sciences
10th Hiroshi and Aya Irisawa Memorial Award for Excellent Papers on Research in Circulation in The Journal of Physiological Sciences

Oral Presentations 

Yoshihiro Ishikawa, Yokohama

Editors

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Ryoko Higa, Yufu
Kenshiro Shikano, Yufu
Miki Watanabe, Yufu
Natsuki Matsuno, Yufu

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Kidney・Urination・Digestion・Absorption
Environmental Physiology
Heart・Circulation 1
Neuron・Synapse
Behavior Science・Biorhythm・Neurochemistry
Oral Physiology・Motor Function
Cell Physiology・Molecular Physiology 1
Pathophysiology
Ion Channel・Receptor 1
Autonomic Nervous・Endocrinology
Ion Channel・Receptor 2
Sensory Function
Muscle Physiology
OthersCNS Function
Nutrition・Metabolism・Thermoregulation
Heart・Circulation 2
Cell Physiology・Molecular Physiology 2
Development・Growth・Aging
Heart・Circulation 3
Heart・Circulation 4

Poster Presentations 
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Ion Channel・Receptor (1)Heart・Circulation (1)Neuron・Synapse (1)Sensory Function (1)Behavior Science・Biorhythm (1)Neurochemistry (1)Autonomic Nervous (1)Muscle Physiology (1)Oral Physiology (1)Endocrinology (1)Kidney・Urination (1)Motor Function (1)Development・Growth・Aging (1)Cell Physiology・Molecular Physiology (1)Environmental Physiology (1)Drug Actions (1)CNS Function (1)Nutrition・Metabolism・Thermoregulation (1)Pathophysiology (1)Physical Fitness・Sports Medicine (1)Respiration (1)Study Methodology (1)Others (1)Ion Channel・Receptor (2)Heart・Circulation (2)Neuron・Synapse (2)Sensory Function (2)Behavior Science・Biorhythm (2)Neurochemistry (2)Autonomic Nervous (2)Muscle Physiology (2)Oral Physiology (2)Endocrinology (2)Kidney・Urination (2)Motor Function (2)Reproduction
Development・Growth・Aging (2)

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Digestion・Absorption
Cell Physiology・Molecular Physiology (2)Environmental Physiology (2)Drug Actions (2)CNS Function (2)Nutrition・Metabolism・Thermoregulation (2)Pathophysiology (2)Physical Fitness・Sports Medicine (2)Blood
Respiration (2)Study Methodology (2)Others (2)Ion Channel・Receptor (3)Heart・Circulation (3)Neuron・Synapse (3)Sensory Function (3)Behavior Science・Biorhythm (3)Neurochemistry (3)Autonomic Nervous (3)Muscle Physiology (3)Oral Physiology (3)Endocrinology (3)Kidney・Urination (3)Motor Function (3)Development・Growth・Aging (3)Cell Physiology・Molecular Physiology (3)Environmental Physiology (3)Drug Actions (3)Membrane Transport
CNS Function (3)Nutrition・Metabolism・Thermoregulation (3)Pathophysiology (3)Physical Fitness・Sports Medicine (3)Respiration (3)Study Methodology (3)Others (3)Plenary Lectures
Special Lectures
Memorial Lectures

Plenary Lecture 1

Plenary Lecture 2（March 17, 11:00〜12:00, Hall 1）（March 18, 11:00〜12:00, Hall 1）

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Serendipities of acquired immunity

From haploid stem cells to blood vessel engineering

Tasuku Honjo（Kyoto University）

Josef M Penninger（ the scientific director of the Institute of Molecular

Acquired immunity emerged sometime at the beginning of vertebrate
evolution as defense mechanism against the infectious diseases causedby pathogens. During this evolution the amazing mechanism including
reassembly of genetic segments was acquired probably by fortuitous
events. We encountered PD-1 in 1992 and found PD-1 is the major
braking system in acquired immunity. In 2002, we discovered PD-1
blockade can cure tumors in mouse models by reactivating the acquired immunity. In 2014, 22 years after PD-1 discovery, the treatmentof cancer by PD-1 blockade is approved by PMDA and FDA, and considered to be revolutionary cancer treatment which is often comparedto the penicillin discovery for the treatment of infectious diseases. The
success of cancer treatment by PD-1 blockade owes to acquired immunity. During the long history of fight against diseases, we almost succeeded to conquer infectious diseases in the last century. Fortunately,
we may be able to overcome cancer the last major life-threatening
disease, by the same mechanism applied to infectious diseases.

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Biotechnology, the head of the Life Sciences Institute of the University of
British Columbia in Vancouver）

We have previously generated murine stem cells with a single set ofchromosomes, termed haploid ES cells. Using such cells we have beenable to rapidly mine essential biological pathway and to use revertible
mutagenesis to identify novel mediators of angiogenesis. Recently wehave expanded our work to engineer human blood vessel organoids
that can be transplanted into mice to establish a fully human vasculartree. We have used this system to model the pathogenesis of diabetes
vasculopathies.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Special Lecture 1

Special Lecture 2（March 17, 15:20〜16:20, Hall 1）（March 18, 13:20〜14:20, Hall 1）

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Osteoimmunology and autoimmunity

Multilayer ionic mechanisms revealed by mathematicalmodeling of iPS derived cardiac myocyte spontaneous
action potentials

Hiroshi Takayanagi（Department of Immunology, Graduate School of
Medicine and Faculty of Medicine, The University of Tokyo）

Bone cells and immune cells share the same microenvironments in thebone marrow, communicating through various cytokines. Osteoblasts,
osteoclasts and osteocytes are not only degrading or forming bone but
have distinct roles in the immune regulation. Thus, much attention has
been paid to the interdisciplinary filed, osteoimmunology, studying theinteraction and shared molecules between bone and immune systems(Physiol Rev. 97:1295-1349, 2017). Here I summarize the recent advancein osteoimmunology and its relevance in the studies on autoimmune
diseases such as rheumatoid arthritis.
Self-tolerance is primarily established by negative selection of selfreactive T cells in the thymus. We found that the transcription factor
Fezf2 plays a critical role in central tolerance by directly regulating
tissue-restricted antigen expression in mTECs independently of Aire
(Cell 163, 975-87, 2015). We have made efforts to understand how Fezf2
and Aire regulate different self-antigens and explored the role of othercell types than mTECs in central tolerance. I will talk about the recent
progress in understanding the mechanism of T cell tolerance in thethymus.

Akinori Noma（BKC Research Organization, Ritsumeikan University）
Undifferentiated myocardial cells (hiPSC-CM) derived from Human induced pluripotent stem cell mostly shows a wide variety of spontaneous
activities. The difference in the configuration and frequency of spontaneous action potential (AP) may be due to variable expression levels of
the ion channels, and thereby the hiPSC-CM may embody mechanismsof various slow diastolic depolarization (SDD), which have been suggested in differentiated cardiac myocytes. To clarify mechanisms of
SDD, we first created ventricular- (V-), atrial- (A-), and nodal- (N-) type
hiPSC-CM mathematical models based on the human ventricular cell
model (Himeno et al., 2015). Then, specific role of each ion channel ingenerating SDD was examined by changing the relative amplitude of
IKr (KV11.1), IK1 (Kir2.1), ICaL (Cav1.2 + Ist, Cav1.3), hyperpolarization-activated current, Iha (HCN4), and IbNSC to determine the rangeof combinations that allow generation of spontaneous action potential.
The lead potential analysis of these action potential well quantified thecontribution of these currents to spontaneous action potentials, and we
could identify two basic mechanisms and two additional mechanisms.
The primary mechanism 1 is the removal of inactivation of IKr on
repolarization (y1 gate) and subsequent deactivation (y2 + y3 gates) during 100 to 200 ms of SDD. The primary mechanism 2 is caused by positive feedback process among inward currents i.e. ICaL and Ist (Cav1.3),
ICaT (Cav 3.1), IN, (Nav1.5), and current of Na+/Ca2+ exchanger, INCX
(NCX1). These primary mechanisms induce the typical sinus node low
membrane potential oscillation. High membrane potential oscillation is
induced by adding the secondary two channel mechanisms; activationand subsequent deactivation of Iha, and unblocking and re-blocking of
IK1 by intracellular factors. The lead potential analysis quantitatively
demonstrated the time-dependent changes in weight of these fourmechanisms along the time course of SDD.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

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Special Lecture 3

Special Lecture 4（March 18, 13:20〜14:20, Hall 2）（March 19, 11:00〜12:00, Hall 1）

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Role of the habenula in addiction and addiction-relateddiseaseSpying on dopamine modulation by developing nextgeneration GRAB sensors

Paul J. Kenny（Ward-Coleman Professor and Chair, Nash Family Department

Yulong Li（School of Life Sciences, PKU-IDG/McGovern Institute for Brainof Neuroscience, Icahn School of Medicine at Mount Sinai, New York.）

The medial habenula (mHb) contains some of the densest concentrations of nicotinic acetylcholine receptors (nAChRs) in the brain and has
emerged as an important region that regulates nicotine consumption.
We have explored the role of the mHb in regulating the motivational
properties of nicotine and tobacco-related disease and also the rolefor septal inputs to the mHb in these processes. We found that thetranscription factor Tcf7l2 is highly enriched in mHb and, using a newline of Tcf7l2 mutant rats, that Tcf7l2 deficiency increases nicotine
self-administration behavior. CRISPR-mediated cleavage of Tcf7l2 inthe mHb similarly increased nicotine self-administration in mice. Using whole-cell electrophysiological recordings and RNA sequencing,
we found that Tcf7l2 regulates the recovery of nicotinic acetylcholine
receptors in the mHb from nicotine-induced desensitization through amechanism involving local cAMP signaling. We also found that dosesof nicotine that stimulate the mHb increased blood glucose levels in rodents, and that repeated exposure to this effect precipitated diabeteslike abnormalities in blood glucose homeostasis. Tcf7l2-deficient rats
were resistant to the diabetes-related actions of nicotine. Relapse rates
are remarkably high in tobacco smokers attempting to quit, particularly during early stages of withdrawal when craving is most intense.
The role of the mHb in nicotine craving and relapse-related behaviors is unknown. We found that neurons in the triangular nucleus of
the septum (TNS) provide excitatory input to the mHb and that nicotine profoundly decreased activity of the TNS-mHb circuit. Doses of
nicotine that disrupt TNS-habenula communication triggered intense
craving-like nicotine-seeking during withdrawal. Chemogenetic stimulation of the TNS-mHb circuit attenuated, whereas inhibition of thiscircuit enhanced, withdrawal-induced craving. These findings suggestthat stimulatory actions of on a mHb-pancreas axis links the addictive
properties of nicotine to its diabetes-prompting actions, and that perturbations in septal communication with the mHb contributes nicotine
craving and relapse.

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Research, PKU-THU Center for Life Sciences, Peking University, Beijing,
P.R.China）

Dopamine (DA) is a central monoamine neurotransmitter involved inmany physiological and pathological processes. A longstanding yetlargely unmet goal is to measure DA changes reliably and specificallywith high spatiotemporal precision, particularly in animals executing
complex behaviors. We very recently reported the development of genetically encoded GPCR-activation-based-DA (GRAB-DA) sensors thatenable these measurements. In response to extracellular DA, GRABDA sensors exhibit large fluorescence increases with subcellular resolution, subsecond kinetics, nanomolar to submicromolar affinities, andexcellent molecular specificity. GRAB-DA sensors can resolve a singleelectrical-stimulus-evoked DA release in mouse brain slices and detect
endogenous DA release in living flies, fish, and mice. In freely behaving
mice, GRAB-DA sensors readily report optogenetically elicited nigrostriatal DA release and depict dynamic mesoaccumbens DA signaling
during Pavlovian conditioning or during sexual behaviors. By tuning
the residues in GFP and GPCRs, we now have developed a second
generation of green GRAB-DA sensors, with over 2-4 fold larger fluorescence responses, 2-5 fold maximum brightness, up to 10-100 foldhigher molecular selectivity (over norepinephrine), more affinity ranges
and distinct pharmacological properties. In parallel, we also generated
promising red GRAB-DA sensor candidates with similar affinity as
green ones. The red sensors are capable of reporting DA dynamics invivo in both flies and in rodents. The new generation dopamine sensors along with other GRAB sensors provide powerful tools to unravel
the in-vivo dynamics of critical neuromodulators for diverse model
systems in physiological and pathophysiological conditions.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Special Lecture 5

Special Lecture 6（March 19, 11:00〜12:00, Hall 2）（March 19, 11:00〜12:00, Hall 3）

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Calcium signaling: Imaging and functional analyses

Recovery from Ischemic Brain Injury by Innate andAdaptive Immunity

Masamitsu Iino（Division of Cellular and Molecular Pharmacology, Nihon
University School of Medicine）
Intracellular Ca2+ signals regulate numerous physiological and pathophysiological functions throughout our body. The advent of fluorescentCa2+ indicators has allowed us to visualize dynamic spatiotemporalchanges in the intracellular Ca2+ concentration. Being intrigued by thespatiotemporal Ca2+ dynamics, we looked into the basic mechanisms
underlying those Ca2+ dynamics, and clarified that regenerative Ca2+
release mechanisms underlie such Ca2+ signals as Ca2+ waves andoscillations. Furthermore, Ca2+ oscillations are found to be an efficientsignaling mechanism minimizing the load to the cells. Based on our molecular understanding of the Ca2+ signaling mechanisms, we have been
searching for new cellular functions that are regulated by Ca2+ signals,
developing and utilizing powerful imaging methods both in vitro andin vivo. Our efforts have identified critical roles of Ca2+ signaling in thecentral nervous system in reaction to insult to the brain. Our researchstrategy can be applied to various organs other than the brain.

Minako Ito, Akihiko Yoshimura（Department of Microbiology and

Immunology, Keio University School of Medicine, Tokyo, Japan）

Stroke including brain ischemia is one of the major causes of deathand disability worldwide. Post-ischemic inflammation is an essential
step not only for the progression of ischemic brain injury, but also forneurological repair. In a mouse stroke model, we have reported that
IL-1β, IL-23 and IL-17 play essential roles in infarct volume growthwithin 1-4 days after brain ischemic stroke model 1, 2). Extracellular
DAMPs (damage-associated molecular patterns) including Peroxiredoxins (Prxs) released from dead cells activate infiltrated macrophages
on day 1 through a Toll-like receptor 2 (TLR2) and TLR4 -dependent
mechanism, then stimulate IL-1β and IL-23 production 2, 3). Then these
inflammatory cytokines induce IL-17 from γδT cells, which exacerbates ischemic brain damage on day 3-4 1). After day 3, DAMPs arecleared by macrophages through type A scavenger receptors, which isan important process for the resolution of brain inflammation 4). Suchneuroprotective macrophages produce less inflammatory cytokines,
but higher amout of tropic factors like IGF-1. At the chronic phase
after a stroke (>14 days), a massive accumulation of T cells, especially
Tregs, occurs in the brain 5). Tregs regulates astroglyosis and reduce
neural damages. Gene expression analysis revealed that brain Tregs
related to Tregs in other tissues such as adipose tissue and muscle,
however, brain Tregs are apparently different from them and express
several unique genes related to the nerve system including serotoninreceptor Htr7. Brain Our findings suggest that macrophages andTregs, and their products may provide new therapeutic opportunities
for neuronal protection against stroke.
References
1) Shichita T, et al. Nature Med. 2009; 15(8): 946-950.
2) Ito M, et al. Nat Commun. 2015; 6: 7360.
3) Shichita T, et al. Nature Med. 2012; 18(6): 911-917.
4) Shichita T, et al. Nature Med. 2017; 23(6): 723-732.
5) Ito et al. Nature. 2019; 565(7738): 246-250.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

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The Sunao Tawara
Memorial Lecture

The Makoto Arita
Memorial Lecture（March 17, 9:30〜10:30, Hall 1）（March 18, 9:30〜10:30, Hall 1）

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ML2

Heart failure as cardiac maladaptation to mechanical stress

Current status of the clinical application of regenerative
medicine of the heart using iPS cells

Issei Komuro（Department of Cardiovascular Medicine, The University ofTokyo Graduate School of Medicine）
Pressure overload induces cardiac hypertrophy by activating many
molecules such as integrin and angiotensin II type1 receptor, and at
first, cardiac function is maintained normal by promoting vascular
growth in the heart through hypoxia-inducible factor-1 (Hif-1)-dependent induction of angiogenic factors. Sustained pressure overload, however, induces an accumulation of p53 that inhibits Hif-1 activity andthereby impairs cardiac angiogenesis and systolic function. We haverecently found that three miRNAs, which are targets of p53, are upregulated in blood of patients showing heart failure within 1 year after
myocardial infarction. Through network analysis of single-cardiomyocyte transcriptomes, we have recently identified gene-modules thatclassify cardiomyocytes and regulate a trajectory of cardiomyocyte remodeling. Upon pressure overload, almost all cardiomyocytes activatedmitochondrial ribosome/metabolism modules, whose activity was correlated with the extent of morphological hypertrophy. Sustained stimuli activated DNA damage signaling module including p53 targets in apart of hypertrophy-stage cardiomyocytes, which induced remodelinginto failing cardiomyocytes and resulted in heart failure. To know how
pressure overload induces expressions of p53, we examined whetherpressure overload induces DNA damage in cardiomyocytes. Comet assay revealed that pressure overload on murine hearts induced single
strand breaks in cardiomyocytes and enhanced accumulation of single
strand breaks in Xrcc1 deficient mice caused heart failure. We haverecently found that heart failure patients with severe DNA damage
do not respond to drug treatment and show poor prognosis. There aremany kinds of cell types in the heart such as cardiomyocytes, endothelial cells, smooth muscle cells and blood cells. We have recently foundthat pressure overload also induces an increase in resident macrophages and robust changes of gene expressions of macrophages in theheart. Depletion of the macrophages easily induced cardiac dysfunction
and we have recently identified the critical molecules and its molecularmechanisms.

S6

Keiichi Fukuda（ Department of Cardiology, Keio University School of Medicine）
Although heart transplantation can drastically improve the survival,
shortage of the donor heart is a serious problem. The regenerative
medicine of the failing heart had been long awaited. To address thisquestion, we used human HLA haplotype homo-iPS cells, which matches to approximately 20% of the Japanese population, to generate ventricular cardiomyocytes. We performed transcriptome of the metabolic
enzymes and fluxome analysis using 13glucose and 13lactic acid on
both ES/iPS cells and cardiomyocytes, and found that their metabolic
pathways were completely different. Moreover, amino acid consumption analysis and metabolome analysis revealed that glutamine is another important energy source for the iPS cells. Based on these findings, we could purify the cardiomyocytes with more than 99% purity.
The transplanted cardiomyocytes did not make teratoma formation in
immuno-deficient NOG mice skin and heart. We transplanted the aggregate (spheroid) cardiomyocytes using our newly developed device.
The transplanted cardiomyocytes could survive in the heart for thelong period, showed physiological cell hypertrophy after transplantation, and could improve cardiac function due to myocardial infarction.
We are now planning to examine the first in human clinical trial totransplant the human regenerated cardiomyocytes to the patients with
HLA-6 class matched dilated cardiomyopathy in the near future.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

The Susumu Hagiwara
Memorial Lecture（March 19, 9:30〜10:30, Hall 1）

ML3
Monoamine and mechanical control of dendritic-spine
synapses and psychiatric disorders
Haruo Kasai（Graduate School of Medicine, The University of Tokyo）
Most excitatory synapses in the cerebral cortex are formed on small
appendages of dendrites called dendritic spines. The spine synapses
exhibit structural plasticity in that glutamate and gamma-butyricacid competitively regulate confined spine enlargement and spreading shrinkage, respectively, resulting in synaptic competition to selectthe efficient synapses by cytosolic Ca2+ signaling. We recently demonstrated that dopamine acts on the dendritic spines in the nucleus
accumbens. We found that dopamine D1 receptors (D1Rs) sensitively
detect the burst of dopamine neuron firing via cytosolic cAMP signaling and play a key role in reward conditioning, with marked stimulus
generalization in awake mice. In contrast, D2Rs detect the short dipof dopamine firing and are involved in discrimination learning, but notin extinction. Interestingly, repeated methamphetamine treatment, ina model for psychosis, hindered the discrimination learning and a D2R
antagonist restored it by enhancement of the plasticity. These findings suggest a novel hypothesis that psychotic symptoms might arise
due to impairment in discrimination learning mediated by D2Rs, which
results in overgeneralization. In addition to the chemical signaling, wefound that the spine enlargement causes mechanical effects on thepresynaptic terminals to facilitate the evoked neurotransmitter releasein a presynaptic F-actin- and SNARE-dependent manner in hippocampal slice cultures. This novel mechano-sensing mechanism (actARE)
is independent of cytosolic Ca2+ and cAMP, and is likely caused byforce-mediated assembly of SNAREs. Thus, spine synapses are capableof “mechanical” transmission to facilitate the electro-chemical learning
processes in the brain.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

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Planned Symposia

Planned Symposium 1
Japan-Canada Joint Symposium
Cutting-edge approaches to the functioning
mechanisms and pathophysiology of ionchannels（March 17, 9:00〜10:50, Hall 2）

PS01-03

Stepwise activation of KCNQ1 and KCNQ1+KCNE1 channel
complexes
David Fedida, Maartje Westhoff, Jodene Eldstrom（Life Sciences Institute, University of
British Columbia, Vancouver, B.C., Canada）

The IKs potassium current is important in regulating the heartbeat as it activates during thelate plateau phase of cardiac systole, and provides a repolarization reserve at times of higher
heart rate to support increased cardiac output. The underlying KCNQ1 tetramers have four
voltage-sensitive activating domains (VSs), a pore that opens to allow current to pass through,and they co-assemble with 1-4 KCNE1 accessory subunits. However, how these components
together gate the IKs complex to open the pore is controversial. The number of activated VSs
required to allow pore opening has been debated extensively, and currently, either a concerted
movement involving all four tetramer subunits, or allosteric regulation of open probability
through sequential activation of individual VSs is thought to precede opening.
By using the locking E160R mutation in KCNQ1 to prevent VSs from moving into activatedconformations, and tracking VS movement, via MTSET modification and fluorescence recordings, we show that E160R-containing VS do not translocate to be detected externally upon
depolarition. E160R, expressed in all four KCNQ1 subunits, produces non-conducting channel
complexes, but if one, two, or three VS contain the E160R mutation, whole cell and single channel currents are still observed in both the presence and absence of KCNE1, and conductance isreduced proportionally with the number of E160R VSs. A model of independent VS movement,
incorporating intermediate and activated states directly coupled to open states can simulate
experimental changes in IKs current kinetics, including changes in limiting slope conductance,
depolarization of the G-V, and tail current acceleration as the number of non-activatable E160R
subunits is increased.
We conclude that KCNQ1 + KCNE1 channels gate like KCNQ1 alone, with movement of each
VS component resulting in increasingly more current in a step-wise manner. This makes for a(COI:No)
uniquely flexible channel complex, tunable for a broad range of responses.

PS01-01

PS01-04

Tomoya Kubota（ Dept Biomedical Infomatics, Grad Sch Med, Osaka Univ, Japan）

Shigetoshi Oiki（Biomed. Imag. Res. Centr., Univ. Fukui, Japan）

Skeletal muscle channelopathy is one of the rare diseases which causes abnormal excitabilityof the sarcolemma. Most patients feel impairment of their quality of life due to myotonia and/or paralysis. In addition, some patients have a life-threatening symptom; cardiac arrythmia
in Andersen-Tawil syndrome, apnea and laryngospasm in infant with myotonia, and so on.
Since the discovery of the causative genes including CLCN1, SCN4A, CACNA1S and KCNJ2,
functional analysis of mutant channels has revealed mechanism of the abnormal membrane
excitability and provided better understanding of structural-functional relationship of protein
and occasionally novel physiological role of ion channels. Since 2005, we have been conducting
genetic analysis of skeletal muscle channelopathies referred from all over Japan and performedin vitro functional analysis of the mutant channels. More than 200 patients were analyzed
with Sanger sequencing and 20 patients with target resequencing or exome sequencing. We
have confirmed the genetic diagnosis for more than 150 patients (74 %). Among them, we
have identified rare, but scientifically significant cases. In this symposium, I am reviewing
skeletal muscle channelopathy cases identified in Japan and discussing the scientific relevancy.
Our data of the skeletal muscle channelopathy in Japan provided important insights to thepathogenesis in both genetic and biophysical aspects of ion channels expressing in skeletal(COI:No)muscle.

The KcsA channel is a prototypical potassium channel, and structure-function relationships
have been examined extensively. The pore domain is shared with a superfamily of thetetrameric cation channel, and the structure of the selectivity filter is well conserved in potassium channels. Thus, elucidating molecular mechanisms of the KcsA channel would help tounderstand other types of potassium channels. We have studied gating and ion permeation
properties of the KcsA channel using lipid bilayer techniques, by which lipid compositions andelectrolyte solutions of both sides of the membrane are readily controlled. Here we examine
K+ permeation through the KcsA channel using lipid bilayer experiments and the molecular
dynamics simulation. For ion permeation through channels, the water flux accompanying
permeating ions is crucially important, which can be measured electrophysiologically by
the streaming potential. The water-ion flux ratio (Jwater/Jion or CRw-i) for various potassiumchannels was 1 at high K+ concentration, which is in accordance with the alternating arraymechanism via transitions between w-i-w-i and i-w-i-w (w: water, i: ion) configurations. On theother hand, we found that the CRw-i value gradually increased over 2 at low K+ concentrations,which cannot be explained by the conventional mechanism. Molecular dynamics simulationof the KcsA channel was performed at different K+ concentrations, and the CRw-i value was
reproduced, warranting further examination. Ion trajectories along the pore were analyzed
using the event-oriented analysis method, which provides relative positional distributions ofions at a given event such as an ion entering the filter (filter-in). We found that the outermost
K+ in the selectivity filter escaped readily towards the extracellular space before a coming
ion entered the filter, rendering only one K+ ion left in the selectivity filter with more watermolecules. Based on these results we provide a queueing mechanism for K+ permeation
through potassium channels. (COI:No)

Functional characterization of mutations identiﬁed in skeletal muscle
channelopathy in Japan

Molecular Dynamics Experiments of Reconstituted Potassium
Channels

PS01-02

Modulation of the voltage sensor domains of KCNQ1 channels by
KCNE subunits
Koichi Nakajo（Div Integr Physiol, Sch Med, Jichi Med Univ, Japan）
KCNQ1 channel is a voltage-gated potassium channel, which is widely expressed in the humanbody including heart, kidney, intestine, inner ear and pancreas. The gating behavior of thechannel is uniquely regulated by a single transmembrane protein called KCNE. There are
five members of KCNE proteins in the human genome. KCNE1, for example, slows gating
kinetics (activation and deactivation) of KCNQ1 and thus underlies the slow K+ current (IKs) inthe heart. KCNE3 (and maybe KCNE2) make the KCNQ1 channel constitutively active (open).
KCNE4 simply inhibits the channel. The properties of KCNQ1 are hugely dependent on which
type of KCNE proteins are co-expressed in the tissue or organ. On the other hand, it is still an
open question of why the modulations of the KCNQ1 channel by similar KCNE proteins are sodiverse. Recent applications of voltage-clamp fluorometry (VCF) to KCNQ1 channels suggest
the voltage-sensing domains (VSD) of KCNQ1 channel are stabilized at a certain state: KCNE1
stabilizes VSD of KCNQ1 at the intermediate state (between the down state and the up state)while KCNE3 stabilizes it at the up state to keep the channel open (Barro-Soria et al., 2014 ＆2015). The next question now could be how the KCNE proteins stabilize the VSD at a certain
state. We recently isolated the zebrafish orthologs of KCNQ1 (zKCNQ1) and three KCNE
genes (zKNCE1, zKCNE3, and zKNCE4). While zKCNE1 and zKCNE4 modulated zKCNQ1 aswell as human counterparts, zKCNE3 failed to make zKCNQ1 constitutively active. We nextcompared the amino acid sequences between mammalian KCNE3 and zKCNE3 and identified
the second half of the transmembrane region of KCNE3 was required to make the channel
constitutively active. This finding could be a good starting point to understand the molecularmechanism of how the KCNE proteins stabilize the VSD of KCNQ1.
(COI:No)S10

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Planned Symposium 2

PS02-03

Vagal regulation of hepatic metabolic and inﬂammatory response
2 1 InFIniti, Kanazawa Univ, japan, 2 Graduate
Hiroshi Inoue1, 2，Yuka Inaba1，Emi Hashiuchi（

Neuro-immune communication andhomeostatic responses（March 17, 9:00〜10:50, Hall 3）

PS02-01

C-type lectin Mincle mediates cell death-triggered sustained
inﬂammation
1 1 Dep of Mol Med and Metab,
Miyako Tanaka1，Yoshihiro Ogawa2，Takayoshi Suganami（

RIEM, Nagoya Univ, Japan, 2 Dep of Med and Bioregulatory Sci, Grad Sch of Med Sci,
Kyushu Univ, Japan）

Accumulating evidence has suggested that chronic sterile inflammation, which contributes tothe pathogenesis of obesity and diabetes, is formed by various factors, such as cell-cell interactions, a variety of cytokines, and innervation. Since the innervation of macrophage activity
had been reported, neuro-immune communication and sterile inflammation have been paid
attention to. We have already elucidated that leptin, a representative hormone derived from
adipocytes, regulates peripheral sterile inflammation through central nervous system. We havealso showed that central leptin signaling controls the development of B lymphocytes in bonemarrow. On the other hand, we have focused on the chronic sterile inflammation, especiallythe microenvironment in obese adipose tissue and revealed that hypertrophied adipocytes andmacrophages form the inflammatory vicious cycles resulted in adipose tissue inflammation.
We have screened and identified Mincle, macrophage-inducible C-type lectin, as a new inflammatory molecule on macrophages that contribute the adipose tissue inflammation. Mincle
is a pathogen sensor for Mycobacterium tuberculosis, but Mincle also can sense cell deathindicating that its involvement in sterile inflammation. Mincle expression was observed in themacrophages which infiltrated into obese adipose tissue and constructed the unique structurestermed crown-like structures (CLSs). It is considered that macrophages receive danger signals
from dead adipocytes and adipocyte–macrophage crosstalk may occur in close proximity
in the CLSs, so CLSs are hallmarks of adipose tissue inflammation. Our data suggests that
Mincle recognizes currently unknown endogenous Mincle ligands from dead adipocytes and isactivated to produce cytokines and chemokines resulted in adipose tissue fibrosis and ectopic
lipid accumulation. Next, we examined the role of Mincle in acute sterile inflammation using
acute kidney injury model. In this symposium, we will provide our up-to-date results anddiscuss the dyshomeostasis caused by cell-cell interaction in the microenvironment. (COI:No)

School of Medical Sciences, Kanazawa Unv, Japan）

The vagus nerve plays an essential role in the organ-crosstalk between the brain and peripheral organs, including the liver. Indeed, The hypothalamus detects whole-body energetic status
and regulates hepatic glucose metabolism through the vagus nerve. Indeed, hypothalamic
sensing of increases in plasma levels of nutrients and hormones results in the suppression of
the vagal activity, which in turn decreases hepatic glucose production (HGP) through alpha 7
nicotinic acetylcholine receptor (A7nAchR) in Kupffer cells. In detail, activation of A7nAchR
suppresses inflammatory cytokines expression in Kupffer cells, including IL-6. The acute suppression of hepatic vagal activity releases Kupffer cells from A7nAchR-dependent suppressionof inflammatory cytokines, resulting in the IL-6 increase from Kupffer cells, followed by HGP
suppression through hepatic STAT3 activation. These suggest that the brain and vagus nerve
regulate hepatic metabolism by their control of hepatic inflammatory cells.
Obesity and insulin-resistance impairs hypothalamic sensing and reduces the fluctuation ofvagus nerve activity according to energetic status. In obese mice, the failure of vagal response
to energetic status results in the smoldering activation of Kupffer cells and blunting of acuteactivation of hepatic IL-6/STAT3 and of suppression of HGP. Furthermore, obesity-induced
failure of vagal control of Kupffer cell can lead to hepatic chronic inflammation. Indeed,
A7nAchR knockout mice reveal nonalcoholic steatohepatitis induced by an atherogenic-diet.
These findings may indicate that autonomic nerves, inflammatory cells and metabolic cells
interact with each other to maintain metabolic and inflammatory homeostasis. (COI:No)

PS02-04

Sterile inﬂammation and neural repair after ischemic stroke
Takashi Shichita（ Stroke Renaissance Project, Tokyo Metro Inst Med）
Inflammation is an essential step for the pathology of ischemic stroke. However, its importancein the process of neural repair after ischemic brain injury has not been clarified. Inflammation
after ischemic brain injury is triggered by the extracellularly released DAMPs (damage-associated molecular patterns) from necrotic brain cells. High mobility group box 1 (HMGB1) andperoxiredoxin (PRX) have been identified as DAMPs in the ischemic brain. Additionally, we
recently identified DJ-1 as previously unknown DAMPs. HMGB1 exaggerates the disruptionof blood brain barrier; on the other hand, PRX and DJ-1 directly activate infiltrating immune
cells through Toll-like receptor 2 (TLR2) and TLR4 to induce the production of inflammatory
cytokines.
These inflammatory DAMPs are removed from ischemic brain through scavenger receptors,
MSR1 and MARCO, which are expressed by microglia and infiltrating macrophages. MSR1
expression levels in these mononuclear phagocytes increase from day 1 to day 3 after stroke
onset. These MSR1-high mononuclear phagocytes efficiently remove DAMPs and promoteneural repair by producing IGF-1, a neurotrophic factor. Sterile inflammation after ischemic
injury will be important for the induction and accumulation of reparative immune cells at
the injured brain region. Novel therapeutic method for ischemic stroke will be developed byclarifying the detailed molecular mechanisms in the reparative process after ischemic stroke.
(COI:No)

PS02-02

PS02-05

Tsuyoshi Inoue（Div CKD Pathophysiol, Grad Sch Med, Univ of Tokyo, Japan）

Masaru Ishii（Dept Immunol Cell Biol, Grad Sch Med, Osaka Univ, Japan）

The kidney is a highly developed organ, and has various functions such as regulation of fluidand electrolyte, regulation of blood pressure, production of erythropoietin, and activation ofvitamin D. Progression to kidney disease is caused by various causes such as diabetes andhypertension, hereditary disease, and glomerulonephritis characterized by urine protein. However, at present, there is currently no drug for chronic kidney disease other than angiotensin II
receptor antagonist (ARB), which has an inhibitory effect on the progression of kidney disease.
Some immune cells have receptors for neurotransmitters and the existence of immune cells
that produce neurotransmitters such as acetylcholine has been clarified, and the mechanismof immune system regulation via the nervous system is being elucidated. Among them,anti-inflammatory reflex through vagus nerve are well-studied. In fact, electrical vagus nerve
stimulation (VNS) has been shown in animal experiments to ameliorate various diseases suchas myocardial infarction, pancreatitis and sepsis. In addition, the effectiveness of the implantable vagus nerve stimulator has been confirmed in Crohn’s disease and rheumatoid arthritis
patients in pilot studies.
We have so far showed the following, 1) VNS protected the kidney from acute kidney injury.
2) α7 nicotinic acetylcholine receptor (α7nAChR) positive macrophages and β2 adrenergic
receptor positive CD4 T cells play an important role in exerting the renal protection effectby VNS. 3) We newly identified Hes1 (hairy and enhancer of split-1) as downstream gene of
α7nAChR by RNA-seq and functional analysis of genes. 4) It was discovered that C1 neuron
stimulation in the medulla by optogenetics had a renoprotective effect. Thus, as the elucidationof the renal protective effect through the neuro-immune system is gradually progressing,the development of further research on the neuro-immune-renal linkage is expected to leadto a new therapeutic option for inflammation-related disorders including kidney diseases.
(COI:Properly Declared)

During the last decade, intravital optical microscopy has launched a new trend in the fieldof biology. By using this advanced imaging technique we have established a new systemfor visualizing in situ behavior of a diversity of living cells within intact tissues and organs.
Among them, we succeeded in visualizing the various dynamic phenomena within bones andjoints, where various kinds of immune cells are produced and functioning although poorly
analyzed by conventional methodology such as histological analyses with decalcified sections.
We have so far identified the real modes of migration, differentiation and function of bonedestroying osteoclasts, special kind of macrophages responsible for bone and joint erosions. In
this presentation I will present the recent update on intravital imaging studies on immune andother systems for clarifying in vivo behaviors of cell and tissue dynamics.
(COI:No)

Neuro-immune axis on kidney disease

Intravital imaging dissecting inﬂammatory cell dynamics in vivo

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S11

Planned Symposium 3
Integration of sensory and metabolic signals
by neuropeptide in hypothalamus(navigates metabolic state.)（March 17, 9:00〜10:50, Hall 4）

PS03-01

Physiological function of NPGL in feeding behavior and energy
metabolism

PS03-03

Visual and endocrine systems mediating green-light effect on ﬁsh
growthAkiyoshi Takahashi1，Daisuke Shimizu2，Satoshi Kasagi1，Kanta Mizusawa1（ 1 Sch Marine Biosci, Kitasato Univ, Japan, 2 FRA Tohoku Natl Fish Res Inst, Japan）
Biological processes of living organisms are affected by environmental photic conditions. Body
color changes adapting individuals to environmental color and hue for camouflage is one ofthe prominent functions observed in bony fish. We recently found that green LED light (peakwavelength 518 nm) stimulated the growth of flatfishes. The potency was greater than that ofblue (464 nm), blue-green (497 nm), red (635 nm), and white (447 nm and 550 nm). Green lightincreased total length, body weight, and food intake of flounders. It is of interest that green
light improved food conversion efficiency. We investigated the roles of the visual and endocrine
systems to clarify pathways evoked by green light. In the eyes of the flounder, several types of
opsins were expressed. These were one ultraviolet opsin, one blue opsin, one blue-green opsin,
two or three green opsins, one red opsin, and one rhodopsin, suggesting that flounders canperceive green light sensitively. In the brain, exposure to green light caused changes in theexpression of melanin-concentrating hormone (MCH) and agouti-related protein (AGRP) genes.
Thus, these neuropeptide hormones are suggested to be associated with food intake undergreen light. In the pituitary, the expression of the somatolactin (SL) gene, which belongs to thegrowth hormone family and is present only in bony fish, was changed with green light. SL issuggested to participate in lipid metabolism under green light. Interestingly, MCH, AGRP, andSL are also representative hormones regarding body color regulation in bony fish. Moreover,association of these hormones with food intake or appetite control is probably common in bonyfish. Here, we suggest that these are key hormones under green light in flatfishes.  (COI:No)

PS03-04

Homeostatic coordination via alteration of hypothalamic energy sensor
by feeding time

Kenshiro Shikano（Dept Neurophysiol, Fac Med, Oita Univ, Japan）

Tetsuya Shiuchi（Dept. Integ Physiol, Tokushima Univ Grad School, Japan）

Hypothalamus is the center of feeding behavior and energy metabolism, and several hypothalamic neural substrates regulate energy metabolism. However, the mechanism of feeding
behavior has not yet totally elucidated. We recently found a novel secretory protein of 80
amino acids and named it neurosecretory protein GL (NPGL). Npgl mRNA expression was
upregulated by fasting and low insulin levels, and NPGL neurons were responded to insulin.
These data suggest that NPGL regulates energy homeostasis. To investigate the function of
NPGL, we performed protein administration and gene overexpression using wild-type rats.
These manipulations showed that NPGL increased lipid accumulation in white adipose tissue(WAT) without remarkable changes in food intake. This adiposity was associated with an
induction of de novo lipogenesis in WAT but not in liver. NPGL did not change the food intake
under the normal chow but increased under the high calorie diets. The food selection test
was conducted because food preference for palatable food was observed by NPGL treatment.
NPGL selectively induced carbohydrate intake. These results show NPGL as a novel neuronal
regulator that drives feeding behavior and fat deposition through de novo lipogenesis in WAT,
and acts to maintain steady fatty level. In the future, the findings of NPGL will reveal the novel
mechanisms of feeding regulation related to lipid metabolism.
(COI:No)

Energy homeostasis is coordinated by hypothalamus, which is central commander forautonomic nervous and endocrine system. Neuron in hypothalamus senses blood substratelevels such as glucose and leptin, thereby regulating feeding behavior and energy metabolism.
However, it had not elucidated how feeding time influence hypothalamic sensor and navigate
coordination of energy homeostasis via neuropeptide function. We separated C57BL/6J micefor 3 feeding scheduled groups; given lab chows freely during dark phase (ZT12-24, Ad-lib
group), first 4-hour in dark phase (ZT12-16; Morning group), and last 4-hour in dark phase(ZT20-24, Evening group). Mice in Evening group showed lower whole-body insulin sensitivity
and energy expenditure despite the smaller food intake than that of Ad-lib group while theseparameters in Morning group were normal. We observed that higher lipid accumulation,
increased gene expression of fatty acid synthesis, decreased fatty acid oxidation and impaired
insulin signals in skeletal muscle in Evening group compared to other groups. Encompassing
analysis revealed that mRNA expression of agouti-related protein (AgRP) was increased in
hypothalamus in Evening group. Inhibition of central AgRP expression by antisense oligo
did not alter insulin sensitivity in whole body and skeletal muscle of Evening group. We
further observed possibilities that glucocorticoid system is a candidate for stimulation of AgRP
expression in Evening group, and that clock time-altered liver by feeding time may control
hypothalamic glucocorticoid sensor by hepatic afferent nerve. These findings suggest thatfeeding time may influence hypothalamic neuronal sensor for glucocorticoid, thereby navigating physiological energy homeostasis mediated by hypothalamic neuropeptides such as AgRP.
(COI:No)

PS03-02

Neural mechanisms of odor-induced feeding behavior
Koshi Murata（ Div Brain Stru Func, Faculty Med Sci, Univ Fukui）
Smell of food can induce appetite and motivated behavior. However, neural mechanisms ofodor-induced feeding behavior are still unclear. We recently found two brain regions which
are supposedly involved in feeding behaviors. One region is the olfactory tubercle (OT). The
OT is part of the olfactory cortex as well as the ventral striatum, which receives olfactory anddopaminergic inputs. Learned odor-induced food seeking behaviors accompanied activation of
dopamine receptor D1-expressing neurons in the anteromedial domain of the OT. Manipulation of the D1 neurons in the anteromedial OT by optogenetics and chemogenetics induced
attractive behavior and feeding behavior. The other region is a newly identified part of theolfactory cortex that send axons to the lateral hypothalamus. We named this area as theventral olfactory nucleus (VON) because it is located beneath the anterior olfactory nucleus.
In this talk, I will introduce our results and discuss how the OT and VON control feeding
behavior.
(COI:No)S12

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Planned Symposium 4
The metabolic modulation by sensory input
from peripheral organs（March 17, 9:00〜10:50, Hall 6）

PS04-01

Analysis of gastro-intestinal autonomic afferents in the mouse dietary
obesity（Dept Physiol 2, Kanazawa Med Univ, Japan）

Mamoru Tanida

The high fat diet (HFD) generates obesity which arises from hyperphagia and decreased
metabolism. Recently, it has been shown that afferent vagal signals in the abdominal organs
sense to nutritional and hormonal stimulations, sending the information to the brain forregulation of appetite and metabolism. For example, CCK, feeding suppressor hormone in
the intestine, stimulates afferent vagal nerve signals in the anesthetized rats. In the preset
study, we used HFD-induced obese mice and examined effects of CCK or leptin, hypophasic
hormone in the adipose tissue on afferent vagal nerve activities in the gastro-intestinal organs
in the anesthetized mice. Intravenous injection of not only CCK, but also of leptin stimulated
afferent vagal nerve activities in the gastric branch, celiac branch and hepatic branch. On theother hand, in the HFD mice, afferent gastric vagal nerve responses to the CCK and leptin
were attenuated, but stimulatory responses of hepatic and celiac afferents were reserved. In
addition, CCK receptor A mRNA and leptin receptor (Ob-Rb) mRNA of nodose ganglion in
the HFD mice did not change significantly. These data suggest that disruption of receptors
function but not receptors expression in vagal afferent pathway may be involved in attenuation(COI:No)of responses of vagal afferents to feeding-related hormones in the HFD mice. 

PS04-02

Thermoregulation via the vestibular system
Chikara Abe, Hironobu Morita（ Dept Physiol, Gifu Univ Grad Sch Med, Japan）
The vestibular system is one of the sensory systems which contributes to the sense of balance and spatial orientation. This also participates in the autonomic nervous response, which
stimulation of the peripheral vestibular organs induces sympathoexcitation. This response isobserved in both rodents and human beings, and we have reported that vestibular system
contributes to the arterial pressure response during postural change as one of the feedforward
control system. In the other autonomic nervous responses, stimulation of the otolith organs in
the inner ear induces hypothermia; exposure to the hypergravity environment decreases bodytemperature by 8 degree Celsius in mice. This response was attenuated by vestibular lesion
or genetic deletion of otolith. In order to elucidate the central mechanism of hypergravityinduced hypothermia, we examined the role of Vglut2, Vgat and ChAT neurons in vestibular
nucleus complex (VNC) on thermoregulation in mice. We used Vglut2-cre, Vgat-cre, and
ChAT-cre mice to manipulate each neuron in VNC. The viral vector was injected in VNC toexpress photo sensors (channelrhodopsin or archaerhodopsin) for optogenetics or hM3D(Gq)for chemogenetics. Unilateral photostimulation of the Vglut2 neurons in VNC induced bodytilt to the ipsilateral side, while photoinhibition induced body tilt to the contralateral side. In
Vgat-cre mice, opposite response was observed compared with Vglut2-cre mouse. Photostimulation of ChAT neurons did not show any responses. Chemogenetics stimulation of Vglut2
neurons showed hypothermia with increasing in activity, while Vgat stimulation increased
body temperature with decreasing in activity. Deletion of Vglut2 neurons in VNC attenuatedhypothermia induced by hypergravity exposure. On the other hand, hypothermia was stillobserved by deletion of Vgat neurons in VNC. Interestingly, chemogenetics stimulation of
Vglut2 neurons in VNC 2 days before hypergravity exposure, the hypothermia was attenuated.
Taken together, hypothermia induced by hypergravity exposure is due to activation of Vglut2
neurons in VNC.
(COI:No)

PS04-03

Molecular Mechanisms Underlying Dietary Fatty Acids and Their
Metabolites-Induced Activation of Brown Adipose Tissue Function
2 1Grad Sch Agric, Kyoto Univ, Japan, 2 C-PIER, Kyoto Univ, Japan）
Tsuyoshi Goto1, （Emerging evidence suggested that the enhancement of thermogenic activity in brown adipocytes or beige adipocytes is an attractive target for the management of obesity. Several
environmental stimuli, such as cold exposure and exercise, activate brown adipose tissue(BAT) function. Moreover, several food factors have also been shown to be effective stimuli toactivate thermogenic BAT function.
Our recent study indicated that treatment with several dietary fatty acids and their metabolites produced by gut microbiota can activates BAT function in mice. We showed fish oil intake
increased oxygen consumption and rectal temperature, with concomitant upregulation of
uncoupling protein 1 (UCP1), the responsible gene for the nonshivering thermogenesis in both
BAT and white adipose tissue (WAT), suggesting that fish oil intake activates BAT function in
mice. Mice fed fish oil diet showed the increase in urinary catecholamine levels, suggesting thatfish oil intake activates sympathetic nervous system (SNS). These effects of fish oil intake were
not observed in transient receptor potential vanilloid 1 (TRPV1) deficient mice. In conclusion,
fish oil intake can induce UCP1 expression in classical brown and beige adipocytes via the
TRPV1-mediated activation of SNS, thereby attenuating fat accumulation and ameliorating
lipid metabolism. We have also showed that KetoA [10-oxo-12(Z)-octadecenoic acid], a linoleic
acid metabolite produced by gut lactic acid bacteria, can activate TRPV1-SNS-BAT pathway.
Actually, dietary KetoA intake ameliorated obesity and obesity-associated metabolic disorders
in WT mice but not in TRPV1 deficient mice. These findings indicated that some of dietary
fatty acids and their metabolites produced by gut microbiota might contribute to the bodyweight management via the activation of TRPV1-SNS-BAT pathway.
(COI:No)

PS04-04
The modulation of thyroid functions by sensory input from pharynx
Harumi Hotta, Kaori Iimura, Harue Suzuki（Dept Auton Neurosci, Tokyo Metropol Inst

Gerontol, Tokyo, Japan）

The thyroid gland, an endocrine organ secretes hormones regulating systemic metabolisms,
receives innervation from sympathetic (cervical sympathetic trunk: CST) and parasympathetic(superior laryngeal nerve: SLN) nerves. We have recently showed that electrical stimulation of
the SLNs increases thyroxine (T4) and calcitonin (CT) secretion into the thyroid venous blood.
Moreover, stimulation of myelinated (presumably afferent) SLN fibers had the same effect asunmyelinated efferent SLN stimulation. From these results, we predicted reflex mechanism ofhormonal secretion from the thyroid via the SLN.
The SLN includes myelinated afferent nerves conveying mechanosensory information from thepharynx and larynx. Accordingly, we applied intermittent mechanical stimuli, as during swallowing food, to the pharynx in anesthetized rats and examined the effect on hormonal secretion
into thyroid venous blood. During stimulation (for a period of 6-9 min), secretion rate of T4
and CT increased two-fold, whereas that of parathormone (PTH) unchanged. The increased
secretion rate was returned to the pre-stimulus control level after the end of stimulation. Such
responses were similar to those during electrical stimulation of the SLNs. The hormonal secretion during pharyngeal stimulation disappeared completely after cutting the SLNs bilaterally.
The pharyngeal stimulation increased parasympathetic efferent nerve activities recorded fromthin thyroid nerves originating in the SLN. These activities disappeared by a ganglionic blocker(hexamethonium, intravenously).
We concluded that sensory input from the pharynx promotes hormonal secretion from thethyroid gland through reflex mediated by SLN, for both afferent and efferent paths. Considering metabolic regulating actions of thyroid hormones and calcitonin, this pharyngeal-thyroid
reflex may help maintain physical and mental health, whenever we intake foods from mouth.
Future studies are needed to clarify whether other stimuli (e.g. vocalization), known to activate
SLN myelinated afferents, can also trigger such a reflex.
(COI:No)

PS04-05

GLP-1 releaser D-Allulose regulates glucose metabolism via〈vagal
afferents – brain〉axis
3 1Grad Sch Life Environm Sci, Kyoto Pref Univ,
Yusaku Iwasaki1，Toshihiko Yada2, （Japan, 2 Integr Physiol, Kansai Elect Power Med Res Ins, 3 Syst Physiol, Grad Sc Med, Kobe
Univ）
Intestinal hormone glucagon-like peptide-1 (GLP-1) is well known as incretin hormone. However,
the endogenous GLP-1 is rapidly cleaved within a few minutes. It is unclear how endogenous
GLP-1 acts on insulin secretion and glucose metabolism. We identified rare sugar D-allulose(Allu) as a GLP-1 releaser, which is a non-metabolizable monosaccharide. Oral administrationof Allu increases GLP-1 secretion, and thereby suppresses food intake via vagal afferents
expressing GLP-1 receptor (GLP-1R) in healthy and hyperphagic obese mice (Y. Iwasaki et al.
Nat. Commun. 2018). In this study, we examined whether endogenous GLP-1 release by Allu
regulates glucose metabolism and insulin secretion/action via [vagal afferents – brain] axis.
Endogenous GLP-1 release by Allu robustly suppressed elevation of blood glucose in ip glucosetolerance test (GTT) in both normal and diet-induced obese (DIO) mice. The early insulin secretion at 15 min of GTT was significantly increased but only modestly, suggesting an additional
mechanism underlying the glycemic control by endogenous GLP-1. Peroral Allu enhanced
insulin action in insulin tolerance test in normal and DIO mice. These effects of Allu were all
blunted by GLP-1R antagonist exendin 9-39, in GLP-1R deficient mice and in mice receiving
the vagal afferent denervation. Therefore, GLP-1 release by Allu promotes glucose tolerance
by enhancing release and action of insulin via [vagal afferents - brain] axis. Additionally, wedemonstrate that endogenous GLP-1 release by Allu effectively corrects hyperglycemia via
enhancement of insulin sensitivity in a glucose-dependent manner. Therefore, GLP-1 releaser
Allu that acts on vagal afferents, may provide a novel category of incretin-based medicine, withhigh efficacy and safety to correct hyperglycemia.
(COI:Properly Declared)

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S13

Planned Symposium 5
Brain function for physical therapy and its
physiological mechanisms（March 17, 9:00〜10:50, Hall 10）

PS05-03

Neural plasticity of the somatosensory cortex that underlies central
post-stroke pain
Kazuaki Nagasaka（ Institute for Human Movement and Medical Sciences, Niigata Univ of
Health and Welfare, Japan）

Central post-stroke pain (CPSP) is an intractable type of chronic pain that may occur several
weeks after the onset of stroke. This pain originates in the thalamus, specifically in the ventral
posterolateral nucleus (VPL). CPSP is characterized by allodynia in which normally innocuous
stimuli are perceived as painful. Developing therapeutic interventions for this abnormal pain
is difficult because the pathogenic mechanisms are unclear. To address this problem, we usedanimal models of CPSP based on the artificial stroke in the VPL, and investigated the neuralplasticity underlying the development and maintenance of CPSP. Here, we describe changes
in brain activity within the somatosensory cortex in rat and macaque models of CPSP. (1) Rat
model: Optical imaging using voltage-sensitive dye (VSD) is a powerful tool for visualizing
stimulus-dependent changes in membrane potential and for revealing the spatiotemporal patterns of neural activity. We performed optical imaging using the VSD RH-795 to investigatechanges in sensory activity evoked by forelimb stimulation in a rat CPSP model. Both thepeak amplitude of the VSD signal and the activation area within the somatosensory cortex
significantly correlated with pain scores, which represent the severity of allodynia. (2) Macaquemodel: In recent years, we have established a model of CPSP using the macaque. We applied
this animal model in the functional magnetic resonance imaging (fMRI) to elucidate the changes
in brain activities that underlie allodynia. The fMRI analysis demonstrated that the activationof sensory cortices, including the primary and secondary somatosensory cortices, was related
to the development of allodynia. These findings support the validity of the animal models forstudying the plastic changes in neural functions underlying CPSP caused by a VPL stroke.
(COI:No)

PS05-01

PS05-04

Ryota Asahara1，Kei Ishii1，Nan Liang2，Hidehiko Komine1，Kanji Matsukawa3

Naofumi Otsuru, Hideaki Onishi（Dept Phys Ther, Niigata Univ health and Welfare, Japan）

Central cardiovascular control during physical exercise（ 1 AHFRC, AIST, Tsukuba, Japan, 2 Dept Human Health Sci, Grad Sch Med, Kyoto Univ,
Kyoto, Japan, 3 Dept Integ Physiol, Grad Sch Biomed and Health Sci, Hiroshima Univ,
Hiroshima, Japan）
Physical exercise requires coordination of motor control with simultaneous and concomitantcontrols of the cardiovascular system necessary to respond to the rapid increases in metabolic
demands of the exercising muscle. Accumulating evidence suggests that a feedforward signal
descending from higher brain centers (termed central command) plays an important rolein mediating the cardiovascular adjustments to exercise. However, little is known regarding neural circuit(s) or region(s) of central command. Understanding the central commandmechanisms would be helpful to design effective rehabilitation programs for patients withcerebro- and cardio- vascular disease. We have conducted a series of healthy human studies
measuring oxygenations (as index of regional tissue blood flow) of the cerebral prefrontal
cortex and skeletal muscle during voluntary exercise using near-infrared spectroscopy. The
oxygenated-hemoglobin concentration in the prefrontal cortex started to increase approximately 5s prior to the onset of voluntary exercise with arbitrary start. However, such increasein the prefrontal oxygenation was absent when exercise was forced to start by cue. When
comparing the muscle oxygenation responses, the increase in muscle oxygenation was greater
during voluntary exercise with arbitrary start than with cued start. These results suggest thatthe prefrontal cortex may play a role in driving neuronal circuits controlling the cardiovascularsystem, which may contribute to vasodilation in the skeletal muscle. In the symposium, we
would like to introduce our recent updates on 1) neural circuit(s) responsible for controllingthe cardiovascular system during exercise and 2) central control of muscle blood flow during(COI:No)
exercise and discuss future directions and clinical implications.

Changes in corticospinal excitability by interoceptive information
Interoception refers to a sensation that develops from within the body such as visceral feelingsof vasomotor activity. Studies have shown that interoceptive information (mainly investigatedusing heartbeat) reaches various regions of the motor cortex including the insula, anterior
cingulate cortex, prefrontal cortex, and sensorimotor cortex. These studies show that interoceptive information reaches these regions within 200–600 ms of the R wave on an electrocardiogram. Therefore, studies have reported that auditory, visual, somatosensory, and nociceptive
perceptions were altered by interoceptive information within this time window. However, theinfluence of interoceptive information on the motor system (corticospinal pathway) has notbeen extensively studied. Moreover, the cortical activities evoked by heartbeat varied according to individual interoceptive accuracy as evaluated using the heartbeat perception task. We
investigated whether interoceptive inputs modulate corticospinal excitability within a specifictime window after the R wave. We also tested whether this modulation differs between goodand poor interoceptive perceivers using transcranial magnetic stimulation (TMS).
In the first experiment, we found that at 200 ms after the R wave, there was a significantly
positive correlation between corticospinal excitability and interoceptive accuracy. On theother hand, there was a significant negative correlation between corticospinal excitability andinteroceptive accuracy at 400 ms after the R wave. These results indicated that interoceptive
information from the heartbeat reached the motor cortex.
Based on the results from the first experiment, we hypothesized that this phenomenon is used
for paired associative stimulation (PAS). PAS is used for facilitating time-dependent plasticity
in the motor cortex using repetitive somatosensory inputs (exteroception) combined with TMS
over the motor cortex. Therefore, we investigated whether similar plasticity can be inducedby combining TMS over the motor cortex with interoceptive inputs. As a result, we found thatthe excitability of the motor cortex may be influenced by interoceptive PAS.
(COI:No)

PS05-02

Transcutaneous spinal direct current stimulation (tsDCS) increases
corticospinal transmission and facilitates ballistic movement of leg
muscles in humans
Tomofumi Yamaguchi（Dept Physical Therapy, Yamagata Pref Univ, Japan）
Transcutaneous spinal direct current stimulation (tsDCS) is a non-invasive neuromodulation
technique that can alter excitability in spinal and supraspinal circuits in animals and humans.
However, it is still unclear whether the effects of tsDCS on corticospinal transmission andmotor performance. Here we investigated whether tsDCS may enhance descending activationof spinal motor neurons and facilitate voluntary ballistic activation of the leg muscles in healthy
adults.
The experiment comprised five studies designed to evaluate the immediate effects of tsDCS
over thoracic-lumbar on corticospinal transmission and voluntary motor output. In experiment 1 and 2, we investigated potential dose-response relationships between tsDCS stimulus
intensity (experiment 1) and duration (experiment 2) on corticospinal transmission probed
by transcranial magnetic stimulation (TMS). In experiment 3, we investigated the effects of
tsDCS on voluntary motor functions using a ballistic force production task involving the ankle
muscles. In experiment 4 and 5, we explored the system-level neural targets of tsDCS by conditioning Soleus H-reflexes with subthreshold TMS (experiment 4), and by comparing evoked
potentials from TMS over the leg primary motor cortex and from electrical stimulation of thecorticospinal tract at the cervicomedullary junction (experiment 5), i.e. motor evoked potentials(MEPs) and cervicomedullary motor evoked potentials (CMEPs) respectively.
Cathodal tsDCS significantly increases voluntary EMG and force production during ballistic
plantarflexion. This is accompanied by increased MEPs and short-latency facilitation of the
Soleus H-reflex induced by TMS, without any changes in the H-reflex or CMEPs.
This is a strong indication that cathodal tsDCS at lumbar level enhances cortical excitability
and thereby promotes motor performance. These findings suggest that cathodal tsDCS may
be an effective strategy to promote rehabilitation training after spinal cord injury and stroke.
(COI:No)S14

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Planned Symposium 6
Recent Advancement of the Multidisciplinary
Approaches to the Pathophysiology
Underlying Arrhythmia（March 17, 15:20〜17:10, Hall 2）

PS06-03

iPS cell-Based Disease Modeling and Therapeutic Approach toInherited Arrhythmias
Takeru Makiyama（ Dept Cardiovasc Med, Grad Sch Med, Kyoto Univ, Japan）
Human induced pluripotent stem cell (hiPSC) technology is a promising tool for regeneration
therapy, drug safety testing, and the investigation of pathogenic mechanisms. The use of
hiPSC-derived cardiomyocytes (CMs) for disease modeling of inherited arrhythmias provides
advantages over primary cells, while, hiPSC-CMs have some limitations: CMs with mixed
subtypes (atrial, ventricular and nodal), immature electrophysiological characteristics such as
shallow diastolic membrane potentials, automaticity even in ventricular-type CMs, etc. To overcome these limitations, we employ dynamic clamp-based approach of IK1 injection and in-silico
model of hiPSC-CM for our research. In this session, I will talk about our current challengesof hiPSC-based disease modeling and novel therapeutic approaches to inherited arrhythmias.
Long-QT syndrome (LQT) is an inherited arrhythmia characterized by delayed ventricularmyocardial repolarization and an increased risk for life-threatening ventricular arrhythmias
and sudden cardiac death. There is no decisive medication therapy to treat LQT and thedevelopment of new therapeutic approaches is highly needed.
A missense mutation, E1115K in CACNA1C, encoding L-type Ca2+ channels (LTCCs)
is located in the crucial site of Ca2+ selectivity (Nature. 1993). Recently, the mutation wasreported to be associated with overlap of LQT and Brugada syndrome. Using patient-derived
hiPSC-CMs, we demonstrated impaired ion selectivity of LTCC resulting in action potential
prolongation and we identified that late sodium current blockers might be candidates to rescue
QT prolongation in this mutation.
Recently, calmodulin is reported to be associated with severe LQT and we generated
calmodulin-related LQT15-hiPSCs carrying a CALM2 mutation. LQT15-hiPSC-CMs exhibited
significant lower beating rate and prolonged action potential durations. We performed genetherapy by allele-specific knockout using the CRISPR-Cas9 system and successfully rescued
the electrophysiological abnormalities in the mutant allele-specific knockout CMs.
Thus, our goal is to develop a genotype-specific treatment, i.e. “Precision Medicine” that will
(COI:No)
improve the prognosis of patients with inherited arrhythmias.

PS06-01

PS06-04

Masatoshi Yamazaki（Dept Bioeng, The University of Tokyo, Japan）

Yukiko Himeno1，Yosuke Okamoto2，Hirohiko Kojitani3，Kyoichi Ono2，Akinori Noma1，
1 1 Dept Bioinformatics, Col Life Sci, Ritsumeikan Univ, Japan, 2 Dept Cell
Akira Amano（

Experimental visualization of rotor as a mechanism of arrhythmia using
ex-vivo optical mapping
Atrial fibrillation (AF) is the most common sustained atrial arrhythmia and affects more than
1 million patients in Japan. It is very important to establish effective therapy for patients withAF, who are increasing explosively in number, in our highly stressed society, where population
aging is accelerating under tight health economic conditions in Japan. Despite of many years of
research, the precise mechanisms of underlying the initiation and maintenance of AF remain
poorly understood. Since about 40 years ago, many theoretical and experimental studies havesuggested that spiral-wave reentry:rotor rotating around a functional obstacle, like hurricane
and tornado, is the major mechanisms of AF. Recently, an attempt to treat persistent/chronic
atrial fibrillation by radiofrequency ablation of rotors was proposed in patients, however, its
efficacy has not been fully established. As it is difficult to detect rotors and perform rotor
ablation with low-resolution (2-3 cm) electrode mapping used in clinical electrophysiologicalstudies, it remains difficult to accurately evaluate the efficacy of rotor ablation for reverting AF
to sinus rhythm. We have developed a multiple electrode mapping system with simultaneous
high-resolution optical mapping system (0.1 mm), which can reliably detect rotors. Thus, theexperimental models in Langendorff-perfused rabbit heart clearly demonstrated that highfrequency rotors exist in arrhythmia.
(COI:Properly Declared)

Mechanisms of Pathophysiology Underlying Arrhythmia Analyzed byComputer Simulations
Physiol, Grad Sch Med, Akita Univ, 3 Dept Cardiovasc Med, Grad Sch Med, Kyoto Univ）

Computer simulation is one of the powerful tools to analyze electrical activities of the excitable
cells such as cardiomyocytes. By using the simulator, it is possible to understand the mechanisms of the cellular functions quantitatively in an integrative manner and test hypotheses forconsistency. It is now well known that Ca2+, as well as ionic channels and transporters, play arole in inducing irregular rhythm to trigger arrhythmia. In order to clarify the causality of theCa2+-induced arrhthmogenic activities of cardiomyocytes, we have developed a mathematicalmodel of Ca2+ releasing unit (CaRU), which represents the function of the structure of dyad
consists of ryanodine receptor channels on the membrane of sarcoplasmic reticulum andL-type Ca2+ channels on the plasma membrane or T-tubules facing to each other. In thisCaRU model, various parameters such as volumes, concentrations, diffusion constants, densities and so on, are required for each Ca2+ diffusion compartment to reproduce its stochastic
behavior. In other words, by changing those parameters, the CaRU model could be successfully
adopted to various kind of cell types to reproduce various physiological and pathophysiologicalphenomenon exhibited by the cells in electrophysiological experiments. The importance ofunderstanding spatio-temporal regulation of local Ca2+ concentrations will be discussed in thepresentation.
(COI:No)

PS06-02

Identiﬁcation of cardiac connexin syndrome using whole-exome
sequence and in-vitro functional assay
3 1Omics Research Center, NCVC,
Taisuke Ishikawa1，Akiko Seki2，Naomasa Makita1, （Osaka, Japan, 2 Dept Cardiovascular Medicine, Tokyo Women’s Medical University,
3 Research Institute, NCVC）

Progressive cardiac conduction defect (PCCD) is a rare inherited arrhythmia sometimes accompanied by sudden death. Genetic causes for PCCD include mutations in cardiac sodium
channel alpha subunit SCN5A with affecting atrioventricular (AV) and ventricular conduction
on the His-Purkinje fiber. Cardiac connexins, Cx40, Cx43, and Cx45, had been suspected as
candidate genes for PCCD because connexins forming gap junctions (GJ) are responsible forthe intercellular permeability of ions and small molecules to transfer the rapid signaling on
the cardiac conduction system. These cardiac connexins are tissue-dependently expressedin distinctive combinations and relative quantifies. Recently we identified Cx40 and Cx45
mutations, and delineated the gene-specific clinical and electrophysiological phenotypes in thefamilial PCCD cases.
The first inheritable mutation of cardiac connexins is Cx40-Q58L found in a PCCD family withsudden death. Cx40 is primarily expressed at the Purkinje fiber, and each mutation carriers
demonstrated the impaired AV and ventricular conduction before adolescence. In vitro study
revealed Cx40-Q58L impairing Cx40 assembly to form GJ. More recently, we reported thefirst Cx45 mutation Cx45-R75H in two unrelated families with distinct ethnicities. Afflictedindividuals had progressive AV block and atrial standstill without ventricular conduction abnormalities and sudden death. In vitro study indicated that impaired intercellular permeabilityof Cx45-R75H affected the automaticity and conduction of sinoatrial and AV node where Cx45
is highly expressed. Patients shared extracardiac phenotypes comprising brachyfacial pattern,
finger deformity, and dental dysplasia. In summary, cardiac connexin mutations confer theisoform-specific clinical phenotypes, and the identification of Cx45-R75H suggest the novel
PCCD entity of conduction defects confined to atrium.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S15

Planned Symposium 7
The emerging roles of pallidal nuclei
in the basal ganglia circuitry（March 17, 15:20〜17:10, Hall 3）

PS07-03

A role for Enkephalin-expressing ventral pallidal neurons in controlling
aversive Pavlovian Conditioning
Tom Macpherson1，Hiroyuki Mizoguchi2，Akihiro Yamanaka2，Takatoshi Hikida1（ 1 Lab for Advanced Brain Functions, IPR, Osaka Univ, 2 RIEM, Nagoya Univ）
The ventral pallidum (VP) is a critical component of the limbic loop of the basal ganglia, andhas been implicated in the regulation of incentive motivation. However, the precise role of the
VP in controlling Pavlovian conditioning of cues paired with appetitive or aversive outcomesis still unclear.
Here we used a Tet-Tag AAV virus system, in which designer receptors exclusively activatedby designer drugs (DREADDs) were expressed in a population of VP neurons containing the
peptide enkephalin, known to be highly expressed within the VP, to investigate the possible
role of the VP in appetitive and aversive Pavlovian associative learning. During acquisitionof an appetitive autoshaping task, hM3Dq DREADDs were activated by administration of
CNO, leading to increased activity in enkephalin-expressing VP neurons. Mice treated with
CNO did not significantly differ from saline-treated controls in their ability to acquire thetask, as indicated by an increase in Pavlovian approach behavior to a reward-associated cue
over the course of 6 daily sessions. However, in a passive avoidance task measuring aversive
Pavlovian conditioning, administration of CNO during conditioning of an aversive foot-shockupon entering a dark chamber resulted in a decreased latency, in comparison to saline-treatedcontrols, to enter the shock-associated chamber when tested 24 hours later.
Here, we demonstrate that activity in enkephalin-expressing VP neurons disrupts aversive, but
not appetitive, Pavlovian conditioning. Thus, decreased activity in enkephalin-expressing VP
neurons is necessary for aversive learning, supporting previous evidence from our group thatneurotransmission blocking in nucleus accumbens D2-neurons, likely leading to disinhibitionof downstream VP neurons, similarly inhibits aversive Pavlovian conditioning in a passive(COI:No)
avoidance task. 

PS07-01

PS07-04

Hiromi Sano1, 2，Indriani Dwi Wahyu1，Satomi Chiken1, 2，Atsushi Nambu1, 2

Yoshihisa Tachibana（ Div Syst Neurosci, Grad Sch Med, Kobe Univ, Japan）

Regulation of voluntary movements by signals through the externalsegment of the globus pallidus（ 1 Div System Neurophysiol, NIPS, Japan, 2 Dept Physiol Sci, SOKENDAI, Japan）
The basal ganglia receive cortical inputs and play a crucial role to execute voluntary movements. The motor cortex sends information to the input station of the basal ganglia, i.e., thestriatum and subthalamic nucleus (STN). The striatum is composed of two types of projection
neurons, i.e., striatonigral direct pathway and striatopallidal indirect pathway neurons. Striatonigral neurons send afferents to the substantia nigra pars reticulata (SNr), while striatopallidal
neurons project to the external segment of the globus pallidus (GPe). In the classical modelof the basal ganglia, striatonigral and striatopallidal neurons have opposite effects on motor
activity. Striatonigral neurons suppress the activity of the SNr and increase motor activity by
disinhibiting thalamic and cortical activity, on the contrary, striatopallidal neurons increase theactivity of the SNr through the striato-GPe-STN-SNr pathway and suppress motor activity. To
elucidate the mechanism of regulation of voluntary movements, it is essential to investigate
information flow along the basal ganglia circuit. We performed electrophysiological recordings
to understand relation between neuronal activity of the basal ganglia and motor activity underawake state. In this symposium, we will show the relation between neuronal activities in
the GPe and motor activity in the following three groups and discuss the role of the GPe on
regulation of voluntary movements.
1) We selectively ablated striatopallidal neurons by applying immunotoxin-mediated cell targeting method to transgenic mice. These mice showed increased motor activity.
2) We investigated pathophysiology of L-DOPA-induced dyskinesia (LID) by applying L-DOPA
to Parkinson’s disease (PD) model mice. PD and LID model mice showed decreased motor
activity and involuntary movements, respectively. We also examined physiological effects of ananti-parkinsonian drug on LID.
3) We investigated electrophysiology of catalepsy model mice induced by dopamine D2 receptor antagonist haloperidol. (COI:No)Is the external globus pallidus a simple relay of indirect pathway in thebasal ganglia circuitry?
The external globus pallidum (GPe) is located at a central position of the basal ganglia (BG)
circuitry and plays important roles in the physiology and pathophysiology of the BG. Classically, the GPe has been thought to be a relay station of striatal indirect pathway: the nucleus
receives direct inputs from the striatum and sends outputs to the subthalamic nucleus (STN),
and finally to the internal globus pallidus (GPi). However, anatomical studies demonstratethat the GPe receives reciprocal inputs from the STN. To support this idea, we have shownthat electrical stimulation of the primary motor cortex induces the triphasic response (early
excitation, inhibition, late excitation) in the GPe as well as the internal globus pallidum (GPi),and that the early excitation in the GPe is blocked by the muscimol inactivation of the STN
or intrapallidal injection of antagonists of ionotropic glutamate receptors. This reciprocal STNGPe transmission is also important for the generation of abnormal oscillations observed in BG
neurons in the parkinsonian primate model. To investigate the GPe firing in the behavioral
paradigm, we have recorded single unit activity in the GPe using a reward-biased saccade task.
A half of GPe neurons show inhibitory response during saccadic behavior, which may reflectstriatal excitatory response through the striato-GPe GABAergic transmission. On the other
hand, the remaining GPe neurons show excitatory response, which may reflect STN excitatory
response through the STN-GPe transmission. Additionally, our recent anatomical data indicatethat a population of GPe neurons project back to the striatum. Taken together, the GPe may
balance the cortico-STN-GPi ’braking system’ and the cortico-striato-GPi ’accelerator system’(COI:No)through the dual reciprocal connections with the STN and striatum.

PS07-02

Ventral Pallidum mediates acquired aversive tastes
Tadashi Inui（Department of Psychology and Program in Neuroscience, Florida State
University）
The ventral pallidum (VP) plays a critical role in the consumption of palatable substances. We
investigated its involvement in the decrease of a palatability of a solution caused by conditioned
taste aversion (CTA). The rats received a pairing of either of “sweet” saccharin sodium or“bitter” quinine hydrochloride solution as a conditioned stimulus (CS) with an intraperitoneal
injection of lithium chloride as an unconditioned stimulus. They were microinjected with bicuculline (a GABAA receptor antagonist) into the VP immediately before the CS test. The drug
treatment increased the intake of the saccharin CS but not quinine. Bicuculline alters the palatability of the saccharin CS from aversive to ingestive. Our study addressed the involvement
and impact of endogenous GABA in the VP on CTA. We measured the extracellular GABA
level before and after presenting saccharin or quinine in conditioned and non-conditioned
animals. Only the conditioned rats that were given saccharin showed an elevated GABA
level, suggesting that the VP GABAergic transmission signals decreased the palatability of anormally preferred sweet, but not aversive bitter, solution. We next investigated the nucleus
accumbens (NAc) that densely innervates the VP, by using a manganese-enhanced magnetic
resonance imaging (MRI) technique. Conditioned or non-conditioned rats were presented witheither saccharin (CS) or water and microinjected with manganese chloride as an MRI enhancer
and activity-dependent anterograde tracer into the NAc. The aversive CS increased manganese movements toward the VP, suggesting an augmented activity of the NAc-VP projection.
Another study demonstrated that the stimulation of mu-opioid receptors in the VP attenuatedaversion to the saccharin CS. Therefore, we conclude that the GABAergic and opioidergictransmissions in the VP, which may derive from the NAc, mediates learned aversive tastes(COI:No)and suppresses their consumption.

S16

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Planned Symposium 8

PS08-03

Regulation of ion channel expression in brainstem auditory circuit
Hiroshi Kuba（ Dept Cell Physiology, Grad Sch Med, Nagoya Univ, Japan）

Systems biology of hearing:from the inner ear to the brain（March 17, 15:20〜17:10, Hall 4）

PS08-01

In vivo detection of sub-nanoscale motion of outer hair cells in guinea
pig cochlea by a high performance optical coherence tomography
Fumiaki Nin（Dept Mol Physiol, Grad Sch Med Dent, Niigata Univ, Japan）
Sound evokes sub-nanoscale vibrations within the sensory epithelium in the mammalian cochlea. The epithelium includes not only immotile cells but also contractile outer hair cells that
actively shrink and elongate in synchronization with sound frequency. Vibrometrical studies
of cochlear mechanics have revealed the active vibrations in these cells. However, the real
motion of outer hair cells remains uncertain due to low spatial resolution of imaging systems
and low reflectivity of the cells. In this study, we dramatically improved performance of acommercial spectral-domain optical coherence tomography that can carry out vibrometry andexamined the sensory epithelium of live guinea pigs. With the tomography of this system wevisualized outer hair cells and separated them from other cellular components. When the
animals were exposed to stimuli at moderate sound pressure, we detected that, in the hair cells,
the amplitude of the vibrations on the apical edge exceeded that on the basal edge. In addition,
the phase of the motion in the former region led that in the latter. These characteristics may
contribute to high sensitivity and broad intensity range of normal hearing.
(COI:No)

Avian nucleus magnocellularis is a homologue of mammalian anteroventral cochlear nucleus,
and represents temporal information of sound with great precision for a wide frequency
range. This precision is accomplished in part because the expression of voltage-gated K+
channel, Kv1.1, is differentiated tonotopically within the nucleus; it increases toward highercharacteristic-frequency regions. However, the mechanisms underlying this differentiation
remained elusive. We examined the development of tonotopic differentiation of Kv1.1 channel
expression, using in-vivo and in-vitro preparations of chickens. Our findings were; (1) the
dependence of Kv1.1 expression on auditory input got strengthened specifically in neurons
tuned to higher-frequency sound at a late period of maturation, creating the difference in Kv1.1
expression among tuning frequencies; (2) attenuation of auditory input suppressed the differentiation in a level-dependent manner; (3) elevation of auditory input during earlier periods
could not reproduce the differentiation; (4) the differentiation of Kv1.1 completely disappearedin the organotypic culture, in which peripheral input was totally absent; (5) treating neurons
in the culture with a high K+ media augmented the expression of Kv1.1 only in those fromhigher-characteristic-frequency regions. Our results indicated that the capability of neurons to
express Kv1.1 via auditory input develops in a cell-specific manner and directs differentiation,highlighting the importance of neuronal capability as well as the level of input in the frequency
tuning of auditory circuits.
(COI:No)

PS08-04

Neural correlates of tinnitus in the auditory cortex of rat
Hirokazu Takahashi, Naoki Wake（Dept Mechano-Informatics, Univ Tokyo, Japan）
Tinnitus is the phantom perception of sound without an external source. The auditory cortex
is a putative neural origin of tinnitus because it plays critical roles in subjective perception ofsound. Specifically, a number of past studies demonstrated that the subjective percepts areassociated with neural synchrony in the sensory cortex. Furthermore, the auditory cortex
exhibits rich map plasticity depending on auditory learning and environments. Because tinnitus
is usually associated with hearing loss, we hypothesize that partial hearing loss induces distortion of the tonotopic map in the auditory cortex, and this distorted map causes aberrant neuralsynchrony of spontaneous activity, i.e., in silence. To address our hypothesis, we attempted toquantify tinnitus percepts in rats and explore the neural correlates of tinnitus in the auditory
cortex. Following unilateral exposure of 10-kHz, 125 dB-SPL tone for 60 min, behavioral tests
of prepulse and gap inhibition showed that rats exhibited a sign of tinnitus of 32 kHz tones
without hearing loss around 32 kHz. In these exposed rats, electrophysiological characterization in the auditory cortex demonstrated that the tonotopic map gained the areal proportionof exposed frequency of 10 kHz and that neural synchrony increased between 10 kHz and 32
kHz regions, i.e., the exposed and tinnitus frequency regions. These measures of hearing-lossinduced plasticity were significantly correlated with the behavioral index of tinnitus. Thus, thepresent results support our hypothesis that partial hearing loss induces the distorted tonotopic
map and aberrant neural synchrony, which are possible neural correlates of tinnitus.
(COI:No)

PS08-02

Regulation of glutamate release by Bassoon and Otoferlin at the innerhair cell ribbon synapse
Hideki Takago（Dept Rehab for Sensory Functions, Research Inst, National Rehab Center for
Persons with Disabilities, Saitama, Japan）

Sound encoding relies on Ca2+ -triggered exocytosis at the afferent synapse between the innerhair cell (IHC) and type I spiral ganglion neurons (SGNs). This auditory first synapse exhibits
wide variations in excitatory postsynaptic current (EPSC) size and shape. The IHC contains
a specialized structure called synaptic ribbon, which tethers plenty of synaptic vesicles forsecurely transferring continuous auditory signals. In the IHC active zones, the scaffold protein
Bassoon and the hair cell-specific putative Ca2+ sensor Otoferlin regulate synaptic ribbon
formation and synaptic vesicle cycle, respectively. Here we aimed to elucidate structural andfunctional roles of Bassoon and Otoferlin in IHC neurotransmission. Immunohistochemical
analysis revealed that most of, but not all of, Bassoon immunopuncta are coupled to synaptic
ribbons, whereas those of Otoferlin are widely distributed in IHCs. Postsynaptic patch-clamp
recordings showed that disruption of Bassoon or Otoferlin drastically reduced EPSC frequency
during IHC depolarization. Moreover, large amplitudes of EPSCs persisted in both mutants
despite lowered EPSC frequency. These results support the hair cell-specific hypothetical release mechanism (i.e. univesicular release) that a single vesicle fusion might produce EPSC sizeand shape diversity, and suggest that Bassoon and Otoferlin are involved in this mechanism.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S17

Planned Symposium 9
Scientific basis of the oriental medicine:mechanisms improving muscle bloodflow at rest
（March 17, 15:20〜17:10, Hall 9）

PS09-03

Contribution of vasodilators to the increase in muscle blood ﬂow after
manual acupuncture
Hisashi Shinbara（ Dept Acp Mox Ther, Fac Hlth Promo Sci, Tokoha Univ, Japan）
Increase in muscle blood flow (MBF) induced by acupuncture is considered to contributeto muscle recovery by washing out waste and algesic substances. However, the underlying
mechanism is unclear. Thus, we studied this mechanism using radiolabeled or fluorescent
microspheres, which are able to quantitatively and noninvasively estimate MBF. Additionally,
we used receptor antagonists and enzyme inhibitors to clarify the involvement of vasodilators.
Manual acupuncture (MA) with the sparrow pecking technique significantly increased MBF of
the hindlimb in rats. The increase was observed only in the muscles where the acupunctureneedle was inserted. The increase was not affected by acute denervation (cutting of thesciatic nerve). However, the increase was significantly but incompletely suppressed by topicalinjection of hCGRP, a CGRP receptor antagonist. Moreover, the increase was not observedin capsaicin-treated rats. These results suggested the involvement of the axon reflex. Nevertheless, the increase was also observed in chronically denervated rats.
The increase was significantly but incompletely suppressed by theophylline, a non-selective
adenosine receptor antagonist; suramin, a purine 2 receptor antagonist; or L-NAME, a nitricoxide (NO) synthase inhibitor. In contrast, the increase was not affected by indomethacin, a
cyclooxygenase inhibitor.
Our studies suggest that the increase in MBF after MA is caused by the following vasodilators:
1) CGRP, which is released from afferent nerve endings via the axon reflex, 2) ATP whichis leaked from muscle cells damaged by acupuncture needles, and 3) ADP and adenosine
which are the degradation products of ATP. As a result, the binding of these vasodilators totheir respective receptors facilitates the production of NO in the vascular endothelium, which
relaxes the vascular smooth muscle. The contribution of other vasodilators is suggested, butvasodilatory prostaglandins do not seem to be involved.
(COI:No)

PS09-01

PS09-04

Masataka Sunagawa（ Dept Physiol, Sch Med, Showa Univ, Japan）

Kenichi Kimura（ Dept Health Sci, Kansai Univ Health Sci, Japan）

The blood flow depends on the cardiovascular system condition and blood characteristics.
For instance, the cardiac contractile force and elasticity and the inner diameter of blood vessels influence the flow as cardiovascular system conditions, and the blood coagulation ability,
platelet aggregation activity, fibrinolytic activity, erythrocyte deformability and blood viscosity
affect it as blood characteristics.
In Eastern medicine, the pathophysiologic concept of blood stasis is called Oketsu. Some Kampo
medicines (Japanese traditional herbal) and acupuncture treatment are empirically known tobe effective in ameliorating Oketsu. Crude drugs such as Peach kernel (Latin=Persicae Semen/
Japanese=Tonin), Moutan bark (Moutan Cortex/Botanpi), and Rhubarb (Rhei Rhizoma/Daio)
have the strong effect of improving Oketsu, while Japanese angelica root (Angelicae Acutilobae
Radix/Toki), Cnidium rhizome (Cnidii Rhizoma/Senkyu), and Peony root (Paeoniae Radix/
Shakuyaku) have a mild effect. Kampo medicines including these agents, such as Tokakujokito,
Keishibukuryogan, Tokishakuyakusan, Tsudosan, Jidabokuippo, and Daiobotanpito, have beenused for patients with Oketsu. We focused on the blood fluidity influenced by changes inblood characteristics and the platelet aggregation activity. In rats administered Tokakujokito,
Keishibukuryogan, or Tokishakuyakusan, the blood fluidity improved, and the platelet aggregation activity decreased. Furthermore, in patients diagnosed with Oketsu-type disease, thedecreased blood fluidity and exacerbated platelet aggregation activities improved following theadministration of these medicines.
We next performed acupuncture treatment at relevant acupoints in rats, such as LI-14(Chinese=Hegu/Japanese=Gokoku), SP-6 (Sanyinjiao/Saninko), and ST-36 (Zusanli/Ashi-sanri).
As the result, the blood fluidity improved, and the platelet aggregation activity decreased.
However, the acupoints PC-6 (Nei Guan/Naikan) and BL-23 (Shenshu/Jin-yu) interestingly
showed no influence on the condition.
These findings suggest that acupuncture and Kampo medicine treatments are effective for
improving the blood flow.
(COI:No)

Acupuncture improves local muscle blood flow (MBF), which may flush out algesic or sensitizing substances to relieve pain. Therefore, acupuncture has been used to treat musculoskeletal
impairments such as shoulder stiffness and muscle pain. The calcitonin gene-related peptide(CGRP) and substance P (SP) released from sensory nerve terminals may be partially responsible for the mechanism underlying the improvement in local MBF elicited by acupuncture.
Recent studies have indicated that nitric oxide (NO) and adenosine are also involved in theincreased MBF induced by acupuncture. In addition to local blood flow, acupuncture may
influence distant blood flow through the vasomotor nerve. Muscle sympathetic nerve activity(MSNA) is thought to primarily represent vasoconstrictor fiber activity, which innervates theskeletal blood vessels, thereby playing an important role in regulating the MBF. We previously reported that locally applying heat induced an increase in MBF at the heated area by
inhibiting MSNA. The increased MBF following acupuncture may also help suppress MSNA.
However, whether acupuncture confers sympatho-inhibitory effects on MSNA in humans
remains unknown. We examined whether MSNA plays a role in the increased MBF following
acupuncture and found that acupuncture did not alter the MSNA at rest. Thus, suppressing
MSNA did not mediate the increased MBF. Therefore, the increased MBF following acupuncture may be mainly due to vasodilators such as CGRP, NO, and adenosine rather than MSNA
suppression. (COI:No)

Improvement of the blood ﬂow following acupuncture and Kampo
herbal medicine treatments

Neural mechanism of the improvement of muscle blood ﬂow elicited byacupuncture or local heat application

PS09-02

Mechanosensing and regulation of blood ﬂow in vascular endothelialcells
2 1 Lab System Physiol, Grad Sch Med, U Tokyo, Japan,
Kimiko Yamamoto1，Joji Ando（
2 Lab Biomed Eng, Sch Med, Dokkyo Med U ）The endothelial cells (ECs) that line the lumens of blood vessels are constantly exposed to a
fluid dynamic force, the shear stress generated by the blood flow. The ECs sense shear stressand transduce it into intracellular biochemical signals, resulting in changes in the morphology, functions, and gene expression profiles of the cells. These EC mechanoresponses are
critical for the homeostasis of the circulatory system, and their impairment can lead to thedevelopment of various vascular diseases such as hypertension, aneurysm, thrombosis, andatherosclerosis. Ca2+ signaling via a P2 purinoceptor P2X4 plays a significant role in thismechanotransduction which leads to the regulation of vascular functions, including the controlof blood pressure, blood flow-induced vasodilation, and flow-dependent vascular remodeling.
The ECs release ATP in response to shear stress and then the released ATP activates P2X4,
which results in flow-dependent intracellular Ca2+ responses; however, the mechanism bywhich the shear stress evokes ATP release remains unclear. Here we report that the cellular
mitochondria play a critical role in this process. Cultured human pulmonary artery ECs were
exposed to controlled levels of shear stress in a flow-loading device, and the changes in the
mitochondrial ATP levels were examined by real-time imaging using a fluorescence resonanceenergy transfer-based ATP biosensor. Immediately upon the exposure of the cells to flow, themitochondrial ATP levels increased, which was both reversible and dependent on the shear
stress intensity. Inhibitors of the mitochondrial electron transport chain and ATP synthaseabolished the shear-stress-induced mitochondrial ATP generation, resulting in the loss of the
ATP release and intracellular Ca2+ responses. These results indicate the novel role of endothelial mitochondria as mechanosignaling organelles that can transduce shear stress information
into ATP generation, triggering ATP release and purinoceptor-mediated Ca2+ signaling which
results in regulation of vascular functions.
(COI:No)S18

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Planned Symposium 10
Japan-China Joint Symposium
Cutting edge of cell fate determination andmigration in the developing cerebral cortex（March 18, 9:00〜10:50, Hall 2）

PS10-03

Mechanisms of neocortical organization by neuronal activity of subplate
neurons
Chiaki Ohtaka-Maruyama1，Noe Kaneko1, 2，Ai Fujii1, 2，HIdeya Kawaji3，Kei Yura2, 4（ 1Tokyo Metropol. Inst. Med. Sci., Neural Network, 2 Ochanomizu Univ. Dept. Science,
3 Tokyo Metropol. Inst. Med. sci., Genomic Med., 4Waseda Univ. Sch. Adv. Sci.
Engineering）
In the mammalian neocortex, an enormous number of neurons are precisely arranged in anordered 6-layered structure in an inside-out manner. This structure is formed by the sequential
generation of neurons and their migration toward the brain surface, termed radial neuronalmigration. In order to complete the neocortical layer structure within the limited time periodof embryogenesis, the radial migration process must be controlled precisely and efficiently.
We previously reported that subplate neurons (SpNs), which are one of the earliest bornand matured types of neurons in the developing neocortex, play an important role in theregulation of radial migration. We revealed that SpNs exhibit spontaneous calcium oscillationsat E15, and actively extend processes to contact newly born multipolar migrating neurons.
Electron microscopic observation demonstrated that synapse-like structures were formed atthese contact sites. This synaptic communication leads to the switch from multipolar migration to locomotion. It is known that SpNs are the first cortical neurons to receive sensory
input from thalamic axons. SpNs then project axons toward layer IV neurons to establish atemporal link between thalamic axons and their final target in layer IV. This initial wiringalso depends on SpN activity, suggesting that they function as a conductor for mammalian
neocortical formation by organizing multiple processes. To elucidate the function of SpNs,
we are now characterizing their subpopulations using an SpN-specific transgenic mouse lines(Lpar1-EGFP or Cre-ERT2 line;D1B-Cre). We found that they have different characteristics interms of molecular marker expression. This can be a piece of evidence for SpNs consisting ofheterogeneous cell populations, and supports the idea that each subpopulation has distinct roles
in neocortical formation. We are currently analyzing single cell RNA-seq data obtained from
Lpar1-GFP positive cells using C1 single cell prep system to clarify gene expression profilingof SpNs.
(COI:No)

PS10-01

PS10-04

Yusuke Kishi, Naohiro Kuwayama, Yoshikuni Wada, Yukiko Gotoh（Grad Sch Pharm, Univ

Kazunori Nakajima（ Dept Anat, Keio Univ Sch Med, Japan）

Chromatin regulation during neural developmentof Tokyo, Japan）

Neurons, an essential cell type for brain function, are derived from neural stem/progenitor
cells (NPCs). During the early stage of mouse neocortical development, NPCs proliferate symmetrically to increase their pool size (the expansion phase). They then acquire neurogenic
competence and generate neurons (the neurogenic phase). The transition of the expansionto-neurogenic phase is critical for determining the number of NPCs and neurons and thus
should be strictly regulated. However, it remains unclear how the timing of this transition isregulated, partly due to the difficulty of genetic manipulation of NPCs during the expansion
phase. We therefore developed a new method to express gene-of-interest in NPCs at E8 during
the expansion phase. In addition, we examined the changes in chromatin accessibility andtranscriptional profile during the transition of the expansion-to-neurogenic phase to list up(COI:No)
candidate molecules regulating the transition.

Novel migration mode in the developing cerebral cortex
Neurons and astrocytes are generated directly or indirectly from the progenitors in the ventricular zone (VZ). When we observed mouse cortical slices at embryonic day (E) 17.5 that had
been transfected into the VZ with a green fluorescent protein (GFP) expression vector 2 days
earlier using in utero electroporation, we found that some GFP-positive cells migrated rapidly
and almost randomly within the IZ and CP. These cells divided frequently, and the daughter
cells continued to exhibit the same irregular movement. Since this migration pattern was different from that of well-known radial migration, by which neurons migrate from the IZ to CP,
we named this novel migration mode “erratic migration”. Cells undergoing erratic migration(hereafter referred to as “erratic migration cells”) moved more quickly than those undergoingradial migration. Erratic migration cells frequently changed direction, although they ultimately
tended to move toward the brain surface. To determine the final fate of the erratic migration
cells, we performed electroporation at E15 to label them with a photo-convertible protein,kikGR. After identifying the cells undergoing erratic or radial migration in slices preparedat E16, we irradiated these cells with a 405-nm laser, dissociated the slices, and cultured thecells on cover slips. Most of the erratic migration cells differentiated into astrocytes, whereas
the cells that had moved in a radial migration mode differentiated into neurons. These results
indicated that erratic migration cells derived from the cortical VZ were astrocyte progenitors.
Further analyses of these cells will be presented.
(COI:No)

PS10-02

Regulation of neural differentiation and cell migration during neocortex
development
Qin Shen（School of Life Sciences and Technology, Tongji Hospital, Tongji University,
China）
Neural progenitor cells (NPC) in the developing neocortex undergo precisely controlled proliferation and differentiation to ensure proper generation and settlement of neurons for correct
cortical architecture. Genes expressed in NPCs but downregulated upon differentiation areoften studied for their roles in promoting proliferation and inhibiting differentiation, but it is lessclear whether mis-regulated expression would also disrupt the behavior of their differentiatedprogeny. Zinc finger E-box binding homeobox 1 (Zeb1) is well known as a key regulator of
the epithelial-mesenchymal transition and cancer metastasis. Mutation of Zeb1 is associatedwith human genetic eye diseases and defective brain development. However, its role in neuraldevelopment is less clear. We found that Zeb1 is expressed in NPCs but turned off during
neuronal differentiation in the developing neocortex. Altering Zeb1’s expression in cortical cells
in vivo by in utero electroporation affected neuronal differentiation and migration. Surprisingly, overexpression of Zeb1 did not increase proliferation of NPCs in the germinal zone, but
disrupted the correct positioning of NPCs and differentiating neurons. This was accompanied
by suppression of NeuroD1, a transcription factor important for neuronal differentiation, anddelayed multipolar-to-bipolar transition. Consequently, long-term overexpression of Zeb1
caused severe migration defects and heterotopia bands in the white matter at postnatal stage.
We found that Zeb1 regulated a cohort of genes involved in cell differentiation and migrationand identified CTBP2 as the functional partner of ZEB1 in the embryonic cortex. Similarly,
overexpression of CTBP2 also repressed the neuronal migration. Binding to CTBP2 is required
for ZEB1 to elicit the effect on multipolar-to-bipolar transition but not suppression of NeuroD1.
This study demonstrates that a gene specifically expressed in NPCs can affect aspects ofneuronal behavior if its expression is mis-regulated, providing an example of requirement for
dynamic control of gene expression during cortical development.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S19

Planned Symposium 11

PS11-03

The role of thalamic matrix cells in wake/sleep cycle regulation
Sakiko Honjoh（IIIS, Univ of Tuskuba）

Women scientists in the optogenetic field –the new fact has come to light using optical
approaches（March 18, 9:00〜10:50, Hall 4）

PS11-01

Cell-type-speciﬁc patterned activities specify gene expression patterns
for olfactory circuit formation
Ai Nakashima（ Grad. Sch. of Pharm. Sci., Univ. of Tokyo）
The development of precise neural circuits is initially directed by genetic programmingand subsequently refined by electrical neural activity. The most prevailing model for theactivity-dependent development of neural circuits postulates the interaction between pre- andpost-synaptic neurons. In Hebbian plasticity, the correlated activity of pre- and post-synaptic
neurons strengthens synaptic connections, whereas uncorrelated or lack of activity weakens
them. However, the olfactory map develops even in mutant mice lacking synaptic partners,
suggesting another activity-dependent mechanism for the olfactory map formation. During
development, axons from various olfactory neurons expressing the same olfactory receptor (OR) segregate into specific glomeruli in an activity-dependent manner. We found that
OSNs exhibited OR-specific temporal patterns of spontaneous activities. Moreover, differing
patterns of neuronal activity induced different expression patterns of axon-sorting moleculesthat regulate glomerular segregation. We propose an activity-dependent mechanism that isfundamentally different from the Hebbian plasticity theory in which cell-type-specific patterned
activity contributes to generating the combinatorial code of axon-sorting molecules for the(COI:No)olfactory map refinement. The neocortex and the thalamus are reciprocally connected and are believed to be criticalfor arousal and cognitive function. Thalamic neurons that project to the cerebral cortex are
divided into two neural subpopulations, core and matrix cells, based on their projection patterns. Core cells are enriched in sensory and motor relay nuclei and project mainly to layer 4of specific cortical areas. Matrix cells abound in some intralaminar and medial thalamic nuclei
project diffusely to cortex, primarily to superficial layers. In particular, multiareal type matrix
cells may constitute a veritable thalamic activating system that facilitates effective interactions
among many cortical areas and thereby sustains arousal and consciousness. Here we focusedon ventromedial nucleus (VM), which is comprised of only multiareal type of matrix cells. We
found that VM matrix cell activity is highly correlated with activated EEG, with sustained highfiring during wake and REM sleep and low firing during NREM sleep in freely behaving mice.
VM matrix cells increased firing before cortical activation in both transitions from NREM sleep
to wake or REM sleep, suggesting its role in cortical activation. To investigate its causal role,
we employed optogenetic stimulation and found that high frequency stimulation of matrix cells
elicits cortical activation and behavioral arousal from NREM sleep. However, interestingly,
the identical optogenetic stimulation failed to promote arousal from REM sleep showing thevigilance state-dependent thalamo-cortical interaction. Our analyses revealed that Grangiercausality from thalamus to cortex is the highest during wake and the lowest during REM sleep.
The low causality from thalamus to cortex during sleep could be a potential mechanism for thevigilance state-dependent fluctuation of consciousness and cognition.
(COI:No)

PS11-04

Optical measurements of brain energy dynamics during sleep
2 1Grad Sch Life Sci, Tohoku Univ, Japan, 2 JST, PRESTO）
Tomomi Tsunematsu1, （Mammalian sleep can be classified into two categories, i.e., rapid eye movement (REM) sleep
and non-rapid eye movement (NREM) sleep. However, we still do not know why we haveto sleep despite repeating the behavior every day. One attractive hypothesis is the energy
conservation theory. It is reasonable to conserve energy by sleeping since the brain is surely
an organ that consumes so much energy. Here, we challenged to clarify the energy dynamics
in brain cells during sleep in mice using optical imaging. First, we focused on the change ofintracellular calcium concentration in astrocyte which receives energy from blood vessels
and passes it to neurons. Next, we revealed the dynamics of intracellular ATP, which is ametabolic molecule and used as energy source, concentration in neurons. Our in vivo optical
recording with genetically-encoded calcium and ATP fluorescent sensor demonstrated that
intracellular calcium level of astrocyte and ATP level of neuron globally fluctuated acrosssleep/wakefulness state, especially in REM sleep. These results help us to understand physiological significance of sleep from an energy perspective.
(COI:No)

PS11-02

Glutamatergic input into the dorsal raphe nucleus and aggressive
arousal
Aki Takahashi（Lab of Behav Neuroendocrinol, Univ Tsukuba, Japan）
Escalated aggression, or violence, has been huge problem in public health. In many cases, violence incidents are triggered by social instigation or frustration. Social instigation-heightened
aggression has been observed in several animal models from fish to rodents in which shortencounter with a potential rival escalates aggressive behavior in the subsequent encounter
with an opponent due to increases of aggressive arousal of individuals. We have shown an involvement of excitatory input into the dorsal raphe nucleus (DRN) in the instigation-heightened
aggression in male mice. In vivo microdialysis showed that glutamate release increased in the
DRN during an aggressive encounter, and the level of glutamate was further increased when
the animal was engaged in escalated aggressive behavior after social instigation. Importantly,
the increase of glutamate was observed during the period of social instigation without direct
physical interaction, suggesting that the glutamate input into the DRN may represent a statusof arousal which increases aggressive behavior in the following encounter. We also found thatmicroinjection of L-glutamate into the DRN escalated aggressive behavior of male mice. The
DRN receives glutamatergic projections from many brain areas including the prefrontal cortex,
lateral habenula, and hypothalamus. In this talk, I will talk about our approach to identify theorigin of glutamate input into the DRN which is involved in aggressive arousal and causes anescalation of aggressive behavior by using optogenetics and DREADD techniques. (COI:No)

S20

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Planned Symposium 12

PS12-03

Drug screening using zebraﬁsh models of muscle diseases
Genri Kawahara, Yukiko Hayashi K（Dept PathoPhysiol, Tokyo Med, Univ, Japan）

New frontiers of locomotion research using
zebrafish as a model system（March 18, 9:00〜10:50, Hall 5）

Zebrafish are useful to analyze the pathomechanism of various human diseases. There are some
excellent zebrafish models of muscle diseases including muscular dystrophies, which show
severe abnormalities in skeletal muscle easily detected by birefringence analysis. Moreoverzebrafish are very powerful tools for therapeutic drug discovery because their characteristics(small size, fast development, clear muscle structures and muscle phenotypes in mutant fish)
are ideal for drug screening. Zebrafish muscle disease models can be used in high-throughput
drug screens to identify candidate drugs that improve the muscle disease phenotypes. In thispresentation, we will show characteristics of model fish of muscular dystrophies and results of(COI:No)drug screening to discover future therapy for muscle diseases.

PS12-01

PS12-04

Shin-ichi Higashijima（ NINS, ExCELLS/NIBB, Japan）

2 1 Dept Physiol,
Buntaro Zempo1，Yasuhiro Yamamoto1，Tory Williams2，Fumihito Ono1, （Regulation of locomotor speed and selection of active sets of neurons
by V1 neurons
During fast movements in vertebrates, slow motor units are thought to be deactivated due tothe mechanical demands of muscle contraction, but the associated neuronal mechanisms forthis are unknown. Here, we performed functional analyses of spinal V1 neurons by selectively
killing them in larval zebrafish, revealing two functions of V1 neurons. The first is the longproposed role of V1 neurons: they play an important role in shortening the cycle period duringswimming by providing in-phase inhibition. The second is that V1 neurons play an importantrole in the selection of active sets of neurons. We showed that strong inhibitory inputs comingfrom V1 neurons play a crucial role in suppressing the activities of slow-type V2a and motor
neurons, and, consequently, of slow muscles during fast swimming. Our results thus highlightthe critical role of spinal inhibitory neurons for silencing slow-component neurons during fast
(COI:No)
movements.

Synaptic silencing of fast muscle is compensated by rewired
innervation of slow muscle
Div Life Sci, Faculty of Med, Osaka Medical College, 2 Laboratory of Molecular Physiology,
NIAAA, NIH ）It is generally accepted that the skeletal muscle in vertebrates consists of two types ofmuscle fibers: slow and fast. For decades, numerous studies have proposed that fast muscles
contribute to quick movement, while slow muscles underlie locomotion requiring endurance.
By generating mutant zebrafish whose fast muscles are synaptically silenced, we examinedthe contribution of fast muscles in both larval and adult zebrafish. Zebrafish have various
advantages in studying synapses in slow and fast muscles. Slow and fast muscle cells inzebrafish are spatially segregated and can easily be distinguished by their anatomical andhistological characteristics. In addition, recent studies suggested distinctive compositions of
nicotinic acetylcholine receptors (AChR) in slow and fast muscles of zebrafish. In zebrafish aswell as in mammals, nicotinic AChR expressed in the neuromuscular junction of fast muscles
are pentamers, composed of α, β, δ, and ε (or γ) subunits. γ subunits are expressed inlarvae and change to ε as the animal matures. However, recent studies showed that AChRs
in slow muscles of zebrafish lack γ/ε subunits, and are composed of only α, β and δ
subunits. In the present study, we established knockout zebrafish lines that lacked ε and/or γ subunits, and analyzed their locomotion and synaptic traits. In the larval stage, mutants
lacked the characteristic startle response to tactile stimuli: bending of the trunk (C-bend)
followed by robust forward propulsion. Surprisingly, adult mutants with silenced fast muscles
showed robust C-bends and forward propulsion upon stimulation. Retrograde labeling revealed
that motor neurons genetically programmed to form synapses on fast muscles are instead
re-routed and innervate slow muscles, which led to partial conversion of slow muscles to fastmuscles. Thus, extended silencing of fast muscle synapses changed motor neuron innervationand caused muscle cell type conversion, revealing an unexpected mechanism of locomotary(COI:No)
adaptation.

PS12-02

Swimming characteristics of different zebraﬁsh strains assayed by aswimmill
Hiromi Hirata, Yuma Wakamatsu（Aoyama Gakuin University, Sagamihara, Japan）
Several zebrafish strains such as AB, Tübingen (TU), Wild India Kolkata (WIK) and Tupfel
long fin (TL) have been established for genetic study. Each strain has its morphological andbehavioral traits. Motor traits, however, have not been explored in zebrafish strains. We here
applied a swimmill, which is a treadmill for fish species, and measured swimming capabilityof adult zebrafish by critical swimming speed, which is the maximum water velocity in whichfish can keep swimming. First, we confirmed that swimming capability does not vary between
female and male. Second, we found that the appropriate water temperature for swimming was
between 16 and 30 oC. Third, our fin clip experiments using long-finned zebrafish revealed
that they can exhibit high swimming capability when the caudal fin length was set between
3 and 10 mm, implying that long-finned zebrafish are unfavorable for fast swimming. Finally,
we compared swimming capability of several zebrafish strains and demonstrated that WIK
fish was significantly less capable of swimming despite that they have short caudal fin (~9mm). The offspring of WIK fish were less capable of swimming, while hybrids of WIK and
TU showed high swimming performance. Thus, lower swimming capability of WIK strain is(COI:No)
inheritable as a motor trait.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S21

Planned Symposium 13
Combined approaches of cutting-edge
techniques and structural information toreveal the elaborate dynamics of membraneproteins（March 18, 9:00〜10:50, Hall 9）

PS13-01

Cryo-EM analysis of the volume-regulated anion channel LRRC8
Go Kasuya（ Dept Physiol, Jichi Medical Univ, Japam）
Cell volume regulation against intracellular and extracellular osmotic alterations is a fundamental homeostatic mechanism for all organisms. Various ion channels are involved in cellvolume regulation. The leucine-rich repeat-containing 8 (LRRC8) protein family, composed of
the five isoforms named LRRC8A-E, are considered to serve as a pore-forming component of
the volume-regulated anion channel (VRAC), which is activated by cell-swelling and permeates anions and other organic osmolytes to the extracellular side, thereby facilitating water
efflux to counteract cell-swelling. As the overall sequence of LRRC8 has little similarity to any
other known classes of ion channels, the detailed structural information has been awaited toclarify the molecular mechanisms of the LRRC8 protein family. Here, I present the present
two homomeric structures of human LRRC8A and LRRC8D isoforms, determined by singleparticle cryo-electron microscopy. These two structures show a hexameric assembly, and thetransmembrane region features a topology similar to gap junction channels. The LRR region,
containing 15 leucine-rich repeats, forms a long, twisted arc, and is flexible and mobile, withrigid-body motions, which might be implicated in structural transitions on pore opening andclosing. The channel pore is located along the central axis and constricted on the extracellularside, where the residues at the tip of the extracellular helix contribute to permeability to
substrates. The structural comparisons with the LRRC8A and LRRC8D isoforms, together
with the structure-based electrophysiological analysis, revealed the two key features involvedin substrate permeability: the wider pore constriction on the extracellular side of the LRRC8D
isoform than that of the LRRC8A isoform, and an additional helix facing to the channel pore
formed by the N-terminal residues observed only in the LRRC8D isoform. Overall, this work
provides a framework for understanding the mechanisms of this unique class of ion channels.
(COI:No)

PS13-03

The conformational change of the cytoplasmic region of voltagesensing phosphatase
Akira Kawanabe1, 2，Manami Nishizawa3，Kazuhisa Nishizawa3，Hirotaka Narita4，
Tomoko Yonezawa2，Yuka Jinno2，Souhei Sakata5，Atsushi Nakagawa4，Yasushi Okamura2（ 1 Fac. Med., Kagawa. Univ., Kagawa, Japan, 2 Grad. Sch. Med., Osaka Univ., Osaka, Japan,
3 Fac. Med. Tech., Teikyo Univ., Tokyo, Japan, 4 IPR, Osaka Univ., Osaka, Japan, 5 Fac.
Med., Osaka Med. Coll., Osaka, Japan）
Conformational changes of cytoplasmic regions of membrane proteins play fundamental roles
both in membrane electrical signals and signal transduction. However, detailed analysis of suchconformational change in living cells has been difficult because of the lack of efficient molecular
tools. To overcome this issue, we utilized a cytoplasmic fluorescent-label technique and detected its
stimulation-dependent fluorescent changes. In this presentation, we introduce our recent studies on
voltage-induced conformational changes of cytoplasmic region of VSP (Sakata et al. 2016, Kawanabe
et al. 2018).
Voltage sensing phosphatase (VSP) consists of a voltage sensor domain (VSD) and a cytoplasmic
catalytic region (CCR) which has phosphatase activity toward PI(4, 5)P2 regulated by membranepotential change. Although the X-ray crystal structure of each domain in Ciona intesinalis VSP (CiVSP) has been individually resolved, the coupling mechanism from the voltage sensor to enzymatic
activity has not been fully understood.
From our dynamics simulations of phospholipid bilayer/CCR systems, we found the characteristichydrophobic region on membrane-protein interface in Ci-VSP (Leu284 and Phe285, called “hydrophobic spine”) which may play an important role in the coupling. Systematic mutation screening
in the hydrophobic spine showed the voltage-dependent phosphatase activity depends on thehydrophobicity and presence of aromatic ring in the amino acid side chain.To gain more mechanicalinsights, we attempted to analyze the conformational changes of the cytoplasmic region of Ci-VSP
with unnatural fluorescent amino acid (Anap) introduced at Lys555. Two components of voltagedependent fluorescence changes were detected in K555Anap (smaller fast decrease and larger
slow increase). In contrast, a larger component was enhanced in a higher-activity mutant (L284F/
K555Anap), whereas it disappeared in a lower-activity mutant (L284Q/K555Anap). These findings
indicate that VSP has two-step activation (resting to partially-activated and, then to fully-activatedstate) and the latter transition depends on the nature of the hydrophobic spine.
(COI:No)

PS13-04

Unveiling the lipid bilayer effects on the ion channel function using a
cutting-edge artiﬁcial lipid bilayer technique
Masayuki Iwamoto（Dept Molec Neurosci, Univ Fukui Fac Med Sci）
All the membrane proteins are under the effects of the lipid bilayer properties such as chemical composition of phospholipids and physical force from the lipid bilayer structure. Taking
these factors into consideration would be essential for understanding the functional mechanismof membrane proteins at molecular level. To date, numerous information concerning such
effects has been accumulated and the specific lipid bilayer environment, which is essential
for the activity, has been revealed for various membrane proteins. However, the quantitative
aspect and the molecular mechanism of the lipid bilayer effects remain unclear for many cases
because of difficulties in the functional assay under the strictly regulated lipid bilayer environment. To overcome such problems, we have developed an artificial lipid bilayer technique,the contact bubble bilayer (CBB) method. The CBB method enabled us the single-channel
current recording of ion channels under the completely regulated lipid bilayer composition andtension. Using the CBB method, we have revealed the molecular mechanism of the anionic lipid
effect on the open probability of the KcsA potassium channel, a canonical ion channel protein.
Furthermore, we have showed quantitatively that the lipid bilayer tension was necessary for
full activity in addition to the ligand binding. The lipid bilayer-dependent property found in the
KcsA channel might be a common feature of ion channel proteins.
(COI:No)

PS13-02 (AP-3)

PI(3, 4)P2- and voltage-dependent gating of two-pore Na+ channel 3
2 1 Div Biophys and Neurobiol,
Takushi Shimomura1, 2，Ki-ichi Hirazawa1, 2，Yoshihiro Kubo1, （Natl Inst Physiol Sci, 2 Dept Physiol Sci, SOKENDAI ）Two-Pore Na+ Channels (TPCs) contain two domains (DI and DII) of a functional unit ofvoltage-dependent cation channels. Each domain has its own voltage sensor domain that possesses three positively charged arginine residues in helix S4. Characteristically, TPC3 shows
the shift of the voltage dependence by long depolarization stimulus, so called as “induction”, in
Xenopus oocyte expression system.
The structural basis of this “induction” mechanism was investigated using multiple approaches
based on two-electrode voltage-clamp technique and the structural model of TPC3. We foundthe correlation between PIP2 level and “induction” currents. Simultaneous recordings of TPC3
current and the fluorescence from specific PIP2 sensors showed that PI(3, 4)P2 concentration isincreased by long depolarization stimulus, possibly through any endogenous system in Xenopus
oocytes. The “induction” kinetics of TPC3 is well correlated with the fluorescent change of PI(3,4)P2 sensors, but not with that of PI(4, 5)P2. The PI(3, 4)P2 sensitivity of TPC3 was confirmed
using direct phosphoinositide injection method and the excised-patch membrane. These results
reveal that “induction” is PI(3, 4)P2-induced modulation of voltage dependence of TPC3. We also
found that a cluster of basic amino acid residues in the cytosolic side of DI is critical for PI(3,4)P2 sensitivity. Interpretation of the mutational effects based on the TPC3 structural model
explained how TPC3 selectively recognizes PI(3, 4)P2 in DI region. While DI recognizes PI(3,4)P2, DII is considered to be mainly responsible for voltage-sensing. The voltage-dependent
movement of DII-S4 was verified using the voltage clamp fluorometry, in which the voltagedependent fluorescent change was detected from the fluorophore incorporated into someresidues in DII-S4. These electrophysiological data of various approaches, combined withthe structural model, revealed the detailed voltage-dependent gating mechanism of TPC3.
(COI:No)S22

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Planned Symposium 14

PS14-03

Past, present and future of JPS
Yoshihiro Ishikawa（ CVRI, Grad Sch Med, Yokohama City Univ, Japan）

Past, present and future of JPS（March 18, 15:20〜17:10, Hall 1）

The Journal of Physiological Sciences is the only English Journal issued by the PhysiologicalSociety of Japan and has a history of more than sixty years. The quality and reputation of the
Journal has been improved significantly in the past several years due to the major contributions by our Society members. Its impact factor is now more than 3, which is equivalent tothat of American Journal of Physiology. Thus, our Journal is growing from a major Asian
physiological Journal to a world-class Physiological Journal. In order to achieve the next goalfor us, the Journal will become an open access journal from 2020. The Journal already started
to accept manuscript submission to a new open access Journal. In the Symposium, we will
explain our progress to our young researchers. In particular, we will explain the history of ourJournal as well as how to contribute their work to our Journal successfully. We will also explain
the status of other major physiological Journals such as Journal of Physiology in England.
(COI:No)

PS14-01

About The Journal of Physiology
Yasushi Okamura（ Dept Physiol, Grad Sch Med, Osaka Univ, Japan）
The Journal of Physiology has been run by the Physiological Society in UK as one of the
traditional international journals in the field of physiology since its foundation in 1878. Many
important works in the fields of physiology were published in JP. This includes Sherrington’sconcept of neural networks and reflex, Adrian’s first electrical recording of action potentials,
Erlanger and Gasser’s conduction velocity measurement of peripheral nerves and their classification of nerve fibers, Hodgkin and Huxley’s works of voltage-gated ion channel and Katz’s
works of mechanisms of synaptic transmission including quantum vesicle release. I will talkabout how policies of the Journal have been changed with progress of science and the styleand environments of research activities in the academics. I will also briefly explain current(COI:No)reviewing processes and scope of papers which the Journal now covers.

PS14-02

About Pﬂügers Archiv European Journal of Physiology
Makoto Tominaga（ Div Cell Signaling, Natl Inst Physiol Sci）
Pflügers Archiv European Journal of Physiology is a peer-reviewed scientific journal in the fieldof physiology. A continuation of a journal founded in 1868 by the German physiologist, EduardFriedrich Wilhelm Pflüger, Pflügers Archiv is the oldest physiological journal, and currently
published by Springer with 11 issues per year. Editor-in-Chief is Dr. Armin Kurtz. Averagedates from submission to first decision are 23 days, and average dates from acceptance topublication are 15 days. Pflügers Archiv European Journal of Physiology publishes the resultsof original research considered likely to further the physiological sciences in their broadest
sense. Topics include pathophysiological or methodological issues when these can be used asa tool for further investigation of physiological mechanisms. Papers should give mechanistic
insights into physiological functions at the molecular and cellular level. Priority will be given to
manuscripts that provide conceptual novelty. Pflügers specifically aims at publishing work on
ion channels, transporters, cardiac electrophysiology, and sensory physiology on a molecularand cellular level. It should be noted that Drs. Erwin Neher and Bert Sakmann, Nobel laureates
for Physiology or Medicine in 1991, published the first description of the patch-clamp technique(COI:No)in this journal.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S23

Planned Symposium 15
Leading-edge approach for regulated
exocytosis in neural system（March 18, 15:20〜17:10, Hall 2）

PS15-01

Quantal glutamate release organized by supramolecular assembly atpresynaptic terminals
Kenzo Hirose（Dept Pharmacol, Grad Sch Med, Univ Tokyo）
Synaptic transmission is described by the quantal release mechanism which was proposed by
Bernard Katz more than a half century ago. According to the quantal release mechanism, thesynaptic transmission should be stochastic and discrete rather than deterministic and continuous. These features of quantal release should have great impact on overall brain computation.
However the actual features of synaptic transmission and the regulatory mechanism at thesingle synapse level remain unclear level. We have addressed the problem by combining two
imaging techniques: a glutamate imaging technique and a superresolution imaging technique.
We developed a fluorescent glutamate probe named eEOS [enhanced E (glutamate) OpticalSensor] and used it to visualize glutamate in cultured hippocampal neurons. The glutamate
imaging revealed stochastic nature of glutamate release at individual synaptic terminals which
is predicted by the quantal release mechanism. Mathematical analysis of the imaging data
provided detailed features of the quantal release mechanism at the single synapse level. We
found that each synaptic terminal has distinct multiple release sites for vesicular exocytosis
and the number of the release sites per synapse varies from only a few to more than ten.
These variability in the number is intriguing because the number provides unique synaptic
weights on presynaptic side. Next we used STORM, a superresolution imaging technique, toexplore molecular and structural correlates of the release sites. We found that Munc13-1, apresynaptic protein essential for vesicular exocytosis, forms nanosized molecular assemblies
at the presynaptic terminal and the Munc13-1 assemblies serve as the release sites. Thesefindings provide new insight into the synaptic weighting mechanism which might afford unique(COI:No)features important for brain computation. 

PS15-03

Mechanosensing of the enlargement of dendritic spines by presynaptic
terminals in rat hippocampus
Hasan Ucar1，Satoshi Watanabe3，Jun Noguchi3，Sho Yagishita2，Yuicihi Morimoto2，
2 1 International Research Center for Neurointelligence
Noriko Takahashi4，Haruo Kasai1, （(WPI-IRCN), UTIAS, The University of Tokyo, Japan, 2 Laboratory of Structural
Physiology, Graduate School of Medicine, The University of Tokyo, 3 National Center of
Neurology and Psychiatry, Tokyo, Japan, 4 Department of Physiology, Kitasato Univ. Schoolof Medicine, Tokyo, Japan）
In this work, we have identified clues for a novel phenomenon where presynaptic activity isenhanced by sensing the mechanical pressure emerging from spine enlargement. We used the
Schaffer collateral (SC) innervating CA1 pyramidal neurons in hippocampal slice cultures. First, wemeasured SNARE assembly in presynaptic boutons of SC by using a probe which measured Förster
resonance energy transfer (FRET) between t-SNARE (Syntaxin1A) and v-SNARE (VAMP2) byutilizing fluorescence lifetime imaging (FLIM) and time-correlated single photon counting (TCSPC)
method. When we pushed single presynaptic boutons by a fine glass pipette FRET value wasincreased, indicating that the mechanical pushing of the plasma membrane towards the vesicle
membrane can induce the trans-SNARE formation. Second, we measured glutamate release in
responses to single action potentials from individual boutons which expressed iGluSnFR. We foundthe pushing augmented the glutamate release in a good correlation with the FRET increase. The
presynaptic FRET increase was independent of cytosolic Ca2+, but dependent on the assembly of
SNARE proteins and actin scaffolds in the presynaptic terminals. Similar enhancement of SNARE
assembly and glutamate release was observed by a low hypertonic sucrose solution. Lastly, to test
the physiological relevance of the mechanical effect, we induced spine enlargement by spike-timingdependent plasticity (STDP) at the dendritic spine using 2-photon glutamate uncaging. We foundthat spine enlargement caused an increase in the trans-SNARE formation and glutamate release
when spines pushed the presynaptic bouton. However when spines twitched and did not exert pushing, even though spines enlarged, FRET or glutamate release enhancements were not observed.
Thus, we have found a new mechanosensing mechanism, involving SNARE proteins and actin fibers
in the presynaptic terminals, which can respond to fine alterations in the postsynaptic structures.
(COI:No)

PS15-04

Lysosomal exocytosis in neurons: mechanisms and its possible roles
Michisuke Yuzaki（Dept Physiol, Keio Univ Sch Med）
Lysosome is a membrane-bound organelle containing hydrolytic enzymes, such as cathepsin B(CatB), which can degrade a variety of biomacromolecules inside animal cells. Ca2+-dependent
exocytosis of lysosomes occurs in hematopoietic lineage cells and some specialized cells.
However, physiological roles of lysosomal exocytosis and its underlying mechanisms haveremained largely unclear in neurons. We found that a synaptic organizer Cbln1, which plays
crucial roles in formation of synapses between cerebellar granule cells (GCs) and Purkinje cells,
was released from lysosomes located at axons, but not dendrites, of developing GCs. Cbln1
exocytosis required a physiological range of repetitive GC action potentials (300 pulses at 2.5-40
Hz), indicating that it serves as a leaky integrator of neuronal activities over time. Exocytosed
Cbln1 was mostly retained in partially fused vesicles or recovered by vesicles on a time scaleof minutes, leaving a small fraction of Cbln1 retained on GC axons by binding to its cell-surface
receptor neurexin. Cbln1 was co-released with CatB from GC axons and inhibited by lysosomal
inhibitors. Cbln1 release did not involve VAMP7, as well as other TeNT-insensitive R-SNAREs,
but was sensitive to Q-SNAREs SNAP29 and Stx4, indicating involvement of unique SNARE
complexes. Inhibition of release of Cbln1 and CatB reduced axonal bouton formation of developing GC axons in vivo, implying that lysosomal exocytosis of Cbln1 and CatB serve as a
new mechanism of activity-dependent coordinated synapse modification. Recently, we havefound that activity-dependent lysosomal exocytosis also occurs from dendrites of hippocampal
neurons via distinct mechanisms and plays crucial roles for synaptic plasticity. In this talk,I would like to discuss mechanisms and possible physiological roles of neuronal lysosomal
exocytosis in dendrites and axons.
(COI:No)

PS15-02

Ca-Transmitter release coupling at hippocampal mossy ﬁber synapses
Takeshi Sakaba（ Doshisha University）
Hippocampal mossy fiber (hMF) –CA3 synapses exhibit presynaptic forms of short- and longterm presynaptic plasticity. The presynaptic mechanisms of transmitter release and synaptic
plasticity still remain to be studied. In order to determine basic properties of hMF-CA3
synapses, we have used simultaneous recordings of pre- and postsynaptic compartments, andused presynaptic capacitance and EPSCs for measuring the kinetics of transmitter release
quantitatively. We have obtained basic information such as release probability, the number ofreadily releasable synaptic vesicles, and the rate of synaptic vesicle replenishment. Comparedto other model synapses such as the calyx of Held and the synapses in the cerebellum, a largepool of readily releasable vesicles is a unique feature of this synapse type, which allows flexibility of synaptic strength at this synapse. Implications in short-term and long-term synaptic
plasticity will be discussed. (COI:No)S24

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Planned Symposium 16
New Insights into Vascular Aging
Mechanisms and Therapeutic Targets（March 18, 15:20〜17:10, Hall 4）

PS16-03

An updated overview on TRP channels involved in cardiovascularinﬂammatory/ﬁbroproliferative diseases
1 1 Dept Physiol, Fukuoka
Ryuji Inoue1，Lin Kurahara H.2，Keizo Hiraishi1, 2，Yuanyuan Cui（
Univ Sch Med, 2 Dept Cardiovasc Physiol, Kagawa Univ Sch Med）

Inflammatory/fibroproliferative disorders are a major cause of morbidity and mortality inthe modern world. The pathogenic mechanisms underlying these disorders involve very
complex and interwoven networks of numerous biochemical mediators and signaling pathways including cytokines/growth hormones and their receptors, proteolytic enzymes, kinases,
phosphatases, transcription factors, ion channels, and other regulatory molecules. Recently,
considerable evidence has accumulated for the essential significance of stress-responsive Ca2+/
Na+-permeable cation channel superfamily, the transient receptor potential (TRP) protein, in
these processes. Notable examples include several TRP channels such as TRPC3, TRPC6,
TRPV1, TRPA1 and TRPM7, which likely contribute to a diverse array of fibroproliferative
disorders in the brain, lung, heart, liver, kidney, bowel and blood vessels. This symposium talkattempts to provide an updated view on this topic, with particular emphasis on cardiovasculardiseases such as atrial fibrillation and pulmonary arterial hypertension.
(COI:No)

PS16-01

PS16-04

James Pearson1, 2，Jennifer Ngo1，Hirotsugu Tsuchimochi1，Takashi Sonobe1，
1 1 Dept Cardiac Physiol, Res Inst, NCVC, Japan, 2 Dept
Mark Waddingham3，Huiling Jin（

Lars Jørn Jensen（Dept. of Veterinary and Animal Sciences, Faculty of Health and Medical

Early loss of coronary regulation by endothelium-derived
hyperpolarization factors in diabetes
Physiol, Monash Univ, 3 Dept Adv Medical Res Pulm Hypertension, NCVC）

States of chronic vascular inflammation and excess oxidative stress develop early in prediabetes, metabolic syndrome and hypertension and contribute to accelerated vascular aging anda progressive decline in coronary function. However, the time course of changes in coronary
vasodilators, vasoconstrictors, transcription factors and markers of senescence is poorly understood. Moreover, the potential influences of the cardiomyocytes and the extracellular matrix
on vessel aging is rarely considered. Utilising synchrotron based microangiography on rodents
we are examining coronary macro- and microcirculation function changes in young and aging
rodents. Gene and protein expression changes are examined with rtPCR and Western Blotting.
To date, our findings suggest that there is a decline in production of endothelium derived
hyperpolarization factors (EDHF) in small coronary arterial vessels ahead of impaired nitricoxide (NO) signaling associated with insulin resistance. Rho-kinase is upregulated in various
vascular disease states, in particular by hyperglycemia and hyperlipidemia associated withinsulin resistance. Oxidative stress and upregulation of Rho-kinase activity have been shownto increase expression of TRPC6 channels leading to increased vasoconstriction and permeability and diminished NO signaling. Moreover, an important EDHF, the large conductanceCa2+-activated potassium channels (BKCa) in coronary vessels are negatively regulated by
Rho-kinase. Here, we examine the relations between NO signaling, BKCa expression, Rho-kinase
activation, senescence and vasomotor regulation in vivo in rodent models of diabetes, hypertension and aging.
(COI:No)

Changes in small artery tone regulation and structural remodeling in
aging, hypertension and obesity/diabetes. Do they have a common
underlying cause?
Sciences, University of Copenhagen）

We investigate the interplay between myogenic tone (MT), flow-mediated vasodilatation(FMVD), and structural remodeling in small arteries and arterioles controlling mean arterial
blood pressure (MAP) and organ blood flow. Low voltage-activated T-type calcium channels
are abundantly expressed in the microcirculation, but their physiological role is still debated.
In mice deficient in the CaV3.1 T-type channel we found that MT was abolished at low
arterial pressures (40-80 mm Hg) but strongly activated at higher pressures (80-120 mmHg).
In contrast, young mice deficient in the CaV3.2 T-type channel isoform had increased MTand decreased FMVD. In old mice these effects of CaV3.2 deletion were abolished, and theexpression of CaV3.1 T-type channels was dramatically reduced. In the same study, we foundthat MT and noradrenaline-induced constriction was increased in old mice. We later showedthat the arterial expression of Rho-kinase 2 isoform (ROCK2) was increased in middle-aged
mice, coinciding with an increased MT and increased effect of the ROCK2-specific inhibitor
KD025. The mechanisms responsible for the age-dependent contributions of T-type channels
and ROCK2 will be pursued in future studies.
In 32-week old rats fed a high-fat/high-fructose diet for 28 weeks (DIO rats), we found thata decreased expression of arterial SKCa, IKCa, and Kir2.1 K+ channels was associated witha decreased FMVD, acetylcholine- and K+-induced vasodilatation and an increase in MAP.
This was interpreted as the loss of EDH-type vasodilatation due to the metabolic syndrome.
In humans, obesity and diabetes leads to hypertrophic remodeling of resistance arteries. We
found that outward hypertrophic remodeling in small arteries from obese and diabetic Göttingen minipigs was correlated with increased fasting plasma levels of glucose and cholesterol,
and with the mRNA expression levels of Rho-kinase 1 and Transglutaminase 2. The data are(COI:No)
discussed in relation to the metabolic syndrome and vascular aging in general.PS16-02

The functional role of vascular microRNAs in diabetes-induced
microangiopathy of the heart
Shruti Rawal1，Pujika Emani Munasinghe1，Isabelle van-Hout1，Sean Coffey2，Michael J Williams2，
1 1 Department of Physiology,
Patrick Manning2，Philip Davis3，Costanza Emanueli4，Rajesh Katare（
HeartOtago, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand, 2 Departmentof Medicine, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand, 3 Department of
Cardiothoracic surgery, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand,
4 National Heart and Lung Institute, Imperial College, London, United Kingdom）

Diabetes adversely affects the function and number of endothelial cells in the microvessels of
the heart, resulting in a condition called diabetic microangiopathy. Compelling evidence fromclinical and pre-clinical animal studies confirm the regression of microvasculature in the diabetic
heart. We, along with others, have shown that molecular dysregulation starts much earlier in the
diabetic heart leading to functional and structural deterioration in the later stage. MicroRNAs(miRNAs) are key molecular regulators playing an important role in regulating a wide range ofphysiological and pathological processes. In the current study, we tested the role of miRNAs indiabetes-induced microangiopathy of the heart. miRNA microarray studies in human type-2 diabetic heart showed significant dysregulation of pro- (miR-126 and -132) and anti-angiogenic (miR92a and -206) miRNAs. Using the type-2 diabetic mouse model, we identified that downregulationof miR-126 and -132 (angiomiRs) start from the early stages (8wks of age) in the diabetic heart
while the changes in the vascular profile were not observed until 20wks of age. Downregulationof angiomiRs was associated with the upregulation of anti-angiogenic target protein P120RasGAP
and SPRED-1. Overexpression of miR-126 and -132 in in vitro cultured diabetic endothelial cells
improved the migration and survival and enhanced the angiogenic ability of the diabetic endothelial cells. Finally, to determine if in vivo overexpression of angiomiRs prevent diabetes-induced
microangiopathy in the diabetic heart, we overexpressed miR-126 expression via intramyocardial
injection of miR-126 mimic in the type-2 diabetic mouse heart at 10wks of age. Results showedpreserved capillary and arteriole density and reduced endothelial cells apoptosis in the diabetic
heart. This eventually preserved cardiac contractility. Altogether, our study showed that diabetes
induces dysregulation of vascular miRNAs and that therapeutic restoration of these miRNAs(COI:No)
could be a novel tool to prevent/treat diabetes-induced microangiopathy.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S25

Planned Symposium 17
Cardiac mechano-physiology: physiology andpathophysiology of the mechano-electricalcoupling（March 18, 15:20〜17:10, Hall 6）

PS17-01

Role of mechanically induced crosstalk between organelles inmyocardial mechanics
Gentaro Iribe（Dept Physiol, Asahikawa Med Univ, Japan）
Cardiac muscle adapts to acutely altered mechanical load by a rapid change in contractile force.
Therefore, it is not surprising if the heart is regulated by feedback from its mechanical state
to functions of excitation contraction coupling (ECC) modules. Recent observations demonstrated that myocardial stretch hyperpolarizes mitochondrial membrane potential (Δψm) andincreases NADPH oxidase (NOX)2-derived reactive oxygen species (ROS) production, whichcauses an immediate increase in Ca2+ sparks. However, physiological roles and underlying
mechanisms of these responses remain unknown. In the present study, we hypothesized thatstretch-induced nucleotide signaling (extracellular nucleotide release via pannexin hemichannels followed by an activation of purinergic P2 receptors) is involved with these responses toadapt increased preload. To probe the possibilities of our hypothesis, enzymatically isolatedmouse ventricular myocytes were subject to stretch using carbon fiber technique to study
stretch-induced changes in Δψm and ROS production, and single cell mechanics. Applying
stretch significantly hyperpolarised Δψm, while the response was abolished in the presenceof carbenoxolone (CBX), an inhibitor of pannexin hemichannels. Stretch-induced increase in
ROS production was abolished in the presence of either Pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid (PPADS), a P2 purinergic antagonist, or CBX. Stretch did not affect any
morphological parameters of Ca2+ transient in C57BL/6 wild type (WT) mice, while the stretch
significantly delayed the time to maximal rising rate of [Ca2+] i in NOX2 knock out (KO) mice.
The resultant cellular contractility in NOX2 KO group was significantly lower than WT group.
The present results suggest that stretch-induced extracellular ATP release via pannexin
hemichannels may reduce intracellular ATP, therefore, stimulates electron transport chain to
hyperpolarize Δψm. And then the released ATP activates P2 to NOX2 pathway for stretchinduced ROS production, which facilitates Ca2+ release from the sarcoplasmic reticulum tomaintain cellular contractility against increased preload.
(COI:No)

PS17-03

Pannexin-1 Contributes to Maintenance of Cardiac Function against
Acute Pressure-overload and Rapid Ventricular Pacing
Kensuke Ihara1，Tetsuo Sasano2，Kentaro Takahashi1，Masahiro Yamazoe1，
1 1 Dept Bio-inform Pharm, MRI, TMDU, Japan, 2 Dept Cardiovasc,
Tetsushi Furukawa（

TMDU, Japan）

Pannexin is a member of gap-junction channels and consists of 3 isoforms, pannexin-1, -2, and-3. Ventricles of hearts mainly express pannexin-1, while atrium express pannexin-2. Unlike
connexin, pannexin expresses only on the surface of membrane, not between a cell and a cell,and transports small molecules including ATP between inside and outside of the cell. Mechanical stimuli change the conformation of the C-terminus of pannexin-1, thereby inducing releaseof ATP. For example, it was shown that ATP release from erythrocytes when they travel
through narrowed vessels can dilate vessels and compensate disturbed blood circulation, as aprotective mechanism against ischemia. Here, we examined the roles of pannexin-1 in hearts
using global pannexin-1 knock-out (Panx1-/-) mice. After transverse aortic constriction (TAC)
surgery, survival rate was significantly lower in Panx1-/- than in WT mice. Movement of ventricular walls was severely deteriorated in Panx1-/- mice, not in WT mice, immediately after
TAC operation. We also examined the effects of rapid pacing in WT and Panx1-/- mice. Rapid
pacing at 900 bpm caused circulatory collapse in Panx1-/- mice immediately after start of pacing, WT mice could tolerate it for at least 30 minutes. After pacing with slower rate (750bpm),
left ventricular fractional shortening (%FS) was significantly reduced in Panx1-/- mice, but not
in WT mice. During pacing, coronary flow velocity was significantly less in Panx1-/- mice thanin WT mice. The average diameter in both left and right coronary arteries, measured after
hearts had been chemically made transparent, was significantly smaller in Panx1-/- mice thanin WT mice. Capillary density was also significantly lower in Panx1-/- mice than WT mice.
From these data, we conclude that pannexin-1 acts as a protective mechanism against pressure
overload and tachycardia in hearts via maintaining coronary circulation.
(COI:No)

PS17-04

Electropharmacological analysis of precordial percussion pacing in a
cardiac standstill animal model: Clinical efﬁcacy of knocking the chest
as a “bridge to pacemaker”
Atsushi Sugiyama1, 2，Mihoko Hagiwara-Nagasawa1，Ryuichi Kambayashi1，Ai Goto1，
2 1 Dept Pharmacol,
Koki Chiba1，Yoshio Nunoi1，Hiroko Izumi-Nakaseko1，Akio Matsumoto（

Faculty Med, Toho Univ, Japan, 2 Dept Aging Pharmacol, Faculty Med, Toho Univ, Japan）

Benefits of precordial percussion pacing (PPP) during cardiac standstill are unknown. We created a cardiac standstill model with microminipig by inducing complete atrioventricular blockwith a catheter ablation technique. The efficacy of cardiopulmonary resuscitation by standard
chest compressions (S-CPR), PPP and ventricular electrical pacing in this model were analyzed
in series. To assess the mechanism of PPP, a non-selective stretch-activated channel blocker
amiloride was administered during PPP. Peak systolic and diastolic arterial pressures during
S-CPR, PPP and ventricular electrical pacing were statistically similar. However, the durationof developed arterial pressure with PPP was comparable to that with ventricular electrical
pacing, and significantly greater than that with S-CPR. Amiloride decreased the induction
rate of ventricular electrical activity by PPP in a dose-related manner. Each animal survived
without any neurological deficit at 24, 48 h and 1 week, even with up to 2 h of continuous
PPP. In microminipig with cardiac standstill, PPP can become a novel means to significantly
improve physiological outcomes after cardiac standstill or symptomatic bradyarrhythmias
even without cardiac pacing. Activation of the non-selective stretch-activated ion-channels may
be involved in the mechanophysiological effects of PPP. Thus, PPP was shown to effectively
provide a stable heart rate and blood pressure without inducing lethal ventricular arrhythmias,and to prevent neurological deficits during cardiac standstill. The efficacy and safety of PPP as
shown in pigs have been recently demonstrated in a patient with a cardiac standstill, indicatingthat PPP may also have significant utility for patients with life-threatening bradycardia until
more definitive therapies are available.
(COI:No)

PS17-02 (AP-6)

The role of TRPC3 and TRPC6 in a stretch-induced slow force
response in cardiomyocytes
1 1 Dept Physiol, Med,
Yohei Yamaguchi1，Gentaro Iribe1，Keiji Naruse2，Akira Takai（

Asahikawa Med Univ, Japan, 2 Dept Cardio Physiol, Grad Sch Med, Okayama Univ, Japan）

An increase in preload induces a biphasic active force enhancement in the heart. The shortterm increase in preload rapidly augments contractile force due to the Frank-Starling mechanism, which accelerates Ca2+ sensitivity of contraction proteins. A further long-term increasein preload for several minutes to hours causes the increase in [Ca2+] i, leading to a slow force
response to stretch (SFR), and a further increase in the contractile force. The stretch-induced
release of angiotensin II has been implicated in the SFR, to raise intracellular Na+ levels,
followed by an increase in intracellular Ca2+ levels via the Na+/Ca2+ exchanger. However,
the extracellular cation influx pathway is poorly understood. To better understand the cationinflux pathway, we focused on TRPC3 and TRPC6, receptor-operated, mechanosensitive nonselective cation channels. In our studies, cardiomyocytes were enzymatically isolated frommouse hearts, and both cell ends were held by two carbon fibers to apply stretch to the cells.
Using this stretch device, we found that TRPC3 and TRPC6, regulated by the angiotensin II
type 1 (AT1) receptor via diacylglycerol produced by phospholipase C, played a pivotal rolein the SFR. Our recent data also showed that stretch-induced activation of TRPC3 increased
intracellular Ca2+ influx, causing the accumulation of Ca2+ in the sarcoplasmic reticulum,
which in turn increased Ca2+ release and, consequently, the twitch force, suggesting that afunctional sarcoplasmic reticulum (SR) is necessary for the SFR. Other data indicate that amyocardial stretch stimulated the AT1 receptor, followed by endothelin release to increase
nicotinamide adenine dinucleotide phosphate oxidase (NOX)-derived reactive oxygen species,
regulating TRPC3 and TRPC6 to increase Na+ influx, leading to the SFR. These data suggest
the possibility that two AT1 receptor-operated cation influx pathways via TRPC3 and TRPC6,
causing the enhancement of Ca2+ release from the SR, may cooperate in the SFR.  (COI:No)

S26

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Planned Symposium 18
Inter-organ communication:
molecular mechanism and pathophysiology
induced by its disruption（March 18, 15:20〜17:10, Hall 8）

PS18-01

A Sarcoplasmic Reticulum Localized Protein Phosphatase Regulates
Phospholamban Phosphorylation and Promotes Ischemia Reperfusion
Injury in Heart2 1 Dept Cell Physiol, The Jikei Univ,
Toru Akaike1，Susumu Minamisawa1，Yibin Wang（

Japan, 2 Dept Anesthesiology, Univ of California, Los Angeles, USA）

Phospholamban is a key regulator of sarcoplasmic reticulum calcium uptake in cardiomyocyte,
its inhibitory activity to SERCA is regulated by phosphorylation. Phospholamban hypophosphorylation is a common molecular feature in failing heart. We have discovered that protein
phosphatase 2Ce (PP2Ce) is a novel serine/threonine protein phosphatase specifically targeted
to sarcoplasmic reticulum membrane, is a specific and potent phospholamban phosphatase in
cardiomyocytes. However, its function has not unknown yet. Here we assessed the hypothesis that PP2Ce is an important player in calcium cycling and cardiac remodeling in heart.
PP2Ce expression was elevated in failing human heart and induced acutely at protein levelby β-adrenergic stimulation or oxidative stress in cardiomyocytes. PP2Ce protein stronger
dephosphorylated β-adrenergic stimulated increase of phospholamban phosphorylation at
threonine 17 site than at serine 16 site in heart. PP2Ce expression reduced β-adrenergic stimulated intracellular calcium transient in isolated adult rat ventricular myocytes, and promotedhydrogen peroxide and ionomycin induced cell death in cultured neonatal rat ventricular
myocytes. Transgenic mice with cardiac specific expression of PP2Ce showed no significant
basal phenotype. However, in isolated perfusion heart preparation, we observed significantly
larger infarct sizes and more impaired functional recovery following global ischemia/reperfusion injury in the transgenic hearts comparing to wild type controls. Therefore, PP2Ce is a
novel component of sarcoplasmic reticulum calcium regulatory network that has a potentially(COI:No)
important role in cell death regulation and cardiac contractility.

PS18-03

Pathogenic factors of diabetic neuropathy: how pancreatic ß cell
dysfunction affects peripheral nervous system?
Kazunori Sango, Hideji Yako, Naoko Niimi, Shizuka Takaku（Diabetic Neuropathy PJ, Tokyo
Met Inst Med Sci, Tokyo）

Peripheral neuropathy is one of the major chronic complications of diabetes mellitus, andits prevalence correlates closely with the degree and duration of hyperglycemia caused bypancreatic ß cell dysfunction and impaired insulin secretion. Although its precise pathogenesis
remains unclear, metabolic disorders of neurons and Schwann cells (e.g. augmentation of
the polyol and other collateral glucose-utilizing pathways, formation of advanced glycation
endproducts, oxidative and endoplasmic reticulum stress) and microvascular abnormalities
are assumed to play key roles in progressive neural dysfunction and irreversible nerve fiber
damage. Streptozotocin (STZ), a compound that has a preferential toxicity toward pancreatic
β cells, is widely utilized to induce diabetes in rodents, and the STZ-diabetic animals exhibit
peripheral neuropathy that partially mimics human disorders. However, recent studies haveindicated that several factors other than continuous hyperglycemia (e.g. recurrent short-term
hypoglycemia and hyperglycemia, altered lipid metabolism, and impaired insulin actions onneurons and Schwann cells) may also contribute to the development of diabetic neuropathy. In
this presentation, we introduce up-to-date studies regarding the neurological disorders in close(COI:No)
association with the disruption of metabolic regulatory mechanisms.

PS18-04

Towards Molecular Pathology Control for Lifestyle-Molecular Dissectionof Hypertension and Atherosclerosis focusing on Eating habit
Tomoaki Ishigami（Dept MS and CR, Grad Sch Med, Yokohama City Univ, Japan）
Both hypertension and atherosclerosis are global health burden for healthy longevity especiallyamong developed countries. Life with daily dietary excess of salt, fat and carbohydrate ischaracteristics for modern developed countries, and definite contributions to cardiovasculardiseases and impairment of healthy longevity. However it is well known that there is farm
relationship between the quality of daily meal and cardiovascular diseases, underlying molecular mechanisms are not known well. Detailed molecular pathophysiological analyses for these
diseases should be essential for handling the subjects appropriately. For hypertension, it was
shown that primary molecular abnormalities existed in tubular sodium transports by molecular genetical analyses for human hereditary and familial hypertension. We have analyzed
molecular pathophysiological dissections for sodium sensitivity and hypertension focusing
on Nedd4-2 which is ubiquitin ligase for epithelial sodium channels located in aldosterone
sensitive distal nephron. Using Nedd4-2 C2 domain knock-out mice, we discovered oral sodium
sensitive enhancements of sodium reabsorption via ENaC in the distal nephron. In addition,
the commensal microbes-derived atherosclerosis via the metabolism-independent mechanisms
have received increasing attention. However, whether the effect of the intestinal microbiota on
atherosclerosis is mediated by immune mechanisms remain uncertain. Our reports provided
evidence for a pathway of immune activation in commensal microbes-derived atherosclerosis,
which driven by high fat and high carbohydrate diet with enhancement of substantial population of splenic B2 cell activation.
(COI:No)

PS18-02TAK-242, an antagonist of toll-like receptor 4, attenuates cardiac
remodeling induced by infusion of Porphyromonas gingivalis
lipopolysaccharide in mice
Yoshiki Ohnuki, Kenji Suita, Satoshi Okumura（ Dept Physiol, Tsurumi Univ Sch Dent Med,Japan）

Porphylomonas gingivalis (PG) is one of the most frequent pathogens in periodontal disease (PD)and its endotoxin, lipopolysaccharide (LPS), circulates systemically in over 50% of PD patients.
Circulating low systemic PG-LPS has been reported to induce cardiac dysfunction; however,the underlying mechanisms remain elusive. In order to elucidate the contribution of toll-likereceptor 4 (TLR4) to the PG-LPS-induced cardiac dysfunction, we investigated the effects ofco-administration of TAK-242, an antagonist of TLR4, with PG-LPS on cardiac function in mice.
Eight-week-old male mice (C57BL/6) were divided into four groups, control (vehicle), LPS (0.8mg/kg/day, i.p.), TAK (3 mg/kg/day, i.p.) and LPS+TAK groups. After the completion of eachtreatment for 4 weeks, echocardiographic measurements were performed under anesthesia,
and then the hearts were excised and used for later analyses. Compared to control group,
left ventricular ejection fraction was significantly decreased in LPS group, but the decrease
was blunted in LPS+TAK group. On the other hand, the number of apoptotic myocytes andthe area of fibrosis were significantly increased in LPS group, but not in LPS+TAK group,compared to control group. In addition, phosphorylation levels of calmodulin kinase II (CaMKII,
Thr-286) and Ca2+-handling proteins, phospholamban (PLB, Thr-17) and ryanodine receptor 2(RyR2, Ser-2814) were also increased in LPS group, but not in LPS+TAK group, compared tocontrol group. These results suggest that low systemic PG-LPS induces cardiac dysfunction inassociation with apoptosis and fibrosis by Ca2+ leakage from sarcoplasmic reticulum through
CaMKII-mediated phosphorylation of PLB and RyR2 in TLR4-dependent manner.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S27

Planned Symposium 19
Progress and perspectives of neuroscience
with data-driven intelligence（March 18, 15:20〜17:10, Hall 10）

PS19-03

Acquisition of Large Volume EM Data Set and 3D Reconstruction with
Automated Segmentation Application
2 1 Natl. Inst. Physiol Sci., Okazaki, Japan, 2 SOKENDAI ）Yoshiyuki Kubota1, （An electron microscopy (EM)-based reconstruction of neuronal circuits from serial ultrathin
sections method was introduced about 30 years ago. It had been achieved all the steps
manually: cutting the serial ultrathin sections using ultramicrotome, image capturing withtransmission electron microscope (TEM), reconstruction using cardboard of the selected
profiles of neural structures to provide impression of depth. A computer software for thereconstruction was introduced to make it more efficient in 1990s. This technology had not been
popular because of a high skill demand in electron microscopy, however, the reconstruction
analysis method offered significantly valuable results. Then, we started to adapt new EM
technologies using scanning electron microscopy (SEM) for the neural network analysis using
the reconstruction method in early 2000. It has been modified and improved them vigorously
and a lot of noteworthy results were published in this decade. EM volume data sets were
getting larger year by year, and the volume size could be huge in size. The success of large
volume EM acquisition using these new EM systems has created an issue, i.e., how to process
large image data sets thus obtained. Soon it became obvious that it was difficult to handle
large EM volume data sets using conventional 3D reconstruction image processing computer
applications. To achieve segmentation easily and efficiently, automated segmentation computer
applications have been developed and used. Segmentation performance has increasingly been
improved and reached to account for more than 90% of the volume. I would like to introduce
our recent improvement for the segmentation of the large volume EM data set acquired with
ATUM-SEM using the automated segmentation applications Flood Filling Network (FFNs).
We found it works better for the EM data set with 30 nm thick sections than the one with 50(COI:No)nm thick sections.

PS19-01

PS19-04

Yasuhiro Tanaka R.1, 2，Yoshito Masamizu2，Yasuyo Tanaka H.2，Takanori Shinotsuka2，
2 1 Brain Science Institute, Tamagawa University, Japan, 2 Dept Physiol,
Masanori Matsuzaki（

Jumpei Matsumoto1，Hisao Nishijo1，Koki Mimura2，Kenichi Inoue3，Yasuhiro Go4，5 1 System Emotional Sci, Grad Sch Med, Univ of Toyama, Japan,
Tomohiro Shibata（

Decoding animal behavior from cellular-resolution neural data
Grad Sch Med, the University of Tokyo, Japan）

Recently, more and more cellular-resolution neural data have been recorded with theadvancement of technology, such as two-photon microscopy or silicon probes. At the sametime, the extraction of animal behavior from movies has been automatized with deep learningtechniques. With such technical progress, neural decoding, a machine-learning method to infer
animal sensory input and motor output from neural data, is becoming essential in neurosciencestudy. We will first clarify what the neural decoding is like and why we use it both frommachine-learning and neuroscience perspectives. Secondary, we will share our knowledge
about evolving neural coding in the primary motor cortex (M1) of mice during motor learning
as a practice of neural decoding. We recorded neuronal ensembles with two-photon microscopyin M1 of the mice that were trained to get water reward by pulling the lever with their rightforelimb. We found layer-specific changes in the stability of the neural representation. In layer
2/3, the accuracy of neuronal ensemble prediction of lever trajectory remained unchanged
globally, with a subset of individual neurons retaining high prediction accuracy throughout thetraining period. By contrast, in layer 5a, the ensemble prediction accuracy steadily improved,and one-third of neurons evolved to contribute substantially to ensemble prediction in thelate stage of learning. Thirdly, we will show the application of a decoding-related techniqueto estimate neuronal dynamics. We recorded the neuronal activities of thalamic axons with
two-photon microscopy in M1 of the mice that were trained similarly as above. In layer 1axons, we found the sequence of the neuronal activity during lever-pull movements. Finally,
we would like to discuss the recent study and future direction. (COI:No)

Utilization of animal markerless motion capture in neuroscience
2 National Institutes for Quantum and Radiological Science and Technology, 3 Primate
Research Institute, Kyoto Univ, 4 Exploratory Research Center on Life and Living Systems,
National Institutes of Natural Sciences, 5 Grad Sch of Life Science and Systems Engineering,
Kyushu Institute of Techonlogy）

Animal behavior analyses are fundamental in neuroscience. The video-based analyses areimportant to measure animal’s complex interactions with the environment and/or the otheranimals (e.g., social interactions), and to estimate animal’s internal states based on the carefulinspection of its motion and posture (e.g., freezing behavior). In the conventional video analyses,
human observers visually inspect images or simple computer-based systems track binary
images of animals. Visual inspection methods have often problems in reproducibility and objectivity, while it is difficult for the latter method to analyze complex behaviors. On the other hand,motion capture systems are useful to analyze detailed motions and postures quantitatively.
However, this method has also problems; tracking markers must be attached to the animal’s
body in the conventional systems and the markers often disturb natural behavioral expression.
Recent advances in 3D depth camera and deep learning have allowed image-based markerless
motion capture, overcoming those limitations of the conventional approaches. In this paper,
those new markerless motion capture tools and their applications are introduced. We will also
show our plan to further improve those tools for a wider range of applications. Finally, we will
discuss how to further utilize the “motion big data” obtained by motion capture. Markerless
motion capture can be of great help in understanding the brain functions that underlie theadaptive behaviors of animals in complex real-world situations, as well as in understanding
various neurological and neuropsychiatric disorders.
(COI:No)

PS19-02

PS19-05

Hidetoshi Urakubo（Grad Info, Kyoto Univ, Japan）

Masakazu Agetsuma1, 2, 3，Issei Sato4，Yasuhiro Tanaka R.5，Atsushi Kasai6，Yoshiyuki Arai3，
Miki Yoshitomo1，Hitoshi Hashimoto6，Junichi Nabekura1，Takeharu Nagai3

Automated 3D reconstruction from a 2D stack of neuronal EM images
Recently, there has been a rapid expansion in the field of micro-connectomics, which targets thethree-dimensional (3D) reconstruction of neuronal networks from a stack of two-dimensional(2D) electron microscopic (EM) images. The spatial scale of the 3D reconstruction grows
rapidly owing to deep neural networks (DNNs) that enable automated image segmentation.
Several research teams have developed their own software pipelines for DNN-based segmentation. However, the complexity of such pipelines makes their use difficult even for computer
experts and impossible for non-experts. We here introduce a new software program, called
UNI-EM, that enables DNN-based EM image segmentation, including ground truth generation,
training, inference, postprocessing, proofreading, and visualization. Non-computer experts canconduct EM image segmentation based on 2D/3D DNNs, such as 2D U-Net, 2D ResNet, 2D
HighwayNet, 2D DenseNet, and 3D flood-filling networks (FFNs). We would like to show how
we can obtain benefits from such automated segmentation on the software UNI-EM, together
with the principles of DNN-based segmentation algorithms. We would also report the current
status of a collaborative study with Prof. Yoshiyuki Kubota for 3D segmentation of a large-scale
neuronal EM images.
(COI:No)

S28

Optical and computational dissection of neural circuit for fear memory（ 1 Div Homeostatic Development, NIPS, Aichi, Japan, 2 Japan Science and Technology
Agency, PRESTO, Kawaguchi, Japan, 3The Institute of Scientific and Industrial Research,
Osaka University, Ibaraki, Japan, 4Graduate School of Frontier Sciences, The University ofTokyo, Tokyo, Japan, 5 Brain Science Institute, Tamagawa University, Machida, Japan,
6 Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan）

For efficient and accurate information processing in cerebral cortex, neural circuit dynamics
must be spatially and temporally regulated with great precision. Medial prefrontal cortex(mPFC) of rodents has been shown important for various types of learning and memory,
including fear memory, and related to various psychiatric diseases. However, it has been challenging to understand the computational mechanism in the mPFC, of which major problems
are the complexity and heterogeneity of the prefrontal networks. Here we investigate this bychronic two-photon Ca2+ imaging from populations of neurons in mouse mPFC in vivo, whichallows us to 1) record activities simultaneously from large number of neurons at the single
cell resolution with high temporal resolution, and 2) investigate changes of neuronal responses
over the learning process. We investigated the change in responses of mPFC neurons during
Pavlovian fear conditioning and memory retrieval using a new device to perform them underthe microscope with a head fixed mouse. We used two types of tones: one of them (CS+) was
associated with the aversive foot shock while the other (CS-) was not. Tone responsive cells
were observed even from the early stage of learning process, and the ratio of different types(CS+ specific, CS- specific, responsive to both, non-responsive) was not changed over the learning process, suggesting that emergence of specific function in individual neurons was unlikely
the basis to encode fear memory. In contrast, population responses were significantly changed
during the learning process and maintained enhanced during memory retrieval, suggesting
possible emergence of novel population codes. By further analyses based on machine learning,
we detected neural ensembles crucial to decode the conditioned response (i.e. freezing behavior
during CS+ after the learning). These results suggest that population coding may be a critical(COI:No)basis for the fear memory in mPFC. 

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Planned Symposium 20
Neural Mechanism of Psychological Stress:
Molecules, Circuits and Disorders（March 19, 9:00〜10:50, Hall 2）

PS20-01

Central circuit mechanisms of sympathetic and behavioral responses to
psychological stressNaoya Kataoka, Yuta Shima, Kazuhiro Nakamura（ Department of Integrative Physiology,
Nagoya University Graduate School of Medicine, Nagoya, Japan）

Psychological stress elicits a variety of sympathetic and behavioral responses in mammals,
which help animals cope with stressors. Sympathetic stress responses include increases inbody temperature, heart rate and blood pressure. However, the central circuit mechanisms
for psychological stress responses are unknown. The initial purpose of our stress research
project was to elucidate the brain network that evokes stress-induced sympathetic responses.
We have reported that psychological stress induces thermogenesis in brown adipose tissue(BAT), hyperthermia and tachycardia by activating a monosynaptic neural pathway from thedorsomedial hypothalamus (DMH) to the rostral medullary raphe. Recently, we discovered thatthe DMH receives glutamatergic excitatory stress inputs from the dorsal peduncular cortex
and dorsal tenia tecta (DP/DTT), located at the ventral limit of the medial prefrontal cortex. To
determine how important the DP/DTT–DMH neural pathway is for the drive of sympathetic
stress responses, we selectively lesioned DP/DTT–DMH projection neurons in rats by Credependent expression of a genetically engineered caspase-3 with a combination of anterograde
and retrograde adeno-associated viruses injected into the DP/DTT and DMH, respectively.
Lesions of DP/DTT–DMH neurons abolished BAT thermogenesis and hyperthermia induced
by social defeat stress, a psychosocial stress model. We further tested the effect of optogenetic
inhibition of the DP/DTT–DMH pathway on stress-induced social avoidance. Wistar rats that
underwent social defeat stress avoided interaction with the dominant Long-Evans rats that
defeated the Wistar rats. Intriguingly, optogenetic inhibition of DP/DTT–DMH projection
neurons in the stressed Wistar rats, which expressed iChloC-mCherry, a chloride-conducting
channelrhodopsin, restored social interaction, and also eliminated sympathetic stress responses
including hyperthermia and tachycardia. These results demonstrate that DP/DTT neurons
transmit a glutamatergic excitatory stress input to the DMH, which is a master signal to drivea variety of autonomic and behavioral stress responses.
(COI:No)

PS20-03

Neuronal hypertrophy dampens stress responsiveness during
habituation
Wataru Inoue（Robarts Research Institute, Western University, Canada）
Encountering a stressor immediately activates the hypothalamic-pituitary-adrenal (HPA) axis,
but this stereotypic stress response also undergoes experience-dependent adaptation. How thebrain adjusts stress responsiveness in the long-term remains poorly understood. We studied
hypothalamic corticotropin-releasing hormone neurons that form the apex of the HPA axis ina mouse model of repeated restraint. Using patch-clamp electrophysiology, we found that the
intrinsic excitability of these neurons substantially decreased after daily repeated stress in atime course that coincided with their loss of stress responsiveness in vivo. This intrinsic excitability plasticity co-developed with an expansion of surface membrane area, which increaseda passive electric load, and dampened membrane depolarization in response to the influx ofpositive charge. Multiphoton imaging and electron microscopy revealed that repeated stressaugmented ruffling of the plasma membrane, suggesting an ultrastructural plasticity that may
efficiently accommodate the membrane area expansion. Overall, we report a novel structurefunction relationship for intrinsic excitability plasticity as a neural correlate for habituation of(COI:No)the neuroendocrine stress response.

PS20-04

Mechanisms for regulation of fear memory after retrieval
Satoshi Kida（ Grad Sch Agricul Life Sci, Univ Tokyo, Japan）
Memory retrieval is not a passive process. Retrieval of contextual fear memory by shortor long reminders initiates reconsolidation and extinction, respectively; reconsolidation maintains or enhances fear memory, while extinction weakens it. We have tried to understandmechanism for regulation of fear memory after retrieval at the molecular, cellular and circuits
levels using contextual fear conditioning and inhibitory avoidance tasks. Our previous studies
showed that reconsolidation of fear memory requires the activation of gene expression in
the hippocampus, mPFC and amygdala, while long-term extinction requires gene expression
activation in the mPFC and amygdala. Based on these findings, we are trying to identify andcharacterize fear and extinction engram neurons in the hippocampus, mPFC and amygdala.
We identified fear engram neurons in the hippocampus and examined the function of theseneurons using c-fos-tag system. Fear engram neurons were labeled by ChR2 or ArchT andeffects of optogenetic activation and inactivation of them were examined during memory
retrieval. Inactivation of fear engram neurons during retrieval erased contextual fear memory,
perhaps, by blocking memory reconsolidation, whereas activation of these engrams during
retrieval blocked acquisition and consolidation of fear memory extinction. These results suggest that modulation of fear memory engram cells are crucial processes to determine the fateof memory; reconsolidation or extinction.
We also identified fear and extinction engram neurons in the amygdala and mPFC. Interestingly, distinct fear and extinction engram neurons were observed in the amygdala, while
single neuronal fear/extinction engram neurons was observed in the mPFC. We are now(COI:No)
characterizing these engram neurons using optogenetics. PS20-02

Multiple roles of social stress-induced inﬂammatory responses in thebrain
Tomoyuki Furuyashiki（Div Pharmacol, Grad Sch Med, Kobe Univ, Japan）
Stress due to adverse and demanding conditions may alter mental and physical functions,and is thought to precipitate various diseases including depression. Depressive subjects show
elevated levels of proinflammatory molecules in the blood and an inflammatory marker in thebrain. However, the causal relationship between inflammation and stress-related pathology ofdepression remained elusive. Using social defeat stress, a mouse model for depression research,
we have revealed roles of inflammatory responses in the brain for stress-induced depressivelike behaviors. Repeated social defeat stress induces microglial activation in the medial
prefrontal cortex (mPFC) through innate immune receptors TLR2/4, thereby leading to neuraland behavioral changes through proinflammatory cytokines IL1α and TNFα. HMGB1, a
non-histone nuclear protein that can act as an endogenous TLR2/4 ligand upon its extracellularrelease, is translocated from the nuclei of mPFC neurons upon repeated social defeat stress,
and promotes depressive-like behaviors. In parallel, repeated social defeat stress attenuates
mPFC dopaminergic response to increase stress susceptibility through prostaglandin (PG) E2
and its receptor EP1. Social defeat stress upregulates PGE2 synthesis in subcortical regions
in a manner dependent on monoacylglycerol lipase that catalyzes 2-arachidonoylglycerol tofree arachidonic acid and cyclooxygenase 1, a PG synthase expressed in microglia. Notably,
this PGE2 synthesis also depends on TLR2/4 integrity. Collectively, we propose that multiple
microglia-derived inflammatory responses upon social stress are coordinated by innate immune
receptors and promote depressive behaviors through distinct, but cooperative, mechanisms.
(COI:Properly Declared)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S29

Planned Symposium 21

PS21-03

Rules for medical research on human subjects in Japan
Tsunakuni Ikka（ National Cancer Center, Japan）

The ethics, laws, and guidelines forhuman and animal researches

I am going to speak on the following points.
1) The basic idea of Reserch Ethics; Protection of human subjects.
2) Ethical guideline for medical research on human subjects.
3) Clinical trial act.

(COI:No)（March 19, 9:00〜10:50, Hall 4）

PS21-01

Law for the Humane Treatment and Management of Animals - past,present and future perspectives Naoko Kagiyama（ Central Institute for Experimental Animals）
The Law for the Humane Treatment and Management of Animals (Law) was enacted in 1973.
The law protects mammals, birds and reptiles, and emphasizes respect for life and well-beingof animals. It specifies the responsibility of the owner of the animal, and calls for the alleviationof pain and distress as well as the humane death of animals used for scientific purposes.
Based on the law, the Standards Relating to the Care and Management of Experimental
Animals (Standards) were specified in 1980. In the same year, the Science Council of Japan(SCJ) advised the government to prepare administrative guidance for the use of animals forscientific purposes.
The law specifies the 3R-Principle (Replacement, Reduction and Refinement) of animal experimentation and adopts the institutional self-regulation system because life science should
not be regulated only for the aspect of humane treatment of animals, and “Replacement” and“Reduction” were considered as the items to be discussed the animal experiment protocols
prepared. In addition, appropriate animal experimentation could be better accomplished basedon guidelines and not by stringent legislation.
Since 2006, animal experimentation has been conducted based on the institutional regulation
system following the Law, Standards, and basic policies on animal experimentation (MEXT,MHLW, MAFF) as quasi-regulations. The items to be investigated when drafting an animal
experiment protocol are listed in the detailed guidelines formulated by SCJ. As proposed by
SCJ, adequate self-regulation should be validated by an outside organization and such validation
improves the social transparency of animal experimentation. However, there is still disagreement over whether we should aim toward more stringent regulations, or continue the current
self-regulation system. The author feels some limitations in the Law for balancing scientificjustification of procedures and animal well-being. It is time to discuss more appropriate guiding
systems not only for scientists but for the public.
(COI:No)

PS21-04

Recent trends in global neuroethics: an overview
Norihiro Sadato（Nat’l Inst Physiol Sci）
The purpose of neuroscience research on humans is to clarify how the brains of healthypeople work. Obtaining new knowledge about brain function is essential scientifically, andit also leads to the elucidation of the pathological conditions of various brain diseases andthe development of treatments. As the number of patients with dementia and movement
disorders will increase in an aging society, such research has social significance. On the other
hand, accurately communicating the new knowledge gained about the work of the brain to
the general public is essential to gain trust from society and to recognize the social usefulness
and significance of the research. Care should be taken not to cause human rights violations as
a result of non-invasive brain function studies. With recent advances in artificial intelligence(AI) and robots, and the development of neuro-intervention technology, international discussions on the above ethical aspects in neuroscience are increasing. In particular, there are
increasing concerns about individual identity as basic human rights (i.e., physical and mental
integrity), behavioral independence (ability to choose behavior), and the possibility of infringing
on privacy. The importance of explanation and consent in participating in experiments as
a procedure for protecting human rights is emphasized. Also, sufficient attention must be
paid to the changes in social norms and the possibility of new forms of discrimination causedby specific cognitive enhancement using neural intervention techniques. As neuroscience is
now a global effort, neuroethics must be equally ready to address global values. The groupof internationally recognized brain projects (the International Brain Initiative, IBI) forms the
Neuroethics Workgroup that organized The Global Neuroethics Summit in 2017 through 2019.
The trends of global neuroethics will be overviewed by introducing the series of summit
meetings. (COI:No)

PS21-02

Guidelines for the Care and Use of Laboratory Primates in
Neuroscience and Behavioral Research
Katsuki Nakamura（ Primate Res Inst, Kyoto Univ）
In Japan, the Ministry of the Environment published Act on Welfare and Management of
Animals and Standards related to the Care and Keeping and Reducing Pain of Laboratory
Animals for Appropriate Care and Keeping of Animals. The Ministry of Education, Culture,
Sports, Science and Technology published Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities on Academic Research Institutions for Appropriate Useof Laboratory Animals. In addition to these, the Science Council of Japan published Guidelines
for Proper Conduct of Animal Experiment. Then each university or Institution should haveits own guidelines for animal experiments. When we conduct animal research, the researchshould adhere to these laws and guidelines. In a university, the president is the responsible
person for all animal experiments. The university should have the animal care and use committee and education and training program. Information disclosure about animal experiments
is demanded. Our system is, so-called Institution or University Management System. We think
this system works very well.
However, once we think international collaboration or data sharing, our system is very complicated. Two years ago, the Japan Neuroscience Society set up an ad-hoc committee to publish
Guidelines for the Care and Use of Laboratory Primates in Neuroscience and Behavioral
Research. We will publish these guidelines both in Japanese and English. In this talk, I am
going to introduce the guidelines.
(COI:No)S30

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Planned Symposium 22
Challenge for the unsolved mechanism ofarrhythmias; from the view of physiologists
and cardiologists（March 19, 9:00〜10:50, Hall 5）

PS22-03

Possibility of recent balloon technologies for persistent atrial ﬁbrillation
ablation
Shiro Nakahara（ Dept Cardiology, Dokkyo Medical University Saitama Medical Center）
Ablation of persistent and long-standing persistent atrial fibrillation (AF) is a potentiallycomplex procedure that carries a lower success rate than ablation of paroxysmal AF. Atrial
substrate modification is required for a successful outcome in most patients with persistent
AF. Substrate modification is considered when AF persists despite effective elimination of
the pulmonary vein (PV) arrhythmogenicity by an extra-ostial PV isolation (PVI), antral PVI,
or wide area circumferential ablation. Currently, we favor a posterior wall isolation, carefulattention to low-voltage areas, and non-pulmonary vein trigger mapping for the persistent andlong-standing persistent AF population. The balloon ablation system is a safe and predictable
toolset to generate a reliable large-area pulmonary vein antral modification for the treatment
AF. Further, hot balloon ablation of AF makes use of a compliant balloon that optimizes the
PV contact. This feature provides for both a wide antral ablation and ablation of the left atrial(LA) posterior wall, which is implicated in the perpetuation of AF. To achieve this effect, wedeveloped a hot balloon-based wide antral ablation (HBWA) protocol and have since applied itin patients with persistent and longstanding persistent AF. In this session, the techniques andoutcomes of the current substrate modification in patients with persistent AF will be reviewed
and discussed.
(COI:No)

PS22-01

PS22-04

Masao Yoshinaga（ National Hospital Organization Kagoshima Medical Center）

Kunichika Tsumoto1，Takashi Ashihara2，Narumi Naito3，Takao Shimamoto3，
1 1 Dept
Akira Amano3，Yoshihisa Kurachi4，Yuichi Kuda1，Mamoru Tanida1，Yasutaka Kurata（

There remained issues to be solved in infants, children, andadolescents with long QT syndrome
Huge progress has been achieved in understanding the pathogenesis, diagnosis, and treatment
of inherited arrhythmia syndrome, particularly of long QT syndrome (LQTS) after discovery
of LQTS-causative genes and rapid advances in DNA sequencing technologies. Knowledge
gained from the progress includes genotype-phenotype relationships and gene-specific riskmanagement strategies; however, exercise-related cardiac events occur not only in patientswith LQT1 but in those with LQT2 and LQT3. Cardiac events by emotion, during rest or sleep
without arousal occur in both patients with LQT2 and LQT3, although the underlying causative genes for ion channels are different. Additionally, a Japanese study also showed circadian
distribution of cardiac events, suggesting the effect of autonomic nervous activity. Details ofgenetic and physiological backgrounds in terms of circadian distribution and autonomic effects
remain unclear.
In Japan, a school-based ECG screening is mandatory for all first, seventh, and tenth gradersfrom 1995. Our data showed that the cumulative risk of symptom in patients with definite
LQTS considerably decreased from the 2005-2011 to 2012-2018 periods, suggesting that the
program had a profound impact on the improvement in the outcome. At the same time,
the program uncovered many patients with a definite LQTS without symptoms. Pediatriccardiologists in Japan should consider an additional guideline for asymptomatic patients who
are diagnosed through this program.
Finally, one of the most important issues in pediatric fields is prevention of sudden infant
death syndrome (SIDS). SIDS is multi-factorial in origin, but genetic analysis showed thatapproximately 10% of SIDS victims carry functionally significant mutations in LQTS genes.
Up to 83% of SIDS occur during night-time sleep. Number of SIDS was 109 in 2016 in Japan;
however, number of out-of-hospital cardiac arrest with unknown etiology in infancy was 428 in
2016. Association between QT dynamics and sleep should be thoroughly investigated.
(COI:No)

PS22-02 (AP-4)

Molecular characterization of the arrhythmogenic trigger unique topulmonary vein cardiomyocytes
Yosuke Okamoto1，Yoshinobu Nagasawa2，Aung Naing Ye3，Yukiko Himeno4，
1 1 Dept Cell
Akinori Noma4，Akira Amano4，Daichi Takagi1，Kuniaki Ishii5，Kyoichi Ono（

Physiol, Grad Sch Med, Akita Univ, Japan, 2 Dept Pharmacol Ther, Fac Pharm Sci, Toho
Univ, Japan, 3 Dept Pathol Diagn, Fac Med, Yamagata Univ, Japan, 4 Dept Bioinfomat, Coll
Life Sci, Ritsumeikan Univ, Japan, 5 Dept Pharmacol, Fac Med, Yamagata Univ, Japan）
Pulmonary veins (PVs) are the major origin of atrial fibrillation. We have reported that IP3R2
in rat PV cardiomyocytes cooperates with Na+-Ca2+ exchanger (NCX) on T-tubule in triggering the norepinephrine (NE)-induced automaticity, and identified a unique hyperpolarization
activated Cl－ current, ICl, h, that potentially facilitates the automaticity. The mathematicalmodel which incorporated the interaction between IP3R2 and NCX, and electrophysiologicalcharacteristics of ICl, h successfully reproduced the NE-induced automaticity. Here, we show
two further topics for the properties of rat PV cardiomyocytes in relation to its arrhythmogenicity. The first one is that a Ca2+-stimulable adenylyl cyclase (AC) is involved in theNE-induced automaticity. Microarray, RT-PCR and immunohistochemistry uncovered that oneof Ca2+-stimulable AC was expressed regionally in the supraventricular area including PV. In
particular, enriched expression of the AC was detected along T-tubule of PV myocytes, whileatrial myocytes hardly displayed T-tubules. HEK293 cells exhibited sustained Ca2+ oscillationin response to UTP under isoproterenol pre-application. Gene-knockout of our interest in
the cells impaired the ability to keep the Ca2+ oscillation. The NE-induce automaticity in PV
cardiomyocytes was reversibly arrested by AC inhibitor. The second topic is related to thestructural basis of ICl, h. Mass spectrometry identified HSPA8 as the CLCN2 interacting proteinfrom rat PV. The auxiliary subunit of CLCN2 was subcloned and introduced into PC12 cell.
With co-expression of the HSPA8, CLCN2 current exhibited the unique voltage-dependency
similar to ICl, h. All these findings suggest that unique molecular interaction among NCX,
IP3R2, and the AC along T-tubule potentiates the arrhythmogenicity of rat PV, and that rat
heart possessed HSPA8 as the auxiliary subunit of the hyperpolarization activated Cl－ channel.
(COI:Properly Declared)

Development of fatal arrhythmias mediating subcellular Na+ channel
expression changes: in silico study
Physiol, Kanazawa Med Univ, Japan, 2 Dept Med Info Biomed Eng, Shiga Univ Med Sci,
Japan, 3 Dept Bioinfo, Coll Life Scis, Ritsumeikan Univ, Japan, 4Global Center Med Eng
Info, Osaka Univ, Japan）

Cardiac voltage-gated sodium (Na+) channels play key roles in the action potential (AP)initiation and propagation. Numerous experimental studies have reported that the Na+ channel
expression was changed within each of the myocytes in congenital and acquired heart diseases
such as Brugada syndrome and myocardial infarction. We hypothesized that an alterationin subcellular Na+ channel expression caused fatal arrhythmias. To test this hypothesis, wehave proposed physiologically relevant in silico human ventricular myocardial strand and ringmodels where myocytes were electrically connected by both gap junctions and an electric fieldmechanism, the latter of which involves an interference effect between intercalated discs (IDs),
in which the electrical communication between myocytes is mediated by the extracellularpotential changes elicited in the intercellular cleft space facing the IDs. We investigated therelationship between the altered subcellular Na+ channel expression and arrhythmogenicity
through computer simulations of AP propagation in the myocardial strand and ring models.
In this symposium, we will present our recent simulation results and would like to discuss theproarrhythmic effects of alteration in the subcellular Na+ channel expressions.
(COI:No)

PS22-05

Inherited arrhythmia in the era of next generation sequencer
Seiko Ohno（Dept.Bioscience and Genetics, NCVC. Japan）
Inherited primary arrhythmia syndromes (IPAS) are diseases mainly caused by mutations
in genes encoding ion channel and related proteins. Long QT syndrome (LQTS) is the mostnotable disease in IPAS. The main cause of LQTS is mutations in genes encoding potassiumchannel (KCNQ1 and KCNH2) or sodium channel (SCN5A). The genotypes caused by thesegenes are classified into LQT1, 2 and 3. Mutations in KCNQ1 and KCNH2 causes decreaseof IKs and IKr, in contrast, mutations in SCN5A increase the late INa. These genotype andelectrophysiological relationships have been progressed complementary. Since the early 2000s,
next generation sequencer (NGS) system has been developed, and we can now access to thehuge genetic data in short time. Until now, 17 genes have been reported in LQTS, and NGS
enabled us to screen all the genes at once. We recently identified that the frequency of LQT8,
caused by CACNA1C mutations was higher than we expected. The identification of novel
mutations in CACNA1C encoding alpha subunit of L type calcium channel will help us understand calcium handling in the cardiomyocytes. Catecholaminergic polymorphic ventriculartachycardia (CPVT) is one of the IPAS, and it was caused by mutations in RYR2 encoding
cardiac ryanodine receptor. Although RYR2 is a huge gene, we can now screen RYR2 easily
and new disease entity caused by RYR2 mutations has been shown. KCND3 encode KV4.3, analpha subunit of Ito. We recently identified a novel KCND3 mutation in a patient with early
repolarization syndrome. The mutation increased the Ito, and simulation study proved themechanism of the early repolarization. Thus, new genetic discovery in IPAS using NGS lead
us to find a new mechanism of cardiac electrophysiology. These progress in both fields will be(COI:No)
indispensable to explore the effective treatment for IPAS.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S31

Planned Symposium 23

PS23-03

Lymphatic vascular development and embryonic edema in mice
Masanori Hirashima（ Div Pharmacol, Grad Sch Med Dent Sci, Niigata Univ, Japan）

Angiology evolving into new research fields（March 19, 9:00〜10:50, Hall 6）

PS23-01

Sequential phosphoinositide conversion is required for TGFβ-induced
receptor endocytosis and endosomal receptor signaling in endothelialcells
1 1 Department of Physiology
Sho Aki1，Kazuaki Yoshioka1，Noriko Takuwa1, 2，Yoh Takuwa（

Kanazawa University School of Medicine, Japan, 2 Department of Health Science, Ishikawa
Prefectural University, Japan）

Phosphatidylinositol (PI) 3-kinases (PI3Ks) are a family of enzymes that phosphorylate membrane inositol phospholipids at the 3’-position of the inositol ring and comprise three classes.
Class I PI3Ks, which mainly generate PI(3, 4, 5)P3 are activated by RTK and GPCR to mediate
activation of Akt and Rac and stimulation of cell proliferation and migration. Class III PI3K,
Vps34, generates PI(3)P to regulate autophagy. In contrast to the well characterized class I
and III PI3Ks, physiological roles of class II PI3Ks were not well understood. We previously
demonstrated that class II α isoform (PI3K-C2α), which generates PI(3, 4)P2 by phosphorylating PI(4)P, is required for endocytosis of angiogenic receptors for TGFβ, VEGF and S1P
and endosomal signaling of these receptors, by which PI3K-C2α contributes to angiogenesis.
However, it is unknown how the PI3K-C2α substrate PI(4)P is derived and the PI3K-C2α
product PI(3, 4)P2 is catabolized, and which enzymes are involved in the phosphoinositideconversions. TGFβ induced rapid decreases in PI(4, 5)P2 at the plasma membrane (PM) withincreases in PI(4)P, followed by increases in PI(3, 4)P2, in a TGFβ receptor ALK5－dependent
manner. siRNA－mediated knockdown studies showed that PI3K-C2α, the 5’-phosphatasesynaptojanin1 (Synj1), and the 4’-phosphatase INPP4B were required for TGFβ－induced
receptor endocytosis. TGFβ induced the recruitment of both Synj1 and PI3K-C2α to the PMwith their substantial colocalization. Interestingly, PI3K-C2α knockdown abolished not only
TGFβ–induced PI(3, 4)P2 increases, but also Synj1 recruitment to the PM, PI(4, 5)P2 decreases.
Finally, the phosphoinositide conversions were necessary for Smad2/3 signaling, participating
in angiogenic actions of TGFβ. These observations demonstrate that the sequential phosphoinositide conversions mediated by Synj1, PI3K-C2α, and INPP4B are essential for TGFβ
receptor endocytosis and its signaling. (COI:No)

Lymphatic vessels comprise a secondary vascular system in mammals and play importantroles in tissue fluid homeostasis, immune response, and fat absorption. We have been studying lymphatic vascular patterning in mouse embryonic skin and found that Aspp1 plays
an important role in lymphatic vessel assembly. Aspp1－/－ mice show embryonic edemawith delayed lymphatic network formation and are growth-retarded during early postnatal
periods. However, Aspp1－/－ mice survive mostly to the adulthood without any overt
abnormalities including lymphedema. We also found a similar transient embryonic edemawith enhanced vascular permeability in haploinsufficient mice for Flt1/VEGFR1, a decoy
receptor for VEGF-A. To address whether regulation of fluid homeostasis is crucial for embryonic development, we crossed these two mutant mice exhibiting a transient embryonicedema. Flt1+/－; Aspp1－/－ double mutant mice showed greater degree of embryonic edema,compared to either single mutant, and turned out to be lethal in large part. These results
indicate that embryos are very sensitive to a change of fluid homeostasis. Embryonic edema inmice is very similar to the increased nuchal translucency, a sign of embryonic edema found by
ultrasonography during human pregnancy. Here I would also like to discuss our novel genetic
screen for gene mutations causing embryonic edema in mice to investigate the pathogenesis,(COI:No)risk, and prognosis of dysregulated fluid homeostasis.

PS23-04

Light and dark sides of aPKC in endothelial cells
Masanori Nakayama（ MPI HLR, Germany）
Cell polarity is critical for tissue and organ architecture and its loss is frequently associatedwith overproliferation and tissue expansion. The polarity protein atypical protein kinase C
lambda/iota (aPKCl) is an oncogene and is associated with cell proliferation through unknown
mechanisms. In endothelial cells, suppression of aPKCl impairs proliferation despite hyperactivated vascular endothelial growth factor (VEGF) signaling. Here we show that aPKCl
phosphorylates the DNA binding domain of forkhead box O1 (FoxO1) transcription factor, agatekeeper of endothelial growth. Although VEGF signaling is known to exclude FoxO1 fromthe nucleus, consequently increasing c-Myc abundance and proliferation, aPKCl controls c-Myc
expression without affecting its localization. We find this pathway is strongly activated in themalignant vascular sarcoma, angiosarcoma, and aPKC inhibition reduces c-Myc expression
and proliferation of angiosarcoma cells. Moreover, FoxO1 phosphorylation by aPKC and aPKC
expression correlates with poor patient prognosis. Our findings may provide a new therapeutic(COI:No)
strategy for treatment of malignant cancers, such as angiosarcoma.

PS23-02

Intravital imaging analysis of the substance discharge through avascular wall in microcirculation using noninvasive and nonlinear
optical microscopy
2 1 Dept Med Physiol, Hamamatsu Univ, Japan, 2 PRESTO, JST）
Naoki Honkura1, （The functional blood circuit always provides oxygen, nutrients and endogenous hormonal
molecules to our organs and tissues from blood vessels. These are essential to keep biologicalhomeostasis of multicellular organisms. The capillary vessels, constructed mainly by endothelial cells, are known to undergo changes in blood flow, blood pressure, diameter, permeability
in response to various stimuli on the local environment. The underlying mechanisms andinterdependences of these responses in different vessel types are largely unknown. Here I
report a non-invasive technique to study dynamic events in different vessel categories using
nonlinear optical microscopy and an image analysis tool, RVDM (relative velocity, direction andmorphology), which allows the classification of vessel categories by the calculated distortionparameters of flowing red blood cell (RBC) captured with slow frame rate. Additionally, oxygen
release from RBCs in blood vessels could be visualized by high spatio-temporal resolution
images with multiphoton excitation of fluorescent molecules and harmonic generation thatoriginated from biological molecules, which enabled us to analyze the location as well as themechanism of oxygen release. Moreover, the mouse who expresses Claudin5 promoter-driven
green fluorescent protein (GFP) is used to distinguish capillary subtypes by its fluorescencesignal in blood vessels in in vivo. Intradermal injection of vascular endothelial growth factor A(VEGFA) into ear skin appeared to induce leakage of circulating fluorescent dye conjugateddextrans, with vessel-type dependent different kinetics, from only capillaries and venules
devoid of GFP expression. VEGFA-induced leakage in capillaries coincides with vessel dilation
and reduced flow velocity. Thus, intravital imaging of non-invasive stimulation combined withnonlinear optical effects and RVDM analysis appeared beneficial for recording and quantification of very rapid events taking place in the blood circuit.
(COI:No)S32

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Planned Symposium 24
What is ‘data-driven’ science（March 19, 14:10〜16:00, Hall 1）

PS24-03

Pharmacological research development based on medical big data(disease name, Lab tests, medication, etc.) of 2.4 million patients at
Nihon University Hospital for 15 years
Satoshi Asai（Divi Pharma, Sch Med, Nihon Univ, Japan）
Nihon University, having been striving for the further improvement of medical progress andmedical services, is engaged in research concerning the utilization of information from dailyclinical practice, and in 2004 has constructed the clinical data warehouse (CDW) known as “Nihon University School of Medicine’s Clinical Data Management System” (NUSM’s CDMS) As
of March 2019 for 15years, this system stores over 2.43 million patient profiles, medical history
data for 1.27 mil. patients (24 mil. records), drug prescription data of 690,000 patients (40 mil.
records), injection medicine prescription data of 270,000 patients (17.5 mil. records), and clinicaltest data of 800,000 patients (440 mil. records). This system possesses a sufficient amount of
information for highly reliable statistical analysis. We have announced to international journals
our research thus far that utilizes this abundance of information, which includes studies of theadditional effects in hypertensive drugs (add-on effects), and of the side-effects that occur inthe combined use of antithrombotic drugs. NUSM’s CDMS is a healthcare database based on
information from daily clinical practice and furthermore constructed on the premise of use
in research, already at a sufficiently practical level. In observational studies using NUSM’S
CDMS, the drug effects, or so-called “effectiveness”, in actual clinical settings, which cannot be
understood through randomized clinical trials (RCTs) carried out in limited environments, areable to be verified and helpful evidence provided.
(COI:Properly Declared)

PS24-01

PS24-04

Yoshihiro Yamanishi（Dept Biosci Bioinf, Fac Comp Sci Syst, Kyushu Inst Tech, Japan）

Shuji Kaneko（Dept Mol Pharmacol, Grad Sch Pharm Sci, Kyoto Univ）

Recent developments in biotechnology have contributed to the increase in the amounts ofhigh-throughput data in the genome, transcriptome, proteome, metabolome, phenome and diseasome. These biomedical big data can be useful resources for drug discovery and healthcare.
Drug repositioning, or the identification of new drug indications (new applicable diseases ofexisting drugs), is an efficient strategy for drug development. The drug repositioning approachhas received remarkable attention in pharmaceutical industry, because it can increase the success rate of drug development and to reduce the cost in terms of time, risk, and expenditure.
In this study, we developed novel machine learning methods (basic technologies of artificial
intelligence) for predicting potential target proteins and new indications of drug candidate
compounds toward automatic drug discovery and repositioning. We performed the predictionof unknown therapeutic effects based on various large-scale omics data of drugs, compounds,
genes, proteins, and diseases in a framework of supervised network inference. Our results
show that the proposed method outperforms previous methods in terms of accuracy and applicability. We performed a comprehensive prediction of new indications of all approved drugs
and bioactive compounds for a wide range of diseases defined in the International Classificationof Diseases. We show several biologically meaningful examples of newly predicted drug indications for cancers and neurodegenerative diseases. The proposed methods are expected to be
useful for various applications in drug discovery and healthcare. (COI:No)

Adverse drug reactions have been utilized to make animal models of human disease since
there are some similarity between the human symptoms and adverse drug reactions. In thiscontext, accumulating case reports of human adverse events would be attractive clinical evidence, if we could statistically identify a concomitant medication that reduces the occurrenceof an adverse event. This simply enables ’drug repositioning’ that proposes a practical reliefof adverse reaction. Moreover, the drug-drug interaction may provide important clues to thediscovery of new molecular mechanism of adverse effects, and hopefully, of human diseases.
The FDA Adverse Event Reporting System (FAERS) is a public database that accumulateshuge self-reports of adverse events. In nearly half of the cases, multiple drugs are prescribed,
so that potential drug-drug interactions are to be analyzed. Focusing on adverse reactions
relating to diabetes mellitus (DM) caused by an atypical anti-schizophrenic drug, quetiapine,
we found that concomitant use of vitamin D analogs significantly suppresses the occurrenceof the quetiapine-induced DM in FAERS. Experimental validation using mice revealed that
quetiapine acutely caused insulin resistance, which was mitigated by dietary supplementation with cholecalciferol. In a gene expression database, several genes downstream of insulin
receptor were downregulated by quetiapine in the liver. Further experiments clarified that a
PI3K regulatory protein gene, pik3r1, was downregulated by quetiapine, which was reversedby cholecalciferol in mouse skeletal muscle. In addition, the insulin-stimulated glucose uptake
into cultured myotubes was inhibited in the presence of quetiapine, which was reversed by
pretreatment with calcitriol in a PI3K-dependent manner. These results suggest that vitamin
D prevents the atypical antipsychotic-induced hyperglycemia and insulin resistance by upregulation of PI3K function. This new strategy will pave the way for drug repositioning andclarifying unknown disease mechanisms.
(COI:No)

Data-driven drug discovery and healthcare by artiﬁcial intelligence

Drug repositioning and target ﬁnding based on clinical evidencePS24-02

A data-driven approach to identify drugs against ischemia-reperfusion
injuryYuhei Nishimura（ Dept Integrative Pharmacol, Mie Univ Grad Sch Med, Japan）
Recent technological advances have enabled us to obtain large amounts of multi-level omics
data. The increasing volume of multilevel omics data continues to create larger and more
complex datasets that are publicly available and can be used to generate disease-associatedbiological networks and to identify potential therapeutic targets within the networks. Further
progress in computational methodology combined with improved disease models will facilitate
the prioritization of therapeutic targets in the networks. Data-driven approaches, such as theintegration of public omics database, bioinformatics tools, and disease models, can provide astrong foundation for deciphering the complex mechanism of various diseases and for the identification of novel therapeutics. In this symposium, I would like to demonstrate an example ofour data-driven approach to identify potential therapeutic drugs against ischemia-reperfusion(COI:No)
injury. 

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S33

Planned Symposium 25

PS25-03

Molecular and Neuronal substrates that regulate nutrient appetite
Tsutomu Sasaki（Nutrition Chemistry Lab, Div Food Science Biotechnol, Grad Sch Agr,

Mechanisms of experience- or metabolismdependent behavioral changes（March 19, 14:10〜16:00, Hall 2）

Kyoto Univ, Japan）

Nutrition is defined as the process of ingesting food for the purpose of growth, metabolism, andrepair. In other words, food is ingested to obtain nutrients that are necessary for maintaining
homeostasis. However, the studies on the homeostatic regulation of appetite have focused
mostly on energy balance. Macronutrients (carbohydrate, fat, and protein) generates energy,
but they cannot be distinguished among each other if we only think about calorie. It is empirically known that animals have appetite for nutrient (called “nutritional wisdom”), yet themolecular and neuronal substrates that regulate nutrient appetite remain largely elusive.
Our works on the homeostatic regulation of appetite by NAD+-dependent protein deacetylase
SIRT1 unexpectedly revealed a mechanism that regulates simple sugar appetite. Simple sugar
ingestion induces secretion of the hepatokine FGF21, which activates hypothalamic oxytocin
neuron, and suppresses simple sugar appetite. Therefore, FGF21-oxytocin axis is a negative
feedback system that regulates simple sugar appetite.
My lab is currently working on fat appetite and protein appetite, and the updates on macronutrient appetite will be presented at the talk.
(COI:No)

PS25-01

PS25-04

Taiju Amano（Dept Pharmacol, Grad Sch Pharm, Hokkaido Univ, Sapporo, Japan）

Hirotaka Sakamoto（Ushimado Marine Inst (UMI), Okayama Univ, Japan）

The medial preoptic area (MPOA) is thought to be the most important region for pup directedbehavior. Previous study revealed that lesion of the MPOA blocked parental behavior andalso initiated the aggressive behavior toward pups. However, the potent input sources into
the MPOA to control the behavioral pattern is not well addressed. In this study, we exploredthe neural nucleus that projects to the MPOA and addressed the function and modulatory
mechanisms. We infused retrograde tracer into the MPOA and checked the expression patternof neural activity marker c-Fos in the mice experienced the exposure of pups. Among thesources of the input into the MPOA, amygdalohippocampal area (AHi) showed double positive
neuron of retrograde tracer and immunoreactive to c-Fos. It is reported that AHi is one of
the areas expressing oxytocin receptor (OXTR), which is important for affiliative behavior.
Therefore, we performed whole-cell patch-clamp recording from the AHi neurons projecting
to MPOA. Application of oxytocin increased the frequency of the spontaneous inhibitory post
synaptic currents (sIPSC). Next, to address the role of the AHi neurons projecting to MPOA
on the behavior toward pups, we infused adeno-associated virus to express hM3Dq. Activationof the AHi neurons projecting to MPOA resulted in the disruption of parental behavior andpromoted the aggressive behavior toward pups. These evidences contribute to understand the(COI:Properly Declared)
circuit-based mechanism to control the parenting behavior.

Male sexual activity is activated by copulatory experience. However, it remains unclearthat effects of copulatory experience on neural circuits in the central nervous system. The
purpose of this study is to reveal effects of copulatory experience on neural circuits controlling
male sexual activity in rats, focusing on the oxytocin-oxytocin receptor (OTR) system. To
visualize OTR-expressing neurons, we generated an OTR promoter-human diphtheria toxin
receptor-2A-channelrhodopsin-2-enhanced yellow fluorescent protein (ChR2-EYFP) BAC transgenic (Tg) rat line and studied effects of copulatory experience on neural circuits controlling
male sexual activity by examining the EYFP fluorescence. OTR-expressing neurons werefirst localized by using these Tg rats, and the medial preoptic area, bed nucleus of the stria
terminalis and ventrolateral part of the ventromedial nucleus (VMHvl) were clearly labeledwith EYFP fluorescence. These nuclei are reported to express OTR in rats. Subsequently,
effects of copulatory experience in males on OTR expression were examined, and copulatory
experience increased in the expression levels of OTR in these 3 brain areas. Because theincrease in the expression level of OTR (EYFP) in the VMHvl was remarkable, the toxin
receptor cell-knockout (TRECK) and optogenetic analyses were performed in the VMHvl of
the copulatory experienced males. By microinjecting diphtheria toxin into the VMHvl, thelocal lesion of OTR-expressing neurons significantly decreased the number of mounts during male sexual behavior. In contrast, the local optogenetic stimulation of OTR-expressing
neurons in the VMHvl significantly attenuated ejaculation reflexes, but did not affect any
pre-ejaculatory behaviors during normal sexual activity. In conclusion, these results suggestthat copulatory experience reorganizes neural circuits expressing OTR, and consequently, the(COI:No)enhanced oxytocin-OTR system in the brain may facilitate male sexual activity.

The role of the amygdalohippocampal area neurons that projects to themedial preoptic area

Effects of copulatory experience on neural circuits controlling malesexual activity via the oxytocin-oxytocin receptor system in rats

PS25-02

The role of oxytocin in behavioral changes induced by social defeat
stressAyumu Inutsuka, Masahide Yoshida, Yuki Takayanagi, Tatsushi Onaka（ Dept Physiol, Med,
Jichi Med Univ, Japan）

Stress affects various behaviors including social interactions. It has been reported that oxytocin
modulates anxiety- and stress-related behaviors. The prefrontal cortex (PFC) is innervated byoxytocin neurons and densely expresses the oxytocin receptor (OXTR). In this study, we investigated the physiological role of the OXTR of the PFC in behavioral changes induced by social
defeat stress. After social defeat stress, expression of c-Fos protein was increased in oxytocin
neurons of the bed nucleus of the stria terminalis, supraoptic nucleus, and paraventricular hypothalamic nucleus. Using virus vectors employing GFP-dependent Cre, we genetically labeled
and traced Venus-expressing neurons in the PFC of Oxtr-Venus knock-in mice. We found that
OXTR-expressing neurons in the PFC consist of two populations: one is somatostatin-positive
interneurons and the other is projection neurons in the layer 2/3 that send their axons to
the amygdala. Chronic social defeat stress increased anxiety-related behaviors and induces
social avoidance in C57BL/6J mice. We performed stereotaxic injections of Cre expressing
adeno-associated virus vectors into the PFC of Oxtr-floxed mice and investigated the behavioral
changes after chronic social defeat stress. We will discuss the behavioral differences between(COI:No)the test mice and the control mice. 

S34

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Planned Symposium 26
New insights of sensory and brain functions
affecting feeding behaviors: circadian,swallowing, taste and pain
（March 19, 14:10〜16:00, Hall 3）

PS26-03

Molecular mechanism for the suppressing effect of low pH on the sweet
receptor sensitivity
2 1 Sect of Oral Neurosci,
Keisuke Sanematsu1, 2，Yuzo Ninoimya2, 3，Noriatsu Shigemura1, （Grad Sch of Dental Sci, Kyushu Univ, Japan, 2 R and D Ctr for Five-Sense Devices, Kyushu
Univ, Japan, 3 Monell Chemical Senses Ctr, USA）

Taste information influences on feeding behaviors. The sweet taste is involved in maintaining homeostasis and mediated by the taste G-protein-coupled receptors (GPCR), TAS1R2 +TAS1R3. It is well known that a glycoprotein, miraculin induces sweet taste by acidification.
Our previous study suggests that the protonation of the sweet receptor was also required
for the sweet-inducing effect of miraculin, praising a possibility that pH affects the sweet
receptor sensitivity. Therefore, we examined responses to sweet compounds under low pH
conditions by using the sweet receptor assay. We found that responses to saccharin werereduced when its solutions were acidified. This effect was species-specific to humans but
not to mice. Using mixed-species pairings of human and mouse sweet receptor subunits andchimeras, we determined that the suppressing effect of low pH on saccharin responses wasmediated by the transmembrane domain of human TAS1R3. Point mutation analysis revealed
the key residues for this effect. These were consistent with the data from molecular dynamics
simulation, indicating that saccharin strongly binds to the negative allosteric moderator site byacidification and suppresses its agonistic activity. Our results suggest that the function of thesweet taste receptor is regulated by the environmental pH and provide new insights into thefunctional characterization of other GPCR.
(COI:No)

PS26-01

PS26-04

Wataru Nakamura（ Dept Oral Chrono-Physiol, Grad Sch Bio-Med, Nagasaki Univ, Japan）

Masamichi Shinoda（ Dept Physiol, Sch Dent, Nihon Univ, Japan）

Some people can quite accurately time the end of their night’s sleep without using an alarm
clock, demonstrating that it might be controlled by biological clock. The regulation of daily
activity onset has been thought to be embedded in circadian rhythms controlling the hormone
release, body temperature, and resulting consciousness. Normally, the release of ACTH andcortisol increases during late stages of sleeping, reaching a daily maximum at the time of
behavioral activity onset. In some rodents, schedules of restricted timed daily feeding cansubstantially modify the temporal distribution of an animal’s behavioral and physiological activities. When food availability is restricted to a short temporal window in the day, animals display
pre-feeding locomotor activity and feeding-associated physiological changes known as foodanticipatory activity (FAA). In the present study, we clarified the property of food anticipatory
activity during temporally restricted feeding (RF) and investigated the effects of aging on theassociated behavioral characteristics. Molecular clock–deficient mice provide crucial evidence
for the involvement of molecular clock genes in circadian regulation of behavioral FAA, andthey may be important models for furthering our understanding of behavioral regulation in
aging by interaction between time measurements and circadian rhythms.
(COI:No)

Feeding behavior is known to be not only essential for life support but also important for
maintaining quality of life. Once orofacial pain such as tooth, tongue, periodontal tissue or
temporomandibular joint pain develops, feeding disorders are immediately caused, resulting
in lower quality of life. Despite burning mouth syndrome (BMS) is more common among
orofacial pain inducing feeding disorders, its pathophysiologic mechanisms remain incompletely
understood. We have found that the Artemin (Artn) mRNA expression in the tongue mucosa
of BMS patients was enhanced compared with nomal subjects. Therefore, we developed the
BMS mouse model by 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) application onto the tongue
dorsum to assess the involvement of Artn in the tongue mucosa in tongue pain hypersensitivity. Persistent, week-long tongue heat hypersensitivity with no signs of tongue histologicalchanges and Artn hyperexpression in the tongue mucosa were induced by the tongue TNBS
application. The consecutive transient receptor potential vanilloid 1 (TRPV1) antagonist or
anti-Artn neutralizing antibody into the tongue mucosa suppressed the persistent tongue heat
hypersensitivity by the tongue TNBS application. Moreover, the TRPV1 expression in the
glial cell line-derived neurotrophic factor family receptor α3 (GFRα3)-positive trigeminal
ganglion (TG) neurons terminating the tongue was potentiated via p38 signaling cascades. Its
TRPV1 hyperexpression was depressed by successive anti-Artn neutralizing antibody into the
tongue mucosa. The tongue TNBS application enhanced the TRPV1 agonist-induced inward
current in acute dissociated TG neurons terminating the tongue, which was inhibited by Artnneutralization. These findings indicate that Artn overexpression in tongue mucosa in BMS
patients potentiates the excitability TG neurons terminating the tongue due to TRPV1 overexpression attributable to the enhancement of GFRα3 signaling via p38 signaling cascades,
resulting tongue heat hypersensitivity. Together with recent studies, we would like to discuss(COI:No)
pathophysiologic mechanisms associated with BMS hampering feeding behaviors.

Meal Timing, Aging, and Circadian Rhythm

Tongue pain hypersensitivity hampering feeding behaviors

PS26-02

Peripheral mechanisms of mechanically evoked swallows
Takanori Tsujimura, Makoto Inoue（Div Dysp Reha, Niigata Univ Grad Sch Med Dent Sci,
Japan）

Swallowing has a vital function for airway protection and is one of the initial steps in foodingestion. Various non-noxious stimuli, including mechanical, taste, and thermal stimuli, may be
involved in natural swallowing. Because impaired laryngeal mechanical sensation is associatedwith food bolus aspiration, it is important to know how the laryngeal mechanosensory systemregulates swallowing initiation. This study was performed to clarify the neuronal mechanism of
mechanically evoked swallows. Urethane-anaesthetized Sprague–Dawley male rats were used.
A swallow was identified by electromyographic burst of suprahyoid and thyrohyoid muscles.
The swallowing threshold was measured by von Frey filament or electrical stimulation of thelarynx. The number of swallows induced by upper airway distension or capsaicin application(0.03 nmol, 3 µl) to the laryngeal mucosa was counted. The effects of topical application ofepithelial sodium channel (ENaC) and acid-sensing ion channel (ASIC) blockers (amiloride,
benzamil, and dimethylamiloride), ASIC inhibitors (mambalgine-1 and diminazene), and gadolinium (0.3–30 nmol, 3 µl) to the laryngeal mucosa on swallowing initiation were evaluated. A
nerve transection study indicated that afferents carried by the superior laryngeal nerve playa primary role in the initiation of laryngeal mechanically evoked swallows. The mechanicalthreshold of swallowing was increased in a dose-dependent manner of amiloride analogues
and gadolinium, but not ASIC inhibitors. The increased swallowing threshold by amiloride
analogues or gadolinium was diminished following saline washout. The number of swallows
by upper airway distension was significantly decreased by benzamil application. However,
the initiation of swallows evoked by capsaicin or electrical stimulation was not affected bybenzamil application. We speculate that ENaC is involved in the initiation of mechanicallyevoked swallows in the larynx.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S35

Planned Symposium 27
Pathophysiology of ion channels andtransporters in tumor growth（March 19, 14:10〜16:00, Hall 5）

PS27-01

Patho-physiological signiﬁcance of Ca2+-activated K+ channels in tumor
microenvironment
Susumu Ohya, Junko Kajikuri, Hiroaki Kito（Dept Pharmacol, Grad Sch Med Sci, Nagoya

City Univ, Japan）
Ca2+-activatedK+

channels (especially, KCa1.1 and KCa3.1) overexpressing in several solidcancers promote cancer cell proliferation and migration. Hypoxic tumor microenvironmentis a common characteristic of solid cancers and elevated hypoxia-inducible factor (HIF) underhypoxic condition is associated with tumor metastasis and poor patient prognosis. HIF signaling pathways also promote cancer cell stemness and chemoresistance. Our studies revealed
the patho-physiological significance of KCa channel up-regulation in hypoxic solid-tumor
microenvironment in vitro using ultra-low attachment cultureware making three-dimensional
cell spheroid formation. The anti-inflammatory cytokine, interleukin-10 (IL-10) is an immunosuppressive factor involved in tumorigenesis, and plays a crucial role in escape from tumor
immune surveillance. We also describe that KCa3.1 activators are a possible therapeutic option
to suppress the tumor-promoting activities of IL-10. (COI:No)

PS27-03

Novel pathophysiological properties of Na+, K+-ATPase in humancancer cells
1 1 Dept. Pharm.
Takuto Fujii1，Takahiro Shimizu1，Hiroshi Takeshima2，Hideki Sakai（

Physiol., Grad. Sch. Med. Pharm. Sci., Univ. Toyama, Japan, 2 Dept. Biol. Chem., Grad.
Sch. Pharm. Sci., Kyoto Univ., Japan）
Cardiac glycosides, potent inhibitors of Na+, K+-ATPase, have been used in treatment ofcongestive heart failure as positive inotropic agents. In addition, epidemiological studies andin vitro studies using cancer cell lines suggested the anti-cancer effects of cardiac glycosides.
Cardiac glycosides at submicromolar concentrations can block cancer cell growth without
affecting enzyme acivity of Na+, K+-ATPase, however, these mechanisms have not been fully
understood. We reported recently that Na+, K+-ATPase α1-isoform is associated with volumeregulated anion channel (VRAC), an anion channel responsible for cell volume regulation andcell death, in membrane microdomains of human cancer cells. Nanomolar concentrations ofcardiac glycosides, such as ouabain, digoxin, and digitoxin, are considered to interact with
Na+, K+-ATPase α1-isoform in membrane microdomains to activate VRAC via productionof reactive oxygen species, thus producing anti-proliferative effects. These cardiac glycosidesinduced effects were not observed in non-cancer cells. On the other hand, we have found thatnanomolar concentrations of cardiac glycosides drastically inhibit glucose uptake in humanliver cancer cells by decreasing the expression level of glucose transporter GLUT1 whichis overexpressed at the plasma membrane in many types of cancer cells. Cardiac glycosides
induce the clathrin-dependent endocytosis of GLUT1 via activation of Ca2+/calmodulindependent protein kinase II, and the endocytosed GLUT1 is degraded in lysosome. In thismechanism, cardiac glycosides may affect on Na+, K+-ATPase α3-isoform which is abnormally
expressed in intracellular compartments of various human cancer cells including liver cancer
cells. Our findings uncovered novel anti-cancer mechanisms in which cardiac glycosides andNa+, K+-ATPase are involved.
(COI:No)

PS27-04

Roles of leucine transporters in cancer and neural development
Hiroyuki Sakurai（Dept Pharmacol, Sch Med, Kyorin Univ, Japan）
Because branched chain amino acids, especially leucine, stimulate cell proliferation via
mTORC1 pathway activation, BCAA transport proteins are considered to be important
regulatory molecules for rapidly dividing cells such as embryonic or cancer cells. One of
BCAA transport proteins, LAT1 encoded by SLC7A5 gene, is expressed relatively specific for
cancer cells and its expression increases as the cancer evolves to more malignant phenotype.
Inhibition of LAT1 reduces leucine uptake into cancer cells, which leads to growth arrest and/or apoptosis via mTOR inhibition. In fact, a specific inhibitor for LAT1, JPH203, is reported tosuppress cancer cell proliferation in vitro and in vivo.
In this talk, I will present relatively unexplored roles of LAT1 and other leucine transporters
in cancer and development.
1.It has been shown that LAT1 is expressed in the placenta and early embryo. Its importancein early development is evident as lat1 knockout mice is embryonically lethal. We investigatedits expression and function in Xenopus embryo. Xenopus lat1 expresses in the notochord,the inducer of neural tissues, and in the eye. Its inhibition by morpholino injection resulted in
defective neural tube closure, primary neurogenesis, and eye formation.
2.Some prostate cancer cells express LAT3 when their growth is dependent on androgen. We
established androgen independent prostate cancer cell line from androgen dependent LNCaP
cells. The newly established cell line expresses y+LAT2, a sodium dependent large neutralamino acid transporter. Its knockdown by siRNA inhibited cell growth.
3. Although the expression levels of LAT1 was comparable between 2 breast cancer cell lines
MDA-MB-231 cells were more resistant to JPH203 treatment than T-47D cells. We are trying
to elucidate JPH203 resistant mechanism by transcriptome analysis using T-47D cells in thepresence or absence of JPH203. (COI:No)

PS27-02

Overexpression of constitutively active K+ channel (KCNK9) and cell
survival
Yoshihiro Konno1, 2，Makoto Sato1，Ikuo Norota1，Yutaro Obara1，Kuniaki Ishii1（ 1 Dept Pharmacol, Faculty Med, Yamagata Univ, japan, 2 Dept. Radiology, Faculty Med,
Yamagata Univ, Japan）
KCNK9, a member of the two-pore K+ channel family, is responsible for background K+
currents that regulate cell resting membrane potential and excitability. KCNK9 channel
is overexpressed in several types of human carcinomas, such as breast cancers and lungcancers, and is generally thought to be an oncogenic channel. However, little is known about
the mechanisms by which KCNK9 promotes oncogenesis. Here we assessed how ectopic
overexpression of KCNK9 affects cell survival in human pancreatic cancer (PANC1) cell. Stable
KCNK9 expression led to hyperpolarization of the resting membrane potential, which did notalter cell proliferation and cell cycle. However, the stable KCNK9 cells acquired resistance tocell apoptosis induced by hyperosmotic stress with enhanced expression of Bcl-2 gene. On theother hand, PANC1 cells transiently transfected with KCNK9 died by caspase-3/7-dependent
apoptosis, which was accompanied by activation of p38 MAPK. One possible explanation forthese paradoxical results is the difference in KCNK9 expression level: stable low expression
is anti-apoptotic and transient high expression is pro-apoptotic. Our data suggest that p38
MAPK pathway is involved in KCNK9-induced apoptosis, but it is not known whether p38
MAPK is activated by KCNK9 stable expression. Although further study is necessary toreveal the mechanisms underlying the oncogenic property of this channel, p38 MAPK might
be a candidate key molecule. In this presentation, I would like to talk about the effects of(COI:No)
overexpression of KCNK9 on cell survival and the possible mechanisms involved.

S36

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Planned Symposium 28
Basic and applied researches to progress theknowledge and therapy for heat stroke（March 19, 14:10〜16:00, Hall 6）

PS28-01

Exercise capacity in hot and humid environmental conditions -Possible
brain mechanisms and cooling strategiesHiroshi Hasegawa（ Grad Integ Arts ＆ Sci, Hiroshima Univ, Japan）

PS28-03

Central neural pathways for thermosensory information to promote heatloss responses and heat avoidance behavior
Kazuhiro Nakamura（ Dept Integrative Physiol, Nagoya Univ Grad Sch Med, Japan）
Thermal homeostasis is maintained in hot environments by autonomic facilitation of heat loss
and by behavioral avoidance of heat. These autonomic and behavioral responses for heat
defense are driven by central thermoregulatory circuits stimulated by sensory informationthat is derived from skin thermoreceptors sensing ambient temperatures. We have revealed
the ascending central neural pathways conveying cutaneous thermosensory information
for autonomic and behavioral thermoregulation. Cutaneous warm-sensory and cool-sensory
excitatory signals are separately transmitted from the spinal cord to the dorsal and external
parts of the lateral parabrachial nucleus (LPB), respectively. Then, the warming-activated andcooling-activated neurons in the LPB separately transmit excitatory signals to the thermoregulatory center in the preoptic area. The LPB-mediated thermosensory pathways are required
to elicit autonomic thermoregulatory responses including cutaneous vasomotor responses aswell as to drive thermoregulatory behaviors including heat avoidance. Interestingly, lesionsof the thalamus that ablate the spinothalamocortical pathway mediating thermal perception
do not affect autonomic or behavioral thermoregulatory responses to environmental thermal
challenges. Our results show that thermal homeostasis in hot environments requires thermosensory transmission mediated by the spinal–LPB–preoptic thermosensory pathways, distinct
from the central thermosensory process for thermal perception by the spinothalamocortical(COI:No)
pathway.

PS28-04

Heat tolerance, not related to heat loss responses
Kei Nagashima, Yuta Masuda（ BTFL, Fac Human Sci, Waseda Univ）

Hyperthermia has been demonstrated as an important factor limiting endurance performancein hot and humid environmental conditions in both human and animal studies. While temperature can affect individual peripheral physiological systems such as muscle contraction
characteristics directly, a dominant role for central mechanisms for exercise impairment has
been proposed over the past two decades. Exercise-induced hyperthermia may have a direct
effect on the central nervous system such as brain temperature, cerebral blood flow, brain
activity, cognitive function, brain neurotransmission and neuromuscular activity. In turn, thesechanges may affect not only the physiological capacity for exercise, but also the athlete’s
perception of heat stress, motivation for exercise or pacing strategy. Many major sport events
are held in extremely hot environments. The recent summer Olympic games are no exception, and these took place under high ambient temperatures (such as in Atlanta 1996; Athens
2004; Beijing 2008). This trend is likely to continue as athletes begin to prepare for what will
likely be Olympics and Paralympics in Tokyo 2020. Several specific approaches including fluid
intake, heat acclimation, pre, per and post cooling and other practical applications have been
investigated (Hasegawa and Cheung, 2013). This presentation focuses on the possible brain
mechanisms at high body temperature that influence exercise performance, and the variablestrategies such as body cooling for preventing hyperthermia during exercise in hot and humid
environmental conditions.
(COI:No)Heat stroke is one of big health problems in Japan. Besides climate changes within these
10-20 years, one of the reasons may be that residents in Japan have less ability of heat tolerance. However, it seems that many studies have considered heat tolerance as physiologicalresponse to minimize the increase in core body temperature during heat exposure and/or
exercise, especially heat loss responses such as elevated skin blood flow and sweat rate. In this
symposium, we introduce two studies evaluating heat tolerance in other point of view. Exp
1, male C57BL6 mice were expose to heat of 33℃ for 14 days with free access to horizontal
running wheel. On the first and last days of the exposure period, abdominal temperature wasmonitored with rotation counts of the running wheel. The rotation counts increased on the last
day compared with those on the first day. Abdominal temperature was greater on the last day
than that on the first day. These results may indicate that thermal input from the periphery
determines the counts of the running wheel and prevents increase of core body temperaturein heat. However, continuous heat exposure may blunt the effect of the peripheral thermal
input. Exp 2, young male subjects were immersed in 40℃ water up to the subclavian level for
40 min. Rectal temperature, skin blood flow and sweat rate of the forehead, and metabolic rate
were continuously monitored. Rectal temperature increased by ~2.5℃, which were various
among subjects. There were no relationships between the increase of rectal temperature andthe skin blood flow and sweat rate; however, negative relationship was found between rectal
temperature and metabolic rate. The results may suggest that metabolic response to heat is(COI:No)also a factor preventing increase in core body temperature. PS28-02

PS28-05

Blood ﬂow distribution in hyperthermia
Manabu Shibasaki（Nara Women’s Univ.）
Climate change has had a widespread impact on humans and natural systems. In recent
decades, a number of severe heat waves have occurred throughout the Northern Hemisphere.
The frequent occurrence of heat waves and the urban heat island phenomenon poses a
significant threat to human health. When exposed to a hot environment for a long time, we feeldizziness or light-headedness. These symptoms indicate orthostatic intolerance. Orthostatic
intolerance is considered to be markedly influenced by the regulation of blood pressure andcerebral blood flow during heat stress. While heat stressed, skin blood flow can increase from~300 mL/min upwards to ~8 L/min and is associated with marked decreases in systemic
vascular resistance. In order to maintain the blood pressure, cardiac output must increase
along with increases in vascular resistance, but 50% or more of cardiac output can be directedto the skin during severe heat stress. Thus, control of skin blood flow becomes an importantfactor for orthostatic tolerance in heat-stressed humans. A factor contributing directly to orthostatic tolerance may be cerebral blood flow or its regulation. As well as other non-cutaneous
vascular beds, heat stress also reduces cerebral blood flow. However, not only the distributionof blood but also hypocapnia due to hyperthermia-induced hyperventilation causes a reductionin cerebral perfusion. In this symposium, I will summarize the regulation of skin and cerebral(COI:No)
blood flow in hyperthermic individuals.

Possible central mechanism of acquired heat tolerance in heatacclimated rats
Kentaro Matsuzaki, Md Emon Hossain, Osamu Shido（Dept Environmental Physiol, Facult
Med, Shimane Univ, Japan）

In humans and rodents, chronic heat exposure has been well known to induce heat acclimationthat improves heat tolerance. We have reported that heat exposure promotes progenitor cellproliferation and neural differentiation in the hypothalamus and the inhibition of hypothalamic
neurogenesis impairs the ability of heat tolerance in rats. To elucidate the mechanisms for heatacclimation, we investigated the effects of direct heat exposure on the proliferation and differentiation of cultured neural stem/progenitor cells (NSCs/NPCs). The NSCs/NPCs isolated fromthe brain of 14.5-day-old rat fetuses were propagated as neurospheres at either 37.0℃ (control)
or 38.5℃ (heat exposure) for four days. The effects on proliferation were investigated by cell
viability assay, measurement of neurosphere diameter, and counting the total number of cells.
The mRNA expressions of heat shock proteins (HSPs) and brain-derived neurotrophic factor(BDNF), cAMP response element-binding (CREB) protein and Akt phosphorylation levels, andintracellular reactive oxygen species (ROS) levels were analyzed using real time PCR, Western
blotting and CM-H2DCFDA assay, respectively. Heat exposure under proliferation condition
increased NSC/NPC viability, neurosphere diameter, and cell count. BDNF mRNA expression,
CREB phosphorylation, and ROS level were also increased by heat exposure. Heat exposure
increased HSP27 and HSP70 mRNA expressions concomitant with enhanced phospho-Akt
level. Moreover, LY294002, a PI3K inhibitor, diminished the effects of heat exposure on NSC/
NPC proliferation. Furthermore, heat exposure under differentiation conditions increased the
proportion of cells positive for Tuj1 (a neuronal marker). These findings suggest that mild heat
exposure increases NSC/NPC proliferation, possibly through activation of the Akt pathway,
and also enhances neuronal differentiation. Direct effects of temperature on NSCs/NPCs may
be one of the mechanisms involved in hypothalamic neurogenesis in heat-acclimated rats.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S37

Planned Symposium 29
Recent advances in muscle physiology（March 19, 14:10〜16:00, Hall 8）

PS29-03

Analysis of cardiac sarcomere dynamics by in vivo nano-imaging
Fuyu Kobirumaki-Shimozawa1，Togo Shimozawa2，Jia Li3，William E. Louch3，
1 1 Dept Cell Physiol, Sch Med, Jikei Univ, Japan, 2 Tech
Shinichi Ishiwata4，Norio Fukuda（

Dev, Sch Sci, The Univ Tokyo, Japan, 3 Inst Clin Med, Fac Med, Univ Oslo, Norway, 4 Dept
Phys, Fac Sci and Eng, Waseda Univ, Japan）

Cardiac contractility is highly dependent on sarcomere length (SL), and a change of merely~100 nm causes a dramatic change in contractile performance (see Kobirumaki-Shimozawa
et al., J Physiol Sci, 2014). Therefore, for full understanding of the mechanism(s) of the heart’s
pump function, it is imperative to analyze the dynamics of cardiac sarcomeres at high precisionin vivo. In the present study, we expressed α-actinin-AcGFP in the Z-disks in cardiomyocytesof the left ventricle in adult mice, and applied SL nanometry (as in Shintani et al., J Gen
Physiol, 2014; Kobirumaki-Shimozawa et al., J Gen Physiol, 2016) for the measurement of SL
displacement at high spatial (20 nm) and temporal (10 ms) resolution under a fluorescencemicroscope, simultaneously with the measurements of left ventricular pressure (LVP) andelectrocardiogram. First, we found that the magnitude of correlation between the movementof each sarcomere and entire myofibrillar (ventricular) contraction (as quantified by “correlation index”, CI) varied between cardiac cycles. In other words, sarcomeres alternately
contributed to ventricular function (average CI, ~65%). Our analysis revealed that the longer
the sarcomeres during diastole, the higher the likelihood they contributed to ventricular
contraction in the subsequent beat. Second, in contrast to a traditional view, a sarcomere in a
myofibril repeated shortening and lengthening independently from parallel neighbors. Third,
CI was coupled with LVP, indicating that ventricular contractility depends on the populationof contributing sarcomeres. The present cardiac nano-imaging system has a broad range of application possibilities for unveiling sarcomere dynamics at high precision in relation to a change
in LVP in health and disease. At the meeting, we will discuss (1) sarcomere synchronization
as a key determinant of the ventricular function and (2) technical aspects of the application of
nano-imaging to the heart in vivo under true physiologic conditions.
(COI:No)

PS29-01

PS29-04

Shuya Ishii1, 2，Kotaro Oyama1, 2, 3, 4，Fuyu Kobirumaki-Shimozawa1，Shinichi Ishiwata5，
1 1 Dept Cell Physiol, Sch Med, Jikei Univ, Japan, 2 Sch Adv Sci Engn, Waseda
Norio Fukuda（

2 1 Dept Physiol, Gifu Univ, Grad Sch Med,
Koji Obata1，Hironobu Morita1，Miyako Takaki（

Microscopic heat pulses induce activation of thin ﬁlaments in striatedmuscleUniv, Japan, 3 QST, 4 PRESTO, JST, 5 Fac Sci Engn, Waseda Univ, Japan）

Cardiac mechanoenergetics in excised, cross-circulated whole heart
preparation under the alteration of thermal condition – The role of
TRPV1 –
Japan, 2 Nara Medical Univ, Japan）

During excitation-contraction coupling of striated muscle, sarcomeres are activated via thinfilament structural changes, i.e., from the “off” state to the “on” state, in response to a rise in theintracellular Ca+2 concentration. We systematically investigated the effects of rapid heating byinfra-red (IR) laser irradiation on the sliding of thin filaments reconstituted with human cardiac
α-tropomyosin (Tm) and bovine ventricular troponin (Tn), or rabbit fast skeletal Tm-Tn complex in the in vitro motility assay. Temperature was varied from 25℃ up to ∽45℃ within 2-10
s. With increasing temperature, the sliding velocity of F-actin and thin filaments reconstituted
with cardiac Tm-Tn was accelerated in the presence of ATP and Ca+2, with a temperature
coefficient (Q10) of ∽2 (between 25℃ and ∽41℃). In the absence of Ca+2 and in the presence of
ATP at 25℃, thin filaments reconstituted with cardiac Tm-Tn did not move; however, IR laser
irradiation elicited movements of the reconstituted thin filaments with a Q10 of 5.5. Likewise,
IR laser irradiation elicited movements of thin filaments reconstituted with fast skeletal Tm-Tn
with a Q10 of 11.0, showing higher sensitivity to temperature. The heating-induced acceleration
was observed in the presence of Ca+2 for cardiac and fast skeletal thin filaments, with thetemperature dependency >2-fold less in both cases. These findings suggest that 1) the “on-off”
equilibrium of the cardiac thin filament state is partially shifted toward the “on” state indiastole at the body temperature, enabling rapid and efficient myocardial dynamics in systole,
and 2) the higher temperature dependency for fast skeletal thin filaments is optimized for themuscle’s physiological properties in vivo.
(COI:No)

Myocardial temperature sensitively affects cardiac contractility and energy metabolism. Here,
we introduced our recent studies concerning the effects of altering cardiac temperature andthe role of TRPV1 on left ventricular (LV) myocardial mechanical works and energetics,
mechanoenergetics using our unique experimental model, the excised, rat whole heart preparation with cross-circulation system. We analyzed the LV end-systolic pressure–volume relationship (ESPVR) and linear relationship between myocardial oxygen consumption per beat (VO2)and systolic pressure–volume area (PVA; total mechanical energy per beat) in isovolumicallycontracting rat hearts during hypo- (32℃), normo- (37℃), and hyperthermia (42℃) in the presence of capsazepine (CPZ, a TRPV1 antagonist) or capsaicin (Cap, a TRPV1 agonist). The slopeof VO2–PVA linear relation represents the O2 cost of PVA, reciprocal contractile efficiency, andits VO2 intercept is composed of O2 consumption for Ca2+ handling in excitation–contraction(E-C) coupling and basal metabolism.
LV ESPVR shifted downward with increasing cardiac temperature, which was suppressed by
CPZ. In Cap-treated hearts, LV ESPVR shifted downward, similar to hyperthermia. The VO2intercepts of VO2–PVA linear relation did not change during increasing cardiac temperature
because of decreased E-C coupling VO2, and inversely increased basal metabolic VO2, which was
suppressed by CPZ, though the VO2 intercepts in Cap-treated hearts significantly decreased
due to the decreased E-C coupling VO2. Logistic time constants evaluating LV relaxation time
were significantly shortened with increasing cardiac temperature related to the accelerationof the detachment in cross-bridge (CB) cycling, indicating increased myosin ATPase activity.
Western blotting analysis revealed that the levels of phosphorylated phospholamban decreased
significantly in hyperthermia-hearts, as well as Cap-treated hearts.
We concluded that thermal intervention could modulate cardiac inotropism, which is, at leastin part, mediated through TRPV1 signaling pathway by changing CB cycling, Ca2+ handling,
and basal metabolism in rat hearts.
(COI:No)

PS29-02

PS29-05

Designing of sarcomere structure and direct visualization of myosin
force generation using DNA nano device
2 1 BDR, RIKEN, Japan, 2 Grad Sch Front Biosci, Osaka Univ, Japan）
Mitsuhiro Iwaki1, （To elucidate how muscle works, extensive experimental and theoretical works have proposed
the swinging lever-arm model. However, the dynamic features of how the myosin head swings
the lever-arm, how myosin initially interacts with actin, and how the swings coordinate witheach other are not well understood even though they are essential for the force generation,
contraction speed, heat production, and response to mechanical perturbations of muscle. Thisis because myosin heads during force generation have not been directly visualized. Here, we
engineered thick filaments comprising DNA origami and human muscle myosin and are optimized for nanometer-precision single-molecule imaging to directly visualize the heads duringforce generation. We found that when a head diffuses, it weakly interacts with actin and thenstrongly binds preferentially to the forward region as a Brownian ratchet. Upon strong binding,the head cooperatively swings its lever-arm in a two-step manner and occasionally reverses
direction. These results can explain the mechanical characteristics of muscle contraction andsuggest that our DNA origami-based assay system can be used to dissect the mechanistic(COI:No)details of molecular motor assembly.

S38

Cross-scale study of beating heart by using a multi-scale heart
simulator
Takumi Washio1, 2，Jun-ichi Okada1, 2，Xiaoke Cui1，Seiryo Sugiura1，Toshiaki Hisada1（ 1UT-Heart Inc., 2 University of Tokyo）

In a healthy beating heart, the left ventricular pressure rises more slowly than the sharp riseof intracellular free Ca2+ concentration, and falls much more rapidly than the slow decay of
free Ca2+ ions. These are important properties to avoid the risk of mechanical damage ofblood vessels due to sudden rise of the blood pressure in the systolic phase, and to obtainsufficient blood-filling in the diastolic phase. In this talk, we focus on the roles of mechanicalcooperativities of the myosin molecules affecting on these properties. By using a multi-scale
heart simulator, we analyze how the myosin molecules communicate mechanically along thethin filament, along the myofibril, and along the fiber in the cardiac muscle to achieve these(COI:No)
properties.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Symposia

Symposium 1
Deeper insights into smooth muscle
physiology using natural products（March 17, 9:00〜10:50, Hall 5）

S01-03Natural products induced disruption of actin polymerization modulatemechanical responses of skinned smooth muscle through various
pathways
Masaru Watanabe, Satoko Mihashi（Grad Sch Health Sci, Tokyo Met Univ, Japan）
Cytochalasin D, a fugal metabolites, and latrunculin B, a sponge toxin, are known to inhibit actin
polymerization, and also to suppress smooth muscle contraction. To clarify the force inhibitory
mechanisms of cytochalasin D and latrunculin B in detail, we examined the compounds effects
on the myosin light chain phosphorylation-dependent and -independent contraction of beta
escin skinned taenia cecum and carotid artery from guinea pig. Both cytochalasin D andlatrunculin B inhibited the maximal Ca ion induced force at around 1 micro M, but enhanced
sub-maximal force development induced by lower Ca ion concentrations. On the other hand,these compounds only suppressed high Mg ion induced force development. These results
suggest that actin filaments disruption by cytochalasin D and latrunculin B enhances Ca ion
sensitivity force through modulation of thin filaments linked Ca ion dependent pathways.
(COI:No)

S01-01

S01-04 (AP-7)

Akira Takai（Dept Physiol, Asahikawa Medical Univ, Japan）

1 1 Dept Cardiovasc
Rin Kurahara1，Keizo Hiraishi1, 2，Yaopeng Hu2，Ryuji Inoue2，Katsuya Hirano（

Effects of inhibition of protein phosphatases 1 and 2A on smoothmuscle contraction
During the last three decades since okadaic acid was shown to be an inhibitor of type 1
and type 2A protein phosphatases (PP1 and PP2A), inhibitory effects on these enzymes havebeen reported for other various naturally occurring substances with different relative affinities
to PP1 and PP2A. They are now widely used as valuable tools in physiological as well asbiochemical research fields. It was the force-enhancing effect of okadaic acid on Triton-X100skinned smooth muscle fibers that provided an initial clue to the discovery of its inhibitory
action to PP1 and PP2A. However it was soon shown that, when applied at 30 – 37℃ topreparations with intact cell membrane, relatively low concentrations (<3 µM) of okadaic acidstrongly inhibited rather than enhanced contractions induced in various smooth muscles byagonist stimulation or high K+ depolarization. At higher concentrations (>10 µM), in contrast,
okadaic acid has been constantly shown to produce or enhance contractions in intact smoothmuscle preparations as well as in permeabilized preparations. The contractile effects of okadaicacid can most simply be explained by inhibition of myosin light chain phosphatase whichcontains PP1 as the catalytic subunit. On the other hand, recent findings obtained by experiments using highly specific PP2A inhibitors have led to the conclusion that the relaxant effectis attributable to inhibition of PP2A. An important implication is that PP2A, as well as PP1, issomehow involved in the regulation and/or maintenance of the contractility of smooth muscles.
Several pieces of evidence suggest that the site of action of PP2A is some rather downstream
step of smooth muscle contraction such as cross-bridge cycling. Further studies are expected
to elucidate the detail of the function of PP2A in smooth muscles.
(COI:No)

Daikenchuto, a traditional herbal medicine, ameliorates ﬁbrosis byactivating TRPA1 channel in intestinal myoﬁbroblasts
Physiol, Sch Med, Kagawa Univ, Japan, 2 Dept Physiol, Sch Med, Fukuoka Univ, Japan）

Background: Daikenchuto (DKT) is a traditional oriental herbal medicine, widely used tomitigate post-operative ileus and constipation. In this study, we investigated the anti-fibrotic
effect of DKT in a murine chronic colitis model and elucidated the role of transient receptor
potential ankyrin 1 (TRPA1) channels of intestinal myofibroblasts in colonic fibrosis.
Methods: A murine chronic colitis model was established by weekly intrarectal administrationof trinitrobenzene sulfonic acid (TNBS). Inflammatory and fibrotic changes were evaluated byhistopathological examination. An intestinal myofibroblast cell line (InMyoFibs) was stimulatedwith TGF-β1, and subsequent intracellular signaling and pro-fibrotic factors were investigated.
Samples from non-stenotic and stenotic regions of Crohn’s Disease (CD) patient’s intestines
were used for pathological analyses.
Results: In TNBS chronic colitis model mice, the extents of inflammation and fibrotic changes
were more prominent in TRPA1－/－ knockout than in wild-type mice. One-week enema administration of DKT suppressed fibrotic lesions in wild-type mice, but not in TRPA1 knockout mice.
Active ingredients of DKT, i.e. hydroxy α-sanshool and 6-shogaol induced Ca2+ influxes in
InMyoFib, which were antagonized by co-treatment with a selective TRPA1 channel blocker
HC-030031. DKT counteracted TGF-β1-induced expression of Type 1 collagen, α-SMA,
N-cadherin, the phosphorylation level of Smad-2 and p38-MAPK and the expression levelof myocardin, a well-known master transcription factor regulating fibrosis signaling at thedownstream of TGF-β1 receptor. Importantly, a 24-hour incubation with another DKT active
ingredient Japanese Pepper increased the mRNA and protein expressions of TRPA1, which inturn negatively regulated collagen synthesis in InMyoFibs. TRPA1 expression in the stenotic
regions of CD patient’s intestine was significantly greater than that in the non-stenotic regions.
Conclusions: DKT suppresses intestinal fibrosis by upregulating the expression and activating
the channel function of TRPA1. This putative mechanism underlies the reported beneficial
actions of DKT on inflammatory bowel disease.
(COI:No)

S01-02Relaxing effects of rubratoxin A on smooth muscles by inhibiting PP2A
Kosuke Takeya（ Dept Biochem, Vet Med, Okayama Univ Sci, Japan）
The discovery of okadaic acid (OA) opened a new era in the smooth muscle research filed. OA
was first introduced as a potent smooth muscle constrictor. Later on, Dr. Takai and colleagues
discovered that it constricted smooth muscles by inhibiting Ser/Thr phosphatases. Few years
later, okadaic acid was reported to have relaxing effect on intact smooth muscles at lower
concentration. This unique dual effects have been attributed to different inhibition potency
between PP1 and PP2A. Based on Ki values for each phosphatase, OA inhibits PP2A about
5,000-times more potently than PP1. In intact smooth muscle tissues, however, the effective
range is much narrower, that is, it relaxes smooth muscles at < 1 µM while it constricts themat >10 µM. This narrow gap made it difficult to study the molecular basis of relaxing effectof okadaic acid by PP2A inhibition. Recently we introduced a novel PP2A specific inhibitor, rubratoxin A (RubA), to clarify the role of PP2A in smooth muscle contractions. RubA
relaxed carbachol-induced contractions as well as ionomycin-induced ones in intact bovine
ciliary muscle and guinea pig taenia cecum. It should be noted that RubA did not show any
contractile effects at the concentrations we examined. These results suggest that PP2A alters
Ca2+-sensitivity of smooth muscle contraction. We then examined its effect on β–escin skinned
guinea pig carotid artery. RubA at 1 µM and 10 µM significantly inhibited contraction at any
Ca2+ concentrations. The data fitting to the Hill equation in [Ca2+]-contraction relationshipindicated that RubA decreased Fmax-Ca2+ and increased [Ca2+]50, indices of Ca2+ sensitivity forthe force and myosin-actin interaction, respectively. These results suggest that PP2A inhibition
causes downregulation of the myosin light chain phosphorylation and direct interference withmyosin-actin interaction.
(COI:No)S40

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Symposium 2
Warm-blooded cool animals:
hibernation and torpor physiology（March 17, 9:00〜10:50, Hall 7）

S02-01

Role of sulﬁde metabolism in hypoxia tolerance of deep hibernators
Fumito Ichinose（Dept Anesthesia, Mass General Hosp, Harvard Med School, Boston, USA）
Small-bodied hibernators such as ground squirrels successfully and repeatedly execute cycles
of metabolic depression, tissue ischemia-reperfusion, and severe global hypoxemia withoutharm. Despite decades of intensive research, detailed mechanisms that permit such extreme
mammalian physiology remain unknown. One of the many physiological mysteries of smallbodied hibernators is their marked tolerance to severe hypoxia, which occurs during rewarming arousal from deep torpor bouts. Hydrogen sulfide (H2S) is an evolutionarily conserved O2
sensor that importantly modulates metabolism and signaling in modern cells. In mice, severe
hypoxia is associated with an acute increase in H2S in the brain. Pre-exposure to moderate
hypoxia or low dose inhaled H2S upregulates the capability to metabolize H2S in the mousebrain, enabling them to survive lethal hypoxia. While H2S has recently been studied as apotential method to confer a “suspended animation state” to non-hibernators, its link withhypoxia tolerance has not previously been explored in a natural hibernator. Interestingly,
ground squirrels express markedly higher levels of enzymes that synthesize or catabolize H2S
in their brain compared to mice. Enhanced capacity to metabolize H2S may enable the hibernators to use it as an organic substrate for energy production when O2 supply is limited and/or
to mitigate the inhibition of mitochondrial complex IV by hypoxia-induced H2S production; thiswould be a novel mechanism of hypoxia tolerance that has not previously been reported in anatural mammalian system. (COI:No)

S02-03Development of a new pharmacological method to induce the long-term
synthetic torpor in mice
Miho Sato-Hashimoto（Dept Lab Sci, Gunma Univ Grad Sch Health Sci, Japan）
Synthetic torpor is defined as a reversible metabolic depression that is induced artificially.
Some attempts to induce synthetic torpor pharmacologically have been made so far, oneof which is the potent method targeting adenosine receptor A1 (A1AR). Administration of
the A1AR agonist, N6-cyclohexyladenosine (CHA), induces hypothermia and bradycardia
dramatically both in non-hibernators (mice and rats) and hibernators (Syrian hamsters andground squirrels). Moreover, one study in arctic ground squirrels showed that the sensitivityof A1AR was increased during hibernation. We report here the pharmacological method to
induce the long-term torpor using CHA combined with treatments of PPAR agonists in mice.
We found that pretreatments of PPARα or -γ agonists prolonged hypothermia induced by
CHA. After treatments of these PPAR agonists, a single CHA injection induced hypothermia
at 20±1 ℃ for over 24 hours, and the heart beat also decreased by around 120 from 700 bpm.
According to results from the dose-response experiments, the pretreatment with a PPARαagonist increased the sensitivity of A1AR five times higher than that in control mice, while
a PPARγ agonist also enhanced the sensitivity but not as much as a PPARα agonist. Thismodel seemed to reproduce some traits in natural torpor including hibernation, because upregulation of PPAR gene expression was reported in hibernating animals. Furthermore, PPAR
agonist treatments have been known to have the protective effects against cardiovascular andbrain diseases. In this synthetic torpor model, thus, the PPAR activation may contribute toat least two changes similar to those during hibernation, that are prolonged hypothermia via
enhancing the sensitivity to CHA and protection from damages caused by low temperatureand hypoxia. (COI:No)

S02-04

Low Temperature Tolerance of a mammalian hibernator, Syrian
hamsters
Yoshifumi Yamaguchi（Institute of Low Temperature Science, Hokkaido University）
Mammal hibernation is a strategy for surviving during the harsh season with cold and foodshortage by reducing metabolisms and body temperature for energy-sparing. Many homeothermic mammals are unable to hibernate as they suffer from organ damage under prolonged
hypothermia that is experienced during hibernation, whereas some mammals including groundsquirrels, chipmunk, and hamsters can survive for long periods at low body temperature. Thishypothermia tolerance in hibernating animals should be based on low-temperature tolerance
at the cellular level, but its molecular mechanism is unclear yet. To elucidate the molecularmechanism of hibernation in mammals, we have studied a small mammalian hibernator, Syrian
hamsters, which can be induced to hibernate irrespective of season under an appropriate
condition. We previously suggested that Syrian hamsters remodeled the set-point of bodytemperature and white adipose tissues by prolonged short photoperiodic and cold condition.
Independently from these seasonal systemic remodeling, we found that primary hepatocytesof Syrian hamsters have tolerance against low temperature at the cellular level. The lowtemperature tolerance was observed not only in animals that hibernate but also animals thatwere kept at warm condition and did not hibernate, suggesting that the low temperature tolerance is an intrinsic property of the Syrian hamster. Surprisingly, however, the low temperature
tolerance in primary hepatocytes was diminished by changing environmental conditions. We
will discuss potential mechanisms of such environment-dependent low temperature tolerancein Syrian hamsters.
(COI:No)

S02-02

S02-05

Takeshi Sakurai（Faculty of Med, Univ of Tsukuba）

Genshiro Sunagawa A.（BDR, RIKEN, Japan）

Some mammals actively lower their body temperature to reduce energy expenditure whenfacing food scarcity, a state known as hibernation. Hibernating animals fully recover to anormal condition with no organ or tissue damage. Because a hypometabolic state could be
beneficial for many medical applications, this ability has evoked great interest. Here, we
identified a novel chemically-defined neuronal population, which resides in the periventricularhypothalamic nuclei in mice, excitatory manipulation of which induced a marked and very
long-lasting hypothermic state, similar to hibernation. In this state, the set-point of bodytemperature of mice was significantly lowered, but their behaviour and metabolism werestill actively regulated, showing stark contrast to states induced by anesthesia. Functions ofthese cells are also necessary for daily torpor and circadian control of body temperature. Thisfinding opens the door to the development of methods to induce a hibernation-like state in
non-hibernating mammalian species including humans.
(COI:No)

Some mammals enter a hypometabolic state either daily torpor (minutes to hours in length) or
hibernation (days to weeks), when reducing metabolism would benefit survival. The metabolic
rate is reduced to 1~30% of normal rates, and the animal results in severe hypothermia,
surprisingly without any tissue damage. The mechanisms for such hypothermia-resistance andhypometabolism-resistance is not understood. In 2016, we developed a method to induce torporstably in mice (Sunagawa GA and Takahashi M, Sci Rep, 2016) and this introduced modern
techniques in genetics such as genetical engineering to the field of mammalian hypometabolism
research. Recently, we found that two genetically close inbred mouse strains C57BL/6J (B6J)
and C57BL/6N (B6N) have distinct torpor phenotypes. This led us to hypothesize that the
torpor phenotype in mice is regulated by relatively few genes or gene loci. We analyzed thetranscriptome of soleus muscles from 38 B6J mice in torpid and non-torpid conditions andidentified 287 torpor-specific genes. Among the torpor specific genes, a transcription factor
ATF3 was found highly expressed during torpor deprivation and that its binding motif wasenriched in torpor-specific promoters (Sunagawa GA, et al., bioRxiv 374975, 2018). In addition,
the results of torpor phenotyping of atf3-KOs will be presented. Our results demonstrate thatmouse daily torpor combined with powerful genetic tools have the potential to study active(COI:No)hypometabolism.

Induction of synthetic hibernation-like state by manipulating
hypothalamic neuronal circuts

Daily torpor in mice as a model of active hypometabolism:
transcriptome analysis of skeletal muscle during torpor

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S41

Symposium 3
Frontiers of biophotonics in physiology:
Ion environments and molecule behaviors
in the nano-space detected by
Raman spectrum（March 17, 9:00〜10:50, Hall 8）

S03-01

Non-invasive sample measurement derived from live tissue by Raman
spectroscopic microscopy
4 1 HORIBA Ltd., 2 Res Inst Clin Physiol, Kyoto
Sakiko Akaji1，Yoshinori Marunaka2, 3, （Indust Health Adoc, Kyoto, 3 Res Organ Sci Technol, Ritsymeikan Univ, Kusatsu, 4 Ins Res
Center Food Nutrit Safety, Jiangsu Univ. Ahenjiang）
Raman spectroscopic technology is enable to measure samples derived from live tissue noninvasively. The research for this technology is focused as the obtained Raman spectrum ishaving the advantages of acquiring the molecular structural information and also for building
the spectral imaging.
One example is the detection of disease-derived cells like cancer. The pathological method, oneof the conventional methods, is available, however it takes time and many procedures to detect
those cells. Recently, some studies have shown that Raman spectroscopic method is able todetect cancerous cells without any pretreatments.
Based on the strength point of Raman spectroscopy described above, we have tried to measure
the samples derived from live tissue in order to prove the possibility of using the non-invasive
measurement method for physiological phenomenon. The visual diagnosis is difficult for themolecular structural change in the samples derived from tissue. Therefore, we are going todetect the cell differentiation stage, and the difference between benign and malignant tumor
by using Raman spectroscopic.
In this presentation, we would like to show the relation between the visual diagnosis andstructural change detection of Raman spectroscopic regarding the cell condition.
(COI:No)

S03-03

Raman microscopic analysis of biological tissues: toward constructionof Raman histopathology
Yoshinori Harada, Hideo Tanaka（ Dept Pathol Cell Reg, Grad Sch Med, Kyoto Pref Univ
Med, Japan）

Pathological histology mainly depends on morphological analysis of tissues, and requires
experts for recognition of tissue status. Raman scattering light measurement can directly
visualize biomolecules by acquiring their vibrations in an unlabeled and nondestructive way.
Raman microscopic imaging has potential to provide objective information on the biochemical
environment as well as morphological appearance of tissues. Here, we introduce our recent
studies of Raman scattering light measurements in heart and liver tissues. Evaluation of
myocardial viability is important for determining the strategy for revascularization therapy.
We have conducted basic studies on myocardial viability evaluation by Raman scattering lightmeasurement. We found that it is useful for in-situ assessment of both very early myocardialischemia and reversibility of myocardial viability in rat hearts without labeling. This wasshown to be realized by mainly sensing redox states and damages of myocardial mitochondrial
respiratory chain. As for studies of liver diseases, using rat nonalcoholic fatty liver disease(NAFLD) models, we incorporated machine learning with Raman spectroscopic imaging to
objectively study liver tissue specimens. We extracted biochemically different regions in livertissue, enabling quantification of chemical components. Using Raman microscopic informationon the chemical composition enabled us to group tissues, providing helpful information forhistopathological examination. In addition, Raman imaging combined with machine learningenhanced the diagnostic power of nascent stages of NAFLD in which morphological features
were not yet apparent. We expect that Raman microscopic analysis of biological tissues canprovide additional valuable microchemical information in relation to pathogenesis, taking(COI:No)advantage of its molecular discrimination ability.

S03-04High-throughput cell analysis by high-speed Raman microscopy
2 1 Dept Appl Phys, Grad Sch Eng, Osaka Univ, Japan, 2 Dept Pathol,
Yasuaki Kumamoto1, （Grad Sch Med, Kyoto Prefect Univ Med, Japan）

Raman spectroscopy is becoming a viable tool for probing a physiological condition of abiological sample as it has a high sensitivity to microenvironment of a molecule. However,its combination with optical microscopy, namely Raman microscopy, is not widely used. Thisis because Raman scattering is extremely inefficient as well as Raman spectral measurement uses a detector with a large number of pixels in general, and consequently the spectral
measurement time consisting of signal accumulation time and detector readout time often
becomes as long as several tenth minutes or even one hour in Raman microscopic imaging ofa single cell. Here I talk about high-speed Raman microscopy for high-throughput cell analysis
and imaging. One of the strategies to accelerate Raman imaging measurement is to conductsimultaneous detection of a number of spectra for reducing the effective signal accumulation
time required for obtaining one spectrum. The simultaneous spectral detection is enabled
by a line illumination slit-scan confocal Raman microscope. It reduced the effective signal
accumulation time by 2 to 3 orders of magnitude. To further increase the imaging speed, I
conducted a narrowband and low-spectral resolution Raman measurement. With this measurement, readout time of the detector was reduced in comparison to a correspondent numberof single-spectrum measurements by 2 orders of magnitude. Furthermore, the low-spectral
resolution measurement increased the number of photons incident to each pixel of the detector,
allowing shortage of the signal accumulation time by 4 times not in trade-off with signal tonoise ratio of spectra. Overall, with the narrowband and low-spectral resolution measurement,
Raman imaging of more than 200 cultured cells with a sub-500-nm spatial resolution took only
20 minutes. I will discuss the potential of this high-speed Raman microscope for physiologicalstudy.
(COI:No)

S03-02

Surface enhanced Raman spectroscopy probe for nanometer-scalemeasurement of pH and hydrogen peroxides on the outer membrane of cells
Leonardo Puppulin1, 2，Shigekuni Hosogi3，Hideo Tanaka4，Yasuaki Kumamoto5, 4，Eishi Ashihara3，
7 1 Department of Nanometrology, WPI Nano Life Science Institute,
Yoshinori Marunaka2, 6, （Kanazawa University, 2 Research Center for Drug Discovery and Pharmaceutical Development
Science, Research Organization of Science and Technology, Ritsumeikan University, 3 Departmentof Clinical and Translational Physiology, Kyoto Pharmaceutical University, 4 Department of
Pathology and Cell Regulation, Graduate School of Medical Science, Kyoto Prefectural Universityof Medicine, 5 Department of Applied Physics, Osaka University, 6 Research Institute for ClinicalPhysiology, Kyoto Industrial Health Association, 7 International Research Center for Food
Nutrition and Safety, College of Food and Biological Engineering, Jiangsu University）

Regulation of intracellular pH and reactive oxygen species (ROS) is critically important for manycellular functions. In the peculiar case of cancer cells, it has been demonstrated that pH homeostasis
is crucial for biological functions such as cell proliferation, metastasis, drug resistance and apoptosis.
As compared to normal cells, the extracellular surface pH of cancer cells is expected to be more
acidic, mainly due to elevated cellular glycolytic activity (Warburg effect). In addition to proton
concentration, also ROS play a key role in cell metabolism. In particular, hydrogen peroxide (H2O2) isrecognized as one of the main second messenger molecules. In fact, it modulates signaling pathways
activating downstream proteins that control cell metabolism. Endogenous H2O2 molecules involvedin cell signaling are primarily generated on the extracellular space by superoxide dismutases that
rapidly convert superoxide anions (O2-) produced by membrane-associated NADPH oxidases (NOXs
enzymes). Several studies demonstrated that during tumorigenesis H2O2 stimulates cell proliferationand preservation of the transformed state. For this reason, a characteristic feature of malignant
cells is upregulation of NOXs (i.e., mainly NOX1, NOX2 and NOX4), which promotes the productionof H2O2 in the extracellular milieu. Since the most significant gradients of protons and H2O2 canbe observed only in a very shallow region of the extracellular fluid in contact with the cell, weshow here the development of a new sensor of nanometer size that can be anchored to the outermembrane of cells. The newly designed sensor is based on gold nanoparticles functionalized with
pH- or H2O2-sensitive compounds that can be detected and quantified using surface enhanced Raman
spectroscopy. We report here the results of quantitative analyses conducted on MKN28 gastriccancer cells, HepG2 human liver cancer cells, A549 adenocarcinomic human alveolar basal epithelial
cells and human epidermal keratinocyte (HEK).
(COI:No)S42

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Symposium 4

S04-03

Estimating neural dynamics based on data-driven approach
Toshiaki Omori（Grad Sch Eng, Kobe Univ, Japan）

Neural mechanisms unveiled by combinedin vitro and in silico approaches（March 17, 9:00〜10:50, Hall 9）

S04-01

Perimeter release model: a nanoscale topographical arrangement of
Ca2+ channels and synaptic vesicles in the active zone
Yukihiro Nakamura（ Dept Pharmacol, Jikei Univ Sch Med, Tokyo, Japan）
Synaptic efficacy and precision are influenced by the coupling of voltage-gated Ca2+ channels(VGCCs) to synaptic vesicles. Accumulating evidence indicates that presynaptic voltage-gated
Ca2+ channels are clustered within the active zone, but the spatial distribution of readilyreleasable pool vesicles has not been elucidated. To investigate this, we performed local Ca2+
imaging and patch pipette perfusion of EGTA at the calyx of Held giant presynaptic terminalin the auditory brainstem. Reaction-diffusion simulations of presynaptic Ca2+ constrained by
the result of these experiments and following release simulations suggest that Ca2+ sensors forvesicular release are located in the range of several tens of nanometers from the perimeterof VGCC clusters and predict that VGCC number per cluster determines vesicular releaseprobability without altering release time course. This “perimeter release model” provides aunifying framework accounting for developmental changes as well as fast and slow release
components observed at this fast central synapse.
(COI:No)

Elucidating neural dynamics is one of the important subjects in neuroscience. Recent developments in recording technologies enable us to access spatiotemporal neural responses. We
have proposed data-driven approaches for estimating latent dynamics of neural systems suchas neuronal intrinsic dynamics and network dynamics. In particular, combined methods withcomputational models and data-driven methods are employed to estimate not only latentvariables but also underlying biophysical parameters. We also discuss methods to extract only
substantially important nonlinear membrane currents from a number of candidate membranecurrents, and estimate neuronal dynamics under nonlinear observation in imaging recording
by means of combined framework with model-driven and data-driven approaches.  (COI:No)

S04-04Computational neurobiology of axonal spike signaling
Haruyuki Kamiya（ Dept Neurobiol, Grad Sch Med, Hokkaido Univ, Japan）
Axonal spikes are generated at the initial segment and propagate to the terminal without
attenuation. Spike propagation along the axon is classically thought as stable binary codefor neuronal computation. However, recent studies by subcellular recordings from the axonsor the terminals have suggested that axonal spikes are subject to fine-tuning in an activitydependent manner. Towards a comprehensive understanding of dynamic control of axonal
spike signaling, a complementary approach by computer simulation has been widely used,since it covers the limitations of the experiment due to technical difficulty in recording froma small structure like the axon. We have developed a simple model of the hippocampal mossy
fiber axon implemented with the microstructure of typical en passant morphology as well as
the ionic conductance recorded experimentally from the axon terminals. The model enables
realistic simulation of membrane potentials and currents from any compartments of axon
arbor, and therefore can be adopted for evaluation of detailed membrane properties difficult
to obtain by experiment. We have used the model to address the mechanisms underlying
axonal afterdepolarization following an action potential. The hypotheses derived from subcellular recoding experiment were tested accordingly with simulation in the model. Anotherapplication of combined experimental and computational analysis will be also discussed on
ectopic spiking of distal axons during epileptic bursts. Thus, mutual feedback of in vitro andin silico approaches helps to convince our understanding of axonal spike signaling and its(COI:No)use-dependent fine-tuning.

S04-02

In vitro modeling of structure-function relationships in neuronal
networks
2 1WPI-AIMR, Tohoku Univ, Japan, 2 RIEC,
Hideaki Yamamoto1，Ayumi Hirano-Iwata1, （Tohoku Univ, Japan）

In vitro experiments using dissociated neurons take irreplaceable roles in molecular andcellular neuroscience. However, its use in the systems-level study has been limited due tothe substantial difference in the network organization from the actual brain. Such structuraldifference in the network results in the generation of an atypical dynamics in cultured neurons,
such as the globally synchronized bursting. Here, we employed surface engineering technology
to prepare guidance cues for the development of cultured cortical neurons and investigated
how the modulation of network structure at the mesoscopic scale influences the network
dynamics [1, 2]. We focus on the modular organization of brain networks, characterized by
the presence of densely-connected subsystems, or modules, that are weakly interacting witheach other. Induction of modular organization was found to suppress coherent bursting andpromoted coexistence of coherent and incoherent states [2]. The result demonstrates that
surface micropatterning provides a unique tool to constructively study the structure-function
relationships in living neuronal networks.
References:[1] H. Yamamoto et al., Phys. Rev. E 94, 012407 (2016).
[2] H. Yamamoto et al., Sci. Adv. 4, eaau4914 (2018).

(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S43

Symposium 5

S05-03

Mitochondrial structures and function under stress
Yoichiro Kusakari（Dept Cell Physiol, Jikei Univ, Japan）

Mitochondrial function and its roles incellular physiology/pathophysiology

Mitochondria are highly dynamic organelles with high plasticity that can transiently andrapidly coordinate their shape, distribution and size. However, the diverse functions of mitochondria in cellular metabolism and stress responses continue to emerge. We will discuss
mitochondrial structures and functions under mechanical stress. (COI:No)（March 17, 15:20〜17:10, Hall 6）

S05-01

Characteristics of the substrate-free mitochondria
2 1 Department of
Chae Hun Leem1，Jeong Hoon Lee1，Quynh Mai Ho1，Jae Boum Youm（

Physiology, University of Ulsan College of Medicine 88 OlumpicRo43Gil SongpaKu, Seoul,
KOREA, 2 Department of Physiology, College of Medicine Inje University, BokjiRo75,
BusanjinKu, Busan, Korea）

S05-04

Pathological role of non-MCU calcium-inﬂux mechanism in the cardiac
mitochondria
Jin O-Uchi（Department of Medicine, Cardiovascular Division, University of Minnesota）

Mitochondria are critical organelle to convert the metabolites to the life-essential chemicals,ATP. For serving the role, the mitochondria have very peculiar steps from metabolites to ATP
formation. Usually the Ψm was regarded as zero without the mitochondrial substrate or in
the presence of uncoupler. However, we found there still considerable Ψm was existed even in
those conditions. So basic scheme of the generation of Ψm is hampered and the question how
to explain this phenomena remains. For attacking these problems, we used a multiparametricmicrofluorometric system to monitor NADH, FAD, and TMRE fluorescence, simultaneously
and developed a measurement method of Ψm quantitatively. The Ψm in substrate free conditions was around -60 mV. The application of ATP generated considerable hyperpolarization.
The application of KATP channel opener, diazoxide (DZX), could depolarize Ψm. When K+
was replaced with meglumin, the Ψm was slightly hyperpolarized and the effect of DZX
was abolished. The KATP channel blockers did not have any effect. The application of ATP
hyperpolarize Ψm, however, ADP or AMP didn’t. Oligomycin blocked the effect of ATP.
Interestingly, oligomycin depolarized the resting Ψm considerably. The effect of ATP was
not affected by DZX. The replacement K+ with meglumin slowed the ATP-induced changeof Ψm and made it transient. The return to K+ medium recovered the effect of ATP. Fromthese results, the Ψm in substrate-free conditions was not zero and K+ may particpate in theformation of the resting Ψm. The F1, F0-ATPase may participate on the formation of the resting Ψm. K+ may be an important modulator for F1, F0-ATPase acitivity. The F1, F0-ATPase
and K+ flux might contribute the formation of the resting Ψm but it still need further study
on them. (NRF-2016M3C1A6936605) (COI:No)

Malignant hyperthermia (MH), frequently associated with the ryanodine receptor type 1 (RyR1),
is a pharmacogenetic disorder of skeletal muscles that exhibits hypermetabolic responses toanesthetic gases. However, sudden cardiac death (SCD) in the MH families are also reported
even though under the conscious condition without anesthesia. We previously reported that
small amount of RyR1 is expressed in the mitochondria, but not in the sarcoplasmic reticulumin the hearts, which serves as an additional mitochondrial Ca2+ (mtCa2+) influx mechanism
distinct from the main mtCa2+-influx pathway, mtCa2+ uniporter (MCU). Therefore, wehypothesize that MH-associated mutant RyR1s form “leaky channel” at cardiac mitochondria
and induce mtCa2+ overload, followed by an increase in the mitochondrial reactive oxygen
species (mROS) generation, which alters the cellular Ca2+ handing in cardiomyocytes. Using
knock-in mice carrying a MH-related RyR1 mutation Y522S (YS), we found that YS hearts exhibit disrupted mitochondrial morphology and develop significantly higher number of multiple
ventricular extrasystoles by β-adrenergic stimulation compared to wild-type (WT) hearts.
We also found that YS mitochondria possess higher basal mtCa2+ concentration ([Ca2+] m) and
depolarized mitochondrial membrane potential. Moreover, pretreatment with RyR1 blocker
dantrolene decreased basal [Ca2+] m and hyperpolarized mitochondrial membrane potential in
YS mitochondria. We next showed that YS myocytes had significantly higher basal cytosolic
Ca2+ concentration as well as slower cytosolic Ca2+ clearance compared to WT. Pretreatment
of dantrolene normalized the Ca2+ handing profiles in YS cardiomyocytes to the levels in WT.
Finally, we confirmed that YS cardiomyocytes exhibited higher mROS level and excessive
cellular oxidation. These data suggest that chronic mtCa2+ overload via leaky mutant mRyR1
increases mitochondrial and cellular oxidation, which may alter cytosolic Ca2+ handling andincrease the arrhythmogenic events in MH. The outcome of this study will lead us to designnovel strategies for the pharmacological management and risk stratification of SCD in MH
patients.
(COI:No)

S05-02

S05-05Characteristics of Ca2+ efﬂux from mitochondria
Ayako Takeuchi, Mohammed Moinul Islam, Satoshi Matsuoka（ Integr. Physiol. Fac. Med.

Exploring the regulation of ER-mitochondria contact and its
physiological roles in mammals

Sci. Univ. Fukui）

Yusuke Hirabayashi（Dept Chem. and Biotech., Grad Sch Eng, Univ Tokyo, Japan）

Mitochondrial Ca2+ is determined by the influx mainly via mitochondrial Ca2+ uniporter (MCU)
and the efflux via Na+-Ca2+ exchange (NCXmit) and H+-Ca2+ exchange (HCXmit), with the NCXmit representing the major component in excitable tissues, whereas the HCXmit being dominant
in non-excitable tissues. Previously we reported that NCLX, which belongs to a Na+/Ca2+-K+
exchanger family and was identified as the NCXmit, participates in regulating various cellularfunctions including excitable cells such as HL-1 cardiomyocytes and non-excitable cells suchas B lymphocytes, by supplying Ca2+ to sarco/endoplasmic reticulum. However, contributionof NCLX to the overall Na+-dependent Ca2+ efflux remains unclear, especially in tissues whereplasma membrane Na+-Ca2+ exchanger (NCX) family exists also in mitochondria. In addition,
electrophysiological characteristics of NCXmit are entirely unknown. We studied these issues
in mouse isolated mitochondria.
Immunoblot showed that both NCLX and NCX1 are expressed in mitochondria of heart, brain
and kidney. Application of Ca2+ to isolated mitochondria rapidly decreased extra-mitochondrial
Ca2+, which was detected by Calcium Green-5N, via Ca2+ uptake through MCU into mitochondria. Subsequent MCU inhibition by Ru360 increased extra-mitochondrial Ca2+, by uncovering
the Ca2+ efflux, which was accelerated in the presence of Na+. This NCXmit activity wasprominent in brain and heart and less in kidney. It was inhibited by an NCLX blocker CGP37157, but was insensitive to an NCX1 blocker, SEA0400, suggesting that NCXmit is mediatedmainly via NCLX. Finally, we performed voltage clamp experiments using mitoplast preparedfrom isolated heart mitochondria. We succeeded in recording the extra-mitochondrial Na+
dependent inward currents with Ca2+ in the pipette, as well as the extra-mitochondrial Ca2+
dependent outward currents with Na+ in the pipette. These currents were inhibited by CGP37157, demonstrating a direct evidence that NCXmit via NCLX is electrogenic.
(COI:No)A network of contact sites between the membranes of different organelles are emerging
as critical platforms for various forms of intracellular signaling. The interface between
ER and mitochondria is of particular interest as a signaling hub because it is thought toplay critical physiological functions such as regulation of Ca2+ homeostasis, lipid biogenesis
and mitochondrial fission. In addition, changes in the number of these contacts have beenreported in various mouse models of neurodegenerative disease, as well as in patients withneurodegenerative diseases. However, despite the fact that multiple proteins are enriched at
ER-mitochondria contacts sites, the molecular mechanisms underlying ER-mitochondria tethering are still largely unknown in metazoans.Here, we will report the identification of PDZD8
as a novel protein required for ER-mitochondria tethering protein (Hirabayashi et al. Science2017). Pdzd8 is an integral ER proteins present at ER-mitochondria contact sites. Using 3D
FIB-serial EM reconstructions, we demonstrate that PDZD8 is required for the formation of
ER-mitochondria contacts in mammalian cells. Using a series of functional loss-of-function andrescue experiments, we found that PDZD8-dependent ER-mitochondria contacts are requiredfor proper Ca2+ exchange between ER and mitochondria in mammalian cells. In dendrites ofcortical pyramidal neurons, PDZD8 is required for Ca2+ uptake by mitochondria following
synaptically-induced Ca2+-release from ER and thereby regulated cytoplasmic Ca2+ dynamics.
Thus, PDZD8 represents a critical ER-mitochondria tethering protein, which is involved in theregulation of dendritic Ca2+ dynamics in mammalian neurons.
(COI:No)S44

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Symposium 6
Crosstalk in modifiable and non-modifiablerisk factors for cardiovascular disease（March 17, 15:20〜17:10, Hall 8）

S06-01

Effects of Endurance Exercise on Arterial Stiffening with Aging
Shigeki Shibata（ Faculty of Health Science, Kyorin University）
A notable consequence of sedentary ageing is large-vessel arterial stiffening. This pathophysiological process is characterized by the development of fibrosis and collagen cross linked products in the arterial wall. Central arterial stiffening increases the risk of cardiovascularrelated
morbidity and mortality in older adults and thus, the development of strategies to forestall
age-associated arterial stiffness has important clinical implications. Sustained, regular endurance exercise training is thought to be one such favorable strategy. In this field, the presenter’s
previous findings are summarized as follows.
i) Masters athletes who have performed near-daily (6-7 sessions per week) vigorous endurance
exercise training plus competition for the majority of their adult lives maintains youthfulcompliant large arteries. ii) More than 4-5 weekly sessions of committed lifelong exercise is associated with a more compliant large arteries in the elderly as compared with their peers. iii) A
lower frequency of lifelong exercise (2-3 sessions per week) is associated with improved carotid
artery compliance and decreased left ventricular afterload. iv) Large arterial stiffening withaging was not substantially improved in previously sedentary healthy seniors even after one
year of progressive and vigorous endurance exercise training when compared with life-longvigorous exercise training by Masters athletes. v) Breaking of advanced glycation end productcross links with Alagebrium was safe, and slowed but did not reverse age related large arterial
stiffening in the elderly without an additive effect of exercise training.
This presentation as a part of symposium “Crosstalk in modifiable and non-modifiable risk
factors for cardiovascular disease” plans to focus on effects of endurance exercise training onlarge arterial stiffening with aging based on recent literature and presenter’s previous findings.
(COI:No)

S06-03

Understanding the inﬂuence of the menopause on shear stress mediatedcerebral artery dilation at rest and during static handgrip exercise
Stefanie Ruediger1，Jeff S. Coombes1，Shigehiko Ogoh2，Daniel J. Green3，Tom G. Bailey1（ 1 School of Human Movement and Nutrition Sciences, The University of Queensland, St
Lucia, Brisbane, QLD, Australia, 2 Department of Biomedical Engineering, Toyo University,
Saitama, Japan, 3 School of Sport Science, Exercise, and Health, The University of Western
Australia, Crawley, Western Australia, Australia）
Background: Declines in circulating-oestrogen during the menopause has a wide-range of
detrimental effects on vascular structure and function. These changes, coupled with elevatedblood pressure, may be associated with reductions in cerebral blood flow (CBF), and cognitive
decline in post-menopausal women (PMW). Exercise-induced elevation in blood flow and shear
stress induces peripheral vascular adaptation. If CBF and shear stress could be enhancedwith exercise, this would likely improve cerebrovascular function in PMW. However, PMW
have reduced sensitivity to shear stress, and exhibit exaggerated blood pressure responses
to exercise, which may limit increases in CBF. Whether exercise-induced increases in bloodpressure alter flow to the brain in PMW is unknown. The aim of this study is to compare
the CBF response to acute exercise in PMW, to pre-menopausal women. Secondly, we aim tounderstand the influence of the menopause on CBF and shear stress flow-mediated-dilation(FMD), as a marker of cerebrovascular function.
Methods: 15 post- and 15 pre-menopausal women (40-65 years), will be recruited. Participants
will undergo a static handgrip-exercise for 3 minutes at 30% of their maximum voluntary
contraction. CBF velocity of the middle and posterior cerebral artery will be assessed continuously, using Transcranial Doppler. We will use Duplex ultrasound to measure internal carotid(ICA) and vertebral artery (VA) blood flow, and shear stress at each artery will be estimated.
At baseline, cognitive function, brachial and cerebral FMD will be assessed.
Discussion: We hypothesize that cerebral FMD will be reduced in post- compared with premenopausal women. Further, the CBF and shear stress response to static handgrip-exercise
will be attenuated in post- compared with pre-menopausal women, and this will be associatedto larger blood pressure responses in PMW. These results will help to understand differencesin brain vascular function at rest, and in response to acute static exercise in PMW.
Data collection is currently underway and will be analysed in February 2020.
(COI:No)

S06-04

Exercise pressor reﬂex in type 2 diabetes-the role of insulin in
circulatory control during exercise
2 1Chubu Univ, Japan, 2 UT Southwestern Medical Center, USA）
Norio Hotta1，Masaki Mizuno（

Type 2 diabetes mellitus (T2DM) is one of the diseases in the world requiring prevention anda medical cure. Insulin resistance is a principal feature of T2DM. Exercise therapy has been
widely known to be effective in improving insulin resistance in T2DM. However, patients with
T2DM display an exaggerated blood pressure response to physical exercise. The potentiatedpressor response to exercise increases cardiovascular event risks such as heart attack andstroke, resulting in a reduction of safety of exercise prescription. To date, the mechanism
underlying T2DM-induced augmented cardiovascular response to exercise has not been fully
investigated. Central command originating from the higher centers and the exercise pressor
reflex originating from active muscles play a crucial role in cardiovascular regulation during
exercise. We have recently reported that both renal sympathetic nerve activity and the bloodpressure response to electrical stimulation of the mesencephalic locomotor region and theisolated L4 and L5 ventral roots were significantly augmented in the combination of high-fat
diet-fed and low-dose streptozotocin-treated T2DM rats as compared with control (Kim et al.,Am J Physiol Regul Integr Comp Physiol, 317:R270-R279, 2019). This suggests that central
command and the exercise pressor reflex generate the exaggerated rise in sympathetic activity and blood pressure during exercise in this disease. Furthermore, evidence suggests that
hyperinsulinemia precedes the development of T2DM and insulin receptors are expressedin the peripheral nerve and dorsal root ganglia neurons. To this end, we hypothesized that
insulin sensitizes thin-fiber muscle afferents mechanically and metabolically, contributing toheightened exercise pressor reflex in T2DM (Hotta et al., J Physiol, doi: 10.1113/JP278527).
Here we discuss alterations in the exercise pressor reflex function in T2DM at tissue and celllevels. Further, we demonstrate evidence suggesting an association between insulin resistanceand exercise blood pressure in non-diabetic elderly subjects.
(COI:No)

S06-02

S06-05

Shigehiko Ogoh（Department of Biomedical Engineering, Toyo University）

Erika Iwamoto（Dept Health Sci, Sapporo Med Univ, Japan）

Ageing is the primary risk factor for cognitive deterioration that is considerable to be
associated with onset of dementia. However, the mechanism of ageing-related cognitive
deterioration remains unclear. On the other hand, cerebral blood flow (CBF) or regulation of
cerebral circulation is attenuated in the elderly. Also, CBF regulation associated with cerebral
metabolism thus likely plays an important role in the preservation of cognitive function. Indeed,
our recent study demonstrated that cognitive function was more strongly affected by changes
in cerebral metabolism than by changes in CBF during exercise. Thus, it could be expectedthat ageing-induced cognitive deterioration may be affected by a decrease in CBF as a resultof brain ischemia and energy depletion. Exercise is a potential therapeutic tool to postpone
or prevent the onset of cognitive decline. It has been reported that onset execise and chronic
exercise training improved cognitive function. This may be associated with exercise-induced
modification in CBF regulation. However, there is a lack of sufficient discussion regarding
the effect of exercise via CBF regulation on age-induced alteration in cognitive function. In
this presentation, I would like to summarize current knowledge on previous investigations
providing the possibility of effect of exercise on cognitive function in elderly compared withthat in young people.
(COI:No)

Endothelial dysfunction is associated with cerebrovascular events such as chronic stroke andvascular dementia. In peripheral conduit arteries, endothelial function is commonly assessedby a noninvasive method, ischemia-induced flow-mediated dilation (FMD). Interestingly, recent
studies measured carotid shear-mediated dilation using hypercapnia to assess the cerebrovascular function. Analogous to the FMD in the peripheral conduit arteries, the decreased
carotid shear-mediated dilation could serve as a potentially useful measure of cerebrovascularendothelial function.
There is a considerable amount of evidence reporting that aging represents the major risk
factor for the development of cardio and cerebrovascular diseases. We previously reported
that carotid shear-mediated dilation is attenuated with healthy aging (Iwamoto E et al., Am J
Physiol Heart Circ Physiol. 2018). The enhanced sympathetic tone may be one of the potential
causes of blunted sheer-mediated dilation with aging (Iwamoto E et al, J Appl Physiol. 2018).
Moreover, the change in sex hormones is a natural part of aging. In women, menopause results
in a rapid loss of estradiol. In our recent data, we found that there is a positive relationshipbetween serum estradiol concentrations and carotid shear-mediated dilation in pre-, peri-, andpostmenopausal women. In contrast, exercise-induced increases in shear stress release thevasoactive substance from the endothelium and have beneficial effects on vascular function.
In fact, exercise training has been shown to improve endothelial function in cardiovascularpatients. In addition, exercise training increases resting cerebral blood flow in ischemic lesions
and improve cognitive function via endothelium-dependent mechanisms. However, it requires
further investigation to elucidate the optimal mode of exercise (aerobic or resistance exercise,continuous or interval exercise) and intensity to improve cerebrovascular function. In this
symposium, I would like to show our ongoing data investigating the effects of aging, sex(COI:No)
hormone, and mode and intensity of exercise on carotid shear-mediated dilation.

Cognitive function and cerebral blood ﬂow; Effects of age and exercise

Cerebrovascular endothelial function: Aging, sex hormone, and exercise

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S45

Symposium 7

S07-03

Hypothalamic regulation of cortical activity for appetitive behavior
Ikue Kusumoto-Yoshida, Jihao Ma, Ran Yamaguchi, Tomoyuki Kuwaki（Dept Physiol, Grad

Molecular and Neural basis for appetiteand food preference（March 17, 15:20〜17:10, Hall 10）

S07-01

Molecular mechanisms to generate various types of taste cells
Makoto Ohmoto（Bio-Center, Tokyo Tech, Japan）
Taste substances are detected by taste cells in taste buds in the oral epithelium. Individual
taste cells contribute to evoke one of five taste qualities: sweet, umami, bitter, sour, and salty
tastes. They are continuously replaced every few weeks by new ones derived from local
epithelial stem cells. We have been interested in the molecular mechanisms underlying thegeneration of various types of taste cells. We found that a POU homeodomain protein Skn-1a
is expressed in sweet, umami, and bitter taste cells and that Skn-1a knockout mice lack thesecells in taste buds. These results suggest that Skn-1a is a determinant to sweet, umami, andbitter taste cells and that some transcription factors other than Skn-1a would also be involvedin the generation and differentiation of taste cells. In this symposium, I will show our recent
works focused on the transcriptional factors expressed in taste tissues to study how various(COI:No)
types of taste cells are generated.

Sch Med Dent, Kagoshima Univ, Japan）

Appropriately regulating food intake is essential for health maintenance. Rising rates ofobesity, diabetes, and cachexia pose significant threats to health, and thus understanding brain
regions that underlie aberrant food intake is critically important. It is well known that the hypothalamus regulates both food intake and energy homeostasis. However, interactions between
the hypothalamus and other brain areas such as cortical regions require further elucidation.
We have been exploring neural circuits downstream of hypothalamic orexin neurons using histological and genetic approach. In study of restricted feeding induced c-fos mapping, increased
c-fos signal positive neurons were observed not only hypothalamic orexin neurons but also in
the insular cortex. During the restricted feeding paradigm, mice showed increased locomotion
just before feeding time that resemble food anticipation similar to the previous study. The
insular cortex is known as the higher order sensory cortex that integrate multiple modalities
and play an important role in establishing homeostasis within the body. These results suggest
an important role of correlated activity for insular and orexin neurons in food anticipatory
behavior. Optogenetic stimulation of orexin neuron axon terminals in the insular cortex onfood intake showed the increase of both food intake and c-fos expression in the insular cortex,
suggesting an important role for the insular cortex in orexin-regulated food intake. (COI:No)

S07-04

Neuronal SIRT1 regulates simple sugar selection through FGF21 andoxytocin signalling in mice
2 1 Div Food Sci. Biotechnol, Grad Sch Agr, Univ Kyoto, Japan, 2 Lab. of
Sho Matsui1, （Metabolic Signal, IMCR, Univ Gunma, Japan）

Diet affects health through ingested calories and macronutrients, and macronutrient balance
affects health span. The mechanisms regulating macronutrient-based diet choices are poorly
understood. Previous studies had shown that NAD-dependent deacetylase sirtuin-1 (SIRT1)
in part influences the health-promoting effects of caloric restriction by boosting fat use inperipheral tissues.
First, we analyzed diet selection behavior in neuron-specific Sirt1 overexpression (NS-OE) andknockout (NS-KO) mice. The results indicated that neuronal SIRT1 promotes fat preference,
whereas it suppresses sucrose preference. Therefore, neuronal SIRT1 shifts diet choice fromsucrose to fat in mice, matching the peripheral metabolic shift.
Next, we identified that SIRT1 positively regulates the expression of oxytocin (Oxt), which
is known to specifically suppress the preference to simple sugar, but not fat. We proved
the necessity of oxytocin signaling for the regulation of sucrose preference by SIRT1 withpharmacological blockade of Oxt receptor and pharmacogenetic inhibition of Oxt neuronal
activation. We also analyzed Oxt neuron-specific Sirt1 overexpression (OS-OE) and knockout(OS-KO) mice to prove that SIRT1 in Oxt neurons is sufficient for regulating sucrose preference in mice. Therefore, SIRT1-mediated suppression of simple sugar preference requires
oxytocin signalling, and SIRT1 in oxytocin neurons drives this effect.
Finally, we searched for the metabolic signal that represents simple sugar ingestion. We
found that the hepatokine FGF21 acts as an endocrine signal to oxytocin neurons, promoting
neuronal activation and Oxt transcription and suppressing the simple sugar preference. SIRT1
promotes FGF21 signalling in oxytocin neurons by up-regulating the expression of beta-klotho,a co-receptor for FGF21, and stimulates Oxt transcription.
Taken together, these data suggest that SIRT1 suppresses simple sugar selection by potentiating the negative feedback by FGF21 via Oxt.
(COI:No)

S07-02

Hypothalamic neuronal circuits regulating hunger-induced taste
modiﬁcation
Ou Fu（Nips, Okazaki, Japan）
The gustatory system plays a critical role in sensing appetitive and aversive taste for theevaluation of food quality. Although taste sensitivity and preference are known to change
depending on internal states, such as hunger, the mechanistic insight remains unclear. Here
we examine the neuronal mechanisms regulating hunger-induced taste modification in themouse brain.
Starved mice exhibit an increased preference for sweet taste and tolerance for aversive
taste. This phenomenon is recapitulated by activating the orexigenic AgRP neurons in the
hypothalamus, especially that projecting to the lateral hypothalamus. Glutamatergic neurons
in the lateral hypothalamus innervate AgRP neurons and they are sufficient and necessary tomodulate taste preferences. Furthermore, two distinct pathways from Lateral hypothalamus to
the lateral septum or the lateral habenula regulate the preference for appetitive and aversive
tastes, respectively. Our results suggest that these hypothalamic circuits for taste modification(COI:No)
would be important for optimizing feeding behavior under energy deficiency. 

S46

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Symposium 8

S08-03

Effect of temperature on seasonal adaptation mechanism in medaka ﬁsh
2 1WPI-ITbM, Nagoya Univ, Japan, 2 Grad Sch Bioagricult Sci,
Takashi Yoshimura1, （Physiological importance of environmental
temperatures in ectotherms（March 18, 9:00〜10:50, Hall 6）

S08-01

Ambient temperature sensing with TRPA1 channel in mosquito
Makoto Tominaga（ Div Cell Signaling, Natl Ist Physiol Sci, Japan）
Temperature and odors profoundly affect the behavior of animals. Transient receptor potential
channel, subfamily A, member 1 (TRPA1) functions as a polymodal nociceptor for sensing both
vital environmental cues in insects. Mosquitoes are recognized as disease vectors, and manyefforts have been devoted to investigations of their host-seeking behaviors and repellents.
However, the physiological characteristics of mosquito TRPA1 have not been systematicallystudied. We identified multiple alternative splice variants of the TrpA1 gene from Anopheles
gambiae (Ag), Anopheles stephensi (As), Aedes aegypti (Aa) and Culex pipiens pallens (Cp)mosquitoes. And we performed comparative analyses of the responses of mosquito TRPA1s
to heat or chemical stimuli with calcium-imaging and whole-cell patch-clamp methods. Comparison of TRPA1 among four mosquito species from different thermal niches revealed that
TRPA1 of mosquitoes inhabiting the temperate zone had a lower temperature threshold forheat-evoked activation, which was supported by the in vivo heat-avoidance test. Notably, thechemosensitivity of mosquito TRPA1 channels revealed differences not only between variants
but also among orthologues. Moreover, we discovered 3 novel mosquito TRPA1 agonists. We
described that thermal niches and evolutionary trajectories significantly affect the functional
properties of mosquito TRPA1, which represents a hallmark of the behaviors that may permit
the design of improved mosquito control methods.
(COI:No)

Nagoya Univ, Japan）

The appropriate timing of various seasonal processes, such as reproduction, migration andhibernation, is crucial to the survival of animals living in temperate regions. However, underlying mechanisms of seasonal adaptation are not well understood.
Medaka fish (Oryzias latipes), an excellent model for studying seasonal adaptation, are active
and exhibit clear phototaxis in conditions simulating summer, but remain at the bottom of
the tank and failed to exhibit phototaxis in conditions simulating winter. Mate preference
tests using virtual fish created with computer graphics demonstrated that medaka are more
attracted to orange-red-colored model fish in summer than in winter. Transcriptome analysisof the eye reveals dynamic temperature-dependent seasonal changes in the expression ofgenes encoding photopigments and their downstream pathways, suggesting that plasticity inphototransduction pathway is crucial for the seasonal changes in color perception.
Depression is considered an adaptation to a harsh environment. Seasonal changes in environment also lead to depression-like behaviors in animals and humans. We observed decreasedsociability and increased anxiety-like behavior in medaka exposed to short day and cool temperature, winter-like conditions. Whole brain metabolomic analysis revealed seasonal changes
in 68 metabolites, including serotonin and glutamate. Transcriptome analysis identified 3,306
differentially expressed transcripts, including circadian clock genes. A broad-spectrum chemical screen identified a drug that reverse the winter behavior. Our chemical genomics study
provides insights into winter induced depression-like behaviors.
(COI:No)

S08-04

Molecular mechanisms underlying temperature-dependent sex
determination in reptiles
Shinichi Miyagawa（ Dept Bio Sci Tech, Faculty Indust Sci Tech, Tokyo Univ Sci, Japan）
Sex determination is a critical element in development that greatly influences the individualon multiple levels, including physiological, reproductive and behavioral phenotype. In contrast
to sex determination based on intrinsic genotypic factors, as commonly seen in many vertebrates, certain reptiles, including the crocodilians and turtles, display temperature-dependent
sex determination (TSD), in which the temperature of the surrounding environment during
embryonic development determines the sexual fate of the individual. However, much of thedetails concerning its underlying molecular mechanism remain to be elucidated, such as how
the developing embryo initially detects the external temperature signals and directs the
gonadal fate accordingly. We have investigated several thermosensory factors, and particularly
focused upon transient receptor potential (TRP) channels as main initiation candidate. Functional characterization of alligator TRPV4 channel reveals that it is activated in temperatures
proximate to alligator TSD. We also found that selective inhibition and activation of TRPV4channel induces both down and upregulation, respectively, of male gene expression cascade,and higher prominence of Müllerian duct in males by TRPV4 inhibition. In addition, we haveinvestigated the effects of several TRP agonists and antagonists on the turtle gonadal differentiation. Our findings provide several insights to genetic framework underlining TSD, andour potential novel findings serve as a basis for further understanding gonadal fate pathway(COI:No)
during vertebrate sex determination.

S08-02

Thermo-sensing mechanisms underlying temperature preference infruit ﬂies
2 1Cell Signaling, NIPS, Japan, 2 Thermal Biology, ExCELLS, Japan）
Takaaki Sokabe1, （Every animal actively seeks for favorable temperatures in dynamic thermal landscapes
depending on their innate preferences. It has been a long-lasting question as to how we sense
the environmental temperatures and a subset of TRP channels have been identified as aphysiological thermosensor in the last two decades, whose activity is directly regulated bytemperature changes. This machinery appears to be conserved among a wide range of species
including endotherms and ectotherms. Due to the small body size, insects equilibrate theirbody temperature with the environments quickly, which differs from mammals with their autonomous body temperature regulation. Therefore, insects are highly sensitive to temperaturefluctuation and show a variety of temperature-dependent physiological behaviors.
We recently found that fruit fly (Drosophila melanogaster) larvae could discriminate subtle
temperature differences and displayed a development-dependent shift in their thermal preference. This thermal preference switch includes rhodopsins, lipid signaling and TRPA1. TRPA1
has been known to respond to temperature changes directly, however, thermo-sensing occurs
upstream of TRPA1, possibly through rhodopsins, in this context. These signaling components
are expressed together in the central and the peripheral nervous system to regulate behaviors.
The roles of rhodopsins outside of phototransduction have been reported in other sensory
modalities or other animals, implying that the unconventional role of the light sensor may be
evolutionarily conserved. I will also discuss our recent concepts including functional roles ofmembrane lipids in sensory processes.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S47

Symposium 9
Reverse engineering the brain functions forthe control of adaptive behaviors（March 18, 9:00〜10:50, Hall 7）

S09-03 (AP-1)

The local network in the striatum tail contributes to the behavioral
switching
2 1 Faculty of Med, Univ Tsukuba, Tsukuba, Japan,
Jun Kunimatsu1, 2，Okihide Hikosaka（
2 National Eye Institute, NIH, MD, U.S.A.）

Although, in our daily life, the object values may change in different environments and we canswitch our behavior accordingly, underlying neuronal mechanism is unclear. To address it, we
devised a new value procedure: scene-based value task. The monkey viewed 8 fractal objects
in 2 scenes (A and B); 4 of them were good (with large-reward) in scene A and bad (withsmall-reward) in scene B, while the other 4 were good in scene B and bad in scene A. After
experiencing this procedure repeatedly, the monkey became able to choose whichever objects
were good. Since scenes A and B were presented in a random sequence, the monkey’s choice
was switched abruptly depending on the scene-context.
We then recorded neuronal activity in striatum tail while the monkey passively viewed theseobjects in different scenes. We found differences between medium spiny neurons (MSNs) andfast spiking interneurons (FSIs). Many of MSNs responded to the fractal objects differently
depending on their values. Importantly, this object-value coding was stronger in either scene
A or B. In contrast, FSIs showed no object-value coding. Instead, many of them responded tothe scenes selectively (stronger to scene A or B). These results suggested that the object-value
coding of MSNs, which is basically stable, is modulated by the inhibitory inputs from thescene-selective FSIs.
To test the causal role of FSI, we locally injected IEM-1460, an inhibitor of GluA2-lacking
AMPARs, in the recording sites to selectively block the excitation of FSIs but not MSNs.
After injection, monkeys were unable to learn new scene-object value association. On the other
hand, object-value learning (no scene) was not affected. This result indicated that the local
network of striatum tail regulates the scene-object association learning. These mechanisms
may support the monkey’s flexible switching based on stable long-term experiences of various
environments.
(COI:No)

S09-01

S09-042 1 Hakubi
Tomohiko Takei1, 2，Stephen Lomber G.3，Douglas Cook J.2，Stephen Scott H.（
Cent, Kyoto Univ, Japan, 2 Cent Neurosci, Queen’s Univ, Canada, 3 Dept Psychol, Western
Univ, Canada）

Yoshihisa Tachibana（ Div Syst Neurosci, Grad Sch Med, Kobe Univ, Japan）

Cryogenic approaches to reveal neural mechanisms for sophisticatedfeedback motor control

Feedback corrections of goal-directed motor action are surprisingly fast and complex, butlittle is known how such flexible feedback motor actions are generated in the central nervous
system. Neurophysiological studies implicate a broad network in fronto-parietal cortices inthese feedback corrections, but the specific role of each region is unknown. Here we investigated the function of dorsal premotor cortex (PMd) and parietal area 5 (A5) in feedback
control by combining a neural deactivation (cooling deactivation) in non-human primates witha model simulation. To give functional implications to the behavioral results, we generated
an optimal feedback control model to observe how deactivations (i.e. reductions) of model
parameters impacted feedback responses. Results showed that deactivation of the “feedback
controller” impaired both response speed and accuracy, whereas deactivation of “state estimator” impaired only accuracy but not speed of the response. Next, we trained a rhesus monkey
to perform a unilateral arm postural task, in which the monkey was required to maintain arm
posture while responding to mechanical perturbations. Under normal conditions, the monkey
made a quick and accurate perturbation response to return to the original position. When we
deactivate PMd, the monkey showed impairments in both response speed and accuracy. On
the other hand, when we cooled A5, monkey showed impairment of response accuracy, but not
response speed. These results suggest that PMd and A5 have different functions in feedback
control: feedback controller and state estimation, respectively. This study demonstrates forthe first time that feedback processing for voluntary control involves cortical circuits beyond
primary motor cortex.
(COI:No)

Functional and anatomical dissociations between corticostriatal andcorticosubthalamic neurons
The cortico-basal ganglia (BG) circuits are important for motor, cognitive, and motivational
control of our actions. To achieve these functions, cortical information deriving from the motor,
associative, and limbic cortices is processed in BG local networks and finally transmitted tothalamocortical networks and brain stem networks. In these circuits, the activation of corticalneurons is considered to have opposing effects on neuronal activity of BG output nuclei (i.e.,the substantia nigra pars reticulata and the internal globus pallidus): the excitatory modulation
through the corticostriatal projection and the inhibitory modulation through the corticosubthalamic projection. However, anatomical and functional dissociations between corticostriatal andcorticosubthalamic neurons have not been fully elucidated. In this talk, first, I would like totalk about the viral tracing study to test whether the same or different populations of corticalneurons in the primary motor cortex (M1) of mice project to the striatum and subthalamic
nucleus. Second, I would like to talk whether the optogenetic manipulation of M1 corticostriatal
and corticosubthalamic neurons has opposing effects on motor behavior using self-initiatedlever-pull task. Finally, I would like to discuss whether such cortical neurons fire differentiallybased on the data obtained from different layers of M1 using in vivo two-photon calcium(COI:No)imaging. 

S09-02

S09-05Michiaki Suzuki（Neural Prosthesis Project, Dementia and Higher Brain Function, Tokyo

Koji Toda1，Saya Yatagai1，Kota Yamada2，Kohei Yamamoto1，Katsuyasu Sakurai3，
4 1 Dept. Psychol., Keio Univ., Japan, 2 Dept. Psychol., Keio Univ.,
Warren Meck 4，Henry Yin（

Ventral striatum as a potential therapeutic target for functional recovery
after spinal cord injury
Metropolitan Inst of Med Sci）

Neuronal mechanisms underpinning functional recovery after spinal cord injury (SCI) havebeen investigated widely in humans and animal models. Following SCI at the mid-cervicalsegment in non-human primates, plastic changes in motor cortices and spinal circuits areassociated with recovery of dexterous finger movements. However, the contribution of
structures up-stream of the motor cortices to recovery after SCI remains unclear. Recently,
we demonstrated that the ventral striatum (VSt), which is largely known as a key subcorticalnode for processing motivation and reward, causally contributes to functional recovery of
dexterous finger movements after SCI in monkeys. In addition, with brain imaging study using
positron emission tomography we clarified that the neuroplastic functional reorganization of
the VSt-motor networks occurs after SCI. These results suggest that the VSt is the pivotal
node of the cortical reorganization required for functional recovery of finger dexterity, and that
the VSt could be a critical target for therapeutic interventions that aim to promote functional
recovery. To test whether the VSt is a useful target for facilitation of motor performance, highfrequency electrical stimulation was delivered to the VSt while an intact monkey performed
the force-tracking task. VSt stimulation enhanced performance such as number of trials. This
result suggests that the VSt might be a key target for the therapeutic intervention to promote(COI:No)functional recovery after SCI.

S48

Neural mechanism of time perception
3 IIIS, Univ. Tsukuba., 4 Dept. Psychol. ＆ Neurosci., Keio Univ.）

Time perception is a subjective experience observed across many species. Animals need tocreate the subjective sense of time based on the integration of multiple sensory-motor information of self and perceivable objective events in the external world. Although many researchers
have tried to understand the psychological and neurobiological mechanisms of the subjective
experience of physical time, theories of time perception have been controversial, contradictory, and confusing. Here we designed a novel experimental setup that combined behavioral,
neurobiological, and computational approaches in investigating interval timing. Head-fixed
mice were trained on a fixed-time schedule Pavlovian conditioning task. We administeredsucrose solution every 10s. No external conditioned stimulus was presented throughout theexperiment. Mice could learn to anticipate the timing of the scheduled reward delivery. We
found that the pattern of anticipatory licking is significantly modulated by motivational state.
In addition, we also used a peak procedure task, in which regular trials are mixed with probe
trials with non-rewarded long interval, to assess the internal representation of the expected
time of reward delivery. Mice showed a peak response around the trained 10s after the previous reward delivery. To investigate the neurobiological substrates for the timing behavior
observed, we used integrative approach that combined immunohistochemistry, chemogenetics,
and optogenetics. This novel multi-disciplinary apparoach paves a way forward to study theneural mechanism of the timing behavior.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Symposium 10
A new world of physiology developed bypeptide hormones（March 18, 9:00〜10:50, Hall 8）

S10-01

Searching of the novel bioactive peptides using various methods

S10-03

Intervention effect on glucose intolerance by supplementation withmethyl modulator
Takahiro Nemoto（Department of Physiology, Nippon Medical School）
According to the Developmental Origins of Health and Disease (DOHaD) theory, low birthweight infants due to malnutrition in the prenatal period acquire a thrifty phenotype. Whenthese babies grow up in a eutrophic environment, there is a mismatch between constitution
and environment, and there is a risk of developing various metabolic diseases. We aimed todevelop an interventional method by creating a rat model that is low birth weight due to low
carbohydrate-caloric restriction during pregnancy and presents hyperinsulinemia with a highfat diet after growth. Metabolomic analysis of rat blood revealed various metabolic changes,
and we investigated whether to improve insulin resistance caused by environmental mismatch
caused by methyl modulator intervention. Offspring were obtained from dams fed a low
carbohydrate-calorie restricted diet (LC) throughout the gestation period. Methyl modulator
diet was prepared according to previous reports and fed to dams during late pregnancy orlactating. Each of the offspring was divided into a high fat diet (HFD) group and a standard
chow group at the age of 4 weeks, and was reared for 18 weeks. Even if the methyl modulator
diet was given to pregnant and postpartum lactating dams, there was no significant increasein body weight or body fat with a HFD-exposure. The blood insulin concentration after oGTT,
which was significantly higher in HFD-exposed LC rats, was reduced by methyl modulator
supplementation. Insulin receptor expression levels in liver and adipose tissues, which weresignificantly lower in HFD-fed LC offspring, were normalized by supplementation with methyl
donor during pregnancy or after birth. Methyl donor supplementation during pregnancy orafter birth in rats with low-calorie intake during fetal life resulted in metabolic changes andnormalized gene expression regulation abnormalities. Currently, we are analyzing the effects(COI:No)on the next generation offspring.

S10-04

Regulatory mechanism of neuropeptide “PACAP” on exocrine system

Takanori Ida（ Frontier, Miyazaki Univ, Japan）

Tomoya Nakamachi（Lab Regul Bio, Grad Sch Sci Eng, Univ Toyama, Japan）

G-protein-coupled receptors (GPCRs) constitute a large protein superfamily that shares a
7-transmembrane motif as a common structure. Human genome sequencing has identified
several hundred orphan GPCRs for which ligands have not yet been identified. GPCRs play
crucial roles in cell-to-cell communication involved in a variety of physiological phenomena
and are the most common target of pharmaceutical drugs. Therefore, the identification ofendogenous ligands for orphan GPCRs will lead to clarification of novel physiological regulatory
mechanisms and potentially facilitate the development of new GPCR-targeted therapeutics.
But in the last 10 years there has been little discovery of novel bioactive peptides for orphan
receptors. To break this situation, we searched for model organisms and discovered several
novel bioactive peptides. Various applied researches are in progress for these peptides. In
parallel, a new peptide extraction method is currently under development. We want to further
develop peptide search research.
(COI:No)

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide belonging to the vasoactive intestinal polypeptide (VIP)/secretin/glucagon superfamily. PACAP
and VIP bind to PAC1 receptor (PAC1R), VPAC1 receptor (VPAC1R), and VPAC2 receptor(VPAC2R). Mammalian PACAP has the highest binding affinity for PAC1R, almost 1000 times
greater than that for VPAC1R or VPAC2R.
Our study was started based on the finding of a new phenotype in PACAP null mice, whichshow dry eye-like symptoms, corneal keratinization and tear reduction. PACAP and its receptor mRNA were expressed in mouse lacrimal glands. PACAP immunoreactivity was mergedwith parasympathetic neuronal marker, and PAC1R immunoreactivity was observed in basalside of acinar cells. PACAP eye drop significantly elevated tear secretion and it was canceledby co-treatment with PAC1R antagonist in mouse. By signaling analysis, PACAP eye drops
stimulated tear secretion via an adenylate cyclase/cAMP/PKA cascade. PACAP stimulated
phosphorylation of aquaporin 5 (AQP5), and its translocation from the cytosol to the membranein lacrimal acinar cells. Moreover, AQP5 siRNA treatment to lacrimal gland attenuated
PACAP-induced tear secretion. These results suggest that PACAP act as an endogenous
regulator of tear secretion through AQP5 translocation.
AQP5 is also expressed in salivary gland and sweat gland. Therefore, we investigated the effect of PACAP on saliva and tear secretion. In three major salivary gland, PACAP mRNA wasdominantly expressed in sublingual gland and submandibular gland. VPAC1R is dominantly
expressed in the three major salivary gland. Intravenous administration of PACAP significantly increased saliva secretion and co-treatment with VPAC1R antagonist suppressed theeffect. Meanwhile, sweat gland expressed PAC1R and subcutaneous administration of PACAP
significantly increased sweat secretion. The PACAP-induced sweat secretion was suppressedby co-treatment with PAC1R and VPAC2R antagonist. These data suggest that PACAP hasa stimulating effect on exocrine glands that express AQP5 via various PACAP receptors.
(COI:No)

S10-02

S10-05

Daisuke Yamanaka1，Haruka Nagata2，Yuka Toyoshima3，Hiroki Nishi2，Lila Otani4，Yuki Goda2，
1 1 Dept
Fumihiko Hakuno2，Asako Takenaka5，Hisanori Kato4，Shin-Ichiro Takahashi2，Koichi Ito（

Sayaka Nagata（ Faculty of Medicine, Univ of Miyazaki, Japan）

Regulation of insulin-like activities in response to each amino acid
Vet Med Sci, Grad Sch Agri Life Sci, Univ Tokyo, Japan, 2 Dept Appl Ani Sci, Grad Sch Agri Life
Sci, Univ Tokyo, Japan, 3 Dept Bioreg, Inst Adv Med, Nippon Med Sch, Japan, 4 Dept Appl Bio
Chem, Grad Sch Agri Life Sci, Univ Tokyo, Japan, 5 Dept Agri Chem, Sch Agri, Meiji Univ, Japan）
Insulin-like growth factors (IGF) and insulin are peptide hormones highly homologous in structure and function. IGF and insulin synthesis/secretion is controlled by quality and/or quantityof protein in diets, leading to regulation of growth and metabolism. We have investigated rolesof amino acids in IGF/insulin systems using growing rats fed a low protein (LP) diet.
It is known that these animals show reduction in gene expression of IGF-I in liver, a major
IGF-producing organ. Our recent analysis using a cultured hepatocyte cell line showed thatdeprivation of essential amino acids (EAAs) decreased IGF-I mRNA levels, indicating that
EAAs are required for upregulation of IGF-I gene expression. Diet-induced insulin secretionis also decreased in LP-fed rats; however, insulin secretion was restored to normal levels
by supplementation of the LP diet with three branched chain amino acids, suggesting animportant role of BCAAs in insulin secretion. On the other hands, neutral lipids (triglyceride)
were accumulated in the liver of LP-fed rats. Analysis using single amino acid-deficient diets
indicated that arginine- or threonine-deficient diets increased hepatic triacylglyceride content.
Our further research showed that LP and arginine-deficient (dArg) diets regulated lipid accumulation through different mechanisms; insulin signaling and de novo lipid synthesis wereenhanced in LP-fed rats, while triglyceride release from liver was attenuated in dArg-fed rats.
To investigate the underlying mechanisms, we cultured hepatocytes in amino acid-sufficient or
deficient medium. Surprisingly, intracellular triacylglyceride level was increased by amino aciddeficiency without addition of any lipids or hormones, indicating that hepatocytes themselves
monitored the extracellular amino acid concentrations to induce lipid accumulation in a cellautonomous manner.
Taken together, we concluded that each amino acid play distinct roles in regulation of insulin/
IGF systems and induction of insulin-like activities, coordinately controlling growth andmetabolism.
(COI:No)

Development of adrenomedullin derivative
Human adrenomedullin (hAM) is a hypotensive peptide hormone that exerts powerful antiinflammatory effects. We recently showed that hAM significantly reduces the clinical severityof the 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis in animal models. Furthermore, ina clinical study, hAM treatment reduced the Disease Activity Index in ulcerative colitis.
However, these therapies required continuous administration of hAM as the half-life of native
hAM is quite short in blood.
To resolve this problem, we developed two kinds of AM derivatives.
Frist, we conjugated the hAM N-terminal with 60 kDa polyethylene glycol (PEG-AM). A single
subcutaneous administration of PEG-AM reduced the total inflammation score in mice with thedextran sulphate sodium (DSS)-induced colitis. In addition, the plasma half-life of PEG-AM wasmuch longer than native hAM. Moreover, we examined the effect of PEG-AM on four-vessel
occlusion model rats, which exhibit vascular dementia. PEG-AM treatment prevented memory
loss and learning disorders in dose-dependent manner.
Furthermore, we designed of human IgG1 Fc fusion protein containing hAM (Fc-AM). Sufficient concentrations of Fc-AM was observed in blood two days after a single subcutaneousadministration. Fc-AM stimulated cAMP production in HEK-293 cells stably expressing theAM1 receptor. Treatment with Fc-AM inhibited blood pressure increase in spontaneously hypertensive rats. In addition, Fc-AM reduced total inflammation scores in the DSS colitis model.
These data indicate that PEG-AM and Fc-AM are a possible therapeutic agent for the treatment of inflammatory bowel disease or vascular dementia.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S49

Symposium 11
AMED support for medical R&D:
From basics to practice（March 18, 9:00〜10:50, Hall 10）

S11-03Development of human arterial graft with mechanically functional
extracellular matrices
Utako Yokoyama1, 2，Tomoyuki Kojima2, 3，Junichi Saito2，Takanori Yoda2，
Takashi Nakamura2，Yoshinobu Sugo3，Kentaro Kurasawa3，Etsuko Miyagi3，
2 1 Dept Physiol, Tokyo Med Univ, Japan, 2 Cardiovasc Res Inst,
Yoshihiro Ishikawa（

Yokohama City Univ, Japan, 3 Dept Ob/Gyn, Yokohama City Univ, Japan）

Elasticity and stiffness are necessary for vascular integrity. Therefore, biological tissueengineered blood vessels with mechanically functional extracellular matrices are desired.
Fibronectin fibrillogenesis plays a critical role for assembling elastic fibers and collagen fibers
which provide elasticity and stiffness. Recently, we found that periodic hydrostatic pressure(PHP) with extremely low frequency promoted fibronectin fibrillogenesis on the surface ofvascular cells. We successfully fabricated implantable human arterial graft by PHP, andexamined PHP-induced mechanosensing mechanisms in vascular smooth muscle cells. We
seeded human umbilical arterial smooth muscle cells (hUASMCs) on cell culture disk to makethe first cell layer. Twenty-four hours after seeding, cells were exposed to PHP for 24 h, andthen cells for the next layer were seeded on the top of the first layer, followed by repeating
the same procedure ten times to construct multi-layer cell sheet. The burst pressure of theconstruct was over 1200 mmHg. The multi-layered construct was trimmed as a patch graft.
The graft was sutured at the abdominal aorta of nude rat in which the same size of aortic
tissue was resected. Five months after implantation, echocardiography confirmed complete
patency and histological analysis revealed that all patch grafts were completely endothelialized.
Host-derived cells markedly infiltrated into the graft. To examine the PHP-responsive genes,
we performed RNAseq analyses and found that angiopoietin-like 4 (ANGPTL4) and insulin-likegrowth factor binding protein 5 (IGFBP5) that binds to fibronectin were highly increased by
PHP. In conclusion, implantable human arterial graft consisting of vascular smooth muscle cells(COI:No)was constructed by exposure to PHP. 

S11-01

S11-04James Pearson1, 2，Hirotsugu Tsuchimochi1，Takashi Sonobe1，Jennifer Ngo1，
4 1 Dept Cardiac Physiol, Res
Mark Waddingham3，Masaki Wakabayashi4，Manabu Shirai（

Keita Saku（Dept Cardiovasc, Grad Sch Med, Kyushu Univ, Japan）

Relations between coronary microvascular dysfunction and thedevelopment of diastolic dysfunction in prediabetic and diabetic rats

Development of Japan originated neuro-modulation system treating
cardiovascular disease

Diastolic dysfunction of the left ventricle is an early factor in the onset of heart failure. Here
we investigated the relations between coronary and myocardial function in the development
of diastolic dysfunction associated with diabetes and the roles played by chronic inflammationand oxidative stress. Currently we are utilizing synchroton based microangiography, in vivo
cross-bridge dynamics and immunoblotting assays to investigate the role of these factors in
non-obese, insulin resistant Goto-Kakizaki rats and obese, hypertensive diabetic stroke-prone
SHR rats. Further, the gene expression of sedentary control and GK rats is being comparedto aged-matched rats following regular, medium-intensity exercise training using RNAseq
analysis. Results to date show that both severe hypertension and diabetes impair cardiomyocyte contractile function and relaxation through myofilament posttranslational modifications
independent of coronary endothelial dysfunction but is exacerbated by such progressive(COI:No)changes in coronary endothelial and smooth muscle function.

The imbalance of autonomic nervous system, disproportional sympathetic upregulation andparasympathetic downregulation, is involved in the pathogenesis of various cardiovasculardiseases, such as hypertension, myocardial infarction (MI) and heart failure. Based on the solid
preclinical evidence of therapeutic effects, several devices to restore the autonomic balance bystimulating or denervating the nerves have been developed in worldwide. We recently develop
the several neuro-modulation devices for cardiovascular disease.
The infarct size in MI predicates survival outcomes, therefore therapies that reduce infarct
size beyond early coronary reperfusion are critical to prevent the subsequent development ofheart failure. Vagal verve stimulation, which directly activates parasympathetic nervous system, has been reported to exert multiple cardio-protective effects. Despite attractive preclinicalstudies of vagal nerve stimulation for acute MI, its clinical translation remained unestablished.
We have been developing an intravenous vagal nerve stimulation (iVNS) catheter system
which can stimulate the right vagal nerve with minimally invasive technique. We confirmedthat iVNS during acute MI markedly reduces the infarct size and prevents subsequent heart
failure in a dog model of ischemia-reperfusion. We are now proceeding the strategies to obtain
the regulatory approval of iVNS catheter system as a medical device.
In this session, we will introduce our challenges in device development and address thebarriers against the development of invasive medical devices in Japan by referring to ourexperiences. (COI:No)

S11-02

S11-05

2 1CVRI, Grad Sch Med, Yokohama City University,
Yoshihiro Ishikawa1，Utako Yokoyama（

3 1 Dept Anesthesiol
Kenji Shigemi1，Osamu Nagata2，Yuka Matsuki1，Yoshihiro Ogino（Inst, NCVC, Japan, 2 Dept Physiol, Monash Univ, 3 Dept Adv Medical Res Pulm
Hypertension, NCVC, 4Omics Center, NCVC）

A new biomarker for abdominal aortic aneurysm
2 Dept Physiology, Tokyo Medical Univ）

Abdominal aortic aneurysm is a common disease among elderly people, and is most commonly
found in men with smoking history. Epidemic survey has shown that the disease may befound in four to six percent of men above age sixty five. The majority of such patients were
found during physical examinations using abdominal ultrasound. Alternatively, in some cases,
the presence of aneurysm is not found until it ruptures and the patient is transferred tohospital under critical conditions. Thus, it is important to establish an easy method of detecting
abdominal aneurysm. We have identified myosin heavy chain 11 as a biomarker of abdominal
aortic aneurysm. We will explain how it was identified and what is the potential use of this
(COI:No)
biomarker in the future.

S50

The research and development of a robot that manages anesthesia
Reanimatol, Sch Med, Fukui Univ, Japan, 2 Dept Anesth, Ctr Hosp Natl Ctr Global HealthMed, 3 Nihon Kohden Corp）

University of Fukui, Center Hospital of the National Center for Global Health and Medicine(NCGM), and Nihon Kohden Corporation are jointly researching and developing a robot device
that manages general anesthesia which is necessary for surgical operation. This study was
accepted by Japan Agency for Medical Research and Development (AMED) as a three-year
project from 2018, and the results are scheduled to be applied for regulatory affairs at
Pharmaceutical and Medical Devices Agency (PMDA) in 2021. From the induction of generalanesthesia to the recovery from general anesthesia, this device under development controls
the intravenous infusion rate of sedative (propofol), analgesic (remifentanil), and muscle relaxant(rocuronium) based on information from the electroencephalogram (BIS processor QE-910P,
Nihon Kohden Co., Tokyo), the muscle relaxation monitor (TOF Watch SX, Nihon Kohden Co.,Tokyo) with three syringe pumps (TE-SS835T and TE-SS800N, Terumo Co., Tokyo) througha personal computer. The required dose of propofol is estimated on the dose-response curve
which is determined with the relationship between BIS value and the effect site concentration(Ce) of propofol. The dose of remifentanil is determined with the balance of the doses of
propofol and remifentanil. Rocuronium is administered to maintain the level of surgical muscle
relaxation (%T1 from 3% to 10%). This device is fundamentally based on the closed-loop drugdelivery system to maintain sedation, analgesia and muscle relaxation appropriately, and is
expected to prevent human error and drug overdose/underdose, and to improve anesthesia
safety as well as to contribute to doctors’ work style. (COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Symposium 12
Control of endogenous regulatory responses
in glial cells in neuroinflammation- From embryonic development topathogenesis of brain diseases（March 18, 15:20〜17:10, Hall 7）

S12-03

Periodontal disease bacteria-induced neuroinﬂammation and
Alzheimerʼs disease
2 1 Dept .Phamacol, Grad Sch Dent, Kyushu Univ, Japan, 2 OBT Research
Zhou Wu1, （Center, Grad Sch Dent, Kyushu Univ, Japan）

Dementia is increasing rapidly in Japan with the super-aged society. Alzheimer’s disease (AD),which accounts for 70% of dementia, could not been treated yet since its onset, resulting
in enormous medical costs and a serious social burden. The cognitive decline in AD due
to neuronal death which is caused by amyloid β (Aβ) accumulation, excessive tau phosphorylation in neurons, and microglia- dependent neuroinflammation accumulates Aβ and tau
phosphorylation. On the other hand, the positive correlations between AD and periodontitis(the most common periodontal disease) have been reported by the western clinical studies,and P. gingivalis (Pg) LPS, the major periodontal disease bacteria, has been detected from the
autopsy brain of AD patients. The involvement of periodontitis in AD has been attracting
attention, because periodontitis is treatable oral disease.
In exploring the impacts of systemic inflammation on brain functions constantly, we havefound that chronic systemic inflammation induces an age-dependent neuroinflammation, whichcauses age-dependent cognitive decline. In investigating the involvement of periodontitis in
AD, we have found that chronic exposure to PgLPS induces AD-like pathological phenotypes,
including microglia-dependent neuroinflammation, Aβ accumulation in neurons and learning
memory impairment in the middle-aged mice. Cathepsin (Cat) B, a lysosomal proteolytic
enzyme, has been determined as a related enzyme in the PgLPS-induced AD-like pathogenesis. Furthermore, we have revealed that CatS is an enzyme related to the Pg LPS- induced
systemic inflammation. In the present symposium, I will explain the involvement mechanism of
periodontitis in AD onset and pathological processes, and introduce the clinical research for improvement of cognitive functions by regulating systemic inflammation and neuroinflammation.
The early intervention in oral/systemic inflammation is a feasible medical approach for prevention or delaying the onset and progression of AD. It is also important to develop the specificinhibitors of CatB and CatS for regulating systemic inflammation and neuroinflammation.
(COI:Properly Declared)

S12-01

S12-04

Kazuya Miyanishi（Dept Mol Cell Physiol, Grad Sch Med, Ehime Univ, Japan）

Shuei Sugama（ Dept Physiol, Nippon Med School, Japan）

Microglia are not resting cells even in normal mature brains, but they constantly move theirramified processes to monitor the activity states of neural circuits. The physiological functionsof microglia are yet obscure. We have found that microglia in substantia nigra pars reticulata(SNr) display more activated morphology rather than those in the SN pars compacta in a
Parkinson’s disease rat model. Microglia may compensate the disordered neurotransmissions
by eliminating glutamatergic syanapses in the SNr from hyperactive subthalamic nuclei.
Furthermore, microglia were found to be weakly activated around the onset of sleep. The
weakly activated microglia may contribute to sleep/wake cycle by phagocytosing synapses.
The activation signals may be mediated by JAK/STAT/IRF -dependent signaling pathway.
These findings suggest that neuroinflammatory processes may be responsible for activationof microglia even in the normal mature brains. We have further noticed that microglia may
affect social interaction between males and females. Behavioral tests have revealed that male
Wistar rats prefer females that are rather inactive and anxious, but friendly. We divided theovariectomized female rats into two groups “Winner” and “Loser” by examining the preferenceby male rats; Winners were more popular among male rats than Losers. In the Winners’
prefrontal cerebral cortex, higher expression of mRNA for phagocytosis-related proteins were
found relative to that in the Losers’. Flow cytometrical analyses showed that microglia from
Winners displayed higher forward scatter values than those from Losers. Inactive microglia
were also found in a rat strain that shows characteristic behaviors similar to cases with attention deficit/hyperactive disorder. In conclusion, microglia may play a role in the behavior bymodulating neural circuits while neuroinflammatory processes may affect microglial function(COI:No)even in the normal mature brain.

The involvement of microglia in neuroinflammatory responses has been extensively demonstrated. Recent animal studies have shown that exposure to either acute or chronic stressinduces robust microglial activation in the brain. In this presentation, we tried to investigate
the underlying mechanism of brain microglial activation by acute stress. At first, we lookedat the spetial distribution of noradrenaline-synthesizing enzyme, DBH, in comparison with
noradrenaline receptors, b1, b2, and b3 adrenergic receptors after which we examined theeffects of the beta-blocker propranolol and alpha-blockers prazosin and yohimbine on stressinduced microglial activation. Finally, we compared stress-induced microglial activation
between wild-type (WT) mice and double-knockout (DKO) mice. The results demonstrated
that microglial activation occurred in most studied brain regions, including hypothalamus,
thalamus, and hypothalamus; that b1- and b2-, but not b3-, adrenergic receptors are colocalizedwith microglial cells, as observed by laser scanning microscopy; that beta-blocker treatment
inhibited microglial activation, with alpha-blocker showing no such effect; and that unlike
WT mice, DKO mice exhibited substantial inhibition of stress-induced microglial activationin the brain. We demonstrate here that neurons/microglia may interact with noradrenaline
via b1- and b2-adrenergic receptors. In this symposium, we discuss a possible mechanism for(COI:No)stress-induced microglial activation. 

Microglia affect animal behaviors; neuroinﬂammatory processes in thenormal mature brain

Stress-induced microglial activation occurs through beta-adrenergic
receptors: Noradrenaline as key neurotransmitter in microglial activation

S12-02

Activating a non-neuronal cardiac cholinergic system consolidates the
blood brain barrier associated with upregulation of anti-inﬂammatory
actionYoshihiko Kakinuma1，Shino Oikawa1，Yuko Kai1，Asuka Mano1，Shuei Sugama1，
2 1 Dept Bioregulatory Sci
Naoko Mizoguchi2，Masayuki Tsuda3，Kazuyo Muramoto（(Physiol), Grad Sch Med, Nippon Medical School, Japan, 2 Dept Physiol Sch Dentistry,
Meikai University, 3 Inst Lab Anim Res, Kochi Medical School）
We previously reported that the heart-specific choline acetyltransferase (ChAT) gene overexpressing mice (ChAT) demonstrate beneficial phenotypes, i.e., tolerance against ischemia and
CNS stress . In this study, we focused on mechanisms responsible for systemic and localized
anti-inflammatory phenotypes of ChAT. ChAT were resistant to systemic inflammationinduced by lipopolysaccharides due to an attenuated cytokine response. In addition, ChAT,
equipped with less reactive Kupffer cells, were refractory to brain cold injury, with decreased
blood brain barrier (BBB) permeability and reduced inflammation. This is because ChAT
brain endothelial cells expressed more claudin-5, and their astrocytes were less reactive withdecreased hypertrophy. Moreover, reconstruction of the BBB integrity in vitro confirmed theconsolidation of ChAT BBB. ChAT were also resistant to 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) neuronal toxicity due to lower mortality rate, fewer neuronal loss of
substantia nigra and attenuated BBB disruption, as evident from reduced sodium fluoresceinlevels in the brain parenchyma. The activated central cholinergic pathway of ChAT leadto anti-convulsive effects like vagus nerve stimulation. However, DSP-4, a noradrenergic
neuron-selective neurotoxin against the locus ceruleus, abrogated the beneficial phenotype
and vagotomy attenuated expression of claudin-5, suggesting the link between the cholinergic
pathway and BBB function. These findings indicate that ChAT possess an anti-inflammatory
response potential, associated with upregulated claudin-5, leading to the consolidation of BBB
integrity. These characteristics protect ChAT against systemic and localized inflammatory
pathological disorders, which targets the CNS.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S51

Symposium 13
New strategies for understanding functions
and mechanisms of ion channels/transporters/
pumps in the upcoming post-structure era（March 18, 15:20〜17:10, Hall 9）

S13-03Structural and molecular basis of Na+/D-glucose co-transporters
hexose speciﬁcity
2 1 Dept Mol Physiol and Biophys, Fac Med,
Kazuyo Kamitori1, 2，Yuichiro Fujiwara1, （Kagawa Univ, Japan, 2 Int Inst Rare Sugar Res and Ed）

Na+-dependent sugar transporters utilize the transmembrane sodium gradients for cellularsugar uptake. Human Na+/D-glucose cotransporters (SGLTs) are abundant in small intestineand kidney where they contribute to the D-glucose absorption and reabsorption, respectively.
Tumor cells highly express SGLTs to facilitate D-glucose uptake and glycolysis, resulting in
the unlimited tumor growth. These physiological and pathological functions made SGLTs thetargets for diabetes or cancer treatment, and several SGLT2 inhibitors are already used asanti-diabetic drugs. Each SGLT transports specific hexoses in specific tissues, therefore thehexose specificity should be precisely regulated depending on their roles. This specificity isdetermined by the structure around the hexose binding pocket. Understanding structural
basis of SGLTs hexose specificity would provide important information for the drug design
targeting these molecules. Although crystal structures of SGLTs has not been reported, here
we performed homology modeling of human SGLT1 using the template structure of Vibrio
parahaemolyticus Na+/D-galactose cotransporter.
We have reported human SGLT1 transport capacity for various hexoses using the twoelectrode voltage-clamp method in Xenopus laevis oocytes. We observed that both D-glucose
and D-galactose were cotransported with Na+ as previously reported, and that none of otherhexoses were transported. This suggests that SGLT1 has tolerance for the orientation of OH
group in the 4-position but intolerance for the 2-, 3-positions. We performed mutation studies
based on the structure model, toward the understanding of hexose specificity. Interestingly,
some of the point mutants at amino acids in the sugar binding pocket changed the substratespecificity; that is, D-mannose and D-allose were transported, or transportation of D-glucose
or D-galactose was diminished. These results shed light on the relationship between the sugar
orientation and the hexose specificity in SGLT1. Present analyses would contribute to the clinical strategies targeting SGLTs, on the points of controlling efficacy and side effects. (COI:No)

S13-01

S13-042 1 Div. Biophys and Neurobiol, NIPS, Okazaki, Japan,
I-Shan Chen1，Yoshihiro Kubo1, （2 1CeSPI, Nagoya Univ, Japan, 2 Grad Sch Pham Sci, Nagoya Univ）
Kazuhiro Abe1, （Roles of the pore helix in the regulation mechanisms of gating of GIRK
channels
2 Physiol Sci. SOKENDAI, Hayama, Japan）

G-protein-gated inwardly rectifying K+ (GIRK) channels control various physiological functions.
For example, GIRK1/2 heterotetramers in the brain regulate neuronal excitability; GIRK1/4
heterotetramers in the heart regulate heart rate. We previously identified a novel GIRK activator, ivermectin, and more recently a novel GIRK inhibitor, terfenadine. We reported that a
GIRK1-specific amino acid residue located in the pore helix behind the selective filter, Phe137,
is critical for the terfenadine-mediated inhibition. Here, by electrophysiological analyses anddocking simulations, we present our observations of mutants at the pore helixes, including
Phe137 in GIRK1 subunit and the corresponding residue Ser148 in GIRK2 subunit, and someamino acid residues which are close to this region. We found that the pore helix residue
plays multiple roles in the function of GIRK channels as follows: (1) it couples the terfenadine
induced conformational change with the channel closure and its side chain volume is crucial;(2) it contributes to the gating behaviour upon membrane potential hyperpolarization; and (3) it
regulates the ion selectivity and the polarity of the residue is important. The results provide us
with a novel insight into the gating mechanisms of GIRK channels by the pore helix. (COI:No)

Structural basis of the proton extrusion mechanism of the gastric protonpump
The gastric proton pump, H+, K+-ATPase is a P-type ATPase that is responsible for acidifying
the gastric juice down to pH 1, and is thus an important drug target for treating gastric
acid-related diseases. It mediates electro-neutral exchange of H+ and K+ coupled with ATPhydrolysis, but with an as yet undetermined transport stoichiometry. Here we present the
crystal structures of the H+, K+-ATPase in complex with two acid blockers, vonoprazan and
SCH28080, in the luminal-open E2P state. These drugs have partially overlapped, but clearly
distinguishable, binding modes, which are defined in the middle of a conduit running from thegastric lumen to the cation-binding site. The crystal structures also revealed a conserved lysine
residue that points to the juxtaposed carboxyl residues in the cation-binding site. The unusual
configuration of the cation-binding site enables the extrusion of a single proton even into thepH1 solution of the stomach (Abe et al., 2018, Nature).
We also show crystal structures of E2-P transition state, in which the counter-transporting
K+ is occluded. We found a single K+ bound to the cation-binding site of H+, K+-ATPase,
indicating an exchange of 1H+/1K+ per hydrolysis of one ATP molecule. This fulfils theenergy requirement for the generation of a six pH unit gradient across the membrane. The
structural basis of K+ recognition is resolved, supported by molecular dynamics simulations.
These results establish how the gastric pump overcomes the energetic challenge to generatean H+ gradient of more than a million-fold - the highest cation gradient known in any mammalian tissue – across the membrane (Yamamoto et al., 2019, eLife).
(COI:No)

S13-02Analysis of the Inactivation mechanism in a Mutant of the VoltageGated Potassium Channel Kv1.2
Hiroko X. Kondo1，Norio Yoshida2，Gen Masumoto3，Matsuyuki Shirota4, 5, 6，Yu Takano7，
8 1 Fac Eng, Kitami Inst Tech, Japan, 2 Grad Sch Sci, Kyushu Univ,
Kengo Kinoshita5, 6, （Japan, 3 RIKEN ISC, 4Grad Sch Med, Tohoku Univ, Japan, 5 Grad Sch Info Sci, Tohoku
Univ, Japan, 6ToMMo, Tohoku Univ, Japan, 7 Grad Sch Info Sci, Hiroshima City Univ,
Japan, 8 IDAC, Tohoku Univ, Japan）

Voltage-gated potassium (Kv) channels conduct potassium ions selectively in response to membrane depolarization, and ion permeation through the pore domain is dynamically regulated
by several types of gating mechanisms. “C-type” inactivation is one of well-defined inactivation
processes in the Kv channels. This inactivation is primarily caused by conformational changes
around the selectivity filter (SF) and/or outer mouth of the channels. The W366F mutant of
Kv1.2 channel (corresponding to W434F in Shaker) is known as a fast-inactivating mutant andis in a C-type inactivated state at a depolarizing membrane potential. In order to clarify the
structural properties of C-type inactivated protein, we performed molecular dynamics simulations of the wild-type and W366F mutant of the Kv1.2-2.1 chimera channel and carried out thestructural analysis by using the statistical methods. In our simulation the W366F mutant wasin a nearly nonconducting state with a depolarizing voltage and recovered from inactivation
with a reverse voltage. The result of the principal component analysis for SF structures showsa conformational difference at V375 and G376 between channels in the ʻactive’ and ʻinactive’
states. To evaluate a correlation between SF conformation and ion occupancy state in SF, weanalyzed the probability distributions of potassium ions in SF of the WT and W366F mutant byusing the three-dimensional reference interaction site model (3D-RISM) theory. Our simulations
and 3D-RISM analysis suggested that structural changes in the SF upon membrane depolarization trap potassium ions around the inner mouth of the SF and prevent ion permeation. This
pore restriction is involved in the molecular mechanism of C-type inactivation. (COI:No)S52

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Symposium 14
The new sunrise of zinc physiology andzinc pathophysiology（March 19, 9:00〜10:50, Hall 7）

S14-01

Integrated omics analysis revealed mechanisms underlying
improvement of mouse hippocampal function with zinc-enriched
breeding over generations
3 1 Res Promo Inst, Fac Med, Oita Univ, Japan,
Kumiko Sakai1，Seiichi Chiba2，Kenji Sakai（

2 Dept Anatomy, Fac Med, Oita Univ, Japan, 3 Dept Biosci Biotech, Grad Sch Biore Bioenvtl

Sci, Kyushu Univ, Japan）

Zinc is a necessary element for neuromodulation in brain and its homeostasis is strictly
controlled under healthy conditions. Relationships between zinc-deficiency and neurologicaldisorders have been well-investigated to date, however, little is known about the implicationof an excess but physiologically normal level of zinc for neuronal metabolism in the brain. In
this study, integrated analysis of metabolomics and transcriptomics were performed improve
comprehensive understanding of the molecular mechanism of brain zinc homeostasis and its
neurological and psychological function.
We continued to administer zinc 3 times the normal level to mice over generations. Behavioral experiments of their offspring showed increases in some brain functions such as social
behavior, learning and memory levels compared to control mice. Metabolomic profiling of
hippocampus tissue revealed distinguishable profiles of metabolites and the pathway analysis
indicated specific relationship to catechol amine biosynthesis by the chronic zinc treatment.
Microarray analyses that were conducted to identify differential expressed genes and their
Gene Ontology analyses using Metascape highlighted genes with functional terms of Behavior,
GPCR, Transmembrane, Neuronal System, etc. We also applied an integrated analysis of themetabolomics and transcriptomics data together with the term Zinc using the system biology
tool IPA to analyze the significantly altered molecular and bio function networks. The integrated omics profiles indicated that the function categories of Nervous System Development
and Function and Cell-To-Cell Signaling and Interaction were strongly facilitated, whereas
the diseases categories of Neurological Disease and Psychological Disorder were significantly
inhibited by zinc-related molecules and genes. This comprehensive study of neurological functions and networks would provide new insights of zinc into advanced and healthy functions ofbrain.
(COI:No)

S14-03

Molecular basis on lung pathogenesis in cystic ﬁbrosis - dysregulated
splicing switches of zinc transporter ZIP2 and anti-inﬂammatory
molecule SIGIRR
Tsuyoshi Shuto（Global Cent Nat Resources Sci, Grad Sch Pharm Sci, Kumamoto Univ,
Japan）

Airway mucus hyperproduction and hyperinflammation are important hallmarks of cystic
fibrosis (CF). Dysregulated expression of airway ion transporters CFTR and ENaC havebeen implicated as causes of CF-associated mucus hypersecretory and hyperinflammatory
phenotypes. However, the roles of Zn2+ and Zn2+ transporters as well as anti-inflammatory
molecules in the regulation of CF airway pathogenesis remain unelucidated. First, we identified
a novel connection between CFTR/ENaC expression and the intracellular Zn2+ concentrationin the regulation of mucin MUC5AC. CFTR-defective and ENaC-hyperactive airway epithelial
cells specifically and highly expressed a unique, alternative splice isoform of the zinc importerZIP2/SLC39A2 (ΔC-ZIP2), which lacks the C-terminal domain. Importantly, ΔC-ZIP2 levels
correlated inversely with wild-type ZIP2 and intracellular Zn2+ levels. Moreover, the spliceswitch to ΔC-ZIP2 as well as decreased expression of other ZIPs caused zinc deficiency, which
is sufficient for induction of MUC5AC. We next explored the possible involvement of single
immunoglobulin interleukin-1 receptor (IL-1R)-related molecule (SIGIRR), a membrane protein
essential for suppressing TLRs- and IL-1R-dependent signals, in the regulation of CF airway
hyperinflammation. We showed that cell surface expression and anti-inflammatory functionof SIGIRR were specifically and remarkably down-regulated in CFTR-defective CF airway
epithelial cells compared to non-CF cells. Notably, CF airway epithelial cells specifically andhighly expressed a unique, alternative splice isoform of SIGIRR that lacks exon 8 (Δ8-SIGIRR),
which results in the production of truncated form of SIGIRR protein. Δ8-SIGIRR protein was
expressed intracellularly and its over-expression abolished plasma membrane expression andfunction of SIGIRR in non-CF cells, indicating that the splice switch to Δ8-SIGIRR is sufficient
for reduced surface SIGIRR expression. Taken together, we demonstrate the unique splicing
switches of ZIP2 and SIGIRR genes control mucus hypersecretory and hyperinflammatory
phenotypes in cystic fibrosis airway epithelial cells, respectively.
(COI:No)

S14-04

Innovative insight into defense against progressive neurodegeneration
caused by synaptic Zn2+ dysregulation
Atsushi Takeda（ Dept Neurophysiol, Sch Pharmaceutical Sciences, Univ Shizuoka, Japan）
The causes of progressive neurodegenerative disorders, i.e., Alzheimer’s disease (AD) andParkinson’s disease (PD) are unknown. The basal (static) concentration of intracellular Zn2+
is estimated to be approximately 100 pM and is extremely lower than that of intracellular
Ca2+ (approximately 100 nM), suggesting that intracellular Zn2+ homeostasis is crucial forneural function. Moreover, the basal concentration of extracellular Zn2+ is estimated to beapproximately 10 nM and is age-relatedly increased based on age-related increase in brain
extracellular Zn. We postulated that progressive neurodegeneration is due to age-related intracellular Zn2+ dysregulation, which is induced by rapid influx of extracellular Zn2+. Neuronalamyloid β1-42 (Aβ1-42) accumulation is considered an upstream event in the AD pathogenesis. Here we report that Zn-Aβ1-42 oligomers formed in the extracellular compartment aresynaptic activity-independently taken up into neurons, followed by rapid intracellular Zn2+
dysregulation. PD is characterized by selective loss of dopaminergic neurons in the substantia
nigra pars compacta of the brain. Here, we report a unique mechanism of nigral dopaminergic
degeneration, in which rapid intracellular Zn2+ dysregulation via the production of reactive
oxygen species, especially hydrogen peroxide causes PD in rats, which is induced by paraquartand 6-hydroxydopamine. I will talk about novel defense strategy against progressive neurodegeneration by controlling intracellular Zn2+ dysregulation.
(COI:No)

S14-02

Zinc complex to treat type 2 diabetes with improving insulin resistance2 1 Dept Anal Bioinorg Chem, Kyoto Pharm
Hiroyuki Yasui1，Yuki Naito1，Yutaka Yoshikawa（

Univ, Japan, 2 Kobe Women’s Univ, Japan）

We have synthesized and investigated transition metal complexes including manganese (Mn),copper (Cu), zinc (Zn), and platinum (Pt) for the purpose of new drug development treating
intractable diseases, based on bioinorganic medicinal chemistry. Especially, Zn(II) compounds
have been clinically used for the treatment of skin injury, gastric ulcer, Wilson disease, andvery recently hypozincemia resulting from low zinc status. Additionally, Zn(II) ion attracts
higher attention because of both the various physiological functions and discovery of manyzinc transporter proteins.
In this paper, we would like to introduce our research of potent Zn(II) complexes to treat type2 diabetes mellitus (DM) with improving insulin resistance. It is well known that the numberof DM patients increases gradually in the world. Therefore, the prevention and treatment
for DM with new kinds and novel actions of medicines are much needed such as GLP-1 likeanalogs, DPP4 and SGLT2 inhibitors. Before now, we have synthesized over 250 kinds ofZn(II) complexes with different chemical-structures of ligans/coordination modes of atoms, andevaluated the in vitro insulin-mimetic/enhancing actions, pharmacokinetics (PK), and anti-DM
therapeutic effects with respect to rational drug design concept.
From the results, we first found that zinc complexes coordinated with both sulfur and oxygen
atoms exhibit high insulin-mimetic activities, preferable PK properties after oral administration, desirable bio-distribution to targeted organs, and effective therapeutic action for DM
including the protection of pancreatic islets. Additionally, we address the action mechanism foranti-diabetic activities of Zn(II) complexes, which directly activate intracellular PI3-K within
the insulin signaling pathway in an insulin-independent manner. Then, it is concluded that ourdeveloped Zn(II) complexes have high potential to treat type 2 DM with improving insulin
resistance in peripheral organs, and that Zn(II) complexes can be insulin-enhancer suppressing(COI:No)
insulin requirements.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S53

Symposium 15
Aging-related changes in physiological
functions induced by the space environment（March 19, 9:00〜10:50, Hall 8）

S15-01

Vestibular plasticity and orthostatic hypotension
Chikara Abe, Hironobu Morita（ Dept Physiol, Gifu Univ Grad Sch Med, Japan）
Daily activity-induced changes in the gravitational vector one of the major disturbances that
affect the cardiovascular system. For example, a postural change from a recumbent to an
upright position induces an increase in the hydrostatic pressure gradient, a footward fluid
shift, reduced venous return and cardiac output, and reduced arterial pressure (AP). This
reduction in AP is sensed by baroreceptors in the blood vessels, and AP is thought to bestabilized by the arterial baroreflex, an important negative feedback process. On the other
hand, postural changes stimulate the peripheral vestibular organs. Stimulation of the peripheral
vestibular organs by head movement or changes in gravitational forces is known to induce
sympathoexcitation (vestibulo-sympathetic reflex). We have reported that the combination ofboth the baroreflex and the vestibulo-sympathetic reflex is important for maintaining AP
during postural change. Daily gravitational change is important for maintaining the vestibularfunction. Elderly people and astronauts are known to show a plastic alteration of the vestibular
system because of reduction in vestibular inputs (less activity in elderly people and no gravity
in astronauts). This plasticity might participate in the increase in risk of orthostatic hypotension in elderly people and astronauts. This possibility will be discussed in this symposium.
(COI:No)

S15-03

Roles of Dkk2 in the effects of gravity change on the interactions
between muscle and bone in mice
1 1 Dept Physiol ＆ Regene Med, Kindai
Naoyuki Kawao1，Hironobu Morita2，Hiroshi Kaji（
Univ Fac Med, Japan, 2 Dept Physiol, Gifu Univ Grad Sch Med, Japan）

Long term space flight simultaneously induces muscle and bone loss, and the interactions
between muscle and bone have been recently noted. Previous studies indicate that inhibitorsof canonical Wnt signal, Dkks, play crucial physiological and pathophysiological roles in bonemetabolism. Although canonical Wnt signal is involved in aging-related muscle atrophy, itsroles in the interactions between muscle and bone have remained unclear. We therefore examined roles of Wnt signal-related factors in the effects of gravity change on the muscle/bonerelationships in mice. Mice were exposed to 3 g hypergravity environment using centrifuge.
Hindlimb unloading was achieved by the tail suspension. Mice were kept in hypergravity and
HU for 4 and 3 weeks, respectively. In a comprehensive DNA microarray analyses, Dkk2 was
identified as a soluble factor whose expression was most increased and reduced by hypergravity and HU in the soleus muscle of mice, respectively. Hypergravity significantly reduced
levels of Dkk2 mRNA in the soleus muscle and serum Dkk2 in mice, although HU increased
the levels of Dkk2 in serum and the soleus muscle of mice. Moreover, shear stress reduced
Dkk2 expression in mouse C2C12 myotubes. Simple regression analysis showed that serum
Dkk2 levels and Dkk2 expression in the soleus muscle were negatively related to trabecularbone mineral density in mice. As for the effects of Dkk2 on bone cells, Dkk2 decreased theexpressions of osteogenic genes, alkaline phosphatase activity, and mineralization in mouseprimary osteoblasts, although it enhanced RANKL expression. In conclusion, we showed that
Dkk2 contributes to the interaction between muscle and bone in response to gravity change
and mechanical unloading in mice. An increase in Dkk2 expression in the skeletal muscle may(COI:No)be involved in microgravity- and disuse-induced bone and muscle loss.

S15-04

Mechanical load-regulated expression of periosteal Osteocrin promotes
CNP-dependent bone formation
Haruko Takano 1，Hiroki Ochi2，Shigetomo Fukuhara3，Yasuhiro Sawada4，Shingo Sato2，
1 1 Dept Cell Biol., Natl. Cerebral and Cardiovasc. Ctr.
Akihiro Yasoda5，Naoki Mochizuki（
Res. Inst., 2 Dept. of Physiol. and Cell Biol., Grad. Sch of Med. and Dent. Sci. Tokyo Med.
and Dent. Univ., 3 Dept. of Mol. Pathophysiol., Inst. of Adv. Med. Sci., Nippon Med. Sch.,

4 Dept. of Rehab. for Motor Func., Natl. Reha. Ctr. for Persons Disabil., 5 Clin. Res. Ctr.,

Natl. Hosp. Org. Kyoto Med. Ctr.）

Genetic regulation followed by mechanical stimuli including loading after birth affects long bone
growth. However, little is known about how mechanical factors modulate bone growth. Here,
we show that a periosteal osteoblast-derived secretory peptide, Osteocrin (OSTN), is inducedby mechanical loading and promotes long bone growth. OSTN expression in tibial periosteum
was suppressed by mechanical unloading and restored by subsequent re-loading. The micelacking OSTN showed less bone mass in trabecular and cortical regions of long bones than thecontrol mice, suggesting the contribution of OSTN to bone development. We found that OSTN
regulated growth plate and trabecular bone formation by inducing proliferation and maturation of chondrocytes through enhancing CNP-dependent signaling. Besides chondral growth,
OSTN together with CNP induced osteoblast differentiation of periosteum-derived multipotent
progenitor cells leading to bone hypertrophy. OSTN-dependent bone growth was ascribed toenhancement of CNP signaling by inhibiting of CNP clearance of NP receptor 3 (NPR3) by
OSTN, because OSTN bound to NPR3. Collectively, we demonstrate that OSTN functions as amechano-transducer that links mechanical loading to CNP-dependent bone growth. (COI:No)

S15-02

Ameliorating effect of noisy galvanic vestibular stimulation on bodybalance
Chisato Fujimoto, Tatsuya Yamasoba, Shinichi Iwasaki（Dept Otolaryngol Head Neck Surg,

Grad Sch Med, Univ Tokyo, Japan）

Noisy galvanic vestibular stimulation (nGVS) is a procedure that applies electrical current aszero-mean current noise to the vestibular system through electrodes placed over the bilateral
mastoid process. An optimal level of nGVS facilitates the processing of subthreshold stimuli
in neural systems, such as the autonomic, motor or postural control systems. With regard to
the postural control system, stability in standing posture and gait performance were improved
during the application of imperceptible optimal level of nGVS in healthy subjects and inpatients with bilateral vestibulopathy (BV). On the other hand, a further increase of nGVS
intensity degrades stability of standing posture. The proposed mechanism behind these effects
is stochastic resonance, in which the existence of an optimal amount of noise can enhancesubthreshold signals in a non-linear system. We recently found that a 30-min application ofnGVS at optimal intensity led to a post-stimulation ameliorating effect of the postural stability
that lasts for several hours in healthy elderly adults and BV patients, even after the cessationof the stimulus. nGVS is a promising candidate for a novel treatment of refractory postural
instability due to vestibulopathy. Further clinical studies are needed to increase the evidence(COI:No)level of the therapeutic effects of nGVS.

S54

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Symposium 16
State-of-the-art physiology in urinary
continence（March 19, 9:00〜10:50, Hall 9）

S16-03

Corticotrophin-Releasing Hormone neurons of Barringtonʼs nucleus:
Probabilistic, spinally-gated control of bladder pressure and micturition
Hiroki Ito（Dept. Urology, Yokosuka Kyosai Hospital, Kanagawa, Japan）
Micturition requires the co-ordinated control of bladder and urethral sphincter via lumbosacral
parasympathetic, sympathetic and somatic motoneurons. In adult mammals, this involves aspino-bulbar-spinal loop and a critical site in this chain of command is Barrington’s nucleus in
the pons (Barr). The afferent signal arising from the bladder is conveyed to the spinal cord via
A delta and C fibre of the pelvic nerve. The spinal cord relays the signal to the periaqueductal
gray (PAG) in the midbrain where it is integrated. Upon the initiation of voiding, this signal
is summoned to Barr which transmits motor commands to the lumbosacral parasympathetic
neurons controlling the bladder and urethral sphincter motoneurons. Lesion of Barr or acutetransection of the pons abolishes micturition, while supra-collicular decerebration or transection of PAG does not stop reflex voiding in cats and rats. Thus, Barr is believed to be thepre-parasympathetic control center of voiding reflex. The largest cell group within this nucleus
are pontospinal glutamatergic neurons that express corticotrophin-releasing hormone (BarrCRH). While there is agreement that they can generate bladder contractions, it is unclear
whether they act as high-fidelity pre-parasympathetic motor drive enabling to determine thedegree of bladder contractions. Combined opto- and chemo-genetic manipulations along with invivo high-density multiunit recordings in mice shows that BarrCRH neurons provide a timing
signal that probabilistically generates non-voiding contractions during the storage phase or a
voiding (including urethral sphincter bursting) depending on the phase of the voiding cycle.
Additionally, they release CRH at a spinal level that acts as a negative feedback brake on the
excitatory effects of BarrCRH neurons. These findings define the roles of the BarrCRH neurons
in voiding and emphasise the importance of the state of priming of a downstream spinal gating
circuit in determining the response of the lower urinary tract.
(COI:No)

S16-01

S16-04

2 1University of Bristol, UK,
Christopher Fry1，Basu Chakrabarty1，Anthony Kanai（

Hikaru Hashitani, Retsu Mitsui（Dept Cell Physiol, Grad Sch Med Sci, Nagoya City Univ,

Cyclic nucleotides such as cGMP and cAMP are recognised as smooth muscle relaxants
and regulators of intracellular [Ca2+] regulation. However, it is increasingly understood thatthey also determine, directly or indirectly, several other functional bladder properties that,when these are deranged, are associated with conditions such as detrusor overactivity. For
example, cGMP and cAMP regulate several purinergic pathways, such as ATP release frompostganglionic parasympathetic nerves to detrusor smooth muscle or from the urothelium; aswell as afferent nerve firing. Thus, they can influence the ability of the bladder to void andalso to respond to bladder filling. Cellular cyclic nucleotide levels can be manipulated either
by altering the extent of production, or the rate of degradation and both offer attractive
routes for drug development. This talk will consider how cyclic nucleotides regulate functional
properties of bladder wall tissues and how their metabolism can be manipulated as potential
drug targets. Cyclic nucleotides play a fundamental role in other tissue processes such as
generation of fibrosis, a fundamental problem in the bladder that decreases its contractile
ability and increases its stiffness. Strategies to reverse this pathological condition will also be
considered in the context of cyclic nucleotide regulation.
(COI:No)

During the storage phase, the urinary bladder does not remain relaxed, rather it maintains
a basal tone by developing micromotions resulting from ’asynchronous’ spontaneous phasic
contractions (SPCs). Importantly, spontaneous ’transient’ rises in the intravesical pressurearising from SPCs appear to have a much larger influence than the baseline pressure rises onafferent nerve firing. Thus, increased SPCs would cause urinary urgency reported in patientswith bladder storage dysfunction known as an overactive bladder (OAB). Besides detrusor
smooth muscle (DSM), the major contractile element in the bladder wall, the contractility of
bladder mucosa predominantly arising from muscularis mucosa (MM) has become evident.
The MM expressing α-smooth muscle actin immunoreactivity forms a discontinuous layer
in the lamina propria that is clearly distinct from DSM. The MM generates bursting spontaneous action potentials, and is capable of developing approximately 8 times greater SPCs
per section area compared to DSM. However, nerve-mediated contractions of the MM arerather modest. Considering such contractile properties of the MM as well as its much smaller
volume compared with DSM, it is unlikely that the contractility of MM contributes to bladdervoiding contractions. The MM may function to prevent excessive stretching of the bloodvessels and urothelium during bladder filling. Nevertheless, it is envisaged that the MM havea larger mechanical impact on afferent nerves compared to the DSM because of its anatomical
proximity. Thus, aberrant SPCs in the MM could be a primary cause of the hyperactivity ofmechanosensitive afferent nerves. Unlike the DSM, the MM receives inhibitory innervation,
namely nitrergic as well as CGRP-containing afferent nerves that may stabilise the MM excitability. On the contrary, the sensitivity of the MM to angiotensin II is more than 100 times
higher compared with the DSM. Neurohumoral modulation of the MM contractility could be a
(COI:No)novel therapeutic target of OAB.

Cyclic nucleotide-dependent pathways as targets for managing bladderdysfunction
2 University of Pittsburgh, USA）

Mechanical stimulation of afferent nerves as a cause of bladder storage
dysfunction
Japan）

S16-02

Circadian micturition rhythm coordinated by the bladder clock
2 1 Dept Urology, Univ Tsukuba, Japan, 2 Dept Urology, Grad
Hiromitsu Negoro1, 2，Jin Kono（

Sch Med, Kyoto Univ, Japan）

Nocturia (micturition at night) is one of the most bothersome lower urinary tract symptoms.
The cause of nocturia is multifactorial, but it can be divided into three factors, including
sleep disorder, increased urine production rate in the kidney and decreased functional bladdercapacity (FBC) at night. In healthy individuals, the urinary bladder increases its functional
volume at night compared with that at daytime. The mechanism of day-night changes of FBC
was unknown, but it has been elucidated gradually after the discovery of the circadian clock
system. The center of the circadian clock exists in the suprachiasmatic nucleus of the brain,
which orchestrates the circadian clocks in most organs and cells, termed peripheral clock. We
reported that the peripheral clock also exists in the bladder, and that connexin43 (Cx43), notonly a major gap junction protein in the bladder detrusor muscle but also a hemichannel for
ATP release in the urothelium, is one of the clock-controlled genes to act as a regulator of
FBC. Urothelial cells sense changes in stretch and transmit mechanotransduction signals to theafferent nerve by releasing various neurotransmitters such as ATP. ATP activates purinergic
receptors expressed by afferent nerves in the suburothelium, whereas ATP activates various
purinergic receptors on the urothelium in an autocrine/paracrine manner to form a positive
feedback loop of ATP secretion. The Cx43 function varied according to the circadian rhythmin the urothelium as well as in the bladder detrusor muscles, which can be involved in thecoordination of circadian micturition rhythm. In the urothelium, circadian expressions ofmechano-sensor molecules have been also reported, and ATP concentration in patients withoveractive bladder is known to be increased. These findings can shed a light on the physiologyof day-night change in micturition. Modulating the circadian clock can be a novel approach totreat nocturia. (COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S55

Symposium 17
Current advance in endocrine disruptor
research（March 19, 9:00〜10:50, Hall 10）

S17-03

Estrogen receptor activation or inhibition induced by next-generation
bisphenols
Ayami Matsushima（ Dept Chem, Fac Sci, Kyushu Univ, Japan）
Bisphenol A (BPA) is a raw industrial material for polycarbonate plastics and epoxy resins;
however, a series of studies showed that BPA induces adverse effects on experimental animals
as endocrine-disrupting chemicals (EDCs) even at low doses. The abnormal aspect of BPA isreported not only on reproductive organs but in their nervous systems, although the molecularmechanisms of these low-dose effects remain unknown. EDCs including BPA are presumed
to directly bind nuclear receptors such as estrogen receptors (ERs), and elicit their harmful
effects. Forty-eight nuclear receptors are reported in humans, with all of these representing potential targets of EDCs. These findings result in avoiding the usage of BPA in daily
products including baby bottles. As a result, emerging number of bisphenol derivatives, (i.e.,
next-generation bisphenols) are utilized as substitutes for BPA, although few methodical risk
assessments on next-generation bisphenols have been carried out. BPA and most of newgeneration bisphenols have two phenol groups in their chemical structures. A biphenyl moiety
is a common privileged structure in the field of drug discovery, therefore we considered thata bisphenol moiety is a privileged structure for nuclear receptors. Indeed, we found that BPA
bound strongly to estrogen-related receptor γ. To find new ligands for ERα, we therefore
screened a library of ca. 200 bisphenol derivatives and found that 20 compounds bind to ERα
with higher affinity than BPA. Most of these activate ERα as its agonists; however, four
compounds, including bisphenol M and bisphenol P act as novel antagonists. These structures
harbor three benzene rings in tandem with terminal hydroxy groups, that is, tricyclic bisphenol
structure, representing a novel privileged structure for an ERα antagonist. The tricyclic
bisphenol structure has a potential to be new mother structure for a new breast cancer drugs.
(COI:No)

S17-01

S17-04

Noriyuki Koibuchi1，Miski Khairinisa A.1，Winda Ariyani1，Wataru Miyazaki3，Asahi Haijima4，
2 1 Dept Integrative Physiol, Gunma Univ. Grad Sch Med,
Izuki Amano1，Yoshito Toshima（

Kei Nomiyama1，Hazuki Miazukawa2，Akifumi Eguchi4，Shouta Nalayama3，
1 1CMES, Ehime
Yoshinori Ikenaka3，Mayumi Ishizuka3，Rumi Tanoue1，Tatsuya Kunisue（

The effect of perinatal exposure to Gadolinium-based contrast agentson cognitive function of the offspring
Japan, 2 Dept Diagnostic Radiol ＆ Nucl Med, Gunma Univ. Grad Sch Med, Japan, 3 Dept
Biosci Lab Med, Hirosaki Univ Grad Sch Health Sci, Japan, 4 Dept Health Sci ＆ Social
Welfare, Sch Human Sci, Waseda Univ, Japan）

Gadolinium-based contrast agents (GBCA) are commonly used to enhance the image during
magnetic resonance imaging (MRI). Although its exact adverse effect in the developing central
nervous system has not yet been reported even after repetitive injection during pregnancy, theaccumulation of GBCA in various brain regions such as the dentate gyrus has recently beenreported. To examine the effect of GBCA exposure during perinatal period, several different
types of GBCA was intravenously administered to pregnant or lactating mice. Measurementof gadolinium of the whole brain showed an accumulation of gadolinium in the offspring whose
mother received gadolinium either prenatally or postnatally, indicating gadolinium can crossthe placenta and excreted in the milk. Behavioral analysis revealed that these animals showed
an increased anxiety, cognitive impairment and motor coordination defect. In the primary culture of rodent cerebellum, GBCA disrupted dendrite arbolization of Purkinje cell. In transienttransfection-based reporter gene assay, GBCA augmented thyroid hormone receptor-mediatedtranscription with lower dose and suppressed it with higher dose. Throughout the experiment, GBCAs containing a linear chelate structure showed relatively greater effect than thosecontaining a macrocyclic chelate structure. These results indicate that GBCA, particularly
linear GBCA, may cause an adverse effect in the developing brain. It may be administeredduring pregnancy or lactating period only when the benefit significantly outweighs the risk of(COI:No)
exposure.New developments of thyroid hormones analysis for the ﬁeld ofenvironmental chemistry and environmental toxicology
Univ, Japan, 2 Grad Sch Agr, Ehime Univ, Japan, 3 Sch Vet Med, Hokkaido Univ, Japan,
4 Cent Prev Med Sci, Chiba Univ, Japan）

Thyroid hormones (THs) play critical roles in neural development, protein synthesis, and thenormal growth of bones in addition to regulating energy and lipid metabolism.
Recent studies have reported that exposure to endocrine disrupting chemicals such as polychlorinated biphenyls (PCBs) alter TH levels in the blood. THs are involved in the regulation ofearly brain development. Since the fetal thyroid gland is not fully functional until week 18–20
of pregnancy, neuronal migration and other crucial early stages of intrauterine brain development largely depend on the supply of maternal TH. Therefore, it is important to accurately
measure the maternal THs concentrations.
In addition, recent updates to OECD developmental/reproductive toxicology guidelines andother regulatory guidelines and guidance require the measurement of TH levels in the bloodof mammalian laboratory species during development. However, preliminary analyses indicatethat there is a wide variability across laboratories in the methods being used to measure
THs in young rodents, as well as in the success of obtaining reliable data. Until now, themeasurement of THs has mainly been immunoassay. However validity, accuracy, sensitivity
and reproducibility of the assays are issues of concern.
This presentation will introduce a high-sensitivity and high-accuracy THs analysis method
using LC-MS/MS that can be measured regardless of animal species, and discuss the development of THs analysis in the fields of environmental chemistry and environmental toxicology.
(COI:No)

S17-02

Direct Actions of Xenoestrogens on Thyroid Hormone Receptors
2 1 Dept Biosci Lab Med, Grad Sch
Wataru Miyazaki1，Ariyani Winda2，Noriyuki Koibuchi（

Health Sci, Hirosaki Univ, Japan, 2 Dept Integrative Physiol, Grad Sch Med, Gunma Univ,
Japan）

Isoflavones (genistein, daidzein), bisphenol A (BPA), and 4-nonyl-phenol (4NP) are known as
xenoestrogens which modulate the actions of estrogen receptors (ERs). These compounds also
act on the other nuclear receptors including thyroid hormone (TH) receptors (TRs). THs haveessential roles for the development and functional maintenance of many organs, and the actionsof THs are directly or indirectly modulated by xenoestrogens. These compounds may affectTH system at multiple levels such as synthesis, secretion, transport and degradation of THs,
expression of TRs and TR-mediated transcription. Isoflavones have been considered as healthy
supplements that reduce risk of the specific diseases, for example, coronary heart disease, cancers, and hot flashes. However, in the clinical field, the effects of isoflavones are controversial.
Under certain conditions, isoflavones may worsen the symptoms of subclinical hypothyroidism,
whereas they may improve thyroid functions in Hashimoto disease patients. Recently, we havereported that isoflavones can bind to TRs directly, and augment TR-mediated transcription.
On the other hand, synthetic xenoestrogens such as BPA and 4NP have been considered
as toxic endocrine-disrupting chemicals, even though the similar augmentation was also
induced by 4NP and BPA in recent studies. In our presentation, we summarize the effects of
xenoestrogens in thyroid hormone action, and discuss why such clarification was established.
Although we need further investigation of the relationships of ERs-mediated actions with TRs,
the direct modulation of xenoestrogens is one of the clues to prevent the adverse effects of(COI:No)
these compounds.

S56

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Symposium 18
History and Up-to-date stories on themechanism of smooth muscle contraction in
health and disease（March 19, 14:10〜16:00, Hall 4）

S18-01Novel signaling molecules which regulate both Ca2+-sensitization ofvascular contraction and cancer cell migration
Sei Kobayashi, Bochao Lyu, Min Zhang, Ying Zhang, Hiroko Kishi, Tomoka Morita（Dept. of Molecular and Cellular Physiol., Yamaguchi Univ. Grad. Sch. of Med.）Previously we found for the first time that in the complete absence of cytosolic Ca2+, Rho-kinase (ROK) can induce contraction of membrane-permeabilized vascular smooth muscle(VSM) and myosin phosphorylation. The Rho-kinase (ROK)-mediated Ca2+-sensitization of VSM
contraction contributes to abnormal vascular contractions, such as vasospasm, which is oneof the major causes of the sudden death. As the causative molecule for the ROK-mediatedCa2+-sensitization of VSM contraction, we discovered sphingosylphosphorylcholine (SPC)
and further identified the “SPC/Fyn/Rho-kinase” pathway as the pathogenic pathway. The
translocation of Fyn and Rho-kinase from the cytosol to the cell membrane activates thesekinases and thereby induces the Ca2+-sensitization of VSM contraction.
We also found that eicosapentaenoic acid (EPA), a component derived from fish oil, can inhibit
the translocation and activation of Fyn induced by SPC and specifically suppress abnormal
vasocontraction without affecting physiological Ca2+-dependent contraction of VSM. Indeed,
EPA abolished cerebral vasospasm after subarachnoid hemorrhage of the patients. However,
because EPA is lipid-soluble, it cannot be either administrated intravenously or used for
emergency treatment. Therefore, we tried to identify water-soluble food components as analternative to EPA. After extensive screening, we found that food extracts/components andChinese traditional medicine (CTM) selectively inhibit the SPC-induced Ca2+-sensitization of
VSM contraction without affecting the physiological Ca2+-dependent contraction of VSM.
Recently we found that the “Fyn/Rho-kinase” pathway also mediates cell migration, which is
critical for cancer invasion and metastasis. We found that food components and CTM also can
suppress cancer cell migration remarkably.
In summary, our results suggest that food extracts/components and CTM would be novel
protective and therapeutic agents for the most lethal diseases, vasospasm and cancer.
(COI:No)

S18-03

Role of CPI-17, an endogenous myosin phosphatase inhibitory protein
in gastrointestinal motility
Qunhui Yang（Dept Vet.Pharmacology, Grad Sch Agr. and Life Sci, the Univ of Tokyo,
Japan）We reported that CPI-17, an endogenous myosin phosphatase inhibitory protein, played anessential role to maintain blood pressure using wild type mice (WT), CPI-17 deficient mice (KO)and phospho-resistant mutant of CPI-17 at threonine 38 (Thr38) to alanine knocked-in mice
(TA). Aim of this study is to clarify role of CPI-17 in gastrointestinal motility. High concentration of KCl (64.5 mM)-induced contractions were not different among WT, KO and TA of ileal
and colonic circular muscles strips. In contrast, carbachol (5 µM)-induced contractions weredecreased in KO and TA than WT. Gastric emptying rate measured by the 13C-octanoic acidbreath test and intestinal transit measured by oral administrated FITC-dextran solution movement did not change between WT, KO and TA. While colonic transit measured by colonicbead expulsion test revealed that colonic transit in KO and TA was significantly slower thanthat in WT. The colonic transit in TA was same with in KO. CPI-17 protein expression wasmuch higher in large intestine than small intestine. In a conclusion, The CPI-17 phosphorylation
signaling at Thr38 is important for maintenance of colonic transport ability than in uppergastrointestinal tract.
(COI:No)

S18-04

Role of Vascular Smooth Muscle PPARγ, a Transcriptional Factor
Masashi Mukohda（ Lab Vet Pharm, Faculty Vet Med, Okayama Univ of Science）
Synthetic agonists of peroxisome proliferator-activated receptor γ (PPARγ), thiazolidinediones(TZDs) are currently prescribed to patients with type 2 diabetes to improve insulin resistance.
TZDs have been shown to protect against vascular diseases such as atherosclerosis and lower
blood pressure. In contrast, loss of PPARγ function in patients carrying PPARγ mutations
causes insulin resistance and hypertension. These evidences suggest PPARγ has protective
effect on cardiovascular diseases. Sigmund laboratory (University of Iowa) generated transgenic mice (S-DN) expressing dominant negative (DN) mutant form of PPARγ (P467L) specifically in vascular smooth muscle cell (SMC) (Cell Metab. 2008). S-DN mice are hypertensive,
exhibit severe vascular dysfunction, and display reduced expression of a novel PPARγ target
gene, RhoBTB1. In addition, S-DN mice exhibited exacerbated atherosclerosis associated withelevated NF-κB-mediated inflammatory markers when bred with ApoE-deficient mice (Am J
Physiol Regulatory. 2013). In this study, we examined the mechanisms by which vascular SMC
PPARγ protects cardiovascular diseases. First, we hypothesized that PPARγ has protective
effect on vascular disease through inhibiting inflammation in SMC. To test this, S-DN werebred with mice expressing luciferase controlled by an NF-κB–responsive promoter. NF-κB
activity induced by tumor necrosis factor α (TNF-α) was increased in vessels from S-DN
mice. Next, we hypothesized that RhoBTB1 plays a protective role in vascular function that is
disrupted in S-DN mice. To test this, we generated triple transgenic mice (S-RhoBTB1/S-DN)
expressing DN PPARγ in SMC along with tamoxifen-inducible, Cre-dependent expression of
RhoBTB1 in SMC. S-DN exhibited severely impaired vasodilation to acetylcholine and sodium
nitroprusside and hypertension, which was reversed by restoration of RhoBTB1 in SMC. We
conclude that 1) SMC-PPARγ has anti-inflammatory effects mediated through inhibition of
NF-κB activity, and 2) a novel PPARγ target gene, RhoBTB1, functions in SMC to facilitate
vasodilation and mediates a protective anti-hypertensive effect.
(COI:No)

S18-02

S18-05

Ko Momotani, Kumiko Sakai（Facl of Pharm Sci）

Rin Kurahara1，Keizo Hiraishi1, 2，Ying Zhang3，Aya Yamamura4，Hiroko Kishi3，
1 1 Dept Cardiovasc Physiol, Sch Med, Kagawa
Sei Kobayashi3，Ryuji Inoue2，Katsuya Hirano（

Too many links indeed linking the missing link in the Ca2+-sensitization
pathway regulating vascular smooth muscle contractions
It is well known that the Ca2+-sensitization pathway is a significant contributor to vascularsmooth muscle (VSM) contractions. The pathway is initiated by multiple agonists throughG protein-coupled receptors (GPCRs), which further activate RhoA and Rho-kinase. Active
Rho-kinase then inhibits Myosin light chain (MLC) phosphatase, resulting in an increase in MLC
phosphorylation and thereby inducing VSM contractions. The player(s) between the GPCRsand RhoA in this signaling cascade had been unidentified for a long time. Here, we presenta brief history of how this missing step has been elucidated and the resulting impact on theunderstanding of vascular physiology. Multiple RhoA GTP exchange factors (GEFs), some are
to do with pathological outcomes, in between GPCRs and RhoA have been reported in recent
years. A good example is leukemia-associated RhoGEF (LARG) in salt-dependent hypertension reported by Offermanns group. The other is p63RhoGEF. Its role in VSM contractions
was first reported by our group. Our unpublished data further point to direct activation of
p63RhoGEF in response to an increase in the internal pressure of small vessels mimicking
physiological cues of vascular tone, a major regulator of blood pressure. The involvement ofanother GEF, PDZ-RhoGEF in VSM contractions, has also been suggested. Thus, the implication of multiple GEFs has been reported, which indeed leads to another layer of complexity
in regulatory mechanisms of VSM contractions. Why different GEFs are necessary for signaltransduction with a single outcome, VSM contraction in the end. One of the current viewssuggests that each GEF is responsible for a specific physiological and possibly pathological cue.
In other words, multiple GEFs are backing diversity in physiological responses in vasculatures.
If this hypothesis stands, it opens the doors to develop specific drags each for a specificpathophysiological condition by targeting a particular GEF while circumventing unwantedside-effects.
(COI:No)

The therapeutic effects of EPA on pulmonary hypertension partly
manifest via inhibition of tyrosine kinase FYN
Univ, Japan, 2 Dept Physiol, Sch Med, Fukuoka Univ, Japan, 3 Dept Mol Cell Physiol, Grad
Sch Med, Yamaguchi Univ, Japan, 4 Dept Physiol, Aichi Med Univ, Japan）

Background and Purpose: Pulmonary arterial hypertension (PAH) is a multifactorial diseasecharacterized by pulmonary arterial remodeling. The Src family non-receptor tyrosine kinases
including Fyn play critical roles in vascular function. Eicosapentaenoic acid (EPA) is known to
inhibit Fyn kinase activity. In this study, we investigated the therapeutic potential of EPA andits metabolite resolvin E1 (RvE1) for PAH.
Method: Cardiodynamic parameters of the monocrotaline-induced PAH rats (MCT rats) wereassessed with echocardiography. Contractile responses of isolated pulmonary arteries were
evaluated by the isometric tension measurement. Proliferation of human pulmonary arterysmooth muscle cells (hPASMCs) derived from PAH patients were evaluated by the MTT
assay. Endothelial to mesenchymal transition (EndoMT) in HPAECs and STAT3 phosphorylation in hPASMCs were examined by immunofluorescence staining and western blot analyses,
respectively.
Results: Administration of EPA to MCT rats significantly improved the pulmonary arterial
thickening, right ventricle dysfunction and cardiovascular fibrosis. Pulmonary arteries isolated
from MCT rats exhibited enhanced contractile responses to serotonin compared with thoseseen with control rats. EPA administration attenuated the enhanced contractile responses in
MCT rats. EPA or RvE1 treatment decelerated the enhanced proliferation of PASMCs derived
from the patients with PAH. Knockdown of Fyn with siRNA prevented TGF-β2-induced
EndoMT in HPAECs and IL-6-induced STAT3 phosphorylation in HPASMCs. EPA and RvE1
suppressed Src family activity by modulating phosphorylation status.
Summary: EPA significantly improved PAH-associated pathophysiology and cardiac dysfunction. These therapeutic effects are likely mediated at least in part via Fyn inhibition. Fyn issuggested to be a potential target for the treatment of PAH.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S57

Educational Programs
Lunchtime Sessions
Luncheon Seminars

EP1-1

Educational Program 1

Educational Program 3

Educational Lecture 1

Model Lecture on Physiology（March 18, 9:00〜10:50, Hall 3）（March 19, 9:00〜10:50, Hall 3）

Diversity of Ca 2 ＋ entry mechanisms associatedwith vascular functions

EP3-1

Noriatsu Shigemura

Ryuji Inoue
Department of Physiology, Fukuoka University School of
Medicine

EP1-2

Regulation of body ﬂuid and electrolytes:
Physiological signiﬁcance of interstitial ﬂuid

Section of oral neuroscience, graduate school of dental
sciences, Kyushu University

EP3-2

EP1-3

Team-Based Learning（ TBL ）for Education of
Physiology
Mariko Miyata

Yoshinori Marunaka
Research Institute for Clinical Physiology, Kyoto Industrial
Health Association / Research Center for Drug Discovery andPharmaceutical Development Science, Ritsumeikan University/ International Research Center for Food Nutrition and Safety,
Jiangsu University

The sense of taste

Tokyo Womenʼs Medical Univ., Dept. of Neurophysiology

EP3-3Homeostatic Control of blood glucose
Michiko Tanaka
Miyazaki Prefectural Nursing University

Circadian clocks based on basic to appliedresearch
Shigenobu Shibata
Advanced Science and Engineering, Waseda University

EP2-1

Educational Program 2

Educational Program 4

Educational Lecture 2

Educational Workshop（March 18, 15:20〜17:10, Hall 3）（March 19, 14:10〜16:00, Hall 10）

Fun to study body temperature regulation: fromthe observation of the whole-body response
Kei Nagashima
Waseda University

EP2-2

Why humans get obese?
- An introduction to nutrient metabolism
Tomohiro Tanaka
Nagoya City University

EP2-3

Physiology of Pain
Jun Sato
Chubu University

S60

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Lunchtime Session 1

Luncheon Seminar 1

History and future directions of physiology inassociation with biology and medicine[ Supported by Medtronic Co., Ltd. ]（March 17, 12:10〜13:10, Hall 6）

LS1-1

Beautiful Harmony in Physiology: From Molecule
to Body and Behavior
Yoichi Ueta

Acquiring knowledge of the heart failurefrom physiological points of view
（March 17, 12:10〜13:10, Hall 2）

LCS1-1

Ehime University Graduate School of Medicine, Organand Morphology Department of Cardiology, Pulmonology,
Hypertension and Nephrology

Department of Physiology, School of Medicine, University ofOccupational and Environmental Health

LS1-2

Osamu Yamaguchi

Research on the physiological functions of basal
forebrain cholinergic neurons
Sae Uchida
Department of Autonomic Neuroscience,
Tokyo Metropolitan Institute of Gerontology

Lunchtime Session 2

Luncheon Seminar 2

Now is the time to make use of your power！
Importance of diversity in research life

[ Supported by Nippon Boehringer Ingelheim Co., Ltd. ]（March 18, 12:10〜13:10, Hall 10）

LS2-1（March 17, 12:10〜13:10, Hall 3）Letʼs us understand unconscious bias
Yukari Date
Vice President, Director, University of Miyazaki

LS2-2Management of Atrial Fibrillation Based on
the Extent of Atrial Fibrosis

LCS2-1

Takanori Yamaguchi
Department of Cardiovascular Medicine, Saga University

Steps for your own successful career
Yumiko Oishi
Department of Biochemistry and Molecular Biology, Nippon
Medical School

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S61

Luncheon Seminar 3

Luncheon Seminar 5[ Supported by Mandom corp. ][ Supported by Bayer Yakuhin Co., Ltd. ]Thermosensitive TRP channels and their
physiological functions

Acquiring knowledge of the heart failurefrom physiological points of view

（March 17, 12:10〜13:10, Hall 4）（March 18, 12:10〜13:10, Hall 2）

LCS3-1

Makoto Tominaga

LCS5-1

Thermal Biology Group, Exploratory Research Center on Lifeand Living Systems, National Institutes of Natural Sciences
Division of Cell Signaling, National Institute for Physiological
Sciences, National Institutes of Natural Sciences
Department of Physiological Sciences, SOKENDAI,（ The Graduate University for Advanced Studies ）LCS3-2

Naohiko Takahashi
Department of Cardiology and Clinical Examination, Faculty of
Medicine, Oita University

Kaori Otsuka-Saito
Fundamental Research Institute, Mandom Corp.
Osaka University, Graduate school of Pharmaceutical
Sciences

Luncheon Seminar 4

Luncheon Seminar 6[ Supported by The Japanese Plasmalogen Society ][ Supported by Century Medical, Inc. ]Plasmalogen: A new frontier of the researchin brain and cardiac diseases

Anatomically Left atrial appendage
management（March 17, 12:10〜13:10, Hall 5）（March 18, 12:10〜13:10, Hall 3）

LCS4-1

Toru Maruyamai
Department of Hematology, Oncology and Cardiovascular
Medicine, Kyushu University Hospital

S62

LCS6-1

Hidenori Sako
Oita Oka Hospital Cardiovascular surgery

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Luncheon Seminar 7

Luncheon Seminar 9[ Supported by TSUMURA & CO. ][ Supported by DAIICHI SANKYO Co., Ltd. ]The physiological role of Oxytocin onsocial behavior

Brugada syndrome and Early repolarization
syndrome（March 18, 12:10〜13:10, Hall 4）（March 19, 12:10〜13:10, Hall 2）

LCS7-1

Takashi Maruyama

LCS9-1

Naohiko Takahashi

Department of Physiology, University of Occupational andEnvironmental Health

Department of Cardiology and Clinical Examination, Faculty of
Medicine, Oita University

Luncheon Seminar 8

Luncheon Seminar 10[ Supported by Nepa Gene Co., Ltd. ][ Supported by Kracie Pharmaceutical Co., Ltd. ]Principles and applications ofelectroporation(NEPA21) in genome
editing gene analysis

Ninjin’yoeito stimulates appetite center
NPY neurons and counteracts anorexia andweight loss: anti-frailty strategy（March 18, 12:10〜13:10, Hall 5）（March 19, 12:10〜13:10, Hall 3）

LCS8-1

Kazunori Hirakawa
Regional General Manager, Kyushu, NEPA GENE CO., LTD.

LCS10-1 Toshihiko Yada
Kansai Electric Power Medical Research Instituter, Center for
Integrative Physiology /Kobe University Graduate School of Medicine, Division of
Diabetes, Metabolism and Endocrinology

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S63

Award Presentations（ March 18 , 14 : 20 〜 15 : 20 ）AP-1〜AP-2

21 st Promotion Award of the Physiological Society ofJapan for Young Scientists

AP-3〜AP-6

10 th Hiroshi and Aya Irisawa Memorial Promotion
Award for Young Physiologists

AP-7

10 th Aya Irisawa Memorial Promotion Award for
Excellence by Women Physiologists

AP-8

10 th Hiroshi and Aya Irisawa Memorial Award for
Excellent Papers in The Journal of Physiological
Sciences

AP-9

10 th Hiroshi and Aya Irisawa Memorial Award for
Excellent Papers on Research in Circulation in
The Journal of Physiological SciencesAP-1 (S09-03)

AP-4 (PS22-02)

2 1 Faculty of Med, Univ Tsukuba, Tsukuba, Japan,
Jun Kunimatsu1, 2，Okihide Hikosaka（

Yosuke Okamoto1，Yoshinobu Nagasawa2，Aung Naing Ye3，Yukiko Himeno4，
1 1 Dept Cell
Akinori Noma4，Akira Amano4，Daichi Takagi1，Kuniaki Ishii5，Kyoichi Ono（

The local network in the striatum tail contributes to the behavioral
switching
2 National Eye Institute, NIH, MD, U.S.A.）

Molecular characterization of the arrhythmogenic trigger unique topulmonary vein cardiomyocytes

Although, in our daily life, the object values may change in different environments and we canswitch our behavior accordingly, underlying neuronal mechanism is unclear. To address it, we
devised a new value procedure: scene-based value task. The monkey viewed 8 fractal objects
in 2 scenes (A and B); 4 of them were good (with large-reward) in scene A and bad (withsmall-reward) in scene B, while the other 4 were good in scene B and bad in scene A. After
experiencing this procedure repeatedly, the monkey became able to choose whichever objects
were good. Since scenes A and B were presented in a random sequence, the monkey’s choice
was switched abruptly depending on the scene-context.
We then recorded neuronal activity in striatum tail while the monkey passively viewed theseobjects in different scenes. We found differences between medium spiny neurons (MSNs) andfast spiking interneurons (FSIs). Many of MSNs responded to the fractal objects differently
depending on their values. Importantly, this object-value coding was stronger in either scene
A or B. In contrast, FSIs showed no object-value coding. Instead, many of them responded tothe scenes selectively (stronger to scene A or B). These results suggested that the object-value
coding of MSNs, which is basically stable, is modulated by the inhibitory inputs from thescene-selective FSIs.
To test the causal role of FSI, we locally injected IEM-1460, an inhibitor of GluA2-lacking
AMPARs, in the recording sites to selectively block the excitation of FSIs but not MSNs.
After injection, monkeys were unable to learn new scene-object value association. On the other
hand, object-value learning (no scene) was not affected. This result indicated that the local
network of striatum tail regulates the scene-object association learning. These mechanisms
may support the monkey’s flexible switching based on stable long-term experiences of various
environments.
(COI:No)

Physiol, Grad Sch Med, Akita Univ, Japan, 2 Dept Pharmacol Ther, Fac Pharm Sci, Toho
Univ, Japan, 3 Dept Pathol Diagn, Fac Med, Yamagata Univ, Japan, 4 Dept Bioinfomat, Coll
Life Sci, Ritsumeikan Univ, Japan, 5 Dept Pharmacol, Fac Med, Yamagata Univ, Japan）

AP-2 (1P-035)

AP-5 (O03-3)

Synaptic plasticity at cortico-striatal pathway in functional recovery after
cortical damage
Susumu Jitsuki1，Sayaka Kogami1，Aoi Jitsuki-Takahashi2，Tomomi Yamanoue1，
1 1 Dept Physiol, Grad Sch Med, Yokohama City Univ, Japan, 2 Dept
Takuya Takahashi（
Biochem, Sch Med, Tokyo Women’s Med Univ, Japan）

Brain damage such as stroke is a devastating neurological condition that may severely compromise patient quality of life. Restoration of motor impairment after brain damage is considered
to be the result of compensative neural plasticity in intact brain regions, mediated by thereorganization of cortical motor maps. Experience-dependent synaptic AMPA (α-amino-3hydroxy-5-methyl-4-isoxazole-propionic-acid) receptor (AMPAR) delivery underlies behaviors
that require neural plasticity such as learning. We have previously found that rehabilitationdependent synaptic delivery of AMPAR in the peri-injured area facilitate functional recovery
after cortical damage. For execution of appropriate motor function, it requires involvementof motor-related brain regions such as striatum, thalamus or brain stem. However, it remains
unclear that the role of other brain region in recovery after brain damage. Here, using corticalinjury rat model, we found that AMPAR-mediated miniature EPSC (mEPSC) of the layer 5
pyramidal neurons in the periinjured cortex were positively correlate with recovery rate offorelimb reaching motor performance after rehabilitative training. On the other hand, mEPSC
of the medium spiny neurons in the periinjured striatum were negatively correlate withrecovery rate of motor performance. Furthermore, mIPSC of the periinjured cortex showed nocorrelation with motor performance. These results suggest that changes of excitatory input inthe peri-injured region could contribute functional recovery after cortical damage. (COI:No)

AP-3 (PS13-02)

PI(3, 4)P2- and voltage-dependent gating of two-pore Na+ channel 3
2 1 Div Biophys and Neurobiol,
Takushi Shimomura1, 2，Ki-ichi Hirazawa1, 2，Yoshihiro Kubo1, （Pulmonary veins (PVs) are the major origin of atrial fibrillation. We have reported that IP3R2
in rat PV cardiomyocytes cooperates with Na+-Ca2+ exchanger (NCX) on T-tubule in triggering the norepinephrine (NE)-induced automaticity, and identified a unique hyperpolarization
activated Cl－ current, ICl, h, that potentially facilitates the automaticity. The mathematicalmodel which incorporated the interaction between IP3R2 and NCX, and electrophysiologicalcharacteristics of ICl, h successfully reproduced the NE-induced automaticity. Here, we show
two further topics for the properties of rat PV cardiomyocytes in relation to its arrhythmogenicity. The first one is that a Ca2+-stimulable adenylyl cyclase (AC) is involved in theNE-induced automaticity. Microarray, RT-PCR and immunohistochemistry uncovered that oneof Ca2+-stimulable AC was expressed regionally in the supraventricular area including PV. In
particular, enriched expression of the AC was detected along T-tubule of PV myocytes, whileatrial myocytes hardly displayed T-tubules. HEK293 cells exhibited sustained Ca2+ oscillationin response to UTP under isoproterenol pre-application. Gene-knockout of our interest in
the cells impaired the ability to keep the Ca2+ oscillation. The NE-induce automaticity in PV
cardiomyocytes was reversibly arrested by AC inhibitor. The second topic is related to thestructural basis of ICl, h. Mass spectrometry identified HSPA8 as the CLCN2 interacting proteinfrom rat PV. The auxiliary subunit of CLCN2 was subcloned and introduced into PC12 cell.
With co-expression of the HSPA8, CLCN2 current exhibited the unique voltage-dependency
similar to ICl, h. All these findings suggest that unique molecular interaction among NCX,
IP3R2, and the AC along T-tubule potentiates the arrhythmogenicity of rat PV, and that rat
heart possessed HSPA8 as the auxiliary subunit of the hyperpolarization activated Cl－ channel.
(COI:Properly Declared)

Drp1-mediated mitochondrial dynamics in cardiac remodeling
Akiyuki Nishimura1，Kakeru Shimoda2，Tomohiro Tanaka2，Kazuhiro Nishiyama1，
2 1Grad Sch Pharm Sci, Kyushu Univ, Japan, 2 Dept cardiocirculatory
Motohiro Nishida1, （Signal, NIPS, Japan）

Mitochondria are dynamic organelles that interchanges their morphology between fusion(elongation) and fission (fragmentation) form. Proper mitochondrial quality control is indispensable for cardiac homeostasis and defects in mitochondrial dynamics are implicated in
the development of cardiac diseases. Our group has investigated the molecular mechanism
underlying the development of maladaptive cardiac remodeling, especially myocardial
early senescence that is observed in cardiac disease patients and mice models and has beensuggested as a major cause of cardiac dysfunction. We found that defective mitochondrial
dynamics through aberrant interactions between mitochondria and actin cytoskeleton are a
key determinant of cardiac remodeling and fragility. Dynamin-related protein 1 (Drp1), a mitochondrial fission-accelerating protein, was activated in myocardium after myocardial infarction,
which induced mitochondrial fission-associated myocardial early senescence. We also foundthat the actin-binding protein filamin A acted as a guanine nucleotide exchange factor for
Drp1. Hypoxic stress induced the interaction of filamin A with Drp1, leading Drp1 activationand mitochondrial fission-associated myocardial senescence in an actin binding-dependent
manner in cardiomyocytes. We previously reported that polysulfidation of Drp1 at Cys624, a
redox-sensitive cysteine residue, negatively regulates its activity. Drp1-filamin A interaction
was regulated by polysulfidation-depolysulfidation cycle of Drp1 Cys624. Electrophile-mediateddepolysulfidation of Drp1 promoted the interaction with filamin A and induced mitochondrial
hyperfission. In addition, pharmacological perturbation of the Drp1-filamin A interaction bycilnidipine suppressed mitochondrial fission-associated myocardial senescence and improved
chronic heart failure in mice. Our results suggest therapeutic potential targeting pathologydependent Drp1-filamin A interaction for the treatment of chronic heart failure.
(COI:No)

AP-6 (PS17-02)

The role of TRPC3 and TRPC6 in a stretch-induced slow force
response in cardiomyocytes

Natl Inst Physiol Sci, 2 Dept Physiol Sci, SOKENDAI ）1 1 Dept Physiol, Med,
Yohei Yamaguchi1，Gentaro Iribe1，Keiji Naruse2，Akira Takai（

Two-Pore Na+ Channels (TPCs) contain two domains (DI and DII) of a functional unit ofvoltage-dependent cation channels. Each domain has its own voltage sensor domain that possesses three positively charged arginine residues in helix S4. Characteristically, TPC3 shows
the shift of the voltage dependence by long depolarization stimulus, so called as “induction”, in
Xenopus oocyte expression system.
The structural basis of this “induction” mechanism was investigated using multiple approaches
based on two-electrode voltage-clamp technique and the structural model of TPC3. We foundthe correlation between PIP2 level and “induction” currents. Simultaneous recordings of TPC3
current and the fluorescence from specific PIP2 sensors showed that PI(3, 4)P2 concentration isincreased by long depolarization stimulus, possibly through any endogenous system in Xenopus
oocytes. The “induction” kinetics of TPC3 is well correlated with the fluorescent change of PI(3,4)P2 sensors, but not with that of PI(4, 5)P2. The PI(3, 4)P2 sensitivity of TPC3 was confirmed
using direct phosphoinositide injection method and the excised-patch membrane. These results
reveal that “induction” is PI(3, 4)P2-induced modulation of voltage dependence of TPC3. We also
found that a cluster of basic amino acid residues in the cytosolic side of DI is critical for PI(3,4)P2 sensitivity. Interpretation of the mutational effects based on the TPC3 structural model
explained how TPC3 selectively recognizes PI(3, 4)P2 in DI region. While DI recognizes PI(3,4)P2, DII is considered to be mainly responsible for voltage-sensing. The voltage-dependent
movement of DII-S4 was verified using the voltage clamp fluorometry, in which the voltagedependent fluorescent change was detected from the fluorophore incorporated into someresidues in DII-S4. These electrophysiological data of various approaches, combined withthe structural model, revealed the detailed voltage-dependent gating mechanism of TPC3.
(COI:No)

An increase in preload induces a biphasic active force enhancement in the heart. The shortterm increase in preload rapidly augments contractile force due to the Frank-Starling mechanism, which accelerates Ca2+ sensitivity of contraction proteins. A further long-term increasein preload for several minutes to hours causes the increase in [Ca2+] i, leading to a slow force
response to stretch (SFR), and a further increase in the contractile force. The stretch-induced
release of angiotensin II has been implicated in the SFR, to raise intracellular Na+ levels,
followed by an increase in intracellular Ca2+ levels via the Na+/Ca2+ exchanger. However,
the extracellular cation influx pathway is poorly understood. To better understand the cationinflux pathway, we focused on TRPC3 and TRPC6, receptor-operated, mechanosensitive nonselective cation channels. In our studies, cardiomyocytes were enzymatically isolated frommouse hearts, and both cell ends were held by two carbon fibers to apply stretch to the cells.
Using this stretch device, we found that TRPC3 and TRPC6, regulated by the angiotensin II
type 1 (AT1) receptor via diacylglycerol produced by phospholipase C, played a pivotal rolein the SFR. Our recent data also showed that stretch-induced activation of TRPC3 increased
intracellular Ca2+ influx, causing the accumulation of Ca2+ in the sarcoplasmic reticulum,
which in turn increased Ca2+ release and, consequently, the twitch force, suggesting that afunctional sarcoplasmic reticulum (SR) is necessary for the SFR. Other data indicate that amyocardial stretch stimulated the AT1 receptor, followed by endothelin release to increase
nicotinamide adenine dinucleotide phosphate oxidase (NOX)-derived reactive oxygen species,
regulating TRPC3 and TRPC6 to increase Na+ influx, leading to the SFR. These data suggest
the possibility that two AT1 receptor-operated cation influx pathways via TRPC3 and TRPC6,
causing the enhancement of Ca2+ release from the SR, may cooperate in the SFR.  (COI:No)

S66

Asahikawa Med Univ, Japan, 2 Dept Cardio Physiol, Grad Sch Med, Okayama Univ, Japan）

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

AP-7 (S01-04)

Daikenchuto, a traditional herbal medicine, ameliorates ﬁbrosis byactivating TRPA1 channel in intestinal myoﬁbroblasts
1 1 Dept Cardiovasc
Rin Kurahara1，Keizo Hiraishi1, 2，Yaopeng Hu2，Ryuji Inoue2，Katsuya Hirano（

Physiol, Sch Med, Kagawa Univ, Japan, 2 Dept Physiol, Sch Med, Fukuoka Univ, Japan）

Background: Daikenchuto (DKT) is a traditional oriental herbal medicine, widely used tomitigate post-operative ileus and constipation. In this study, we investigated the anti-fibrotic
effect of DKT in a murine chronic colitis model and elucidated the role of transient receptor
potential ankyrin 1 (TRPA1) channels of intestinal myofibroblasts in colonic fibrosis.
Methods: A murine chronic colitis model was established by weekly intrarectal administrationof trinitrobenzene sulfonic acid (TNBS). Inflammatory and fibrotic changes were evaluated byhistopathological examination. An intestinal myofibroblast cell line (InMyoFibs) was stimulatedwith TGF-β1, and subsequent intracellular signaling and pro-fibrotic factors were investigated.
Samples from non-stenotic and stenotic regions of Crohn’s Disease (CD) patient’s intestines
were used for pathological analyses.
Results: In TNBS chronic colitis model mice, the extents of inflammation and fibrotic changes
were more prominent in TRPA1－/－ knockout than in wild-type mice. One-week enema administration of DKT suppressed fibrotic lesions in wild-type mice, but not in TRPA1 knockout mice.
Active ingredients of DKT, i.e. hydroxy α-sanshool and 6-shogaol induced Ca2+ influxes in
InMyoFib, which were antagonized by co-treatment with a selective TRPA1 channel blocker
HC-030031. DKT counteracted TGF-β1-induced expression of Type 1 collagen, α-SMA,
N-cadherin, the phosphorylation level of Smad-2 and p38-MAPK and the expression levelof myocardin, a well-known master transcription factor regulating fibrosis signaling at thedownstream of TGF-β1 receptor. Importantly, a 24-hour incubation with another DKT active
ingredient Japanese Pepper increased the mRNA and protein expressions of TRPA1, which inturn negatively regulated collagen synthesis in InMyoFibs. TRPA1 expression in the stenotic
regions of CD patient’s intestine was significantly greater than that in the non-stenotic regions.
Conclusions: DKT suppresses intestinal fibrosis by upregulating the expression and activating
the channel function of TRPA1. This putative mechanism underlies the reported beneficial
actions of DKT on inflammatory bowel disease.
(COI:No)

AP-8

Overexpression of neuronal K+-Cl− co-transporter enhances dendritic
spine plasticity and motor learning
Kayo Nakamura1, 2，Andrew John Moorhouse3，Dennis Lawrence Cheung3，Kei Eto1，
2 1 National Institutes for
Ikuko Takeda1，Paul Wiers Rozenbroek 3，Junichi Nabekura1, （Physiological Sciences, 2 Sokendai, 3University of New South Wales）

The neuronal K+-Cl− cotransporter KCC2 maintains a low intracellular Cl− concentration andfacilitates hyperpolarizing GABAA receptor responses. KCC2 also plays a separate role in stabilizing and enhancing dendritic spines in the developing nervous system. Using a conditionaltransgenic mouse strategy, we examined whether overexpression of KCC2 enhances dendritic
spines in the adult nervous system and characterized the effects on spine dynamics in the
motor cortex in vivo during rotarod training. Mice overexpressing KCC2 showed significantly
increased spine density in the apical dendrites of layer V pyramidal neurons, measured in vivousing two-photon imaging. During modest accelerated rotarod training, mice overexpressing
KCC2 displayed enhanced spine formation rates, greater balancing skill at higher rotarod
speeds and a faster rate of learning in this ability. Our results demonstrate that KCC2 enhances spine density and dynamics in the adult nervous system and suggest that KCC2 may
play a role in experience-dependent synaptic plasticity.
(COI:No)

AP-9

Epac1 deﬁciency inhibits basic ﬁbroblast growth factor-mediatedvascular smooth muscle cell migration
Yuko Kato1, 2，Utako Yokoyama1，Takayuki Fujita1，Masanari Umemura1，Tetsuo Kubota2，
1 1Yokohama City University, 2 Tokyo Medical and Dental University）
Yoshihiro Ishikawa（
Vascular smooth muscle cell (VSMC) migration and the subsequent intimal thickening playroles in vascular restenosis. We previously reported that an exchange protein activated bycAMP 1 (Epac1) promotes platelet-derived growth factor (PDGF)-induced VSMC migrationand intimal thickening. Because basic fibroblast growth factor (bFGF) also plays a pivotal role
in restenosis, we examined whether Epac1 was involved in bFGF-mediated VSMC migration.
bFGF-induced lamellipodia formation and migration were significantly decreased in VSMCs
obtained from Epac1−/− mice compared to those in Epac1+/+-VSMCs. The bFGF-induced
phosphorylation of Akt and glycogen synthase kinase 3β (GSK3β), which play a role in bFGFinduced cell migration, was attenuated in Epac1−/−-VSMCs. Intimal thickening induced by
the insertion of a large wire was attenuated in Epac1−/− mice, and was accompanied by thedecreased phosphorylation of GSK3β. These data suggest that Epac1 deficiency attenuates(COI:No)bFGF-induced VSMC migration, possibly via Akt/GSK3β pathways.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S67

Oral Presentations

Oral Session 1
Kidney・Urination・Digestion・Absorption（March 17, 15:20〜16:20, Hall 7）

O01-1

The Physiological Roles of Moesin, a Cytoskeletal Protein, in thickascending limb of loop of Henle via NKCC2
1 1 Dept Mol Physiol, Col Pharm Sci,
Kotoku Kawaguchi1，Ryo Hatano2，Shinji Asano（

Ritsumeikan Univ, Japan, 2 Dept Med Physiol, Grad Sch Med, Chiba Univ, Japan）

Tubular reabsorption of electrolytes in the kidney is an essential function in regulating fluidbalance in the body. Na+/K+/2Cl－ cotransporter type 2 (NKCC2) is specifically expressed inluminal cell surface of the thick ascending limb of the loop of Henle (TAL). The reabsorption of
NKCC2 plays important roles in regulating fluid balance. In general, luminal surface expression
level of NKCC2 is regulated by intracellular membrane trafficking involving exocytosis and endocytosis. It is well known that exocytosis of NKCC2 is promoted by the cAMP/PKA pathway
via some hormone stimulation. On the other hand, the molecular mechanisms of endocytosis of
NKCC2 are not clear. Recently, moesin, which is a crosslinker between membrane proteins andactin cytoskeleton, was reported to interact with NKCC2. However, the physiological roles ofmoesin in the kidney remain unclear. Here, we examined the roles of moesin in the regulationof renal function in vivo by using male moesin-null (Msn－/y) mice.
To evaluate the cell surface expressions and endocytosis of NKCC2, we analyzed thesechanges using protein biotinylation in tubular suspension including TAL. We investigated the
distribution of NKCC2 in lipid raft. To examine the renal physiological roles, we performed
biochemical analysis of plasma and urine.
In these results, we found that apical surface expression of NKCC2 was significantly increasedin Msn－/y TAL. Internalized NKCC2 was significantly reduced in the Msn－/y TAL. Lipid raft
expression of NKCC2 was significantly decreased in Msn－/y mice. Increase of plasma Na+ andCl－ concentration was observed in Msn－/y mice. Urinary absolute excretions of Na+ and Cl－ in
Msn－/y mice were lower than those of WT mice. These results suggest that moesin regulates
the apical surface expression level of NKCC2 by targeting NKCC2 to lipid raft and plays(COI:No)
important roles in the renal electrolyte handling.

O01-2

Elucidation of pace maker cells of spontaneous activities in detrusor
smooth muscle
Kentaro Kawagoe1，Eljamal Kareman1，Tomoko Maki2，Shinsuke Nakayama3，
4 1 Dept Clin Pharmacol, Grad Sch
Shunichi Kajioka1，Toshiyuki Sasaguri1，Masatoshi Eto（

O01-3

Common Ca2+-dependent inactivation (CDI)-based mechanism may
exist in FSGS mutations in the N-terminal domain of TRPC6 channels
Masayuki Mori X.1，Onur Polat K. 2，Ryo Okada1，Ayaka Suzuki1，Sayo Ohmura1，
3 1 Dept Biomaterials and Chemistry, Sch Med, UOEH, Japan, 2 Dept Synthetic
Ryuji Inoue（and Biological Chem, Grad Sch Engr, Kyoto Univ, 3 Dept Physiol, Sch of Med, Fukuoka
Univ）

Transient Receptor Potential Canonical member 6 (TRPC6) is a receptor activated nonselective cation channel, and its mutations are known to be associated with focal segmental
glomerulosclerosis (FSGS), a glomerular disease leading to the end-stage renal failure. Our recent finding that impairment of Ca2+-dependent inactivation (CDI) in TRPC6 FSGS-associatedmutations (Polat OK et al., JASN, 2019) raises the question as to whether disrupted CDI could
be a unifying mechanism to explain the dysfunctionality of FSGS mutations in the coiled-coilC-terminus domain which dimerization is presumed to critical for Ca2+/calmodulin binding.
To address this point, we examined the N-terminal mutations (in the ankyrin domains) as wellas those on the C-terminal, out-side of the coiled-coil domain, by using whole-cell patch-clamp
and Ca2+ imaging experiments. The newly tested FSGS mutations had also significant delayof inactivation, including L780P, reportedly attenuated Ca2+ response phenotype in C-terminal.
Therefore, the CDI mechanism could have substantial potential to unify the pathogenesis of
FSGS mutations, and may thus deserve further investigation in the future.
(COI:No)

O01-4

Study of Spatiotemporal electrical activity in the colon of mice withhypoganglionosis
Shinsuke Nakayama1，Kouichirou Yoshimaru2，Takayoshi Yamaza3，Shunichi Kajioka4（ 1 Dept Cell Physiol, Grad Sch Med, Nagoya Univ, Japan, 2 Dept Pediatric Surg, Kyushu
Univ, Grad Sch Med Sci, 3 Dept Mol Cell Biol and Oral Anat, Kyushu Univ, Grad Sch Dent
Sci, 4 Dept Clin Pharmacol, Kyushu Univ, Grad Sch Med Sci）
Hypoganglionosis, such as Hirschspring’s disease (HSCR) and its allied disorders, interpreted byprenatal deficiency of tropic factors with the detailed etiology still unclear, causes refractory
alimentary disorders associated with functional ileus. In this study, in order to explore possible
effective therapy, we characterized functional alterations in JF1 mice with hypoganglionosis.
JF1 mice are piebald mice, which possess the classic piebald s allele of Ednrb with two
nucleotide substitutions, and reduce the expression of endothelin receptor type B (EDNRB)
in the colon, causing a mild case of megacolon. Spatio-temporal electrical activity is the basisof smooth and elaborate motility in the gastrointestinal tract including the colon. We thus
monitored the field potential in isolated muscle sheets of the proximal colon, using 8 × 8
microelectrode array (MEA) covering ~1 × 1 mm2 area. In wild-type B6 mice, samples fromthe proximal colon displayed basal slow electrical oscillations with the period of ~3-4 s in a
majority of sensing electrodes, and occasionally large synchronized potentials occurred. On theother hand, the proximal colon of JF1 mice, basal rhythmic oscillations were negligible, andelectric complexes consisting of rapid and slow potentials occurred frequently. It is noted thatthe rapid component of prolonged bursting was a characteristic feature, and the bursting rapid
potentials propagated only in limited regions of MEA recording area. The frequency of rapid
potentials was 1-2 Hz. Also, the magnitude of electric potentials was significantly smaller in
JF1 mice than in wild-B6 mice. In organ bath experiments, isolated colon samples exhibited
spontaneous contractions corresponding to the electrical activity measured with MEA. In thelight of histological and blood examinations, we discuss possible mechaisms that account for theprominent differences in electrical and mechanical activity.
(COI:No)

O01-5

Fibroblast growth factor-23 and high calcium exposure inhibits intestinal
calcium absorption in Caco-2 monolayer
Mayuree Rodrat, Kannikar Wongdee2, 4，Narattaphol Charoenphandhu1, 2, 3

Med, Kyushu Univ, Japan, 2 Spinal Injuries Center, 3 Department ofCell Physiology,（ 1 Department of Physiology, Faculty of Science, Mahidol University, Thailand, 2 Center ofCalcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Thailand,
3 Institute of Molecular Biosciences, Mahidol University, Thailand, 4 Faculty of Allied Health
Sciences, Burapha University, Thailand）

Pacemaker cells of spontaneous activities of bladder smooth muscle were believed to c-kit+ interstitial cells of Cajal (ICC) like cells as well as gastrointestinal. Recently, however, PDGFRα+but c-kit negative interstitial cells have been described in bladder wall as a new candidate ofpace maker cells. Thus, class of pace maker cells of bladder is still under controversial. The aimof this study is to clarify the pathological modification of probable pace maker cells of overactive bladder using bladder outlet obstruction (BOO) mouse. In micturition reflex, the numberof urination per day significantly increased in BOO compared to sham. The daily urination
volume and one void volume were significantly decreased in BOO compared to sham. The
bladder weight was significantly larger in BOO compared to sham. There was no significant
difference in water intake between the two groups. In cystometry, BOO mice’s non-voiding
contractions significantly increased. In BOO, mRNA expression of c-kit, CD34 and PDGFRα
was significantly elevated, however interestingly immunostaining indicated significant reduction of PDGFRα+ cells. In MEA recording system, sham mice were hardly seen spontaneous
activities. Small waves of spontaneous action potential followed by relatively slow large waves
could be seen in BOO mice. Spontaneous activities were enhanced with 0.3 µM carbacholand completely suppressed with 2μM nifedipine.BOO model mice show significant differencein voluntarily voiding behavior analysis among urination frequency, daily urination volume,and single void volume. The result of significant reduction of PDGFRα+ cells despite of theobvious increase of its mRNA expression suggests that BOO might interrupt protein synthesesof PDGFRα or that the reduction of PDGFRα by BOO might provoke the increase of its
mRNA. In MEA recording, we demonstrated that lower urinary tract obstruction increases
the spontaneous electrical activity in bladder smooth muscle.
(COI:No)

Excessive calcium intake and intestinal calcium hyperabsorption often lead to a variety ofadverse effects such as vascular calcification, nephrolithiasis, and dementia. Therefore, theintestinal epithelial cells probably possess a negative feedback mechanism to prevent an excessive calcium transport. Although 1, 25-dihydroxyvitamin D3 [1, 25(OH)2D3] and parathyroid
hormone (PTH) are well recognized as calcium-regulating hormones, a few of endocrine andparacrine factors can inhibit or counterbalance calcium absorption. Previously, fibroblast
growth factor (FGF)-23 has been reported to abolish the 1, 25(OH)2D3-enhanced intestinal
calcium absorption in mice. We herein hypothesized that FGF-23 locally produced by theintestinal epithelial cells regulated calcium absorption as part of negative feedback mechanism.
The present study also aimed to determine whether FGF-23 was able to alter the changes
in epithelial electrical properties and electrogenic ion transport in 1, 25(OH)2D3-treated Caco-2monolayer, as indicated by short-circuit current (Isc) and transepithelial resistance (TER) in
Ussing chamber. The results demonstrated that 1, 25(OH)2D3 significantly increased Isc acrossCaco-2 monolayer, suggesting an increase in the electrogenic ion transport, while TER was
decreased in 1, 25(OH)2D3-treated monolayer. In addition, calcium transport across intestinal
epithelium-like Caco-2 monolayer enhanced by 1, 25(OH)2D3 disappeared after prolonged
exposure to high apical ionized calcium. Apical and/or basolateral exposure to FGF-23 completely abolished the 1, 25(OH)2D3-enhanced calcium transport as well as epithelial electricalparameters, i.e., Isc and TER, while pretreatment with FGF-23-neutralizing antibody could
restore the 1, 25(OH)2D3-enhaced calcium transport even in the presence of high apical calcium.
Moreover, FGF-23 was found in both apical and basolateral culture media of 1, 25(OH)2D3- andCaCl2-treated groups, but not the untreated group. In conclusion, prolonged exposure to highapical calcium and excessive calcium absorption probably induces local production of FGF-23
as a part of negative feedback loop, which, in turn, inhibits calcium transport, and modulates
epithelial integrity and electrogenic transport of ions across the intestinal mucosa. (COI:No)

Graduate School of Medicine, Nagoya University, 4 Department of Urology, Graduate Schoolof Medical Sciences, Kyushu University）

S70

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Oral Session 2
Environmental Physiology（March 17, 16:20〜17:20, Hall 7）

O02-3

Mental stress does not impair aerobic exercise performance after wholebody cold exposure
Daiki Imai1, 2，Ryosuke Takeda1，Eriko Kawai2，Kosuke Saho2，Akemi Ota2，Emiko Morita2，
2 1 Res Ctr for Urban Health ＆Yuta Suzuki1, 2，Hisayo Yokoyama1, 2，Kazunobu Okazaki1, （Sports, Osaka City Univ, Osaka, Japan, 2 Dept of Environ Physiol for Exerc, Osaka City
Univ Grad Sch of Med, Osaka, Japan）

Whole body cold exposure or mental load applied before exercise is known to decrease exercise performance. However the effects of simultaneous application of these stresses on exercise
performance remain unknown. We investigated the effects of simultaneous application of wholebody cold exposure and mental load on aerobic exercise performance compared with cold exposure alone. Seven healthy young male participants underwent the stroop color word test (500
stimuli/set × 2) as mental stress trial (MS), while watched a documentary DVD as non-stress
trial (NS) during 60 min of cold exposure using a water-perfused suit. Water temperature in thesuit was maintained at 15℃ for 10 min then at 10℃. They undressed the suit then performeda cycling test at 80% VO2max to the exhaustion. Esophageal and skin temperatures (Tes andTsk, respectively), plasma adrenaline ([Ad] p), noradrenaline ([Nord] p), and cortisol ([Cortisol] p)
concentrations, and subjective fatigue index (SFI) were measured before and immediately after
mental stress and exercise. Exercise duration was evaluated as aerobic performance. We foundthat Tsk was lower after cooling than baseline, while Tes remained unchanged. Tsk and Tes
were not different between trials. A significant effect of interaction (time × trial) was found on[Ad] p (p=0.023), [Nord] p (p=0.047), and SFI (p=0.040), while not on [Cortisol] p. Exercise duration
was not different between trials (p=0.762). In conclusion, the simultaneous application of mentalload accompanied with whole body cold exposure exacerbated tiredness via the enhanced
sympathetic nerve activity compared with cold exposure alone, while did not impair aerobic(COI:No)
exercise performance.

O02-1

O02-4

Yasukazu Nakahata1，Rezwana Ahmed1, 2，Atsushige Ashimori1, 2，Kazuyuki Shinohara1，
2 1 Dept Neurobiol and Behavi, Nagasaki Univ, Japan, 2 Gene Regulation,
Yasumasa Bessho（

Yuta Masuda, Kaito Kawashima, issei Kato, Shuri Marui, Kei Nagashima（ Body Temperature

Replicative senescent human cells possess altered circadian clocks
with a prolonged period and delayed peak-time
Dev Biol Sci, NAIST, Japan）

Over the last decade, a wide array of evidence has been accumulated that disruption of
circadian clock is prone to cause age-related diseases and premature aging. On the other hand,aging has been identified as one of the risk factors linked to the alteration of circadian clock.
These evidences suggest that the processes of aging and circadian clock feedback on eachother at the animal level. However, whether these two processes influence each other at thecellular level is still largely unknown. Our lab has recently revealed that the primary fibroblast
cells derived from Bmal1-/- mouse embryo, in which circadian clock is completely disrupted,
do not demonstrate the acceleration of cellular aging, i.e., cellular senescence. Hence, in mystudy I asked the reverse question, i.e. whether cellular senescence affects the circadian clock.
Interestingly, we found that senescent cells possess a longer circadian period with delayed
peak-time and that the variability in peak-time is wider in the senescent cells compared totheir proliferative counterparts, indicating that senescent cells show alterations of circadianclock. From these results, it is also reasonable to propose that investigation at cellular level is a
powerful and useful approach to dissect molecular mechanisms of aging in the circadian clock.
(COI:No)

O02-2

Thyroid hormone (TH) induced cell proliferation and migration throughTH receptor (TR)-dependent and -independent pathways
1 1 Department of
Ariyani Winda1，Wataru Miyazaki1, 2，Izuki Amano1，Noriyuki Koibuchi（

Integrative Physiology, Graduate School of Medicine, Gunma University, Japan,
2 Department of Bioscience and Laboratory Medicine, Graduate School of Health Sciences,
Hirosaki University, Japan）

Thyroid hormone (TH) plays an important role for controlling organ development and functional
maintenance. During development, TH may promote cellular proliferation and migration. The
actions of TH are mainly mediated by TH receptor (TR)-dependent pathway, which requires
binding of TH to TR that may localize in the nucleus, mitochondria, and plasma membrane.
TH also exert the actions through TR-independent pathway through binding to membraneprotein, such as integrin αVβ3. However, the mechanism of TH action on cell proliferationand migration have not yet been clearly determined. In this study, we examined the effects of
TH derivatives (T3, T4, rT3, and Triac) on cell proliferation and migration. A short exposure of
THs induced cell proliferation in C6 and Neuro-2A cells. Flow cytometry showed that total cellnumber in G2 phase increased after THs exposure. The expression of p-ERK1/2 proteins andCcnd, Ccna, Ccnb, Cdk1, Cdk4, and c-Fos mRNA increased by TH derivatives. TH derivatives
also increased F-actin cytoskeleton remodelling, that leads to cell migration in C6 and 3T3 cells.
Our study also showed that THs exposure induced F-actin rearrangement overlapped withthe upregulation of p-Akt, p-Rac1/cdc-42, Rho A, Cdc42, or Rac1/2/3 protein expression level.
Knockdown of TRs by RNAi or co-exposure integrin αVβ3 with XJ735, a selective integrin
αVβ3 inhibitor, reduced the cells proliferation and migration. These results indicate that THs
exposure exerts their action in cell proliferation and migration through both TR–dependentand –independent signaling pathway.
(COI:No)An Examination of The Factor of Heat Tolerance During Passive HeatStressand Body Fluid Lab, Fac Human Sci, Waseda Univ, Japan）

Introduction: It has been reported that many factors such as age, gender, and exercise habit,
etc. affect heat tolerance of individuals; however, the mechanism remains unclear yet. Previous
studies have suggested that factors involved in heat dissipation such as sweating are importantin determining heat tolerance, although the clear evidences have not been presented. In thepresent study, we tested the hypothesis that reduction of metabolic heat production is oneof mechanisms increasing heat tolerance. Therefore, we assessed thermoregulation duringbathing in a hot water, where responses of heat dissipation are largely suppressed.
Methods: Healthy male participance (age, 23.73 ± 2.89 y ; bw, 65.75 ± 8.58 kg; and % fat, 17.18± 6.14 %)immersed in water of 41℃ to the clavicle level, where room temperature was at 33℃
with 50% relative humidity. We continuously measured rectal temperature (Trec), temperatureof 4 skin sites, metabolic heat production (Hprod) by calorimetry, sweat rate (SR) at back, andskin blood flow (SkBF)at face. The duration of bathing was determined till Trec=39.0℃ or for
60min if Trec <39.0℃.
Results: Trec increased during bathing in all participants and reached 38.25 ± 0.51℃ ranging
from 36.85 to 39.04℃ at 30 min after the onset of bathing. The value of cumulative Hprod iscorrelated with the increase in Trec (P<0.01, r = 0.83). There was no correlation between theincrease in Trec, and meanTsk and cumulative values of SR and AUC-SkBF and % body fat.
Conclusion: Metabolic heat production during bathing is different among participants. In addition, as lower the metabolic heat production was, increase in body core temperature wassuppressed. The results may suggest that suppression of heat production is one of mechanisms(COI:No)
involved in heat tolerance. 

O02-5

Chronic treatment with the synthetic glucocorticoid methylprednisolone
in Sprague-Dawley rats induces anhedonic behaviour anddysregulation of the circadian tryptophan hydroxylase mRNA
expression proﬁle in the dorsal and median raphe nuclei
Nidia M. Reyes-Prieto1，Mitsuhiro Yoshimura2，Yvonne M. Kershaw1，
1 1University of
Christopher A Lowry3，Becky L. Conway-Campbell1，Stafford L Lightman（
Bristol. United Kingdom, 2 University of Occupational and environmental Health. Japan,

3 University of Colorado. USA）

Stress-related neuropsychiatric disorders represent a major worldwide problem. Hypothalamic-pituitary-adrenal (HPA) axis dysregulation and serotonergic dysfunction have both been
implicated in the development of depression, however the relationship between the two is
not fully understood. Here we show that expression of tryptophan hydroxylase (tph2) mRNA,
which codes for the rate-limiting enzyme in the biosynthesis of serotonin in the raphe complex, fluctuates significantly over the 24 hour period. The lowest levels of tph2 expression
coincided with the circadian nadir in adrenal glucocorticoid secretion, while the highest levels
coincided with the initial rise in circulating glucocorticoids preceding the onset of the rats’active phase. Administration of the synthetic glucocorticoid methylprednisolone in drinking
water suppressed adrenal glucocorticoid secretion, and induced a pronounced phase shift inglucocorticoid receptor (GR) activation in serotonergic neurons throughout the raphe complex.
The circadian rhythm of tph2 mRNA expression was markedly altered in the MPL treatedrats, particularly for nadir and peak expression levels throughout the dorsal (DRD), ventral(DRV), ventrolateral/ventrolateral periaqueductal grey (DRVL/VLPAG) and median (MnR)
raphe nuclei. MPL treated rats exhibited a significantly reduced sucrose preference, consistent
with anhedonic behaviour and symptomatic of a depressive phenotype. Taken together, ourdata demonstrate that the endogenous daily rhythm in tph2 expression throughout the raphecomplex and the associated affective state of the individual are highly sensitive to HPA axis
disruption, in particular shown here with synthetic glucocorticoid treatment. Moreover, ourfindings provide a potential mechanism underpinning the adverse side effects on mood and(COI:No)
behaviour reported by patients treated with synthetic glucocorticoids. 

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S71

Oral Session 3
Heart・Circulation 1（March 18, 13:20〜14:20, Hall 3）

O03-3 (AP-5)

Drp1-mediated mitochondrial dynamics in cardiac remodeling
Akiyuki Nishimura1，Kakeru Shimoda2，Tomohiro Tanaka2，Kazuhiro Nishiyama1，
2 1Grad Sch Pharm Sci, Kyushu Univ, Japan, 2 Dept cardiocirculatory
Motohiro Nishida1, （Signal, NIPS, Japan）

Mitochondria are dynamic organelles that interchanges their morphology between fusion(elongation) and fission (fragmentation) form. Proper mitochondrial quality control is indispensable for cardiac homeostasis and defects in mitochondrial dynamics are implicated in
the development of cardiac diseases. Our group has investigated the molecular mechanism
underlying the development of maladaptive cardiac remodeling, especially myocardial
early senescence that is observed in cardiac disease patients and mice models and has beensuggested as a major cause of cardiac dysfunction. We found that defective mitochondrial
dynamics through aberrant interactions between mitochondria and actin cytoskeleton are a
key determinant of cardiac remodeling and fragility. Dynamin-related protein 1 (Drp1), a mitochondrial fission-accelerating protein, was activated in myocardium after myocardial infarction,
which induced mitochondrial fission-associated myocardial early senescence. We also foundthat the actin-binding protein filamin A acted as a guanine nucleotide exchange factor for
Drp1. Hypoxic stress induced the interaction of filamin A with Drp1, leading Drp1 activationand mitochondrial fission-associated myocardial senescence in an actin binding-dependent
manner in cardiomyocytes. We previously reported that polysulfidation of Drp1 at Cys624, a
redox-sensitive cysteine residue, negatively regulates its activity. Drp1-filamin A interaction
was regulated by polysulfidation-depolysulfidation cycle of Drp1 Cys624. Electrophile-mediateddepolysulfidation of Drp1 promoted the interaction with filamin A and induced mitochondrial
hyperfission. In addition, pharmacological perturbation of the Drp1-filamin A interaction bycilnidipine suppressed mitochondrial fission-associated myocardial senescence and improved
chronic heart failure in mice. Our results suggest therapeutic potential targeting pathologydependent Drp1-filamin A interaction for the treatment of chronic heart failure.
(COI:No)

O03-1

O03-4

Tatsuya Sato, Nobutoshi Ichise, Takeshi Kobayashi, Hiroya Yamazaki, Yoshinori Terashima,
Noritsugu Tohse（Dept Cell Physiol, Sap Med Univ, Japan）

1 1Cardiovascular Physiol, Graduate
Chen Wang1，Jian Chen2，Keiji Naruse1，Ken Takahashi（

Mitochondrial aerobic respiration in the fetal heart is activated just after
initiation of the heartbeat

Backgrounds: We demonstrated that the heartbeat in rat fetal heart initiates at aroundembryonic day 10.0 (E10.0) with a calcium transient via extracellular calcium influx being
preceding to muscle contraction. We also showed that the pathway of gene expressions related
to glucose metabolism assessed by transcriptome analysis in the heart just after initiation of
the heartbeat was the secondly highest next to the pathway related to muscle contraction,suggesting that changes in energy metabolism play pivotal roles in initiation of the heartbeat.
Although mitochondria are central site in energy metabolism in the adult heart, it remains
unclear whether mitochondrial respiration is involved in energy metabolism in the fetal heartat the beginning of the heartbeat.
Methods: The embryos at E10.0 in Wistar rats were divided into two groups according to thehearts without (pre-) or with (post-) heartbeat. Comprehensive metabolites of the fetal heart
were evaluated by metabolome analysis, and real time mitochondrial oxygen consumption rate(OCR) for enzymatically isolated fetal rat cardiomyocytes was assessed by an extracellular
flux analyzer.
Results: According to the principal component analysis in the metabolomic analysis, fructose
6-phosphate, fructose 1, 6-bisphosphate, which are metabolites in glycolytic pathway, weremajor factor loading components in pre-heartbeat group, whereas GTP, reduced glutathione,
and ATP, which are associated with mitochondrial respiration, were major determinants inpost-heartbeat group. OCR in isolated cells at baseline was extremely low in both groups;
however, maximal OCR induced by the addition of an uncoupler (FCCP) showed a significant
increase only in cells from post-heartbeat group, suggesting the presence of proton gradient
between the inner mitochondrial membrane and the matrix after initiation of the heartbeat.
Conclusions: The findings suggest that mitochondrial aerobic respiration is activated just after
initiation of the heartbeat presumably in response to ATP demand in the fetal beating heart.
(COI:No)O03-2

Acute and Temporary Overstretch Destructed Inner Mitochondrial
Membrane Without Subsequent Cardiac Dysfunction in Rat Hearts
Naritomo Nishioka1, 2，Hiroki Bochimoto3，Ping Yu Xiong4, 5，Shunsuke Baba1, 6，Jun Tanihata3，
1 1 Dept Cell Physiol, Jikei Univ Sch Med, Tokyo,
Susumu Minamisawa1, 3，Yoichiro Kusakari（
Japan, 2 Dept Cardiac Surgery, Jikei Univ Sch Med, Tokyo, Japan, 3 Division of Aerospace Med,

Dept Cell Physiol, Jikei Univ Sch Med, Tokyo, Japan, 4 Dept Med, Queen’s University, Kingston,
ON, Canada, 5 Dept Biomed and Molecular Sciences, Queen’s University, Kingston, ON, Canada,
6 Dept Pediatrics, Jikei Univ Sch Med, Tokyo, Japan）
Introduction: We reported last year that acute overstretch destructed inner mitochondrial membrane with preserved sarcomere structure of rat papillary muscle. However, it is unclear whether
acute overstretch-induced destruction of mitochondria would impair cardiac function or not.
Purpose: We made two different models for acute overstretch to investigate the changes incontractility and organelle structures in cardiac muscle.
Methods: 1) in vivo study: We ligated pulmonary artery (PA) of male SD-rats (BW>350g) for 30
seconds to make a model of right ventricle (RV) expansion, then released ligation and followed by 30
min. Cardiac function was monitored by transthoracic echocardiogram (TTE) and the intracellularultrastructure of RV free wall was observed by transmission electron microscope (EM). 2) in vitro
study: to mimic in vivo model, the RV papillary muscle of male SD-rats was overstretched within 2
seconds up to 120% of Lmax, and the overstretched status was kept for 5 minutes, then returned
to Lmax (1Hz, 36℃).
Results: In in vivo study, TTE revealed PA ligation made the area of diastolic phase enlarged to300%. However, there was no significant difference in RV ejection fraction and volume, and Tricuspid Annular Plane Systolic Excursion (TAPSE) between before and 30 min after ligation (n=9).
In EM analysis, focal vacuolated mitochondria with decrease in electron density of matrix appeared,
despite the sarcomere kept normal structure in cardiomyocytes of the RV expansion model. In
in vitro study, the active tension was reduced to 34.6±6.3 % of Lmax during overstretching andrecovered up to 101.0±21.7% of Lmax after returning to Lmax (n=5).
Conclusions: Acute and transient overstretch did not subsequently impair cardiac function, although it disrupted inner mitochondrial membrane. This study indicates the functional robustnessof myofilaments and structural fragility of mitochondria to mechanical stress in cardiac muscles.
(COI:No)

S72

Roles of the TRPM4 channel in mitochondrial function, ROS
generation, and calcium releasein myocardial ischemia–reperfusion
injurySch Med, Okayama Univ, Okayama, Japan, 2 Harbin Medical University）

Ischemic heart disease is one of the most common causes of death in the current era. Mitochondrial dysfunction, reactive oxygen species (ROS) generation, and calcium (Ca2+) overload
are three crucial factors that trigger myocardial ischemia–reperfusion (I/R) injury. Inhibition of
TRPM4, a calcium-activated nonselective cation channel, protects the rat heartfrom I/R injury,
but the specific mechanism is unclear.
In this study, we investigated the mechanism of cardioprotection against I/R injury via TRPM4,
using two types of I/R injury models (500μmol-hydrogen peroxide[H2O2] and 24h of hypoxia[2% O2] followed by2h of reoxygenation[H/R]). We knocked out the TRPM4 gene in the rat
cardiomyocyte cell line H9c2 using CRISPR/Cas9. With H2O2 treatment, both intracellular
calcium levels and ROS production increased in wild-type (WT) cells, whereas these changes
were mitigated in knock-out (KO) cells. Additionally, with this treatment, mitochondrial membrane potential and intracellular ATP levelsreduced in WT cells, whereas these changes were
mitigated in KO cells. Furthermore, these effects of H2O2 in WT and KO cells were similarly
observed underthe H/R condition.
The findings suggest that blockade of the TRPM4 channel might protect the myocardium from
I/R injury by maintaining mitochondrial membrane potential and intracellular ATP levels,
possibly owing to the prevention of aberrant increases in intracellular calcium and ROS.
Key words: Myocardial ischemia-reperfusion injury, TRPM4, Mitochondrial membrane potential, ATP, Reactive oxygen species, Calcium, CRIPSR/Cas9
(COI:No)

O03-5

Heart rate variability analysis of parents and infants during a hug
2 1 Dept Anatomy, Faculty Med, Toho Univ, Japan,
Sachine Yoshida1，Hiromasa Funato1, （2 WPI-III, Univ Tsukuba, Japan）

Parents often hold their infants firmly in their arms to express affection and joy. Such physicalcontact is well-known as a hug, but the physiological effects on infants and parents remain
unclear. Here, we examined the heart rate variable of infants and their parents during theparent-infant hug with different month ages of the first-year infants. We found several parameters of infant heart rate variability changed around four months, indicating the pronounced
parasympathetic activity. We examined changes in the R-R interval (RRI) in infants under andover four months old during being held, hugged, and hugged tightly by their parents. The RRI
increase rate during a hug changed depending on the ages and activity levels of infants. Suchcontext-dependent RRI changes were absent during holding and tight hugs. The parent-infant
hug also decreased heart rates in the parents. A hug seems to function as one of the nonverbal
communication methods between parents and infants after around four months. These findings
will contribute to the understanding of typical and atypical development.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Oral Session 4

O04-3

Neuron・Synapse

Univ, Japan）（March 18, 13:20〜14:20, Hall 4）

Excitability plasticity and behavioral modulation by acute inﬂammationof the cerebellum
2 1 Hakubi center, Kyoto Univ, Japan, 2 Dept Biophysics, Grad Sch Sci, Kyoto
Gen Ohtsuki1, （Cerebellar dysfunction is related to various psychiatric disorders, including autism-spectrum
disorders and schizophrenia. However, the physiological mechanism has not been well understood. Recently, I have investigated the immune-triggered hyperexcitability in the cerebellum.
Activated microglia via exposure to bacterial endotoxin lipopolysaccharide or heat-killed
Gram-negative bacteria induced potentiation of the intrinsic excitability in Purkinje cells (PCs),
which was suppressed by microglia-activity inhibitor or microglia-depletion. An inflammatory
cytokine, tumor necrosis factor-α (TNF-α) released from microglia via toll-like receptor 4
triggered this plasticity. Region-specific inflammation in the cerebellum in vivo showed depression- and autistic-like behaviors. Furthermore, both TNF-α-inhibition and microglia-depletion
reverted such behavioral abnormality. Resting-state functional MRI revealed overconnectivity
between the inflamed cerebellum and prefrontal neocortical regions. Thus, immune activity inthe cerebellum induces neuronal hyperexcitability and disruption of psychomotor behaviors inanimals (Yamamoto et al./ *Ohtsuki, 2019 Cell Reports).
Excitability plasticity of the dendrites may be related to the selection of electro-conductivity
of synaptic transmission along dendritic processes (Ohtsuki et al., 2012 Neuron; Ohtsuki andHansel, 2018 iScience). In the later part of the talk, I will present the evidence of discordanceof concurrent EPSCs between soma and dendrite of PCs. My results suggest that this discordance contributed to form a cluster of the synaptic transmission. Both of the induction of
intrinsic plasticity and inhibition of SK channels reduced the number of clusters, implying thatthe heterogeneous excitability among PC dendrites may endow neurons with branch-specificcomputational power at dendrites (*Ohtsuki, in revision). I will also discuss the modulation of
the electro-conductivity of PC dendrites by immune activation. Taken together, the plasticity of
the intrinsic excitability of PCs may be associated with impaired neuronal information processing in a disease state, like as in the acute cerebellar inflammation.
(COI:No)

O04-1

O04-4

Ayane Ninomiya1，Nobutake Hosoi2，Michifumi Kokubo1，Izuki Amano1，Asahi Haijima3，
1 1 Dept. Integrative Physiol, Grad
Wataru Miyazaki4，Hirokazu Hirai2，Noriyuki Koibuchi（

Rei Yamada, Hiroshi Kuba（ Dept Cell Physiol, Grad Sch Med, Nagoya Univ, Japan）

A Novel Role of Thyroid Hormone Receptor in Synaptic Plasticity inCerebellar Purkinje Cells
Sch Med, Gunma Univ, Japan, 2 Dept. Neurophysiology and Neural Repair, Grad Sch Med,
Gunma Univ, 3 Sch Human Sciences, Waseda Univ, 4 Dept. Bioscience and Laboratory
Medicine, Grad Sch Health Sci, Hirosaki Univ）

Thyroid hormone (TH) is essential for the development and the maintenance of the brain
function. TH action is mediated by TH receptor (TR). TR binds to a specific DNA sequence
on TH-target genes and thus functions as a ligand-dependent transcription factor. In thyroid
diseases such as congenital hypothyroidism or resistance to TH (RTH), TH-TR binding is
dominantly disrupted, leading to the various symptoms such as motor deficits. However, thespecific mechanism has not been cleared, besides that proper motor coordination is deeply
related to long term depression (LTD) of synaptic transmission from parallel fiber (PF) to
Purkinje cell (PC) in the cerebellum (Ito, 1989). Therefore, we examined the involvement of TR
in synaptic plasticity at PF-PC synapses by using transgenic mice (Mf-1 mice) which express
dominant-negative TR specifically in PCs. Since Mf-1 display the impairment of motor coordination and motor learning, decrease in TR signaling in PCs may alter synaptic plasticity andcontribute to motor incoordination. A whole-cell patch clamp recording of Mf-1 PCs revealed
the inhibition of LTD but instead the induction of long term potentiation (LTP) of the synaptictransmission at PF-PC synapses. This indicates that the intracellular calcium dynamics may
be disrupted in Mf-1 PCs since LTD requires a higher elevation of the intracellular calcium
concentration in PCs than LTP does. In addition, single-PC qPCR showed that the mRNAlevels of some important molecules for the intracellular calcium dynamics in PCs (SERCA2,
IP3R, and P/Q-type calcium channel) decreased in Mf-1 PCs, indicating the possible TH-target
genes. Taken together, the present study suggested a novel possible role of TR in synaptic
plasticity at PF-PC synapses by regulating the expression of some important genes for LTD
occurrence in the cerebellum. This finding could give a new insight into the mechanism of
motor deficits in thyroid diseases.
(COI:No)

O04-2

Presynaptic inhibition of GABA release from striatal medium spiny
neurons onto cholinergic interneurons by M1 muscarine receptors
Toshihiko Momiyama, Etsuko Suzuki（Dept Pharmacol, Jikei Univ Sch Med）
We used transgenic mice with restricted expression of channelrhodopsin-2 (ChR2) in the
striatal medium spiny neurons (MSNs). Whole-cell patch-clamp recordings were made fromstriatal cholinergic interneurons in P10-17 mice brain slices. Neurons were voltage clamped at-60 mV. Light stimulation (470 nm, 5 ms duration) evoked postsynaptic currents in the presenceof glutamate and glycine receptor antagonists. These postsynaptic currents were blocked by
GABAA receptor antagonist, bicuculline, suggesting they were GABAA receptor-mediatedinhibitory postsynaptic currents (IPSCs). A muscarinic acetylcholine receptor agonist, carbachol (1 µM), suppressed IPSCs by 49.5 ± 7.8%, (n = 5). To examine the changes in GABA
release probability, we calculated coefficient of variation (CV) at baseline, after applicationof bicuculline, after washout of bicuculline and after application of carbachol. CV at baseline
and after application of bicuculline were 0.2 ± 0.02 and 0.2 ± 0.03, respectively. The CV was
not increased after application of bicuculline, suggesting the action site of bicuculline waspostsynaptic GABAA receptors. On the other hand, CV after application of carbachol was
significantly increased (0.5 ± 0.06, p = 0.004), suggesting that GABA release probability waschanged by carbachol. In addition, carbachol (10 µM), wich strongly inhibits light-evoked IPSCs,
did not affect inward currents evoked by puff-applied GABA (100 µM). These results suggestthat activation of M1 muscarine receptors presynaptically inhibits GABA release from MSNs
(COI:No)
onto cholinergic interneurons.

Dendritic synapse clustering facilitates interaural time difference coding
for low frequency soundSynaptic clustering at dendrites enhances nonlinearity of integration and hence has strong impact on the input-output relationship of neurons. However, how the synaptic distribution shapes
specific brain functions are not well understood. Auditory coincidence detection is a neuronalbasis of encoding the interaural time differences (ITDs) for sound localization. In birds, neurons
in nucleus laminaris (NL) receive binaural excitatory inputs on separate dendrites and mediate
auditory coincidence detection. The dendrtic structure of NL is differentiated tonotopicallyand neurons with low tuning frequency (low-CF neurons) have prominently long dendrites. In
this study, we examined the dendritic location of synapses in low-CF neurons. We analyzed
the distribution of excitatory synapses mainly using focal glutamate uncaging and found thatsynaptic terminals were clustered at distal dendrites. We recorded voltage responses at soma
and found that responses generated at distal dendrites were strongly attenuated particularly
at the strong stimulation. Model study revealed that the clustered inputs at distal dendrite
generated large depolarization at the site, which decreased driving force of synaptic currents
and increased shunting conductance of K+ channels, then increased the extent of attenuationin an intensity-dependent manner. This sublinear summation prevented unilateral firing andincreased the dynamic range of ITD coding. We concluded that the synaptic clustering at distal
dendrite would be a cellular basis to accomplish the sound localization for wide intensity ranges(COI:No)of low frequency sound.

O04-5

Sustained synaptic input regulates depotentiation and LTP suppression
via the protein phosphorylation / dephosphorylation under theactivation of NMDAR and mGluR-IP3R
Jun-Ichi Goto1，Satoshi Fujii1, Kenya Kaneko1，Hiroki Fujiwara1，Yoshihiko Yamazaki1，
3 1 Department of Physiology, Yamagata University School of
Katsuhiko Mikoshiba2, （Medicine, Japan, 2 Laboratory for Developmental Neurobiology, Center for Brain Science,
RIKEN, Japan, 3 Present address: Shanghai Institute for Advanced ImmunochemicalStudies, Shanghai Tech University, China）
We examined the role for very low frequency (less than 0.1 Hz) firing of hippocampal CA1 pyramidal neurons under the in vitro slice preparations by examining the induction of depotentiationand LTP suppression. Electrophysiological recordings were obtained from CA1 region of guinea
pig hippocampal slices. The monitoring stimuli were applied every 20 seconds to the Schaffer
collateral - CA1 pyramidal cell pathway via a bipolar stimulating electrode to obtain field EPSPs
and population spikes.
In the experiments of depotentiation, LTP was induced by tetanic stimulation (100 Hz, 100 pulses)
and low frequency stimulation (2 Hz, 1000 pulses) was applied 30 minutes after the tetanus toinduce depotentiation. The monitoring stimuli were halted for 20 minutes right after the tetanusor the low-frequency stimulation, then the induction of depotentiation was inhibited.
In the experiments of LTP suppression, low frequency stimulation (1 Hz, 1000 pulses) was applied
60 minutes before the tetanus (100 Hz, 100 pulses). This preconditioning normally suppresses theinduction of LTP, while the monitoring stimuli were halted for 10 minutes right before thetetanus, the LTP suppression was almost totally blocked (normal amplitude of LTP was induced).
Pharmacological studies indicated that the activation of NMDAR, mGluR, IP3R and calcineurin
during not only the period of inductive stimuli but also the period of test pulse stimuli is critical
for the induction of depotentiation and LTP suppression.
These results indicate that not only the inductive stimuli but also monitoring stimuli may contribute to the establishment of depotentiation and LTP suppression, probably via the regulationof postsynaptic molecular mechanisms such as calcium dependent protein phosphorylation /dephosphorylation events. The authors declare no conflict of interest regarding this presentation.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S73

Oral Session 5
Behavior Science・Biorhythm・
Neurochemistry（March 18, 13:20〜14:20, Hall 5）

O05-3

The Protective Effect and Mechanism of CoA-Cl in acute phase after
spinal cord injury
Issei Sakamoto1, 2，Naoyuki Himi1，Norito Hasyashi1, 2，Emi Maruyama1，Osamu Miyamoto1（ 1 Dept Physiol 2, Kawasaki Sch Med, Okayama, Japan, 2 Dept of Orthopedics Surgery,
Kawasaki Sch Med, Okayama, Japan）

Spinal cord injury (SCI) induces severe motor and sensory dysfunction. Previous studies in
our laboratory showed the neuroprotective effects of COA-Cl, a novel synthesized adenosine
analog, in a rat stroke model. We evaluated the neuroprotective effects of COA-Cl in acuteand subacute phase of SCI in the present study. Male Sprague-Dawley rats aged 8 weeks
were used. SCI model was produced at T8/9 level by using drop device (20 g x 25 mm). Rats
were randomly divided into 4 groups. Acute group and subacute groups were administrated
at a dose of 6 mg/kg COA-Cl in saline for 5 days from just and 4 days after SCI, respectively.
As control groups, acute and subacute vehicle group were injected same volume of saline as
COA-Cl groups. Motor function was evaluated using the Basso-Beattie-Bresnahan Locomotor
Score (BBB score) and inclined plane test at 7, 14, 21, 28 and 31 days after SCI. The cavity
volume of spinal cord was evaluated by HE stained tissue after the final motor assessment. At
7 days after SCI, apoptotic cells and pERK/ERK were also evaluated by TUNEL staining andWestern blot, respectively. BBB score and inclined plane test were significantly improved in
the acute group, while the subacute group did not show any improvement in motor functions.
Furthermore, significant decreases in both the cavity volume and TUNEL positive cells in the
spinal cord of the acute group were observed compared with the subacute group. In addition,
the level of pERK/ERK was increased in acute group at 7days after SCI. Our findings suggestthat COA-Cl exert neuroprotective effect via MAP kinase cascade at acute phase after SCI,
resulting in the recovery of motor functions. COA-Cl could be a novel therapeutic agent for theacute phase of SCI.
(COI:No)

O05-1

O05-4

1 1 Dept Physiol, Hyogo Col Med,
Nozomu Nakamura1，Masaki Fukunaga2，Yoshitaka Oku（

Hideki Hida, Atsunori Hattori, Naoki Tajiri, Yoshitomo Ueda, Akimasa Ishida,
Takeshi Shimizu（Dept Neurophysiol ＆ Brain Sci, Nagoya City Univ Grad Sch Med Sci,

Respiratory phase transition modulates cognitive performance during
the retrieval process
Japan, 2 Div Cereb Intg, Nat’l Inst Physiol Sci, Japan）

Increasing evidence provides that the timing of respiration alters and shapes cognitive
performance in the temporal resolution of sub-seconds. We previously demonstrated that, in
healthy subjects performing a matching-to-sample visual recognition task, the accuracy was
remarkably decreased when the retrieval process contained expiratory-to-inspiratory phase(EI) transition (or the onset of inspiration; Nakamura et al., PLoS ONE 13: e0204021, 2018).
Meanwhile, immediately before the EI transition, the inspiratory activity in the brainstem
starts and the expiratory activity has its peak; the period right before the EI transition might
have another effect on performance even though the retrieval process does not contain the
EI transition. Furthermore, it remains unclear about the neural basis of respiratory-dependent
cognitive decline using fMRI. Here we modified the behavioral paradigm so as to be able to
be extended to fMRI studies.
Our results showed that the accuracy was decreased in the retrieval process containing the
EI transition, and unchanged in the retrieval process containing the inspiratory-to-expiratory
phase transition. Moreover, the accuracy was significantly decreased when the retrieval
process (or final choice) was completed immediately before the EI transition. We will discuss
about the EI transition dependent effect on cognitive performance and its neural basis of theretrieval process(COI:No)

Enriched environment affects neuronal dendrite morphology andoligodendrocyte differentiation in neonatal white matter injury model
Japan）

Hypoxia-ischemia (H-I) in preterm infants occasionally results in neonatal white matter injury(NWMI) associated with neurodevelopmental disabilities. Although we previously reported
the improvement of disturbed motor function in NWMI by enriched environment (EE) that
contains increased motor activity, social interaction and exploration, the cell dynamics in EE
effect on NWMI model is still unclear. To investigate whether EE during the period of development can change microenvironment of NWMI model affecting on cell morphology and celldifferentiation, a rat NWMI model made by H-I at P3 was grown in either condition of EE or
standard environment (SE) from P25 to P70, followed by morphological and immunohistochemical assessments. In the NWMI model under SE, significantly complex dendrites with manybranching were shown in H-I side of the cortex compared in the contralateral side. However,similar extension pattern was observed by Sholl analysis in both side of the cortex in the
NWMI model under EE, which is comparable to the pattern in Sham groups. Interestingly, thedendrite complexity in NWMI-EE group was normalized to those in sham group. In addition
to morphological changes, oligodendrocyte (0L) differentiation was induced by EE: significant
increase of mature OL was shown on the upper area of ipsilateral motor cortex, and stronger
MBP intensity was also detected in NWMI-EE group. These data suggested that EE during
the period of development has significant effects on disturbed microenvironment in a rat
NWMI model, affecting on neuronal dendrite morphology and OL differentiation. (COI:No)

O05-2

O05-5

1 1WPI-IIIS,
Takeshi Kanda1，Takehiro Miyazaki1，Hideitsu Hino2，Masashi Yanagisawa（

Akio Inoue1，Yuriko Inoue2，Hiromitsu Ezure2，Naruhito Ohtsuka2，Akitoshi Inoue3，
5 1 Human Brain Res. Cent., Grad Sch Med, Kyoto
Yoshinobu Manome4，Koichi Shiraishi（

Dynamics of local cortical connectivity during sleep revealed by twophoton imaging and graphical modeling
Univ Tsukuba, Japan, 2 Institute of Statistical Mathematics）

Sleep supports brain functions through its modulatory effects on the cerebral cortex. Whether
sleep modulates functional connectivity in the cortical local circuits, however, is poorly understood. Here we investigated functional connectivity, that is, covarying activity between
neurons, during spontaneous sleep/wake states using two-photon calcium imaging of identified
excitatory/inhibitory neurons in the motor cortex. Functional connectivity was estimated witha statistical learning approach graphical lasso and quantified by “the probability of establishingconnectivity (sparse/dense)” and “the strength of the established connectivity (weak/strong)”.
Local cortical connectivity was sparse in non-rapid eye movement sleep (NREM) sleep anddense in REM sleep, which was similar in both excitatory and inhibitory neurons. Sparseand dense connectivity during NREM and REM sleep could decrease and increase synaptic
efficacy, respectively. Sleep deprivation is known to disrupt learning and memory. To understand the underlying mechanisms, we next examined if sleep deprivation and its recovery
sleep affect local cortical connectivity. Sleep deprivation induced strong excitatory/inhibitoryand dense inhibitory, but not excitatory, connectivity. Subsequent NREM sleep after sleep
deprivation exhibited weak excitatory/inhibitory, sparse excitatory, and dense inhibitory connectivity. These findings indicate that local cortical connectivity becomes quite dense without
NREM sleeps, which could cause saturation of learning ability such as synaptic potentiation. In
addition, post-sleep deprivation NREM sleep might contribute to restoration of learning ability
through depotentiation of synaptic efficacy. (COI:No)

S74

Trapping of Mn ions in nerve vesicles by Ryanodine receptor antagonist
Dantrolene for Mn-MRI method
Univ, Japan, 2 Dep.Anat., Showa Univ.Sch.Med, 3 Med. Chem., Kansai Med. Sch, 4 Div.Mol.
Cell.Biol., res.Cent.Med.sci., Jikei Uni.Med, 5 Dev. Med. Eng. Jikei Univ. Med）
As nerve cells uptake Mn ions through Ca channel depending on nerve activity, and Mn ions
induce the increase of T1 signal of MRI, Mn-MRI is used to monitor the brain activity in vivo.
Then, we studied Ca and Mn ions inside the cultured Hippocampal neurons using fluorescent
Ca indicator Fluo4, the fluorescence of which is increased bu Ca ions and is reduced by Mnions. When nerve cells were activated by glutamate, Ca ions entered into nerve cells. And
Mn ions also entered into nerve cells slowly. When Fluo4 was charged after Mn ion uptake,the glutamate activation induced Ca entry into the cells followed by reduction of fluorescence
due to release of Mn ions from vesicles. This result indicates that Mn ions inside the cells
were uptaken to the vesicles inside the cells and Mn ions were released from the vesicles by
the Ca induced manner. When Mn ions charged cells were treated several timed with withglutamate, Mn ions inside the cells disappeared, and the flurescence of Fluo4 was not reduced
by glutamate activation, Therefore, Mn ions inside the cells were released by nerve activation.
Ca ions are considered to be released from vesicles through Ryanodine receptor, RyR, as.
RyR activator, 4-Chloro-m-cresol, induced the release of Ca ions from the vesicles, while RyR
antagonist, Dantrolene, reduced the increase of cellular Ca ions ater Glutamate activation. We
prevented the release of Mn ions in the vesicles by Dantrolene, and measured the high quality
Mn-MRI using Bruker 9.4T MRI machine with cryoprobe.
(COI:No)I have no COI with regard to the presentation. 

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Oral Session 6
Oral Physiology・Motor Function（March 18, 13:20〜14:20, Hall 6）

O06-3

Distinct limb preference in forelimb-movement representations in the rat
motor and parietal association cortices
Shogo Soma1, 2，Akiko Saiki2，Junichi Yoshida2，Shigeki Kato4，Yukari Takahashi5，
Satoshi Nonomura2, 3，Yae Sugimura K.5，Alain Rios2, 3，Masanori Kawabata2, 3，
3 1 Dept Mol Cell
Kazuto Kobayashi4，Fusao Kato5，Yutaka Sakai2，Yoshikazu Isomura2, （Physiol, Grad Sch Med, Kyoto Pref Univ Med, Japan, 2 Brain Sci Inst, Tamagawa Univ,
Japan, 3 Dept Physiol and Cell Biol, Grad Sch Med Dent Sci, Tokyo Med Dent Univ, Japan,
4 Dept Mol Genet, Fukushima Med Univ, Japan, 5 Dept Neurosci, Jikei Univ Sch Med, Japan）
The voluntary movements are expressed via motor information processing of distinct corticalareas. This processing are conducted not only in the primary and secondary motor cortices(M1 and M2) but also in the parietal association area (e.g., the posterior parietal cortex, PPC).
To comprehensively understand the neuronal basis of controlling multiple limb movements, wedeveloped a novel behavioral task to monitor movements of the right and left forelimbs separately
and recorded the neuronal activity in the M1, M2, and PPC with cutting edge multi-neuronal
recording techniques. We differentiated between intratelencephalic (IT) and pyramidal tract (PT)neurons in the motor cortices using optogenetically evoked spike collision in rats expressing channelrhodopsin-2 (Thy1-ChR2 transgenic rats) and found that M1-PT neurons exhibited a preferred
spike activity during contralateral forelimb movement, whereas M2-IT neurons were associatedwith both contralateral and ipsilateral forelimb movements (bilateral spike activity). Surprisingly,
the PPC neurons preferentially represented ipsilateral forelimb movements. To test the causality between neuronal activity and forelimb movements, we attempted to reproduce muscular
movements by optogenetic activation of these cortical areas using Thy1-ChR2 transgenic rats.
Consistent with the correlation between neuronal activity and forelimb movements, the optogenetic M1, M2 and PPC activation evoked contralaterally, bilaterally, and ipsilaterally biased
forelimb movements, respectively. Finally, we examined the effects of optogenetic manipulation
on task performance. In the VGAT-Cre rats, PPC or M1 inhibition by optogenetic GABA releaseshifted the behavioral limb preference contralaterally or ipsilaterally, respectively. In addition,
weak optogenetic PPC activation, which was insufficient to evoke motor responses by itself,
shifted the preference ipsilaterally; although similar M1 activation showed no effects on task
performance. Our findings suggest that the motor information processing that controls forelimb
movement rely on the orchestration of distinct cortical and projecting cell population. (COI:No)

O06-1

O06-4

2 1 Dept Physiol,
Junko Fujita-Yoshigaki1，Osamu Katsumata-Kato1, 2，Megumi Yokoyama1, （Mayu Takahashi, Yoshikazu Shinoda（ Dept Systems Neurophysiol, Grad Sch Med, Tokyo

Exocrine glands such as salivary glands have two secretory pathways: regulated and constitutive pathways. The mechanism to separate secretory proteins to the two pathways in salivary
gland cells remains to be clear. To analyse the proportion of secretory proteins transported
into the two pathways in salivary acinar cells, we prepared expression system of the HaloTagproteins that fused with full-length cystatin D (fCst5H), which is one of salivary proteins, andits signal peptide sequence (ssCst5H). HaloTag is a reporter protein that is designed to forma stable covalent bond with fluorescent ligands, thus can be used for pulse-chase analysis. At
24 h after the transfection of the genes into primary culture of parotid acinar cells, HaloTagproteins were labeled with TMR ligand. After washout of unbound ligands, the cells were
cultured for another 8 h. We collected culture medium and harvested cells to measure theamounts of HaloTag-fused proteins that were released to the medium and retained in thecells. After labeling samples with another HaloTag ligand, AlexaFluor 660 (AF660) ligand,samples were separated by SDS-PAGE and fluorescence intensities of the two ligands were
measured. Although the ratios of released and retained TMR-labeled fCst5H and ssCst5H
were comparable, the ratio of released and retained AF660-labeled fCst5H was lower thanthat of ssCst5H. To explain the difference between the two proteins, a mathematical model
was formulated. We defined kc and 1-kc as the proportions of transport into constitutive andregulated pathways, respectively, and assumed that constitutive and regulated pathways take60 min and 120 min to release secretory proteins. Simulation analysis revealed that kc of fCst5H
was lower than that of ssCst5H, which indicates that fCst5H was transported into regulated(COI:No)secretory pathways more efficiently than ssCst5H.

Sensory signals for eye movements (visual and vestibular) are initially coded in different framesof reference but finally translated into common coordinate and share the same final common
pathway, namely the same population of extraocular motoneurons. It is assumed that thesaccade system uses the horizontal and vertical Cartesian coordinates, based on the findingsthat the lesions in the PPRF and the riMLF cause impaired horizontal and vertical saccades,
respectively. Neural pathways for generating horizontal saccades from the superior colliculus(SC) were understood well, but those for generating vertical saccades have not been identified
yet. This study analyzed synaptic connections from the SC to vertical ocular motoneurons andtectoreticular saccade neurons (TRNs) in the opposite SC in anesthetized cats. TRNs in the
rostromedial SC activated superior rectus and inferior oblique motoneurons and TRNs in the
rostrolateral SC activated inferior rectus and superior oblique motoneurons via the riMLF.
These innervation patterns are similar to those in the VOR from the anterior canal and theposterior canal, respectively. Stimulation of the SC and recording postsynaptic potentials in
TRNs in the opposite SC showed that the inhibitory commissural connection exists between
the medial (lateral) SC representing upward (downward) oblique saccades on one side andthe lateral (medial) SC representing downward (upward) oblique saccades on the other side.
This pattern of reciprocal inhibition between the SCs is very similar to that seen between thebilateral vestibular nuclei in the oblique eye movements evoked from the anterior semicircular
canal on one side and the posterior semicircular canal on the other side. These similaritiesof the motoneuronal innervation patterns and the commissural inhibitions in the VOR andsaccade systems strongly suggest that both systems use the common semicircular canalcoordinate. (COI:No)

O06-2

O06-5

Masashi Shin1，Aya Matsushima2，Shihomi Mori1, 3，Fujio Okamoto1，Hiroshi Kajiya1，
1 1 Dept Physiol, Fukuoka Dent Col,
Hidemitsu Harada4，John Bartlett D.5，Koji Okabe（

Naoki Takada1, 2，Masaki Takasugi1，Akiyoshi Uezumi3，Hiroaki Nakamura2，Naoko Ohtani1

Simulation analysis of selective transport of cystatin D-fused HaloTagproteins to regulated and constitutive secretory pathways
Nihon Univ Dent Sch at Matsudo, Matsudo, Japan, 2 Res Inst Oral Sci, Nihon Univ Dent Sch
at Matsudo, Japan）

Time Lapse Imaging of Mouse Ameloblasts
Japan, 2 Fukuoka Dent Col, Japan, 3 Dept Oral Surg, Fukuoka Dent Col, Japan, 4 Dept Anat,
Iwate Med Univ, Japan, 5 Div Biosci, Ohio Sta Univ, Col Dent, USA）

Purpose: Tooth enamel is the hardest tissue in the body. Ameloblasts form enamel as they
move away from the dentin enamel junction. Each ameloblast forms an enamel rod and eachrod is the mineralized trail of the ameloblast as it migrates. However, how the ameloblasts
move relative to each other to form the enamel rod pattern is unknown. We have generated amouse model in which ameloblasts are labeled with a fluorescent protein (tdTomato) expressedby the amelogenin promoter. This mouse model enables us to monitor the movement of live
ameloblasts and allows us to isolate stage specific ameloblasts.
Materials ＆ Methods: The mandibular ameloblasts of AmelX-promoter-tdTomato (AT) mice
were cleared of fluorescence by immersion in CUBIC reagents and the cleared tissue wasobserved by light-sheet microscopy. Time-lapse imaging was performed ex vivo by use ofmandibular incisors from AT mice.
Results: tdTomato positive ameloblasts from AT mouse incisors were developmentally staged
and were quenched of tdTomato fluorescence. Next, mandibular incisors were dissectedfrom AT mice and live ameloblast movement was observed by time-lapse imaging ex vivo.
tdTomato positive cells elongated, arranged radially, and showed collective migration toward
the incisor tip.
Conclusion: For the first time, the dynamic movement of living mouse ameloblasts could
be observed and analyzed. This method will provide new insights in understanding how
ameloblast movement promotes enamel formation and calcification processes.
(COI:No)

Common coordinate of eye movements shared by saccadic andvestibulooculomotor systems
Medical and Dental Univ, Japan）

Mechanism of intramuscular ectopic fat formation in sarcopenic obesity（ 1 Dept Pathophysiol, Grad Sch Med, Osaka City Univ, Japan, 2 Dept Orthop. Surg, Grad Sch
Med, Osaka City Univ, Japan, 3Tokyo Metropolitan Institute of Gerontology）
Coexistence of sarcopenia and obesity, called sarcopenic obesity, is a major public health problem because its condition increases the risk of metabolic impairment and physical disability.
In sarcopenic obesity, the formation of intramuscular fat has attracted attention as one of thecauses of decreased skeletal muscle mass and quality. Platelet-derived growth factor receptoralpha (PDGFRα) positive mesenchymal progenitors have recently been identified and studied
as the origin of ectopic fat in skeletal muscle. PDGFRα-positive mesenchymal progenitors
rapidly proliferate following muscle injury, and contribute to the differentiation of satellite
cells, and promote skeletal muscle regeneration. Normally, PDGFRα-positive mesenchymal
progenitors that proliferate during muscle regeneration eventually undergo apoptosis. On theother hand, in pathological conditions such as aging or neuromuscular diseases, it has beenreported that some progenitors do not undergo apoptosis and differentiate into adipocytes.
In this study, we aimed to establish a mouse model to elucidate the mechanism of obese
sarcopenia. High fat diet-fed mice and genetically obese mice showed the ectopic fat cell formation, that should not be observed in normal situation, during muscle regeneration following
muscle injury and loss of muscle mass. The intramuscular ectopic fat was confirmed to be
derived from PDGFRα-positive mesenchymal progenitors by using PDGFRα-CreER mice forgenetic lineage tracing. In addition, we compared the gene expression of PDGFRα-positivemesenchymal progenitors before and after muscle injury between high-fat-diet and normal
diet-fed mice by using RNA-sequencing analysis. The analysis of the results indicated thatthe interaction with the extracellular matrix might be important for the differentiation of
PDGFRα-positive mesenchymal progenitors into adipocytes. In this meeting, we will show thedetails of the results and the ideas about the mechanism of the ectopic fat formation in skeletal
muscle.(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S75

Oral Session 7
Cell Physiology・Molecular Physiology 1（March 18, 13:20〜14:20, Hall 7）

O07-3

Chloride intracellular channel protein 2 prevents distant metastasis ofmalignant cells
Akihiro Umakoshi, Junya Tanaka（ Dept Physiol, Grad Sch Med, Ehime Univ, Japan）
We have established a distant metastasis model using immunocompetent Wistar rats bytransplanting rat glioma cell line C6 cells in the back of neonatals. The transplanted cells
formed a visible tumor mass in the back within two weeks, and the cells metastasized into thelung by 5 weeks. The most transplanted rats died by 7 weeks due to the distant metastasis.
Using this model, we transplanted C6 cells expressing enhanced green fluorescence protein(EGFP). Five weeks later, tumor masses were dissected from the back and the lung. Cells
with EGFP fluorescence were isolated from the both tumor masses using a cell sorter 5
weeks after transplantation. Cells from the two tumor masses were transplanted again into
the back of neonatal rats. Then, the cells from the lung tumor metastasized more frequently
and earlier than the other ones. Total RNA purified from the cells and analyzed by RNA-seq.
Consequently, chloride intracellular channel protein 2 (CLIC2) was found to be more highly
expressed by the cells from the back tumor than those from the lung one. Then, C6 cells
expressing CLIC2 at a higher level were established (C6-CLIC2 cells). C6-CLIC2 cells were notdifferent from the control C6 cells in terms of proliferation and migration, while their invasive
activity through Matrigel containing various extracellular matrix proteins was much weaker
than the control cells. When transplanted into the back, the metastasis by C6-CLIC2 cells weremuch suppressed and the survival periods of animals were much prolonged. The mechanisms
underlying the effect of CLIC2 expression are still to be elucidated, CLIC2 may strengthen
the cell-cell contacts by increasing expression adhesion molecules, leading to prevention of(COI:No)
intravasation of cells or hematogenous spread. 

O07-1

O07-4

1 1 Dept Mol
Tomonori Okazaki1，Kotoku Kawaguchi1，Takashi Nakahari2，Shinji Asano（

Chiaki Katagiri1, 3，Taku Kaitsuka2，Chigusa Shimizu O.4，Kazuhiko Tomizawa2，
1 1 Dept Molecul. Cell physiology, Grad Sch Med,
Chitoshi Takayama4，Masayuki Matsushita（

Pathophysiological Roles of an Actin-Binding Protein Moesin in Primary
Mouse Microglia
Physiol, Col Pharm Sci, Ritsumeikan Univ, Japan, 2 Res Unit for Epithelial Physiol, Res
Org of Sci and Tech, Ritsumeikan Univ, Japan）
Introduction: Microglia (MG) are immune cells in the central nervous system. In resting state,
MG with highly branched processes survey around cells whereas they retract their processes,
migrate into injury sites and remove neuronal debris by phagocytosis in response to injury.
They also secret many inflammatory cytokines and neuroprotective cytokines. Here, we focus
on the roles of an actin-binding protein moesin which is involved in morphological changes
through regulation of small GTPase; Rac, Rho and Cdc42 by using primary MG prepared frommoesin-knockout (Msn-KO) mice.
Methods: Primary MG were prepared from whole brain of newborn mice by the shaker
method. We examined phenotypes accompanying morphological changes and reorganizationof the actin cytoskeleton. Process retraction induced by LPS stimulation was observed under
microscope. Phagocytosis was evaluated by counting the numbers of FBS-coated fluorescencebeads incorporated in the cells in absence or presence of UDP. Migration was evaluated bycounting the number of migrated cells toward ADP by using Boyden chamber.
Results: The Msn-KO MG showed decreased rate of process retraction stimulated by LPS
compared with the WT MG. The numbers of FBS-coated fluorescence beads were significantly
decreased in the Msn-KO MG in the UDP-stimulated phagocytosis. The numbers of migrated
cells were significantly decreased in the Msn-KO MG in the ADP-stimulated migration assay. The Msn-KO MG treated ADP or UDP showed decreased numbers of ruffle membranecompared with the WT MG. However, the Msn-KO MG retained their activity to synthesize
and secrete tumor necrosis factor-α (TNF-α) and nitric oxide (NO) in response to LPS.
Conclusions: Moesin is not indispensable but involved in processes retraction, phagocytosis andmigration accompanying with actin-remodeling and membrane ruffling. However, moesin is not
involved in TNF-α nor NO secretion. (COI:No)

O07-2

Glutamate modulates activities of microglia through JAK/STAT/IRFdependent signaling pathway
Haruna Takeda, Kazuya Miyanishi, Kota Kanehisa, Kanta Mikami, Choudhury ME,
Hajime Yano, Junya Tanaka（ Dept Mol Cel Physiol, Grad Sch Med, Ehime Univ, Japan）
Glutamate is the most abundant excitatory amino acid neurotransmitter in the CNS. There
have been literatures describing that glutamate affects morphology and functions of microglia,
suggesting a possibility that glutamate mediates the interactions between neurons andmicroglia. Yet, the effect of glutamate on microglia has not been fully elucidated. Glutamate
upregulated mRNA levels for several activation markers, such as F4/80, CX3CR1, MMP2, Cathepsin S, MerTK and MFG-E8 in primary rat microglia culture. We investigated theeffects of glutamate on phagocytic activity of microglia by incubating microglia cells (BV2; amurine microglial cell line and primary rat microglia) with fluorescent beads. The internalized
bead fluorescence was evaluated by FACS. Glutamate enhanced uptake of florescent beads
or microglial phagocytosis. When incubated with glutamate, enhanced phosphorylation of
STAT1, STAT3 and expression of IRF1 protein was observed. Furthermore, siRNA-induced
knockdown of JAK1, STAT1, or IRF1 abolished the stimulating effects of glutamate on mRNA
expression that encoded CX3CR1, Cathepsin S, MFG-E8 and so on. However, glutamate did
not affect the expression of mRNA for inflammatory mediators, such as IL-1β, IL-6. Thus it issuggested that glutamate-induced activation of JAK-STAT pathway is responsible for phagocytosis rather than proinflammatory reactions. We further examined the effects of compounds
that suppress the JAK-STAT pathway on the actions of glutamate. Tofacitinib, a JAK1/3
inhibitor, suppressed glutamate-induced phosphorylation of STAT1, STAT3 and expression of
IRF1. Taken together, glutamate may activate JAK1 in microglial cells, causing activation of
the downstream signaling pathway. These results suggest a possibility that microglia change
their activities in response to activities of glutamatergic excitatory neurons. It is necessary to
determine whether the effects of glutamates are mediated by glutamate receptors expressedby microglia. (COI:No)

S76

kinase activity of TRPM7 involvement in the regulation of lipid transfer
Univ of Ryukyus, Japan, 2 Dept Molecul. Cell physiology, Grad Sch Med, Kumamoto Univ,
Japan, 3 Dept Neurosurgery, Grad Sch Med, Univ of Ryukyus, Japan, 4 Dept Molecul
anatomy, Grad Sch Med, Univ of Ryukyus, Japan）

TRPM7, a member of TRP family of cation channels, is a unique bifunctional protein containing an ion channel and a C-terminus kinase domain. However, the physiological functions of
TRPM7 and its kinase activity in vivo remain elusive. We generated kinase-inactive mutant
mice and analyzed their phenotype. TRPM7 mutant mice show normal ion channel activity without noticeable kinase function in cells isolated from adult animals. These mice havenormal body weight, food intake and general locomotor activity. Screening of serum clinicalparameters showed that serum Ca2+ and Mg2+ levels were not altered, but serum triglycerideand total cholesterol were significantly decreased. TRPM7 kinase deficient mice fed high-fat
diet increased the accumulation of fat and sclerosis of the liver compared to wild type mice.
Our findings define TRPM7 kinase activity as a key cell signaling component that regulates(COI:No)
lipid homeostasis in the liver.

O07-5

Temporal regulation of IL-1β induced JNK signaling dynamics
Taichiro Tomida, Kimitaka Yamaguchi, Masanori Ito, Yoshinori Mikami, Daisuke Ohshima,
Satomi Adachi-Akahane（Dept Physiol, Fac Med, Sch Med, Toho Univ, Japan）
Background: JNK (c-jun N-terminal Kinase) is a multi-functional kinase mediating stress andinflammatory response involved in cell death, proliferation, and cytokine production in pathologies of a wide variety of inflammatory disorders. It has been known that the duration of JNK
signaling determines the specificity of downstream cell functions that leads to either cell-deathor survival, but how the dynamics of JNK are regulated in cells remains largely unknown.
Aim: We aimed to understand how inflammatory cytokine IL-1β regulates JNK signaling
dynamics in living cells and unveil the impact of JNK dynamics on downstream gene expression that determines cell fates.
Methods: JNK reporter based on FRET was engineered to analyze JNK activation dynamics
in living HeLa cells. Periodic pulsatile IL-1β stimuli to cells at variable frequencies were employed to analyze frequency-response of JNK activation, which is subjected to systems-analysis.
Gene expression profiles were obtained by conducting microarray and qRT-PCR analyses.
Results: We succeeded in the quantitative evaluation of JNK dynamics at single cell resolution
upon IL-1β stimulation by FRET imaging. The frequency-response of JNK exhibited a typicalnegative-feedback-type regulation. We found that a phosphatase expressed downstream ofp38 MAPK functions as a negative regulator. Such regulation forces JNK to be transiently
activated even when cytokines are given continuously. In contrast, JNK activity was repeatedly re-activated when cytokines were repetitively applied as short pulsatile stimuli at interval>2hr. Consistently, we found that the expression of some JNK dependent genes are efficiently
induced by the repetitive short stimuli depending on the frequency rather than the total
duration of cytokine exposure.
Conclusion: Our novel approach combining FRET imaging and systems-analysis revealed akey mechanism that governs JNK dynamics in living cells. Such regulation may contribute to
avoid excessive inflammatory response while properly inducing adequate response throughgene expression in a temporally regulated manner.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Oral Session 8
Pathophysiology（March 18, 13:20〜14:20, Hall 8）

O08-3

Mitochondrial antidotal machinery against N6-isopentenyladenosine isessential for sustaining glioma-initiating cells
Atsushi Fujimura1, 2，Takahiro Yamamoto3，Fanyan Wei4，Kazuhito Tomizawa4（ 1 Dept Physiol, Grad Sch Med, Dent, Pharma, Okayama Univ, Japan, 2 Neutron Therapy
Research Center, Okayama Univ, Japan, 3 Dept Neurosurgery, Faculty Life Sci, Kumamoto
Univ, Japan, 4 Dept Mol Physiol, Faculty Life Sci, Kumamoto Univ, Japan）
Mitochondria are pivot points in various biological phenomena. In glioma, mitochondria control energy production and cellular metabolism to adapt to tumor microenvironment such as hypoxia, andthus contribute to sustaining cell viability and stemness, which further resulted in poor prognosis.
Mitochondrial function is sustained by genomic DNA-encoded proteins as well as mitochondrial
DNA-encoded proteins, whose expressions are tightly regulated by intra-mitochondrial translational
machinery. Previously, we identified CDK5RAP1 as one of the key regulators of intra-mitochondrial translation in skeletal and cardiac muscles. CDK5RAP1 converts N6-isopentenyladenosine to
2-methylthio-N6-isopentenyladenosine at the anticodon-loop of the several mitochondrial tRNAs,and consequently potentiates the accuracy and efficiency of the intra-mitochondrial translation.
Therefore, our starting hypothesis in this study was: CDK5RAP1 regulated cell viability and stemness by controlling intra-mitochondrial translation. To demonstrate this, we performed loss-of- andgain-of-function study with patient-derived glioma-initiating cell (GICs). As we expected, CDK5RAP1
was required to sustain GIC-related traits such as self-renewal capacity, tumor-propagating potential,
undifferentiated markers. However, to our great surprise, these phenomena were independent fromCDK5RAP1 regulation of intra-mitochondrial translation. In GICs, loss-of-CDK5RAP1 did not induce
the translational deficiency and respiratory deficit, but attenuate the GIC-related characteristics.
We found that CDK5RAP1 deficit resulted in the accumulation of N6-isopentenyladenosine, which
acted as cytotoxic agent in GICs. Mechanistically, the accumulated N6-isopentenyladenosine triggered autophagic programs as validated by autophagosome formation, AMPK activation, and mTOR
signaling pathway inhibition, which further resulted in loss-of-GIC related traits. By conversion of
N6-isopentenyladenosine to 2-methylthio-N6-isopentenyladenosine in mitochondria, CDK5RAP1
abrogated the antitumor effect of N6-isopentenyladenosine. Moreover, we demonstrated that hypoxic
condition activated CDK5RAP1 to promote amelioration of the tumor-suppressive effect of N6isopentenyladenosine. Our work shows that GICs utilize the detoxification mechanism to abrogate
endogenous N6-isopentenyladenosine, and indicated that the mechanism might be a good target todevelop anti-tumor strategy. (COI:No)

O08-1

O08-4

1 1 Johmoh
Yusuke Takatsuru1，Shunichi Motegi2，Keita Yonemochi3，Noriaki Hattori（
hospital, 2 Department of Radiology, Josai Clinic, 3 Gunma Prefectural College of Health
Sciences）

Youko Inukai, Satoshi Iwase, Motohiko Satou（Dept Physiol, Sch Med, Aichi Med Univ, Japan）

Preliminarily study on functional impairment of swallowing induce byscent stimulation

Segmental or unilateral hyperhidrosis accompanied by anhidrosis
in another area may be compensatory: estimated based on themechanism of the similar efferent phase of the physiological skinpressure−sweating reﬂex

In recently, number of elder people is extremely increase in Japan compared to other country.
Especially, number of “Kohki-kohreisha (over 75 years old people)” is increasing and patient
who suffered with geriatric syndrome is most popular and problem case in clinical field. Oneof the geriatric syndromes, disfunction of swallowing is most difficult condition because of it
inducing many diseases due to low nutrition. We actually have several supportive meals, butthey sometimes induce farther health problem (Takatsuru et al., 2019. https://doi.org/10.6890/
IJGE.201909_13(3).0017). Thus, to improve the swallowing function is important to keep elder
people in healthy and good quality-of-life condition. We previously found the patient who
improved the swallowing function by preference scent stimulation (data not published). In
this study, we planning to confirm the effect of preference scent on brain function including
swallowing. Healthy volunteer answered the questionnaire: preference scent (maple, peppermint, apple, coconuts, and Darjeeling tea. Both under blind and open manner question),food-intake-behavior (i.g., custom on meal intake, lively on food-intake such as visiting famous
restaurant and cook the new dish after watching cooking program on TV), and knowledge-onfood (i.g., knowledge on seasonal food, recipe of cooking). Part of them also perform functional
magnetic resonance imaging (fMRI) test with preference scent stimulation. We found that
scent detection score was decreased dependent on age. On the other hands, preference scentcould detect without depend on age. This result indicated that preference scent potentiallysuitable for stimulant on brain function without age. We also found that group of high scoreon the scent detection showed high score on the knowledge-on-food questionnaire. We next
analysis the result of fMRI to confirm the relationship between the brain area which activatedby preference scent and result of questionnaire on food-intake-behavior/knowledge-on-food.
(COI:No)

Background: Segmental or unilateral hyperhidrosis is a form of the sweating disorder. Most
cases with localized unilateral hyperhidrosis are considered idiopathic. Some of the secondary
localized segmental or unilateral hyperhidrosis are caused by direct overactivity of sympathetic neurons. While, the other cases of these disorders which are accompanied by anhidrosisor hypohidrosis in another area may be compensatory; it is likely caused by underlying lesions
in the areas with anhidrosis, but the precise mechanism remains unclear. This hyperhidrosis
often occurs on contralateral to the same dermatomes with anhidrosis, and ipsilateral rostraland caudal dermatomes adjacent to those of anhidrosis. The similar efferent phase of thephysiologicalskin pressure－sweating reflex: might be associated with these mechanisms. This reflex
is primarily due to inhibition of ipsilateral sweating by unilateral skin pressure; secondarily
sweating is increased on the contralateral same dermatome and ipsilateral adjacent otherdermatomes.
Objective: Pathophysiology of unilateral or segmental hyperhidrosis was estimated based on
experimental findings of the skin pressure－sweating reflex.
Study 1: Microneurography indicated that unilateral skin pressure reduced the amplitude ofsudomotor nerve activities ipsilaterally and increased this contralaterally. But those synchronisms were not changed.
Study 2: Studies using the ventilated capsule method during heating showed that sweating
decreased on the upper body and increased on the lower body by pressure on the skin ofbilateral back by lying supine. Central sudomotor sympathetic outflow in response to bodytemperature was simultaneously hyperactivated, indicating that sweating is enhanced incompensation to maintain a constant total sweating rate.
Conclusion: Segmental or unilateral hyperhidrosis in segments not directly affected may becompensatory.
(COI:No)

O08-2

O08-5

Mami Noda, Tetsushi Niiyama, Kosuke Aoi（Lab Pathophysiol, Grad Sch Phar Sci, Kyushu

Ching-Yi Tsai（Institute for Translational Research in Biomedicine, Chang Gung Memorial

Thyroid hormones (THs) are essential for the development and function of the central nervous
system (CNS). In the CNS, circulating thyroxine (T4) crosses blood-brain barrier via specifictransporters and is taken up to astrocytes, becomes L-tri-iodothyronine (T3), an active formof TH, by type 2 de-iodinase (D2). T3 is released to the brain parenchyma from astrocytes(glioendocrine system). In adult CNS, both hypo- and hyper-thyroidism, the prevalence infemale being 10 times higher than that in male, may affect psychological condition and potentially increase the risk of cognitive impairment and neurodegeneration including Alzheimers
disease (AD). We have reported that non-genomic effects of T3 on microglial functions and its
signaling and sex- and age-dependent effects of THs on glial morphology in the mouse brains ofhypo- and hyper-thyroidism. Behavioral changes also showed sex-dependence. The significant
effect of THs on synaptic spine in male and females hyperthyroidism was analyzed as well.
These results may help to understand physiological and pathophysiological functions of THs
in the CNS and how hypo- and hyper-thyroidism affect psychological condition and cognition.
(COI:No)

Acute hepatic failure is a devastating consequence of hepatotoxic liver injury that can leadto the development of neurological complication called hepatic encephalopathy (HE) and isassociated with 50-90% mortality without liver transplantation. Despite a highly challenging
clinical problem, systematic evaluations of the cellular mechanisms of HE-related mortality
are still lacking. Clinical studies showed that the degree of baroreflex dysregulation is related
to the severity of HE. At the cellular level, oxidative stress resulting from mitochondrial
dysfunction in brainstem nuclei in the baroreflex circuit, including nucleus tractus solitarii(NTS) and rostral ventrolateral medulla (RVLM) are known to result baroreflex dysregulation.
This study investigated whether the same mechanism underlies the high mortality in HE. An
azoxymethane (AOM)-induced acute liver failure model of HE employing C57BL/6 mouse wasused. Diffusion tensor imaging (DTI) of the brainstem was performed, together with bloodpressure, heart rate and indices of baroreflex recorded by radiotelemetry. Animals died within
20-36 h after AOM (100 µg/g, ip) injection. DTI further revealed that the connectivity between
the NTS and nucleus ambiguus (NA), the origin of the vagal innervation of the heart, wasprogressively disrupted though sustained, concurrent with impaired but persistent cardiac
vagal baroreflex. On the other hand, the connectivity between NTS and RVLM was progressively disrupted until its disappearance, coincidental with the abolition of baroreflex-mediatedsympathetic vasomotor tone that signifies brain death clinically. Results from JC-1 staining of
the ventrolateral medulla showed a decrease of mitochondrial membrane potential during HE,
alongside an elevation of reactive oxygen species and necrotic cell death and a reduction in
ATP level in the NTS or RVLM. Our results suggested that impairment of baroreflex takes
place during the progression towards death in HE, and mitochondrial dysfunction induces(COI:No)bioenergetic failure, oxidative stress and necrotic cell death are the culprits.

Cellular mechanism of psychiatric symptom and behavioral change
induced by thyroid dysfunction
Univ, Japan）

Contributions of mitochondrial dysfunction to baroreﬂex dysregulation in
hepatic encephalopathy
Hospital, Kaohsiung, Taiwan）

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S77

Oral Session 9

O09-3

Molecular mechanism of ion selectivity through the KcsA K+ channel
2 1 NanoLSI, Kanazawa Univ, 2 Facult
Takashi Sumikama1，Kenichiro Mita2，Shigetoshi Oiki（

Ion Channel・Receptor 1（March 18, 13:20〜14:20, Hall 9）

Med Sci, Univ Fukui）

Selectivity of the K+ channels is critical to maintain the resting potential. Conventional explanation for the selectivity is usually based on the difference in affinity: the affinity of K+ to the K+
channels is higher than that of Na+, so K+ preferentially permeates the K+ channels comparedwith Na+. However, our recent study (Sumikama and Oiki, JPS 2019) showed that the affinityof K+ to the KcsA K+ channel is low (Kd of a third K+ ion to the channel holding two ions is approximately 50 mM), posing a question for the explanation. Here, we examined Na+ permeation
through the KcsA K+ channel by molecular dynamics simulations and electrophysiologicalmeasurements. The measured Na+ current through the K+ channel at 350 mV was 1.2 pA(by experiments at 2 M) and 0.14 pA (by simulations at 1 M), and the conductance ratio (γK/γNa) was 78.3 (by experiments) and 38.5 (by simulations). That is, the selectivity is not so highas expected, and Na+ ions really but slowly permeate the K+ channel. The analysis on the ion
trajectories in the simulations indicates that the difference in the free energy barriers at both
intracellular and extracellular entrances causes the difference in the permeation rates between
K+ and Na+. Especially, the barrier at the extracellular side is high for Na+, blocking the influxof Na+. Thus, it was revealed that the selectivity is not originated by affinity, but by kinetics.
(COI:No)

O09-1

O09-4

Regulation of phosphoinositide distribution by voltage-sensing
phosphatase in mouse spermatozoa

Effects of removing mucus on HCO3 and CO2 transport in guinea-pig
pancreatic duct

Takafumi Kawai, Yasushi Okamura（ Grad. Sch. of Med., Osaka Univ, Japan）

Libin Liu（Dept Human Nutrition, Grad Sch Med, Nagoya Univ, Japan）

Voltage-sensing phosphatase (VSP) shows phosphoinositides phosphatase activity that iscoupled to membrane potential. In the present study, we report that VSP is activated in mice
sperm flagellum and generates unique subcellular distribution pattern of PtdIns(4, 5)P2. We
already found that VSP protein is expressed in matured sperm and the VSP convert PtdIns(4,5)P2 into PtdIns(4)P by MS/MS analysis. VSP-deficient sperm show severe defect in theirmotility after capacitation, but not before capacitation, resulting in significant reduction in success rate of fertilization in in vitro fertilization experiment. This was because abnormal Ca2+
influx occur in tail of VSP-KO sperm. The abnormal Ca2+ signal appears to be caused by theenhanced K+ conductance. Electrophysiological analysis indicates that K+ current that would
be derived from Slo3, sperm specific K+ channel is enhanced in VSP-/- sperm. However, thehigh PtdIns(4, 5)P2 affinity of Slo3 may not account for the altered K+ channel activity in sperm
flagellum, assuming that PtdIns(4, 5)P2 level in sperm flagellum is similar to other cell types,
such as neurons. Most interestingly, freeze-fracture electron microscopy analysis indicates that
normal sperm have much less PtdIns(4, 5)P2 in the principal piece than in the midpiece of theflagellum, and this polarized PtdIns(4, 5)P2 distribution disappeared in VSP-deficient sperm.
Thus, VSP appears to optimize PtdIns(4, 5)P2 distribution of the principal piece. Here we(COI:No)
discuss how such specialized PtdIns(4, 5)P2 distribution regulate the Slo3 activity.

In the exocrine pancreas, goblet cells and some duct cells produce mucin. When mucin issecreted out of the cells and hydrated, mucin forms mucus. Mucus layer is thought to protect
ductal cells from the harmful contents of the pancreatic juice. However, the physiological roleof mucus in pancreatic duct is largely unknown.
we examined the effects of removing mucus on HCO3－ and CO2 transport in guinea-pig
pancreatic duct cell. Interlobular pancreatic duct segments were isolated and the lumen was
microperfused separately from the bath. We tried to remove luminal mucus by applying
N-acetyl-L-cysteine (acetylcysteine, 6 or 12 mg/ml) for 6 min to the luminal perfusate. Intracellular pH (pHi) was measured in duct cells loaded with BCECF.
When ducts were luminally perfused with 125mM HCO3－-5% CO2 solution, luminal applicationof acetylcysteine caused (p<0.05) pHi increase by 0.14±0.02 (n=6). To examine HCO3－ and CO2transport across the apical membrane, bath and lumen were first perfused with HCO3－- CO2free Hepes-buffered solution and the luminal perfusate was switched to 125mM HCO3－-5% CO2
solution. pHi rapidly decreased due to CO2 diffusion into the cells and cells remained acidified.
Initial pHi decline was ~26% faster (p<0.05) after luminal treatment with acetylcysteine (n=6).
To examine HCO3－ permeability of the apical membrane, duct cells were stimulated by forskolin (1 µM) and depolarized by applying 70mM K+ to the bath. When the luminal perfusate
was switched to 125mM HCO3－-5% CO2 solution, pHi transiently dropped and then increased
due to HCO3－ entry into the cells via CFTR. The rate of pHi increase was ~43% faster (p<0.05)
after treatment with acetylcysteine (n=6).
These data suggest that removal of luminal mucus enhances HCO3－ and CO2 permeability of
the apical membrane in guinea-pig pancreatic duct cell.
(COI:No)

O09-2

O09-5

Involvement of Pannexin in the trigeminal ganglion in trigeminal
neuropathic pain
Ryoko Kurisu1, 2, 3，Masamichi Shinoda3，Yoko Yamazaki2，Masahiko Shimada1, 2，
3 1 Dept Orofacial pain management, Grad Sch Med Dent Sci, Tokyo Med Dent
Koichi Iwata（

Univ, Tokyo, Japan, 2 Dept Orofacial Pain Clinic, Dent Hosp, Tokyo Med Dent Univ, Tokyo,
Japan, 3 Dept Physiol, Nihon Univ Sch Dent, Tokyo, Japan）
Trigeminal nerve injury occasionally causes orofacial neuropathic pain, which is difficult todiagnose and treat. It is essential to know the mechanisms underlying orofacial neuropathic
pain to develop the appropriate treatment of these patients. The pannexin (Panx) which facts
predominantly as a transmembrane channel connecting the intracellular and extracellularspace is reported to be involved in the regulation of the excitability of ganglionic cells. In
this study, we examined the involvement of Panx in the trigeminal ganglion (TG) in orofacial
neuropathic pain following partial infraorbital nerve ligation (pIONL) in rats.
Male Sprague-Dawley rats (200g-260g) were used in this study. We established pIONL model
rats by tight ligation with 6-0 silk of one-third thickness of the left infraorbital nerve underdeep anesthesia. Mechanical head-withdrawal threshold (MHWT) of left whisker pad skin wasmeasured using von Frey filament every other day before and 14 days after pIONL. On
day 14, Panx expression in TG was also assessed immunohistochemistry. Moreover, MHWTs
were measured after pIONL with the continuous intra-TG administration of pannexin inhibitor(10Panx; 1 microl, 100 mM).
The MHWT of the whisker pad skin ipsilateral to pIONL was significantly decreased on day
1 after pIONL, and the decrease of MHWT persisted until day 14. Panx was expressed in TG
neurons and satellite glial cells in TG, and the number of Panx-immunoreactive TG neurons innervating the whisker pad skin was increased on day 14. The intra-TG 10Panx administration
significantly recovered the decreased MHWT after pIONL.
The present findings suggest that Panx expressed in TG neurons and satellite glial cells in TG
is involved in orofacial mechanical allodynia associated with pIONL.
(COI:No)

S78

Secretory reﬂex pathway of short chain fatty acids in the rat distal colon
Daiki Harata1，Shinji Asano2，Atsukazu Kuwahara3，Yuko Kuwahara3，Ikuo Kato4，Toshio Inui5，
7 1 Dept Mol Physiol, Grad Sch Life Sci, Ritsumeikan Univ, Japan,
Yoshinori Marunaka3, 6, （2 Dept Mol Physiol, Col Pharm Sci, Ritsumeikan Univ, Japan, 3 Res Unit for Epithelial Physiol,
Res Org of Sci and Tech, Ritsumeikan Univ, Japan, 4 Dept Med Biochem,Col Pharm Sci, Kobe
Pharm Univ, Japan, 5 Saisei Mirai Clinics, Japan, 6 Res Inst for Clin Physiol, Kyoto Ind Health
Assoc, Japan, 7 Dept Mol Cell Physiol, Grad Sch Med Sci, Kyoto Pref Univ Med, Japan）

Propionate, a short chain fatty acid, induces biphasic ion transport, K+ secretion followed by
Cl－/HCO3－ secretion in rat distal colon. These secretory responses are mediated via activationof an enteric neural reflex circuit constituted by afferent, inter- and efferent neurons. Previous
study showed that efferent neurons stimulated by propionate is cholinergic, however, afferent
neurons stimulated by propionate still remained uncertain. Propionate stimulates free fatty
acid receptor 2 and 3 (FFA2/3) expressed in enteroendocrine L cells. Stimulation of thesereceptors leads to release of neuropeptides such as GLP-2 from L cells. As gastrointestinal
tract is innervated by intrinsic and extrinsic neurons, we hypothesized that released GLP-2
stimulates intrinsic and extrinsic afferent neurons, and the stimulated afferent neurons releasetachykinins. Therefore, in the present study, the contribution of these neuropeptides to afferent
neuronal reflex pathway stimulated by propionate, and in addition, the involvement of intrinsic
and extrinsic neurons to the neural reflex circuit were studied. We prepared muscle-strippedmucosa-submucosa preparation of rat distal colon and measured short-circuit current (Isc) as
an indicator of net ion transport using Ussing chamber. Pretreatment with tetrodotoxin (TTX)or a blocker of Nav1.8, TTX-insensitive voltage-dependent Na+ channel, A803467, alone had
no effect on ion transport stimulated by propionate, however, simultaneous pretreatment with
TTX and A803467 suppressed Cl－/HCO3－ secretion, suggesting a synergistic contribution of
intrinsic and extrinsic neurons within the neural reflex circuit. GLP-2(3-33) and GLP-2(11-33),
GLP-2 antagonists, suppressed K+ secretion stimulated by propionate. Pretreatment with
CP96345, an NK1 antagonist, suppressed Cl－/HCO3－secretion, and on the contrary, osanetant,an NK3 antagonist, enhanced Cl－/HCO3－ secretion stimulated by propionate. In conclusion,
it is considered that propionate-induced colonic ion transport is mediated via activation ofneural reflex circuit constituted by intrinsic and extrinsic neurons and the afferent pathway is
mediated via GLP-2 and tachykinins.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Oral Session 10
Autonomic Nervous・Endocrinology（March 18, 13:20〜14:20, Hall 10）

O10-1

Nicotinic cholinergic modulation of olfactory bulb function
Sae Uchida, Fusako Kagitani（Dept Auton Neurosci, Tokyo Metropol Inst Gerontol, Japan）
Olfaction is known to severely decline in the preclinical stage of Alzheimer’s disease, in addition to the age-related impairment. The olfactory bulb, the first olfactory center, receives
cholinergic basal forebrain input, as does the neocortex. Our previous study showed that
nicotinic acetylcholine receptors (nAChRs) in the brain play a crucial role in vasodilation in the
neocortex that is induced by basal forebrain cholinergic activation or nicotine injection. In thisstudy, we examined cholinergic regulation of blood flow in the olfactory bulb, using adult rats
anesthetized with urethane.
Focal chemical stimulation by microinjection of L-glutamate into the horizontal limb of the
diagonal band of Broca (HDB) in the basal forebrain which is the main source of cholinergic
input to the olfactory bulb increased extracellular ACh release in the ipsilateral olfactory bulb.
When the regional cerebral blood flow was measured using laser speckle contrast imaging, thefocal chemical stimulation of the HDB did not significantly alter the blood flow in the olfactorybulb, while increases were observed in the neocortex.
Next, we investigated the effect of nAChR activation on the odor-induced olfactory bulb
blood flow response. Odor stimulation increased olfactory bulb blood flow, without changes in
neocortical blood flow and systemic blood pressure. Intravenous injection of nicotine (30 µg/kg), a nAChR agonist, significantly augmented the odor-induced increase response of olfactory
bulb blood flow. This nicotine-induced augmentation was negated by α4β2-preferring nAChR
antagonist.
We conclude that basal forebrain cholinergic activation increases extracellular ACh release in
the olfactory bulb. The increased ACh does not produce vasodilation at rest in the olfactorybulb. Activation of nAChRs in the brain potentiates the odor-induced blood flow response in(COI:No)the olfactory bulb.

O10-3

Protective role of orphan nuclear hormone receptor COUP-TFII in acutekidney injury
Sumiyasu Ishii, Noriyuki Koibuchi（Dept. Integr. Physiol, Gunma Univ. Grad. Sch. Med,Japan）

Background and Aims: An orphan nuclear hormone receptor chicken ovalbumin upstream
promoter-transcription factor II (COUP-TFII) plays essential roles in organogenesis of embryos. Recently COUP-TFII is also implicated in several diseases in adults. Here we focus on
the role of COUP-TFII in cisplatin-induced acute kidney injury (AKI).
Methods: Tissue distribution of COUP-TFII was analyzed by RT-qPCR. Male tamoxifeninducible COUP-TFII-knockout mice or control mice were intraperitoneally treated with 30
mg/kg body weight of cisplatin at 12 weeks old to induce AKI. The kidney samples were
subject to morphological studies, TUNEL assay, immunohistochemistry and RT-qPCR.
Serum levels of creatinine, blood urea nitrogen and tumor necrosis factor alpha (TNF-α)
were measured. Depletion of COUP-TFII using siRNA followed by RT-qPCR and chromatin
immunoprecipitation were done in VA-13 cells.
Results: COUP-TFII was the most abundantly expressed in the kidney among organs.
Administration of cisplatin induced a more severe AKI in adult COUP-TFII-knockout mice.
An increase in dead cells in both proximal tubules and thick ascending limb of Henle’s loop
(TAL) was observed in the knockout mouse kidney. The expression levels of COUP-TFII
decreased in the TAL by cisplatin administration. There was no difference in the expression
levels of transporter mRNAs responsible for cellular cisplatin uptake between control andknockout mouse kidney. COUP-TFII-knockout mice and COUP-TFII-depleted cells exhibited
an elevation in TNF-α levels, suggesting the involvement of the TNF-α pathway. Chromatin
immunoprecipitation showed that COUP-TFII was enriched in the potential binding site,
suggesting that COUP-TFII might directly suppress the TNF-α gene at transcriptional level.
Conclusions: These results indicate the involvement of COUP-TFII in the pathophysiology of
AKI and COUP-TFII may be a potential therapeutic target for AKI.
(COI:No)

O10-4

Effects of Aquilaria subintegra leaves extract on learning and memoryof ovariectomized rats
Khachanan Inthiwong, Onrawee Khongsombat, Pornnarin Taepavarapruk（ Department of
Physiology, Faculty of Medical Science, Naresuan University）

Aquilaria subintegra (AS) is one of Aquilaria species which able to produce agarwood. Mostof the agarwood is processed into oil which is used in perfumes and in the production of
traditional medicine as anti-asthma, anti-stress, painkiller, and many other diseases. AS leaf
extracts are also known to possess antipyretic, laxative and antimicrobial activities. Data froman in vitro study suggested the potential of AS to treat neurological related disease such as
Alzheimer’s disease (AD) due to its ability to inhibit acetylcholinesterase and its anti-oxidant
properties.
Aim of the study: This study aimed to investigate the effect of AS leaves extract on learningand memory of ovariectomized rats that has been widely used as an animal model of AD.
Materials and method: Female Sprague Dawley rats were subjected to bilateral ovariectomy(OVX). Following a full recovery, AS leaves extracts (10, 100, 1000 mg/kg B.W.) were orally
administered for 60 days. The negative control group received RO water and the positive
control group received donepezil. Novel object recognition (NOR) and Morris water maze
(MWM) tests were employed to evaluate their cognitive functions. The levels of superoxide
dismutase (SOD), malondialdehyde (MDA), catalase (CAT), as well as acetylcholinesterase(AChE) activities in the hippocampus were measured.
Results: OVX rats received AS leaves extract for 60 days showed a significant improvement
in both object recognition and spatial memories when compared to the OVX control group.
Treatment with AS leaves extract also induced significant increases in the activities of SOD,
CAT and AChE and reduced the level of MDA in the hippocampus when compared to the
OVX group.
Conclusions: AS leaves extracts could improve cognitive and memory impairments in female
OVX rats by the antioxidant activities and acetylcholinesterase inhibition of the leaves, therefore AS may be useful in reducing the risk of AD in postmenopausal women.
(COI:No)

O10-2

O10-5

Satoshi Koba, Nao Kumada, Tatsuo Watanabe（Div Integr Physiol, Tottori Univ Fac Med,

Sirion Aksornthong1, 2，Wacharaporn Tiyasatkulkovit2, 3，Nattapon Panupinthu1, 2，
5 1 Department of Physiology, Faculty of Science,
Narattaphol Charoenphandhu1, 2, 4, （Effect of ionotropic glutamate receptor blockade in rat RVLM on
sympathomotor activation by mesencephalic locomotor neurons
projecting to the RVLM
Japan）

Our preliminary data showed that mesencephalic locomotor neurons sending axonal projections to the rostral ventrolateral medulla (RVLM) (MLR-RVLM pathway) are capable of eliciting sympathomotor activation in rats, suggesting that the MLR-RVLM pathway is a part ofbrain circuitries for central command. This study examined the effect of ionotropic glutamate
receptor blockade in rat RVLM on sympathomotor activation by the MLR-RVLM pathway.
Male rats received microinjections bilaterally in the RVLM with a retrograde adeno-associatedvirus that encoded a channelrhodopsin with green fluorescence protein. Under anesthesia,
473 nm wavelength laser illumination of the MLR after administration in the RVLM with acocktail of AP5 and CNQX, NMDA and AMPA glutamate receptor blockers, respectively,
elicited 29% less (P<0.05) renal sympathoexcitation than that after saline administration (n =6). Likewise, ventral root excitation in response to laser illumination of the MLR seen aftersaline administration in the RVLM of decerebrate, nonanesthetized rats was suppressed to a
65% extent (P<0.05) by glutamate receptor blockers administered preliminarily (n = 6). These
results indicate that ionotropic glutamate receptor blockade in rat RVLM reduced sympathomotor activation caused by excitation of the MLR-RVLM pathway. Glutamatergic neurons in
the MLR-RVLM pathway likely play a significant role in generating central command function.
(COI:No)

Elevated Blood Pressure induced by High-Salt-Diet Consumption
Caused Negative Calcium Balance and Bone Loss in Rats
Mahidol University, Bangkok 10400, Thailand, 2 Center of Calcium and Bone research,
Faculty of Science, Mahidol University, Bangkok 10400, Thailand, 3 Department of Biology,
Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand, 4 Institute of
Molecular Biosciences, Mahidol University, Nakhon Pathom 73170, Thailand, 5The
Academy of Science, The Royal Society of Thailand, Bangkok 10300, Thailand）

With the change in dietary consumption in the modern lifestyle especially the exceeding amountof salt, high-salt-diet (HSD)-induced hypertension will become worldwide problem. Beside the riskof cardiovascular diseases, clinical studies demonstrated low bone mass and high risk for bone
fracture in hypertensive patients. However, investigation of association between hypertension
and bone loss is still controversy. In vitro study also showed direct effect of high NaCl environment on enhancing osteoclast differentiation and bone resorption. Nevertheless, data from invivo model of HSD-induced hypertensive model have been limited. Therefore, this study aims toinvestigate whole-body calcium balance and bone micro-structure in HSD-induced hypertensive
rats. The male Sprague-Dawley rats received HSD (HSD; 8% NaCl) or normal diet (Control; 0.8%
of NaCl) for 5 months. As expected, rats that received HSD showed significant elevation of bloodpressure with the onset at first month and remained hypertensive throughout the study (SBP
132.22±18.5 mmHg and DBP 115.04±2.96 mmHg) with marked hypertrophy of cardiac myocytes
shown by histology. Next, calcium balance study was performed monthly. We found drasticallycalcium loss via urine and feces, together with decreased fractional calcium absorption leading tonegative calcium balance, especially 3-month after HSD induction. Micro-computed tomography
analysis demonstrated significant decreased trabecular bone mineral density (BMD) of tibia andthe increasing medullary area of tibia diaphysis at 5 month of HSD induction. Consistent with thenegative calcium balance and impaired bone microstructures, bone histomorphometric analyses
showed a significant decrease in osteoblast number and osteoid volume in rats receiving HSD for5 months. These findings suggest HSD-induced hypertension caused negative calcium balance(COI:No)and reduction in BMD via impaired bone formation.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S79

Oral Session 11

O11-3

Functional Coupling of Metabolic Sensors, TRPM2 and Sirtuin
1 1 Dep Physiol, Aichi Med Univ,
Makiko Kashio1，Makoto Tominaga2, 3，Satoru Masubuchi（

Ion Channel・Receptor 2（March 19, 11:00〜12:00, Hall 4）

Aichi, Japan, 2 Div Cell Signal, NIPS, Okazaki, Aichi, Japan, 3Thermal Biology Group,
ExCELLS, Okazaki, Aichi, Japan）

TRPM2 is a thermosensitive non-selective cation channel expressed in various tissues including brain, spleen and pancreatic b-cells where TRPM2 is continuously affected by core bodytemperature. TRPM2 activity at body temperature could be regulated along with metabolicstate because its activity is affected by intracellular factors reflecting cellular metabolism suchas NAD+ metabolites, Ca2+ and redox signal. Therefore, TRPM2 is suggested to function as
body temperature/metabolic sensor.
Additional metabolic sensors, sirtuins, are a group of NAD+-dependent enzymes to regulateenergy homeostasis, circadian rhythm and longevity, etc. Because sirtuins generate o-acetyl
ADPR (OAADPR), a TRPM2 activator, along with its enzymatic activity, we are interested infunctional coupling of TRPM2 and sirtuins, especially in SIRT1 which is present in cytoplasm
to interact with TRPM2 in plasma membrane.
Intracellular Ca2+-imaging of TRPM2/SIRT1-expressing HEK293T cells has revealed that a
SIRT1 activator leads TRPM2 activation and intracellular Ca2+-elevation. Moreover, immunoprecipitation studies have clarified physical interaction between TRPM2 and SIRT1 whichis enhanced by phorbol ester (PMA)-treatment. Analysis of phosphoproteins using phos-tag
SDS-PAGE has shown that PMA-treatment also increases TRPM2 phosphorylation, suggesting an PKC-mediated effective coupling of TRPM2 and SIRT1.
We’d like to discuss possible regulatory mechanisms and physiological functions of metabolic(COI:No)sensors, TRPM2 and SIRT1.

O11-1

O11-4

Fumitaka Fujita1, 2, 3, 4，Yoshiro Suzuki7，Kaori Saito1, 2, 3，Masayuki Takaishi1, 4，
6 1 Mandom corporation, 2 Advanced
Fumihiro Okada1, 2，Makoto Tominaga4, 5，Ken J. Ishii 3, （Prakash Arullampalm, Jean-Sébastien Rougier, Lijo Cherian, Hugues Abriel（University ofAluminum ion blocks hTRPV1 and hTRPA1 activities
Cosmetic Science, Graduate School of Pharmaceutical Sciences, Osaka University, 3 Center
for Vaccine and Adjuvant Research (CVAR), National Institutes of Biomedical Innovation,
Health and Nutrition (NBIOHN), 4Thermal Biology Group, Exploratory Research Center onLife and Living Systems National Institutes of Natural Sciences, 5 Department of
Physiological Sciences, SOKENDAI, 6The Institute of Medical Science, The University ofTokyo, 7 Iwate Medical University）
Aluminum is utilized in human life from ancient time. Aluminum potassium sulfate has beenused as an antiperspirant from the Roman period. In modern medicine, aluminum compounds
are formulated in several vaccines as adjuvants which enhance immune responses at vaccination. On the other hand, aluminum pottassium sulfate shows the anti-inflammatory effect inJapanese spa. Putting all this together, aluminum compounds have several effects on humanbody including opposite one. We hypothesize that aluminum shows different effects in the
different states. For clarification of the aluminum effect, we focused on thermo-sensitive Transient Receptor Potential (TRP) channels because we already found alumminum ion activateshTRPM4. TRP channels expressed in sensory neuron were evaluated whether aluminum ion
affects their activities. In calcium imaging experiments, aluminum ion blocked hTRPV1 andhTRPA1 activities, but not hTRPM8, hTRPV3 or hTRPV4 ones. IC50 values of aluminum
potassium sulfate at pH 5.0 on hTRPV1 and hTRPA1 were 103 µM and 246 µM, respectively.
Futhermore, aluminum potassium sulfate inhibited hTRPV1 and hTRPA1 even at the neutral
pH 7.4. These results indicated that aluminum plays a role as an analgesic agent at the ionicstate, suggesting aluminum compounda used as vaccine adjuvant might have the analgesic roleat vaccination. (COI:No)

Deletion of Trpm4 alters the function and expression of NaV1.5 channel
in mouse cardiac myocytes
Bern, Institute of Biochemistry and Molecular Medicine）

Transient receptor potential melastatin member 4 (TRPM4) encodes a Ca+-activated nonselective cation channel and expressed in several tissues including the heart. Pathogenic
mutants in the TRPM4 gene have been reported in patients with inherited cardiac diseases
including conduction blocks and Brugada syndrome but its role in cardiac channelopathy is still
unclear. To study the functional consequences on cardiac electrical activity due to the deletionof Trpm4 (Trpm4-/-) in mice, we performed perforated patch-clamp and immunohistochemistry studies on isolated atrial and ventricular cardiac myocytes. We demonstrate that Trpm4
is expressed in atrial and ventricular cardiac myocytes and Trpm4 deletion reduces the peakNa+ current in the myocytes.
Furthermore, we performed ex-vivo pseudo-surface (sECG) and intracardiac (iECG) electrocardiogram on a Langendorff setup. The heart is perfused retrogradely by the coronary arteries.
We used two silver electrodes on the heart for pseudo surface ECG (sECG) and inserted an
octapolar intracardiac catheter via the right atrium to the apex of right ventricle to record
ex-vivo intracardiac ECG (iECG).
In iECGs four out of five Trpm4-/- mice hearts show intraventricular conduction delay to reach
impulse from apex of right ventricle to 3mm proximal part of right ventricle. We did not see
any delay in Trpm4 +/+ mouse heart. Moreover, we challenged those hearts with mexiletine(Sodium channel blocker). Surprisingly Trpm4-/- mice hearts are less sensitive to mexiletine.
This study provides the first evidence that Trpm4 shows directly impact on Na+ current inmouse cardiac myocytes and Trpm4 prevents sensitivity of mexiletine. In the further, we will
apply our newly identified mouse-specific Trpm4 blockers and some other INa blockers tounderstand more about the role of Trpm4 regulating Na+ current in mouse heart. (COI:No)

O11-2

O11-5

Yaopeng Hu1，Daniela Ross Kaschitza2，Maria Essers2，Prakash Arullampalam2，
1 1 Dept Physiol, Sch Med, Fukuoka Univ,
Yuanyuan Cui1，Hugues Abriel2，Ryuji Inoue（

1 1 Dept Synthetic
Akito Nakao1，Yoshihiro Matsunaga1，Takafumi Miki2，Yasuo Mori（
Chemistry and Biological Chemistry, Grad Sch Engineering, Kyoto Univ, Japan, 2 Grad Sch
Brain Science, Doshisha University, Japan）

Recent evidence indicates that enhanced activity of TRPM4 contributes to acquired arrhythmic changes under stressed conditions. It is reported that TRPM4 channel activity isstrongly modified by Ca2+/calmodulin, but the mechanism underlying it remains unknown.
We therefore investigated it by noting a potential significance of CaMKII-mediated TRPM4channel overactivation under disrupted Ca2+ homeostasis by both cellular experiments andnumerical simulations.
All experiments were performed in TRPM4 expressing HEK293 cells and HL-1 cardiomyocytes.
The possible protein-protein interaction was tested by the Duolink immunoassay. In order toquantitatively evaluate how CaMKII modifies TRPM4 activation, the average relationshipsof steady state open probability potential and time constant for voltage-dependent activation
versus membrane potential at different [Ca2+] i were reconstructed. Numerical descriptions for
TRPM4 channel gating both with and without treatment of a CaMKII inhibitor KN-62 wereobtained as well. In TRPM4 expressed HL-1 cells, the incidence of early afterdepolarizations(EADs) was increased after incubation with AngII which also activated the CaMKII signaling.
Both TRPM4 channel blocker 9-phenanthrol and KN-62 could suppress this arrhythmic change.
Mathematical simulation indicated that a prominent increase in TRPM4 current density
induced EADs at the late repolarization phase. Intriguingly, incorporation of altered rate
constants after treatment with KN-62 could significantly alleviate these abnormal excitations.
In this study, an important mechanism underlying CaMKII-mediated TRPM4 channel regulation under stressed condition was displayed. The simulation described here could facilitate
our understanding about electrophysiological changes induced in remodeled cardiomyocytes.
(COI:No)

The dynamics and localization of voltage-dependent Ca2+ channel (VDCC) complexes are crucial
for neuronal excitability, neurotransmitter release, and Ca2+-induced gene regulation. Recently,
VDCC beta-anchoring and -regulatory protein (BARP) was identified as a novel regulator of
VDCC activity via the interaction with VDCC β subunits. However, its molecular mechanismand physiological significance have been largely unknown. In this study, we demonstrated
that BARP reduced the surface expression level of α1 subunit, resulting in the decrease ofcurrent density of VDCC. Fluorescence recovery after photobleaching assay revealed that
BARP accelerated the fluorescence recovery of α1 and β subunits, presumably because
BARP increases the dynamics of VDCC subunits for the switching and targeting of VDCC
complexes. In cultured hippocampal neurons, BARP targeted α1 and β subunits to axonal anddendritic protrusions. Electron microscopic study showed that hippocampal neurons of BARP
knockout mice had a lower density of spines and shorter-necked spines. These results suggestthat BARP is a key regulator of the localization and dynamics of VDCC complexes for various(COI:No)
physiological events such as proper formation of dendritic spines.

A theoretical approach to investigating the arrhythmogenecity of
TRPM4 channel overactivation via CaMKIIJapan, 2 Biochem Mol Med, Bern Univ, Switzerland）

S80

BARP is a key regulator of the localization and dynamics of voltagedependent Ca2+ channel complexes

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Oral Session 12
Sensory Function（March 19, 11:00〜12:00, Hall 5）

O12-3

Presynaptic feedback system of oxytocin-ergic neurons in the
hypothalamus of adjuvant arthritic rats
Takanori Matsuura1, 2，Teruaki Fujitani1，Makoto Kawasaki1，Hitoshi Suzuki1，
1 1 Department ofHaruki Nishimura1, 2，Kazuhiko Baba1, 2，Yoichi Ueta2，Akinori Sakai（

Orthopedics, School of Medicine, University of Occupational and Environmental Health,
Kitakyushu, Japan, 2 Department of Physiology, School of Medicine, University ofOccupational and Environmental Health, Kitakyushu, Japan）
Background and aims: Oxytocin (OXT) is a neurohypophysial hormone that is synthesized in
the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus. Recently, it has
been suggested that OXT plays a role in sensory modulation. It was shown that OXT was
up-regulated by acute and chronic nociception. However, the up-regulated mechanism is little
known.
Methods: In the present study, we examined excitatory postsynaptic currents (EPSCs) in OXTergic neurons in the PVN after chronic inflammation from an adjuvant arthritis (AA) model
rat using the whole cell patch clamp recording. We used transgenic rats that expressed OXT
and the monomeric red fluorescent protein 1 (mRFP1) fusion gene to visualize the OXT-ergic
neurons. To induce AA, OXT-mRFP1 transgenic rats were intracutaneously injected heat-killed
Mycobacterium butyricum (1 mg/rat) in paraffin liquid at the base of their tails. Furthermore, thefeedback system of synthesized OXT was also examined by OXT receptor antagonist (L-368,899).
Results: We found that the frequency of miniature (m)EPSCs and spontaneous (s)EPSCs in
OXT-mRFP1 neurons in the PVN has significantly increased in AA rats. Further, L-368,899 dosedependently even more increased the frequency of sEPSCs and mEPSCs in the neurons in AA
rats. In bath application of GABAA receptor antagonist (picrotoxin) and cannabinoid receptor
1 antagonist (AM 251), L-368,899 still increased the frequency of mEPSCs. However, under thebath application of NOS inhibitor (L-NAME), L-368,899 did not change the frequency in AA rats.
Conclusions: It is suggested that OXT-ergic neurons are up-regulated by increasing glutamate
release in the AA rats, and up-regulated OXT neurons have a feedback system by released
OXT. It is possible that NO but not GABA may contributes to the feedback system of up(COI:No)
regulated OXT neurons.

O12-1

O12-4

2 1 Dept Neurosci, Grad Schl Med, Kyoto Univ, Japan,
Masatoshi Kasai1，Tadashi Isa1, （Yuto Tei1, 2，Yoshinori Mikami1，Masanori Ito1，Taichiro Tomida1，Daisuke Ohshima1，
1 1 Department of Physiology, Faculty of Medicine,
Yuichi Hori2, Satomi Adachi-Akahane（

Effect of light isoﬂurane anesthesia on orientation selectivity in mousesuperior colliculus
2 ASHBi, Kyoto Univ, Japan）

Activation of astrocytes in the trigeminal nucleus associated with ocular
neuropathic pain in a rat dry eye modelSuperior colliculus (SC) is a brain stem center which plays key roles in generating spatialattention and mediating the signal for sensory-motor translation. Recent studies wide filed or
two-photon calcium imaging techniques reported that the superficial layer of the SC (sSC) has
orientation columnar like functional structures (Feinberg ＆ Meister, 2014) or the global spatial
segregation of direction selective (DS) or orientation selective (OS) cells in accordance with its
retinotopic locations (Malmazet et al., 2018). But it remains unclear what kind of rules regulate
this functional structure.
Here we investigated effects of anesthesia on DS and OS in the mice sSC. We delivered
GCaMP6f calcium sensor to the sSC neurons by injecting Adeno-associated virus (AAV) vector(AAV1-hSyn-GCaMP6f) after making a small suction in caudo-medial part of the cortex. To
achieve long-term optical access to the SC, we implanted a small glass cube on the sSC.
The Neuronal population responses were recorded by in vivo two photon calcium imaging.
Direction and orientation selectivities of the sSC neurons were tested by applying moving bar
stimuli (width: 3–5 deg, speed: 10–20 deg/s, 12 directions) and calculating global direction ororientation selectivity index.
First, we test the effect of isoflurane. At normal maintenance level of isoflurane administration(2–3%) caused strong response suppression as seen in the sensory cortices. But during the
combination of light isoflurane (0.8–1%) with chlorprothixene change the preferred orientation
or direction in similar angles. On the other hand, the ketamine-xylazine anesthesia did notchange the selectivity at the population level. These results suggest that the DS and OS
could modulated by GABAergic inhibitory network, which is activated by isoflurane. NMDA
receptor mediated excitatory network, which suppressed by ketamine, have little effect on DS
and OS modulation in the sSC.
(COI:No)

Toho University, 2 Department of Ophthalmology, School of Medicine, Toho University）

O12-2

O12-5

Kazuhiro Horii1，Hiroki Shimaoka1，Yuuki Horii1，Takahiko Shiina1，Yasutake Shimizu1, 2

2 1 Dept Dev Neurophysiol, Grad Sch Med,
Takeshi Imai1, 2，Shigenori Inagaki1，Ryo Iwata（

Inhibitory effect of oxytocin on the regulation of colorectal motility via
descending pain inhibitory pathways in hyperalgesic rats
（ 1 Lab Vet Physiol, Unit Grad Sch Vet Sci, Gifu Univ, Gifu, Japan, 2 Center for Highly
Advanced Integration of Nano and Life Sciences, Gifu University (G-CHAIN)）

We have previously demonstrated activation of the descending pain inhibitory pathways by
noxious stimuli in the colorectum enhances colorectal motility in rats. It has been known thatthe descending pain inhibitory pathways are modulated during development of hyperalgesia.
However, it remains unclear whether the development of hyperalgesia also impacts on thepathways regulating colorectal motility. Thus, the purpose of this study is to examine whether
the regulation of colorectal motility via the descending pain inhibitory pathways are modulated
in an experimental model of hyperalgesia. To induce hyperalgesia in rats, we injected complete
Freund’s adjuvant into left hind paw two days before experiments. Anesthetized rats werecannulated in the colorectum, and changes of intraluminal pressure and expelled volume were
measured. Intrathecal administration was performed by a spinal cannula located at the L6-S1
level of the spinal cord. Administration of a noxious stimulant capsaicin into the colorectal
lumen enhanced colorectal motility in male rats. However, in hyperalgesic male rats, capsaicin
administration failed to enhance colorectal motility. In the case of female rats, administration ofcapsaicin had no effects on colorectal motility not only in hyperalgesic but also intact conditions.
When an inhibitor of oxytocin receptor was intrathecally injected into the spinal cord, capsaicinenhanced colorectal motility both in hyperalgesic male and female rats. The oxytocinergic
inhibitor had no effects on colorectal motility in intact males and females. Our findings suggest that oxytocin in the lumbosacral spinal cord negatively impacts the colorectal motility inboth sexes. Considering that the inhibitory effects of oxytocin on the colorectal motility are
manifested exclusively in the hyperalgesic condition, oxytocinergic descending pathway may
be activated only when potent painful stimuli are applied. The oxytocinergic pathways would
be involved in pathological mechanisms of defecation disorder associated with inflammatory
diseases such as colonic and bladder inflammation.
(COI:No)

Chronic dry eye symptom is associated with not only corneal epithelial disorder but also
ocular neuropathic pain. However, its molecular mechanism remains to be elucidated. The
aim of this study was to elucidate the molecular mechanism underlying the progression of dry
eye-induced ocular pain by focusing on the involvement of glial cells in the trigeminal nucleus.
The unilateral dry eye was induced by left extraorbital and intraorbital lacrimal gland excision(LGE) in 6-week-old SD rats. For sham surgery, incisions were made and glands were partially
exposed on the right side. Spontaneous tear volume was reduced from the day after LGE. One
week after the LGE operation, the spontaneous eye blink rates and the corneal epithelial injury
score evaluated by fluorescein staining were significantly higher on the LGE side than thesham side. The sensitivity of the cornea to hypertonic stimulation was significantly higher on
the LGE side compared with the sham side, indicating that the LGE-induced corneal epithelial
defects progressed to neuropathy. Based on these results, we performed experiments at two
weeks and eight weeks after LGE as acute phase and chronic phase of dry eye-induced
neuropathic pain, respectively. The glial cell activation in subnucleus interpolalis/subnucleus
caudalis of the trigeminal nucleus (Vi/Vc) was analyzed by immunostaining. The density ofNissl staining and immunostaining indicated that the density of neurons was significantly lower
on the LGE side than the sham side. The intensity of GFAP-immunopositive regions in the Vi/
Vc on the LGE side was enhanced in both two and eight weeks after surgery. These results
suggest that reactive astrocytes are already increased in the acute phase in the trigeminal
nucleus of the LGE side. The dry eye-induced corneal epithelial damage may develop ocular
neuropathic pain by activation of astrocytes.
(COI:No)

Widespread inhibition and modulation in the mouse olfactory sensory
neurons in vivo
Kyushu Univ, Japan, 2 RIKEN CDB）

Odor recognition starts from olfactory sensory neurons (OSNs), but this process is yet to be
fully understood in physiological conditions in vivo. Here, we performed two-photon calcium
imaging of mouse olfactory sensory neurons in vivo and found that odors produce not only
excitatory, but also inhibitory responses at their axon terminals. Robust inhibitory responses at
OSN axon terminals remained in two independent mutant mice, in which all possible sourcesof presynaptic lateral inhibition were eliminated. Therefore, we examined the responses in theolfactory epithelium in vivo, and found widespread inhibitory responses at the level of OSN
somata. Moreover, responses to odor mixtures demonstrated extensive mutual modulation(both suppression and enhancement) in OSNs. An in vitro assay demonstrated that someodorants act as inverse agonists to some odorant receptors. The bidirectional nature of OSN
responses may be useful for robust odor coding under noisy sensory environment. (COI:No)

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S81

Oral Session 13
Muscle Physiology（March 19, 11:00〜12:00, Hall 6）

O13-1

Roles of extracellular vesicles secreted from mouse muscle cells inmuscle-bone interactions
Yoshimasa Takafuji, Kohei Tatsumi, Masayoshi Ishida, Naoyuki Kawao, Kiyotaka Okada,
Hiroshi Kaji（Dept Physiol and Regene, Med, Kindai Univ, Japan）
Purpose: The interactions between muscle and bone via humoral factors have been recently
noted. It is known that various muscle-derived humoral factors (myokines), such as myostatin,
irisin, follistatin and osteoglycin, affect bone metabolism. Extracellular vesicles (EVs) containvarious proteins or micro RNA, and play a vital role in physiological and pathophysiologicalprocesses, such as cell proliferation, differentiation and metastasis, by transferring their contents to the distant tissues. Recently, several evidences suggest that EVs are crucial mediators
which is responsible for the cell-cell communications in bone tissue. However, roles of EVs inthe muscle-bone interactions have still remained unknown. In the present study, we investigated the effects of EVs secreted from mouse C2C12 myoblasts (Myo-EVs) on osteoclasts
and osteoblasts in mice. In addition, the effects of loading of mechanical stress or treatment
with endocrine factors on C2C12 myoblasts on the bioactivity of Myo-EVs were investigated.
Results: Myo-EVs were isolated from the conditioned medium of C2C12 cells by ultracentrifugation. Myo-EVs suppressed osteoclast formation and mRNA expressions of osteoclast-related
genes induced by receptor activator of nuclear factor κB ligand (RANKL) in mouse bonemarrow cells. Moreover, Myo-EVs suppressed oxygen consumption and mRNA expressions ofmitochondrial biogenesis markers induced by RANKL in mouse bone marrow cells. Fluid flow
shear stress on C2C12 myoblasts enhanced the suppressive effects of Myo-EVs on osteoclast
formation and the mitochondrial biogenesis, but treatment with 1, 25-dihydroxyvitamin-D3
or dexamethasone on C2C12 myoblasts didn’t enhanced these effects of Myo-EVs. On theother hands, Myo-EVs didn’t affect the phenotypes and the mitochondrial biogenesis of mouseprimary osteoblasts.
Conclusion: The present study first showed that Myo-EVs suppress osteoclast formation andthe mitochondrial energy metabolism in mouse bone marrow cells. EVs secreted from skeletal(COI:No)muscles might be a crucial mediator for the muscle-bone interactions.

O13-3

Increase in phospholamban content in mouse skeletal muscle after
denervation
2 1 Dept Inst Analysis,
Tsutomu Nakada1, 2，Masatoshi Komatsu2，Mitsuhiko Yamada（

RCSAS, Shinshu Univ, Japan, 2 Dept Mol Pharmacol, Shinshu Univ Sch Med, Japan）

It is well-known that denervation of motor nerves induces atrophy and decreases contractile
force of the skeletal muscle. However, it is not completely understood how denervation alters
calcium handling in the skeletal muscle. We investigated the effect of denervation on theexpression and function of proteins involved in calcium handling. Two weeks after denervation of the right sciatic nerve in mice, we observed a significant decrease in mass andcross-sectional area of the ipsilateral tibialis anterior (TA) and flexor digitorum brevis (FDB)
muscles. Also, we observed a significant decrease in the specific tetanus contractile force
in the ipsilateral TA muscle. Calcium imaging of the ipsilateral FDB showed that the peak
twitch and tetanus calcium concentrations were significantly decreased due to a decrease incalcium content of the sarcoplasmic reticulum (SR). Denervation reduced the maximum rateof sarcoplasmic/endoplasmic calcium ATPase (SERCA) activity. Interestingly, the amount ofphospholamban (PLN), but not its transcripts, was increased in the ipsilateral vs. contralateralside after denervation, suggesting that denervation impairs constitutive regulation of PLN.
Immunohistochemical analysis revealed increased PLN in all major fiber types in TA with IIx
fibers showing a threefold higher expression than the contralateral side. These results suggestthat the abnormal increase in PLN in the ipsilateral fast-twitch fibers may be involved indecreased SERCA activity, SR calcium content, peak calcium transients, and contractile forcesof denervated muscles.
(COI:No)

O13-4

The role of calpain activation and vimentin cleavage in the signaltransduction of abnormal vascular smooth muscle contraction
meditated by SPC/Fyn/ROK pathway
Hiroko Kishi, Qian Lu, Tomoka Morita, Ying Zhang, Bochao Lyu, Min Zhang, Nan Li,
Minhui Xu, Sei Kobayashi（Dept Mol Cell Physiol, Grad Sch Med, Yamaguchi Univ, Japan）
Rho-kinase (ROK)-mediated Ca2+-sensitization of vascular smooth muscle (VSM) plays a criticalrole in abnormal VSM contractions such as vasospasm. Previously we identified sphingosylphosphorylcholine (SPC)/Fyn/ROK pathway as a novel signaling pathway for abnormal
VSM contraction. As possible downstream targets of Fyn tyrosine kinase, we identified
vimentin by focused proteomics in which tyrosine-phosphorylated proteins were concentratedusing 4G10 antibody and identified by tandem mass spectrometry. Interestingly, western blot
analysis revealed that SPC induced limited proteolysis of vimentin not only in human coronary
artery smooth muscle cells (CASMCs) but also in VSM strips of the porcine coronary artery.
Since vimentin is reported as the target of calpain, we examined the possible involvement of
calpain. In CASMCs, SPC increased calpain activity, which was blocked by PD150606, a calpain
inhibitor. Furthermore, PD150606 inhibited the SPC-induced abnormal VSM contraction bothin porcine coronary and mouse basilar arteries. PD150606 also inhibited U46619-induced VSM
contraction in porcine coronary arteries. PD150606 did not inhibit Ca2+-dependent contraction
induced by high K+ depolarization. Mass spectrometric analysis showed that calpain cleaved
vimentin at its N-terminus. These findings suggest the possible involvement of calpain in thesignal transduction of Ca2+-sensitization of VSM contraction induced by SPC and U46619.
(COI:No)

O13-2

O13-5

Jun Tanihata, Susumu Minamisawa（ Div. Aerospace Med, Dept Cell Physiol, The Jikei

Ying Zhang1，Bochao Lyu1，Tomoka Morita1，Dan Cui2，Hiroko Kishi1，Min Zhang1，
1 1 Dept Mol Cell Physiol, Grad Sch Med,
Qian Lu1，Nan Li1，Eiji Ikeda2，Sei Kobayashi（

Is N-terminal titin fragment a new urinary biomarker to detect muscle
atrophy?
Univ, Japan）

Introduction: Many studies have attempted to determine the associations between blood andurine biomarkers and muscle damage and atrophy. However, poor correlations between thechanges in biomarker levels and the magnitude of muscle damage and atrophy have beenreported. Recently, the N-terminal fragment of titin(N-titin), a giant sarcomeric protein that is
involved in muscular passive tension and viscoelasticity, has been reported to detect muscle
damage in patients with skeletal muscle dystrophy and in healthy volunteers with endurance
exercise.
Purpose: To investigate whether urinary N-titin is changed during the muscle atrophic period
and whether its increase reflects muscle atrophy.
Methods and Results: C57BL/6J mice (male, 10 weeks of age) were used of this study. Urine
samples were obtained after sciatic nerve removal surgery (denervation) to induce muscle
atrophy. We measured the urinary levels of N-titin with a highly sensitive ELISA system.
11 days after denervation, dissection was performed, and the limb muscle was weighed. The
weight of 4 types of limb muscles were significantly decreased at 11 days after denervation.
Although skeletal muscle weights were predominantly reduced and mRNA levels of Atrogin-1
and MuRF-1 such as markers of muscle atrophy, were increased, there was no significant
change in urinary titin level. On the other hand, mRNA levels of titin in soleus muscles, atypical slow twitch muscle, were increased by muscle atrophy.
Conclusion: These results suggest that it is difficult to identify the muscle atrophy with urinary
titin levels. We would like to identify a urinary biomarker that detect muscle atrophy other(COI:No)
than titin.

S82

Role of paxillin in sphingosylphosphorylcholine (SPC)-induced Ca2+sensitization contraction of vascular smooth muscle
Yamaguchi Univ, Ube, Japan, 2 Dept Pathol, Grad Sch Med, Yamaguchi Univ, Ube, Japan）
Rho-kinase (ROK)-induced Ca2+-sensitization contraction of vascular smooth muscle (VSM) isa major cause of cardiovascular and cerebrovascular vasospasm. We previously demonstrated
that sphingosylphosphorylcholine (SPC) induced this abnormal contraction via a SPC/Fyn/
ROK pathway. However, Fyn could not directly activate ROK in vitro/in vivo experiments.
The combination of pull down assay and MALDI-TOF mass spectrometry made us identify
paxillin as the possible downstream molecule of Fyn. Until now, no direct evidence is provided
to prove paxillin involved in SPC-induced contraction. In the present study, we examined therole of paxillin in SPC-induced Ca2+-sensitization contraction of VSM. First, paxillin knockdown
inhibited SPC-induced contraction in human coronary artery smooth muscle cells. Then weused Cre-loxP system to generate tamoxifen-inducible and smooth muscle-specific paxillin
knockout mouse and showed that targeted deletion of paxillin in VSM inhibits SPC-induced
Ca2+-sensitization of VSM contraction in thoracic aorta. Paxillin knockout also inhibited
SPC-induced myosin light chain phosphorylation. These results indicate that paxillin plays animportant role in SPC-induced Ca2+-sensitization contraction of VSM. (COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Oral Session 14
Others（March 19, 11:00〜12:00, Hall 7）

O14-3

A dual orexin receptor blocker suvorexant inhibits hypercapnic
ventilatory augmentation
1 1Clin
Isato Fukushi1, 2，Shigefumi Yokota3，Kotaro Takeda1, 4，Jiro Terada1, 5，Yasumasa Okada（

Res Ctr, Murayama Med Ctr, Musashimurayama, Japan, 2 Fac Hlth Sci, Iryo Sosei Univ, Iwaki,
Japan, 3 Dept Anat and Neurosci, Shimane Univ, Izumo, Japan, 4 Fac Rehabili, Sch Healthcare,
Fujita Hlth Univ, Toyoake, Japan, 5 Dept Respirology, Grad Sch Med, Chiba Univ, Chiba, Japan）

Suvorexant (Belsomra ®), a dual orexin receptor antagonist, inhibits the arousal system in thecentral nervous system, and has been widely used for the treatment of insomnia. Because manyof sleep-inducing drugs suppress ventilation, concerns could be raised whether suvorexant
affects breathing. However, the effects of suvorexant on ventilation have not been well clarified.
To address this issue, we firstly analyzed orexin receptor (OX2R) expression in neurons in thebrainstem respiratory regions with putative respiratory neuron markers, i.e., with Phox2b in
the parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN), and NK1R/somatostatinin the preBötzinger complex (preBötC) of the mouse, respectively. Secondly, we analyzed ventilatory parameters in room air, hypercapnic (5% CO2) and hypoxic (10% O2) conditions before
and after injection of two doses of suvorexant (10 mg/kg and additional 90 mg/kg). Experiments were performed in unanesthetized adult male mice. Respiratory flow was non-invasively
measured by whole body plethysmography. Respiratory parameters (tidal volume, respiratory
rate, and minute ventilation) were calculated from the measured respiratory flow. The oxygen
concentration in the chamber was monitored with an oxygen concentration analyzer. In
results, immunohistological analysis demonstrated that OX2R is expressed in Phox2b andNK1R/somatostatin immunoreactive neurons in the pFRG/RTN and preBötC, respectively,
suggesting the involvement of orexin in respiratory control. Either dose of suvorexant did notaffect ventilation in room air or hypoxic ventilatory responses. However, hypercapnic ventilatory augmentation was significantly attenuated in the suvorexant-administered conditions ascompared to the control (before suvorexant-administered) condition (two factor within-subject
ANOVA). None of the mice died in even after high dose (total cumulative dose 100 mg/kg)
of suvorexant administration. We conclude that suvorexant inhibits hypercapnic ventilatory
augmentation. Suvorexant may have to be carefully used in patients with hypercapnic respiratory failure or sleep related hypoventilation.
(COI:No)

O14-1

O14-4

3 1 Res Ctr for Urban Health ＆Kazunobu Okazaki1, 2，Nanae Shirai3，Yasushi Enomoto（Takanori Matsubara1，Takayuki Yanagida2，Noriaki Kawaguchi2，Takashi Nakano2，
Junichiro Yoshimoto2，Shin-ichiro Horigane3，Shuhei Ueda3，Sayaka Takemoto-Kimura3，
1 1 Dept Neurosci II, RIEM, Nagoya Univ, Japan,
Akihiro Yamanaka1，Takayuki Yamashita（

Aerobic capacity is associated with blood volume or total hemoglobin
mass in female runners
Sports, Osaka City Univ, Osaka, Japan, 2 Dept of Environ Physiol for Exerc, Osaka City
Univ Grad Sch of Med, Osaka, Japan, 3 ARIHHP, Univ of Tsukuba, Tsukuba, Japan）

Bidirectional remote control of deep neuron activities using X-rays

2 NAIST, 3 Dept Neurosci I, RIEM, Nagoya Univ, Japan）

High aerobic capacity (maximal oxygen uptake, VO2max; and lactate threshold, LT) and low
oxygen cost (O2 cost) are the fundamental physiological factors for the high endurance athletic
performance. It has been well known that VO2max is closely correlated with blood volume(BV) and total hemoglobin mass (THb) in endurance athletes. On the other hand, the increased
venous return to the heart with the increased BV would enhance muscle blood flow andtherefore reduce lactic acid accumulation and enhance LT during exercise. Therefore, it isthought that LT is also associated with BV and THb in endurance athletes. In this study,
we investigated that relationships between aerobic capacity and blood volumes or blood
constituents in female runners. In fifteen female collegiate middle and long distance runners,
BV, THb (optimized CO rebreathing method) and hematological values were determined atrest, and VO2max, velocity at VO2max, LT (velocity at 3 mM/L of lactic acid concentration inblood), and O2 cost were determined during a graded maximal treadmill running test. We
found that there are significant correlations between VO2max and BV (R = 0.72, P = 0.002) or
THb (R = 0.75, P = 0.001), and between LT and BV (R = 0.66, P = 0.008) or THb (R = 0.71, P= 0.003). On the other hand, there were no significant correlations between O2 cost and BVor THb, and between VO2max or LT and hematological parameters (red blood cell counts,
hemoglobin concentration, hematocrit). These results suggest that the increase in BV and THb
are required to achieve high endurance athletic performance through the increase in VO2max
and LT in female runners.
(COI:No)

Recent advances in material science in close conjunction with neuroscience have made it
possible to achieve remote/wireless optogenetic control of neuronal activities in living animals.
Light-sensitive proteins called opsins are activated only by visible light, which has a low tissue
penetration depth. Therefore, for stimulating a large population of neurons in the deep brain, it
would be advantageous to employ up-converting phosphors which emit visible light in response
to more tissue-penetrating near-infrared (NIR) light (Chen et al., Science 359: 679–684, 2018;
Miyazaki et al., Cell Reports, 26: 1033-1043, 2019.). However, the tissue penetration depth of
NIR is limited to several millimeters and NIR illumination can cause abrupt heat generationin body surface. To overcome these issues, we utilize inorganic scintillators that exhibit visible
luminescence called scintillation when excited by X-ray. We show that a certain yellowemitting scintillator can efficiently activate red-shifted opsins upon X-irradiation. With thesescintillator/opsin combinations, we successfully activated and inhibited dopamine neurons in
the ventral tegmental area of freely moving mice to wirelessly drive related behaviours underX-irradiation. The scintillator crystal was bio-compatible and safely implantable. X-ray irradiations during the behavioural test caused reduction of the number of proliferating cells in thebrain and possibly in the other tissues as well but did not affect body weight and home-cage
behaviour of the mice. These results demonstrate that X-ray-induced scintillation can be used
for wireless optogenetics.
(COI:No)

O14-2

O14-5

Tomoyuki Ito（Dept Rehab Med, Tanabe Memorial Hosipital, Kyo-Tanabe, Japan）

Sean Holroyd（Weill Cornell Medicine Qatar）

ActiGraph is a clock-type accelerator that is attached to the wrist or trunk and can measurephysical activity during walking from three-dimensional acceleration. Recently, physical activity during propulsion of a wheelchair by bilateral arms has been reported using ActiGraphattached to the wrist and/or trunk. However, there is no report of physical activity of one-sided
upper and lower limb propulsion by ActiGraph seen in hemiplegic patients. In this study, we
compared the relationship between the oxygen intake (VO2) and the vector magnitude (VM)
measured by ActiGraph when the wheelchair propelled by one hand and one foot.
Twelve healthy individuals (7 male, 5 female, age 35.2 ± 9.4 years, weight 61.4 ± 12.0 kg; mean
± SD) propelled wheelchair around one lap of 40m counterclockwise. After 3 minutes of rest,
3 minutes of slow wheelchair propulsion (3 km / h for male and 2.5 km / h for female) was
made followed by of fast wheelchair propulsion (5 km / h for male and 4 km / h for female).
ActiGraph GT3X-BT were worn on the right wrist and the right ankle.
VO2 at rest, slow, fast wheelchair propulsion were 3.6±0.1, 7.7±1.5, 12.0±2.8 ml/min/
kg, respectively, upper limb VM at slow, fast wheelchair propulsion were 11264.6±543.8,
2068.5±750.7 counts, respectively, and lower limb VM: 820.4±187.6, 1134.0±372.4 counts, respectively, and total VM at slow, fast wheelchair propulsion were 2085.0±489.3, 3202.5±1079.7
counts, respectively. There was a significant positive correlation between VO2 and total VM(r = 0.95).
Since there was a positive correlation between VO2 and total VM, the physical activity ofwheelchair propulsion by one hand and one foot can be measured using ActiGraph.  (COI:No)

The use of online videos as learning supplements by medical students has escalated over the
past decade1. With much of the available material being of varying quality it is of concern thatstudents may not be able to critically appraise online videos. In this study the responses ofpremedical students (n=44) to an assignment where they evaluated the physiological content
of three YouTube videos on the “Mechanics of Breathing” were analyzed. Students had alreadybeen exposed to this subject in lectures. The 3 videos met the following criteria; Video 1 – correct and professionally presented, Video 2 – major errors but professionally presented, Video
3 – major errors and unprofessionally presented. Students were asked to rank the videos inorder from best to worse as well as to judge them based on the following: were they accurate/
factual; was the source of information reputable; could they be used for revising physiologicalconcepts. Interestingly, 52% of the students selected Video 2 as their top-ranking video, 48%
selected Video 1, none selected Video 3. Many students commented on the difficulty in choosing
between Video 1 and 2 with only 16% of students identifying the major errors in Video 2.
Furthermore 64% of students would use Video 2 for revision, with over 50% of these choosing
this video alone. Finally, 55% of students chose Video 2 as having come from a reputable source
based on the popularity (number of views) of the video and the fact it was associated with a
website. The results from this study suggest that a sizeable percentage of premedical students
do not have the adequate training to be able to choose appropriate study material from online
videos. It is suggested that faculty should either produce their own online material or direct
their students to specific online videos or websites.
1. O’Malley et al. Adv Physiol Educ 43:383-391, 2019
(COI:No)

Evaluation of physical activity in wheelchair propulsion by one hand
one foot using ActiGraph accelerator

Can premedical students critically appraise YouTube “physiologicaleducation” videos?

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S83

Oral Session 15
CNS Function（March 19, 11:00〜12:00, Hall 8）

O15-3

Midbrain dopamine neurons drive whisker movements associated withreward processing
Takayuki Yamashita1，Kohta Mizutani1, 2，Takashi Nakano3，Yasutaka Mukai1，
3 1 Dept Neurosci II, RIEM,
Takatoshi Hikida2，Akihiro Yamanaka1，Junichiro Yoshimoto（
Nagoya Univ, 2 Inst Protein Res, Osaka Univ, 3 NAIST）

Animals including rodents express their internal states as facial movements. However, thecellular and neural circuit mechanisms underlying facial expressions of animals are still
enigmatic. Investigating mice performing an auditory Go/No-Go task, we found that notablewhisker protractions were induced immediately after Go cue presentation and that reward
acquisition evoked a shift of whisker set-point toward a more protracted angle and often
triggered whisking. Such reward-related whisker movements were commonly observed
among mice we studied, so that behavioral outputs of the mice performing the task could be
accurately decoded and predicted only by time plots of their whisker angle. The cue-locked
whisker protraction developed over the course of task learning, whereas whisking probability
after reward acquisition was not correlated with task proficiency. These properties of rewardrelated whisker movements were reminiscent of activities of dopamine neurons in the ventral
tegmental area (VTA). We therefore tested for optogenetic stimulation of VTA dopamine
neurons and found that transient excitation of VTA dopamine neurons induced whisking.
Temporal association between an auditory cue and rewarding optogenetic stimulation of VTA
dopamine neurons elicited both cue-locked whisker protraction and stimulation-locked whisking, suggesting that licking-associated facial movements are not involved in reward-related
whisker movements. Furthermore, virally induced, CRISPR/Cas9-mediated knock-out of Drd1
(or D1R) in the nucleus accumbens attenuated whisker movements induced by activationof VTA dopamine neurons. Our findings thus suggest that midbrain dopamine neurons, via
accumbal D1R neurons, trigger whisker movements expressing expectation and acquisition ofa reward.
(COI:No)

O15-1

O15-4

Munenori Ono（Dept Physiol 1, Kanazawa Med Univ）（ 1 Division of System Neurophysiology, National Institute for Physiological Sciences, 38,
Nishigonaka, Myodaiji, Okazaki, Aichi, 444-8585, Japan, 2 Department of Physiological
Sciences, SOKENDAI (The Graduate University for Advanced Studies), 38, NIshigonaka,
Myodaiji, Okazaki, Aichi, 444-8585, Japan）

Simultaneous in vivo recording of the local ﬁeld potential and the signalof ﬂuorescent calcium indicator in the hippocampus of Altzheimerʼsmouse model
Alzheimer disease (AD) patients have severe learning disabilities. In the early stage of AD,
several studies have suggested that soluble amyloid beta induced the disorder in the neuralactivity and the intracellular calcium dynamics, which are very likely to be relevant to thelearning disability as well as the progression of cell death in AD. However, the neurophysiological disorder in early stage of AD has not been fully elucidated. In this study, to examine thedisorder in the neural activity and calcium dynamics in vivo, we utilized a photometric patch
electrode (PME), with which simultaneous recording of fluorescence and electrical signals wereavailable. Using a PME, we recorded the local field potential (LFP), spike activities, and thesignals of fluorescent calcium indicator in the hippocampus, where is closely related to learning
ability and spatial cognition. The recordings were performed in dorsal hippocampal CA1 regionof model mice of AD (3xTg), with which spatial cognitive defects were confirmed in the earlystage of AD. During the PME recording, the mouse was placed on a treadmill to monitor the
movement, and head-fixed via a metal rod. The spatial learning ability of the individual animal
was assessed by Morris Water Maze (MWM) test in advance of the PME recording. The PME
recordings showed that the calcium signal increased when the theta oscillation dominated in
LFP during locomotion. The transient rise of the calcium signal was also seen sporadicallywhile the animal was stationary. At the rise of the calcium signal, the fast gamma oscillationin the LFP was observed. Compared with the WT animals, AD model animals had less powerin the high frequency oscillation at the rise of the calcium signal. Interestingly, the degradationin the high frequency oscillation was correlated with the disorder in the spatial learning test.
(COI:No)

O15-2

Effects of bilateral lesions of amygdala on cardiovascular responses
during treadmill running in rats
1 1 Dept Physiol, Grad Sch Health ＆ Sports
Kei Tsukioka1, 2，Ko Yamanaka1，Hidefumi Waki（

Sci, Juntendo Univ, Chiba, Japan, 2 JSPS Research Fellow）

It has been reported that the amygdala regulates cardiovascular system in response toemotional events. In our previous study, we observed that maximal exercise performance wasgreater after bilateral amygdala lesions of rats. Therefore, our hypothesis was that amygdala
lesions increased maximal exercise performance by modulating the cardiovascular responses
during exercise . To examine this, arterial pressure (AP) in rats were chronically measured
by radio-telemetry during treadmill running. Rats were subjected to forced running for 60
min (≦ 20 m / min) per day for 3 days for treadmill habituation. Thereafter, an incremental
exercise test was conducted until exhaustion (pre-test). Exercise intensity was initiated at 10m / min, and the velocity was increased by 2 m / min at every 3 min. In the amygdala lesion
group, bilateral lesions in the amygdala were induced by electrical microstimulation. In thesham group, the tip of the electrode was inserted into the amygdala and immediately removed
without the electrical microstimulation. After surgery, re-training was conducted similarly
as the habituation protocol, and the incremental exercise test was repeated (post-test). We
found that the mean AP (MAP) in both groups of animals exhibited a gradual increase duringsubmaximal exercise (low/moderate intensity) followed by a drastic increase during high/maximal-intensity of exercise. However, comparing with the sham group, the MAP responsein the amygdala lesion group was significantly lower during submaximal exercise whereas it
was greater during high/maximal-intensity of exercise, suggesting that the amygdala may
have a role in limiting exercise performance via modulating autonomic cardiovascular control.
(COI:No)

S84

Weak correlated activity of pallidal neurons during task performance innormal and mild parkinsonian monkeys
Woranan Wongmassang1, 2，Taku Hasegawa1, 2，Satomi Chiken1, 2，Atsushi Nambu1, 2

The globus pallidus is a part of the basal ganglia and divided into the external (GPe) andinternal (GPi) segments. Both GPe and GPi neurons change their activity during voluntary
movements, suggesting that they play a crucial role in control of voluntary movements. Although not only firing rates of neurons but also firing correlations between neurons have beenreported to encode neuronal information, GPe/GPi neurons do not exhibit correlated activity
at rest in the healthy state. On the other hand, oscillatory and synchronized activity has been
observed in the GPe/GPi in movement disorders, such as Parkinson’s disease (PD). To examine
the possibility that neuronal correlations in the GPe/GPi contribute to control of movements
and pathophysiology of movement disorders, we simultaneously recorded activity of multiple
neurons in the GPe/GPi of Japanese monkeys (Macaca fuscata) during hand reaching task byusing the multi-channel electrodes and analyzed their cross-correlations. We found that only
a limited number of GPe/GPi neurons showed correlated activity in relation to task events inhealthy monkeys. Next, we generated a mild PD monkey by injecting 1-methyl-4-phenyl-1, 2,
3, 6-tetrahydropyridine (MPTP), a dopaminergic neurotoxin. In the mild PD monkey that canperform the motor task, correlated activity was not increased. These results suggest thatindependent activity of GPe/GPi neurons is essential to normal information processing within
the basal ganglia and appropriate control of voluntary movements.
(COI:No)

O15-5

Glutamatergic and GABAergic control of monkey subthalamic activity
during motor task
Zlata Polyakova1, 2，Nobuhiko Hatanaka1, 2，Satomi Chiken1, 2，Atsushi Nambu1, 2（ 1 Div System Neurophysiol, Natl Inst Physiol Sci, Okazaki, Aichi, Japan, 2 Dept Physiol Sci,
SOKENDAI, Okazaki, Aichi, Japan）
The subthalamic nucleus (STN) receives cortical inputs through the cortico-STN hyperdirect
and cortico-striato-external pallido (GPe)-STN indirect pathways and drives the internal segment of the globus pallidus, the output nucleus of the basal ganglia. Activity of STN neurons
is modulated in relation to voluntary limb movements, and its abnormal activity has beenreported in movement disorders, such as Parkinson’s disease. However, the control mechanismof STN activity by cortical inputs is still not clear. To address this issue, we recorded neuronal
activity of STN neurons in awake monkeys (Macaca fuscata) in combination with blockade ofneurotransmission by local drug injections. Stimulation of motor cortices induced early andfollowing late excitations in STN neurons. Pharmacological blockade showed that corticallyinduced early and late excitation in STN neurons are mediated by the hyperdirect and indirect
pathways, respectively, and that cortical inputs to the STN are mainly mediated by N-methylD-aspartate receptors. Next, we recorded STN neuronal activity during performance of go/stop reaching task at three different targets. The results showed that the motor region in the
STN is involved in both motor execution and cancellation. Task-related STN activity was also
controlled through the hyperdirect cortico-STN glutamatergic and indirect GPe-STN GABAergic inputs. Stop-related activity was mainly transmitted through the hyperdirect pathway thatcaused facilitation in the STN, while the role of the indirect pathway was minor. Some neurons
with stop-related activity showed their activity in the specific direction, while other neurons
showed their activity in all directions. (COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Oral Session 16
Nutrition・Metabolism・Thermoregulation（March 19, 11:00〜12:00, Hall 9）

O16-3

A combination of high fat diet and nicotine enhances CB1
endocannabinoid receptor in hypothalamic nuclei in mice
Tingting Guo1, 5，Mami Matsumoto2，Takashi Yagi1，Hiroyuki Koyama1，Daisuke Aotani1，
Kazunobu Sawamoto2，Yasuhiko Minokoshi3，Hiroaki Masuzaki4，Nobuya Inagaki5，
1 1 Dept Gastro and Metab, Grad Sch Med, Nagoya City
Kazuwa Nakao6，Tomohiro Tanaka（

Univ, Japan, 2 Devel Regen Neuro, Inst of Brai Sci, Grad Sch Med, Nagoya City Univ, Japan,
3 Divis of Endo and Metab, Nat Ins for Phys Sci, Japan, 4 Divis of Endo, Diab and Metab, Hema,
Rheum, Grad Sch Med, Ryukyus Univ, Japan, 5 Dept of Diab, Endo and Nutri, Grad Sch Med,
Kyoto Univ, Japan, 6 Med Innov Cent, Grad Sch Med, Kyoto Univ, Japan）
Background: Although combinatorial effect of dietary fat intake and tobacco smoking on bodyweight has been suggested, underlying mechanisms have not been fully elucidated. Cannabinoid receptor 1 (CB1R) is a GPCR expressed widely in the brain and may play a role both inobesity and tobacco addiction, since its pharmacologic blockade is effective for the both. Here,precise distribution of CB1R within the appetite center and potential changes in a murine
model of obese smokers have been examined.
Methods: We analyzed mRNA levels of CB1R in micro-dissected hypothalamic nuclei as wellas CB1R immunoreactivity in brain slices from male C57BL/6 mice. Mice kept on a highfat diet (HFD) for four weeks were administered with nicotine (12ug/g body weight/day)intraperitoneally, and body weight, food intake and CB1R mRNA levels were examined.
Results: CB1R mRNA and protein were detected in hypothalamic nuclei as well as hippocampus. CB1R mRNA in the arcuate (ARC) and lateral nuclei (LH) of the hypothalamus werehigher than paraventricular (PVN) and ventral-dorsal medial nuclei (V/DMH). Nicotine suppressed food intake either under standard or HFD by 29±5% and 53±13%, respectively. HFD
or nicotine alone slightly lowered CB1R levels in the PVN or LH, respectively. By contrast,
treatment of HFD-fed mice with nicotine led to an increase in CB1R levels in ARC, PVN, V/
DMH and LH by 45%, 65%, 34% and 50%, respectively.
Conclusions: CB1R mRNA was widely distributed in multiple hypothalamic nuclei. The
expression of CB1R was augmented only when mice were treated with HFD and nicotine
in combination. These data suggest a potential combinatorial effect of the two insult and thatan exposure to nicotine in obese subjects may provoke an enhanced central endocannabinoid
activity and may lead to the development of cross addiction at the level of the hypothalamus.
(COI:No)

O16-1

O16-4

Tomoe Nakamura-Nishitani.1，Shu Nakao2，Hirotsugu Tsuchimochi1，James Pearson1，
3 1 Dept Card Physiol, Natl Cereb ＆ Cardiovasc Ctr, Japan, 2 Stem
Shigeo Wakabayashi（

Atsushi Fukushima, Miyako Furuta, Hitomi Fujioka, Toshiya Funabashi（Dept Physiol, St

A novel regulatory mechanism of energy metabolism mediated by
Neuronal calcium sensor-1 and its relation to exercise therapies
Cell Regen Med Lab, Col Life Sci, Ritsumeikan Univ, Japan, 3 Dept Pharm, Osaka Med Col,
Japan.）
Obesity is a risk factor of life-threatening diseases such as stroke and myocardial infarction,
thus it is important to identify novel mechanisms of its regulation and prevention. Neuronal
Ca2+-sensor-1 (NCS-1) is an EF-hand Ca2+- binding protein, playing important roles in excitabletissues via activation of Ca2+-signals. We have previously discovered that NCS-1 deficient (KO)mice become obese with age. Analyses using the metabolic cages indicated that both foodintake and locomotor activity were not different from those in wild-type (WT) mice, but energy
metabolism was significantly decreased in KO mice. At the cellular level, mitochondrial respiration rate and the levels of proteins involved in mitochondrial thermogenesis and biosynthesis,
as well as its Ca2+-dependent upstream regulators were all decreased in the brown adipocytes
in KO mice. These results suggest that decrease in mitochondrial respiration followed bydecrease in energy metabolism are major mechanisms of obesity observed in KO mice.
Furthermore, we investigated whether exercise therapy would prevent the metabolic disorderinduced by NCS-1 deficiency. Eight weeks treadmill training less than lactate threshold level
inhibited both increase in body weight and adipocyte hypertrophy in KO mice, while amountof skeletal muscle were increased. Taken together, these results suggest an existence of anovel Ca2+- dependent pathway regulating energy metabolism mediated by NCS-1, and aerobic
exercise can prevent obesity even that was induced by genetic background difference such as(COI:No)
NCS-1 deficiency.

O16-2

Withdraw

Effects of Omega-3 fatty acids feeding on high-fat induced impairmentof glucose tolerance and behavioral performances
Marianna Univ Sch Med, Kawasaki, Japan）

It is well known that a high-fat (HFD) or Western diet increases body weight, impairs glucose
tolerance and results in obesity. Since a high-fat diet consumption is suggested to have a
profound impact on the brain, learning and memory processes may alter. Omega-3 fatty acids,on the other hand, attenuate the inflammatory response induced by such as HFD. The present
study examined whether or not omega-3 fatty acids have beneficial effects on HFD-induced
exacerbation. C57BL6 male mice were divided into 3 groups: Fed with an ordinal control dietgroup, HFD group fed with 45% fat diet, and HFD+omega-3 group fed 45% fat diet but half of
fat is from Menhaden oil, instead of Lard. Feeding for 10 weeks had no impact on inhibitory
avoidance task and open field test. In HFD+omega-3 group, Y-maze task performed tendency
to lowering because intraperitoneal glucose tolerance test was impaired. Next, we examinedomega-3 feeding after HFD. This means mice were fed for HFD 5 weeks and switched feeding
HFD containing omega-3 for additional 5 weeks. This treatment had no impact on inhibitory
avoidance task and open field test. However, omega-3 supplement improved attenuation ofalternation in Y-maze task by HFD and significantly recovered impaired glucose tolerance
by HFD. We conclude that omega-3 fatty acids is beneficial effects on some HFD-induced
impairment.
(COI:No)

O16-5

The impact of chronotype on the effect of dietary restriction and serum
level of sRAGE among obese patients - preliminary report
Zofia Orzechowska1，Dominika Kanikowska1，Katarzyna Korybalska1，Joanna Grzelak1，Rafał Rutkowski1，
1 1 Department
Agnieszka Zawada2，Aldona Juchacz2，Maki Sato3，Andrzej Bręborowicz1，Janusz Witowski（

of Pathophysiology, Poznan University of Medical Sciences, Poznan, Poland, 2 Department of Internal Diseases,
Metabolism and Nutrition, Poznań University of Medical Science, Poznań, Poland, 3 Department of Physiology,
Institutional Research, Aichi Medical University School of Medicine, Aichi Medical University, Aichi, Japan）

A chronotype is an individual, internal biological rhythm, concerning different preferences of thetime of day, which depends equally on the properties of the individual biological clock and lifestyle. It
is divided into the morning (“lark”), evening (“owl”), or mixed type. It has been reported in previous
studies that evening chronotype is closely associated with obesity. This study aimed to assess thechronotype’s impact on the effect of low-calorie diet treatment and serum level of Soluble Form of
Receptor for Advanced Glycation End Products (sRAGE) in patients with excessive accumulation of
adipose tissue in the body.
The study group consisted of 70 obese patients. These individuals underwent a three-week diettreatment. Patients used a diet with a reduction in the daily supply of calories by 25-30% in relation
to the total energy demand calculated using the Harris and Benedict formula and the physical activity rate. The patient’s weight and serum levels of sRAGE were collected before and after dietarymodification. Blood lipids and glucose levels were also assessed. Furthermore, a chronotype evaluationquestionnaire was carried out to evaluate the biological rhythm of each patient. The control groupconsisted of 200 donors from the Regional Blood Donation Center in Poznań (100 men and 100 women),among whom a survey assessing chronotype was also carried out, the anthropometric parameters
were measured, and the body mass index (BMI) was determined.
A majority of respondents in both the experimental and control group presented a “lark” chronotype.
Preliminary results presented in this study suggest that the highest BMI values were found it the socalled “owl” individuals. At the same time, the low-calorie diet allowed the most effective results to beachieved in this group, which included both weight reduction (mean of 5.3 kg) and average fat loss. For
larks it was 4, 6 kg and for intermediate type 4, 7 kg. Furthermore, we found a significant statisticaldifference in BMI between lark and intermediate chronotype (before 42, 8 vs 38, 6; after 41, 0 vs 37, 2).
The initial results presented are based on an incomplete group of patients. The study is currently
being continued and will ultimately include a group of 200 individuals. We believe that the final results
will allow us to clearly show the correlation between a given chronotype type and the effectiveness(COI:No)of a low-calorie diet.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S85

Oral Session 17
Heart・Circulation 2（March 19, 11:00〜12:00, Hall 10）

O17-3

Molecular mechanisms of contraction rhythm homeostasis in warmed
cardiomyocytes
3 1 Department of Biomedical Sciences,
Seine Shintani A.1，Shinichi Ishiwata2，Norio Fukuda（
College of Life and Health Sciences, The Chubu University, Aichi 487-8501 Japan,
2 Department of Physics, Faculty of Science and Engineering, Waseda University, Tokyo
169-8555 Japan, 3 Department of Cell Physiology, The Jikei University School of Medicine,Tokyo 105-8461 Japan）

It is well established that an increase in body temperature, albeit by a slight magnitude,
results in dramatic changes in the function of various organs, coupled with altered cellular
homeostasis. In the present study, we investigated the effects of infra-red laser irradiation on
sarcomere dynamics in living rat neonatal cardiomyocytes, by taking advantage of sarcomere
length nanometry. We found that a rapid increase in temperature to 38–42 ℃ induced [Ca2+] iindependent high-frequency (~5–10 Hz) sarcomeric auto-oscillations (Hyperthermal Sarcomeric
Oscillations; HSOs). In myocytes with intact sarcoplasmic reticular function, HSOs coexistedwith [Ca2+] i-dependent spontaneous beating in the same sarcomeres, with markedly different
frequencies (~7 and ~1 Hz for the former and the latter, respectively). We simulated HSOs
with a multi-scale, multi-physics heart simulation model. Our simulation predicts that there
exists a “reverse stroke” during the power stroke of myosin, which is vital for HSOs, as well as
for rapid myocardial relaxation for subsequent ventricular filling. Based on these findings, wewill discuss the physiological significance and molecular mechanisms of HSOs.
(COI:No)

O17-1

O17-4

Ryo Fukunaga1，Mariko Omatsu-Kanbe1，Kakeru Shimoda2，Masakazu Agetsuma3，
1 1 Dept Physiol, Shiga Univ Med
Junichi Nabekura3，Motohiro Nishida2，Hiroshi Matsuura（（ 1 Dept Cell Physiol, Grad Sch Med, Jikei Univ, Japan, 2 Dept Cell, Padova Univ, Italy）

Identiﬁcation of ANP promotor-driven AcGFP-expressing cardiac
progenitor cells in mice
Sci, Otsu, Japan, 2 Div Cardiocirculatory Signaling, 3 Duv Homeostatic Development）

The adult mammalian heart comprises heterogeneous cell lineages, including cardiomyocytes,
vascular smooth muscle cells, fibroblasts and cardiac stem or progenitor cells. Atypicallyshaped cardiomyocytes (ACMs) are identified in the culture of cardiomyocyte-removed
fractions obtained from mouse cardiac ventricles as beating cells. Since ACMs show thecharacteristics of ventricular, atrial and SA-nodal cells based on the expression of cell-specificproteins and electrophysiological properties, those cells are likely to be cardiac progenitor
or immature cardiomyocytes. One of the most important characteristics of those cells is theabundant expression of atrial natriuretic peptide (ANP) usually absent in the ventricular myocytes. However, the localization and morphology of these cells in the myocardium has yet to
be clarified. In the present study, we prepared ANP promotor-driven AcGFP-expressing mice
generated by infection of adeno-associated virus (AAV2-9) encoding pANP-AcGFP to visualize
and trace ANP-expressing ACMs. The cells with GFP signal were identified in the interstitialspaces among ventricular myocytes and also GFP-positive beating ACMs were present in theculture. The results indicate that ACMs with ANP-promotor exist in the cardiac ventricles and(COI:No)survive to develop into beating cells in the dispersed culture.

Pitx2c Overexpression in Atrial Cardiomyocytes Impaired Sinus Atrial
Node Function
Shunsuke Baba1，Toru Akaike1，Satoko Shinjo1, 2，Susumu Minamisawa1

Introduction: Sinus atrial node (SAN) dysfunction is associated with fatal diseases. The transcription factor Pitx2c, which is expressed in the left-sided heart may suppress differentiationof SAN cells because Pitx2c knockout mice had aberrant pacemaker cells in the left atria.
Hypothesis: Pitx2c overexpression in the right atria may impair SAN formation and function.
Methods: We generated the mice harboring the atrial cardiomyocyte-specific overexpressionof the Pitx2c by crossing two types of mice; the one has the Cre gene at the endogenous
locus of the sarcolipin gene specifically expressed in the atria, and the other was introduced
Pitx2c gene between loxP sequences using the CAG promoter. Then, we examined the cardiac
phenotypes of Pitx2cflox/cre+ (over expression:OE) and Pitx2cflox/cre- (control:CON) mice.
Results and Conclusions: The expression levels of Pitx2c mRNA were more than 100
times higher in the right atrium of OE than CON mice. OE mice were born with normal
Mendelian ratios. Echocardiography showed no change in the OE heart (n=7). Telemetryelectrocardiography showed the standard deviation of RR interval was significantly larger
in OE (15.3±8.40msec) than CON (10.3±5.91msec) mice(n=5). Ectopic atrial beats were foundin OE mice. The expression levels of TBX3, SCN5A and Kir3.1 mRNAs were significantly
downregulated, and those of podoplanin were significantly upregulated in the right atria of OE(n=7). We assumed these transcriptional alternations involved in SAN formation and channel
activities could induce electrical remodeling in the SAN and right atrium of OE mice. In conclusion, atrial cardiomyocyte-specific Pitx2c overexpression decreased heart rate and increased
its variability, indicating that the downregulation of Pitx2c in the right atrium is required to(COI:No)
maintain SAN function and electrical modeling of the atria.

O17-2

O17-5

Mariko Omatsu-Kanbe, Ryo Fukunaga, Hiroshi Matsuura（ Dept Physiol, Shiga Univ Med

Hiroko Izumi-Nakaseko1，Koki Chiba1，Mihoko Hagiwara-Nagasawa1，Ai Goto1，
Yoshio Nunoi1，Ryuichi Kambayashi1，Akio Matsumoto2，Yasunari Kanda3，
2 1 Dept Pharmacol, Faculty Med, Toho Univ,
Atsuhiko Naito T.4，Atsushi Sugiyama1, （Multinucleation of cardiac progenitor cells by cell fusion and nuclear
division
Sci, Otsu, Japan）

The adult mammalian heart comprises several cardiac stem or progenitor cells, thoughcardiomyocytes do not actually multiply to substitute new cells for damaged ones. Atypicalmyocardial cells (ACM) derived from mouse cardiac ventricles that develop into beating cells
are likely to be cardiac progenitor cells rather than stem cells. Although most of the ACMs
possess multiple nuclei, these cells are not observed to be proliferated. In the present study, we
prepared ACMs from Fucci2 (fluorescent, ubiquitination-based cell cycle indicator 2) transgenic
mice, of which nuclei were fluorescently labelled, to investigate the cell cycling and cocultured
these cells with those from wild-type mice. The dynamics of the labelled nuclei were observed
using live cell imaging system, and the precise morphology and the protein expression of thenucleus were examined. We found that the cell fusion and nuclear division without cytokinesis
occurred in these cells in the culture. The results suggest that the multinucleation process ofACMs, different from that in the myogenesis in skeletal muscle, is close to that observed in the(COI:No)
neonatal heart stage.

S86

Physiological properties of excitation-contraction relationships in humaninduced pluripotent stem cell-derived cardiomyocytes sheetsjapan, 2 Dept Aging Pharmacol, Faculty Med, Toho Univ, Japan, 3 Div Pharmacol, NIHS,
Japan, 4 Dept Physiol, Div Cell Physiol, Toho Univ Sch Med, Japan）

Currently available human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs)
have been known to show a negative force-frequency relationship as one of their immature
properties. In this study, we explored several potential physiological factors which may
overcome such limitation of the contraction movement. For this purpose, we prepared one
layered, higher cell-density sheets of hiPSC-CMs, and simultaneously recorded the motion
vectors and field potentials. First, we analyzed the contraction and relaxation processes underspontaneous activity. Next, we controlled the direction of cell movement by electrical pacing
and also tested the effect of supplying the higher oxygen tension on the cell sheet. Under
spontaneous activity, an electrical excitation propagated unidirectionally over the cell sheet,
but a synchronous movement consisted of multiple contractions which started from various
sites. During electrical stimulation, the contraction started around the pacing electrodes andwe observed the positive force-frequency relationships in contraction as well as relaxation
along with the frequency-dependent shortening of the field potential durations. The use of
fractional analysis of motion vectors demonstrated that contraction as well as relaxation
processes consisted of fast and slow phases. Increase in oxygen tension from 20 to 95% in
mixed gas accelerated the fast phase of relaxation. β-Stimulation accelerated the timing offast phase of relaxation, whereas a tyrosine kinase inhibitor dasatinib delayed it. Thus, theseobservations can indicate that the currently proposed procedure may become a new tool for
integrating the drug-induced biological phenomena in vitro extrapolating to clinically observed(COI:No)
cardiac efficacy and adverse effects.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Oral Session 18
Cell Physiology・Molecular Physiology 2（March 19, 14:10〜15:10, Hall 7）

O18-1

Differential roles of class II PI3-kinase-C2α and -C2β in clathrinmediated ﬂuid phase endocytosis in vascular endothelial cells
1 1 Dept. Physiol., Grad. Sch.
Kazuaki Yoshioka1，Sho Aki1，Noriko Takuwa1, 2，Yoh Takuwa（

Med., Kanazawa Univ., Japan, 2 Dept. Health Sci., Ishikawa Pref. Nursing Univ., Japan）

O18-3

CTLA4-Ig (Abatacept) Directly Inhibited Osteoclastogenesis by
Interfering With Intracellular Calcium Oscillations via Fc receptor gamma
Hiroyuki Okada1，Hiroshi Kajiya2，Yasunori Omata1，Shunichi Sudo2，Masashi Shin2，
Fujio Okamoto2，Jun Hirose1，Koji Okabe2，Takeshi Miyamoto3, 4，Sakae Tanaka1（ 1 Dept Orthopaedic Surgery, Grad Sch Med, Tokyo Univ, Japan, 2 Dept Physiological Science ＆Molecular Biology, Fukuoka Dental College, Japan, 3 Dept Orthopaedic Surgery, Faculty Life
Sciences, Kumamoto Univ, Japan, 4 Dept Orthopedics, Keio University School of Medicine, Japan）
CTLA4-Ig (cytotoxic T-lymphocyte antigen 4-Ig; abatacept) is a biologic drug for rheumatoid
arthritis. CTLA4 binds to the CD80/86 complex of antigen-presenting cells, and blocks theactivation of T cells. Previous reports showed that CTLA4-Ig directly inhibited osteoclastdifferentiation, the whole inhibitory mechanism of CTLA4-Ig for osteoclast differentiation
remained unclear.
Bone marrow macrophages (BMMs) from wild-type mice were cultured with M-CSF andRANKL with or without the recombinant mouse chimera CTLA4-Ig. Intracellular calcium
oscillations of BMMs with RANKL were detected by staining with calcium indicator Fura-2
immediately after administration of CTLA4-Ig or after one-day treatment. Calcium oscillations’ change with the acute administration were analyzed with wavelet time-series analysis.
Finally, LPS-induced osteolysis of calvarial bone was used to examine the negative effect on
osteoclastogenesis in vivo. Wild-type and Fc receptor gamma (FcRγ)-deficient C57/BL6 mice
were used for these experiments.
CTLA4-Ig inhibited osteoclast differentiation and reduced the expression of the nuclear factor
of activated T cells NFATc1 in BMMs in vitro. Calcium oscillations in BMMs were suppressedby CTLA4-Ig both immediately after administration and after one day of treatment. CTLA4-Ig
did not affect osteoclastogenesis and did not cause remarkable changes in calcium oscillationsin FcRγ-deficient BMMs. In vivo, CTLA4-Ig rescued LPS-induced osteolysis in wild-type mice,and the improvement did not occur in FcRγ-deficient mice.
In conclusion, CTLA4-Ig inhibited intracellular calcium oscillations via FcRγ, down-regulated
NFATc1 expression, and directly inhibited osteoclastogenesis in vitro and in vivo. This is thefirst report of co-factor (CTLA4 - CD80/86) around co-stimulatory signal (FcRγ) in osteoclastogenesis.
(COI:No)

O18-4

The Ultrastructural physiology of Bile canaliculus in Porcine Liver
Donated after Cardiac Death and Preserved with Machine Perfusion
Preservation
Hiroki Bochimoto1, 2，Yo Ishihara2, 3，Daisuke Kondoh4，Hiromichi Obara5，Naoto Matsuno2

Phosphatidylinositol 3-kinases (PI3Ks) are a family of lipid kinases that phosphorylate the 3’-OH
group at the inositol ring of phosphoinositides (PIs) on the plasma membrane and intracellularorganelle membranes. PI3Ks regulate a diverse array of dynamic membrane events. Whileclass I and class III of PI3K are well-characterized, functional roles of class II PI3K isoforms(PI3K-C2α, -C2β and -C2γ) were largely unknown. We previously demonstrated that global
C2α-knockout (KO) mice were embryonic lethal due to severe defects in angiogenesis. The
inducible endothelial cell (EC)-specific C2α deletion resulted in a similar phenotype. Mechanistically, C2α was required for ligand-induced endocytosis of the angiogenic receptors including
vascular endothelial growth factor receptor-2 (VEGFR2), sphingosine-1-phosphate receptor-1(S1P1) and transforming growth factor-β receptor (TGFβR), and thereby receptor-mediatedendosomal activation of Rho, Rac, and Smad-2/3. We studied the possible involvement of C2α
and C2β in clathrin-mediated fluid-phase endocytosis (pinocytosis) in EC. C2α and C2β wereboth required for uptake of FITC-dextran, i.e. pinocytosis. FITC-dextran uptake was partiallydependent on both clathrin and dynamin, and both PI3K-C2α and PI3K-C2β were requiredfor clathrin-mediated, but not clathrin-non-mediated, FITC-dextran uptake at the step leading
up to its delivery to early endosomes. Both C2α and C2β were co-localized with clathrincoated pits and vesicles. However, actin filament staining showed that C2β but not C2α washighly co-localized with actin filament-associated clathrin-coated pits and vesicles and required
for actin filament formation at the clathrin-coated structures. The C2β-binding protein, ITSN1,
was required for C2β co-localization at actin filament-associated clathrin-coated structuresand pinosome-associated actin filament formation. These results indicate that C2α and C2β
are involved in clathrin-mediated endocytosis with cell type-specific functional compensations.
(COI:No)（ 1 Div Aerosp Med, Dept Cell Physiol, Jikei Univ Sch Med, Japan, 2 Dept transpl technol
therapeut develop, Asahikawa Med Univ, Japan, 3 Shonan Kamakura Gen Hosp, Japan, 4 Lab
Vet Anat, Obihiro Univ Agricul Vet Med, Japan, 5 Dept Mech Eng, Tokyo Metropol Univ,
Japan）

O18-2

O18-5

Identiﬁcation of ROS induced Na+-independent Mg2+ efﬂux in rat
ventricular myocytes
1 1 Dept Physiol, Tokyo
Michiko Tashiro1，Hana Inoue1，Masato Konishi2，Utako Yokoyama（

Background: The effects of each type of machine perfusion preservation (MP) of liver grafts
donated after cardiac death on the bile canaliculi of hepatocytes remain unclear. Here weanalyzed intracellular three-dimensional ultrastructures of the bile canaliculi and endomembrane systems in hepatocytes after warm ischemia followed by successive MP under various
temperature conditions.
Methods: Porcine liver grafts after warm ischemia for an hour were perfused for four hours
with modified University of Wisconsin gluconate solution under hypothermic (HMP, n = 4)or warm MP (WMP, rewarming up to 22℃, n = 5) conditions. Transmission and osmiummaceration scanning electron microscopy were utilized to evaluate the ultrastructure of thebile canaliculi.
Results: Scanning electron microscopy revealed that lumen volume of the bile canaliculi
decreased after warm ischemia. In the liver grafts preserved by HMP, bile canaliculi tended
to recover in terms of lumen volume, although their microvilli were regressed. In contrast, theWMP preserved functional form of microvilli of bile canaliculi.
Conclusions: MP potentially restored the bile canaliculus lumen and alleviated the cessation ofcellular endocrine processes due to warm ischemia. In addition, WMP prevented the retraction of microvilli of bile canaliculi, suggesting further mitigation of damage of bile canaliculi.
(COI:No)

Imaging analysis of spatiotemporally regulated crosstalk between
thrombus formation and its lysis
Yuko Suzuki, Hideto Sano, Naoki Honkura, Tetsumei Urano（Dept Med Physiol, Hamamatsu

Med Univ, Tokyo Japan, 2 Admission center, Tokyo Med Univ, Tokyo Japan）

Univ Sch Med, Japan）

Intracellular Mg2+ may function to prevent Ca2+-overload caused by ROS (reactive oxygen
species) in ventricular myocytes. Although the regulation mechanism of intracellular-free
Mg2+ concentration ([Mg2+] i) remains unclear, [Mg2+] i should be regulated by the balance of
Mg2+ influx and efflux. Na+-gradient dependent Mg extrusion has been widely recognized asa Mg2+-efflux system.
To study effects of ROS on Mg2+ homeostasis in rat ventricular myocytes, we measured[Mg2+] i using a fluorescent indicator, mag-fura-2 in Ca2+-free Tyrode solution. [Mg2+] i(1.05±0.03 mM, n=9) was decreased to 0.82±0.03 mM (p=0.0005) in 3 min, after 5-min treatment of 500 µM H2O2. The lowered [Mg2+] i was maintained up to ～20 min, and then recovered
to the basal level at 50 min after application of H2O2.
Similar experiments were repeated (i.e., 5-min application of H2O2) in various extracellularsolutions. 1) In the absence of Mg2+, [Mg2+] i was decreased from 1.09±0.04 to 0.80±0.07 mM
in 3 min (n=5, p=0.03), but was not recovered, indicating lack of Mg2+ influx. 2) In the absenceof Na+ (substitution of NMDG for Na+), [Mg2+] i was decreased from 0.92±0.06 to 0.75±0.03mM
in 20 min (n=9, p=0.03), and was subsequently recovered. 3) When the cells were first loadedwith Mg2+ in a low-Na+ and high-Mg2+ solution, the raised [Mg2+] i (1.67±0.05 mM, n=3) was
rapidly decreased to 0.54±0.1 mM in 3 min (p=0.013).
These results suggest that Na+-independent Mg2+ efflux in rat ventricular myocytes, which isactivated by ROS, is newly identified. There is a possibility that the ROS-induced Mg2+ efflux
causes further damage to cells.
(COI:No)

Background: Recent advances in genetic engineering and optical instrumentation have allowed
us to analyze the spatiotemporal regulation of thrombus formation and its lysis. Employing
human platelet-containing normal plasma (plt-NP), we demonstrated the initiation of tissue
factor-triggered clot formation from phosphatidylserine exposing platelets and an unevenfibrin network was formed due to focal generation of thrombin. We also found that the lysisof clot was initiated from activated platelets where both tissue-type plasminogen (tPA) andplasminogen accumulated.
Aim: We further analyzed the mechanisms of crosstalk between coagulation and fibrinolysis
by focusing on thrombin activatable fibrinolysis inhibitor (TAFI) which can be activated bythrombomodulin (TM)-bound thrombin.
Methods: 1) Employing plt-NP or normal platelet-containing TAFI deficient plasma (plt-TAFIDP), effects of neutralizing antibody of TM (TM-Ab) and activated TAFI inhibitor (TAFIaI)
were determined as tPA-induced clot lysis time (CLT) using turbidimetric method. 2) Fibrin
network formation and lysis were visualized by confocal microscopy and the localizationof fluorescence labeled plasminogen and TAFI were analyzed. 3) Activation of TAFI was
evaluated under flow condition employing a microchip-based flow chamber system (Total
Thrombus-formation Analysis System).
Results: 1) CLT in plt-NP was prolonged in a platelet concentration-dependent manner, which
was restored by either TM-Ab or TAFIaI. In plt-TAFI-DP, however, no prolongation of CLT
was shown, suggesting that plasma TAFI is essential for platelet-dependent activation of
TAFI. 2) Time required for plasminogen accumulation, considered as TAFI activation marker,
was shortened by either TM-Ab or TAFIaI. Labeled TAFI localized to both fibrin fiber andactivated platelets, in which the former was Lys-binding site dependent- but the latter was
independent-manner. 3) TAFIaI significantly prolonged occlusion of flow chamber in the presence of tPA, suggesting that TAFI was efficiently activated under flow condition.
Conclusion: Spatiotemporal analysis enabled us to understand the precise crosstalk between
coagulation and fibrinolysis mediated by TAFI.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S87

Oral Session 19
Development・Growth・Aging（March 19, 15:10〜16:10, Hall 7）

O19-3

Stress-induced premature senescence caused by inhibition ofproteostasis in human ﬁbroblasts
Yasuhiro Takenaka1, 2，Ikuo Inoue2，Takanari Nakano3，Masaaki Ikeda4，
1 1 Dept Physiol, Nippon Med Sch, Japan, 2 Dept Diabetes andYoshihiko Kakinuma（

Endocrinol, Saitama Med Univ, Japan, 3 Dept Biochem, Saitama Med Univ, Japan, 4 Dept
Physiol, Saitama Med Univ, Japan）

To investigate how prolonged disturbances of proteostasis is involved in cellular senescenceprocess in proliferating cell, we developed a model in which young normal human fibroblasts
MRC-5 were treated with either of a reversible proteasome inhibitor, MG-132, and V-ATPase
inhibitor, Bafilomycin A1. After 5-days drug treatment, cells showed senescent phenotypes
such as a flattened morphology, permanent cell cycle arrest, expression of senescenceassociated β-galactosidase, and upregulation of p21 and p53. Induction of γH2A.X, a marker
of DNA damage response, was detected especially during post-treatment period of either drug.
Levels of intracellular reactive oxygen species (ROS) such as hydroxyl radicals and hydrogen
peroxide also significantly elevated during and after drug treatment, which possibly caused deleterious damage to the nuclear DNA. To clarify the source of ROS, we evaluated mitochondrial
function in the course of senescence induction. Mitochondrial signals detected by fluorescencedye and the copy number of mitochondrial DNA gradually increased during and after drug
treatment, whereas mitochondrial membrane potential was temporarily downregulated during
drug treatment, indicating partial loss of mitochondrial function. Transient upregulation of
PGC-1α and sustainable increase in TFAM protein levels were confirmed by immunoblots
whereas expression of mitophagy-associated protein Parkin was suppressed during and after
drug treatment. Addition of rapamycin to the culture media containing MG-132 or Bafilomycin
A1 rescued induction of premature senescence and recovered cellular proliferation potential
possibly by suppressing generation of excess ROS. Transport of SOD2 and GPx4, antioxidant
enzymes, into the mitochondria was inhibited on only early period of drug treatment (day 1).
It is concluded that temporal deterioration of mitochondrial transport could be a trigger of
initial generation of ROS and subsequent induction of mitochondrial biogenesis and cellularsenescence.
(COI:No)

O19-1

O19-4

Keisuke Ikegami1, 2，Kazumasa Saigoh3，Atsuko Fujioka2，Mamoru Nagano2，
Chihiro Satou4，Ken Kitajima4，Susumu Kusunoki3，Satoru Masubuchi1，
2 1 Dept Physiol, Sch Med, Aichi Med Univ, Japan, 2 Dep Anato,
Yasufumi Shigeyoshi（

Wendy Hempstock, Fumiya Kurihara, Noriko Ishizuka, Hisayoshi Hayashi（Dept Physiol, Sch

Effects of expression variation in ﬂanking genes on phenotypes ofSt8sia2-knockout mice

Intestinal nutrient absorption and barrier function in SAMP1
senescence accelerated aged mice
Food and Nutr, Univ of Shizuoka, Japan）

The induction of null mutations by means of homologous recombination is a powerful techniquefor validating gene function, but the genetic background and closely linked genes flanking
the targeted locus often affect phenotypic changes in the null mutant. ST8 alpha-N-acetylneuraminide alpha-2, 8-sialyltransferase 2 (ST8SIA2) synthesizes polysialic acid (PSA), whichis essential for brain development. Although previous studies reported that St8sia2-deficient
mice that have a mixed 129 and C57BL/6 (B6) genetic background showed mild and variablephenotypes, the reasons for this remain unknown. We hypothesized that this phenotypic difference is caused by diversity in the expression or function of flanking genes of St8sia2. A
genomic polymorphism and gene expression analysis in the flanking region revealed reduced
expression of insulin-like growth factor 1 receptor (Igf1r) on the B6 background than on thatof the 129 strain. This observation, along with the finding that administration of an IGF1R
agonist during pregnancy increased litter size, suggests that the decreased expression of Igf1r
associated with ST8SIA2 deficiency caused lethality. This study demonstrates the importanceof gene expression level in the flanking regions of a targeted null allele having an effect onphenotype.
(COI:No)

Aging is an inevitable universal biological process, which can be characterized by a generaldecline in physiological function with the accumulation of diverse adverse changes and increased probability of death. In the intestine, it is widely believed that with aging, the intestinal
barrier function decreases, which leads to “leaky gut”. Leaky gut is connected to a wide
variety of diseases, including inflammatory bowel disease and colon cancer, which affect manyelderly people all over the world. In addition, it is thought that nutrient absorption efficiency
is decreased in the aged intestine, which may contribute to malnutrition and muscle wastingin the elderly. As a first step toward understanding the details of cellular senescence in theintestine, we conducted experiments using senescence accelerated mouse prone 1 (SAMP1),
which is a strain of mice developed by Jackson Laboratories and the University of Kyoto.
SAMP1 mice mature normally and then experience accelerated aging. SAMP1 mice have alifespan around half that of normal mice, which means they reach old age at around 8-9 monthsof age, with an average lifespan of 9.7 months. To investigate the effect of aging on intestinal
nutrient absorption and barrier function we measured glucose and peptide absorption as well
as dilution potential using Ussing chambers. Tissue was also collected for quantitative RT-PCR
and immunofluorescence analysis. SAMP1 mice were compared to age-matched SAMR1 mice,
which are mice of the same background that do not have the accelerated aging phenotype(average lifespan: 16.3 months). Preliminary results suggest that the large intestine becomes
more permeable, or leaky. However, surprisingly, the small intestine of SAMP1 mice seems
to become tighter compared to SAMR1 controls. Thus this may be a mechanism by which
nutrient absorption is decreased in aged mice.
(COI:No)

O19-2

O19-5

Kindai Univ Fac Med, Japan, 3 Dep Neuro, Kindai Univ Fac Med, Japan, 4 Lab Ani Cell
Fun, Biosci Biotech Center, Nagoya Univ. Japan）

Orthotopic Transplantation of Human iPSC-derived Liver Tissue with
Lumenized Blood Vessels is an Effective Cell Therapeutic for Liver
Cirrhosis
Tomomi Tadokoro1，Yoshiki Kuse2，Megumi Matsuo3，Yutarou Uchida1，Kohei Kaida1，
Koudai Kimura1，Akira Higashibata4，Toko Hashizume4，Yasuharu Ueno2，Satoshi Okamoto1，
2 1 Dept Regen Med, Grad Sch Med, Yokohama City
Soichiro Murata1，Hideki Taniguchi1, （Univ, Japan, 2 Div of Regen Med, Inst of Med Sci, The Univ of Tokyo, Japan, 3 Dept of Gen
Srg, Grad Sch of Med, Chiba Univ, Japan, 4 JEM util ctr, Human Spaceflight tech dir, JAXA,
Japan）

Organoid research is making remarkable progress and a partial reconstitution of complex
organ system has been achieved by organoids. We previously reported in vivo vascularizationof human induced pluripotent stem cell (hiPSC)-derived liver organoids and its therapeutic
effect on mouse acute liver failure. To treat end-stage liver diseases, we sought to develop thecell culture technology for in vitro liver tissue reconstruction including lumenized blood vessels and the novel orthotopic transplantation on liver surface. In this study, 3D imaging of liver
bud formation at mouse embryonic stages revealed that vascular network is formed outsideof the hepatic cluster by static interactions between hepatic, endothelial and mesenchymal
cells. As hepatic progenitors derived from anterior intestinal portal and lateral endoderm
are fused to form liver bud, we statically fuse small liver buds derived from hiPSC to mimic
embryonic liver bud formation, and successfully generate liver tissue including lumenizedblood vessels. We also showed novel orthotopic transplantation of rat embryonic livers canrescue liver cirrhosis concomitant with maturation of donor organ. Finally, we demonstrated
that hiPSC-derived liver tissue improved survival rate and the blood biochemical parametersof liver cirrhosis model, and ameliorated liver fibrosis. It is speculated that recovery fromliver fibrosis is achieved via MMPs secreted from hiPSC-liver tissue and anti-fibrotic factors
derived from macrophages accumulated after transplantation. Taken together, in vitro tissuereconstruction technology could be a promising tool for regenerative medicine when combinedwith liver surface transplantation.
(COI:No)

S88

Exendin-4 promotes neurite outgrowth, Schwann cell survival/migration,and myelination in vitro
Kazunori Sango, Shizuka Takaku, Naoko Niimi, Hideji Yako（Diabetic Neuropathy PJ, Tokyo
Met Inst Med Sci, Tokyo）

Besides its insulinotropic actions on pancreatic β cells, the localization of glucagon-like peptide-1 receptor (GLP-1R) at the nervous system suggests neuroprotective activities of GLP-1.
Previous studies have suggested the efficacy of a GLP-1 receptor agonist exendin-4 (Ex-4) for
functional repair after brain and sciatic nerve injury, and amelioration of Parkinson’s disease
and diabetic neuropathy. However, the underlying mechanisms for these Ex-4 effects remain
unclear. In this study, the effects of Ex-4 on neurite outgrowth of 8-week-old female Wistar rat
dorsal root ganglion (DRG) neurons, survival and migration and of immortalized adult Fischer
rat Schwann cells IFRS1, and myelination in the DRG neuron-IFRS1 co-culture system were
investigated. Ex-4 dose-dependently (1 nM < 10 nM < 100 nM) promoted neurite outgrowth of
DRG neurons, and these effects were attenuated by co-treatment with PI3 kinase (PI3K) inhibitor LY294002 (5 µM and 25 µM). By MTS and scratch wound assays, 100 nM Ex-4 significantly
enhanced survival and migration of IFRS1 Schwann cells, respectively. Moreover, immunofluorescence and western blot analyses performed at 21 days of co-culture revealed that 100
nM Ex-4 significantly increased the number of myelin protein 22 (PMP22)-immunoreactive
IFRS1 cells surrounding βIII tubulin-immunoreactive neurites and up-regulated the protein
expression of PMP22 and myelin protein zero (MPZ). Western blotting performed at 1 day of
co-culture resulted in Ex-4-induced phosphorylation of a serine/threonine kinase AKT, suggesting that Ex-4 accelerates the myelination process in the co-culture via activating PI3K/
AKT pathway. Because GLP-1R is expressed in both DRG neurons and IFRS1 Schwann cells,and Ex-4 promotes DRG neurite outgrowth and IFRS1 Schwann cell survival and migration,
Ex-4 may act on both cells to promote myelination. These findings imply the efficacy of Ex-4 inaccelerating axonal regeneration and remyelination following peripheral nerve injury, as wellas preventing and restoring diabetic and other peripheral neuropathies.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Oral Session 20
Heart・Circulation 3（March 19, 14:10〜15:10, Hall 9）O20-3

Sphingosine kinase-2 is required for autophagic lipid degradation in
macrophage and inhibits atherosclerosis
Yoh Takuwa1，Kazuhiro Ishimaru1，Kazuaki Yoshioka1，Noriko Takuwa1, 2，Yasuo Okamoto1（ 1 Dept Physiol, Kanazawa Univ Sch Med, Japan, 2 Dept Health Sci, Ishikawa Pref Nursing
Univ, Japan）

Atherosclerosis, which underlies ischemic coronary heart diseases, is characterized by the
infiltration of cholesterol-accumulating macrophages in the intima of the vasculature. Althoughsphingolipids are implicated in atherosclerosis as both membrane components and lipid mediators, the precise role of sphingolipids in atherosclerosis remains elusive. We show that genetic
disruption of sphingosine kinase-2 (SphK2) but not SphK1 aggravates the formation of atherosclerotic lesions in mice with ApoE deficiency. Bone marrow chimaera experiments show
the involvement of SphK2 expressed in myeloid cells. In macrophages, deficiency of SphK2,
a major SphK isoform in this cell type, increases cellular levels of sphingosine and ceramides.
SphK2-deficient macrophages have increases in lipid droplet-containing autophagosomes
and autolysosomes and defective lysosomal degradation of lipid droplets via autophagy with
impairment of luminal acidification and proteolytic degradation in the lysosomes. Transgenic
overexpression of SphK1 in SphK2-deficient mice prevented aggravation of atherosclerosis
and abnormalities of autophagosomes and lysosomes in macrophages, which suggested that
two SphKs have at least partially overlapping actions. These results collectively demonstratethat SphK2 is necessary for autophagosome- and lysosome-mediated catabolism of intracellularlipid droplets to prevent the development of atherosclerosis; therefore, SphK2 may be a novel
therapeutic target of atherosclerosis.
(COI:No)

O20-1

O20-4

Meihua Li, Can Zheng, Toru Kawada, Kazunori Uemura, Masashi Inagaki, Masaru Sugimachi

Hirohiko Kohjitani1，Shigeya Koda2，Yukiko Himeno2，Takeru Makiyama1，Yuta Yamamoto1，
Yimin Wuriyanghai1，Daisuke Yoshinaga3，Asami Kashiwa1，Akira Amano2，Akinori Noma2，
1 1 Dept Cardiovasc Med, Grad Sch Med, Kyoto Univ, Japan, 2 Dept Physiol,
Takeshi Kimura（

Donepezil Treatment Prevents the Progression of Chronic Heart Failure
and Improves the Prognosis in Spontaneously Hypertensive Rats with
Myocardial Infarction（Dept cardiovascular dynamics, NCVC, Osaka, Japan）

Multi-layer Cardiac Pacemaker Mechanisms demonstrated in Human
Induced Pluripotent Stem Cells by Developing Mathematical Cell Models

Introduction: We have demonstrated that acetylcholinesterase inhibition by donepezil
improves long-term survival of chronic heart failure (CHF) rats with myocardial infarction(MI). This study aimed to investigate whether donepezil is applicable to the treatment of CHF
complicated with hypertension.
Methods: CHF was induced by permanent extensive MI in 7-week-old male spontaneously
hypertensive rats. After one-week recovery, we implanted a blood pressure transmitter formonitoring daily hemodynamics. Survived animals were randomly assigned to untreated (UT,n = 23) or donepezil treated (DT, n = 22) group. In the DT group, rats received average doseof 5 mg/kg/day of donepezil in drinking water (50 mg/L). The dose of donepezil was chosen inorder to decrease the heart rate by 20–30 bpm. At the end of 7-week treatment, the impactsof donepezil were evaluated by hemodynamics, neurohumoral states, immunohistochemistry,morphology, and 50-day survival rate.
Results: Compared with UT, DT significantly decreased the heart rate (305 ± 12 vs. 335 ±11bpm, P<0.05), but did not change the mean blood pressure. Although there was no significant
difference in the MI size between the two groups, DT improved 50-day survival (76% vs. 43%,
P = 0.006), through suppressing the progression of cardiac hypertrophy (3.83 ± 0.05 vs. 4.09
± 0.07g/kg, P<0.05) and cardiac dysfunction (cardiac index: 101 ± 4 vs. 89 ± 4 ml/min/kg,
P<0.05; LVEDP: 12 ± 3 vs. 22 ± 2 mmHg, P<0.05). Additionally, DT not only decreased plasma
levels of norepinephrine, BNP and AVP, but also improved the systemic inflammation.
Conclusions: Donepezil treatment prevented the progression of cardiac remodeling, cardiac
dysfunction and improved the prognosis of CHF in spontaneously hypertensive rats withMI, suggesting donepezil may be used as a new pharmacotherapy for patients with CHF
complicated with hypertension.
(COI:No)

Facl Life Sci, Sch Life Info, Ritsumeikan Univ, Japan, 3 Dept Ped Med, Grad Sch Med, Kyoto
Univ, Japan）

O20-2

O20-5

Kenji Suita, Yoshiki Ohnuki, Satoshi Okumura（ Dept Physiol, Tsurumi Univ Sch Dent Med,

2 1 Faculty of Science and Medicine, University ofJean-Pierre Montani1，Bruce N. Van Vliet（

Occlusal disharmony increased vulnerability to atrial ﬁbrillation via
sympathetic activation in mice
Yokohama, Japan）

Background: Autonomic nervous system plays an important role to improve cardiac function against acute change of hemodynamics. The β-adrenergic receptor (β-AR) signaling is
one of the most important regulators to maintain cardiac function by sympathetic activity.
However, persistent sympathetic activation is known to induce cardiac remodeling, leading
to cardiovascular diseases such as heart failure and arrhythmias. Atrial fibrillation (AF) isthe most prevalent arrhythmia and a source of considerable morbidity and mortality. Tooth
loss or incorrect positioning causes occlusal disharmony. Furthermore, tooth loss and AF
are both risk factors for ischemic stroke and coronary heart disease. Also, it is shown that
occlusal disharmony has adverse effects on other systemic organs via stress-induced autonomic
imbalance. Here, we hypothesized that the occlusal disharmony may be a risk factor of AF.
Results: Occlusal disharmony was induced by means of bite-opening (BO) for a period of 2
weeks. The duration of AF induced by transesophageal atrial burst pacing were significantly
elongated in BO mice than control. Atrial myocyte apoptosis and fibrosis were significantly
increased by BO as compared to control mice. Oxidative DNA damage in the myocardium wassignificantly greater in BO-treated mice than control. Consistent with the atrial remodeling,
the CaMKII phosphorylation (threonine-286), CaMKII oxidation (methionine-281/282) and RyR2
phosphorylation (serine-2814) were significantly increased in BO-group than control. The indicators of apoptosis such as Bax/Bcl-2 ratio, activated caspase-9 and activated caspase-3 weresignificantly increased by BO. Akt phosphorylation (serine-473), an anti-apoptotic factor, wassignificantly decreased by BO. Co-treatment of propranolol, a β-AR antagonist, significantly
prevented BO-induced alterations in the atrial myocytes.
Conclusion: Occlusal disharmony may be an inducer of increased AF vulnerability throughpersistent activation of sympathetic nervous system.
(COI:No)

Undifferentiated myocardial cells derived from Human induced pluripotent stem cell (hiPSCCM) mostly shows spontaneous activity. The difference in the configuration and frequency of
spontaneous action potential (AP) may be due to variable expression levels of the ion channels,
and thereby the hiPSC-CM may embody mechanisms of various slow diastolic depolarization(SDD). To clarify mechanisms of SDD, we first created whole 3 types of hiPSC-CM mathematical models. Then, specific role of each ion channel in generating SDD was examined by changing the relative amplitude of delayed rectifier K+ current (IKr), inward-rectifier K+ current(IK1), L-type Ca2+ current (ICaL + Ist), hyperpolarization-activated current (Iha), and IbNSC
to determine the range of combinations that allow generation of spontaneous action potential.
The lead potential analysis well quantified the contribution of these currents, and we could
identify two basic mechanisms and two additional mechanisms. The primary mechanism 1 isthe removal of inactivation of IKr on repolarization (y1 gate) and subsequent deactivation (y2 +y3 gates) during 100 to 200 ms of DD. The primary mechanism 2 is caused by positive feedback
process among inward currents i.e. ICaL and Ist, T-type Ca2+ current (ICaT), inward Na+
current (INa), and current of Na+/Ca2+ exchanger (INCX). These primary mechanisms induce
the typical sinus node low membrane potential oscillation. High membrane potential oscillation
is induced by the secondary two channel mechanisms; activation and subsequent deactivationof Iha, and unblocking and re-blocking of IK1 by intracellular factors. These mechanisms work
between the two primary mechanisms, but gradually relax by themselves and evoke relatively
lower rhythm of SDD. Finally, IbNSC is mainly responsible for determining the negative limitof the maximum diastolic potential, while IKr, IKur, IKto induces repolarization from theplateau potential. If these repolarizing reserves are limited, the oscillation is stopped at theplateau potential near -20 mV.(COI:No)

Regulation of Blood Pressure and Cardiac Output during Chronic
Alpha1-Adrenergic Stimulation
Fribourg, Switzerland, 2 Division of Basic sciences, Memorial University of Newfoundland,
St. John’s, Canada）

Background: The alpha1-adrenergic receptor plays a key role in cardiovascular regulation.
The main objective of this study was to evaluate the long-term control of blood pressure
and cardiac output during chronic vasoconstriction induced by alpha1-adrenergic stimulation.
Methods and results: We infused phenylephrine (PE) intravenously (1 microg/kg/min) for 10
days in 7 dogs housed in metabolic cages and maintained on a fixed sodium intake. Mean arterial pressure (MAP) and cardiac output (CO) were monitored continuously 20 hours/day. On
the first day, PE caused a rapid increase in MAP from a control value of 91 ± 4 mmHg to 113± 8 mmHg, and decreased both CO and heart rate from 2.32 ± 0.10 L/min and 69 ± 3 beats/
min to 1.77 ± 0.06 L/min and 51 ± 2 beats/min, respectively. The initial increase in MAP was
not sustained as MAP stabilized then at a value of 99 ± 4 mmHg (days 8-10) whereas CO andheart rate remained decreased at 1.74 ± 0.09 L/min and 54 ± 2 beats/min, respectively (days
8-10). Total peripheral resistance increased initially by 62% and remained elevated by ~50%
throughout the infusion. However, due to an increase in hematocrit from 37.9 ± 1.9 to 52.2 ±
3.6, arterial oxygen delivery, estimated by the product of CO and hematocrit, was maintained.
Summary and conclusions: Chronic vasoconstriction led to a mild hypertension with asustained decrease in heart rate and cardiac output. However, despite the low cardiac output,
oxygen delivery to the tissues was maintained secondary to an increase in hematocrit. Ourdata suggest that arterial oxygen delivery rather than cardiac output itself is a strongly
regulated variable.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S89

Oral Session 21

O21-3

Heart・Circulation 4

Eriko Kawai1，Daiki Imai1, 2，Kosuke Saho1，Emiko Morita1，Yuta Suzuki1, 2，
2 1 Dept of Environ Physiol for Exerc, Osaka
Hisayo Yokoyama1, 2，Kazunobu Okazaki1, （（March 19, 15:10〜16:10, Hall 9）

The effects of fragrance inhalation of Lavender essential oil on bloodpressure and muscle sympathetic nerve activity
City Univ Grad Sch of Med, Osaka, Japan, 2 Res Ctr for Urban Health ＆ Sports, Osaka City
Univ, Osaka, Japan）
Introduction: Previous animal studies reported that fragrance inhalation of Lavender essential
oil decreased sympathetic nerve activity as well as blood pressure (BP). We recently foundin humans that fragrance inhalation of Grapefruit essential oil increased diastolic BP which
was associated with an increase in muscle sympathetic nerve activity (MSNA). The purposeof this study was to investigate the effect of the fragrance inhalation of Lavender essential oil
on BP and MSNA.
Methods: Young healthy subjects were participated in this study (men; n=7, woman; n=1).
During subjects in a sitting position, they inhaled brank air via face mask for 10 min as baseline(BL), then inhaled air included fragrance components of Lavender essential oil collected in a
Douglas bag for 10 min. Beat-by-beat BP and heart rate (HR), and MSNA (peroneal nerve,
microneurography) were continuously recorded during experiment.
Results: Diastolic BP (BL, 56.4 ± 5.9 mmHg; inhalation, 55.0 ± 5.6 mmHg) and MSNA burst
frequency (BL, 18.0 ± 6.2 bursts/min; inhalation, 15.8 ± 6.4 bursts/min) decreased significantly
during fragrance inhalation of Lavender essential oil compared to BL. On the other hand,systolic BP and HR remained unchanged with fragrance inhalation.
Conclusion: The fragrance inhalation of Lavender essential oil has the effects to decrease(COI:No)
diastolic BP and MSNA.

O21-1

O21-4

Toru Kawada1，Takashi Sonobe2，Takuya Nishikawa1，Meihua Li1，Tsuyoshi Akiyama2，
1 1 Dept Cardiovasc Dynamics, Natl Cereb CardiovascJames Pearson2，Masaru Sugimachi（

Though comprehensive works have established the pressure-volume (PV) analysis for cardiac
functions, the potential application of this technique in the urinary bladder, which functions
resemble to the heart as it is continuously filled with fluid and periodically contracts to propel
fluid against resistance, has not been established.
In forty urethane-anesthetized (1.2 g/kg) female Sprague-Dawley rats, cystometry was
conducted to record voiding contractions induced by a constant saline infusion (0.04 ml/min);and the PV relationship was established by plotting intra-vesical pressure (IVP) against intravesical volume (IVV).
In a micturition cycle, PV data point moved counterclockwise and the PV trajectory shaped
an enclosed loop in the plane. PV loops coincided well and the loop-derived urodynamic parameters kept stable under a constant saline infusion (0.04 ml/min).
Enhancing preload by elevating infusion rates (to 0.08 and 0.12 ml/min) increased the area
enclosed by the PV loop (Apv) and shifted loops to the right and slightly upward. Augmenting
afterload by increasing outlet resistance (to 1/4 and 1/2 urethra clamping) increased Apv andshifted loops to the right and upward. Without affecting Apv, muscarine (0.01 and 0.1 mM)induced inotropic states enhanced peak and voiding pressures but reduced end-filling and fillingvolumes that was as opposed to the atropine (0.01 and 0.1 mM)-induced anti-inotropic state.
Collectively, PV analysis not only reliably assayed baseline bladder function but also validly
assessed modified bladder functions both in response to altered external environment andbladder contractility itself; thereby could provide a novel dimension in cystometry exploring(COI:No)
bladder functions/diseases. Contribution of afferent stimulation to hemodynamic and myocardial
acetylcholine release during vagal nerve stimulation
Ctr, 2 Dept Cardiac Physiol, Natl Cereb Cardiovasc Ctr）

Purpose: Although vagal nerve stimulation (VNS) is considered to be a new treatment strategy
for heart failure, clinical trials of VNS showed diverse efficacy results. This study aimed toelucidate the contribution of afferent stimulation to hemodynamic and myocardial interstitialacetylcholine (ACh) release during VNS.
Methods: We measured arterial pressure (AP), heart rate (HR), and ACh responses to 10-min
right VNS in anesthetized Wistar-Kyoto rats. The experiment was performed in the rats with
intact vagi (INT, n=7) and the rats whose right vagus was sectioned at the afferent portion of
the stimulation site (VAX, n=6). VNS was tested with a stimulation voltage of 3 V or 10 V anda stimulation frequency of 5 or 20 Hz. The pulse duration was fixed at 0.1 ms.
Results: The high-voltage VNS (10 V, 5 Hz or 20 Hz) produced significant initial reductionsin AP and HR in less than 10 s in the INT group. These initial AP and HR responses were
not observed in the VAX group. The high-frequency VNS (20 Hz, 3 V or 10 V) significantly
increased myocardial interstitial ACh levels in both the INT and VAX groups.
Conclusion: The initial hemodynamic responses, observed during high-voltage VNS in the
INT group, were attributable to afferent stimulation. Unmyelinated afferent C fibers may be
responsible for these initial hemodynamic responses because high-voltage VNS was required
for the responses. Myocardial interstitial ACh release, observed during high-frequency VNS
in both the INT and VAX groups, was mainly attributable to efferent stimulation. Lightly
myelinated efferent B fibers may be responsible for the myocardial ACh release.
(COI:No)

Pressure-volume analysis of ratʼs micturition cycles in vivo
Tzer-Bin Lin（Dept Physiol School Med, Taipei Med Univ, Taiwan）

O21-2

O21-5

Mark T. Waddingham 1，Hirotsugu Tsuchimochi2，Ryotaro Asano1，Mikiyasu Shirai1，
1 1 Pulmonary Hypertension, NCVC, Osaka, Japan,
James T. Pearson 2，Takeshi Ogo（

（Dept Cardiovascular Dynamics, NCVC, Japan）

Purpose: Pulmonary arterial hypertension (PAH) is associated with right ventricular (RV)
dysfunction. The mechanisms leading to RV dysfunction remain unclear, although overactivation of the sympathetic nervous system (SNS) appears to be a central event. G protein-coupled
receptor kinase 2 (GRK2) overexpression is known to enhance SNS overactivation in heart
failure. GRK2 has also been shown to negatively modulate nitric oxide bioavailability, which canpromote diastolic dysfunction and myofilament dysfunction. However, the connection between
GRK2, the myofilaments and diastolic dysfunction has not been examined in RV dysfunction
in PAH. Therefore, our aim was to evaluate changes in GRK2 expression over the time-courseof RV dysfunction development and relate these to changes in global RV function and myofilament function in a rat model of PAH.
Methods: PAH was induced in rats using the Sugen/10% chronic hypoxia (SuHX) method, andrats were subsequently housed in normoxic conditions for 3 or 6 weeks. We simultaneously
examined global RV function and myofilament function in anesthetized rats using cardiac
catheterization and muscle x-ray diffraction, respectively.
Results: SuHx rats exhibited significantly increased RV end systolic pressure (P<0.05), indicative of PAH. An increase in RV end diastolic pressure and RV tau relaxation constant wasalso observed, suggesting early diastolic dysfunction in PAH rats. Muscle x-ray diffraction
experiments revealed a progressive decline in RV diastolic myosin mass in PAH rats. We
found a progressive decline in relative eNOS phosphorylation and a significant increase in
iNOS (P<0.01) and GRK2 (P<0.01) expression in SuHx rats 6 weeks after induction. Importantly
we found a correlation between GRK2 expression and iNOS (P<0.0001) expression and eNOS
phosphorylation (P<0.05).
Conclusion: These data suggest that GRK2 may drive the development of RV dysfunction,(COI:No)potentially by modulating myofilament function in PAH.

Background: Positive end-expiratory pressure (PEEP) is the mechanical ventilation that applies pressure above atmospheric pressure at the end of expiration. PEEP prevents alveolar
collapse, improves pulmonary congestion, and improves respiratory status in patients withcongestive heart failure (HF). However, PEEP suppresses venous return through an increase
in intrapleural pressure, and reduces cardiac output (CO). The lack of understanding ofquantitative relation between PEEP and hemodynamics makes it difficult to set optimal PEEP.
Therefore, we aimed to quantitatively predict the impact of PEEP on hemodynamics using theframework of circulatory equilibrium.
Theoretical consideration: The framework of circulatory equilibrium defines circulatory
equilibrium as the intersection of cardiac output curve (COC) and venous return surface (VRS).
PEEP increases the intrapleural pressure and shifts COC rightward and VRS upward. Theintersection of shifted COC and VRS represents hemodynamics under PEEP.
Method: Two mongrel dogs were pre-implanted with a flow probe to measure CO. We measured CO, central venous pressure (CVP) and left ventricular end-diastolic pressure (LVEDP)
under closed-chest and then increased PEEP stepwise. Using our proposed framework, we
predicted the hemodynamic impact of PEEP and compared them with those measured. To
reproduce the various pathological conditions, we induced volume overload by the plasma
expanders and HF by embolizing coronary artery with the glass microspheres. We repeatedthe hemodynamic prediction protocol under each condition of volume load and HF.
Result: The estimated CO (r2=0.95, root mean squared error [RMSE] 1.4 ml/min/kg), CVP(r2=0.86, RMSE 0.33 mmHg) and LVEDP (r2=0.88, RMSE 0.17 mmHg) matched well with thosemeasured regardless of the conditions of volume overload or HF.
Conclusion: The framework of circulatory equilibrium can quantitatively predict the hemodynamic modulation by PEEP. It may contribute to the optimal setting of PEEP in patients with
HF.(COI:No)A potential role for GRK2 in the development of right ventricular
dysfunction in pulmonary arterial hypertension
2 Cardiac Physiology, NCVC, Osaka, Japan）

S90

Prediction of the hemodynamic impact of positive end-expiratory
pressure using the framework of circulatory equilibrium in dog
Takuya Nishikawa, Kazunori Uemura, Toru Kawada, Yohsuke Hayama, Masaru Sugimachi

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

Poster Presentations
Day 1（ March 17 , 14 : 20 〜 15 : 20 ）1P-001〜1P-012

Ion Channel・Receptor（ 1 ）1P-013〜1P-024

Heart・Circulation（ 1 ）1P-025〜1P-036

Neuron・Synapse（ 1 ）1P-037〜1P-046

Sensory Function（ 1 ）1P-047〜1P-053

Behavior Science・Biorhythm（ 1 ）1P-054〜1P-056

Neurochemistry（ 1 ）1P-057〜1P-061

Autonomic Nervous（ 1 ）1P-062〜1P-067

Muscle Physiology（ 1 ）1P-068〜1P-072

Oral Physiology（ 1 ）1P-073〜1P-077

Endocrinology（ 1 ）1P-078〜1P-079

Kidney・Urination（ 1 ）1P-080〜1P-085

Motor Function（ 1 ）1P-086〜1P-088

Development・Growth・Aging（ 1 ）1P-089〜1P-095

Cell Physiology・Molecular Physiology（ 1 ）1P-096〜1P-101

Environmental Physiology（ 1 ）1P-102〜1P-104

Drug Actions（ 1 ）1P-105〜1P-111

CNS Function（ 1 ）1P-112〜1P-117

Nutrition・Metabolism・Thermoregulation（ 1 ）1P-118〜1P-123

Pathophysiology（ 1 ）1P-124〜1P-125

Physical Fitness・Sports Medicine（ 1 ）1P-126〜1P-127

Respiration（ 1 ）1P-128〜1P-129

Study Methodology（ 1 ）1P-130〜1P-132

Others（ 1 ）1P-001

1P-004Masayuki Yamashita（ Centr Basic Med Res, Int Univ Health ＆ Welfare, Japan）

2 1 Div Biophys and Neurobiol,
Ki-ichi Hirazawa1, 2，Takushi Shimomura1, 2，Yoshihiro Kubo1, （Anti-integrin antibodies enhance axon extension and electric guidance
Growing axons are directed not only by chemical signals but also by electric fields in a process
known as galvanotropism. The axons of embryonic brain, spinal cord, and retina extend along
the extracellular voltage gradient towards the cathode. In the embryonic central nervous
system, endogenous positive DC potentials are generated by neuroepithelial cells’ sodiumtransport, of which disruption results in erroneous path-finding of newborn neurons’ axons(Yamashita, BBRC, 2013). This means that the electric field is essential for orienting axons during embryonic development. However, there is no experimental evidence for the cell surface
molecule that is responsible for galvanotropism. The axons of embryonic neurons express
integrin, although the role for integrin in axon development was unknown. Here I show that
integrin regulates axon extension and is involved in the electric axon guidance. Retinal strips ofchick embryos were cultured in the electric field of the same strength as that in vivo (15 mV/mm). To maintain the environment for retinal ganglion cell (RGC) axon extension, the retinal
strip was embedded in Matrigel®, since Matrigel® and the inner limiting membrane, onto
which RGC axons extend expressing integrin α6β1, contain the extracellular matrix ligands,
laminin and collagen. RGC axons extended from the retinal strip towards the cathode. A monoclonal anti-chicken integrin β1 antibody TASC significantly enhanced the cathodal growth.
The enhancement of the cathodal growth depended on the concentration of TASC. Anothermonoclonal anti-chicken integrin β1 antibody W1B10 also enhanced the cathodal growth in adifferent dose-dependent manner: it was more effective than TASC at low concentrations (≦50 µg/mL). The negative control isotype antibody mouse IgG1 showed no enhancement of thecathodal growth even at a high concentration (200 µg/mL). TASC and W1B10 also increased
axon extension without electric fields. This indicated that integrin regulates axon extension
itself. (COI:No)

Analysis of the structural dynamics of Two-pore Na+ channel 3
Natl Inst Physiol Sci, 2 Dept Physiol Sci, SOKENDAI ）Two-pore Na+ channel 3 (TPC3) is a voltage-gated Na+ channel. Its single polypeptide has 2
repeats of canonical motif of voltage-gated cation channels, composed of 6 transmembrane
helices (6TM). Each of the 6TMs has one voltage sensor domain (VSD) and one pore domain(PD). Previously we revealed that the 2nd VSD of TPC3 is responsible for voltage sensing,whereas the 1st 6TM contains a phosphoinositide (PI) binding site, which potentiates voltage
sensitivity of TPC3 gating. We also revealed PI dependent potentiation needs a functional
linkage between the 1st 6TM and 2nd 6TM, through one electrostatic interaction between the
1st PD and 2nd PD. These findings suggest that PI binding to the 1st 6TM can modulate voltage
dependent structural change of the 2nd VSD via functional coupling between the 1st 6TM and2nd 6TM. To reveal how this PI binding affects voltage dependent structural changes of the
2nd VSD, we performed voltage clamp fluorometry analysis (VCF) of TPC3 heterologously
expressed in Xenopus oocytes. In VCF, along with TPC3 current, we simultaneously recorded
local structural changes of TPC3 based on the fluorescent intensity from fluorophore attachedto the specific site in TPC3. We successfully detected voltage dependent structural changes ofthe 2nd VSD which could cause pore gating. Furthermore, when we manipulated PI concentration within oocyte by the co-expression of voltage sensitive phosphatase, structural changeof the 2nd VSD was affected by PI concentration change. The effect of PI binding on the 2nd
VSD movement could be further supported by the analysis of one TPC3 mutant which has adeficiency of its PI binding. Therefore, PI binding may regulate the voltage sensitivity of TPC3
gating through potentiation of the movement of the 2nd VSD.
(COI:No)

1P-002

1P-005

Tatsuki Kurokawa1, 2，Jaime Horton S.3，Takuya Shiraishi2，Naghum Alfulaij3，
Andrea Small-Howard L.4，Helen Turner C.5，Yasuo Mori2，Alexander Stokes J.3（ 1 Dept Physiol, Grad Sch Med, Oita Univ, Japan, 2 Grad Sch Eng, Kyoto Univ,

Univ, Japan）

TRPV1 is a physical and regulated component of the natriuretic peptide
signaling system

3 Univ Hawaii, 4 GB Sci Inc, 5 Chaminade Univ）

Activation of the atrial natriuretic signaling pathway is intrinsic to the pathological responses
associated with a range of cardiovascular diseases that stress the heart, especially those involved in sustained cardiac pressure overload which induces hypertrophy and the pathologicalremodeling that frequently leads to heart failure. We identify transient receptor potential
cation channel, subfamily V, member 1, as a regulated molecular component, and therapeutictarget of this signaling system. TRPV1 is expressed in cardiac myocytes, but we understandrelatively little of the potential regulatory coupling of TRPV1 to pathways that control heart
physiology, and the longitudinal impact of TRPV1 inhibition in heart health under conditionsof applied pathology has some attendant controversies. Data show that TRPV1 is a physicalcomponent of the natriuretic peptide A, cGMP, PKG signaling complex, interacting with theNatriuretic Peptide Receptor 1 (NPR1), and upon binding its ligand, Natriuretic Peptide A(NPPA, ANP). TRPV1 activation is subsequently suppressed through production of cGMP
and PKG mediated phosphorylation of the TRPV1 channel. Further, inhibition of TRPV1, withorally delivered drugs, suppresses chamber and myocyte hypertrophy, and can longitudinally
improve in vivo heart function in mice exposed to chronic pressure overload induced bytransverse aortic constriction, reversing pre-established hypertrophy induced by pressureload while restoring chamber function. TRPV1 is a physical and regulated component of thenatriuretic peptide signaling system, and TRPV1 inhibition may provide a new treatment
strategy for treating, and reversing the loss of function associated with cardiac hypertrophy(COI:No)and heart failure.

Analyzing PI(4, 5)P2 sensitivity of GABAA receptor by voltage sensing
phosphatase
Shunichi Sugimoto, Takafumi Kawai, Yasushi Okamura（ Dept Phyiol, Grad Sch Med, Osaka
Ion channels play major roles in physiological processes in diverse biological systems. Recent
studies indicate that many types of ion channels are directly controlled by phosphatidylinositol-4, 5-bisphosphate (PIP2), which is important for diverse cellular processes. However,
PIP2-sensitivity of type γ-aminobutyric acid receptor (GABAAR), major inhibitory pentameric
ligand-gated ion channels in the central nervous system, remains unknown. Interestingly,
recent studies in structural biology found that each GABAAR binds to PIP2 and that there arepotential PIP2-binding sites in the α1 subunit, raising a possibility that function of GABAAR
is regulated by PIP2 (Laverty D, et al. Cryo-EM structure of the human α1β3γ2 GABAA
receptor in a lipid bilayer. Nature. 2019). Furthermore, in that paper, there were significant
differences in the potential PIP2-binding sites among different α subunits. Therefore, we hypothesized that GABAAR is sensitive to PIP2 and that there is a difference in PIP2-sensitivity
among different α subunits. To examined it, we used voltage sensing phosphatase (VSP),
which shows voltage-dependent phosphatase activity against PIP2. We studied Xenopus
oocytes expressing Ciona intestinalis VSP (Ci-VSP) and GABAAR, and evaluated the PIP2sensitivity of GABAAR by depolarization-induced depletion of PIP2. High PIP2-sensitivity of
GABAAR was observed in the Xenopus oocytes expressing some types of α subunits together
with β and γ subunits. We also discuss the relationship of the present results with the(COI:No)
PIP2-binding sites revealed by structural biology.

1P-003

1P-006

Yasutaka Mukai1, 2, 3, 4，Hidenori Aizawa5，Kenji Tanaka F.6，Takeharu Nagai7，
3 1 Neuroscience II, RIEM, Nagoya Univ, Japan, 2 Neural regulation, Grad
Akihiro Yamanaka1, 2, （Natsuki Mizutani, Yasushi Okamura（ Dept Integrative Physiol, Grad Sch Med, Osaka Univ,

Long-lasting calcium increase is induced by dopamine via D1 receptor
in hypothalamic orexin neurons
Sch Med, Nagoya Univ, Japan, 3 CREST, JST, Japan, 4 Research Fellowship, JSPS, Japan, 5 Dept
Neurobiol, Grad Sch Biomed and Health Sci, Hiroshima Univ, Japan, 6 Dept of Neuropsychiatry,
Sch of Med, Keio Univ, Japan, 7 Dept Biomol Sci and Eng, ISIR, Osaka Univ, Japan）

Sleep/wakefulness switches in seconds to hours-time scale in mammals from mice to humans.
Orexin neurons which produce a neuropeptide orexin in the lateral hypothalamic area (LHA)
are important for maintenance of wakefulness. Preceding studies reported several substances
regulate activity of the orexin neurons. However, most of the studies based on electrophysiological recordings measured by in milliseconds to minutes-time scale. Therefore, a mechanism
underlying activity change in range of seconds to hours-time scale which comparable withsleep/wakefulness state change is still elusive. We previously found that dopamine (DA)induced long-lasting increase of intracellular calcium concentration in orexin neurons inacute brain slices for more than 1 hour. Furthermore, we found the DA-induced increase in
intracellular calcium concentration was diminished by dopamine D1-like receptor antagonist,
SCH-23390.
In this study, we introduced in vivo genome-editing to examine receptors implicated in themechanism of long-lasting calcium increase. We used an adeno associated-virus (AAV) encoding both Cas9 (SaCas9) and a short guide RNA (sgRNA) designed to recognize dopamine D1
receptor (D1R) encoding gene (Drd1). To knockout D1R, we unilaterally injected the AAV
into the LHA of transgenic mice expressing a calcium indicator yellow Cameleon-Nano50
exclusively in orexin neurons. Four weeks after AAV injection, we made acute brain slices
and separated them into hemispheres, D1R-knockedout side and D1R-intact side. Then weapplied DA for 2 minutes through perfusion and monitored calcium activity for more than 2hours. As a result, D1R-knockedout side showed diminished calcium increase induced by DA
compared with D1R-intact slice. This result suggested that long-lasting increase induced by
DA is mediated through D1R. We are now trying to find out its physiological roles in vivo.
(COI:No)

S92

Alteration in the coupling between voltage sensor movement andphosphatase activity in voltage-sensing phosphatase with mutation involtage sensor domain
Suita, Japan）

Voltage-sensing phosphatase (VSP) shows phosphatase activity toward phosphatidylinositol 4,
5-bisphosphate (PI(4, 5)P2) upon membrane depolarization. Voltage sensor deficient mutants
lack phosphatase activity and mutations in a short linker between voltage sensor domain(VSD) and the PTEN-like phosphatase domain reduce phosphatase activity, suggesting that
activation of VSD “couples” with phosphatase activity via the linker. However, it is unclear
how VSD regulates phosphatase activity. We have recently found that amino acid mutation
at F234 (two amino acids downstream of the 4th arginine (R4) of S4) in Ciona intestinalis VSP(Ci-VSP) causes a remarkable decrease in the phosphatase activity whereas the gating current is normal. To understand what structural detail of VSD is optimal for inducting enzyme
activity in Ci-VSP, we analyzed both VSD motions and enzyme activities of F234 mutants andother VSD mutants. Mutants of Ci-VSP were expressed in Xenopus oocytes and analyzed by
the two-electrode voltage clamp. We studied the voltage-driven motion of VSD or cytoplasmic
catalytic region with thiol-reactive fluorescent dye or with unnatural fluorescent amino acid,Anap, respectively. Surprisingly, in multiple mutants including F234 mutant, phosphataseactivity remarkably decreased whereas the gating current remained intact. We interpretthese findings based on the hypotheses: (1) VSD cannot be fully activated, leading only partial
phosphatase activity, (2) the structure of the fully-activated VSD differs from that of the normal
full activation state, failing to induce the full phosphatase activity, (3) local membrane structure
or environment facing the cytoplasmic side of the VSD may be altered by the mutations,
possibly preventing the interaction between enzyme region and the plasma membrane which
may be critical for phosphatase activity (the second and the third idea are not exclusive to eachother). Detailed analysis will provide insights into molecular mechanisms by which VSD motion
leads to phosphatase activity in VSP.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

1P-007

1P-0102 1 Div Biophys ＆ Neurosci, NIPS, Japan,
Michihro Tateyama1, 2，Yoshihiro Kubo1, （2 1Thermal Biology Group,
Tomoyo Ujisawa1，Sandra Derouiche1, 2，Makoto Tominaga1, （We previously reported that the THIK-1 channel, a member of the two-pore-domain potassiumchannel family, is activated both by Gi/o- and Gq-coupled receptors. As the primary structureis highly conserved in the THIK channel family, we expected that Gi/o- and/or Gq-coupled
receptors might regulate the THIK-2 channel. Here we examined the effects of receptors on
the activity of the THIK-2 channel expressed in CHO cells by patch clamp recording. As theintracellular N-terminal cytoplasmic residues of the THIK-2 channel is known to inhibit the expression on the plasma membrane, most of the N-terminal residues were deleted (ΔN-THIK-2)
to increase the expression level. Although the current density of the ΔN-THIK-2 channel was
small (nearly 4 pA/pF), the density was increased about 3 or 10 times by stimulation of theco-expressed Gi/o-coupled metabotropic glutamate receptor (mGlu2) or Gq-coupled adrenergic
α1A-receptor (α1A-AR), respectively. The effect of mGlu2 was inhibited by incubation ofcells with pertussis toxin (300 ng/mL) for more than 16 hours, indicating that the effect ofmGlu2 is mediated by Gi/o. The effect of α1A-AR was inhibited by the treatment of cells witha PLC inhibitor, U73312 (2 µM, 10 min), showing that the effect is mediated by PLC. These
results clearly showed that the THIK-2 channel can be activated both by Gi/o- and Gq-coupled
receptors.
(COI:No)

Temperature perception is a critical ability for the survival of all animals in order to determine
viable thermal environments and to avoid damage caused by extreme temperatures. The
thermosensitive transient receptor potential (thermoTRP) ion channels are the molecular
thermal sensors that allow the monitoring of environmental temperature. Each of these ionchannels has a specific range of temperature sensitivity. However, the precise involvementof the thermoTRPs in temperature selection and temperature avoidance behaviors remains
unclear. Several temperature behavioral assays are used to quantify thermal preference suchas a two-plate choice assay or a linear gradient assay. But, these assays lack thermal resolution
and many biases are present. A circular thermal gradient assay using Thermal Gradient Ring
(TGR) was introduced recently. It allows to determine more accurately the thermal selection
behaviors of mice by providing high thermal resolution and by eliminating corner artifacts. We
hope to find features between temperature thresholds at a molecular level and temperature
preference at an individual level, and assess more precise contribution of each thermoTRP tothermal preference by using this new circular gradient assay and several TRP-deficient mice.
(COI:No)

1P-008

1P-011

Activation of the THIK-2 channel by Gi/o and Gq coupled receptors
2 SOKENDAI, Dept Physiological Sci）

Physiological roles of Prrt3, an orphan metabotropic receptor:
Comprehensive behavioral test battery analysis using homozygous fullgene knock-out mice derived from ﬂox mice
Tomomi Yamamoto1，Satoko Hattori 2，Li Zhou3，Rie Natsume3，Kohtarou Konno4，
I-Shan Che1，Masahiko Watanabe3, 4，Kenji Sakimura3，Tsuyoshi Miyakawa2，
1 1 Div Biophys ＆ Neurobiol, Natl Inst Physiol Sci, 2 Div Sys Med Sci, Inst
Yoshihiro Kubo（

Comprehensive Med Sci, Fujita Med Univ, 3 Dept Cellular Neurobiol, Brain Res Inst, Niigata
Univ, 4 Dept Anatomy, Faculty of Med, Hokkaido Univ）
Prrt3 is an orphan metabotropic receptor with a long N-terminus extracellular region, and it
belongs to family C which mGlu and GABAB receptors also belong to. We previously showedby immunohistochemical study that Prrt3 protein is chiefly expressed in the thalamus, cerebellar cortex, hippocampus and substantia nigra in the brain and that it is preferentially expressedin the presynaptic terminal. We previously raised gene knock out (KO) mice using ES cells
obtained from KOMP Repository in which all exons are targeted, but homozygous (homo)
KO mice showed high mortality. By the DNA microarray analysis, the influence of dramaticchange of expression of flanking genes was suspected. Thus, we constructed Prrt3 gene flox
mice and raised, by crossing with ActB-Cre mice, full KO mice whose exon 2 and 3 of Prrt3
genome is lost. A complete loss of Prrt3 protein in the homo KO mice brain was confirmed byimmunohistochemical staining. We performed comprehensive behavioral test battery analysis
using homo flox and flox derived homo KO mice. By the Barnes probe test to analyze thespatial memory, we observed a significant decrease in the retention of spatial memory after
4 weeks interval from the completion of training. The scores of homo KO mice of Latency to
the 1st arrival at the target hole, Error to 1st and Distance to 1st were all significantly higher
than those of homo flox mice. However, the time spent around the target hole did not show
significant difference. The data could be interpreted that that the homo KO mice remember
the target location but do not remember well the way to reach there. These results provide us(COI:No)
with clues toward the understanding of physiological roles of Prrt3. 

1P-009Two aromatic residues in the extracellular loop structure of the
FMRFamide-gated Na+ channel is critical for the channel activation
2 1 Lab Neurobiol, Grad Sch Int Sci Life, Hiroshima Univ,
Yasuo Furukawa1，Iori Tagashira（Japan, 2 Lab Neurobiol, Grad Sch Int Arts ＆ Sci, Hiroshima Univ, Japana）

FMRFamide-gated Na+ channel (FaNaC) is a homo-trimeric peptide-gated sodium channel,
which is activated by a molluscan cardioactive peptide, FMRFamide. FaNaC is a member of
DEG/ENaC family, and the extracellular domain of the DEG/ENaC channels is divided into
five subdomains (Finger, Knuckle, Thumb, Palm, and beta-ball domains).
We previously reported that six aromatic residues in the Finger domain which are conserved
among molluscan FaNaCs are involved in the FMRFamide sensitivity of Aplysia FaNaC,
AkFaNaC, (Furukawa and Tagashira, FAOPS2019). In the same study, we also carried out thedocking simulation, and found that the mutation of those aromatic residues affects the dockingof FMRFamide. These results suggest that the conserved aromatic residues in the Finger
domain may construct the FMRFamide binding site.
In the present study, we further examined the function of two other conserved aromatic
residues (F188, Y189) in the loop structure within the Finger domain. We found that the
FMRFamide sensitivity of AkFaNaC is substantially decreased if either F188 or Y189 ischanged to valine, and that the double mutant (F188V-Y189V) is practically insensitive to
FMRFamide. The FMRFamide docking simulations, however, failed to show obvious differences in the results between the wild-type channel and the mutant channels. These results
may be explained if F188 and Y189 are involved in the activation steps following the initial(COI:No)
ligand binding step.

Temperature preference analysis by using thermal gradient assay
ExCELLS, Natl Inst of Natural Sci, Japan, 2 Div of Cell Signaling, Natl Inst for Physiol Sci,
Natl Inst of Natural Sci, Japan）

Molecular mechanism of the GON domain in maintaining calcium
balance2 1 Dept Physiol, TWMU, 2 TIIMS）
Sawako Yoshina1，Shohei Mitani1, （ADAMTS9 is a metalloprotease that cleaves components of the extracellular matrix and isalso implicated in intracellular protein transport. ADAMTS9/GON-1 has a unique C-terminal
domain called the GON domain. The function of intracellular protein transport is dependent
on the GON domain but independent of protease activity. However, molecular mechanisms of
ADAMTS9 in cells remain unknown.
To investigate the intracellular role of ADAMTS9/GON-1, we searched for genes whose depletion suppressed the gon-1 phenotype. We identified several suppressor genes. To determine
whether the GON domain interacts with the suppressor genes, we performed immunoprecipitation experiments in HEK293 cells transfected with the Flag-tagged GON domain and Myctagged candidate proteins. We found that the GON domain interacts with several suppressor
gene products. The suppressor genes included a molecule involved in ubiquitination of inositol
1, 4, 5-trisphosphate receptor (IP3R). IP3R is an IP3-gated ion channel that releases Ca2+ fromthe ER. Next, we investigated whether ADAMTS9/GON-1 is involved in intracellular calcium
homeostasis. We found that the GON domain depletion increased Ca2+ leak from the ER lumen
to the cytosol. Furthermore, ubiquitination of IP3R was increased by ADAMTS9 depletion.
Now, we are investigating how the GON domain is involved in calcium homeostasis. (COI:No)

1P-012Involvement of thermosensitive TRP channels in temperaturedependent microglia movement
Sandra Derouiche1, 2，Rei Nishimoto3，Kei Eto4，Junichi Nabekura4，Makoto Tominaga1, 2（ 1 Division of Cell Signaling (NIPS), Okazaki Japan, 2Thermal Biology Group (ExCELLS),
Okazaki, Japan, 3 Department of Anesthesiology and Resuscitology, Okayama University
Hospital, Okayama, Japan, 4 Division of Homeostatic development (NIPS), Okazaki, Japan）
Microglia maintain the homeostasis of the central nervous system and migrate via chemotaxis
in their activated state. Interestingly, hypothermia was shown to reduce the microglial migration induced by ischemia, suggesting a modulation of microglia movement by temperature.
Although several ion channels and transporters are known to support the microglia movement,
the molecular mechanisms behind the temperature-dependent movement of microglia have
not yet been elucidated. Some members of the transient receptor potential (TRP) channels
superfamily with thermosensitivity, constitute strong candidates for the mediation of this phenomenon. Here, we investigate the regulation of mouse microglia movement by temperatureand the involvement of thermosensitive TRP channels. All together our in vitro and in vivo
results suggest a role of TRPM2, TRPM4 and TRPV4 channels in the temperature-mediatedmicroglia movement, both in physiological and pathological conditions.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S93

1P-013

1P-016

Tomomi Watanabe-Asaka1，Moyuru Hayashi1，Akane Suzuki2，Satoshi Uemura3，
1 1 Dept Physiol, Sch Med, Tohoku Med
Jun Takai3，Takashi Moriguchi3，Yoshiko Kawai（

2 1 Dept 1 st Surgery, Grad Sch Med, Yamaguchi Univ,
Toshiro Saito1，Junichi Sadoshima（

GATA transcription factors participate in recanalization of the lymphatic
vessels
Pharm Univ, Japan, 2 Sch Med, Tohoku Med Pharm Univ, Japan, 3 Div MedBiochem, Sch
Med, Tohoku Med Pharm Univ, Japan）

Lymphedema occasionally emerges after extensive lymph node dissection as well as sentinel
lymph node biopsy. Meanwhile, mechanism underlying the recanalization of lymphatic vessels
upon lymph node resection has been largely elusive. GATA family of transcription factors
regulate multitude of developmental processes through binding to GATA motif in a seriesof target genes. Recently, it has been suggested that GATA2 and GATA3 are importantfor development of lymphatic vessel and lymph node in embryonic stages. Given this, wehypothesized that GATA2 and GATA3 play a role for recanalization and growth of lymphatic
vessel in adult animal. To address this issue, we examined lymphatic formation and recanalization upon lymph node resection using adult Gata2 heterozygous deficient (Gata2+/－) miceand endothelial cell-specific Gata3 conditional knock-out (ECKO) mice. We found that popliteal
lymph node formation was impaired in the Gata3 ECKO mice, whereas the Gata2+/－ mice
showed normally formed popliteal lymph nodes. We thereafter examined recanalization oflymphatic vessels 3 weeks after the resection of popliteal lymph node in the Gata2+/－ mice.
Recanalization was evaluated by visualization of lymph vessels using Evans Blue dye injection
technique. Of note, the recanalization rate was significantly diminished in the Gata2+/－ mice incomparison with the control group. Our results thus clearly demonstrate that GATA2 plays acrucial role for the lymphatic recanalization upon lymph node excision. Prompt regenerationof lymphatic vessels during wound repair promotes smooth lymphatic circulation and thereby
confers substantial clinical benefits. We believe that further analyses of roles played by GATA2
in the lymphatic regeneration will open new therapeutic avenues.
(COI:No)The central role of Ulk1-dependent autophagy in mediating mitophagyin the heart during ischemia and reperfusion
Japan, 2 Dept CBMM, New Jersey Med Sch, Rutgers Univ, USA）

Removal of damaged mitochondria by mitophagy is essential for cardiac homeostasis.
However, activation of mitophagy and the underlying mechanism have not been clearly demonstrated in the heart during ischemia and reperfusion. Mito-Keima is a pH-sensitive marker
that indicates mitophagy by a high 560nm/440nm signal ratio. We crossed transgenic miceexpressing Mito-Keima with mouse models of autophagy loss-of-function, namely mice withcardiac-specific knockout of atg7 (atg7cKO) or ulk1 (ulk1cKO). Tg-Mito-Keima (Keima-WT), TgMito-Keima-atg7cKO (Keima-atg7cKO), and Tg-Mito-Keima-ulk1cKO (Keima-ulk1cKO) mice
were subjected to 48 hours of fasting. In Keima-WT hearts, the area with high-ratio dots was
1.1±0.1 % of the total heart area at baseline and starvation increased it to 2.3±0.1 % (p<0.05),
indicating stimulation of mitophagy. The area with high-ratio dots during starvation was significantly smaller in Keima-ulk1cKO (0.9±0.2 %, p<0.05) but not in Keima-atg7cKO (2.0±0.2 %),suggesting that mitophagy occurs in an Ulk1-dependent but Atg7-independent fashion. During
ischemia, mitophagy was transiently activated, peaking at 30 minutes after coronary artery
ligation in Keima-WT (3.1±0.2 %) and Keima-atg7cKO (2.7±0.2 %), but it was significantly
blunted in Keima-ulk1cKO (1.1±0.2 %, p<0.05). ulk1cKO mice displayed significantly larger
infarcts (34.9±4.3 %, p<0.05) than WT mice (19.7±2.8 %), suggesting that Ulk1 protects theheart against ischemia. During reperfusion following 30 minutes of ischemia, mitophagy wastransiently augmented in Keima-WT (3.5±0.2 %) and Keima-atg7cKO (3.3±0.2 %), but not in
Keima-ulk1cKO (1.1±0.2 %). These results suggest that Ulk1-dependent autophagy is essential
for the induction of mitophagy in the heart during ischemia and reperfusion.
(COI:No)

1P-014

1P-0173 1 AHFRC, AIST, Tsukuba,
Ryota Asahara1，Kei Ishii1，Nan Liang2，Kanji Matsukawa（

Hiroshi Matsuura1，Akiko Kojima2，Yutaka Fukushima1, 2，Yu Xie1，Xinya Mi1，5 1 Dept Physiol, Shiga Univ Med Sci, 2 Department
Sabirov Ravshan Z3，Yasunobu Okada4, （The increased prefrontal oxygenation prior to and at the onsetof overground walking using multichannel wireless near-infrared
spectroscopy
Japan, 2 Dept Human Health Sci, Grad Sch Med, Kyoto Univ, Kyoto, Japan, 3 Dept Integ
Physiol, Grad Sch Biomed and Health Sci, Hiroshima Univ, Hiroshima, Japan）

Our laboratory has reported using wireless 2-channel near-infrared spectroscopy (NIRS) thatoxygenated-hemoglobin concentration (Oxy-Hb, as index of regional cerebral blood flow) in
the prefrontal cortex (PFC) increased prior to and at the onset of natural overground walking
with arbitrary start. The increase in prefrontal Oxy-Hb prior to and at the onset of walking
was absent when walking was forced to start by cue, suggesting that the initial increasein prefrontal Oxy-Hb may appear in association with central command. However, the focal
location of the initial increase in Oxy-Hb over the PFC during overground walking remained
unsolved. The aim of the present study was to identify by using multichannel NIRS thespecific prefrontal area activating prior to and at the onset of natural overground walking. The
Oxy-Hb responses in multiple prefrontal areas were measured during overground walking
in 15 subjects. Furthermore, we examined to what extent the initial increase in prefrontal
Oxy-Hb during overground walking was dependent on walking speed. Overground walking
with arbitrary start evoked the initial increase in Oxy-Hb of the dorsolateral PFC (DLPFC)
and ventrolateral PFC (VLPFC) but not the frontopolar area. Furthermore, the initial increases
in Oxy-Hb of the DLPFC and VLPFC were independent of walking speed. Taken together, it
is suggested that the DLPFC and VLPFC are activated prior to and at the onset of natural
overground walking and the prefrontal activities may play a role in driving neuronal activity
in the caudal brain responsible for the generation of central command.
(COI:No)

1P-015

Analysis of hypoxic in utero environments that maintain cardiomyocyte
proliferation and its implications for regeneration in adults
Ken Hashimoto1，Aya Kodama1，Momoko Ohira1，Misaki Kimoto1，Akira Hanashima1，
1 1 First Dept. Physiol., Kawasaki Med. Sch., Japan,
Yoshihiro Ujihara2，Satoshi Mohri（
2 Dept. Electr. Mech. Eng., Nagoya Inst. Tech., Japan）

Mammalian cardiomyocytes lose their proliferative capacity shortly after birth, which is a major obstacle for therapeutic heart regeneration in adults. We and others have recently shown
the importance of hypoxic in utero environments for active fetal cardiomyocyte proliferation.
In this study, we found two novel cardiomyocyte cell cycle promoters in mice, Fam64a andNovex3. These two molecules were abundantly expressed in hypoxic fetal cardiomyocyte
nuclei, but this expression was drastically repressed by oxygen exposure, and in postnatal
cardiomyocytes following the onset of breathing and the resulting elevation of oxygen tension.
Sufficient expression of Fam64a and its degradation in M phase by ubiquitin ligase APC/C,
was both shown to be required for the completion of cardiomyocyte mitosis and cytokinesis.
Transgenic mice overexpressing Fam64a in postnatal cardiomyocytes exhibited enhancementof cell cycle activity in neonatal and adult hearts. Novex3 is the short splice variant of the
giant sarcomeric protein connectin (titin). We unexpectedly found that it was localized in the
cardiomyocyte nuclei specifically during the fetal stage, in addition to sarcomeric expression.
Mechanical analysis by atomic force microscopy and microneedle-based tensile tests demonstrated that Novex3 contributes to the elasticity/compliance of the nucleus at interphase andfacilitates cardiomyocyte proliferation, by promoting phosphorylation-induced disassembly of
multimer structures of nuclear lamins. Interestingly, Novex3 knockdown inhibited Fam64a
expression, and vice versa. Therefore, these three molecules (Fam64a, APC/C, and Novex3)
seem to work cooperatively to maximize cardiomyocyte proliferation during hypoxic fetal
stage. We now set out to achieve novel regeneration strategy by introducing these molecules
into adult hearts, which would create cardiomyocytes with high proliferative capacity andcompliant nuclei. (COI:No)

S94

Effect of ATP released from the maxi-anion channel on left ventricular
contractile function in Langendorff-perfused mouse heart modelof Anesthesiology, Shiga University of Medical Science, Otsu, Japan, 3 Institute of Biophysics
and Biochemistry, National University of Uzbekistan, Tashkent, Uzbekistan, 4 Department of
Physiology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan, 5 National
Institute for Physiological Sciences (NIPS), Okazaki 444-8787, Japan）

We have previously shown i) that the organic anion transporter SLCO2A1 constitutes anessential core component of the ATP-conductive large-conductance anion (Maxi-Cl) channel,
and ii) that this Maxi-Cl channel contributes largely to the release of ATP into coronary
effluent observed during and following the oxygen-glucose deprivation in the perfusate. The
present study was undertaken to examine the effect of ATP released via Maxi-Cl channels onleft ventricular contractile function, using a Langendorff-perfused mouse heart model. Isolatedheart was perfused at 37℃ with oxygenated normal Tyrode solution at a constant hydrostaticpressure of 80 mmHg. Left ventricular function was assessed by measuring left ventricularpressure using a fluid-filled balloon (made of plastic film) connected to a pressure transducer.
After the initial 30-min stabilization period, heart was then perfused with oxygen-glucosedeprived (OGD) Tyrode solution for 6 min, which was followed by 10-min perfusion withoxygenated normal Tyrode solution in the absence and presence of the ATPase apyrase
and the A1 adenosine receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX). In theabsence of apyrase and DPCPX, left ventricular developed pressure (LVDP) was decreased
from baseline value of 68.55.2 mmHg to 49.81.7 mmHg at the end of 6-min perfusion with OGD
Tyrode solution, which was followed by a transient increase to 84.03.2 mmHg during subsequent perfusion with oxygenated normal Tyrode solution. On the other hand, in the presence of
apyrase and DPCPX, LVDP was decreased during 6-min perfusion with OGD Tyrode solution
to the same degree but without exhibiting a transient increase in LVDP during subsequent
perfusion with oxygenated normal Tyrode solution. These results strongly suggest that ATP
released via Maxi-Cl channels contributes to the development of transient positive inotopy
during reperfusion after the short-period hypoxia/ischemia in the heart.
(COI:No)

1P-018Analysis on Combinations of Ion Channel Permeabilities of Guineapig Ventricular Myocyte Model that Generate Similar Action Potential
Waveforms
Maho Yamamoto1，Kazuki Okumura1，Tomohisa Kawakami1，Rina Sato2，Yukiko Himeno2，
2 1 Dept Bioinfo, Grad Sch Life Sci, Ritsumeikan Univ, Shiga, Japan, 2 Dept
Akira Amano（
Bioinfo, Col Life sci, Ritsumeikan Univ, Shiga, Japan）

Drug effect estimation system was developed to reduce cost for drug discovery by estimating
the drug effects to specific ventricular myocyte ion channels in silico, which is necessary forevaluating drug safety. The system estimates the effects of drugs on ion channels by using
action potential waveforms (APWs) of ventricular myocytes. APWs are used as input data
which are measured from guinea-pig ventricular myocytes before and after drug administration with suction electrode method. This system estimates the effect of drug on ion channels
by searching for the similar APWs that were generated by various combinations of ion channel
permeabilities. The similarities are evaluated excluding phase 0 and 1, since they are difficult
to measure accurately, and also excluding the end of repolarization phase where the waveform
often becomes unstable. The problem of the system is that the estimation accuracy is low
because similar APWs are generated from different combinations of channel permeabilities.
We analyzed the characteristics of these combinations by searching for the similar APWs toseveral typical waveforms. As a result, there were combinations of potassium channel permeabilities to compensate for changes in sodium channel and/or calcium channel permeabilities.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

1P-019

1P-022

Shiro Kato, Kota Kishida, Akari Sanechika, Akira Amano（Dept Bioinf, Col Lifesci,

Shin Takeo, Yoichiro Kusakari, Naritomo Nishioka, Susumu Minamisawa（ Dept Cell Physiol,

Left Ventricular End-Systolic Pressure and Volume Relation (ESPVR) becomes linear in large
mammals such as human or canine. Emax which is slope of ESPVR, is well known as anindex of cardiac contraction because of its independence of loading conditions of Left Ventricle(LV). Time-Varying Elastance Model (TVEM) was proposed as a mathematical model of LV
by using the linear characteristics of ESPVR. In TVEM, the LV elastance increases duringejection phase, thus each isochronous Pressure and Volume Relation (PVR) lies on a line. On theother hand, detailed cardiac contraction model has been proposed, and by using such model, LV
model is also proposed. Interestingly, the characteristics of isochronous PVRs become different
for these models. Although the linear characteristics of the isochronous PVRs were the same,the slope of isochronous PVRs for the detailed cardiac contraction model slightly decreased
during ejection phase, and the unloaded volume largely decreased. From the mathematicalanalysis, the PVR characteristics of contraction model comes from the combination of well(COI:No)
known force length relation and force velocity relation.

Background: The overstretch of cardiac muscle is known to drastically decrease active force
development. Cardiac muscle can produce greater active force in response to stretching within
an optimal range of sarcomere length (1.8 µm≦SL≦2.2 µm) according to Starling’s law of
the heart. Therefore, repetitive overstretch would further worsen cardiac contractility. How
cardiac contractility recovers when its length is recovered to the optimal range after repetitive
overstretch has not yet been examined, however.
Purpose: We exposed cardiac papillary muscle to repeated overstretch and release to investigate the changes in active tension before and after overstretch.
Methods: We used male Sprague-Dawley rats (BW≧350 g) and dissected the right ventricularpapillary muscles. A papillary muscle was stretched to Lmax, at which point the active tension
reached the maximal level. We then stepwisely stretched it within 2 s up to 120% of Lmax, andstimulated the muscle (1 Hz, 36℃) with tension measurement. Experiments were performed
with rats at 14 weeks and 2 years of age.
Result: The active tension 4 min after overstretch was decreased to 32.8% of Lmax, but recovered to 92.1% of Lmax 20 min after overstretch release. Although the active tension gradually
decreased as the papillary muscle was repeatedly overstretched and released, it remained
around 75% of Lmax after the fourth overstretch release (experimental time 2 h). In contrast,
the active tension of the papillary muscle whose length was kept at Lmax decreased to 10%
of Lmax at 2 h. This phenomenon was much more obvious in younger rats than in older rats.
Conclusion: The contractile force of cardiac muscle that undergoes several overstretchesintermittently was much stronger than that which continuously kept its length within normal
limits. This result indicates that overstretch may be effective in protecting the contractility of(COI:No)
cardiac muscle.

1P-020

1P-023

Changes in Left Ventricular Elastance During Ejection Phase--- Model Study --Ritsumeikan Univ, Shiga, Japan）

The serotonergic system mediates cardiovascular responses evoked bystimulating the lateral habenula of rats
Tri Doan Huu1, 2, 6，Yuma Sato1, 3，Masayuki Matsumoto4, 5，Tadachika Koganezawa1, 5

Preservation of active cardiac force after release from repetitive
overstretch
Med, Jikei Univ, Japan）

Decrease in the forearm muscle oxygenation assists recovery ofarterial blood pressure during supraventricular tachycardia but notduring ventricular tachycardia（ 1 Dept Physiol, Fac Med, Univ Tsukuba, Japan, 2 Dr Prog Biomed Sci, Grad Sch Comp
Human Sci, Univ Tsukuba, Japan, 3 Mstr Prog Med Sci, Grad Sch Comp Human Sci, Univ
Tsukuba, Japan, 4 Dept Cogn Bahav Neurosci, Fac Med, Univ Tsukuba, Tsukuba, Japan,
5 Transborder Med Res Ctr, Univ Tsukuba, Japan, 6 Ctr Adv Trn Clin Sim, Univ Med
Pharm Ho Chi Minh City, Vietnam）

Kei Ishii1，Hidehiko Komine1，Takeshi Machino2，Fumi Yamagami2，Yasuhiro Hatori 1，
Jongseong Gwak 1，Tsubasa Izaki1，Akihiko Nogami 2，Kazutaka Aonuma 2，
1 1 AHFRC, AIST, Tsukuba,
Hideo Tsurushima 3，Satoshi Kitazaki 1，Motoyuki Akamatsu （Neurons in the lateral habenula (LHb) are activated by stressful events such as pain, physicalconstraint, open field exposure and social defeat. Although these events induce cardiovascularresponses, whether and how the lateral habenula regulates the cardiovascular autonomic
nervous system remains unclear. To investigate this issue, here we electrically stimulated
the LHb and examined the effect on cardiovascular responses in anesthetized rats. We foundthat the electrical stimulation of the LHb increased mean arterial pressure (MAP) and decreased heart rate (HR), while stimulating the outside of the LHb at 0.25 mm distance affected
neither of them. Notably, denervating the bilateral vagus nerves, which include the cardiac
parasympathetic nerves, completely suppressed the effect of the LHb stimulation on HR but
did not change that on MAP. On the other hand, administration of propranolol (5-10 mg/kg,
i.v.), a nonselective β adrenergic receptor antagonist, partly attenuated the effect of the LHb
stimulation on MAP but did not affect that on HR. Furthermore, systemic administration of
methysergide (1 mg/kg, i.v.), a nonselective serotonergic receptors antagonist, attenuated the
responses of MAP and HR to the LHb stimulation.
These results indicated that the activation of the LHb neurons induces cardiovascularresponses through both the sympathetic and the parasympathetic nervous systems. The
serotonergic system also mediates the cardiovascular responses. Our findings suggest that the
LHb and the serotonergic system cooperate to induce cardiovascular responses when animals(COI:No)
encounter stressful events.

Rapid hypotension caused by cardiac tachyarrhythmias is counteracted by reflexly-evoked
peripheral vasoconstriction. However, it was still unclear whether peripheral vasoconstriction
does assist recovery of arterial blood pressure (AP) during various types of actual tachyarrhythmia. The degree of AP recovery would differ between paroxysmal supraventriculartachycardia (PSVT) and ventricular tachycardia (VT) because of lesser cardiac output and/or
ability to evoke vasoconstriction in VT cases. We hypothesized that AP recovers via peripheral
vasoconstriction during PSVT but it remains decreased during VT. To estimate peripheral
blood flow responses, forearm tissue oxygen index (TOI) was measured by near-infrared spectroscopy (NIRS). The NIRS and hemodynamic data were collected during electrophysiologicalstudy. Mean AP (MAP) decreased (P<0.05) rapidly in both PSVT and VT, while the arrhythmia
rates were similar (P > 0.05). Thereafter MAP returned to the pre-arrhythmia level at 40 sfrom the onset of PSVT. The decreased MAP was maintained during VT. PSVT caused adecrease in the forearm TOI, which did correlate with MAP recovery (P<0.05). During VT,
there was no such relationship between the TOI response and MAP recovery (P > 0.05).
These relationships were also evident when the data with drugs (e.g., isoproterenol) infused
during EPS was excluded. These results provide a positive suggestion that the decrease in theforearm muscle oxygenation probably via vasoconstriction does recover MAP during PSVT
but fails to recover MAP during VT.(COI:Properly Declared)

1P-021

1P-024

Yukari Takeda, Satoshi Matsuoka（ Dept of Integrative and Systems Physiol, Univ of Fukui）

Megumi Ito1，Yoshihiro Ujihara2, 3，Akira Hanashima3，Takeshi Honda3，Aya Kodama3，
3 1Creative Eng
Shukei Sugita2，Masanori Nakamura2，Ken Hashimoto3，Satoshi Mohri（

Mitochondrial contribution to automaticity of murine sinoatrial nodal
cells
The automaticity of sinoatrial nodal cells (SNCs) attributes to the activities of ion channels at theplasma membrane. There is however growing evidence that spontaneous local calcium release(LCR) from the sarcoplasmic reticulum (SR) regulates the pacemaker activity. Previously, we
have reported that mitochondrial Na+-Ca2+ exchanger, NCXm, is involved in the generation of
LCR and automaticity in murine SNCs. Although it is anticipated that mitochondrial Ca2+ efflux
through NCXm modulates Ca2+ handling of nearby SR, the spatial association between LCR
and mitochondria has never been assessed. In the current study, we investigated the local orientation of LCR in relation to location of mitochondria in murine SNCs. For LCR detection, Ca2+
dynamics were measured by 2D imaging of Calbryte-520™-loaded murine SNCs. Mitochondria
areas were simultaneously identified by co-labeling of SNCs with tetramethylrhodamine ethyl
ester (TMRE). High-speed and high-resolution imaging of the SNCs detected LCRs between
rhythmic firings of Ca2+ transient in all SNCs. The probability of LCR occurrence was foundto be higher at mitochondria area (69.1±13.8%). Moreover, the distance between LCR andadjacent mitochondria area was significantly shorter in LCRs evoked in the early phase of Ca2+transient cycle (average 0.68 µm, median 0.00 µm), when compared to that evoked in the latephase (average 1.52 µm, median 0.16 µm). Taken together, LCRs, especially those in the earlyphase of Ca2+ transient cycle were generated in close proximity to mitochondria in murine
SNCs.
(COI:No)

Japan, 2 Cardiovascular Division, University of Tsukuba, Tsukuba, Japan, 3 Department of
Nuerosurgery, University of Tsukuba, Tsukuba, Japan）

Comparison of passive mechanical properties of rat, chicken, frog andturtle ventricles
Program, Nagoya Inst Tech, Japan, 2 Dept Electric Mech Eng, Nagoya Inst Tech, Japan,

3 First Dept of Physiol, Kawasaki Med Sch, Japan）

Vertebrate hearts have undergone marked morphological changes to adapt to different
environments and lifestyles during evolution. Mammals and birds have the hearts consistingof two atria and two ventricles, while amphibians and most reptiles have the hearts consistingof two atria and one ventricle. However, functional changes in the ventricles during the courseof evolution from one to two ventricles have remained unclear. Here, we compared passive
mechanical properties of the ventricle of frogs and turtles with left ventricle (LV) of rats andchickens. To analyze the passive mechanical properties of the ventricles, we obtained therelationship between the ventricular pressure and the inflow volume when the cardioplegic
solution was introduced into diastolic-arrested ventricle or the LV at a constant speed. To compare different-sized hearts, the inflow volume was normalized by the ventricular weight. The
results showed that LVs of rats and chickens were significantly stiffer than ventricles of turtles
and frogs. Isolated cardiomyocytes in rats were significantly wider than in chickens, turtles andfrogs, suggesting that rat cardiomyocytes were stiffer than others in the longitudinal direction
at a single cell level. Because the extensibility of cardiomyocytes is regulated by the elastic
protein connectin (also called titin) that is striated muscle-specific and the largest protein thatconnects the Z-line to the M-line in half-sarcomeres, we compared the primary structure ofconnectin. We found that the elastic regions, which function to confer extensibility to connectin,were greatly shorter in rat and chicken hearts than in the frog and turtle hearts, indicatingthat connectin in rat and chicken hearts was stiffer than that in turtle and frog hearts. These
results suggested that ventricular extensibility was restricted at the molecular level during thecourse of evolution from one to two ventricles.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S95

1P-025

1P-028Hiroshi Ueno1，Yu Takahashi2，Shunsuke Suemitsu2，Motoi Okamoto3，Takeshi Ishihara22 1 IRCN, UTIAS, Univ.Tokyo, Japan, 2 Dept
Satoru Kondo1，Kohei Kikuta2，Kenichi Ohki1, （Although epilepsy is one of the most common chronic neurological disorders with a prevalenceof approximately 1.0%, the underlying pathophysiological processes remain to be elucidated.
Understanding the molecular and cellular mechanisms involved in the development of epilepsy
is important for the development of appropriate therapeutic strategy. Kindling is widely accepted as a functional model of temporal lobe epilepsy. Pentylenetetrazol (PTZ) is commonlyused in kindling and to study seizure mechanisms, neurobiology of epilepsy, learning andmemory disorders caused by seizures, and to evaluate the effects of new treatments. In thisstudy, we investigated the effects of status epilepticus on astrocytes, microglia, and extracellular matrix (ECM) molecules in the somatosensory cortex and piriform cortex of mice. The
purpose of this study was to reveal changes in the astrocytes and ECM in the cortical region ofexperimental epilepsy models. Activation of astrocytes was observed in many cortices except
the retrosplenial granular cortex after PTZ-induced kindling acquisition in mice. Activatedastrocytes in the cortex were found in layers 1-3 but not in layers 4-6. In the somatosensory andpiriform cortices, no change was observed in the number of parvalbumin (PV)-positive neurons
and PV-positive neurons covered with perineuronal nets. However, the amount of ECM in theextracellular space was increased. The expression of VGLUT1- and GAD67-positive synapses
was also increased. Thus, in the PTZ-kindling epilepsy mice model, an increase in the numberof ECM molecules and activation of astrocytes were observed in the somatosensory cortex
and piriform cortex. These results indicate that PTZ-induced seizures affect not only the
hippocampus but also other cortical areas. Our study findings may provide an opportunity to(COI:No)develop new therapeutic approaches to prevent seizures or their consequences.

Understanding how neurons integrate thousands of synaptic inputs is critical to discern corticalinformation processing. Substantial evidences suggest the importance of spatial arrangementof synaptic inputs onto dendrites for neuronal computation. However, the principle of spatial
arrangement and integration mechanisms of inputs remain largely unsolved.
Recent advances of functional imaging technique enable recordings of activities of individual
spines using various calcium sensors. Due to the low time resolution of calcium signal transient,back propagating action potential (bAP) invades spines and makes the accurate spine analysis
difficult. To solve this problem, we developed a new method to overcome by using inhibitory
optogenetics. We sparsely co-expressed GCaMP6s and inhibitory optogenetic protein withsoma-localized signal in mouse primary visual cortex (V1) by adeno associated virus (AAV)
and recorded visually evoked spine signals from layer 2/3 excitatory neurons. With photoinhibition by targeted one-photon laser, we successfully suppressed the somatic depolarizationand individual spine signal was detected. Firstly, we recorded the visually-evoked signals form
the spines without inhibition followed by with inhibition and compared them. We found thatthe bAP-subtracted method does not always estimate the accurate visual response of spines
especially when the spine shows the simultaneous response with the soma. Next, to investigate
the input-output relationship, we recorded ~1,000 spine responses from individual orientation or direction selective neurons. We found that ~30% of recorded spines were visually
responsive and among them ~90% were selective for either orientation or direction. These
selectively responded spine were distributed all over the dendrites but showed some clusteringof orientation or direction selective inputs that matched with the somatic selectivity. We also
investigated the spatial frequency selectivity of these inputs and their functional organizationon the dendrites.
(COI:No)

1P-026

1P-0292 1 Dept Hlth ＆ Nutr Sci, Fac Human Hlth,
Katsushige Sato1，Yoko Momose-Sato（

3 1 Dept Physiol, Sch Med, Wakayama Med Univ,
Moritoshi Hirono1, 2，Yuchio Yanagawa（

Alteration of astrocytes and extracellular matrix molecules in the cortex
of pentylenetetrazol-kindled mice
（ 1 Dept Med Tech, Kawasaki Univ Med Welf, Japan, 2 Dept Psych, Kawasaki Med Sch,
3 Dept Med Tech, Okayama Univ）

Optical analysis of functional development of the glossopharyngeal
nerve pathway in the mouse fetus brainstem
Komazawa Women’s Univ, Japan, 2 Dept Nutr ＆ Dietetics, Coll Nutr, Kanto-Gakuin Univ,
Japan）The glossopharyngeal nerve (N.IX) transfers motor and sensory information related to visceraland somatic functions, such as salivary secretion, gustation and the control of blood pressure.
N.IX-related neural circuits are indispensable for these essential functions. Compared with thestrenuous analysis of morphogenesis, we are only just starting to elucidate the functiogenesisof these neural circuits during ontogenesis. In the present study, we applied voltage-sensitive
dye recording to the embryonic mouse brainstem, and examined the functional developmentof the N.IX-related neural circuits. First, we optically identified the motor nucleus (the inferior salivatory nucleus (ISN)) and the first-order sensory nucleus (the nucleus of the tractus
solitarius (NTS)). We also succeeded in recording optical responses in the second/higher-order
sensory nuclei via the NTS, including the parabrachial nucleus. Second, we pursued neuronal
excitability and the onset of synaptic function in the N.IX-related nuclei. The neurons in the
ISN were excitable at least at E11, and functional synaptic transmission in the NTS was firstexpressed at E12. In the second/higher-order sensory nuclei, synaptic function emerged ataround E12-13. Third, by mapping optical responses to N.IX and vagus nerve (N.X) stimulation,
we showed that the distribution patterns of neural activity in the NTS were different between
the N.IX and the N.X from the early stage of ontogenesis. We discuss N.IX-related neuralcircuit formation in the brainstem, in comparison with our previous results obtained from chick(COI:No)and rat embryos.

Functional organization of response-selective inputs on dendrites ofmouse primary visual cortex neurons
Phyiol, Grad Sch Med, Univ Tokyo, Japan）

GABA release from cerebellar Purkinje cells is insensitive toendocannabinoid-mediated synaptic modulation
Japan, 2 Grad Sch Brain Sci, Doshisha Univ, Japan, 3 Gunma Univ Grad Sch Med, Japan）

Endocannabinoids (eCBs) act as ubiquitous modulators of synaptic transmission via the activation of cannabinoid receptors (CBRs) in the CNS. Cerebellar Purkinje cells (PCs) project to thedeep cerebellar nuclei (DCN), where they make synaptic contacts with DCN neurons. PCs
also extend axon collaterals that terminate in the vicinity of the PC layer and make synaptic
contacts with Lugaro cells and globular cells. However, the effects of eCBs on GABAergictransmission from PC axon terminals have yet to be investigated thoroughly. In this study, we
examined the modulatory actions of eCBs on the GABAergic transmission at PC-globular cell
synapses and PC-DCN neuron synapses using whole-cell patch-clamp recordings from mouse
cerebellar slices. We showed that the CBR type 1 agonist WIN55212 did not affect either
spontaneous or miniature inhibitory postsynaptic currents (IPSCs) recorded from globular cells
under control conditions and in a state of enhanced synaptic activity. By contrast, another Gi/oprotein-coupled receptor agonist, baclofen, significantly reduced the frequency of miniature
IPSCs in globular cells. Moreover, we showed that WIN55212 had no effects on IPSCs inlarge DCN neurons, the primary targets of PCs. A type 2 CBR agonist, HU308, also had no
significant effects on IPSCs in either globular cells or large DCN neurons. Globular cells andlarge DCN neurons did not elicit depolarization-induced suppression of inhibition (DSI). Taken
together, our results suggest that GABA release from PC axon terminals is insensitive toexogenous CBR agonists and devoid of regulation by DSI, indicating that PCs do not express
functional CBRs at their axon terminals. This is in sharp contrast to the fact that PCs receive
abundant excitatory and inhibitory inputs that are under eCB-mediated presynaptic inhibitory
modulation. The actions of eCBs are selective to distinct synapses and possibly contribute toboth information processes and rigorous signal transmission in the cerebellum.
(COI:No)

1P-027

1P-0302 1 Dept Nutr ＆ Dietetics, Coll Nutr, Kanto-Gakuin
Yoko Momose-Sato1，Katsushige Sato（
Univ, Japan, 2 Dept Hlth ＆ Nutr Sci, Fac Human Hlth, Komazawa Women’s Univ, Japan）

Hiroyuki Yamazaki1，Kenji Hanamura1，Banri Segawa1，Narutoshi Tsukahara2，
1 1 Dept. Neurobiol. Behav. Gunma Univ. Grad. Sch.
Akikazu Murakami2，Tomoaki Shirao（

Prenatal exposure to nicotine disrupts synaptic network formation by
inhibiting spontaneous correlated wave activity

Correlated spontaneous activity propagating over a wide region of the central nervous system
is expressed during a specific period of embryonic development. In a previous study using theoptical imaging technique with a voltage-sensitive dye, we demonstrated that this wave-like
activity, which we referred to as the depolarization wave, plays a fundamental role in the earlyprocess of synaptic network formation. We found that in ovo application of bicuculline/strychnine or d-tubocurarine, which blocked neurotransmitters mediating the wave, significantly
reduced functional synaptic expression in the brainstem sensory nucleus. This result, especiallyfor d-tubocurarine, an antagonist of nicotinic acetylcholine receptors, raised the possibility
that prenatal nicotine exposure associated with maternal smoking affects the developmentof neural circuit formation by interfering with the correlated wave. In the present study,
we tested this hypothesis by examining the effects of nicotine on the correlated wave andassessing the chronic action of nicotine on functional synaptic expression. The application of
nicotine transiently increased electrical bursts and embryonic movements associated with thewave, but subsequently inhibited these activities. Furthermore, chronic exposure to nicotine
in ovo markedly reduced functional synaptic expression in the brainstem sensory nucleus, theparabrachial nucleus. This study suggested that prenatal nicotine exposure disrupts the initialformation of neural circuitry by inhibiting the correlated spontaneous activity.
(COI:No)

S96

Live cell imaging of endogenous drebrin using camelidae singledomain antibody
Med., Maebashi, Japan, 2 Dept. of Parasitol. Grad. Sch. Med., Univ. Ryukyus, Okinawa,
Japan）

Drebrin functions as an F-actin stabilizing factor in dendritic spines, where it regulates spine
morphogenesis and is involved in synaptic plasticity. GFP-fused drebrin (GFP-drebrin) has beenused for many studies, particularly for real-time imaging. However, there are some difficulties
in use of GFP-drebrin. For example, when it is expressed at higher level in neurons, the spinesand filopodia abnormally change their shapes by F-actin-remodeling activity of drebrin. In thisstudy, we made a novel tool for drebrin-imaging using the camelidae antibody that consists ofa single monomeric variable antibody domain. We isolated some “variable domains of heavy
chains of camel heavy-chain antibody” (VHH) by affinity selection to drebrin using VHH
domain-phage library. All selected VHH clones recognized exogenous and endogenous drebrin
in western blotting. A drebrin-VHH clone (3E3) showed a similar immunostaining pattern incultured neurons to an authentic drebrin monoclonal-antibody (clone M2F6). Next, we fused
the VHH to IgG-Fc with GFP to visualize endogenous drebrin in living cells. When GFP-3E3
expression vector was transfected to cultured neurons, GFP signals were observed mainly indendritic spines. The signals translocated to dendrites from dendritic spines by depolarizing
stimuli with high potassium, which is consistent with our previous studies. Furthermore, wedelivered GFP-3E3 into developing mouse neocortex via in utero electroporation and observed
them in living brains of adult mice. In vivo imaging showed that GFP-3E3 normally localized
in dendritic spines, suggesting that the expressed GFP-3E3 were not toxic. These indicate that
(COI:No)GFP-3E3 is useful tool for observation of endogenous drebrin in vitro and in vivo.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

1P-031

1P-034

Takaki Watanabe1, 2，Honoka Suzuki1，Kazuto Sakoori1，Shutaro Inoue1，Tsubasa Akamatsu1，
2 1 Dept ofManabu Abe3，Kenji Sakimura3，Naofumi Uesaka1, 2，Masanobu Kano1, （Univ, Japan）

Protocadherin 10 delays developmental climbing ﬁber synapse
elimination in a subset of aldolase C-positive Purkinje cells in the
cerebellum
Neurophysiol, Grad Sch of Med, Univ of Tokyo, Japan, 2WPI-IRCN, UTIAS, Univ. of Tokyo,
Japan, 3 Dept of Cell Neurobiol, Brain Res Inst, Niigata Univ, Japan）

Accumulating evidence suggests that the cerebellum is involved in autism spectrum disorder(ASD). Here, we focused on protocadherin 10 (Pcdh10), an ASD-associated gene that is expressedin subsets of cerebellar Purkinje cells (PCs), and investigated whether Pcdh10 is involved indevelopmental elimination of early-formed redundant climbing fiber (CF) to PC synapses, a
representative model of synapse pruning in the developing brain. We generated Pcdh10-DIOtdTomato mice and crossed them with GluD2-Cre mice to obtain conditional Pcdh10 knockout(Pcdh10-cKO) mice in which PCs that have endogenous Pcdh10 will express tdTomato insteadof Pcdh10. We found that the expression of tdTomato in Pcdh10-cKO was restricted to a subsetof aldolase C (Aldoc)-positive PCs, which was consistent with a previous report about Pcdh10
expression in PCs. We performed whole-cell recordings from PCs in cerebellar slices at various
postnatal days and examined CF synapse elimination. We found that in the cerebellar vermisof wild-type mice, CF synapse elimination was delayed in Aldoc-positive/Pcdh10-positive PCs
when compared with Aldoc-negative PCs or Aldoc-positive/Pcdh10-negative PCs. This delayof CF synapse elimination in a subset of Aldoc-positive PCs was absent in Pcdh10-cKO. Similardelay of CF synapse elimination was also found in Aldoc-positive PCs in the right cerebellar
hemisphere including Crus I, Crus II, and Lobule simplex of wild-type mice, whereas thedelay was not seen in Pcdh10-cKO. Conversely, ectopic overexpression of Pcdh10 in PCs by inutero electroporation at E12 increased the degree of multiple CF innervation at young adultstage. Pcdh10-cKO mice had a mild motor deficit and exhibited repetitive behavior in marble
burying tests. These results indicate that Pcdh10 maintains CF synapses and counteracts
their elimination in a subset of PCs during postnatal development, and suggest that deletion of
Pcdh10 might be related to abnormal repetitive behavior relevant to a core symptom of ASD.
(COI:No)

Spatio-temporal analysis of synaptic integration in hippocampal
neurons by membrane potential imaging
Masato Morita, Tomoo Hirano, Shin-ya Kawaguchi（Dept Biophys, Grad Sch Sci, Kyoto
Neurons form many synapses and receive lots of inputs there. Synaptic inputs are integrated
spatially and temporally in the somatodendritic compartment, generating output firing at theaxon initial segment. In hippocampal pyramidal neurons, it is well known that synaptic potentials summate supralinearly, however, its spatio-temporal property remains unclear. To study
this issue, using a genetically encoded voltage indicator and rapid multi-spots local glutamate
uncaging, we have quantitatively examined the supralinear summation of EPSPs in cultured
hippocampal neurons. Hippocampal neurons were transfected with an improved version of
ASAP1, which showed larger fluorescence changes upon membrane potential changes (~50%/100 mV). EPSPs upon glutamate uncaging at dendritic spines were recorded by voltage
imaging and/or the whole-cell patch clamp method. We find that simultaneous EPSPs in
response to glutamate uncaging by 405 nm laser illumination at 2 distinct sites exhibit higher
depolarization than the linear sum of each EPSP. The closer the distance between 2 sites
stimuli is, the higher the supralinear summation becomes. Notably, the supralinear augmentation of EPSPs is evident even when the individual EPSP is tiny as ~1 mV. In this poster,
we are also going to show the molecular mechanisms underlying the supralinear dendritic(COI:No)
computation in hippocampal neurons.

1P-032

1P-035 (AP-2)

Megumi Taketo（Dept Cellular Functional Biol, Facult Med, Kansai Med Univ）

Susumu Jitsuki1，Sayaka Kogami1，Aoi Jitsuki-Takahashi2，Tomomi Yamanoue1，
1 1 Dept Physiol, Grad Sch Med, Yokohama City Univ, Japan, 2 Dept
Takuya Takahashi（

Function of metabotropic glutamate receptor 1 in the neonatal
hippocampal marginal zone
Metabotropic glutamate receptors (mGluRs) are distributed in the central nervous systemand play important roles as synaptic plasticity and memory formation in various neuronalprocesses. mGluRs are classified into three groups I to III, and group I mGluRs consist of two
subtypes, mGluR1 and mGluR5. Group I receptors couple to Gq protein and increase intracellular Ca2+ concentration ([Ca2+] i) through the release of Ca2+ from intracellular stores. These
receptors also regulate the channel activity of plasma membranes. Hippocampal marginal zone
contains early-developed neurons including Cajal-Retzius cells (CR cells), which regulate neuronal migration through secretion of glycoprotein, reelin. CR cells also project their dendrites toother hippocampal neurons and regulate network activity. Though the function of the secreted
reelin has been studied considerably, less is known about the regulation of CR cell excitability.
In the present experiments, functional expression of mGluR1 was detected by fluorescence
Ca2+ imaging, in acute slices of neonatal rat hippocampus. In CR cells, [Ca2+] i elevation was
induced by application of group 1 mGluR-specific agonist in the presence of mGluR5 specificantagonist, MPEP. Whereas, the [Ca2+] i elevation was prevented by mGluR1-specific antagonist, CPCCOEt. Characteristics of the Ca2+ mobilization after activation of mGluR1 in neuronsof marginal zone were further investigated. Elimination of extracellular Ca2+ could not preventthe mGluR1-induced [Ca2+] i increase in CR cells. Effects of Ca2+-permeable channel blockers on
mGluR1-mediated Ca2+ mobilization were also determined. Possibility of a cross-talk between
mGluR1 and other receptors expressed in CR cells is under investigation.
(COI:No)

Synaptic plasticity at cortico-striatal pathway in functional recovery after
cortical damage
Biochem, Sch Med, Tokyo Women’s Med Univ, Japan）

Brain damage such as stroke is a devastating neurological condition that may severely compromise patient quality of life. Restoration of motor impairment after brain damage is considered
to be the result of compensative neural plasticity in intact brain regions, mediated by thereorganization of cortical motor maps. Experience-dependent synaptic AMPA (α-amino-3hydroxy-5-methyl-4-isoxazole-propionic-acid) receptor (AMPAR) delivery underlies behaviors
that require neural plasticity such as learning. We have previously found that rehabilitationdependent synaptic delivery of AMPAR in the peri-injured area facilitate functional recovery
after cortical damage. For execution of appropriate motor function, it requires involvementof motor-related brain regions such as striatum, thalamus or brain stem. However, it remains
unclear that the role of other brain region in recovery after brain damage. Here, using corticalinjury rat model, we found that AMPAR-mediated miniature EPSC (mEPSC) of the layer 5
pyramidal neurons in the periinjured cortex were positively correlate with recovery rate offorelimb reaching motor performance after rehabilitative training. On the other hand, mEPSC
of the medium spiny neurons in the periinjured striatum were negatively correlate withrecovery rate of motor performance. Furthermore, mIPSC of the periinjured cortex showed nocorrelation with motor performance. These results suggest that changes of excitatory input inthe peri-injured region could contribute functional recovery after cortical damage. (COI:No)

1P-033

1P-036

Mitsuharu Midorikawa, Mariko Miyata（ Dev Neurophys, Dept Physiol, Sch Med, Tokyo

Hisako Nakayama, Mariko Miyata（ Dept Neurophysiol, Grad Sch Med, Tokyo Women’s

Fast synaptic transmission is important for information processing at central nervous systems,
but how it acquired through the development is largely unknown especially on presynaptic
side. Here, we focused on the presynaptic terminals of afferent fiber at mouse sensory thalamus, lemniscal fiber, where we managed to perform direct patch-clamp recordings from thepresynaptic terminals throughout synaptogenesis to synapse strengthening periods.
Somatosensory information from the maxillary region of rodent is conveyed to the ipsilateraltrigeminal nuclei via the infraorbital nerve and then to relay neurons in the contralateral ventral posteromedial thalamic nucleus (VPM) via medial lemniscal fibers. VPM relay neurons are
innervated by multiple afferent fibers before the maturation, but synapses are eliminated upon
the developmental maturation and eventually dominated by a single strong lemniscal fiber.
The kinetics of exocytosis was examined by membrane capacitance measurements, as well as
by preterminal-postsynaptic neuron paired recordings. With development, the total releasable
pool of the terminal becomes larger during immature periods first, and the enlargement of
the fast component of transmitter release occurs thereafter. The fast component is mediatedby vesicles tightly coupled to the calcium channels. Together with remarkable shortening ofaction potential durations, the developmental changes could fine-tune the speed, reliability, andplasticity of transmitter release in mature synapse, which underlies precise afferent sensory(COI:No)signal transmission.

Synaptic remodeling during the postnatal development is a process to establish functional
neuronal circuits suitable for individual growth environment and sensory experience. The synaptic remodeling usually consists of strengthening of specific synaptic inputs and elimination ofothers. In the developing sensory thalamus, each VPm neuron receives synaptic inputs from
more than three afferents (MLs, medial lemniscal fibers) which convey sensory informationfrom whisker and perioral regions in the brain stem, PrV2 and PrV3, respectively. Among thecompeting synaptic inputs, those arise from one PrV2-ML are selectively strengthened andmaintained but others are eliminated by postnatal day 20 (P20). Those processes have beenshown to depend on sensory experience using whiskers during P12-P14, however, mechanisms
underlying the sensory experience dependent processes are almost unknown. In the present
study, we examined how neuronal activities of postsynaptic VPm neurons have impacts on
the selective strengthening and elimination. Neuronal activities were reduced by injecting
AAV vectors expressing Kir2.1 to thalamic VPm region at P3. We confirmed that excitabilityof the Kir2.1-expressing neurons was reduced at P10. The amplitude of ML-EPSCs elicited in
the Kir2.1-expressing VPm neurons was significantly smaller than that in Kir2.1-unexpressing
VPm neurons at P21-P25. As for elimination, the numbers of discrete steps in the EPSCs in
response to gradually strengthened stimulus in most Kir2.1-expressing VPm neurons were
one as is the case in Kir2.1-unexpressing VPm neurons. Our results suggest that selective
strengthening and elimination during developmental synapse remodeling are differentially(COI:No)
regulated by neuronal activities of postsynaptic VPm neurons.

Direct measurements of transmitter release kinetics at lemniscal ﬁber
terminals in the somatosensory thalamus
Women’s Med Uni, Tokyo, Japan）

Neuronal activities underlying an experience-dependent synaptic
remodeling in the developing sensory thalamus
Medical Univ, Tokyo, Japan）

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S97

1P-037

1P-040

Yo Shinoda1，Yuta Yamada1，Mari Kikuta2, 3，Momoko Sakamoto1，Yayoi Tsuneoka1，
1 1 Dept Env andTsutomu Takahashi1，Eiko Yoshida4，Toshiyuki Kaji4，Yasuyuki Fujiwara（

Yoshitaka Ohtubo, Aiko Miyazaki（Dept Human Intelligence Sys, Grad Sch Life Scie Sys

Immunohistological and behavioral analysis of peripheral nerve injury
in methylmercury-exposed rats
Health, Sch Pharm, Tokyo Univ Pharm and Life Sci, Japan, 2Tokyo Gakugei Univ Int Sec

Sch, Japan, 3 Keio Univ, JST/GSC, Japan, 4 Dept Env and Health, Dept Pharm, Tokyo Univ

Sci, Japan）

Methylmercury (MeHg) is known as the causal substrate of Minamata disease, which induces
central and peripheral nerve injury. MeHg induced neural degeneration have been investigatedmainly by using brain because of the principal pathological phenotype is caused by the impairment of central nervous system (CNS). In contrast, MeHg induced neural injury in peripheral
nervous system (PNS) is not well investigated. To clarify the aspects of neural injury in PNS
induced by MeHg, 9 weeks Wistar male rats were exposed by MeHgCl solution orally for 5days and not exposed for subsequent 2 days (Day 7). This cycle was continued to the next week
again (Day 14). In Day 7 and 14, rats were anesthetized, fixed and their dorsal root ganglion(DRG), motor and sensory fibers were excised and cryosectioned. Co-staining of axonal marker
NF and myelin marker MBP showed axonal degeneration in sensory, but not in motor fibers
in Day 14. The peripheral sensory neurons in DRG were also degenerated especially middle
to large sized neurons in Day 14. In fact, DRG has many different subtypes of neurons thatsense pain, pressure, itch, etc., so that neurons were classified by different markers such as
NF, PLXNC1, TrkA and TH, however, these classified staining did not show any differences ofneural degeneration between each subtypes. We also analyzed another cell type using several
cell-type specific markers such as microglia (Iba1), macrophage (CD68), astrocyte (GFAP),
fibroblast (Vimentin), epithelial cell (CD31) and Schwann cell (SOX10). Microglia, macrophageand Schwann cell were significantly increased in Day 14 DRG and sensory fibers. These results
suggest that MeHg affects peripheral neural degeneration in DRG and axonal degeneration insensory fiber but not in motor fiber with concerting several cells.
(COI:No)

1P-038Transduction of sodium taste in the taste buds
2 1 Dept Mol Cell Physiol, Kyoto Prefec Univ Med, Japan, 2 PRESTO,
Akiyuki Taruno1, （JST）

Immunohistostaining and Ca2+-imaging of tracheal solitarychemosensory cells in miceEng, Kyutech, Japan）

The respiratory tract contains solitary chemosensory cells (SCCs) and chemosensory cellclusters for chemical detection. These cells express taste receptors, Tas1Rs and Tas2Rs, andare thought to use intracellular signaling molecules, such as gustducin and IP3 receptor type
3 (IP3R3), the molecules used in the signal transduction of taste to convert chemical into
biological information. In this study, we investigated the co-expression of gustducin and IP3R3
and performed Ca2+-imaging of SCCs in mouse trachea. Gustducin-immunoreactive SCCs
were observed throughout the trachea. In double immunohistostaining, 75% (632/842 cells) of
IP3R3-expressing cells were also immunoreactive to gustducin, whereas 34% (632/1878 cells)of gustducin-expressing cells were IP3R3 immunoreactive. Before measuring the intracellular
Ca2+ changes with fura-2, fura-2-stained cells were identified with immunohistochemistry,
because these cells were scattered on the tracheae surface. Almost all (98%) fura-2-stained
cells were gustducin-immunoreactive. In a few SCCs, a spontaneous intracellular Ca2+ increase
occurred in physiological saline. Further, some, but not all SCCs responded to 10 mM denatonium benzoate. Bacteria in the tracheae produce chemicals, including bitter substances usedin quorum sensing. SCCs in tracheae increase the intracellular Ca2+ in response to bitter
substances, which may trigger physiological reactions, such as an epithelial inflammatory(COI:No)
response.

1P-041

Correlations of oral capsaicin thresholds and genotypes of capsaicinreceptor gene in our medical students
Yoshihiro Murata1，Kiwamu Shibano1，Masahiro Yamaguchi1，Fumino Okutani2

Sodium as an ion (Na+) is an essential mineral and the major cation in the extracellular fluid,
and sodium taste regulates Na+ intake. Amiloride-sensitive epithelial sodium channel (ENaC)
in taste cells is the Na+ sensor mediating behavioral attraction to sodium salts. However,
how taste cells process oral Na+ downstream of ENaC remains to be defined. I performed
simultaneous patch-clamp and Ca2+ imaging experiments in ENaCα-expressing (ENaCα+)
taste cells acutely dissociated from the fungiform taste buds of mice expressing GCaMP3
under the control of ENaCα promoter. Electrically excitable ENaCα+ taste cells thatgenerated spikes by 2 mM Ba2+ responded to ENaC-mediated Na+ influx by a suprathreshold
depolarization and action potential firing. In the whole-cell voltage-clamp recordings, the same
cells exhibited an amiloride-sensitive ENaC current, a voltage-gated Na+ (Nav) current, and a
voltage-gated non-selective outward current. The outward current was abolished by 10 µM
carbenoxolone, a blocker of CALHM1/3 channel, and knockout of Calhm3, demonstrating thatthe current was mediated by CALHM1/3. As CALHM1/3 is the action potential-dependentneurotransmitter-release channel originally found in sweet-, bitter-, umami-sensing type II taste
cells, it is suggested that taste cells transduce ENaC-mediated Na+ influx directly into a burstof action potential and thereby CALHM1/3 channel-dependent neurotransmission. Meanwhile,
I also found taste cells that express functional ENaCs but neither Nav nor CALHM1/3 channels. Thus, these studies identified functional ENaC expression in two functionally distinct
taste cell populations. Further studies are required to determine how each cell population(COI:No)
contributes to sodium taste perception.

（ 1 Dept Physiol, Kochi Med Sch, Japan, 2 Dept Occup Health, Kochi Med Sch, Japan）

1P-039

1P-042The cells and neurotransmission underlying sodium taste in the tastebuds
2 1 Dept Mol Cell Physiol, Kyoto Prefec
Miho Nakanishi1，Kengo Nomura1，Akiyuki Taruno1, （Univ Med, Japan, 2 PRESTO, JST）

The excessive intake of salt (NaCl) can cause hypertension, and salt intake is regulated by thetaste of sodium. Thus, understanding the mechanism of sodium taste is important. It is known
that sodium-sensing in the tongue is mediated by amiloride-sensitive epithelial sodium channel(ENaC). Our recent electrophysiological studies showed that ENaC function exists in two distinct taste cell populations. One harbors Nav and CALHM1/3 channels, and the other harbors
neither Nav nor CALHM1/3. CALHM1/3 is the action potential-dependent neurotransmitter
release channel. However, it remains unclear how each cell population contributes to sodium
taste perception.
We first performed immunohistochemical examinations to detect ENaCα- and Calhm3-expressing cells. Consistent with the electrophysiological findings, ENaCα-expressing cells weredivided into two distinct populations based on Calhm3-expression. We subsequently carriedout a behavioral taste test, called brief-access lick test, using mice lacking ENaCα in Calhm1expressing cells (ENaC-cKO) and Calhm3-knockout mice. In the test, mice were depleted of
sodium and then presented with various concentrations of NaCl in randomized order, and licksof each solution were counted in 5-sec window. The control mice showed behavioral attraction
to NaCl; they preferred higher concentration of NaCl. In contrast, the attractive responses to
NaCl were strongly attenuated in ENaC-cKO mice, and abolished in Calhm3-knockout mice.
Furthermore, the amiloride-sensitive component of the gustatory nerve responses to oral NaCl
stimuli was absent in both ENaC-cKO and Calhm3-knockout mice.
In summary, ENaC function in CALHM1/3-expressing cells is indispensable for the gustatory
nerve response and behavioral attraction to NaCl, and CALHM1/3 function is also necessary
for taste responses to NaCl. We concluded that the taste cells expressing both ENaC andCALHM1/3 are responsible for sodium taste perception, and the sodium taste cells employ the
CALHM1/3 channel for the neurotransmitter release to the gustatory nerves.
(COI:No)S98

We reported in the previous meetings that detection thresholds to capsaicin at the anterior
part of the tongue showed a bimodal distribution. To explore the mechanisms that underlie
the individual differences, we examined the correlations of oral capsaicin thresholds with single
nucleotide polymorphisms (SNPs) of capsaicin receptor gene TRPV1. Oral capsaicin thresholds
were measured on the anterior part of the tongue by the “filter-paper disc” method. TRPV1
SNPs (rs222747, rs224534, and rs8065080) were detected by TaqMan SNP Genotyping Assays
using real-time PCR (StepOnePlus, Thermo Fisher Scientific). Among 227 subjects with a
median age of 21-year-old from our medical students, we found a haplotype significant for thosewith the higher-threshold of oral capsaicin (haplotype frequency, 0.029 for the “lower” groupvs. 0.141 for the “higher” group; permutation test, P = 0.012; SNPAlyze, dynacom). Individual
differences in oral capsaicin thresholds could be caused by genetic factors. This study wasapproved by the Ethic Committee of Kochi Medical School 23-102 and partly supported by
KAKENHI (19K02296) to Y.M.
(COI:No)

Pharmacological blockade of spinally-sensitized dorsal horn neurons in
a reserpine-induced ﬁbromyalgia model
Toru Taguchi1, 2，Daisuke Uta3，Katsuyuki Tsuboshima4，Hisao Nishijo4，Kazue Mizumura5（ 1 Dept. Phys. Ther., Niigata Univ. Health Welfare, Niigata, 2 Inst. Hum. Mov. Med. Sci,
Niigata Univ. Health Welfare, Niigata, 3 Dept. Appl. Pharmacol., Grad. Sch. Med. Pharm.
Sci., Univ. Toyama, Toyama, 4 Syst. Emot. Sci., Grad. Sch. Med. Pharm. Sci., Univ.
Toyama, Toyama, 5 Dept. Phys. Sch. Dent. Nihon Univ., Tokyo）

Chronic widespread pain such as fibromyalgia (FM) is a major medical problem. Behavioral
pharmacological studies showed that some drugs were effective for reducing pain, yet the
spinal anti-nociceptive mechanisms are poorly understood. The present study was conducted
to examine whether therapeutic drugs for FM (amitriptyline, duloxetine, and pregabalin) could
affect neuronal responses of the superficial dorsal horn (SDH) neurons using a rat model of FM.
Subcutaneous injections of reserpine (1 mg/kg), a depleter of biogenic amines in the nervous
system, were administered to make the FM model (Nagakura et al. Pain, 2009). Under urethane
anesthesia, extracellular recordings in vivo were made from the SDH neurons at the lumbar
segments L4-L5. The SDH neurons showed the higher spontaneous discharges in the FMgroup compared with the control group. Responsiveness to quantitative mechanical stimuli, applied to the receptive field of the SDH neurons, induced stimulus intensity-dependent increases
in the discharge rate, and the response magnitude was significantly greater in the FM groupcompared with the control. Bath application of amitriptyline (0.1～1 mM) and duloxetine (0.1～1 mM) on the surface of the spinal cord remarkably suppressed the increased spontaneousand mechanical discharges in a dose-dependent manner, while that of pregabalin (1 mM) did
not. These results suggest that the SDH neurons are sensitized in an FM model, and that theneurons are the site of analgesic actions for amitriptyline and duloxetine, but not pregabalin,
for the treatment of FM.(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

1P-043

1P-046

Akiko Arata, Hirotaka Ooka, Chiaki Uchida, Sotatsu Tonomura（ Dept Physiol, Hyogo CollFumiyo Toyoda1，Shogo Haraguchi2，Kazutoshi Yamamoto3，Masahiro Matsunaga4，
3 1 Dept Neurophysiol,
Minoru Takase5，Tomoaki Nakada6，Kazuyoshi Tsuitsui3，Sakae Kikuyama（

The gate mechanism of sensation-respiration in the parabrachial
nucleusMed, Nishinomiya, Japan）

The multi-sensational signals were projected to the lateral parabrachial nucleus (LPB) in thepons. LPB has also known as the system of inspiratory-expiratory (I-E) phase switching that
contributes to the control of respiratory rate. Thus, the tight interaction between respirationand pain signals as nociception-respiratory coordination was expected in LPB. In this study,
we investigated the pain-respiratory coordination system using the pons-medulla-spinal cord
preparation intact forelimb isolated from postnatal 0-4 days-rats. The spontaneous inspiratory
activity was recorded from cervical fourth (C4) ventral nerve root, and we examined the
responses of C4 activity when 2% capsaicin was injected into the forelimb. The C4 inspiratory rate increased significantly in the preparation with pons, but not the removal of pons
(P<0.01). Moreover, we examined the properties of LPB neurons in this preparation. First,
the responded area of LPB from C8 dorsal root stimulation was detected by optical imaging
using voltage-sensitive dye; the LPB neurons (n=45) were recorded from the responded area
using whole-cell patch-clamp. I-E neurons, which were synchronized with the I-E phase of C4
ventral root activity, located in the extra LPB. The spontaneous or non-spontaneous firing
neurons, which was not synchronized with the C4 activity called non-respiratory neurons. All
I-E neurons and eight non-respiratory neurons, which existed in the extra lateral PB. Eachneuron tested the current-voltage (I-V) relationship in current-clamp mode. According to the
responses of hyperpolarizing current pulses, post-inhibitory rebound (PIR) was observed in 13
non-respiratory neurons. These results suggested that 1) the pons contribute to the increase of
respiratory rate by noxious stimulation; 2) I-E neurons could directly receive noxious information, so I-E neurons were thought to be the core mechanism of pain-respiratory coordination;3) the non-respiratory LPB neurons which expressed PIR might be contributed to the onset(COI:No)
switching mechanism of the pain-respiratory coordination network.

Steroidal substances derived from the abdominal gland of the malenewt, Cynops pyrrhogaster, may act as female-attracting pheromones
Nara Medical Univ, Japan, 2 Department of Biochemistry, Showa Univ Sch. Med, Tokyo, Japan,

3 Dept Biol, Fac Educ Integr Sci, Waseda Univ, Japan, 4 Dept Health and Psychosocial Med, Aichi

Med Univ Sch Med, Nagakute, Aichi, Japan, 5 Amphibian Research Center, Hiroshima University,
Hiroshima, Japan, 6 Dept Comp Behav Med, Nippon Vet Life Sci Univ, Japan）

Previously, we identified a decapeptide (sodefrin) in the abdominal gland (AG) of the malenewt as a major female-attracting pheromone. Meanwhile, we found that several steroidal
substances are synthesized in the gland and that among them, pregnenolone and androstenedione are released from the gland. We confirmed that both steroids exert a female-attracting
activity. The minimum effective amount were approximately 0.4ng in both cases when they
were subjected to a preference test that was developed by ourselves. When both steroids
were applied in combination, they showed an additive effect. The minimum effective amountof these steroids was estimated to be the amount contained in 10-2 piece of the AG of thesexually developed male newts. The minimum effective amount of sodefrin had previously
been determined to be 10ng, which is contained in 10-3 piece of the gland. Accordingly, we
concluded that sodefrin is the major pheromone exerting a female-attracting activity in thefield during the breeding season. When the combination of 10 ng sodefrin with 0.04 ng ofboth steroids, all of which are equivalent to 10-3 piece of AG contents, was compared withsodefrin alone or both of the two steroids in terms of attracting the female newts, sodefrin in
combination with the steroids exhibited the most potent activity. In order to determine theresponsiveness of the receptor cells in the vomeronasal epithelium of the lateral nasal sinus
region to these steroids, electro-olfactograms (EOGs) were recorded. In the sexually developed
females, the vomeronasal epithelium showed a considerable response to the steroids. Whensubthreshold amount of the steroids was applied together with sodefrin, the EOG response
was markedly enhanced as compared with the response to sodefrin alone. BSA-conjugatedandrostenedione also induced EOG response in the epithelium, indicating that the steroid acts
through the membrane receptors of the vomeronasal cells.
(COI:No)

1P-044

1P-0472 1 Neurorehabilitation research group, AIST, Tsukuba,
Nobuo Kunori1，Ichiro Takashima（

Yu Tahara（ Lab Physiol Pharm, Sch Adv Sci Eng, Waseda Univ, Japan）

An implantable cranial window using a collagen membrane for chronic
voltage-sensitive dye imaging
Japan, 2 Human Informatic Research Institute, AIST）

Incorporating optical methods into implantable neural sensing devices is a challenging approach for brain-machine interfacing. Specifically, voltage-sensitive dye (VSD) imaging is a
powerful tool enabling visualization of the network activity of thousands of neurons at highspatiotemporal resolution. However, VSD imaging usually requires removal of the dura materfor dye staining and thereafter the exposed cortex needs to be protected using an opticallytransparent artificial dura. This is a major disadvantage that limits repeated VSD imaging
over the long term. To address this issue, we propose to use an atelocollagen membrane as
the dura substitute. The membrane used in the present study was optically transparent andenough to pass excitation/emission light for VSD imaging. We made a small cranial chamber
device, which is a tubular structure equipped with a collagen membrane at one end of the tube,
and implanted the device over the sensorimotor cortex of rats. When performing the VSD
imaging 1 week following implantation surgery, we successfully observed the forelimb-evokedneuronal activities in the sensorimotor cortex through the collagen membrane. We also tried
pharmacological modulation of neuronal responses through the implanted-cranial window. Thegabazine, GABAA receptor antagonist, placed into the implanted chamber clearly increased
forelimb-evoked neuronal responses. These results indicate that the atelocollagen membrane
was chemically transparent, allowing VSD staining and pharmacological interventions acrossthe membrane material. Because of its ideal chemical and optical manipulation capability, thiscollagen membrane may be widely applicable in various implantable neural sensors to monitor
the changes of neuronal activity due to variety of reasons such as motor learning and stroke.
(COI:No)

1P-045

Neuronal mechanism of contrast sensitivity modulation via
noradrenergic β receptor
Keisuke Tsunoda（ Grad Sch Frontier Biosci., Osaka Univ, Osaka, Japan）
Noradrenaline (NA) secreted throughout the brain by noradrenergic neurons in the locus coeruleus regulates various brain functions, including vision. Previous electrophysiological studies
on the primary visual cortex (V1) revealed that iontophoretically-administered NA modulates
the visual responses and the modulatory effect differs from neuron to neuron, being facilitative
or suppressive. The NA-induced neuronal response modulation in V1 suggests to modulate
the visual information processing, affecting perceptual visual detectability. However, it has notbeen unclear whether and how the noradrenergic response modulation of V1 neurons affects
perceptual visual performance.
In order to investigate this point, we performed multipoint extracellular recordings fromthe V1 of the rats performing the visual stimulus detection task, and first examined therelationship between task performance and neural activity, and then, tested the effect of anoradrenergic Beta-receptor antagonist. The behavior task was Go/No-go visual detection task.
Grating stimulus with various contrasts was presented on the front monitor of a rat underhead-restrained condition, and the perceptual contrast threshold as a lower contrast limit
enabling to detect the stimulus was estimated. The rat was rewarded by making a response(pulling a lever) to the stimulus presentation. During the rat performed the task, propranolol(Beta-receptor antagonist, PRP) was topically administered on the cortical surface of V1.
About 10% of V1 neurons changed their firing rates corresponding on the task performance,
in which the magnitude of the visual responses was higher in the correct trial than the misstrial. PRP administration significantly lowered the perceptual contrast threshold, suggesting
an improvement of the perceptual detectability for low contrast stimulus. Also, the neuronalsignal-to-noise ratio was improved at low contrast conditions but not at high conditions. Therefore, NA might reduce animal’s visual ability to detect stimuli at low contrast conditions via
Beta-receptor in V1.(COI:No)

Circadian clock disruption in mice with adenine-induced
tubulointerstitial nephropathy
Chronic Kidney Disease (CKD) is increasing in incidence and has become a worldwide
health problem. Sleep disorders are prevalent in CKD patients raising the possibility thatthese patients have a disorganized circadian timing system. Here, we examined the effect ofadenine (0.2%, 2 weeks)-induced tubulointerstitial nephropathy on the circadian system in mice.
Compared to controls, adenine-treated mice showed serum biochemical measures of renal
dysfunction as well as increased expression of renal markers for inflammation and fibrosis. The
CKD mice exhibited fragmented sleep behavior and locomotor activity, with lower amountsof activity compared to controls. On a molecular level, the CKD mice exhibited low amplitude
rhythms in their central circadian clock (suprachiasmatic nucleus) as measured in vitro
PER2::LUCIFERASE driven bioluminescence. Whole animal imaging revealed that the treatedmice also exhibited dampened oscillations in the intact kidney, liver, and submandibular gland.
Consistently, dampened circadian oscillations were observed in several circadian clock genes
and clock-controlled genes in the kidney of the CKD mice. Finally, mice with a geneticallydisrupted circadian clock (Clock mutants) were treated with adenine and compared to WT
controls. The treatment evoked worse kidney damage as higher gelatinases (MMP-2 and 9) andadenine metabolite deposition in the kidney. Adenine also caused non-dipping hypertension andlower heart rate. Taken together, the data indicate that central and peripheral circadian clocks
are disrupted in the adenine-treated mice, and suggest that the disruption of the circadian clock
accelerates CKD progression.
(COI:No)

1P-048Glucocorticoid exposure during the circadian inactive phase inducesaberrant expression of hypothalamic orexigenic and anorexigenic
neuropeptides and alters feeding behaviour in rats
Mitsuhiro Yoshimura1，Benjamin Flynn1，Yvonne Kershaw1，Zidong Zhao1，
1 1THS, BMS, Univ of Bristol,
Becky Conway-Campbell1，Yoichi Ueta2，Stafford Lightman（

UK, 2 Dept of Physiol, Sch of Med, UOEH ）Adrenal glucocorticoid secretion is characterised by both circadian and ultradian rhythmicity.
Here, we have assessed how dysregulated corticosterone (CORT) rhythm affects feeding
behaviour in rats. Adult male SD rats were adrenalectomised, implanted with a jugular cannulaand an intraperitoneal telemetry probe. Rats were kept under 12/12 light/dark cycle andprovided with food and saline ad libitum. They were divided into 2 groups: FORWARD and
REVERSE. We used a programmable pulsatile infusion system to deliver CORT-HBC, which
is a water-soluble form of CORT. In the FORWARD group, the CORT-HBC infusion pattern
was programmed to deliver both circadian and ultradian components of an endogenous CORT
profile determined by automated blood sampling of adrenal-intact animals. In the REVERSE
group, the same dose and pattern of CORT-HBC was infused, except phase-shifted by 12h.
Food and saline intake were measured every 12h and body weight was measured daily for 5
days. Brains, subcutaneous and epididymal fat, and blood samples were collected at zeitgeber
time (ZT) 1 and ZT13. In the FORWARD group, the percentage of food intake was significantly
greater in the dark phase compared to the light phase. In the REVERSE group, food intake
profile was significantly dysregulated. No change in body mass, subcutaneous or epididymal fat
mass was observed between groups. Locomotor activity profiles were similar; however, core
body temperature was altered in REVERSE compared to FORWARD. In situ hybridization
histochemistry revealed that hypothalamic gene expression of the anorexigenic neuropeptides
were significantly upregulated at ZT13, whereas, that of orexigenic neuropeptides were markedly upregulated at ZT1 in REVERSE compared to FORWARD. Clock genes expression in
the suprachiasmatic nucleus was unaffected by infusion pattern. These results suggest thatdesynchronization of light/dark cues and circadian CORT rhythms can induce inappropriate
feeding behaviour by directly altering hypothalamic neuropeptide expression. (COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S99

1P-049

Prefrontal Parvalbumin Interneurons are Essential for Social Behavior
Development
1 1 Dept Psychiatry, Nara
Kazuhiko Yamamuro1，Hirofumi Morishita2，Toshifumi Kishimoto（1P-052

Odor induced anti-pruritic effect on pruritogen-induced scratching
behavior in mice
Tatsuroh Kaneko, Tomoyuki Kuwaki, Hideki Kashiwadani（Dept Physiol, Med, Kagoshima

Medical Univ, Japan, 2 Dept Psychiatry, Icahan School of Medical Sinai Univ, USA）

Univ, Japan）

Social isolation (incl. loneliness experience) during developmental critical windows could behighly detrimental to proper functioning of mature prefrontal cortex (PFC) and establishmentof appropriate adult behaviors. However, the specific circuits that undergo social experiencedependent maturation to regulate social behavior development are poorly understood. Here
we show that juvenile social isolation in mice leads to reduced intrinsic excitability and input
drives of adult parvalbumin-positive interneurons (PVIs) in medial PFC (mPFC), suggesting
juvenile social experience is required for their proper activation in adulthood. In vivo imaging
of mPFC-PVI activity by fiber photometry demonstrated that adult mPFC-PVIs are preferentially activated by social signals. Recapitulating decreased activity of PVIs through acutechemogenetic suppression revealed that normal social behavior requires physiological mPFCPVI activity. Conversely, chemogenetic restoration of mPFC-PVIs activity in the adult animal
rescued juvenile isolation-induced social deficits. Therefore, PVI development in the juvenile m(COI:No)PFC is critically linked to long-term impacts on social behavior.

Pruritus is one of the uncomfortable feelings on the skin and sometimes impairs our quality oflife. Antihistamines are the initial drug of choice for the treatment of pruritus, but there areseveral types of pruritus on which antihistamines do not efficiently work. Thus, the development of treatment for antihistamines-resistance pruritus is one of the pressing issues.
Previously we have shown that odor of linalool, one of the monoterpene alcohols in lavender
extracts, induced analgesic effects triggered by olfactory input. The linalool analgesia significantly attenuate chemical pains induced by formalin or capsaicin, raising the possibility that thelinalool odor may also attenuate antihistamine-resistance itch evoked by chemical pruritogen.
To address the possibility, we observed the scratching behaviors evoked by intradermal
administration of pruritogens in mice under linalool odor exposure.
Male C57/BL6 mice (9 weeks-old) were used in the experiments. As peripheral pruritogens,
serotonin (50ug/50uL/site) and chroloquine (200ug/50uL/site) were intradermaly injected into
the nape of the neck of mice. Immediately after the injection, mice were placed in observationchamber in which was filled with linalool odor or odorless air (as control). Then scratching
behavior was video-recorded for 30 minutes and analyzed.
In serotonin-induced scratching mice model, linalool odor exposure significantly reduced
scratching behavior for first 6 minutes after serotonin injection. Linalool odor also significantly
reduced scratching behavior in chroloquine-induced itchy mice for 20 minutes after chroloquine injection.
In conclusion, linalool odor exposure decreased scratching behavior in peripherally induced
itchy mice models. These data suggest the potential benefit of linalool odor on the control ofpruritus in clinical situation.
(COI:No)

1P-050

1P-053

Daisuke Ono, Hung Chi Jung, Srikanta Chowdhury, Wang Guanyi, Akihiro Yamanaka

Ryohei Umeda1，Kazumasa Hada 2，Kenshiro Shikano1，Ryoko Higa1，Hirotaro Urushibata 2，
1 1 Dept Neurophysiol, Fac Med,Hiroshi Shiraishi 2，Toshikatsu Hanada 2，Reiko Hanada （The suprachiasmatic nucleus regulates wakefulness via CRF neurons
in the hypothalamus（RIEM, Nagoya Univ, Japan）

Physiological function of VRK 2 in zebraﬁsh model
Oita Univ, Japan, 2 Cell Biol, Grad Sch Med, Oita Univ, Japan）

Living organisms exhibit endogenous circadian rhythms and adapt to the 24 hour daily cycles
on Earth. Circadian rhythms are known to organize temporal timing of physiology and behavior such as body temperature, metabolism, and sleep/wakefulness. In mammals, the suprachiasmatic nucleus of the hypothalamus (SCN) functions as master circadian pacemaker including
thousands of neurons that express self-sustained and synchronizing circadian rhythms in firing
activity and gene expression. Nevertheless, little is known about neuronal projections from the
SCN that regulate sleep/wakefulness. Here we show that corticotropin-releasing factor (CRF)
neurons in the paraventricular nucleus of the hypothalamus mediate circadian rhythms in
the SCN. Optogenetic activation of CRF neurons promotes wakefulness. Furthermore, in vivo
Ca2+ recording using fiberphotometory revealed that CRF neurons in the PVN were active
during wakefulness, but not NREM and REM sleep. We also found that neuronal activity of
CRF neurons in the PVN were regulated by the SCN. We will discuss mechanisms by which
the SCN regulated sleep and wakefulness in mice.
(COI:No)

Vaccinia related kinase 2 (VRK2), a serine / threonine kinase belonging to the Vaccinia-related
kinase family, which plays an important role in cell survival and stress response to hypoxia.
Recently, VRK2 gene mutations in humans have been reported to be one of the causes ofschizophrenia. In previous studies, schizophrenia and its related diseases were shown by
Genome-wide association study (GWAS) analysis, and it was found VRK2 as one of candidate
genes of their causes. The functional analysis of VRK2 has been done by in vitro studies using
cells, however in vivo analysis has not been performed yet. Thus, it is not clear the physiological function of VRK2 in vivo. Therefore, we have established a VRK2 gene-deficient zebrafish(VRK2 KO) using the CRISPR / Cas9 system, and analyzed a series of behavior experiments.
At first, their growth conditions such as body length and body weight were analyzed in VRK2
KO and control zebrafish (WT). Since VRK2 gene mutations is reported to be a cause of mental
illness, we next analyzed morphological changes of the brain using HE staining. In addition,
we performed a series of behavioral analysis of aggression, sociality, and anxiety related toschizophrenia using Mirror test, Social interaction test, and Novel tank diving test, respectively.
Here, we will present these data.
(COI:No)

1P-051

1P-054

A corticohypothalamic pathway for psychological stress-induced social
avoidance

The ontogeny of glycine transporter 1 (GlyT1) during development in
the spinal cord

Yuta Shima, Naoya Kataoka, Kazuhiro Nakamura（ Dept Integrative Physiol, Nagoya Univ

Chigusa Shimizu1，Daisuke Omata1，Ryuji Tomoyose1，Kie Okano1，Shiori Kobayashi1，
1 1 Dept.Mol.Anatomy, Grad.Sch.Med, University of the
Akihito Okabe2，Chitoshi Takayama（

Grad Sch Med, Japan）

Psychological stress causes various physiological responses by stimulating sympathetic
mechanisms (e.g., increases in body temperature, heart rate and blood pressure) and behavioral
mechanisms in mammals. Recently, we discovered a group of glutamatergic excitatory neurons in the dorsal peduncular cortex and dorsal tenia tecta (DP/DTT), located at the ventral
limit of the medial prefrontal cortex, and found that they mediate master stress signaling to thedorsomedial hypothalamus (DMH) to drive various sympathetic stress responses. However, it
is unknown whether this DP/DTT→DMH pathway contributes to behavioral changes induced
by stress or emotion. Here, we examined the effect of the DP/DTT→DMH pathway on social
avoidance induced by social defeat stress, an animal model of psychosocial stress. A male
Wistar rat underwent social defeat stress from a dominant, male Long-Evans rat, and was subsequently placed in an open field. Then, the same Long-Evans rat, which was caged, was placedin the field. The stressed Wistar rats mostly stayed at the corners of the field, being away fromthe Long-Evans rat and avoiding interaction. This stress-induced social avoidance contrasted
with active social interaction exhibited by naïve Wistar rats, which had not undergone social
defeat stress. To selectively inhibit the DP/DTT→DMH pathway in the stressed Wistar rats,
the animals beforehand received injections with adeno-associated virus (AAV) to transduce
DP/DTT→DMH projection neurons with iChloC, a photo-activated chloride channel shownto suppress neuronal activity. Photoinhibition of the DP/DTT→DMH pathway completely
reversed the social defeat stress-induced social avoidance: the Wistar rats often entered theinteraction zone surrounding the cage of the Long-Evans rat and actively interacted. These
results demonstrate that the stress signaling mediated by the DP/DTT→DMH pathway isessential to drive stress-induced social avoidance as well as sympathetic stress responses.
(COI:No)

S100

Ryukyus, 2 Seinan Jo Gakuinn University）

In the spinal cord, glycine and Gamma-amino butyric acid (GABA) are inhibitory neurotransmitters. Released glycine and GABA is removed from the synaptic cleft by glycine
transporter1 (GlyT1) and GABA transporter 3 (GAT-3). We have previously investigated thedevelopmental changes in GABA-removal system (Kim et al. 2014). However, the developmentof glycine-removal system in astrocytes remains unclear. The present study aimed to reveal
the ontogeny of the glycine-removal system in the astrocytes by examining the immunohistochemical localization of GlyT1 in the embryonic and postnatal mouse cervical spinal cord.
On embryonic day 12 (E12), GlyT1 was expressed in the mantle layer. GlyT1 was localized
in the process of radial glia. On E14, GlyT1 was localized in the ventral horn. This expression
pattern was different from GAT-3. Weak GAT-3 immunolabeling was localized to several
radial fibers extending from the central canal to the pial surface on E12. On E14, GAT-3 wasdetected in the radial processes. After E18, GlyT1 was colocalized with GAT-3 in astrocytes. In
our previous study, GlyT2, which uptakes glycine in presynaptic terminal, was first detected in
the ventral horn on E16 after GlyT1 was expressed (Sunagawa et al. 2017).
These results suggested that glycine and GABA were uptaken in different region of the radial
glia in E12-14, but after that, both neurotransmitters were removed by the same astrocytes.
Before the formation of glycinergic terminal, glycine-removal system in astrocytes might beready.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

1P-055

Modulatory effects of repeated psychophysical stress on nociceptive
neural activities in the rostral ventromedial medulla (RVM) evoked by
noxious stimulation to the craniofacial tissue in the rats
Mana Hasegawa1, 3，Masayuki Kurose2，Noritaka Fujii1, 3，Kensuke Yamamura2，
2 1 Div Dental Clinic Edu, Niigata Univ Grad Sch Med Dent Sci, 2 Div
Keiichiro Okamoto（

Oral Physiol, Niigata Univ Grad Sch Med Dent Sci, 3 General Dent Clinic Edu Unit, Niigata
Univ Med Dent Hosp）
Dysfunction of the descending pain controls, indicated by neural changes in the rostral
ventromedial medulla (RVM), are known to increase craniofacial nociception under repeatedpsychophysical stress conditions. Previous reports showed that repeated psychophysical stressconditionings impaired serotonergic (5HT) mechanisms in the brain. However, it remains
unclear if neural changes in the RVM, which had facilitatory effects on craniofacial nociception,
could be due to dysfunction of 5HT mechanisms under stress conditions. SD male rats wereassigned to repeated Forced Swim Stress (FST) and sham conditionings for 3 days (10 min/d).
Fluoxetine (selective serotonin reuptake inhibitor) or vehicle (saline) was administered daily
just after each FST. Single unit activities were isolated in the RVM, and units were classified
into three types of units (ON-, OFF- and NEUTRAL-cell) based on the responsiveness tonoxious heating stimulation (52 ℃) to the facial skin over the masseter muscle under generalanesthesia. Heat-evoked neural discharges for those units are quantified in each group after
repeated FST. Further, nociceptive EMG activity evoked by heating stimulation to the facialskin was recorded in the suprahyoid muscle, simultaneously. Repeated FST increased neuralactivities of ON- cells due to increases in prolonged after-discharges. Further, FST also had
modulatory effects on OFF-cell activity indicated by prolonged the pause duration in spontaneous activities. FST-induced changes of response properties in ON- and OFF cells were prevented by the systematic administration of fluoxetine. FST had no effects on neural activities
in NEUTRAL-cell. Nociceptive EMG activities were significantly increased after repeated FST,
which was reduced by daily administration of fluoxetine. Application of fluoxetine alone did
not affect the neural and EMG activities. These results indicated that increases in craniofacial
nociception in the RVM after repeated FST could be due to neural changes in ON- and OFF
cell activities through the changes in 5HT mechanism.
(COI:No)

1P-056The effect of an acupuncture “press tack needle” treatment on orexin
secretion under acute stress
Aki Fujiwara1，Mana Tsukada1，Takuji Izuno1，Hideshi Ikemoto1，Yasunori Takayama1，
1 1 Dept Physiol,
Shi-yu Guo1，Yan-qing Liu1, 2，Tadashi Hisamitsu1，Masataka Sunagawa（

Sch Med, Showa Univ, Japan, 2 Dept of Combined Traditional Chinese and Western Med,
Yangzhou Univ Sch Med, China）
Orexins are neuropeptides secreted mainly from the hypothalamus and are involved in the control of stress responses. We previously reported that press tack needle (PTN) treatment at the
GV20 acupuncture point (Baihui) suppresses orexin A secretion, which is increased by chronic
mental stress. However, this decrease in orexin A secretion is inhibited by electroacupuncture
treatment at the SP6 (Sanyinjiao) and ST36 (Zusanli) acupoints in rats with acute pain. In thepresent study, we investigated whether or not the effects on orexin secretion differ dependingon the types of stress experienced using a rat acute stress model.
Rats were randomly divided into three groups as follows: 1. no stress, 2. acute stress, and 3.
PTN-treated acute stress. In the PTN+Stress group, the PTN (length: 1.2 mm) was fixed at theacupuncture point that corresponds to human GV20. As acute stress, a restraint stress load
was applied for 90 minutes, after which the amount of defecation induced by restraint stressand the plasma orexin A concentration were measured. Compared with the Stress group, theamount of defecation was significantly suppressed in the PTN + Stress group. The plasma
orexin A concentration was significantly decreased in the Stress group compared with theControl group, but in the PTN + Stress group, this decrease was significantly suppressed.
In the chronic stress model, the increased orexin A secretion was suppressed by PTN. In
contrast, in an acute stress model, PTN inhibited the decrease in the orexin A secretion. Thesefindings suggest that PTN treatment is also effective under acute stress and that the effect has(COI:No)a moderating effect on the secretion of orexin A.

1P-057Microinjections of an ionotropic excitatory amino acid receptors
activator L-cysteine identiﬁed a parasympathetic carotid vasodilator
response zone that spans the rat brainstem?
Yumi Takemoto（Neurophysiol, Grad Sch Biomed, Hiroshima Univ, Japan）
Our previous study demonstrated that microinjection of an ionotropic excitatory amino acidreceptors activator L-cysteine into the rostral ventrolateral medulla pressor area (RVLM)
produce equi-pressure common carotid (CA) vasodilation in the superior cervical sympathetic
denervated rat (SCD), different from pressor and CA vasodilator responses to L-cysteine in theventral pons of the salivatory nucleus level. It may suggest any parasympathetic functional relation to the pre-sympathetic area in the rat RVLM. The present study was performed to findthe exact place where L-cysteine stimulation produces carotid vasodilation in the brainstem
around the RVLM. In anesthetized intact rats or SCDs, a window was ventrally opened toaccess the brainstem, then common carotid flow (CAF), arterial blood pressure (AP) and carotid flow resistance (AP/CAF) were monitored. L-Cysteine microinjections produced carotid
vasodilation with equi-pressure or pressor responses at the dorsal and rostral sites from the
RVLM, forming a plate-like response zone in individual rats. The plate atlas varied from rat torat. Vasodilator sites in intact rats and SCDs extended 1.4 mm anterior to and 2.5 mm dorsal to
the RVLM, corresponding to the parvicellular reticular and salivatory nuclei which were identified with dye injections. Vasodilator region was rostral and just adjacent to localized carotid
vasoconstrictor region within the RVLM pressor area in intact rats, but the region included
the whole RVLM pressor area in SCDs which had no carotid vasoconstrictor region. Thus,
L-cysteine microinjections into the brainstem parasympathetic related zone produced common
carotid vasodilation in rats. It may have functionally identified an autonomic region capable ofregulating common carotid flow across the rat brainstem where pre-sympathetic neurons and(COI:No)
parasympathetic related fibers may interact. Further studies are required. 

1P-058

Fos expression in rat MLR neurons projecting to the RVLM following
the voluntary treadmill exercise
Yui Yamane, Tatsuo Watanabe, Satoshi Koba（ Div Integr Physiol, Tottori Univ, Japan）
Neurons in the mesencephalic locomotor region, MLR, send axonal projections to the rostral
ventrolateral medulla, RVLM (MLR-RVLM neurons). Here we tested the hypothesis that the
MLR-RVLM neurons are excited in association with voluntary locomotion. Male rats that hadreceived bilateral microinjections into the RVLM with a retrograde adeno-associated virus vector that encoded GFP were accustomed to the voluntary treadmill exercise for seven-to-ninedays. On the experimental day, they were treadmill exercised at 16 m/min for 40 min (n = 7)or placed on the treadmill for a comparative control period (n=7). The paraformaldehyde-fixed
brains were then processed for double-immunofluorescence microscopy. The MLR of exercised
rats contained more expression of Fos protein in both GFP- and ChAT- immunoreactive (IR)neuronal cells than that of control rats (19 ± 4 vs. 8 ± 2% in GFP-IR cells and 22 ± 5 vs. 8± 2% in ChAT-IR cells, respectively, P<0.05). Interestingly, GFP-IR, RVLM-projecting cells in
the MLR did not correspond with ChAT-IR cells, which were abundantly distributed in thepedunculopontine nucleus within the MLR. These results suggest that a portion of RVLMprojecting MLR neurons, which are unlikely cholinergic, is excited during locomotion.(COI:No)

1P-059The lateral parabrachial nucleus and Kölliker-Fuse nucleus involve inthe reﬂex responses of heart rate to noxious mechanical stimulation of
the hindpaw in anesthetized rats
Hana Nozawa1, 2，Rie Shimoju1, 3，Takamichi Taniguchi1, 2，Hideshi Shibata4，
3 1Grad. Sch. Health ＆ Sci., Int. Univ. Health ＆ Welfare, Otawara 324Mieko Kurosawa1, （8501, Japan, 2 Dept. Occupational Ther., Intl. Univ. Health ＆ Welfare, Otawara 324-8501,
Japan, 3 Center Med. Sci., Intl. Univ. Health ＆ Welfare, Otawara 324-8501, Japan, 4Lab.
Vet. Anat., Ins. Agric., Tokyo Univ. Agric ＆ Tech., Fuchu, Tokyo 183-8509, Japan）

Noxious mechanical stimulation (pinching) of the hindpaw reflexly increases arterial pressureand heart rate via the supraspinal structure in anesthetized rats. We have recently shownthat the lateral parabrachial nucleus (LPBN) and the Kölliker-Fuse nucleus (KF) were involvedin the reflex responses of arterial blood pressure to pinching of a contralateral hindpaw. The
present study was aimed to clarify the involvement of these nuclei in the reflex responses ofheart rate. Heart rate was measured by a pulse rate tachometer. Muscimol, a widely usedneuronal inhibitor, was nanoinjected into the unilateral LPBN or KF. Pinching was applied witha surgical clamp at a force of 3-5 kg to the hindpaw for 20 s. The administration of muscimol
into the LPBN or KF nuclei had no influence on basal values of the heart rate. On the other
hand, the heart rate values at 20 s after pinching of the hindpaw were significantly attenuatedafter the administration of muscimol when pinching was applied to the hindpaw contralateral
to the site of muscimol injection. The heart rate values in response to pinching of the ipsilateralhindpaw were slightly attenuated; however, the attenuation was not statistically significant.
The effects of muscimol injected into the KF on the reflex responses were similar to thoseinjected into the LPBN. The present results demonstrate that both the LPBN and KF are
involved in the reflex responses of heart rate elicited by pinching of the contralateral hindpaw.
(COI:No)

1P-060GABAergic neurons in the rostral medullary raphe nucleus regulate thecardiac parasympathetic system
Taro Minoura, Kazuhiro Nakamura（ Dep Integrative Physiol, Nagoya Univ Grad Sch Med,Japan）

When mammals are exposed to environmental stressors, glutamatergic sympathetic premotor
neurons in the rostral raphe pallidus nucleus (rRPa) of the medulla oblongata are activatedto drive sympathetic responses, such as tachycardia and thermogenesis. We have found that
GABAergic neurons in the rRPa are also activated at the same time. However, the functionsof the GABAergic neurons are unknown. In this study, we sought for their functions. To
optogenetically stimulate GABAergic rRPa neurons, these neurons were transduced with
ChIEF-mCherry, a channelrhodopsin variant by injecting a Cre-dependent adeno-associatedvirus vector into the rRPa of Gad2-ires-Cre mice. It was confirmed that most of neurons expressing ChIEF-mCherry were GABAergic. Furthermore, their axon terminals were found to
be closely associated with cholinergic neurons in the nucleus ambiguus, which were potentiallycardiac parasympathetic preganglionic neurons. This observation suggests that GABAergicrRPa neurons innervate cardiac parasympathetic preganglionic neurons to inhibit them. To
test this hypothesis, we examined the effect of photostimulation of GABAergic rRPa neurons
on heart rate under anesthesia. Photostimulation of either cell bodies in the rRPa or theiraxon terminals in the nucleus ambiguus significantly increased heart rate. Prior intravenous
injection of atropine, a muscarinic receptor antagonist, suppressed the increase in heart ratecaused by photostimulation of cell bodies of GABAergic rRPa neurons. These results suggestthat activation of GABAergic rRPa neurons elicits tachycardic response by inhibiting parasympathetic outflow to the heart. The rRPa is likely to be a brain site that controls the balance
between sympathetic and parasympathetic efferent tones by regulating the glutamatergic and(COI:No)GABAergic neurons therein.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S101

1P-061

1P-064

Ena Yamamoto1，Takatoshi Horiuchi1，Misaki Ichikawa2，Mio Matsuyama1，Nao Suzuki2，
2 1 Dept Biomedical Engineering, Toyo Univ, Japan, 2 Depat Biomedical
Joji Horiuchi1, （1 1 Dept Cell
Tomohiro Nakanishi1，Takako Terui1, 2，Fuyu Kobirumaki1，Norio Fukuda（

Role of orexin neurons during social defeat stress and descending
projections from the hypothalamus in the rat
Engineering, Toyo Univ, Japan）

It is known that the hypothalamus plays an important role in the cardiovascular response
evoked by psychological stress. Orexin (ORX) neurons are localized within the dorsomedial
hypothalamic area (DMH) and the perifornical area (PeF). Therefore, it is suggested that the
ORX neurons are involved in the stress induced-cardiovascular response. However, a neuralnetwork of the stress response is still unknown. In the present study, we investigated the roleof the ORX neurons on the cardiovascular response evoked by social defeat stress (SDS) and
its descending projection from the hypothalamus in conscious rats. A telemetry probe was
implanted into an experimental animal to measure blood pressure (BP) and heart rate (HR),and the rat was exposed to a single SDS (SSDS) or repeated SDS (14 days). In addition, somerats were microinjected anterograde neural tracer, BDA and then exposed to the SSDS. After
both SDS challenges, the brains were removed and stained c-Fos, ORX and BDA. BP and HR
were significantly increased during both SDSs. After the repeated SDS challenge, however,baselines of BP and HR did not change. The number of c-Fos immunoreactive neurons in the
DMH and the PeF increased after both SDSs. In contrast, the number of ORX neurons in theseareas did not change in both SDSs. The percentage of c-Fos-expressed ORX neurons in the
DMH profoundly increased (~40%) in the repeated SDS group compared to that of the SSDS(~20%). The nerve terminals from the DMH were observed in the midbrain ventrolateral (vl)periaqueductal grey (PAG) and the medullary raphe. These results suggest that the ORX
neurons in the hypothalamus play a crucial role in the cardiovascular response evoked by the
SDS, and that the stress response is mediated via neurons in the vlPAG and the medullary(COI:No)raphe.

1P-062

Role of P2Y signals in generating spontaneous contractions in theguinea pig seminal vesicles
Mitsue Takeya1，Hikaru Hashitani2，Ryuhei Higashi3，Kei-ichiro Nakamura4，
1 1 Dept Physiol, Kurume Univ Sch Med., Kurume, Japan, 2 Dept Cell
Makoto Takano（Physiol, Grad Sch Med Sci, Nagoya City Univ, Nagoya, Japan, 3 Advanced Imaging
Research Center, Kurume Univ Sch Med, Kurume, Japan, 4 Dept Anat, Kurume Univ Sch
Med, Kurume, Japan）

In seminal vesicles (SVs) of the guinea pig, the mucosa is essential for generating spontaneous
phasic contractions and corresponding electrical and Ca2+ activity in SV smooth muscles.
Recently, we have reported that subepithelial synchronous interstitial cells (SSICs), distributedin the lamina propria of SV mucosa, generate spontaneous electrical slow waves to trigger
synchronous Ca2+ transients in SV smooth muscles. Dye-coupling between SSICs and SV
smooth muscles were also demonstrated, suggesting that SSICs function as pacemaker cells todrive the spontaneous contractions by sending depolarising signals via gap junctions. Here we
further explored whether purines released from mucosal cells are involved in the SV spontaneous contractions using fluorescent Ca2+ imaging and immunohistochemistry. In mucosa-intact
preparations, SV smooth muscles developed spontaneous Ca2+ flashes and associated contractions at 3.9 ± 0.6 min－1. The frequency of the SV spontaneous contractions were reduced bypartial removal of epithelium but restored by 10 µM ADP (1.8 ± 1.1 min－1 in control perfusate,vs 3.1 ± 1.5 min－1 in ADP, p<0.05). In contrast, mucosa-denuded preparations, SV smoothmuscles were quiescent and failed to contract in response to ADP, suggesting that P2Y signals
within the SV mucosa play a role to accelerate the spontaneous contractions. In the SV mucosa,
SSICs were distributed just beneath the layers of columnar and basal epithelial cells. SSICs andboth types of epithelial cells were immunopositive for P2Y1. 10 µM ADP or 0.1 µM MRS2365, a
P2Y1 agonist, evoked Ca2+ transients in the basal cells and SSICs. However, 1-10 µM MRS2500,
a P2Y1 antagonist, and additional application of suramine, a non-selective P2 antagonist, did not
suppress the generation of either Ca2+ transients in SSICs or SV spontaneous contractions.
These results indicated that purines play a role to facilitate mucosa-dependent SV contractions.
(COI:No)

Effects of omecamtiv mecarbil on the contractile properties of skinned
porcine left atrial and ventricular muscles
Physiol, Jikei Univ, Japan, 2 Dept Anesth, Jikei Univ, Japan）

Background: Omecamtiv mecarbil (OM) is a compound that has been developed to treat
systolic heart failure via targeting cardiac myosin to increase myocardial contractility. The effects, however, have not been systematically investigated in muscles with different expression
profiles of myosin heavy chain (MHC).
Methods and Results: Active force was measured with skinned porcine left atrial (LA; ~100%
α-MHC) and ventricular (LV; ~100% β-MHC) fibers. Sarcomere length was set at 2.1 µm. OM
left-shifted the mid-point of the force-pCa curve (ΔpCa50) by 0.07 and 0.25 pCa units in LA at 0.5
and 1.0 µM, respectively. The magnitude of the shift of ΔpCa50 significantly more pronounced
in LV, in that the values were 0.17 and 0.32 pCa units at 0.5 and 1.0 µM, respectively. In both
LA and LV, the rate of rise of active force (t1/2) became slower in the presence of OM, with themagnitude greater in LV; i.e., ~5% and ~10% at 0.5 and 1.0 µM in LA, as compared to ~20%
and ~30% at 0.5 and 1.0 µM in LV. Finally, we investigated the effects of inorganic phosphate(Pi) on maximal force at pCa 4.5. In LA, Pi decreased maximal force in an inverse-sigmoidal
manner with and without OM up to 20 mM. However, in LV, OM attenuated the depressanteffect of Pi in a concentration-dependent manner.
Conclusions: OM increased Ca2+ sensitivity in both LA and LV, with the effect more pronounced in LV. Provided that OM slowed t1/2 by a greater magnitude in LV than in LA andthe compound attenuated the depressant effect of Pi only in LV, we conclude that OM exerts
its Ca2+-sensitizing effect in cardiac muscle in a MHC isoform-dependent manner.  (COI:No)

1P-065

Transcriptome in fast- and slow-twitch ﬁbers of zebraﬁshSouhei Sakata, Fumihito Ono（Dept Physiol, Faculty Med, Osaka Medical College, Japan）
Skeletal muscle fibers are mainly divided into two different types; fast-twitch fiber and slowtwitch fiber. Fast-twitch fibers generate strong power and are used for bursts of movements
such as sprinting, whereas slow-twitch fibers contribute to long-endurance exercises such as
distance running. A difference of gene expressions in these two types of fibers associated withmyosin activities and metabolism has been reported. Here we performed a comprehensive
gene expression analysis using the next-generation sequencing (NGS) technique in adult zebrafish. We used a transgenic line expressing the red-fluorescent protein, mCherry, to identify
slow-twitch fibers that forms a thin layer in fish trunk. Our sequencing yielded 4.8 million and
4.6 million reads, and 89.71% and 96.87% reads were mapped to the genome for the fast- andslow-twitch fiber, respectively. Gene ontology analysis showed that genes involved in cellularcomponent organization, cytoskeleton organization and oxidation-reduction process among
others were significantly different between two fiber types. Kyoto Encyclopedia of Genes andGenomes (KEGG) analysis showed that gene expressions associated with multiple pathways,
including cell cycle, regulation of actin cytoskeleton and calcium signaling pathway weresignificantly different. We found that many genes, including myosin VI and VII, slow myosin
heavy chains, myoglobin, ryanodine receptor Ia were specifically expressed in the slow-twitch
fiber. On the other hand, genes such as parvalbumins, fast muscle-type troponins or ryanodine
receptor III showed strong expression in the fast-twitch fibers. Our NGS data is expected to(COI:No)
uncover molecular mechanisms that characterize fast- and slow-twitch fibers. 

1P-063

1P-066

Yuttamol Muangkram（Dept Bioinformatics, Life Sciences, Ritsumeikan Univ, Japan）

Ippei Yamato, Tsuyoshi Fukuzawa, Shuichi Soeda, Tetsuro Tamaki（Muscle Physiol ＆ Cell

Mathematical model to understand the exercise-induced skeletal
muscle fatigue during intense exercise
During intense exercise activity, it leads to a drop in maximal voluntary contraction or theinability of contractile and metabolic processes of contracting muscle to sustain the desired
work output defined as muscle fatigue. It is widely accepted that a close relationship between
an increase in lactate concentration and a decrease in pH, and the accumulation of muscle
inorganic phosphate have been regarded as the major causes of skeletal muscle fatigue. Intense
exercise greatly activates ATPase activity and promotes ATP production, thus leading to analteration of the metabolic by-products. However, the biological mechanisms underlying thedevelopment of muscle fatigue remain not fully understood. In this study, we aimed to construct a basic mathematical model which can reproduce the biological processes of metabolic
fatigue in a variety of exercise intensity which is consistent with known experimental data.
The novel model captures the key biological reactions including; 1) the role of creatine kinase
reaction in maintaining ATP homeostasis, 2) the role of bicarbonate buffer system in regulating
blood pH, and 3) the characteristic of metabolic alteration at transition state. Moreover, we
revised our previous hybrid contraction model by introducing the inhibitory effect of metabolic
by-products based on the structural and experimental data. The accumulation of metabolic
by-products reduces the number of attached cross-bridge enhancing the reduction in maximal
contraction. In conclusion, the current model which consists of reliable biological fluxes andmetabolic concentration in good agreement with experimental results can provide a better
understanding of metabolic fatigue during intense exercise. For further study, the combinationof the current model with other mechanism-related fatigue, e.g. the shortage of substrates, may(COI:No)
help elucidate the nature of metabolic exercise-induced metabolic fatigue.

S102

Decreased response of muscle hypertrophy and the expression ofmuscle atrophy-relating factors in the sarcopenia muscle
Biol Unit, Tokai Univ. Japan）

The changes of skeletal muscle atrophy-relating genes were examined in the progressing
sarcopenia muscle in old rat after the strong stimulation of muscle hypertrophy. For thispurpose, the surgical ablation (SA) of the synergistic muscles (gastrocnemius and soleus
muscles) was performed on the right hindlimb, and hypertrophic stimulation was added to theplantaris (PLT) muscles using old (over 2.5-years-old, n=9) and normal adult (10-15-week-old,n=9) male Sprague-Dawley rats. Contralateral-left side was preserved as a control. After 10days of the SA, both side PLT muscles were excised and weighed, then, the progressing
sarcopenia was confirmed by the standard co-relation curve of the body-PLT muscle weight,
which was obtained by over 500 rats at the left control PLT. Expressions of Myogenin,
Atrogin-1 and MURF1 were analyzed by real time qPCR based on the control. Significant
increase of Myogenin (2.5-fold) was observed in the adult group, but there were no changes
detected in remaining two factors. Thus, this the muscle hypertrophy depending increase of
myogenin expression. However, in the old group, quite higher increase (33-fold) of myogenin
was detected associated with the slight increase in Atrogin-1 (1.3-fold) and MURF-1 (1.8-fold).
Interestingly, the old group showed 28-fold increase of Myogenin even in the contralateralcontrol. These results showed that the sarcopenia muscle basically showed an extremely highlevel of Myogenin in the resting state, and this was further accelerated by the hypertrophic
stimulation. However, this is not the muscle hypertrophy depending increase, because of thefollowing increase of Atrogin-1 and MURF-1. This result further supported by the result of ourtime-course study that the compensatory muscle hypertrophy did not occur in the sarcopenic(COI:No)muscle in the old rat after 5 weeks.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

1P-067

A possible of gastric inhibitory polypeptide in the regulation of skeletal
muscle mass
Katsumasa Goto1, 2，Megumi Aoshima1，Arisa Hagiwara1，Tomohiro Yamashita1，
2 1 Dept Physiol, Grad Sch HealthHuascar Pedro Ortuste Quiroga P.1，Shingo Yokoyama（
Sci, Toyohashi SOZO Univ, Japan, 2 Dept Health Sci, Toyohashi SOZO Univ）

Various interorgan communication networks of proteins, peptides, and metabolites have been
proposed. A member of incretin gastric inhibitory polypeptide (GIP), which is synthesizedin and secreted from K cells, regulates nutrient absorption in a gastrointestinal tract. GIP
receptor (GIPR) expresses in not only a gastrointestinal tract but also β cells in the pancreas.
Recently, the expression of GIP in skeletal muscle cells is also confirmed. However, the interorgan communication network between a gastrointestinal tract and skeletal muscle remains
unclear. In the present study, we investigated a physiological role of GIP in the regulationof the skeletal muscle mass using mouse myoblast-derived C2C12 cells. GIPR expressed inboth C2C12 undifferentiated myoblasts and differentiated myotubes. Administration of GIP
stimulates myogenic differentiation of C2C12 cells. Knockdown of GIPR suppressed the mRNA
expression level of MyoD and Pax7 and myogenic differentiation of C2C12 cells. On the other
hand, GIPR-knockdown stimulated proliferation of C2C12 myoblasts. Evidences indicates GIPRassociated intracellular signal(s) play a regulatory role in skeletal muscle mass by maintaining
myogenic differentiative potential. This study was supported, in part, by KAKENHI (Grant
Numbers JP16K13022, JP17K01762, JP18H03160), the Science Research Promotion Fund fromthe Promotion and Mutual Aid Corporation for Private Schools of Japan, and Graduate Schoolof Health Sciences, Toyohashi SOZO University.
(COI:No)

1P-070Behavioral study on nausea induction by serotonin agonists in rats
Jun Sanefuji, Yoshiyuki Hirai, kazunari Hisadome, Mayu Fujita, Shaoyi Su, Nanae Yamazaki,
Makoto Funahashi（Dept. Oral Physiol, Grad Sch Dent, Hokkaido Univ, Japan）
In previous studies to determine whether nausea is induced by serotonin agonists in rats, conflicting results have been shown (Rudd, 1998; Higgins, 1993; Miller and Nonaka, 1992). To clarify
the conflict, we investigated the effect of serotonin agonists as an unconditioned stimulus (US)on the CTA to saccharin. Male Wister rats weighing 200 to 350 g at the start of the study wereused. 0.1% saccharin sodium solution was used as conditioned stimulus (CS). 1-phenylbiguanide(1-PBG; 30 mg/kg i.p.), meta-chlorophenylbiguanide (mCPBG; 10 mg/kg i.p.) or 0.3M LiCl (0.1%
body weight i.p.) were used as US. The volume of saccharin intake was compared before andafter applying the US. CTA experiments were repeated twice in each animal. In the first trialof CTA experiment with 1-PBG or mCPBG, saccharin consumption was slightly decreased but
the acquisition of CTA was not statistically significant (n=5). In the animals that once experience LiCl-induced CTA at the first trial, the second trial of 1-PBG injection significantly induced
CTA to saccharin (n=5). These results suggest that the serotonergic mechanism for inductionof nausea may be boosted by preceding nausea experience. In any case, the nausea-inducing
effect of serotonin agonists is not as strong as LiCl, but it was found to be effective enough to
induce nausea in rats that once experienced nausea induction. The present study may lead tounderstanding anticipatory nausea and vomiting during the repetitive chemotherapy.(COI:No)

1P-068

1P-071

Kinji Kurihara（ Div Physiol, Sch of Dent, Meikai Univ, Japan）

Univ Sch Dent at Matsudo, Japan）

Amylase in saliva is used as a stress marker, but amylase is partially questioned as a stressmarker because it is secreted by various stimulations without stress. MicroRNAs are small
non-coding RNAs of 18-28 nucleotides. We examined a new diagnostic method of stress exhaustion using microRNAs in salivary glands as bio-markers.
We investigated microRNA expression patterns in the salivary glands of ICR mice and identified 39 microRNAs. The expression patterns of these microRNAs in mice treated with various
hormones were analyzed by quantitative real-time PCR.
When adrenaline was administered for 12 days using a micro osmotic pump (Alzet, Model 1002)
continuously and the sympathetic nerve alone was enhanced for a long period, the microRNA
did not change. It is well known that anti-stress hormones (glucocorticoids) are secreted when
the hypothalamus recognizes chronic stress.
When dexamethasone was administered to normal male mice, miR-29b-3p was increased andlet-7c-3p was decreased significantly. Dexamethasone administration to castrated mice showeda more significant effect. These results suggest that glucocorticoids have an additive effectwhen androgens are depleted as PADAM.
Administration of dihydrotestosterone to castrated mice increased miR-21-5p and miR-141-3p
in salivary glands, however had no effect for miR-29b-3p and let-7c-3p.
Dexamethasone administration increased miR-16-5p and miR-451a in serum significantly. Thechange of miRNA in salivary gland by dexamethasone was not correlated to one in serum.
In conclusion, enhancement of sympathetic nerve alone does not change the microRNA patternin salivary gland. Changes in the expression pattern of miR-21a-5p and miR-141-3p are affectedby androgen, whereas let-7c-3p and miR-29b-3p are not. Thus, an increase in the ratio of
miR-29b-3p to let-7c-3p is a selective biomarker of stress via the hypothalamus. Furthermore,unlike amylase, it can be distinguished stress exhaustion from sympathetic nerves acceleration.
(COI:No)This works was supported by JSPS KAKENHI Grant Number 18K19757

Purpose: Secretory granules (SGs) are generated from the Golgi complex. After the generation,
newly-formed SGs become a mature SGs by homotypic fusion, acidification, condensation andmembrane remodeling. It is generally considered that these process is required to acquire thecapacity of stimulus dependent secretion. Here, we examined whether newly-formed SGs havean ability of the secretion.
Material＆Methods: Isoproterenol (IPR) was injected (5mg/kg) into rat abdominal cavity todeplete pre-existing SGs. Newly-formed SGs were purified by Percoll centrifugation. Acinar
cells were dispersed from parotid glands by treatment with collagenase and hyaluronidase.
After stimulation with 1 µM IPR, secretions of amylase and procathepsin B (proCB) into incubation buffer were measured by immunoblot analysis.
Results＆Conclusion: We observed that newly-formed SGs were generated at 5 hours after
injection of IPR. After stimulation with 1 µM IPR, depletion of newly-formed SGs from parotid
gland was observed by electron microscopy. The syntaxin 6 was concentrated in membraneof newly-formed SGs. Because syntaxin 6 is marker of immature SGs, it is supposed the
exocytosis of newly-formed SGs. Moreover, secretion of amylase from newly-formed SGs was
detected in incubation buffer after stimulation with 1 µM IPR for 10 min. And proCB was also
detected in the buffer. But the matured cathepsin B was not detected because proCB activatesafter transport to lysosome. Our results suggests that the newly-formed SGs already has anability of the secretion, and that the aim of membrane remodeling of SGs is the other function(COI:No)
except for acquirement of secretion capacity. 

1P-069

1P-072

Sadao Ohyama1, 2，Suzuro Hitomi3，Asuka Higashikawa1，Wataru Ofusa1，Haruna Toda1，
Sobhan Ubaidus1，Maki Kimura1，Daisuke Nishida4，Toshihide Mizoguchi4，Kentaro Ono3，
1 1 Dept Physiol, Tokyo Dent Coll, Japan, 2 Oral surg Tokyo Metro
Yoshiyuki Shibukawa（3 1 Department of Information andTaichi Akie1，Hideaki Seki2，Hiroko Toshima（Communication Systems Engineering, Student, Chiba Institute of Technology, 2 Department
of Information and Communication Systems Engineering, Chiba Institute of Technology,
3 Department of Food and Nutrition, Toita Wemen’s College）

Diagnosis of stress exhaustion during induction of anti-stress hormonesby hypothalamic response to chronic stress using mouse salivary glandmicroRNAs as biomarkers

Neural communication between odontoblasts and pulpal neurons in
dentinal pain

Komagome Hosp, Japan, 3 Dept Physiol, Kyushu Univ Coll, Japan, 4Oral Health Sci Cent,Tokyo Dent Coll, Japan）

The teeth are composed of enamel, dentin, cementum and dental pulp. Among dental pain,
dentinal pain is caused by the dentinal fluid movement by the various stimuli applied on thedentin surface. Dentinal fluid movement activates mechanosensitive TRP/Piezo channels inodontoblasts, and subsequent intracellular Ca2+ signals elicits release of ATP from the pannexin channel. The released ATP activated the P2X3 receptor in pulpal neurons to generate
and propagate action potentials to generate dentinal pain (Odontoblast hydrodynamic receptor
theory). In the present study, we analyzed rat nociceptive behaviors to examine whether theseneural communication between odontoblasts and neurons was necessary to induce dentinal
pain or not. We prepared rat model which is enable to induce dentinal pain by dentin exposureon the mandibular incisors (pain group). For the pain group rats, we applied a bonding agent
on dentin surface, or administrated antagonists for the piezo channel, P2X3 receptor andpannexin-1 channels. We evaluated nociceptive scores upon cold water stimulation of dentin
surface of the incisors. The nociceptive score was significantly increased in the pain grouprats, while the scores were decreased in the rat with bonding agent treatment as well as in
the rat administrated by the various channel/receptor antagonists. These in Vivo experimental
results indicated that neurotransmission between odontoblasts and neurons is necessary for(COI:No)the development of dentinal pain. 

Secretion of amylase and procathepsin B from newly-formed secretory
granules in rat parotid acinar cells
Osamu Katsumata-Kato, Megumi Yokoyama, Junko Fujita-Yoshigaki（Dept Physiol, Nihon

Evaluation of swallowing function by surface electromyography
recorded by handy electromyographPurpose: Surface electromyography is on of methods for non-invasively evaluating swallowing
function. We had already reported swallowing function evaluation method using a 4-channel
surface electromyogram. We thought that the electromyograph which we had used wasdifficult to use for elderly people at home care. Therefore, we have developed a method forrecording the surface electromyogram during swallowing using a handy type electromyographand measuring the swallowing function index automatically.
Subjects and Method: Subjects were 12 young healthy male (21.7 ± 0.5 years old). Surface
electrode was affixed on the suprahyoid muscle group, a ground electrode was affixed on
the seventh cervical spinous process, and a surface electromyogram during swallowing wasrecorded with Neuropack n1. 3 ml of water, hot water, carbonated water, and jelly-type drink
were used as load drink. Using the software developed independently, the amplitude and peaklatency of the recorded swallowing surface electromyogram were automatically measured.
EMG was recorded 5 times for each beverage, and the average of the 5 measurements wasused as a swallowing function index.
Result: The average peak latency of all subjects when swallowing cold water, carbonatedwater, hot water, and jelly was 473.6 ± 164.0, 433.9 ± 193.3, 450.6 ± 208.3, 666.1 ± 194.4 (msec),and latency that induced by jelly-type drink was delayed significantly (p = 0.016).
The amplitudes were 31.9 ± 10.9, 31.5 ± 9.1, 34.4 ±16.0, and 31.2 ± 8.5 (μV), respectively, andthere was no significant difference among the 4 groups.
Conclusion: It was found that jelly-type drinks are drinks that require long time for swallowing. We thought this was related to aspiration prevention. Since young people did not find any
difference in other drinks, we would like to study the elderly next time.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S103

1P-073

A novel nucleic acid analogue COA-Cl enhances glucose-dependent
insulin secretion
Ikuko Tsukamoto1，Akram Hossain1，Maki Takata1，Liu Wenhua2，Katsuya Hirano1，
1 1 Fac Med, Kagawa
Yasuo Kubota1，Junsuke Igarashi3，Masaaki Tokuda4，Ryoji Konishi（

Univ. Japan, 2 Chinese Academy of Sciences, 3 Morinomiya Univ. of Med. Sci., 4 International
Office, Kagawa Univ.）
COA-Cl is a synthesized nucleoside analogue with the molecular weight of 284. We previously
reported that COA-Cl has angiogenic potency with the promotion of the synthesis and secretion of VEGF. COA-Cl also exhibits neurotrophic/neuroprotective property.
In this study, we explored the effects of COA-Cl on insulin secretion both in vitro and in vivo.
In mouse insulinoma cells (MIN6), COA-Cl enhanced the secretion of insulin only under thehigh-glucose condition while a typical diabetes therapeutic drug Glibenclamide(SU) enhanced
the insulin secretion regardless of glucose concentration. COA-Cl also enhanced the glucoseinduced Ca2+ influx in MIN6. Further, enhancement of the glucose-dependent insulin secretion
was detected with rat pancreatic islet culture.
As for the in vivo study, we performed the OGTT (oral glucose tolerance test) using normal
rats. Increase in insulin concentration in blood by COA-Cl was observed, however the effects
of COA-Cl on blood sugar were unclear. Pharmacodynamics/kinetics and other physiologicaleffects of COA-Cl should be investigated in future. (COI:No)

1P-074

Ni+-sensitive Ca2+ channels are involved in the exocytotic secretionfrom somata/dendrites in vasopressin neurons
Kaori Sato-Numata1, 2，Tomohiro Numata1，Yoichi Ueta3，Yasunobu Okada4, 5（ 1 Dept Physiol, Sch Med, Fukuoka Univ, Fukuoka, Japan, 2 Japan Society for the Promotionof Science, Tokyo, Japan, 3 Dept Physiol, Sch Med, Univ Occupat and Environ Health,
Kitakyushu, Japan, 4 Dept Physiol, Kyoto Prefect Univ Med, Kyoto, Japan, 5 Natl Inst
Physiol Sci, Aichi, Japan）
Arginine vasopressin (AVP) is an important hormone that maintains plasma osmotic homeostasis. It is synthesized by AVP-producing neurons (AVP neurons) located in the hypothalamus,transported to the axon terminals located in the posterior pituitary gland, and then secretedby exocytosis into the systemic circulation. It has been reported that the Ca2+ influx via a
voltage-gated Ca2+ channel (VGCC) is important for exocytotic secretion from axon terminals.
It is known that AVP is secreted not only from the axon terminal but also from the somata/dendrites to the brain stroma. AVP secreted from somata/dendrites was significantly increased by hyperosmotic stimulation. This secretion was almost completely suppressed by an
exocytosis inhibitor tetanus toxin. In addition, somato-dendritic AVP secretion was significantly
suppressed in the presence of Ni+, which is a non-specific Ca2+ channel inhibitor. The Ca2+
channel currents measured in AVP neurons by the patch-clamp technique were sensitive to
Ni+. The currents were also suppressed by an N-type VGCC blocker ω-CgTx and a T-type
VGCC blocker NNC 55-0396. These results suggest that VGCCs, including T-type and N-type
VGCCs, are involved in the mechanism of AVP secretion not only from axon terminals into thesystemic circulation but also from somata/dendrites into the brain stroma by hyperosmotic(COI:No)stimulation.

1P-075

1P-076

Changes in gene expressions of hypothalamic feeding-related
neuropeptides in the streptozotocin-induced diabetic rats with variable
hyperglycemia and hyperphagia
Satomi Sonoda1, 2，Kazuhiko Baba1，Kenya Sanada1，Kazuaki Nishimura1，
Haruki Nishimura1，Kentaro Tanaka1，Mitsuhiro Yoshimura1, 3，Hiromichi Ueno1，
1 1 Dept Physiol, Sch
Takashi Maruyama1，Yosuke Okada2，Yoshiya Tanaka2，Yoichi Ueta（

Med, UOEH, Japan, 2The First Dept of Inter Med, Sch Med, UOEH, Japan, 3Translational
Health Sciences, Sch Med, Univ of Bristol, UK ）Streptozotocin (STZ), which destroys beta-pancreatic cells, is widely used to develop diabetic
animal models. Here, we examined the relationship between expression of the genes encoding
hypothalamic feeding-related neuropeptides in the paraventricular nucleus (PVN) and thearcuate nucleus (ARC) and plasma glucose levels in STZ-administered adult male Wistar rats
exhibiting variable plasma glucose levels. STZ (80 mg/kg) was administered intraperitoneally(i.p.) in adult male Wistar rats. Rats were divided into 3 groups: PG1 (<300 mg/dl at lightperiod), PG2 (≧300 mg/dl at light period and <200 mg/dl after fasting for dark period) and PG3(≧300 mg/dl at light period and >200 mg/dl after fasting for dark period). Two weeks after
i.p. administration of STZ, they were decapitated after fasting for 12 hours. The gene expressions of proopiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART),
neuropeptide Y (NPY), agouti-related protein (AgRP) in the ARC, corticotrophin releasing
hormone (CRH), thyrotropin-releasing hormone (TRH) in the PVN were quantified by using insitu hybridization histochemistry. POMC and CART were significantly decreased in PG2 andPG3 compared to PG1. On the other hand, NPY, AgRP and TRH were significantly increasedin PG3 but not PG2 compared to PG1. CRH was comparable among all groups. The gene
expressions of orexigenic neuropeptides was correlated with the severity of hyperglycemia. In
contrast, the gene expressions of anorexigenic neuropeptides such as POMC and CART was
significantly decreased, exacerbating hyperglycemia. These results suggest that hyperphagia
in STZ-induced diabetic rats may be caused by dynamic regulation of hypothalamic feeding(COI:No)
related neuropeptides associated with plasma glucose levels.

1P-077

Effects of the gene expression of hypothalamic feeding-regulatingneuropeptides after being exposed to different gravity in mice via
vestibular inputs
Yoichi Ueta1，Satomi Sonoda1，Mitsuhiro Yoshimura1, 2，Kazuhiko Baba1，Kenya Sanada1，
Kazuaki Nishimura1，Haruki Nishimura1，Kentaro Tanaka1，Hiromichi Ueno1，
3 1 Dept Physiol, Sch Med, UOEH,
Takashi Maruyama1，Chikara Abe3，Hironobu Morita（
Japan, 2Translational Health Sciences, Sch Med, Univ of Bristol, UK, 3 Dept Physiol, Grad
Sch Med, Gifu Univ, Japan）

The effects of hypergravity on the gene expression of the hypothalamic feeding-related neuropeptides in sham operated and vestibular-lesioned mice were examined by in situ hybridization
histochemistry. Vestibular lesion was conducted by a laser. Adult male C57BL/6J mice werereceived sham operation (Sham) or vestibular lesion (VL) before the experiment. After therecovery, they were divided into 4 groups: Sham-1g, VL-1g, Sham-2g, and VL-2g (n=6 ineach) and exposed to 1g or 2g environment with centrifugation of custom-made gondola-typerotating box for 3 days, 2 weeks, and 8 weeks. At the end of the each time point, they were
decapitated. The gene expression of the corticotrophin releasing hormone (CRH) in the paraventricular nucleus (PVN), propiomelanocortin (POMC), cocaine- and amphetamine-regulatedtranscript (CART), neuropeptide Y (NPY), agouti-related peptide (AgRP) in the arcuate nucleus(ARC), melanin-concentrating hormone (MCH), and orexin in the lateral hypothalamic area
(LHA) were quantified by using in situ hybridization histochemistry. VL did not affect allneuropeptides which we investigated at each time point. CRH in the paraventricular nucleus
was increased significantly in Sham but not in VL mice after 3 days of exposure to a 2g
environment compared with a 1g environment. Significant decreases in POMC and CART
and significant increases in NPY, AgRP and orexin were observed in both Sham and VL
mice. After 2 weeks of exposure, CRH and POMC were increased significantly in Sham but
not in VL mice. After 8 weeks of exposure, the hypothalamic feeding-related neuropeptides
were comparable between Sham and VL mice. These results suggest that the hypothalamic
feeding-related neuropeptides may be affected during the exposed duration of hypergravity via(COI:No)
vestibular inputs.

1P-078

Hypothalamic oxytocin is signiﬁcantly up-regulated after acute osmotic
challenge and acute hypovolemiaSize-selective accumulation of MR tracers in the kidney of the mussel
Mytilus galloprovincialis

Hiromichi Ueno1，Mitsuhiro Yoshimura1, 3，Kenya Sanada1, 2，Takashi Maruyama1，
1 1 Dept physiol, Sangyoika Univ, Fukuoka,
Tetsu Miyamoto2，Yutaka Otsuji2，Yoichi Ueta （Yoshiteru Seo1，Hidefumi Wakashin1，Yoshie Imaizumi-Ohashi1，Mika Yokoi-Hayakawa1，2 1 Dept Regl physiol, Dokkyo Med Univ Scl Med, Tochigi, Japan, 2 Div Marine
Eriko Seo（

Japan, 22nd internal medicine, Sangyoika Univ, Fukuoka, Japan, 3Translational Health

Sciences, Bristol Medical School, University of Bristol, UK ）Background: We generated a transgenic rat line that expresses oxytocin (OXT)-monomeric
red fluorescent protein 1 (mRFP1) fusion protein to visualize the dynamics of OXT. In thistransgenic rat line, hypothalamic OXT can be assessed in diverse physiological and pathophysiological conditions by semi-quantitative fluorometry of mRFP1 fluorescence intensity as
a surrogate marker for endogenous OXT. Hypothalamic vasopressin is known to be increased
after acute osmotic challenges and acute hypovolemia, whereas the dynamics of OXT after
these stimuli are unknown.
Method: The OXT-mRFP1 transgenic rat line was used to observe mRFP1 fluorescent intensity in the supraoptic (SON) and paraventricular nuclei (PVN) in perfusion-fixed brains at 0, 3,
6, 12, and 24h after acute osmotic challenge and acute hypovolemia induced by intraperitoneal(i.p.) administration of 3% hypertonic saline (HTN) and polyethylene glycol (PEG), respectively.
Rats in the experimental groups, but not the control group, were food and water restricted
during the experiment. Fos expression, a marker for neuronal activity, was also assessed byusing immunohistochemistry. We also analyzed the gene expression of OXT and mRFP1
in the SON and PVN at 3 and 6h after i.p. administration of HTN and PEG by using in situ
hybridization histochemistry.
Result: mRFP1 fluorescence intensity in the SON and PVN were significantly increased at 3
and 6h after i.p. administration of HTN and PEG along with robust Fos-like immunoreactivity(co-expression). OXT and mRFP1 gene expression was dramatically increased at 3 and 6h after
i.p. administration of HTN and PEG.
Conclusion: Acute osmotic challenge and acute hypovolemia induced activation of OXT
neurons and increased gene expression of OXT in the SON and PVN. These results suggestthat not only endogenous vasopressin, but also endogenous OXT, has one of the key roles formaintaining body fluid homeostasis to cope with hyperosmolality and hypovolemia. (COI:No)

S104

Life Sci, AORI, Univ Tokyo）

In order to determine the molecular weight dependency of atrial wall filtration into kidneys ofthe mussel Mytilus galloprovincialis, we applied 5 magnetic resonance (MR) tracers: manganeseion (Mn2+; 0.055 kDa), gadolinium ion (Gd3+; 0.16 kD), manganese-ethylenediaminetetraacetic
acid (MnEDTA; 0.35 kDa), gadolinium-diethylenetriamine pentaacetic acid (GdDTPA; 0.55 kDa),
and oligomer-based contrast agent (CH3-DTPA-Gd; 2.1 kDa). After a bolus injection of the
MR tracers into the visceral mass, T1-weighted MR imaging (T1w-MRI) and the longitudinalrelaxation rates (1/T1 = R1) were measured by 7 T MRI at 20℃. One hr after injection, theMR tracers distributed uniformly in the visceral mass. The increase of R1 of kidney (R1K)
was detected by group of injection of Mn2+ and MnEDTA, and urine concentrations were
estimated at 210 and 65 µM from R1K, respectively. No increase of R1K were detected by
GdDTPA and CH3-DTPA-Gd injected mussel. When the mussels were additionally incubatedin seawater with 10 µM MnCl2, R1K was increased in the GdDTPA-injected mussel, but not in
the GdCl3-injected mussel. Therefore, GdDTPA does not interfere renal accumulation of Mn2+,
but, Gd3+ might have inhibited. Since incubation in seawater with 10 µM MnEDTA showed noincrease in the R1K, it is suggested that injected MnEDTA was filtrated as MnEDTA per se,
and not likely separated into free Mn2+. Thus, we concluded that the molecular weight cut-off(MWCO) of the atrial wall of the Mytilus galloprovincialis is around 0.5 kDa, which is almost
1/100 of that for vertebrate animals. In the vertebrate, it is considered that the slit diaphragm
formed by nephrin controls the MWCO for filtration by the glomerulus. We are not surenephrin can cover the wide range of the MWCO. Therefore, it is necessary to investigate themolecular-barrier mechanism in the atrial wall of Mytilus.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

1P-079

1P-082

Taiki Nishizuka1，Junichi Taniguchi2，Akinori Noma1，Akira Amano1，Yukiko Himeno1，
1 1 DeptBioinf, Col Life Sci, Ritsumeikan Univ, Shiga, Japan, 2 Mol.Pharmacol.
Taiki Tahara（

Yoshihide Satoh, Kojun Tsuji（Dept Physiol, Nippon Dent Univ Niigata, Japan）

Quantitative analysis of epithelial transport in proximal tubule withmathematical modelMed. Jichi Med. Univ., Tochigi, Japan）

Proximal tubule (PT) is known to reabsorb about 2/3 of filtered Na+, K+ and water. This factraises several questions about the mechanisms underlying their transport across proximal
tubular wall. The transcellular reabsorption of Na+ is coupled with partner solutes such as
glucose, HCO3-, etc. Total amount of partner solutes in filtrate is at most 40 mM × glomerular
filtration rate (GFR), which is not enough to reabsorb the 2/3 of filtered Na+, i.e., 2/3 × 140
mM × GFR. About 2/3 of filtered K+ is also reabsorbed along the PT against the driving
force generated by Na+, K+-ATPase in basolateral membrane. These contradictions cannot
be explained by assuming only transcellular reabsorption of Na+ and K+. In addition, epithelial reflection coefficients of Na+ and K+ are not very high, though aquaporins, transcellularpathways of water, are highly selective to water. This fact suggested that the water should bereabsorbed via a pathway not very highly selective to water, probably paracellular pathway. In
order to reveal the roles and mechanisms of paracellular reabsorption, we built a mathematical
PT model that enabled us to analyze the transcellular and paracellular transport separately.
Assuming that water passes at tight junction (TJ), our model analysis predicted that highosmolarity in lateral space between neighboring epithelial cells drove much water reabsorption via paracellular TJ water pathway. This water reabsorption was accompanied by K+
solvent drag. In addition to this, the K+ should be electrically reabsorbed at the latter sectionof PT where luminal potential was positive to interstitium. Paracellular backflow of partner
solutes coupled with transcellular Na+ reabsorption accelerated their recycling, which might(COI:No)be important to reabsorb enough amount of Na+.

1P-080Inhibitory Effect of Acupuncture on Long Latency Reﬂex in Humans
Akira Nihonmatsu（Hokkaido College of Oriental Medicine）

Suppression of the swallowing reﬂex by stimulation of thepedunculopontine tegmental nucleus
It has been reported that the swallowing central pattern generator (CPG) is divided into twoparts in the medulla; a dorsal area including the nucleus of the solitary tract and a ventral area
corresponding to the reticular formation around the nucleus ambiguus. Morphological study
has reported that pedunculopontine tegmental nucleus (PTg) projects to the latter area. It istherefore likely that the PTg is involvement in control of swallowing. This study examines
whether the swallowing reflex is modulated by stimulation of the PTg. These experiments
were performed on rats anesthetized by urethane (1.3 g/kg, i.p.). Electromyograms wererecorded from the mylohyoid muscle to identify swallowing event. The swallowing reflex
was evoked by electrical stimulation (0.2 ms duration, 30 Hz) of the superior laryngeal nerve(SLN). Repetitive electrical stimulation applied to the pedunculopontine tegmental nucleus(PTg) (0.2 ms duration, 30 Hz, 150-200 µA). During recording sessions, the SLN and the PTg
were simultaneously stimulated for 10 s. As a control, the SLN was solely stimulated for 10 s
twice before and after the simultaneous stimulation. After each experiment, the stimulus sites
were checked histologically. The PTg stimulation had suppressive effect on the number ofswallowing reflexes. The number of swallows was 8.8 ± 0.9 (mean ± SE) in the pre-control, 4.3± 1.0 during Ce stimulation, and 9.6 ± 1.0 in the post-control (n = 6). The onset latency of thefirst swallow was significantly longer (0.70 ± 0.31 s; mean ± SE, n = 6) than in the pre-control(0.31 ± 0.12 s) or the post-control (0.27 ± 0.18 s). The present study suggests that the RN is
involved in the control of swallowing, and that stimulation of the PTg affects the swallowing(COI:No)
CPG.

1P-083Functional recovery by cell grafts of oligodendrocyte progenitor cells toneonatal white matter injury model in rats

In this study, we examined effects of acupuncture stimulation on short latency reflex (SLR) andlong latency reflex (LLR) to determine the site of action of acupuncture stimulation in modulating motor reflexes. Further, we investigated the relationship between changes in LLR andchanges in the N20 somatosensory evoked potential (SEP) component induced by acupuncture
stimulation and examined the changes in central motor conduction time (CMCT).
Sixteen healthy and right-handed adults (11 males and 5 females; 28.9 ± 6.6 years; upper limb
length 54.9 ± 3.2 cm) participated in this study. The experiments were performed under
three conditions: (1) control (no acupuncture stimulation), (2) acupuncture stimulation of rightsided Hegu (LI4), and (3) acupuncture stimulation of left-sided LI4. An acupuncture needle(0.18 mm in diameter) was inserted up to a depth of 10 mm at the right- or left-sided LI4.
Electrical stimulation was delivered to the median nerve in the right hand joint at a 120%
intensity compared with the threshold to produce an M-wave. SLR and LLR were recorded
from the opponens pollicis muscle of the right hand. The frequency and amplitude ratio of SLR(latency, approximately 20–30 ms) and LLR (latency, approximately 40–70 ms) were analyzed.
We analyzed the mean latency and calculated the CMCT using the mean latencies of LLR,
N20, F-wave, and M-wave.The frequency and amplitude ratio of SLR were reduced by theacupuncture stimulation of left- and right-sided LI4, respectively. LLR frequency and amplitude
ratio were reduced by acupuncture stimulations on either side. A correlation was observed
between changes in the LLR amplitude ratio and changes in the N20 SEP amplitude inducedby acupuncture stimulation. No effect of acupuncture stimulation was observed on CMCT.
These findings suggest that acupuncture stimulation inhibits motor nerve reflexes via both
(COI:No)spinal and supraspinal modulation systems.

Naoki Tajiri1，Atsunori Hattori1，Shino Ogawa1, 2，Akimasa Ishida1，Takeshi Shimizu1，
1 1 Dept Neurophysiol ＆ Brain Sci, Grad Sch Med, Nagoya City Univ, Japan,
Hideki Hida（

1P-081

1P-084

Hiroya Yamazaki, Tatsuya Sato, Nobutoshi Ichise, Yoshinori Terashima, Aoi Kato,
Noritsugu Tohse（Dept Cell Physiol, Sap Med Univ, Japan）（ 1 Dept Health Sci, Fac Nurs, Jpn Red Cross Hokkaido Coll Nurs, Kitami, Japan, 2 Dept
Human Informatics, Fac Sci Engi, Kokushikan Univ, Tokyo, Japan, 3 Dept Exer Physiol,
Fac Med, Hoshi Univ, Tokyo, Japan, 4 Lab Physiol, Dept Phys Ther, Fac Med Sci, Teikyo
Univ Sci, Tokyo, Japan）

Characteristics of blood pressure measurement by wrist-cuff automatedoscillometric measurement at rest and after exercise in healthy college
students

Background: A wrist-cuff automated oscillometric device is portable and useful for selfmonitoring blood pressure (BP) home and outdoors when an upper-arm device is not available.
However, it remains unclear whether physical activity such as exercise affects the wrist BP
measurement or not.
Methods and Results: Ninety-seven healthy college students (median age 20 years ranging
from 19 to 36 years, 70.1% men) were participated. Rest BP measurement at the sitting position on the wrist measured by wrist-cuff automated oscillometric device (Omron HEM-6183)
was compared with BP on upper-arm measured by automated oscillometric device (Omron
HEM-7130-HP) and classical auscultatory method. The median BP (IQR) in wrist, upper-arm
oscillometric and upper-arm auscultatory method were 109 (100 - 118), 108 (101 - 113) and 109(104 - 116) mmHg for systolic BP and 66 (61 - 71), 65 (59 - 72) and 68 (64 - 71) mmHg for diastolicBP, respectively. There were no statistical differences among three groups in both systolic BP
and diastolic BP. To assess the effect of exercise on wrist BP measurement, BP on the wrist
and upper-arm were simultaneously measured with winding oscillometric cuffs on separate
arms before and after Master’s double two-step exercise test. The ratio of systolic BP justafter exercise test to that before exercise on wrist (1.22 ± 0.14) was significantly decreased
compared with upper-arm measurement (1.27 ± 0.14), and the difference was significantly
correlated with exercise-induced increase in pulse rate (Spearman’s rho = 0.23), suggesting the
involvement of sympathetic nerve activity.
Conclusion: The results suggest that wrist BP measurement at rest by wrist-cuff automatedoscillometric device seems generally similar compared with upper-arm BP measurement,
while blunted response of exercise-induced BP elevation should be considered in wrist BP
measurement.
(COI:No)2 Dept Obstet ＆ Gynecol, Grad Sch Med, Nagoya City Univ, Japan）

Hypoxia-ischemia (H-I) in preterm infants occasionally results in neonatal white matterinjury (NWMI) associated with neurodevelopmental disabilities such as paralysis and cognitive
dysfunction. Based on selective vulnerability of late oligodendrocyte progenitor cells (OPCs)
to H-I, we made a rat NWMI model that showed hindlimb motor dysfunction without lossof cortical neurons, hypomyelination in the sensorimotor cortex and disturbed cortical motor
map in the ipsilateral motor cortex. To find out new effective treatment for NWMI, we arechallenging cell therapy to NWMI model using OPCs. In this study, we investigated whether
the grafted OPCs can promote motor function in NWMI model. Male rats that received rightcommon carotid artery occlusion followed by 6% hypoxia for 1 hour at P3, were grafted green
fluorescent protein (GFP)-positive OPCs (2.0 x 105 cells/2μl) into the corpus callosum two days
later. Three groups were prepared weaning from P25: sham-operated controls, NWMI + nongrafted group, NWMI + OPC-grafted group. As behavioral evaluations, hindlimb retraction,
elevated body swing, beam walk ability, rotarod, horizontal ladder tests were performed at 4
and 8 weeks, followed by immunohistochemical investigations. The OPC-grafted group showed
better behavioral recovery in hindlimb retraction, elevated body swing, beam walk ability tests
compare to the non-grafted NWMI group at both 4 and 8 weeks. The differences in rotarod andhorizontal ladder tests were shown at 8 weeks. These data suggest that OPC transplantation
during the period of development has a potency to improve deteriorated motor function in the
rat NWMI model. Histological evaluations for cell survival and differentiation of grafted OPCs,
neurons, astroglial cells, and microglia will be also presented to know the mechanism of OPC
effects on developing NWMI model.
(COI:No)

Inﬂuence of lower leg-bath on upright postural sways: comparison ofartiﬁcial high concentration CO2-water with general tap-water
Noriyuki Yamamoto1，Tadashi Wada2，Fumiko Takenoya3，Masaaki Hashimoto4

In this study, the influence of artificial high concentration CO2-water (CO2≧1000 ppm) lower
leg-bath on standing posture sway was investigated. The healthy female college students(n=12) participated in this study. Postural sway was recorded by detecting the body’s centerof pressure (COP) continuously with a force platform equipped with a data processor. Subjects
were requested to stand on the platform with their feet parallel, gazing at a target mark in
2.0 m distance. The body sways of each subject were recorded for 1min, first with the eyes
open (EO) and next with eyes closed (EC) conditions. The path length of COP and area of COP
trace were registered pre and post lower legs bathing. Each subject immersed both lower legs
in tap- and CO2-water (35℃) up to the tibial point for 10 min. The electrocardiogram (ECG)
was recorded continuously using a multitelemeter system. Cutaneous blood flow (BF) was
measured by laser-Doppler flowmeter in the hand index finger and in the right calf. BF in index
finger was not affected whereas BF in calf significantly increased by CO2-water immersion.
Heart rate read from ECG did not show any difference between these two water-baths. The
path length was significantly decreased by lower legs water immersion in both eye conditions,irrespective of the bath water sort. In the present study, we showed that local water immersion at neutral temperature is able to contribute to a more stable standing posture. Because
of ineffective visual conditions, this effect on postural sways might be due to somatosensory(COI:No)
modification by the local water-immersion.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S105

1P-085

Characteristics of ﬁne motor performance and factors associatedwith motor problems in preschoolers with developmental coordination
disorders
Misaki Mikami1, 2，Shuhei Koeda1，Manabu Saito3，Ayako Osato3，Takahito Masuda4，
1 1Grad Sch Health Sci, Hirosaki
Chihiro Sato1，Kazuhiko Nakamura3, 5，Junko Yamada（

Univ, Japan, 2 JSPS Research Fellow, 3 Grad Sch Med, Hirosaki Univ, Japan, 4 Fac Edu,

Hirosaki Univ, Japan, 5 Research Center for Child Mental Dev, Grad Sch Med, Hirosaki
Univ, Japan）

The main symptom of developmental coordination disorder (DCD) is a motor skill deficit withunknown etiology that significantly interferes with child’s activities of everyday living and academic achievements. Poor fine motor skill is one of the most common problems experienced bychildren with DCD. This study aimed to reveal the characteristics of fine motor performanceand the factors associated with fine motor problems in children with DCD. Thirty nine Japanese children aged five years, 18 with DCD and 21 typically developing (TD) were examined.
A web camera was positioned above children’s hands and recorded during the Movement
Assessment Battery for Children 2 (MABC-2) (Age Band 1) posting coins task. Then average
speed, acceleration and total trajectory length of children’s hand movement were calculated.
We also carried out the grip strength test and the finger to nose test. Differences in all scores
between the DCD and TD groups were analyzed. In addition, correlation analyses were used
between scores on the MABC-2 posting coins task, video tracking data, and other factors. The
average speed was significantly slower, and the total trajectory length was longer in the DCD
group. They also scored worse on average grip strength and the correct answer rate of thefinger to nose test. A significant correlation was found between scores on the MABC-2 posting
coins task and average grip strength. These results suggest that children with DCD exhibit
slowness and extra movement in fine motor performance, weaker grip strength, and problems
in the sense of position and movement. Moreover, overshoot dysmetria appears to point tosome degree of dysfunction in the brain.
(COI:No)

1P-088The role of TRPM7 on neuronal cell differentiation using induced
pluripotent stem (iPS) cells
Shingo Tsuji, Susumu Yoshie, Akihiro Hazama（ Dept Cell Integrative Physiol, Sch Med,
Fukushima Med Univ, Fukushima, Japan）

Background: Transient receptor potential cation channel subfamily M member 7 (TRPM7)
is a nonselective cation channel and is expressed in various cells types. TRPM7 has been
known to play roles in cell adhesion, directionality, and migration. However, the relationshipbetween differentiation and TRPM7 is not elucidated. In this study, we investigated the role of
TRPM7 on neuronal cell differentiation using mouse induced pluripotent stem (iPS) cells andmetergoline, which is an activator of TRPM7.
Methods: We have generated neuronal cells from iPS cells by stromal cell-derived inducing
activity (SDIA) method, with/without metergoline. The differentiated cells from iPS cells
were characterized by morphology, Nanog-GFP reporter system, gene expression, and immunoreactivity.
Results: While the cells without metergoline showed the typical morphology of neuronal cells,
the cells treated with metergoline did not show their morphology and formed the undifferentiated iPS cell colonies. Furthermore, Nanog-GFP reporter system was used to visualize endogenous Nanog (an undifferentiated marker) gene expression. The cells without metergoline did
not show the expression of GFP controlled by the Nanog promoter. On the other hand, GFP
expression was strongly observed in the cells treated with metergoline. Real-time RT-PCR also
showed that the expression levels of undifferentiated markers such as Nanog and Rex1 in thecells treated with metergoline were higher than that of the untreated cells. Additionally, while
TuJ1 (a neuronal marker)-positive cells were appeared in the cells without metergoline, thosecells were not appeared in the cells treated with metergoline.
Conclusion: The activation of TRPM7 is involved in the inhibition of the neural cell differentiation from iPS cells or the maintenance of undifferentiated iPS cells. (COI:No)

1P-086

1P-089Susumu Yoshie1，Ryosuke Nakamura2，Daisuke Kobayashi1，Masao Miyake1，Koichi Omori2，
1 1 Dept Cell Integrative Physiol, Grad Sch Med, Fukushima Med Univ,
Akihiro Hazama（

1 1 Dept Cellular andKanae Ouchi1, 2，Susumu Yoshie1，Masao Miyake1，Akihiro Hazama（

Generation of airway epithelium with CFTR function from iPS cells
Fukushima, Japan, 2 Dept Otolaryngol, Grad Sch Med, Kyoto Univ, Kyoto, Japan）

Background: Primary airway epithelial cells and experimental animals have been used forcure of airway disease such as cystic fibrosis (CF) caused by mutations of cystic fibrosis
transmembrane conductance regulator (CFTR) gene. However, it is not easy to acquire anadequate quantity of cells. Furthermore, CFTR knockout mice cannot mimic human-specific
CF phenotype due to species difference. Therefore, airway epithelium from induced pluripotent
stem cells (iPS cells) are expected to be a useful cell source for cure of CF disease. The aim of
this study is to generate functional airway epithelium with CFTR function.
Methods: We generated airway epithelium from iPS cells based on serum-free conditions andair-liquid interface culture. iPS cell-derived airway epithelium was characterized by RT-PCR,
immunocytochemistry, HE staining, Scanning Electron Microscope (SEM), ciliary movement,
and measurement of CFTR function using yellow fluorescent protein (YFP) molecule sensitive
to halide ions.
Results: RT-PCR and immunocytochemistry indicated that airway epithelium markers andCl- channel markers including CFTR were detected in iPS cell-derived airway epithelium.
Pseudostratified ciliated columnar epithelium like tracheal epithelium was confirmed by HE
staining. Furthermore, SEM analysis indicated that iPS cell-derived airway epithelium had
morphologically cilia. Additionally, iPS cell-derived airway epithelium showed the ciliary movement with a ciliary beating frequency of approximately 10 Hz. Finally, the transport functionof CFTR was successfully confirmed, judging from the change in fluorescence intensity using
YFP molecule.
Conclusion: Airway epithelium generated from iPS cells has CFTR function and will be auseful cell source for cure of CF disease, and goblet cell hyperplasia caused by asthma andcigarette smoking.
(COI:No)

Cl- channels regulate lipid droplet formation via Rab8a expression
during adipocyte differentiation
Integrative Physiol, Fukushima Med Univ Grad Sch Med, Fukushima, Japan, 2 Dept Judo
Therapy, Koriyama Inst Health Sci, Koriyama, Japan）

Cl- channels have been known to play important roles for the differentiation, cell volume regulation, migration, proliferation, and intracellular acidification. However, whether Cl- channels are
involved in adipocyte differentiation from ASCs remains unknown. Thus, we investigated therole of Cl- channels on adipocyte differentiation using adipose tissue-derived stem cells (ASCs)
and Cl- channel blocker. We induced rabbit ASCs into adipocytes using Cl- channel blocker.
The expression levels of adipocyte markers were no significant difference between the cells
treated with a Cl- channel blocker NPPB and untreated cells. However, when the cells weretreated with Cl- channel blockers, lipid droplets (LDs) sizes decreased compared with theuntreated control. Interestingly, the expression levels of Rab8a, which is known as a regulatorof LD fusion, were also decreased in the cells treated with NPPB. Other Cl- channel blockers,
DIDS and IAA-94, also inhibited large LDs formation and Rab8a expression.
Additionally, we examined the role of lysosomal acidification on LDs formation, using Bafilomycin. Interestingly, Rab8a expression levels and LDs sizes were significantly decreased in
the differentiated cells treated with Bafilomycin. Furthermore, intracellular pH value of thedifferentiated cells treated with Cl- channel blocker and Bafilomycin were shifted to acidic side.
Therefore, it is considered that the lysosomal acidification was also involved in LDs formation.
These results demonstrate that Cl- channels do not regulate the adipocyte differentiation, but
do regulate the LDs formation via Rab8a expression. (COI:No)

1P-087

1P-090

Syunta Maruo, Takayoshi Hosono（Dept Bio Med Engng, Grad Sch Osaka Electro-Commun

Takeshi Shimizu1，Hideji Murakoshi2, 3，Hidetoshi Matsumoto4，Akimasa Ishida1，
1 1 Dept of Neurophysiol and Brain Sci, Grad School of Med Sci,
Naoki Tajiri1，Hideki Hida（

Exercise capacity and intelligence assessed in behavioral tests inadults mice after betamethasone given at 4-day-old infancy
Univ）

Purpose: In clinical practice of obstetrics, betamethasone has been administrated to pregnant
women with threatened premature delivery to decrease the possibilities of suffering respiratory distress syndrome, intraventricular hemorrhage and periventricular leukomalacia after
birth. However long-term side effects of betamethasone on the developmental delay or in intelligence and motor ability has not been clarified. We showed that betamethasone administrated
in infancy might deteriorate motor capacity after growth using a small number of rats. In thisstudy we investigated long-term effects of betamethasone in its infancy using mice.
Method: We administered betamethasone of 0.5mg/body weight (kg) on 4-day-old mice (group
B, n=29) and also established control group (group S, n=27) by giving saline. We performed
behavioral tests using 3- to 10-week-old mice in both the groups. We performed suspension
test by hooking mice’ forelimbs on horizontal bar of 40 cm high and measured suspension timeusing 3-week-old mice for consecutive five days to test motor ability. We performed step-down
type passive avoidance test by measuring staying-time on insulted platform using 5-weeks-old
mice after 2-day learning periods to avoid unpleasant electrical shocks. We performed Rotarod
test by placing mice on the rotating rod. We removed brains of mice under deep anesthesia,
and measured brain sizes.
Results: Averaged body weights in B group were significantly smaller in S group. In Rotarod
test the mice in B group stayed on the rotating rod significantly shorter than those in S group.
There were no significant differences in the results of suspension tests, step-down passive
avoidance and the sizes of the brains.
Conclusion: Betamethasone administration to mice infants may cause delay in their motor(COI:No)
activities after growth.

S106

New assay system to detect mechanical force in myelinating
oligodendrocytes using a tension sensor probe
Nagoya City Univ, 2 Supp Cent for Brain Res, Natl Inst for Physiol Sci, 3 Dept of Physiol
Sci, Grad Univ for Advanced Studies, 4 Dept of Mater Sci and Engineer, Tokyo Inst ofTechnol）

Oligodendrocytes (OLs) form myelin sheath around neuronal axons to express higher brain
functions. Although both thick and thin axons exist, the ratio of diameter of axon + myelin toaxon diameter (g-ratio) is adjusted to optimum values for each axon, suggesting a factor that
controls myelin formation in response to the axon diameter. To clarify the mechanism of theconstant g-ratio, we try to investigate mechanical factors depending on its diameter. To visualize the OL generating force during myelination process, a tension sensor based on fluorescenceresonance energy transfer (FRET) was used: the efficiency of FRET decreases when the force
is applied on it, setting two fluorescent molecules apart from each other.
Artificial polystyrene nanofibers with a diameter similar to the axons are firstly prepared tomeasure the mechanical force without neuronal activity, extracting a physical factor that is
purely dependent on the axon diameter. We found that cultured OL could wrap the myelin
sheath around the nanofibers when OLs were cultured on polystyrene nanofibers. The change
of FRET intensity in the myelinating OL depending on the nanofiber diameter was also
observed in our preliminary experiment. In addition, the focal adhesion morphology could be
investigated as the tension sensor is localized at the focal adhesion. Thus, we can examine
the correlation among the following four factors using FRET system: axon diameter - OLgenerating force - focal adhesion morphology - myelin shape, in the same cultured OL cell.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

1P-091

A cytosolic N-terminal region of Transmembrane Channel-like protein 1(TMC1) is cleaved and imported into the nucleus in an overexpression
systemSoichiro Yamaguchi1，Maho Kamino2，Maho Hamamura2，Ken-ichi Otsuguro2（ 1 Lab Physiol, Facul Vet Med, Hokkaido Univ, Japan, 2 Lab Pharmaco, Facul Vet Med,
Hokkaido Univ, Japan）

Transmembrane Channel-like protein 1 (TMC1) has been shown to be the pore-forming subunit of Mechano-Electrical Transduction (MET) channel, which transduces mechanical stimuli
into electrical signals at the top of stereocilia of hair cells in the inner ear. As an unexpected
result, we found that the cytosolic N-terminal regions of mouse TMC1 (mTMC1) which wasoverexpressed in HEK293 cells were localized in nuclei in a small population of the transfected
cells. The accumulation of the N-terminal regions into the nuclei depended on nuclear localization signals within the N-terminal region. Western blot analyses revealed that at least threefragments of the N-terminal region were cleaved and produced from overexpressed mTMC1.
Amino acid residues which were required for each cleavage were identified by site-directedmutagenesis approaches. Using a next-generation sequencer, we performed transcriptome
analyses of the cells which transiently expressed the most dominant N-terminal fragment fromamong the three fragments. In comparison with transcriptomes of the cells in which empty
vectors were transfected, the overexpression of the N-terminal fragment of mTMC1 slightly
altered expression levels of some genes. Although it is still unknown whether these phenomena
happen in vivo, these results suggest that the cytosolic N-terminal fragment of mTMC1 mightwork as a transcription factor in some specific conditions. (COI:No)

1P-092

Mitochondrial localization of ﬂuorescent fusion proteins is inhibited by
fast folding of ﬂuorescent proteins
Sayaka Kashiwagi, Yoichiro Fujioka, Aya O. Satoh, Aiko Yoshida, Mari Fujioka,
Prabha Nepal, Atsushi Tsuzuki, Ozora Aoki, Sarad Paudel, Hitoshi Sasajima, Yusuke Ohba

（Dept. Cell Physiol., Fac. Med. ＆ Grad. Sch. Med., Hokkaido Univ.）

Green fluorescent protein (GFP), its variants, and coral fluorescent proteins (FPs) were
widely used in the field of cell physiology and cell biology. Once they are fused with organelle
targeting and/or retention sequences, the resulting fluorescent fusion proteins (FFPs, ororganelle markers) enable us to visualize cellular organelles and their dynamics in living
cells. However, such FFPs occasionally display behaviors different from that of the native
proteins: i.e. mislocalization due to aggregation, multimerization of FP, or other reasons. Here,we constructed a series of FFPs targeted to different organelles that harbor a variety of FPs
with different color spectra and found that FFPs fused with certain types of FPs were localized
not only to the mitochondria and endosomes, but also to the cytosol and the nucleus. Suchmislocalization was observed for FFPs that included several variants of Aequorea Victoria
GFP (avGFP) and a monomeric variant of the red FP from Discosoma sea anemones (DsRed).
Comparison of the amino acid sequences revealed that the FPs in mislocalizing FFPs containmutations that accelerate protein folding or maturation, indicating that fast folding of FPs
might prevent expected localization of FFPs. In fact, reintroduction of amino acid substitutions
to make the FP sequences identical to those of wild-type avGFP significantly restored the FFP
localization to the expected organelles. In addition, similar amino acid substitutions improved
the mitochondrial localization of the pH-sensitive GFP variant pHluorin that was targeted tothe mitochondria. This improvement appears to be advantageous for the precise monitoringof mitochondrial pH. Taken together, fast folding of FPs, which is generally thought to bebeneficial for fluorescence imaging, might occasionally inhibit the expected localization of FFPs.
We therefore propose that the significance of FP selection to maximize FFP function.(COI:No)

1P-093

Research on the role of CFL1-mediated cytoskeleton remodeling ininvasion and metastasis of gastric cancer cells
Haibo Wang1，Tengyang Ni1，Zewen Chu1，Masataka Sunagawa2，Yanqing Liu1

（ 1 Medical college, Yangzhou University, Yangzhou, China, 2 Showa University）

Objective: To investigate the role CFL1-mediated cytoskeleton remodeling in invasion andmetastasis of gastric cancer cells, and to provide new ideas and experimental basis for researchand development of new anti-metastasis therapies and drugs for gastric cancer.
Method: The expression of CFL1 in 37 clinical samples was analyzed to find out the differenceof CFL1 expression between paracancerous tissue and gastric cancer tissues. Western blot
and PCR were used to detect the expression of CFL1 in cells. Cytoskeleton staining combinedwith immunofluorescence staining was used to analyze the relationship between CFL1 andcytoskeleton activity. TEM and SEM were used to observe the changes of microfilamentson the inner and surface of gastric cancer cells. Lentivirus infection technology is used tooverexpress and interfere with the expression of CFL1 in gastric cancer cells. The subcutaneous transplantation model and peritoneal metastasis model of gastric cancer in nude mice
were used to detect the invasion and metastasis of gastric cancer cells in vivo. Living imaging
system and transwell assay were used to observe the invasion and metastasis of gastric cancer
cells in vitro and in vivo.
Result: The expression of CFL1 in gastric cancer was significantly higher than that in adjacenttissues. The same results were found in the analysis of gastric mucosal cells and gastric cancer
cells; The expression of CFL1 was significantly increased in the cells with active cytoskeleton
remodeling; CFL1 plays an important regulatory role in cytoskeleton remodeling. Invasion andmetastasis experiments showed that inhibiting CFL1 could significantly inhibit the invasion
and metastasis of gastric cancer cells. The same results were obtained by living imaging
system experiments of small animals.
Conclusion: These findings demonstrate that CFL1 induces invasion and metastasis bypromoting cytoskeletal rearrangement. Our results may provide the basis for developing new
anticancer drugs to inhibit CFL1.
(COI:No)

1P-094

Proliferation and migration of NIH/3T3 are suppressed by cesium
Ziasmin Khatun, Natsumi Nishimura, Daisuke Kobayashi, Akihiro Hazama（Dept Cell Integrat Physiol, Grad Sch Med, Fukushima Med Univ, Japan）
Fibrosis, a common feature of epithelial tissue injury is attributable to the excessive accumulation of extracellular matrix such as collagen, produced by excessive proliferation of fibroblast
cells, provoked by injured epithelial cells. Our laboratory previously showed that CsCl inhibitedthe growth of the human cancer cells by inhibition of the glycolytic pathway. In this study,
we investigated the effects of CsCl on fibroblast cell cultures. Mouse fibroblast cells (NIH/3T3)
were grown to form a confluent monolayer in 24 well plates, and artificial wounds werescratched, then treated by 1 mM, 3 mM, and 10 mM CsCl. Gap closure was checked before
and after treatment using fluorescence microscopy. For cell viability assays, cells were plated
at 60 mm dishes at a density of 1 × 105 cells per dish and cell viability was measured at 0,24, 48 and 72 h of treatment with 1 mM, 3 mM and 10 mM CsCl using Trypan blue. The
treatment of cesium inhibited the migration of fibroblast cells compared with the control
and inhibited the migration significantly and a dose-dependent manner. The number of thecells was decreased a dose-dependent manner after treatment, whereas the viability of cells
remained almost unchanged. Above these results suggest that CsCl inhibits the migration andproliferation of fibroblast cells. This study provides a possible therapeutic role of CsCl in thetreatment of tissue fibrosis.
(COI:No)

1P-095

Effects of 405 nm light irradiation by using light emitting diode on HeLa cells
Toshitaka Ikehara1, 2，Mutsumi Nakahashi3，Takahiro Emoto4，Masatake Akutagawa4，4 1 Dept Phys Therapy, Fac Health
Koichiro Tsuchiya5，Akira Takahashi6，Yohsuke Kinouchi（

Welfare, Tokushima Bunri Univ, 2 Inst Health Sciences, 3Tokushima Agri Forest Fish Tech
Center, 4 Dept Electri Electro Engineer, Grad Sch Sci Tech, Tokushima Univ, 5 Dept Med
Pharmacol, Grad Sch Biomed Sci, Tokushima Univ, 6 Dept Med Environ Nutri,Grad Sch
Biomed Sci,Tokushima Univ）

We tested effects of 405 nm wavelength irradiation on cultured HeLa cells. Cells were plated
in plastic dish (3cm diameter) and were maintained for 24-48 hours. The cells were irradiatedwith the light at 146 mW/cm2. Reactive oxygen species (ROS) were monitored by fluorescentprobe, dichlorodihydrofluorescein diacetate (H2DCF-DA).We tried to measure the intra- andextracellular ROS accumulation after 385, 395 and 405nm light irradiation for 15 min. The
ROS accumulation by these lights irradiation indicated a similar tendency and 405nm lightshowed relatively high ROS accumulation compared to other 2 lights. Then, I tested further
about influences of 405 nm light on the cells. We tried again to measure the intracellular ROS
accumulation in the lower glutathione (GSH) cells loaded with 1- Chloro-2, 4 dinitrobenzene(CDNB), GSH and thioredoxin (mentioned below) are known as intracellular physiologicalscavenger. The ROS accumulation in the low GSH cells obtained by addition of CDNB more
rapidly increased compared to normal cells. Moreover, we also irradiated the light to low
GSH cells obtained by preincubation with buthionine sulfoximine (BSO) for 24 hr. The lightirradiation for more than 1.5 hr significantly induced cell death in the low GSH cells. Then, we
tried to measure the intracellular thioredoxin and GSH content. But, GSSG (oxidized GSH) was
only slightly increased with the irradiation and thioredoxin content was increased regardlessof cell death. These results suggest that the intracellular ROS induced by 405 nm light arecontaining singlet oxygen (1O2) and hydroxyradical, and the intracellular glutathione plays animportant role of scavenging these ROS.
(COI:No)

1P-096

Disrupted feeding rhythms reduced serotonin sensitivity to stressand p-CREB/CREB ratio in hippocampus, leading to time-dependent
depressive-like behavior
Atsushi Haraguchi, Miyabi Fukuzawa, Konomi Tamura, Kengo Takahashi, Rina Hirooka,
Tomohiro Yamazaki, Shuhei Sato, Shigenobu Shibata（ Lab. of Physiology andPharmacology, ASE, Waseda Univ, Japan）

The circadian clock regulates the feeding rhythm, and feeding in turn regulates the phaseof peripheral clocks, suggesting a close interaction between the circadian clock and feeding.
Moreover, the circadian clock system also affects mood. We have indicated that disrupted
feeding rhythm, like night eating syndrome (NES), increased 5-HIAA/5-HT in hippocampus,
and then caused depressive-like behavior in the beginning of inactive period. However, we
could not clear why disrupted feeding rhythm caused time-dependent depressive-like behavior.
Therefore, to understand the mechanism by which disrupted feeding rhythm caused timedependent depression-like behavior, we treated NES group with 5-HT receptor agonists andantagonists, and examined p-CREB/CREB ratio in hippocampus as marker of depressive-likebehavior. We prepared two groups, control and NES groups. A normal diet (ND) with adlib-feeding was given to mice in the control group, and a high-fat diet for 5-minute during theinactive period under ND ad lib-feeding was given to mice in the NES group. We injected5-HT receptor agonists or antagonists to mice 30 min before a forced swimming test at ZT
1, after adaptation to each feeding protocol for 4 weeks. As the result, immobility time in
the NES group treated with 5-HT1B agonists was significant shorter than those in the NES
group injected with saline. Moreover, we found that at p-CREB/CREB ratio in hippocampus of
NES group was lower at ZT 1 than that of control group at ZT 1, and similar values in bothgroups at ZT 13. Currently, we are measuring the active rate of some signal kinase related
in 5-HT1B and CREB/BDNF signal. These results indicated that disrupted feeding rhythms
reduced serotonin sensitivity and lowered ratio of p-CREB/CREB in hippocampus, leading to(COI:No)
time-dependent depressive-like behavior.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S107

1P-097

Artiﬁcial organic arsenic compounds promote the expression of Tau
protein in Neuro2a cells
Tomoyuki Masuda1，Junko Itoh1，Mioko Iseki1，Yuka Sugawara1，Yasushi Tomidokoro1，
1 1 Fac Med, Univ
Naruhiko Sahara2，Takeshi Ikeuchi3，Kazuhiro Ishii1，Akira Tamaoka（

1P-100Anorectic action of estrogen replacement is not modiﬁed bygonadotropin releasing hormone (GnRH) and gonadotropins in
ovariectomized rats
Ayana Izumi, Kurumi Iida, Rie Shiroi, Yume Mori, Keiko Morimoto, Akira Takamata

Tsukuba, Japan, 2 Nat Inst Radiol Sci, Japan, 3 Brain Res Inst, Niigata Univ, Japan）（Nara women’s University, Japan）

In 2003, health hazards occurred in Kamisu City, Ibaraki Prefecture by well water contaminated with a high concentration of organic arsenic compound diphenylarsinic acid (DPAA).
Arsenic is known as one of the metalloid elements and exists mainly in the form of both organicand inorganic arsenic compounds in nature. However, DPAA detected from well water is an
artificial organic arsenic compound that would not exist in nature. Due to the health hazards ofenvironmental arsenics, patients exposed to DPAA mainly exhibited neurological symptoms.
When arsenic compounds in the environment enter the body via drinking water, they are
known to pass through the blood-brain barrier and reach the brain. This suggests long-term
intake of DPAA via well water caused the accumulation of DPAA in the brains of patients,
resulting in these health hazards. Tau protein is known as an indicator of neurodegeneration.
In the pathological condition, Tau is over-phosphorylated, causing neurodegeneration. In thisstudy, we focused on changes in the amount of total and phosphorylated Tau proteins in
cultured neurons after the addition of DPAA, and investigated the possibility that DPAA
may be involved in the promotion of neurodegeneration. We used N2a cells (a cell line derived
from mouse neuroblastoma) in which human Tau was overexpressed. DPAA was added at
the concentrations of 0, 0.1, 1.0, 10, and 50 (μg/ml), and cultured for 48 hours after addition.
Cells and medium were collected after culture, and the amounts of total and phosphorylated
Tau proteins were determined by Western blotting. As a result, the both intracellular Tau
proteins in the N2a-MAPT cells were significantly increased compared to those in the control.
Furthermore, the amount of total extracellular Tau also increased significantly compared tothe control. These results suggest that DPAA may be involved in the mechanism of neurodegeneration.
(COI:No)

Estrogen replacement in ovariectomized rats reduces food intake specifically during the lightphase. It suggests that estrogen exerts an anorectic action by modifying circadian feedingrhythm. Ovariectomy, however, elevates the levels of gonadotropin releasing hormone (GnRH)
and gonadotropins, such as luteinizing hormone (LH) and follicle stimulating hormone (FSH) via
the negative feedback system of the hypothalamo-pituitary-gonadal axis. To elucidate whether
GnRH and/or gonadotropins are involved in the anorectic action of estrogen replacementin ovariectomized rats, we examined the effect of GnRH antagonism on feeding behavior infemale rats. Wistar female rats ware divided into ovary-intact (SHAM) groups and ovariectomized (OVX) groups. One week after surgery, rats in the SHAM groups were implanted
subcutaneously with an empty silicon capsule, and rats in the OVX groups were implanted
a silicon capsule containing cholesterol (Veh) or 17βestradiol (E2). And, rats in each groupwere injected subcutaneously with either 1 mg/kg degarelix, a GnRH antagonist, or the sameamount of vehicle (5% mannitol). They were provided free access to water and a standard
chow. Both endogenous and exogenous estrogens attenuated daily food intake, especially foodintake during the light phase, and body weight gain, regardless of the levels of GnRH andgonadotropins. In contrast, GnRH antagonism with degarelix unaltered food intake and weightgain in both the estrogen replete and depleted conditions. The results indicate that GnRH and/or gonadotropins do not a role in the regulation of food intake, suggest that effect of estrogen
replacement on food intake behavior in ovariectomized rats is not modified by the elevated
GnRH or gonadotrophins.
(COI:No)

1P-098

1P-101

One-week-galvanic vestibular stimulation improves arterial pressure
control at the onset of postural change
2 1Grad Sch Health Med, Gifu Univ Med Sci, Japan,
Kunihiko Tanaka1，Akihiro Sugiura（
2 Dept Radiotech, Gifu Univ Med Sci, Japan）

Mild stress inﬂuences estrogen-induced enhancement of sucrose
intake in ovariectomized rats
Kurumi Iida, Ayana Izumi, Rie Shiroi, Yume Mori, Keiko Morimoto, Akira Takamata（Nara Women’s Univ, Japan）

The vestibular system consists of otolith and semicircular canals. It has been known that theotolith stimulation modifies the sympathetic nerve activity. Previously, we demonstrated thatthe otolith organs are playing an important role for controlling arterial pressure (AP) at theonset of head-up tilt (HUT). Furthermore, sub-somatosensory galvanic vestibular stimulation(GVS) arguments AP response at the onset of HUT. In the present study, we examined chronic
effects of sub-somatosensory GVS for AP control.12 healthy subjects were recruited. AP wasmeasured continuously, and the posture was changed from supine position to 60 degrees of
HUT. Center of gravity (CoG) was measured with eye-open and -close. Romberg ratio wasanalyzed. For 6 subjects, sub-somatosensory GVS of white noise was applied 10 minutes per
day for 7 days. The amplitude of GVS was set and monitored at 0.1 mA-lower than somatosensory threshold. At 8th day, AP during postural change and CoG were measured again for theall-12 subjects. The subjects without GVS, both AP change at the onset of HUT and Romberg
ratio of total trajectory length in CoG did not change significantly, compared to those of thefirst day. However, AP at the onset of HUT was significantly higher in the subjects who had
GVS, compared to that before GVS. Romberg ratio was significantly smaller than that before
GVS. The changes were maintained in 9th and 10th days. Thus, one-week-GVS improves both
(COI:No)posture and AP control, and the change was maintained at least for 3 days.

Intake of palatable food or drink is regulated by the hedonic feeding mechanism, and is
influenced by many factors, such as physiological and emotional/psychological conditions.
We previously found that 17β-estradiol (E2) replacement in ovariectomized rats enhanced
progressive increase in 10% sucrose solution, indicating that E2 increases preference for sweet
taste. Furthermore, chronic stress is also known to affect the feeding behavior. In the present
study, we hypothesized that the E2-induced enhancement of sucrose intake is influenced bymild stress with isolation rearing. Seven-week-old female Wistar rats were ovariectomized,
and were implanted subcutaneously with a silicon capsule containing either E2 (E2 group)or cholesterol (Veh group). After the surgery, rats of each group were reared in a normal
condition (control rearing), in which rats were singly housed in a transparent plastic cage witha wire mesh lid, so they can see and hear the neighbor rats, or an isolated condition (isolation
rearing), in which rats were singly housed in the same cage, which was separated by the thickwalls, ceiling and floor, so they cannot see or hear the neighbor rats. Then, rats were allowed
access to 10% sucrose in addition to standard chows and water for 14 days. On the first day,sucrose solution intake was not different among the groups. Sucrose solution intake gradually
increased with time in all groups. However, the increase in sucrose solution intake over the 14
days was greater in the E2-control group than the other groups. Therefore, in the last week,sucrose solution intake in the control rearing was greater in the E2 group than the Veh group,
whereas the intake in the isolation rearing was not different between the E2 and the Vehgroups. These results suggest that isolation stress attenuates the E2-induced enhancement ofsucrose intake in rats.
(COI:No)

1P-099

1P-102

Effects of acceleration (+Gz) on blood pressure and biomarker inanesthetized rats
3 1 Dept
Megumi Tandai-Hiruma1，Satoshi Maruyama2，Yasunaga Shiraishi3，Masanori Fujita（

Physiol, Natl Def Med Coll, Tokorozawa, Japan, 2 Aero Med Lab, JASDF, Tachikawa,Japan, 3 Div Environ Med, Natl Def Med Coll, Tokorozawa, Japan）

Introduction: Acceleration (+Gz) occurs during fighter aircraft maneuvers. It is well known
that the +Gz causes loss of consciousness. We previously reported the effect of the +Gz on
blood pressure in urethane or pentobarbital anesthetized rats. The purpose of this study is toconfirm the effect of the +Gz on blood pressure and to estimate the biomarker of +Gz exposurein rats with the anesthesia recommended in recent years.
Methods: We used 12-week-old male Sprague-Dawley rats. Rats were randomly allocated to
a +2, +3, +4 and +5 Gz exposure groups. All rats were intraperitoneally anesthetized with a
combination of ketamine (75mg/kg) and xylazine (10mg/kg). 30min after the anesthesia, the rat
was exposed to the +Gz load for 2 min using the animal centrifuge system (arm radius 1.15m,
Tomy Seiko Co. Ltd., and Bio Research Center Co. Ltd.). Arterial pressure (AP) and central
venous pressure (CVP) were continuously recorded during +Gz exposure. Blood was sampled
before +Gz exposure (20 min after the intraperitoneal injection) and after +Gz exposure (40 min
and 70 min after the intraperitoneal injection).
Results: In +2 Gz exposure groups, AP rapidly decreased but gradually recovered during theload. In +3 or higher Gz exposure group, AP rapidly decreased but little recovery was seen. AP
and CVP were decreased during +Gz exposure and the recovery of AP during +Gz exposure
was affected by the strength of the load. The value of blood glucose and lactic acid went up
after the +Gz exposure.
Conclusion: In our experimental protocol, the blood pressure regulation during +Gz exposure
was disturbed depending on the strength of the load. The value of blood glucose and lactic acid
were indicated as biomarkers of +Gz exposure. (COI:No)

S108

Complaxity of cardiorespiratory responses to dexmedetomidine in adultrats
Yoichiro Kitajima, Chikako Saiki, Nana Sato, Ryoji Ide, Yukari Nagakura, Toshio Imai

（Dept physiol, Sch of Life Dent. at Tokyo, The Nippon Dent. Univ, Japan）

Introduction: A sedative drug dexmedetomidine (DEX) is known to have depressive effect on
respiration less apparent than that of circulation. In this study, we examined in adult rats theeffect of DEX on the cardiorespiratory indices, including the arterial blood gases.
Materials and methods: Adult Wistar rats (n = 18) had been received cannulation in the tailartery and vein under isoflurane anesthesia. After recovery, they were divided into threegroups and Administered one of the three drugs; vehicle (normal saline, NS group), 5 µg/
kg DEX (Low group), and 50 µg/kg DEX (High group). We measured respiratory indices; i.e.
respiratory rate (f R), tidal volume (VT), minute ventilation (V ’E), mean inspiratory flow (VT/
TI), arterial blood gases (PaO2, PaCO2, and pH), and circulatory indices; i.e. heart rate (HR) andmean blood pressure (MBP).
Results: Compared to NS group, High and Low group animals significantly decreased f R andincreased VT, and maintained VT/TI, with DEX administrations. Mean f R was lower in Highgroup than that in Low group, and significant decrease in V ’E was observed only in Highgroup. PaO2 decreased in Low and High groups, but increase in PaCO2 and decrease in pH wassignificant only in High group. Compared to NS group, High and Low group animals decreased
HR (-27 and -17 %, respectively) and increased MBP (+52 and +30 %, respectively) immediately
after DEX administration. However, the increase in Low group was only temporary andbecame lower than that in NS group several minute after DEX administration. In contrast,
increased MBP in High group was not coming down during the measurement.
Conclusion: Our results suggest that DEX overload causes hypoventilation and paradoxical circulatory changes (hypertension and bradycardia), which could be factors to disrupt functional
cooperation between respiration and circulation.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

1P-103

Contribution of oxytocin to the anti-stress effect of the Kampo medicine
Kamikihito -Part IIMana Tsukada, Hideshi Ikemoto, Naoki Adachi, Yasunori Takayama, Takayuki Okumo,
Wakako Yogi, Mami Kato, Tadashi Hisamitsu, Masataka Sunagawa（ Dept Physiol, Grad Sch
Med, Showa Univ, Japan）

1P-106

Participation of GABAergic mechanisms in the reduction ofnoradrenaline release in the median preoptic nucleus caused byvolume expansion in rats
Junichi Tanaka1, 2，Makoto Takahashi 1, 2，Akihiko Ushigome3，Ken Fujisawa2，
4 1 Dept Disability Sci, Grad Sch Edu, Naruto Univ
Shinichiro Takahashi 1，Yasushi Hayashi （Purpose: Kamikihito (KKT) is a Kampo medicine (Japanese traditional herbal medicine) thatis administered to patients with psychological symptoms, such as anxiety, depression andsleeplessness. Oxytocin, a hypothalamic nonapeptide, is linked to increased levels of social
interaction, well-being and anti-stress effects. We have already reported that KKT exerts
regulatory effects on stress responses under acute stress conditions. We therefore investigatedwhether or not oxytocin contributes to the mechanism underlying such an effect.
Methods and Results: Male Wistar rats were divided into control, acute stress (Stress), andKKT (300 mg/kg/day po)-treated acute stress (KKT+Stress) groups. Rats in the Stress andKKT+Stress groups were exposed to a 90-min restraint stress procedure involving novel
physical stress. During the stress loading, the cerebrospinal fluid (CSF) was collected using
microdialysis. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) were used tomonitor the oxytocin secretion in the CSF. In the Stress and KKT+Stress groups, the CSF
oxytocin levels were increased during stress loading. At 30 and 60 min after stress loading, thelevels in the Stress group were decreased; however, those in the KKT+Stress group remained
higher than before stress loading. Furthermore, the anxiety-like behavior immediately after
the acute stress loading was examined using an open field test. As a result, the total moved
distance in the Stress group significantly decreased; however, the decrease was significantly
inhibited in the KKT+Stress group. Moreover, the effect of KKT was obstructed by the
pre-administration of the oxytocin receptor antagonist (L-368,899 hydrochloride; 10 mg/kg,
intraperitoneal).
Conclusion: These results suggested that KKT has anti-stress activities and that increased
oxytocin secretion may be a mechanism underlying this phenomenon.
(COI:No)

Edu, Japan, 2 Sci Sch Edu, Joint Grad Sch, Hyogo Univ Teach Educ, Japan, 3 Faculty
Human Care, Teikyo Heisei Univ, Japan, 4 Dept Food ＆ Human Nurt, Notre Dame Seishin
Univ, Japan）

1P-104

1P-107

Tomonori Furukawa1，Yoshikazu Nikaido2，Shuji Shimoyama1，Yoshiki Ogata1，
3 1 Dept Neurophysiol, Grad Sch Med, Hirosaki Univ, Japan, 2 Dept
Shinya Ueno1, （Mitsuyuki Ichinose, Yutarou Kurisawa（ Dept BiolSci, Sci ＆ Eng, Iwate Univ, Morioka, Japan）

Chronic diazepam administration impaired hippocampal CA3 LTP andspine morphology in aged mice
Anesthesiol, Grad Sch Med, Hirosaki Univ, Japan, 3 Research Center for Child Mental
Development, Grad Sch Med, Hirosaki Univ, Japan）

Diazepam (DZP, a benzodiazepine), which bind with high affinity to gamma-aminobutyric acidtype A receptors (GABAA-Rs) and potentiate the effects of GABA, is widely prescribed foranxiety, epileptic discharge, insomnia, muscle-relaxing, and anti-convulsants. However, the longterm use of DZP is limited due to adverse effects such as tolerance, dependence, withdrawal
effects, and impairments in cognition and learning. Additionally, some clinical reports haveshown that chronic DZP treatment increases the risk of dementing disorder in the elderly.
Several studies reported that chronic DZP administration could affected neuronal activityof hippocampus, dendritic structure, and cognitive performance. However, effects of chronic
DZP administration on aged mice regarding cognitive function is still incompletely understood.
In the current study, to investigate the effect of chronic DZP administration on cognitive
function, we performed behavioral test, morphological analysis for dendritic spine density,and hippocampal long-term potentiation (LTP) assay in both young (8 weeks-old) and aged (12months-old) mice. DZP was administered chronically by Alzet mini-osmotic pumps. Morris
water maze test was used for spatial learning and memory evaluation. To visualize dendritic
spines and analyze spine density, lucifer yellow was injected into hippocampal neurons. The
retrieval performance was impaired by chronic DZP administration in aged mice but not inyoung mice. LTP was attenuated by DZP administration in CA1 of young mice and CA3 ofaged mice. The spine density of hippocampal neuron was decreased by chronic DZP administration in CA1 of both young and aged mice and CA3 of aged mice. These results suggested
that the effects of chronic DZP were different between young and aged mice. The impairmentof retrieval performance induced by chronic DZP administration was likely to be affected byattenuation of LTP and decrease of dendritic spine density in hippocampal neurons.  (COI:No)

In urethane-anesthetized rats, microdialysis was employed to investigate whether γ–aminobutyric acid (GABA) receptor mechanisms are implicated in the modulation of noradrenaline (NA)release in the median preoptic nucleus (MnPO) in response to changes in extracellular fluid
volume. In intact animals, volume expansion (VE) elicited by intravenous infusion of 4% Ficoll(1% body wt, 0.4 ml/min) significantly decreased the release of NA in the MnPO that accompanied an elevation (18 ± 3 mmHg of baseline at 20 min after the start of the Ficoll infusion) inmean arterial pressure (MAP). The VE-induced decrease in the NA release in the MnPO wassignificantly attenuated by perfusion with either the GABAA receptor antagonist bicuculline(10 µM) or the GABAB receptor antagonist phaclofen (10 µM) through a microdialysis probe.
The amount of the antagonist effects was much greater in the bicuculline-treated group thanin the phaclofen-treated group. In bilaterally-vagotomized rats, no significant changes in the
NA release were observed. These results demonstrate the contribution of the GABAergic
system to the regulation of NA release in the MnPO by neural inputs from the peripheral
baroreceptors, and imply that the GABAergic inhibitory action may be mediated through
GABAA receptors rather than GABAB receptors in the MnPO.
(COI:No)

Physiological effects in CNS and the autonomic nervous system bylistening two kinds of classical music
To investigate physiological effects by listening classical music, especially so-called α-music,
we measured EEG and the autonomic nervous system by measuring heart rate and heart ratevariability. Psychological conditions were monitored by describing two subjective psychologicaltests, i.e. the multiple mood scale (MMS) and the General Arousal Checklist (GACL). Subjects
were young healthy 3 students. The subjects listened the 2 kinds of classic music selected bycommercial CD. Both music belongs to relaxation music. One kind is calm and feel comfortable
but another kind is exciting music or excitement. By listening the music, amplitude of α andδ band in frequency analysis (mFFT) of EEG was changed. Heart rate was reduced. The
sympathetic nervous activity indicated by LF/HF was reduced. These data indicated that listening the music suppressed the sympathetic nervous activity. The parasympathetic nervous
activity indicated by HF was contrarily enhanced. Respiratory interval between inhalationand expiration was reduced during listening the music, the reduction size was depended on
kinds of music and the subjects. The data indicate that the autonomic nervous activities inheart activity and respiration was influenced by the kinds of music. MMS in each subject wasdifferent by two kinds of music. GACL in each subject was also different by two kinds of music.
These data suggested that listening the classic music, especially so-called α-music, rested
general brain activity recorded by EEG, namely leading to relaxation in the central nervous
activity and the autonomic nervous activity, and comfortable feelings in the multiple moodscale and arousal level. However, based on the results of the central and autonomic nervous
activities and psychological effects, responses to two kinds of music were different dependingon the subjects.
(COI:No)

1P-105

1P-1082 1 Dept Systems Physiol, Grad Sch Med, Okinawa
Yutaka Hosokawa 1，Syunji Sugimoto（
Univ, Japan, 2 Dept of Comp, Sci and Eng, Grad Sch Eng, Toyohashi Univ. of Technology,
Toyohashi）

Ryo Sawagashira, Masaki Tanaka（ Dept Physiol, Grad Sch Med, Hokkaido Univ, Japan）

Salicylate-induced changes of the responses to the downward FM
sounds in AI and DC ﬁeld of guinea pigs observed by optical recording

The influence of salicylate on the responses to the downward FM sounds in the primary
auditory cortex (AI) and DC field of the guinea pig were investigated using optical imaging
with a voltage-sensitive dye (RH795). Eight guinea pigs were anesthetized with ketamine (80mg/kg) and xylazine (40 mg/kg). Activity patterns to the downward FM sounds (the linear
sweep: the start and end frequency, 16 and 0.5 kHz in 16-64 ms duration) and tones (0.5-16 kHz,
200 ms duration) at 55-85 dB SPL were recorded from the AI and DC field on both sides before(control) and 0.5-2 hours after the intraperitoneal injection of 300 mg/kg salicylate. When thesound pressure level is high, the active-spots to the downward FM sounds were appeared at
the 16kHz-frequency band (FB) in the AI and DC field with and without salicylate injection.
On the lower sound pressure, the active-spots were separately appeared in the lower FB ofthe each field at an hour after salicylate injection. The active-spots positions were separated
longer when the sound pressure was lower. These results show that the responses to thehigh frequency sound were suppressed by the salicylate injection and the active-spots to thedownward FM sounds appeared at the lower FB. In other words, these results suggest thethreshold increase of the high FB after salicylate injection. Similar results were reported in the
rat auditory cortex (Jiang et al., 2017). (COI:No)The oculomotor foraging task: a novel behavioral paradigm to evaluate
working memory capacity and utility
Working memory deficits are commonly observed in various neurodegenerative disorders,
whereas quantitative evaluations of these deficits are usually difficult in clinical cases. Therefore, developing the above assessments is in urgent need. Recent studies in experimental
animals use low-dose ketamine (NMDA receptor antagonist) to disrupt working memory
partly mimicking pathophysiology of schizophrenia. Here, we developed a novel behavioral
paradigm to assess memory capacity and utility, and adapted it to monkeys with and without
ketamine administration.
In the oculomotor foraging task, the animals were presented with 15 identical objects (0.6ºwhite squares, > 4º apart each other) on the screen. One of the objects was associated witha liquid reward, and monkeys were trained to search for the target by making sequential
saccades for up to 6 seconds. We assume that the rate of revisiting movements to the same
objects may depend on the amount of memory capacity and utility. We constructed the “foraging model” incorporating the following three parameters: 1) memory capacity, 2) exploratory
rate and 3) memory decay, which could explain several features of behavioral data. When we fit
the data obtained from three monkeys with those expected from the model, the goodness-of-fit
evaluated by coefficient of determination was always greater than 0.88. The resultant estimatesof memory capacity ranged from 7-11 items, exploratory rate 18-28% and the memory decay
2-5 chronologic items in memory storage. Following the systemic administration of ketamine(≤ 1.5 mg/kg), all these parameters dramatically changed. In contrast, systemic administrationof medetomidine (α2 adrenoceptor agonist, 10 µg/kg) reduced saccade velocity, but failed toalter the model parameters. Thus, our oculomotor paradigm and the foraging model appear
to be useful to quantitatively evaluate working memory capacity and utility, which might be(COI:No)applicable to clinical testing in neurodegenerative disorders in future studies.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S109

1P-109

Neural correlates of task performance in the mouse anterior cingulate
cortex

1P-112

What determines the set value (i.e., 37) of our core body temperature?
1 1 Div. Integr Physiol, Sch. Med,
Yuki Yoshimura1，Kazuomi Nakamura2，Tatsuo Watanabe（

Hiroshi Nishimaru, Riga Sachu, Yusaku Takamura, Jumpei Matsumoto, Taketoshi Ono,
Hisao Nishijo（System. Emotional Sci., Univ. Toyama, Toyama, Japan）

Fac. Med, Tottori Univ, Japan, 2 Div. Pathol Bioche, Sch. Life Science, Fac. Med, Tottori
Univ, Japan）

Deciding when to start and when to stop a certain behavior is crucial for the animal to survive.
The medial prefrontal cortex (mPFC) is implied in such decision making and controlling the
behavior based on a external and internal cues including reward signals. However, how themPFC dynamically codes the transition between active and inactive states still remains
unclear. Among the mPFC areas, the anterior cingulate cortex (ACC) has been shown to be
involved in a wide range of functions including motor control, emotion and motivation.
In this study, we investigated how the information for initiation and termination of the rewardseeking behavior is coded in the ACC by examining the neuronal signals in mice performing astop-and-go task on a spherical treadmill.
Three male mice were trained to perform a self-paced locomotor task with their head fixed andto run on the spherical treadmill to obtain a reward. In a single trial of this task, the mouse canstart running voluntarily but when it stopped, a visual stimulus was presented, and then it had
to start running again within 2 seconds to obtain sucrose/water solution as a reward. After 1-2
weeks of training to (90-100 trials per day), the success rate of the task exceeded 60% but theinterval between trials varied from several seconds to several minutes. In 423 sorted single
units in the ACC recorded by micro-drive tetrode arrary, the majority of them (273 units, 65%) showed activity related to the task. Among those task-related units, more than half of them(144 units, 53%) increased their firing rate when the mice were running while 24% (66 units)of them were active during the interval. These results indicate that the ACC neurons encode
information related to active/inactive states of the animal during goal directed behaviors.
(COI:No)

Purpose: Core body temperature (Tc) of homeothermal animals is set at around 37℃ ; however,
little is known about what determines the set value (i.e., 37) of Tc. In this study, we examined
the possibility that the Tc in pregnant mice establishes the set value of the Tc in their offspring.
Method: To this end, we cultured fertilized mice embryos in vitro at 37℃ or 38℃, which is
1℃ warmer than common embryo culture temperature, from pronuclear embryo to blastocyst.
Thereafter, we transferred these blastocysts into uteri of pseudo-pregnant mice. In 9-weeks
old male offspring, the Tc was measured using telemetry system. In addition, hypothalamus of
those animals was subjected to RNA-Seq analysis and real-time RT-PCR.
Results and Discussion: The Tc of mice derived from 38℃ cultured embryos (38℃-group)
was significantly lower than that of the control mice (37℃-group). However, there were no
significant differences in body weight and physical activity. The RNA-Seq analysis and realtime RT-PCR revealed that expressions of Insulin-like growth factor (Igf-1) and Igf-binding
protein 2 (Igfbp2) in the hypothalamus of 38℃-group were significantly higher than those of
the control mice. Igf-1 and Igfbp2 were also expressed in the liver; however, there were no
significant differences between the two groups. Judging from the previous reports, it seems
likely that Igf-1 increases the Tc thorough its action in the hypothalamus, and that this effectis being inhibited by Igfbp2. In order to examine whether upregulation of Igfbp2 is responsible
for the hypothermia seen in the 38℃-group, we have just started the new study, in which we(COI:No)are planning to analyze the Tc of the forebrain-specific Igfbp2 KO mice. 

1P-110

1P-113Brain activity measurement during art works viewing using NIRS
2 1Grad Sch HHS, Kyoto Univ, Japan, 2 Tenri Health Care Univ,
Mariko Yamagishi1, （Zymosan-induced fever and brain prostaglandin E2 production in miceare mediated by cyclooxygenase-2 expressed in brain endothelial cells

Japan）

Hiromu Kitagawa（ Dept Physiol, Grad Sch Eng, Osaka Inst Technol, Japan）

In this study, we examined the emotion-induced brain activity when a paired person appreciates art works consisted of various combinations of sounds and lights, using a 2-channel typewearable NIRS (near infrared spectroscopy), HOT-1000 (Hitachi High Technologies Corporation). Compared with PET, fMRI, and other multi channels NIRS using for emotion research,
HOT-1000 is a very small, lightweight, and low restraint measuring device that allows natural
art appreciation and free action. In this experiment, we created a cylindrical 3D art work withlight and sound as motifs, and asked healthy and familiar pair subjects to enter into the work.
During the subjects appreciating changes in lights and sounds, we measured and evaluated thebrain activity obtained using HOT-1000. We also evaluated effects of oral communication witheach other on the appreciative comprehension. As a result, it was suggested that the brain
becomes more active by talking with others during appreciation. On the other hand, several
problems required to be solved were remained to detect subtle changes in the brain activity
induced by changes in lights and sounds. Some of them were caused by the environmental and(COI:No)device-induced anxieties.

Purpose: Inflammation is associated with fever and hyperalgesia. These reactions involve thecentrally produced prostaglandin E2 (PGE2) as a common mediator. In this study we examined
the mechanism of fever and central PGE2 production in a mouse model of peripheral inflammation.
Methods: Mice were subcutaneously injected with zymosan at the plantar of hind paw. PBS
was injected as the control. In some experiments, celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, was administered intraperitonealy just after zymosan injection. Abdominal temperature
was recorded under free moving state. A separate group of mice were euthanized 3 h after
zymosan injection and blood was removed by cardiac perfusion. Their brains were sampled
for PGE2 analysis, immunological detection of COX-2, and ex vivo analysis of PGE2 production.
Results: Body temperature was elevated from 3 h to 9 h after zymosan injection. Celecoxib
significantly suppressed zymosan-induced rise in body temperature. Zymosan injection significantly elevated PGE2 contents of brain tissue compared to PBS. Zymosan induced COX-2
expression in blood vessels, especially in endothelial cells, rather than brain parenchyma as
revealed by immunohistochemistry and western blot. Isolated subarachnoidal blood vessels
and brain parenchyma were separately incubated in HEPES-Ringer solution and PGE2 released into the incubation medium was measured. Blood vessels produced more PGE2 thanbrain parenchyma from both zymosan and PBS injected mice. Furthermore, blood vessels
from zymosan injected mice produced more PGE2 than those from PBS-injected mice. These
results indicate that fever and central production of PGE2 during zymosan-induced peripheral
inflammation are mediated by COX-2 expressed in brain endothelial cells.
(COI:No)

1P-111

1P-114

Makoto Osanai1, 2，Hiroki Tanihira2，Ryo Inagaki1，Satomi Kikuta2, 3，Toshikuni Sasaoka4，
6 1Osaka Univ Grad Sch Med, Japan, 2 Tohoku Univ Grad Sch Med,
Atsushi Nambu5, （Shuhei Koeda1，Kanae Akiyama1，Manabu Saito2，Misaki Mikami2，Tamaki Mikami2，
1 1 Hirosaki Univ Grad Sch Health Sci,
Chihiro Sato1，Kazuhiko Nakamura2，Junko Yamada（The role of dopamine D1 receptor on the whole brain activity and on
the motor function
Japan, 3 Primate Res Inst, Kyoto Univ, Japan, 4 Brain Res Inst. Niigata Univ, Japan, 5 Div

Syst Neurophysiol, Natl Inst Physiol Sci, Japan, 6 Dept Physiol Sci, SOKENDAI, Japan）
Dopamine (DA) is one of the most important neurotransmitters in the central nervous system,
especially in the basal ganglia, and is believed to modulate the neuronal activities. In the striatum, direct pathway neurons express dopamine D1 receptors (D1R), whereas indirect pathway
neurons express dopamine D2 receptors (D2R). It is thought that this dichotomy contributes
to the distinct roles for direct and indirect pathway neurons of the striatum. However, how
DA signals modulate whole brain activities and how those affect behavioral properties has notbeen well explored.
To reveal the role of D1R, we conducted motor performance tests on D1R conditional knockdown (D1R-KD) mice. All behavioral tests indicated that the mice exhibited impaired motor
abilities when D1R expression was suppressed. Next, we conducted the whole brain activity
mapping in D1R-KD mice by quantitative activation-induced manganese enhanced MRI (qAIMMRI; Kikuta et al., 2015). qAIM-MRI is based on the use of Mn2+, which entered through Ca2+
channels in activated neurons, as a marker of neuronal activities. After intraperitoneal injectionof MnCl2, whole brain activities were measured by MRI. The results suggested that reduced
D1R expression led to increase activity in the nucleus accumbens, hippocampus, and thalamus(COI:No)of D1R knock-down mice compared to normal conditions.

S110

The nutrition and food intake of Japanese 5-years autism spectrum
disorder children
Japan, 2 Grad Sch Med, Hirosaki Univ, Japan）

The children with developmental disorders are at risk of having an unbalanced diet because of
sensory problems, such as hyperesthesia. The current study investigated the food and nutrition
intake of Japanese autism spectrum disorder (ASD) children.
Subjects were 994 children who underwent the Hirosaki Five-Year-Old Children Developmental Health Check-up. We investigated children’s nutrition status using the Brief-type
Self-administered Diet History Questionnaire 3 years (BDHQ3y). We divided children into threegroups of ASD group, development disorder (DD) group, typically development (TD) groupby the each diagnosis. The intergroup comparison of the volume of the food and nutrition ateach group was also achieved with the one-way ANOVA (Bonferroni correction was used as
post-hoc analysis). Data were analyzed with the SPSS version 26.0. Probability values p<0.05
were considered statistically significant.
ANOVA revealed statistically significant difference in the K, Vitamin K, Foric acid, Cryptoxanthin (F=3.0-5.4, p<0.05). The result of post-hoc analysis, the ASD group had fewer intakes ofvitamin K, natto and many intakes of the vegetable juice than TD group (p<0.05).
As a result of investigation, it was thought that the deficiency of vitamin K of ASD children
was caused by poor intake of the natto. There are many children who don’t like food of a strong
smell such as the fermented food in the ASD children. Influence of the hyperesthesia is thought
about for this behavior. Additionally, it was known to the ASD children that intestinal bacterial
flora had many evil bacteria, and deficiency of vitamin K was thought about in one of thecauses. And the excessive intake of the vegetable juice seems to be associated with vegetables
phobia. A protector might give it vegetable juice as a substitute of the vegetables phobia of the
ASD children.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

1P-115

Evaluation of postprandial sleepiness by event-related potential P300
3 1 Department of Information andShota Satake1，Hideaki Seki2，Hiroko Toshima（Communication Systems Engineering, Student, Chiba Institute of Technology, 2 Department
of Information and Communication Systems Engineering, Chiba Institute of Technology,
3 Department of Food and Nutrition, Toita Wemen’s College）

1P-118The spatial and time-dependent changes of various miRNAs
expression in the ischemic rat brain
Kana Sugimoto, Chihpin Yang, Yukie Murata, Rina Gono, Yuichiro Hirata, Teiji Harada,
Yohei Miyashita, Kazuma Higashisaka, Kazuo Harada, Ryuichi Katada, Hiroshi Matsumoto（Dept Leg Med, Grad Sch Med, Osaka Univ, Japan）

Purpose: People may feel strong sleepiness after lunch. We thought that this drowsiness wasdue to circadian rhythm, but we couldn’t deny the influence of the meal content of lunch. We
thought that if we were able to reduce afternoon drowsiness, we could improve afternoon
work efficiency. We wanted to find an objective and easy-to-measure sleepiness index in order
to examine the content of meals to reduce sleepiness after lunch. In this research, we focusedon the latency of the event-related potential P300, the response time at the time of P300
recording, and the power in the α band of EEG.
Subjents ＆ Method: Subjects were 10 young healthy male(21.5±0.7years old). Electroencephalograms were recorded using Polymate mini (Miyuki Giken). The related electrodes wereattached at Fz, Cz, and Pz of the international 10-20 method and the insensitive electrode was
attached binaural combined electrode. We have developed our own software for summing andanalyzing evoked potential and for frequency analysis of the power of α band of EEG. We
used high-carbo diet (carbohydrate 97.8 g) and low-carbo diet (46.8 g) as load diets to induce
postprandial sleepiness.
Results: The P300 latency, response time, and α-band power before loading of the highcarbo group and the low-carbo group were (378.7±53.2msec, 272.5±47msec, 15.2±1.2) and(384.0±81.6msec, 291.6±24.5msec, 16.0±1.4). There was no significant difference between
the two groups. After meal-loading thye were (444.7±98.7msec, 289.7±54.9msec, 15.8±2.1),(341.3± 44.1msec, 303.8 ± 31.6msec, 16.6 ± 2.5). Latency of P300 was significantly delyed in
the high-carbogroup (p= 0.041).
Conclusion: We thought that latency of P300 could be applied as a non-invasive objective
sleepiness index.
(COI:No)

Stroke is one of the leading causes of morbidity and disability in the world. However, thetherapeutic strategies for the treatment of this disease remain limited. MicroRNAs (miRNAs)
are small non-coding RNAs that are the key mediators for post-transcriptional gene silencing, and achieved by binding to the 3’untranslated regions of their target mRNAs. In thebrain, miRNAs likely contribute to most, if not all, neuroinflammatory processes, including thegeneration of reactive oxygen species, apoptosis, loss of blood-brain barrier integrity, leukocyte
migration and activation, and edema. However, miRNA-mediated gene regulation in ischemic
stroke and neuroinflammation have not been fully explored. In the present study, we evaluatedthe spatial and time-dependent changes of various miRNAs expression on a transient middle
cerebral artery occlusion (tMCAO) model of rats. Rats subjected to tMCAO were deeply
anesthetized with carbon dioxide at 1, 3, and 7 days postreperfusion (dpr). Tissues from thecerebral cortex representing the three regions: the ischemic core, the peri-infarct tissue andthe contralateral cortex were dissected out, which 750 miRNAs were analyzed using TaqMan
Array MicroRNA Cards. The expressions of mmu-miR-449a-5p, -34b-3p, -434-5p, -598-3p, and-126a-3p greatly changed in the peri-infarct tissue at 7 dpr, and mmu-miR-199a-3p, -155-5p,-21a-5p -376b-5p, 206-3p, and -137-3p in the core region at 7 dpr. These changes may be correlated with the appearance of activated astrocytes in the peri-infarct tissue and activatedmicroglia/macrophages in the ischemic core. Thus, we should elucidate the roles of thesechanged miRNAs in the ischemic stroke, suggesting that it can be explored as a potential
therapeutic option. (COI:No)

1P-116

1P-1193 1 Department of Information andKazuya Yamamoto1，Hideaki Seki2，Hiroko Toshima（

2 1 pLED, Tokushima Univ, Japan, 2 RIEM, Nagoya
Hiroki Takanari1，Satoru Hashimoto（

Effect of salt intake on gastric rhythm recorded by electrogastrogram
Communication Systems Engineering, Student, Chiba Institute of Technology, 2 Department
of Information and Communication Systems Engineering, Chiba Institute of Technology,
3 Department of Food and Nutrition, Toita Wemen’s College）
Purpose: It has been pointed out that there are 3cpm and 6cpm automatic movements inelectrogastrograms. We have found that the powers of these two types of movement alternatewith a regular rhythm. We reported that this rhythmic alternation appears only at night whenparasympathetic function is dominant, the middle disappears, and is affected by the contents
of dinner. The previous report suggested that gastric rhythm was influenced by the amount of
supper salt, so this time we examined the rhythm at night after ingesting two test meals withsignificantly different salinity.
Subjects ＆ Method: The subjects were 5 young healthy men (21.7 ± 0.5 years old). Electrodes(Nihon Kohden, Vitrode V) were affixed at two locations across the epigastric region, theground electrode was affixed under the left clavicle, and an electrogastrogram was recorded
on a portable data recorder (Biolog 2000, S ＆ ME). After starting the recording of electrogastrogram at 19:00, he took a loaded meal, and finished recording at 7:00 the next morning. In a
subject, two kinds of load diets, a high salt diet (8.9 g salt) and a low salt diet (5.1 g salt) were
loaded on different days. The electrogastrogram was frequency analyzed with an originallydeveloped software.
Results: In all subjects, a clear rhythm was observed in the 6 cpm power / 3 cpm curve atnight after a low salt diet, with periods of 0.067 cpm and 0.033 cpm. Only a small rhythm of
0.067 cpm was observed at night after a high salt diet, and the 0.033 cpm swell disappeared.
Discussion: The disappearance of rhythm appeared after 5 hours after dinner, and it wasthought that the rhythm disappeared due to the influence of the supper contents that caused
some liquid change.
(COI:No)

1P-117

Gene expression of hypothalamic feeding related peptides
and neuroendocrine responses in an experimental allergic
encephalomyelitis rat
Kentaro Tanaka1, 2，Mitsuhiro Yoshimura1, 3，Kazuhiko Baba1，Kenya Sanada1，Haruki Nishimura1，
Kazuaki Nishimura1，Satomi Sonoda1，Hiromichi Ueno1，Reiko Saito2，Takashi Maruyama1，
1 1 Dept Physiol, Sch of Med, UOEH,
Tatsushi Onaka4，Koichi Kusuhara2，Yoichi Ueta（
Kitakyushu, Japan, 2 Dept Pediatr, Sch of Med, UOEH, Kitakyushu, Japan, 3 Dept Translational
Health Sciences, Sch of Med, Bristol, UK, 4 Dept Physiol, Sch of Med, Jichi, Shimono, Japan）

Experimental allergic encephalomyelitis (EAE) is a model of human multiple sclerosis (MS). MS
shows various clinical symptoms, including inflammatory anorexia. In the present study, we
examined the changes in body weight and food intake, the expression of hypothalamic feedingrelated peptides and neuroendocrine responses such as the hypothalamo-neurohypophysial
hormones (arginine vasopressin (AVP) and oxytocin) and the hypothalamo-pituitary adrenal(HPA) axis in EAE rats. The weight gain and cumulative food intake in EAE rats weresignificantly lower than those in controls. Gene expression of the hypothalamic orexigenic
peptides (NPY, AgRP) in EAE rats were significantly increased at day 12 and day 18 after theirimmunization compared with controls. The gene expression of the hypothalamic anorectic
peptides (POMC, CART) in EAE rats were significantly decreased at day 12 and day 18 after
their immunization compared with controls. The gene expression of oxytocin but not AVP in
the supraoptic nucleus and the whole paraventricular nucleus (PVN) of the hypothalamus of
EAE rats was significantly increased, along with elevation of plasma oxytocin but not AVP at
day 12 and day 18 after their immunization compared with controls. At day 12 after immunization, CRH gene expression was downregulated and AVP gene expression was upregulated in
the parvocellular division of the PVN compared with controls. Furthermore, gene expressionof POMC in the anterior pituitary was significantly increased, along with elevation of plasma
corticosterone at day 6, 12 and 18 after immunization compared with controls. These results
suggest that inflammatory anorexia in an EAE model induced by immunization may be
associated with dysregulation of hypothalamic feeding-related peptide gene expressions and(COI:No)
activation of both oxytocinergic pathways and HPA axis via AVP but not CRH.

The inﬂuence of KATP channel dysfunction on mitochondria
Univ, Japan）

Background: The ATP sensitive potassium channel (KATP channel) exists on mitochondrial
inner membrane (MIM) to maintain respiratory function of mitochondria via stabilizing MIM
polarity. We explored the influence of gain-of-function mutation of KATP channel on mitochondrial function.
Methods: Human fibroblasts (hFBs) from healthy volunteer and the patient with gain-of-function
mutation on ABCC9 gene, which encodes regulatory subunit of KATP channel, were subjectedto the experiments. The ATP production in each cell was evaluated using IntraCellular ATP
assay kit (Toyo-b-net). Mitochondrial respiratory function was analyzed using extracellular flux
analyzer, XFe24 (Agilent Technologies), by estimating oxygen consumption rate (OCR). The
membrane potential of MIM was evaluated by fluorescent imaging of TMRE.
Results: The IntraCellular ATP assay revealed that total ATP production at baseline was
not changed by KATP channel dysfunction. There was no significant difference in baseline
OCR between wild type and mutated hFBs, either. However, maximum OCR measured
in the presence of 2 µM FCCP was significantly decreased in hFBs with mutated KATP
channel compared with wild type hFBs, suggesting that the reserved respiratory function of
mitochondria was suppressed by KATP dysfunction. Fluorescent microscopy showed that themembrane potential of MIM was lower in hFBs with KATP channel mutation than in wild
type hFBs, suggesting that KATP channel dysfunction caused depolarization of MIM.
Conclusion: Gain-of-function mutation of KATP channel caused the impairment of reserved
respiratory function of mitochondria via depolarization of mitochondrial inner membrane.
(COI:No)

1P-120

Visualization of epileptogenic activities in human hippocampal slices
ex vivo
1 1 Dept Pathol, Brain
Hiroki Kitaura1，Masafumi Fukuda2，Yukihiko Fujii3，Akiyoshi Kakita（

Res Inst, Niigata Univ, Japan, 2 Dept Neurosurgery, Nishi-Niigata Chuo Hosp, 3 Dept
Neurosurgery, Brain Res Inst, Niigata Univ）

Introduction: Mesial temporal lobe epilepsy (MTLE) is the most frequent focal epileptic
syndrome in adults, and the majority of seizures originate primarily from the hippocampus.
The resected hippocampal tissue often shows severe neuronal loss as that referred to hippocampal sclerosis (HS). Accordingly, there is a paradox between the clinical and pathologicalfeatures: why should epilepsy be derived from such degenerated tissue? Here we investigatedepileptiform activities ex vivo using living hippocampal tissue taken from patients with MTLE.
Methods: We prepared acute brain slices from patients with MTLE within 45 min after
resection, and optical imaging or local field potential recordings (LFP) was performed ex vivo.
We also used a brain block corresponding to the mirror surface of each slice and performed
histopathological examination.
Results: We revealed that epileptiform activities developed from the subiculum, regardless ofthe existence of HS. We found spontaneous rhythmic activities in the subiculum and detecteddiscrete component of high frequency oscillations (HFO), a clinical biomarker of the ECoG
suggesting the epileptogenic regions. Immunohistochemistry of the HS tissue revealed loss ofinwardly rectifying K+ channel 4.1 (Kir 4.1) in astrocytes in the subiculum, indicating failure of
the extracellular K+ buffering and possible association with neuronal hyperexcitability.
Conclusion: These results indicate that pathophysiological alterations involving the subiculum
could be responsible for epileptogenesis in patients with MTLE.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S111

1P-121

1P-124

Takaya Yamasaki1, 2，Shunya Nakayama1, 2，Hiroshi Koie1，Chungyu Pai1, 2，
Yasuyo Ito-Fujishiro1, 2，Kiichi Kanayama1，Yoshiko Munesue2，Tadashi Sankai2，2 1 Laboratory of Veterinary Physiology, Nihon
Yasuhiro Yasutomi2，Naohide Ageyama（

Mutsumi Takahashi1，Yogetsu Bando2, 3，Takuya Fukui4, 5，Akiko Maruyama4, 6，Masaaki Sugita7，
1 1 Dept Physiol, Nippon Dent Univ, Niigata, Japan, 2 Grad Sch of
Kojun Tsuji1，Yoshihide Satoh（

Cardiac disease, especially myocarditis and cardiomyopathy are the leading cause of humanmorbidity and mortality. It has been suggested that a part of dilated cardiomyopathy and myocarditis is associated with autoimmune abnormality. Further, the rodent model of experimental
autoimmune myocarditis (EAM) has been reported that the T-cell immune system leading
main cause of autoimmune in cardiomyocytes. These from, we have been developing EAM
model using cynomolgus monkeys, which are closely related to humans, for model of dilated
cardiomyopathy. In this study, we aimed to analyze the immune response in autoimmune
myocarditis with immunohistochemical method using EAM model of cynomolgus monkeys.
We used 5 healthy monkeys which kept same environment in TPRC. We injected to immunize
intradermal on medial femoral with plain myosin, and induced autoimmune myocarditis. We
performed cardio-specific examinations including echocardiography, ECG, chest X-ray andblood tests. After several weeks, we conducted immunohistochemical and histopathologicalexaminations. As a result, histopathological analysis revealed interstitial fibrosis, complicatedmyocardium and infiltrated lymphocytes such as dilated cardiomyopathy. And other medical
exam revealed remarkable augment of cardiac hormone, decrease of ejection fraction, abnormal ECG and regurgitation of cardiac valves. Those findings provided the diagnosis of heart
failure that mimic the human myocarditis. Immunopathologically, infiltrated lymphocytes in
cardiac tissues were mainly CD3 and CD4 positive cells, and some infiltrated cells were stainedwith CD68. In addition, a part of injured cardiomyocytes expressed IL-1β. From these, it wassuggested that EAM model of cynomolgus monkeys also lead the T-cell immune response.
Moreover, these results suggested that the expression of IL-1β and CD68+ macrophagewere related to mechanism of myocardial injury. In conclusion, EAM models of cynomolgus
monkeys could be a mimicked model of human myocarditis and it is ideal models that elucidate(COI:No)
the pathophysiological mechanism of myocarditis and cardiomyopathy.

The aim of this study was to evaluate the relationship between occlusal state and static posture control function in trampoline gymnast. The subjects were 12 male trampoline gymnasts(19.7±1.4 years) and 11 healthy men (20.5±1.1 years). Dental Prescale was used to evaluate
the occlusal state, and the right-and-left difference of the occlusal contact area (occlusal stability) was determined. A single-layer mouthguard was fabricated using a 3.0-mm-thick-sheet.
The static posture control function was evaluated by a gravity center fluctuation meter, andthe area enclosed by the trajectory of gravity center fluctuation (ENV-AREA) as the sizeof center of gravity sway and locus length per unit area (LNG/E-AREA) as fineness of attitude control were used for analysis. The measurement were performed when wearing and
not wearing the mouthguard (wearing-MG/no-MG). The correlations between the occlusal
stability and ENV-AREA, or LNG/E-AREA were analyzed. The difference of ENV-AREA
or LNG/E-AREA depending on wearing-MG or no-MG was analyzed. In the healthy men, asignificant positive correlation was found between the occlusal stability and ENV-AREA, anda negative correlation was found between the occlusal stability and LNG/E-AREA. However,no significant correlation was found in trampoline gymnasts. ENV-AREA was smaller in
trampoline gymnasts and healthy men when wearing-MG than when no-MG. LNG/E-AREA
was greater in trampoline gymnasts when wearing-MG than when no-MG. There were difference between trampoline gymnasts and healthy men in ENV-AREA and LNG/E-AREA
when wearing-MG. The relationship between occlusal state and static posture control differeddepending on the target, and it was clarified that the effect of the equalization of occlusal
contact by wearing a mouthguard on static posture control function was more prominent
in trampoline gymnasts than in healthy men. This work was supported by JSPS KAKENHI(COI:No)
Grant Number JP18K09668.

Immunohistochemical analysis of Experimental Autoimmune
cardiomyopathy Model (EAM) in Nonhuman Primates

Univ, Japan, 2Tsukuba Primate Research Center, NIBIOHN, Ibaraki, Japan）

Examination of relationship between occlusal contact state and static
posture control function: Comparison of trampoline gymnasts andhealthy subjects
Health and Sport Sci, Nippon Sport Sci Univ, Tokyo, Japan, 3 BANDO Dental Clinic, Ishikawa,Japan, 4 Facul of Health and Human Sci, Kanazawa Gakuin Univ, Ishikawa, Japan, 5 JGA, Tra,
Commit, 6 JGA, Tra, Reinforce, 7 Facul of Sport Sci, Nippon Sport Sci Univ, Tokyo, Japan）

1P-122

1P-125

Mikio Hayashi（Dept Cell Physiol, Inst Biomed Sci, Kansai Med Univ, Hirakata, Japan）

Yoshihiro Yamashina1，Kazuyuki Tabira2，Hiroki Aoyama1，Hirofumi Hori1，Emiko Morita1，
1 1 Dept Physical Therapy, Aino Univ, Osaka, Japan,
Nami Sakagami1，Tomoko Hirayama（

Involvement of A2A and A2B adenosine receptors in physiological andpathophysiological functions in pancreas
Introduction: Adenosine is considered to play a role in acini-to-duct signaling in the exocrine
pancreas. A2B adenosine receptors is involved in anion secretion in a human pancreatic
adenocarcinoma cell line. However, the molecular basis of functional adenosine receptors in the
exocrine pancreas remain inconclusive.
Objectives: The present study focused on A2A and A2B adenosine receptors involved in physiological and pathophysiological functions in the pancreas.
Methods: Immunohistochemical analysis was performed in the rat, mouse, and guinea pig
pancreas. The secretory rate and concentration of HCO3－ in pancreatic juice from the rat
pancreas were measured. In silico analysis was performed of data from The Cancer Genome
Atlas database.
Results: The A2A adenosine receptor colocalized with ezrin, an A-kinase anchoring protein, in
the luminal membrane of duct cells in the mouse and guinea pig pancreas. The A2A adenosine
receptor agonist CGS 21680 stimulated HCO3－-rich fluid secretion from the rat pancreas. Low
mRNA expression of ADORA2A, which encodes the A2A adenosine receptor, was associatedwith poor prognosis of human pancreatic adenocarcinoma patients. In contrast, high expressionof ADORA2B was associated with poor prognosis.
Conclusion: These results indicate that A2A adenosine receptors may be, at least in part,
involved in exocrine secretion of pancreatic duct cells via acini-to-duct signaling. The adenosine
receptors may be a potential therapeutic target for cancer as well as exocrine dysfunctions of
the pancreas. (COI:No)

Effects of Exercise Load when Walking in Water on Respiratory Muscle
Strength in Elderly Men
2 Dept Physical Therapy, Kio Univ, Osaka, Japan）

Purpose: The purpose of the present study was to investigate the effects of exercise load when
walking in water on respiratory muscle fatigue in elderly men.
Methods: Eight healthy elderly men (age 67.2±2.6 years)participated in 15-min walking trials inwater at 40%, 50%, and 60% of their predicted maximum heart rate. We measured respiratory
muscle strength by evaluation of maximum inspiratory and expiratory pressure (PImax, andPEmax, respectively) before and after walking trials. PImax and PEmax were evaluated using
a sthenometer attached to the spirometer (AAM337, Minato, Osaka, Japan) and were considered the surrogate indices of inspiratory and expiratory muscle strength, respectively. The
percent changes in PImax and PEmax following the walking in water (Δ%PImax, Δ%PEmax)
were calculated.
Results: After walking at 60% of the predicted maximum heart rate, PEmax decreased significantly than before walking trials (p<0.05); however, no changes in PEmax were observed under
the other load conditions. The Δ%PEmax after walking at 60% of the predicted maximum
heart rate was significantly greater than those in the other load conditions. The PImax did notchange under all exercise load conditions.
Conclusions: We demonstrated that, in elderly men, a greater expiratory muscle fatigue was
induced by walking in water at a 60% load of the predicted maximum heart rate. Our results
indicate that walking in water may be an effective and preferable option for strengthening
expiratory muscles in the setting of cardiopulmonary training or respiratory rehabilitation.
(COI:No)

1P-123

1P-126

Yasuhito Motojima1, 2，Mitsuhiro Yoshimura3, 4，Yoichi Ueta3，Akinori Sakai1

Univ Sch Med, Tokyo, Japan）

Evaluation of surgical incision-induced tissue swelling and Fos-like
immunoreactivity in the spinal cord, hypothalamus, thalamus, andamygdala of Trpv1 knockout and Trpv4 knockout mice
（ 1 Dept Orthopaedic Surgery, Sch Med, UOEH, Japan, 2 Medical Department, Oita HCC,

Japan, 3 Dept Physiology, Sch Med, UOEH, Japan, 4Translational Health Science, Bristol
Med Sch, Bristol Univ, UK ）Pain management is a major concern regarding the treatment of postoperative patients.
Transient receptor potential (TRP) channels are considered to be new therapeutic targets forpain control. However, details of pain pathways which are involved in TRP channels remain
unclear. We evaluated tissue swelling and the number of Fos-like immunoreactive (Fos-LI)
positive cells in the dorsal horn of the spinal cord, paraventricular nucleus of the hypothalamus(PVN), paraventricular nucleus of the thalamus (PVT), and central amygdala (CeA) in wildtype (WT), Trpv1 knockout (Trpv1-/-), and Trpv4 knockout (Trpv4-/-) mice after surgicalincision. Mice were divided into four groups: WT control, WT incision, Trpv1-/- incision, andTrpv4-/- incision. Mice were anesthetized, and only those in the incision groups received asurgical incision to their right plantar hind paw. Changes in paw diameter and in the numberof Fos-LI positive cells in the dorsal spinal cord, PVN, PVT, and CeA were evaluated 2 hours
after the incision. There was no statistically significant difference in the paw diameter among
incision groups. Fos-LI positive cells in laminae I-II of the dorsal spinal cord and PVN was
significantly increased in all incision groups compared to the WT control group. A significant
increase in Fos-LI positive cells was also observed in laminae III-IV of the dorsal spinal cordin Trpv1-/- and Trpv4-/- incision groups compared with the WT incision group. There wasno significant difference in Fos-LI positive cells in lamina V of the dorsal spinal cord, PVT,
and CeA in Trpv1-/- and Trpv4-/- incision groups compared with the WT incision group. Our
results indicate that Trpv1 and Trpv4 might be involved in pain pathways in laminae III-IV ofthe dorsal spinal cord.
(COI:No)S112

Characteristics of the burst generating networks released bydisinhibition in the spinal cord of the neonatal rat
Shu Takahashi, Makito Iizuka, Yoshihiro Mikami, Masahiko Izumizaki（Dept Physiol, Showa
Disinhibition of the spinal cord caused spontaneous seizure-like motor activity. Previous study
suggested that the disinhibition could cause burst activities in the phrenic nerve, and thispropriospinal network could be harnessed to allow diaphragm function after spinal cord injury.
However, the characteristics of this network has not been fully clarified. In the present study,
to characterize this network, the isolated spinal cord from neonatal rats at P0-3 was cut into
blocks with three spinal segments, i.e., C3-5, T6-8, L2-4 and L6-S2, and motor activities wererecorded from C4, T7, L3 and S1 ventral roots (VRs). Under control conditions, there were
few spontaneous activities in all four VRs. When 10 ＆mu;M strychnine, a broad antagonist ofglycine and GABAA receptors, was applied, spontaneous burst activities were observed in all
VRs. This result suggests the burst generating network (BGN) exists in each segment. Next,
using the whole spinal cord preparations from C3 to S2 segments, effects of local disinhibition in the C3-5 segments on the BGNs in other segments were examined. Under control
conditions, spontaneous activities frequently occurred in all VRs, and those amplitudes anddurations were fluctuated. When strychnine was locally applied to the C3-5 segments, the large
spontaneous burst activities concurrently occurred not only in the C4VR but in all other VRs.
The occurrence frequency was much higher than that in the block preparations. The electricalstimulation of the C4 dorsal root evoked reflex response in the C4VR and weak reflex response
in other VRs under control conditions. Under the local disinhibition in C3-5 segments, same
stimulation caused burst activity in all VRs recorded. These results suggest that the BGNs arestrongly connected to each other, and it would be difficult to drive BGN individually. (COI:No)

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

1P-127

Involvement of the Phox2b-positive neurons located in the dorsal
medulla in the sucking rhythm generation
Makito Iizuka1，Keiko Ikeda2，Hiroyuki Igarashi3，Kazuto Kobayashi4，Hiroshi Onimaru1，
1 1 Dept Physiol, Showa Univ Sch Med, Tokyo, Japan, 2 Dept Physiol,
Masahiko Izumizaki（

Int Univ Health and Welfare, Chiba, Japan, 3 Dept Physiol Pharmacol, Schulich Sch Med
Dent, Robarts Res Inst, Western Univ, Canada, 4 Dept Molecular Genetics, Inst Biomed Sci,
Fukushima Med Univ Sch Med, Fukushima, Japan）

We developed a transgenic (Tg) rat in which Phox2b-positive neurons expressed one of channelrhodopsin variants; ChRFR(C167A), and found that the photo-stimulation of the Phox2bpositive neurons from the dorsal skull causes sucking movement in this Tg neonatal rat at freemoving conscious condition. Furthermore, the local photo-stimulation of the dorsal region of the
medulla near the solitary nucleus, where the Phox2b-positive neurons are clustered, caused thesucking motor activity in the phrenic and hypoglossal nerves in the isolated brainstem spinal
cord preparations from the Tg neonatal rats. In the present study, to examine the role of thePhox2b-positive neurons in the sucking rhythm generation, the whole cell recordings were obtained from cells in the dorsal medulla, and pattern of activity during the sucking rhythm was
examined. The recorded cell was stained with 0.2% Neurobiotin for later visualization. Phox2b
immunostaining was also carried out to check whether the recorded cell was Phox2B-positiveor -negative. Many of Phox2b-positive neurons were depolarized by photo-stimulation, but did
not fire in phase with the sucking rhythm. Many of neurons that showed the sucking rhythmic
firing were Phox2b-negative. However, a few neurons that fired synchronous with the sucking
motor bursts were Phox2b-positive. It has been shown that the Phox2b-positive neurons areglutamatergic. To examine the involvement of the inhibitory neurons in the sucking rhythm
generation, a broad antagonist of the glycine and GABAA receptors, 10 ＆mu;M strychnine
was applied. Under strychnine, photo-stimulation still could evoke the sucking motor bursts
without any apparent effects on the rhythm frequency, although the seizure-like activity often
obscured the sucking activity. The present results suggest that the sucking generators consist
from excitatory interneuronal networks, and the Phox2b-positive neurons firing in phasewith the sucking motor bursts could be constituents of the sucking rhythm generator itself.
(COI:No)

1P-130Analysis of the function of bcl-7 in the LIN-28 / let-7 pathway
Luna Izuhara1，Sawako Yoshina 1，Sayaka Higuchi2，Yuji Suehiro1，Shohei Mitani1, 2（ 1 Dept Physiol, TWMU, Tokyo, Japan, 2TIIMS, TWMU, Tokyo, Japan）

The human BCL7 gene family functions as a tumor suppressor, and is involved in cancer
development and progression. However, there are still many unclear points about the mechanisms. We previously reported that the C. elegans bcl-7 gene, which is the only homolog of the
BCL7 gene family, is involved in the regulation of self-renewal ability as stem cells, and actsin the Wnt signaling pathway. However, because several phenotypes of the bcl-7 mutants aredifferent from those of mutants for Wnt signaling, there is a possibility that pathways otherthan Wnt also work with BCL-7.
We previously reported that the C. elegans bcl-7 mutant influences the development of seamcells, which have both self-renewal potential and differentiation capability. So we sought therelationship between bcl-7 and the LIN-28 / let-7 pathway that regulates the seam cell differentiation.
In this study, using RNA interference (RNAi) experiments, we investigated the relationshipbetween bcl-7 and the LIN-28 / let-7 pathway. We found that bcl-7 RNAi alleviated the sterile
phenotype of the lin-28 mutants, while lin-28 RNAi did not drastically affect that of bcl-7
mutants. Also, bcl-7 RNAi alleviated the lethal phenotype of the let-7 mutants.
Next, we investigated the relationship between bcl-7 and regulation of let-7 expression. Whenthe expression level of bcl-7 decreased, the expression stage of let-7, which is a representative
factor, became earlier. And this effect is inherited through generations.
We investigated whther the transgenerational inheritance of parental phenotypes are involvedin epigenetic modifications. Now, we found that bcl-7 may regulate histone methylation.
In the future, we will further clarify the relationship between maintaining the undifferentiatedstate of stem cells and functions of bcl-7, lin-28 and let-7 genes.
(COI:No)

1P-128

1P-1312 1 Div Neurosci, Sch
Yoshiya Matsuzaka1，Teruaki Watabe2，Junko Kawakami2，Kenji Hoshi（

Akinaga Kusano1，Takeshi Sakamoto10, Miyu Mori2，Yuki Yoshida3，Hayata Kimura4，
Shinnosuke Tomita5，Miho Sakuma6，Hiromu Ito3，Risa Tanaka7，Naoki Hashimoto8，
Michinari Kawahara2，Noriko Mukai2，Iori Nishida9，Mion Horie7，Shota Sagasaki4

Application of unsupervised machine learning to analysis of large
scale, multi-dimensional neuronal data
Med, Tohoku Med Pharm Univ, 2 Div Med Pharm Info Sci, Grad Sch Pharm, Tohoku Med
Pharm Univ）

The participation experience to Integrated Laboratory Practice: we havedecided the experimental issues by ourselves

Recent neuroscience studies requires the analysis of increasingly large scale of simultaneously
recorded neuronal population and the number of features obtained for individual neurons
including morphological, immunohistochemical and functional properties. Yet the conventional
analysis protocol depends on criteria subjectively set by human scientists, which leaves open
the possibility of overlooking either important features or relation between features. In thepresent study, we examined the efficacy of unsupervised machine leaning in objectively
categorizing neuronal data each of which is characterized by complex features. Two Japanesemacaques were trained to perform a two choice arm reaching task in which a color cue
instructed the monkeys either to reach to or away from the spatial cue which subsequently
appeared either on the left or the right of the working space. The neuronal data were recorded
from the posterior medial prefrontal cortex (pmPFC), presupplementary and supplementary
motor areas (preSMA and SMA, respectively). Each neuron was characterized by elevenfeatures that were defined by their temporal profile of spiking activity and the areas where
they were recorded. The multidimensional data of 492 neurons were categorized by Kohonen’s
self-organizing map (SOM). The SOM successfully identified several neuronal clusters thatwere either common across or specific to respective cortical area. Also, it objectively measured
and visualized the degree of similarities as well as dissimilarities between the identified neuronal clusters. Finally, we examined how the neuronal clusters were reorganized across different
task conditions. Our preliminary results indicated that machine learning is a viable data-mining
tool for data that is growing both in the size of neuronal population and complexity in future
neuroscience studies.
(COI:No)（ 1 Faculty of medicine, Oita university, Japan, 2 Gifu University Faculty of Medicine,
3 University of Fukui Faculty of Medicine, 4 Chubu University College of Life and Health
Sciences, 5 Fujita Health University Faculty of Medicine, 6Tokyo Women’s Medical
University Faculty of Medicine, 7 National Defense Medical College, 8 Hyogo College ofMedicine, 9 Akita University Faculty of Medicine, 10 Kindai University Faculty of Medicine）

1P-129

1P-132

Assessment of actin cytoskeleton by ﬂuorescent polarization
microscopy
2 1 Mec sci, Fac Sci Tec, Tokushima Univ, Japan,
Yohei Kanemura1, 2，Hiroki Takanari（
2 Institute of Post-LED Photonics）

Background: Cytoskeleton including actin plays an important role in maintaining cell morphology and its movement. In the research field of mechanobiology, proper assessment of
the cytoskeletal response against the external force is important. Subcellular distribution ofactin can be observed by fluorescent microscopy. However, the microstructure or dynamicchanges of cytoskeleton due to mechano-stress can hardly be observed with an ordinary optical
microscope. Polarization microscopy is a technique that is generally used in physical property
analysis. It is also possible to analyze stress applied to a substance by polarization analysis. We
propose a new method for measuring the mechano-stress on the cytoskeleton by polarization
microscopy. To achieve this issue, we first confirmed that the fluorescence-labeled actin fiber
could be observed by polarization microscopy.
Methods ＆ Results: A custom-made polarization microscope was established to observe
fluorescence polarization. A polarizer was placed immediately after the light source and ananalyzer was placed immediately before the detector. Actin cytoskeleton in HeLa cells fixedwith 4% paraformaldehyde was labeled with rhodamine by immunostaining method, andplaced on the microscopic stage. The changes in the intensity of detection light was acquired
while rotating the polarizer, then the fluorescence intensity of rhodamine-labeled actin fiber
was periodically changed. The time phase of the detection light change was shifted between
the vertical fiber and the horizontal fiber with respect to the microscopic field, suggesting that
this was not an artifact, but a change in the detected light accompanying the polarizer rotation.
However, the polarization extinction ratio due to polarizer rotation was only 3.5, it was thoughtthat improvements of the polarizer and optical system were still necessary.
Conclusion: Fluorescent polarization microscope was established, and polarization dependencyof fluorescent labeled actin fiber was confirmed. Fluorescent polarization microscopy could be
a powerful tool for analyzing the microstructure of actin cytoskeleton.
(COI:No)

Purpose: Participation to Integrated Laboratory Practice (August, 2019 at Department of
Physiology Gifu University).
Methods & Results: The participants were from many different Universities with year 1-6,
who were divided into 3 groups. After reading a case sheet describing haemorrhagic shock,group discussion was followed. After deciding the experiments design, day 2-4 was for theexperiments though it was modified after observing/analyzing the results. Main theme wasoxygen and blood pressure (group A), hypertonic solution on shock state (group B), and plasm
outflow to the third space during shock (group C). Monitored issues were blood pressure, ECG,
electrolytes, hematocrit, et ctr. At day 5, the results was presented. The organizer behaved asa tutor (not giving any answer, but questions), The ethics committee made a round. At final
presentation (September), new search results and/or histological analysis were added.
Conclusion: We were happy to decide the experimental protocol by ourselves. The group
discussion was useful/stimulus for the junior students. We all appreciate this project, laboratory experiments without guidebook.
(COI:No)

Concentrations of blood lactic acid and blood glucose levels during
incremental and decremental treadmill exercise
Tadashi Saitoh, Kyuichi Niizeki（Dept Bio-Systems, Grad Sch Sci Eng, Yamagata Univ, Japan）
The aim of this study was to investigate changes in concentrations of blood lactic acid andblood glucose levels during incremental and decremental exercise. One subject who exercised
regularly participated in this study. The protocol of incremental exercise constituted of 5 setsof running between 8 km/h and 16 km/h on a treadmill, with an increase of 2 km/h after
each set. The decremental exercise protocol was the reverse of the incremental exercise
protocol. There was also a 2 min rest period before and after the exercise test and between thesets. During rest, concentrations of blood lactic acid and blood glucose levels were measured
using blood obtained from pricking the subject’s fingertip. Heart rate was calculated during
the exercise test using electrocardiography. Each exercise test was conducted 7 times inthe total study period. The concentrations of blood lactic acid after the subject exercises at
speeds over lactic threshold level in incremental exercise were significantly higher than thatin decremental one. However, the blood glucose level and heart rate were not significantly
different between incremental and decremental exercises. These results suggest that the effectof a warm-up exercise is greatly reflected in the concentration of blood lactic acid. (COI:No)

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S113

Poster Presentations
Day 2（ March 18 , 14 : 20 〜 15 : 20 ）2P-001〜2P-018

Ion Channel・Receptor（ 2 ）2P-019〜2P-033

Heart・Circulation（ 2 ）2P-034〜2P-051

Neuron・Synapse（ 2 ）2P-052〜2P-064

Sensory Function（ 2 ）2P-065〜2P-074

Behavior Science・Biorhythm（ 2 ）2P-075〜2P-078

Neurochemistry（ 2 ）2P-079〜2P-084

Autonomic Nervous（ 2 ）2P-085〜2P-092

Muscle Physiology（ 2 ）2P-093〜2P-098

Oral Physiology（ 2 ）2P-099〜2P-104

Endocrinology（ 2 ）2P-105〜2P-107

Kidney・Urination（ 2 ）2P-108〜2P-114

Motor Function（ 2 ）2P-115〜2P-119

Reproduction

2P-120〜2P-123

Development・Growth・Aging（ 2 ）2P-124〜2P-127

Digestion・Absorption

2P-128〜2P-139

Cell Physiology・Molecular Physiology（ 2 ）2P-140〜2P-145

Environmental Physiology（ 2 ）2P-146〜2P-148

Drug Actions（ 2 ）2P-149〜2P-158

CNS Function（ 2 ）2P-159〜2P-167

Nutrition・Metabolism・Thermoregulation（ 2 ）2P-168〜2P-175

Pathophysiology（ 2 ）2P-176〜2P-178

Physical Fitness・Sports Medicine（ 2 ）2P-179〜2P-181

Blood

2P-182〜2P-184

Respiration（ 2 ）2P-185〜2P-187

Study Methodology（ 2 ）2P-188〜2P-190

Others（ 2 ）2P-001

Expression of Mechanosensitive Ion Channel in Osteoblast
Sayoko Nagai1, 2，Haruna Toda1，Sadao Ooyama1，Wataru Oofusa1，Asuka Higashikawa1，2 1 Dept Physiol, Tokyo Dent Coll,
Maki Kimura1，Yoshiyuki Shibukawa1，Akira Katakura（

2P-004“Knock-off” and “lock-in” of the polyamine block by K+ ions determine
the position along the voltage axis of the current-voltage relationship ofthe Kir2.1 inward rectiﬁer

Japan, 2 Oral Pathobiological Sci Surg, Tokyo Dent Coll, Japan）

Keiko Yanagi-Ishihara（ Dept Physiol, Kurume Univ Sch Med, Kurume, Japan）

Introduction: Mechanical stress is one of the important regulatory factors to regulate bonehomeostasis. Although it has been reported that application of stress stimulation to osteoblasts
elicits an increase in intracellular Ca2+ concentration ([Ca2+] i), their detailed mechanism of themechanosensitive processes remains unclear. We thus investigated the biophysical and pharmacological properties of direct mechanical stimulation-induced [Ca2+] i response in osteoblasts.
Method: Mouse osteoblast-like cells, MC3T3-E1, were cultured for 12 to 24 h in 5% CO2 at
37℃, and loaded with Ca2+ fluorescent indicator fura-2/AM for 1 h. The standard extracellularsolution was Krebs solution, and we measured [Ca2+] i responses during plasma membrane
stretch with hypotonic extracellular solution or direct mechanical stimulation with a glass micropipette, we utilized several mechanosensitive ion channel blockers, Gd3+, GsMTx4, RN1734,
HC030031, and clemizole. To activate Piezo1 channel, we applied Yoda1 to the osteoblasts.
Result: An application of hypotonic Krebs solution and Yoda1 solution increased [Ca2+] i in theosteoblasts. When direct mechanical stimulation was applied, [Ca2+] i was increased and notshowed significant desensitizing effects. Extracellular Gd3+, GsMTx4 and RN1734 reversibly
inhibited mechanical stimulation-induced [Ca2+] i increases. When the concentration of Gd3+
or GsMTx4 were changed, changing in the increases of [Ca2+] i was observed in their dosedependent manner.
Discussion: Gd3+, GsMTx4 and RN1734 are inhibitors of mechanosensitive ion channels,
and significantly suppressed [Ca2+] i increases induced by direct mechanical stimulation tothe osteoblasts. Mechanosensitive ion channels might be involved in the mechanosensitive
processes of osteoblasts.
Yoda1 is an activator of Piezo1 channel while GsMTx4 is an antagonist for it. Piezo1 channel
thus might be the one of the functional mechanosensitive ion channel in osteoblast. (COI:No)

Kir2.1 conductance declines steeply with membrane depolarization (i.e., inward rectification)
due to voltage-dependent pore blockades by intracellular cations, chiefly spermine (polyamine).
It has been suggested that multiple K+ binding sites exist along the long pore of Kir2.1, andthat the negatively charged residues located in the central cavity and in the cytoplasmic porecontribute to the polyamine binding. Importantly, the voltage dependence of the block causing
the inward rectification shifts along the voltage axis with the change in EK when extracellular K+ concentration ([K+] out) is altered, as if the block is coupled to the K+ flow throughthe channel. However, it has been reported that the voltage-dependence does not shift when
intracellular K+ concentration ([K+] in) is changed. Here, we examined the effects of intracellular
K+ and Na+ on the voltage dependence of the spermine block to explore the mechanism of the
EK dependence of the inward rectification. When the effects of [K+] in on the spermine block
were examined by replacing K+ with Na+, the effects of the pore block by intracellular Na+
overlapped with the effects of [K+] in on the spermine block. When [K+] in was altered without
any cation substitution, the “genuine” voltage dependence of the spermine block evaluated byits unblocking rate shifted along the voltage axis by the amount of the change in EK. When
[K+] in was altered by replacing K+ with Na+, the shift became smaller than the change in
EK as [K+] in was lowered and [Na+] in was increased. From these results, we propose thatthe polyamine at the blocking site is “knocked-off” by K+ ion(s) coming from the extracellularsolution (Hille ＆ Schwarz, 1978), and is “locked-in” by K+ ion(s) entering the pore from theintracellular solution and preventing the bound polyamine from dissociating.
(COI:No)

2P-002

2P-005

Riko Ikeda（ Dept Mol Physiol, Col Pharm Sci, Ritsumeikan Univ, Japan）

Yasunori Takayama, Mami Kato, Mana Tsukada, Naoki Adachi, Hideshi Ikemoto,
Masataka Sunagawa（ Dept Physiol, Showa Univ Sch Med, Japan）

Basal ciliary beating enhanced by the temperature-dependent Ca2+
entry in airway ciliary cells of Hochu-ekki-to (TJ-41) treated mice

ANO1 inhibition by the extracts from Glechoma hederacea

The effects of Hochu-ekki-to (TJ-41, Bu Zhang Yi Qi Tang), a Chinese traditional medicine,on airway ciliary beatings were examined. The ciliary beat frequency (CBF) and ciliary bend
angle (CBA) were measured in airway ciliary cells isolated from lungs using a video microscopy
equipped with a high-speed camera. In the test mice, TJ-41-containing water was administeredfor 4-6 weeks (1.8g/kg/day), while in the control mice, only water was administered. The basal
CBF and CBA of the TJ-41 treated mice were enhanced compared with the control mice. In
the control mice, the basal CBF was maintained by both cAMP and Ca2+ signals whereas, in
TJ-41 treated mice, the basal CBF was enhanced and maintained by Ca2+ signal, not cAMPsignal. An [Ca2+] i increased by TJ-41 treatment inhibited a cAMP accumulation by activating
PDE1A in airway ciliary cells. The concentration response studies of acetylcholine and ionomycin exhibited that TJ-41 treatment shifted the CBF concentration-response curves to lower
concentrations. TJ-41 treatment enhanced CBF, CBD and [Ca2+] i stimulated by temperature.
TRPV4 is expressed in airway ciliary cells judging from RT-PCR. We are now on studying
the possibility that in airway ciliary cells, TJ-41 treatment stimulates TRPV4 expression andactivity, leading to enhancement of CBF, CBD and [Ca2+] i under the basal condition. (COI:No)

Anoctamin 1 (ANO1, also called TMEM16A) is a calcium-activated chloride channel expressedin primary sensory neurons of both trigeminal and dorsal root ganglions. Increases in theintracellular free calcium activates ANO1 and the generated chloride efflux evokes a neuronal
excitation in primary sensory neurons. ANO1 activity enhances the physiological functionsof calcium-permeable channels (ex. TRP channel) and G protein-coupled receptors, which are
involved in acute and inflammatory pain sensations. Therefore, ANO1 inhibition would be an
intriguing way to reduce pain in some clinical situations. It has been recently reported that
ANO1 is inhibited by many plant-derived natural compounds, including tannic acid, polyphenols and menthol. Especially, the menthol, a major component in mint (Menthae herba), has astronger inhibitory effect on ANO1 current compared with other natural compounds. Here,
we focused on menthol-containing plant species, and found that the extracts from Glechomahederacea herb (Glechomae herba) also inhibited ANO1 current induced by intracellular freecalcium. In preparation of the extracts, we grinded the dried Glechomae herba and boiled it at
60 ℃ for 1 hr. After centrifugation and filtration of supernatant, we made the stock powder
by evaporation and freeze dehydration. Finally, we applied the diluted solution to HEK293T
cells expressing mouse ANO1 in whole-cell patch-clamp recording. The ANO1 currents were
inhibited by the Glechomae herba-contained solution. This fact indicates a possibility that
Glechomae herba reduces pain sensation. Currently, this plant is not socially used well inworldwide. However, our study suggests that Glechomae herba could be a natural medicine to
reduce pain, for instance oral pain sensation.
(COI:No)

2P-0032P-006

The analysis of novel ATP release channel in the mitochondria
Toshiyuki Ishii, Takumi Akagi, Makoto Kaneda（ Dept. Physiol., Nippon Med. Sch., Tokyo,

Effects of kampo medicine containing ginger on TRPV1-ANO1
interaction

Japan）

Mami Kato, Yasunori Takayama, Mana Tsukada, Masataka Sunagawa（ Dept Physiol, Sch

Adenosine triphosphate (ATP) plays important roles for P2X receptors-mediated signal transmission in sensory system, especially taste and pain sensation. In retina, we have previously
identified the localization of seven subtypes of P2X receptors, and shown that these P2X
receptors modulate visual information. However, the origin of ATP as a neurotransmitter inthe retina has not been well elucidated. Recently, ATP release through calcium homeostasis
modulator (CALHM) channels has been reported in taste buds. We have examined the expression level of mRNA of CALHM1, CALHM2, and CALHM3, and found that signals of CALHM2
were dominantly expressed in the retina. Therefore, in the present study, we investigatedwhether the CALHM2 channel-mediated ATP release can occur in the retina by morphologicaland physiological methods. The immunoreactivity of CALHM2 was detected in the outerplexiform layer and the inner plexiform layer, the synaptic layers of the retina. In the outerplexiform layer, strong immunoreactivity was detected at the terminals of photoreceptors. At
the terminals of photoreceptors, the immunoreactivity for CALHM2 was co-localized with the
immunoreactivity for COXIV, a marker of mitochondria. The immunoreactivity of CALHM2
was detected in mitochondria of the rod spherules and the cone pedicles at the ultrastructurallevel. In HEK293T cells, the immunoreactivity for CALHM2 was also co-localized with the
immunoreactivity for COXIV. An overexpression of CALHM2 in HEK293T cells increased the
ATP concentration of the extracellular solution. On the other hand, knocking down of CALHM2
in HEK293T cells reduced the ATP concentration of the extracellular solution. These results
support the hypothesis that CALHM2 contributes ATP release from mitochondria, and the
ATP release via CALHM2 might be involved in the modulation of visual information.(COI:No)

Daikenchuto (TJ-100) is a kampo medicine, one of the Japanese traditional herbal medicines,
composed of the extracts from ginger (Zingiber officinale), ginseng (Panax ginseng), Japanesezanthoxylum peel (Zanthoxylum piperitum) and maltose. The component percentages are 50,
30 and 20 %, respectively. TJ-100 is widely used in clinical situations to improve the intestinal
motility and blood flow. It has been reported that TJ-100 inhibits a postoperative adhesion
formation and paralytic ileus. TJ-100 contains some pungent components including hydroxy-αsanshool and 6-shogaol, which are agonists of a capsaicin receptor, transient receptor potential
vanilloid 1 (TRPV1) channel. Activation of TRPV1 induces the releases of calcitonin gene
related peptide (CGRP) and substance P. These neuropeptides enhance vasodilation followedby the increase in intestinal blood flow. In this sturdy, we focused on anoctamin 1 (ANO1),a calcium-activated chlorine channel. Previous report suggests that ANO1 promotes neuralexcitation through the interaction with TRPV1. However, TJ-100 effect on ANO1 is unknown.
Here, we show that TJ-100 (100 µg/mL) inhibits ANO1 current induced by 100 nM intracellular
free calcium. We investigated that in HEK293T cells expressing mouse TRPV1 (mTRPV1) andmouse ANO1 (mANO1), or cells expressing either mTRPV1 or mANO1. Interestingly, TJ-100
induced the large currents dependent on TRPV1-ANO1 interaction, although TJ-100 activatedmTRPV1 and inhibited mANO1. These results indicate that TRPV1-ANO1 interaction evokes
the nervous excitation in the oral administration of TJ-100. This could be a novel molecular(COI:No)mechanism to explain the pharmacological effects of TJ-100 in enteric canal.

S116

Med, Showa Univ, Japan）

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

2P-007

Phospholipid transport mechanism of transmembrane protein 16F
Takahiro Shimizu, Kanon Shirai, Syota Nabeshima, Takuto Fujii, Hideki Sakai（Dept Pharm
Physiol, Faculty Pharm Sci, Univ Toyama, Japan）

2P-010

The NH2-terminal region is the key for the differential drug sensitivity of
TRPM7 and TRPM6
Hana Inoue1，Takashi Murayama2，Takuya Kobayashi2，Ryo Mizumoto1，Utako Yokoyama1

Transmembrane protein 16 (TMEM16) family is composed of ten isoforms. Among them,
TMEM16A and 16B have been demonstrated to function as Ca2+-activated Cl－ channels. On
the other hand, TMEM16C, D, F, G, and J have been reported to show Ca2+-activated phospholipid scramblase activities. Especially, TMEM16F has a pivotal role in phosphatidylserine
exposure during blood coagulant. Its mutation causes Scott syndrome showing a blood clotting
disorder. We found previously that human TMEM16F exhibits not only phospholipid scramblase activities but also Ca2+-activated Cl－ channel activities with low Ca2+ sensitivity. In thepresent study, we therefore investigated how these functions are correlated in HEK293T cells
in which human TMEM16F is exogenously expressed. In flow cytometry using annexin Vphycoerythrin (PE), Cl－ channel blockers, which inhibited TMEM16F-dependent Cl－ currents,
significantly decreased Ca2+-triggered phospholipid scramblase activities. The Cl－ currents
measured by whole-cell patch-clamp recordings were closely correlated with the scramblase
activities in some TMEM16F mutants. In tail current analysis, the mutants having larger
scramblase activities exhibited slower tail currents, suggesting that increased gating frequency
of TMEM16F channels enhanced their phospholipid transports. Phospholipid scramblase
activities were temperature-dependent, and they were decreased at low temperature. These
results suggest that structural changes during TMEM16F channel gating are essential for thescramblase activities.
(COI:No)（ 1 Dept Physiol, Tokyo Med Univ, Tokyo, Japan, 2 Dept Pharmacol, Juntendo Univ Sch Med,Tokyo, Japan）

2P-0082P-011Jun Ichikawa, Ryuji Inoue（Dept Physiol, Fukuoka Univ Sch Med, Japan）

2 1 Dept Physiol, Osaka Med College, 2 Div
Yuriko Takeda1，Fumihito Ono1，Koichi Nakajo1, （Implications of the mechanosensitivity of TRPC6 channel in bonemarrow stromal cell cycle progression

Three zebraﬁsh ROMK channels show different pharmacologicalproperties

Several lines of evidence suggest that bone marrow stromal cells (BMSCs) are mechanosensitive. However, how this property operates in physiological settings and what molecular mechanism is involved therein remain entirely unclear. We therefore investigated the mechanicalresponses of BMSCs in some detail by using a digital Ca2+ imaging technique. Sole applicationof 2, 4, 6-trinitrophenol (TNP), a cell membrane-bulging agent, did not induce discernible Ca2+
responses. However, this agent potentiated Ca2+ elevations in BSMCs elicited by UTP, anagonist of the P2Y2 receptor. A similar phenomenon is known in vascular smooth muscle cells,
where synergistic activation of TRPC6 channel was shown to occur by simultaneous receptor
and mechanical stimulations.* Consistent with this mechanism, siRNA knockdown of TRPC6
expression abrogated the potentiation of UTP-induced Ca2+ elevations in BMSCs by TNP.
The flow cytometry analysis in BMSCs indicated that siRNA knockdown of TRPC6 arrests
the cell cycle at the G2/M phase. Furthermore, the cell cycle-specific arrest by synchronizing agents showed a heightened potentiation of UTP-induced Ca2+ responses by TNP in the
M phase compared with the other cell cycle phases. Collectively, these results suggest thatthe mechanosensitivity of TRPC6 may play a facilitatory role for the G2/M progression of
proliferating BMSCs.
*Inoue R. et al., Circ.Res. 104:1399-1409, 2009

TRPM7 and its closest homologue TRPM6 are Ca2+/Mg2+-permeable cation channels withan alpha-kinase domain at their COOH-terminal. It has been reported that TRPM7 and
TRPM6 are differentially affected by 2-aminoethyl diphenylborinate (2-APB) which inhibits
TRPM7, but activates TRPM6. To elucidate the underlying mechanism of its different action,
we investigated the effect of 2-APB using various truncated and chimeric channels. We firsttested the effect of 2-APB on the kinase domain truncated TRPM7 (TRPM7-ΔK). TRPM7-ΔK
current was significantly inhibited by 200 µM of 2-APB (201.9 ± 27.1 pA/pF and 42.8 ± 7.57
pA/pF, before and after 2-APB application, respectively, n = 5), suggesting that the kinasedomain is not the target of 2-APB. Truncation of NH2-terminal region (NTR, 1-644) of TRPM7
impaired the expression, therefore, we constructed a chimeric channel consisted of the NTR of
TRPM6 (1-636) and the TRPM7 transmembrane domain with its kinase domain (645-1863). The
chimeric channel was expressed on the plasmamembrane, though the current was strongly
inhibited by intracellular free Mg2+ with an IC50 value of 11 µM, compared to TRPM7-wild
type (IC50 346 µM). Interestingly, the chimeric channel was not inhibited by 2-APB (57.3 ±4.7 pA/pF and 60.3 ± 4.7 pA/pF, before and after 2-APB application, respectively, n = 5).
These results suggest that the NTR of TRPM7 is involved in the differential effects of 2-APB.
(COI:No)(COI:No)

2P-009Analysis of the molecular mechanism underlying ER Ca2+ sensor
STIM1-dependent suppression of CaV1.2 channel activity
Takuro Numaga-Tomita, Hiroki Takahashi, Mitsuhiko Yamada（ Dept Mol Pharma, Sch Med
Health Sci, Shinshu Univ, Japan）

Activation of phospholipase C-coupled cell surface receptors evoked intracellular Ca2+ increase
via phosphatidylinositol 4, 5-bisphophate hydrolysis. Ca2+ release from Ca2+ store, endoplasmic
reticulum (ER) by inositol 1, 4, 5-trisphosphate receptors causes the reduction of a Ca2+ content
in ER. ER-resident Ca2+ sensor STIM1 senses this reduction, activates Orai1 channels at theplasma membrane, and induces store-operated Ca2+ entry. It is known that STIM1 activation
upon store depletion also suppresses the activity of CaV1.2 L-type voltage-dependent Ca2+
channels at the plasma membrane through a largely unknown molecular mechanism. In thisstudy, we measured the effect of STIM1 on recombinant CaV1.2 channel currents expressedin tsA201 cells in the whole-cell configuration of the patch-clamp technique. Co-expression of
STIM1 significantly suppressed the CaV1.2 current. Under this condition, the expression ofchannel subunits were not reduced as assessed with wester blotting whereas CaV1.2 gating
currents were greatly suppressed, indicating that STIM1 inhibited the membrane expressionof CaV1.2 . The STIM1-dependent suppression of CaV1.2 current was abrogated by the deletionof distal C-terminus (DCT) of CaV1.2. Endogenous STIM1 in tsA201 cells also significantly
suppressed CaV1.2 channel currents when activated by thapsigargin without altering theirsteady-state inactivation or activation. These data suggest that STIM1 induces the endocytosisof CaV1.2 via interaction with DCT, thereby reducing the number of CaV1.2 channels and theamount of their currents upon store-depletion.
(COI:No)

Integrative Physiol, Jichi Med Univ）

Potassium ion homeostasis is important in all organisms. To maintain its concentration at theappropriate level, potassium excretion at the distal nephron is essential, for which ROMK
channels are the main pathway. They are also responsible for recycling potassium ions to help
Na+-K+-2Cl－ cotransporter NKCC2. The genetic defect of either of ROMK or NKCC2 can cause
Bartter syndrome, which exhibits secondary hyperaldosteronism. Fish also need to maintainthe potassium ion homeostasis by excreting potassium ions through the kidney. Distinct frommammals, fish also utilize the gill for potassium excretion. Zebrafish possess seven ROMK
genes in the genome (kcnj1a.1~6 and kcnj1b). We previously isolated all seven genes fromkidney and gill of zebrafish and confirmed that three of them (kcnj1a.1, kcnj1a.2, and kcnj1b)
were functional as ROMK channels. We also identified that they have different sensitivity
against extracellular barium: kcnj1b is the most sensitive to barium and kcnj1a.2 is the leastsensitive to barium. This is partially due to the variety of amino acid side chains at the end ofpore helix. In this study, we examined the recently-developed ROMK-specific inhibitor VU591.
Interestingly, only kcnj1a.1 was sensitive to VU591. VU591 is supposed to bind deep in thepore and presumably interact with Val168 and Asn171 (Swale et al. Biophys J. 2015). All threezebrafish ROMK have different amino acid residues at 168 and 171; Leu and Asn in kcnj1a.1,
Leu and Ser in kcnj1a.2, Ala and Asn in kcnj1b. We conclude that the different sensitivity to
VU591 is due to the variation of amino acid residues in the deep pore region, as opposed tothe case of barium sensitivity in which the shallower pore region may be more responsible.
(COI:No)

2P-012

Investigation of the Analgesic Effect of Saliva of Mosquito with a PatchClamp Method
Tianbang Li（Cell Signaling, NIPS, Japan）
Various tropical diseases are transmitted via mosquito bites, which are barely sensed byhuman beings. The painless blood feeding of mosquito enlightens the development of bionicmicroneedles, which could be applied for diabetes treatment, neonatal care and acupuncture
therapy. It is believed that there are two reasons for humans to be not able to sense theache of puncture: mechanically, the subtle mosquito proboscis could efficiently avoid the pain
spots of the skin and reduce the resistive force. On the other hand, biochemically, the saliva
of mosquito which reaches free nerve endings might contain some compounds which possess
analgesic effects. However, the later mechanism is poorly understood. To address the questionof the analgesic effect of mosquito saliva, we investigated whether it affects human nociceptors.
Transient Receptor Potential (TRP) V1 and TRPA1 play crucial roles in perception of painfulstimuli. Expressing either TRP channel in HEK293 cells and then performing a whole-cellpatch-clamp recording provided us a promising in vitro model to analyze the analgesic effect ofsaliva of Culex pipiens pallens. Surprisingly, 20-fold diluted mosquito saliva sample suppressed
human TRPV1 currents but not human TRPA1 currents. Moreover, such an analgesic effect
was abolished upon heating the saliva sample to 95oC for 20 minutes, indicating that peptides
might be involved in this suppressing. We plan to not only repeat electrophysiology experiments in mouse dorsal root ganglia neurons but also carry out behavioral experiments in amouse model. Mass spectrometric method will be also employed to identify which ingredientof mosquito saliva plays the major role in analgesia. Taken together, this work might shed lightupon the mechanism of the anesthetic effect of mosquito saliva, which could offer support for(COI:No)developing bionic microneedles to achieve painless injection.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S117

2P-013

2P-0161 1 Dept Physiol, Shiga Univ
Xinya Mi1，Wei-Guang Ding1，Akiko Kojima2，Hiroshi Matsuura（

Satomi Kamata1，Asuka Higashikawa2，Maki Kimura2，Sadao Oyama2，Wataru Ofusa2，
1 1 Dept Removable Partial
Haruna Toda2，Yoshiyuki Shibukawa2，Shuichiro Yamashita（

Possible involvement of transient receptor potential canonical channelin ectopic activity of pulmonary vein cardiomyocytes of guinea-pig
Med Sci, Otsu, Japan, 2 Dept Anesthesiol, Shiga Univ Med Sci, Otus, Japan）

Expression of Ca2+ activated K+ Channels in Human Cementoblast
Prosthodont, Tokyo Dent Coll, 2 Dept Physiol, Tokyo Dent Coll）

Pulmonary veins contain a myocardial layer, and its ectopic activity propagates into the left
atrium and underlies atrial arrhythmias such as atrial fibrillation. The ectopic activity in pulmonary vein cardiomyocyte (PVC) appears to arise from multiple ionic mechanisms. The present
study was designed to examine the possible involvement of transient receptor potential canonical 3 (TRPC3) channel in the electrical activity of PVC, enzymatically isolated from guinea-pig
heart/pulmonary vein preparations. Spontaneous action potential was observed in 40.2% of
PVC during exposure to normal Tyrode solution, and firing frequency in these spontaneously
active PVC was 120.0±26.7 /min (n = 6), which was significantly reduced by the selective
TRPC3 blocker Pyr-3 at 0.5 µM (24.0±10.1 /min, 80.8±10.7% reduction). The β-adrenergic
agonist isoproterenol (100 nM) markedly increased the firing rate to 195.5±14.0 /min, which
was also significantly reduced by the concomitant presence of 0.5 µM Pyr-3 (92.5±23.9 /min,
7.0.±8.5% reduction). Western blot experiments using anti-TRPC3 antibody detected mostabundant immunoreactive band at ~85 kDa, which is close to the expected molecular mass
for the TRPC3. Immunocytochemistry experiments using anti-TRPC3 antibody detected theexpression of TRPC 3, predominantly in the transverse tubule. These results strongly suggestthat TRPC3 channel is expressed in PVC and functionally contributes to ectopic electrical(COI:No)
activity of PVC in guinea-pig. 

Purpose: The function of the cementum is to provide an attachment site for collagen fibers thatconnect to and fix the alveolar bone teeth. Cementum is formed by cementoblasts. Transmembrane signaling associated with cell membrane ion transport regulates various physiologicalprocesses in the cell, but the detailed properties of cementoblast ion signals have not yet beenclarified. The purpose of this study was to investigate the biophysical and pharmacologicalproperties of currents generated by ion channels expressed in human cementoblasts (HCEM).
Materials ＆ Methods: We measured ionic currents using conventional whole-cell patch-clamp
recording. Krebs solution was used as a standard extracellular solution (standard-ECS). Standard intracellular solution (standard-ICS) was composed as followings (in mM) ;140 KCl, 10 NaCland 10 HEPES. we prepared Cs-ICS solution by equimolary replacing K+ in the ICS with Cs+.
We used non-selective K+ channel blocker TEA and Ca2+ -activated K+ channel blocker IbTX,
apamin and TRAM-34 to examine pharmacological properties of the ionic current recorded.
Results ＆ Conclusion: Depolarizing voltage steps from holding potential of -70 mV with 10 mV
increments evoked outward currents under the ECS/ICS condition. When the same depolarization stimulation was applied to the cells with Cs-ICS, the outward currents almost disappeared.
When TEA and IbTX were administered under the ECS/ICS, the current densities of theoutward currents significantly and reversibly decreased. However, apamin and TRAM-34 had
no effects on the current density of the outward currents. These results suggested that HCEM
expresses large-conducutance Ca2+-activated K+ channels.
(COI:No)

2P-0142P-017Involvement of the C-terminal domain in cell surface expression andG-protein coupling of mGluR6
Atsushi Shimohata1，Dilip Rai1, 2，Takumi Akagi1，Toshiyuki Ishii1，Mie Gangi1，
1 1 Dept Physiol,
Takuma Maruyama1，Yuko Kiyama1，Ikuo Ogiwara1，Makoto Kaneda（

Nippon Med Sch, Japan, 2Visual Funct Sec, Depat Rehabil Sens Funct, Nat Rehabil Center,
Japan）

Metabotropic glutamate receptor 6, mGluR6, is expressed in the dendritic tips of retinal ONbipolar cells and activates G-protein-dependent signaling cascades upon binding to glutamate
released from photoreceptors. We herein investigated the role of the mGluR6 C-terminal
domain (CTD) (residues 840-871) in cell surface localization and G-protein coupling using immunocytochemical, biochemical, and electrophysiological approaches involving 293T cells andprimary hippocampal neurons. We tested C-terminally truncated mGluR6, and showed that theremoval of up to residue 858 did not affect surface expression or glutamate-induced G-proteinmediated responses, while a 15-amino acid deletion (Δ857-871) impaired these functions. However, a 21-amino acid deletion (Δ851-871) restored surface expression and glutamate-dependent
responses, which were again attenuated when the entire CTD was removed. We investigatedother mGluR6 deletions, in which amino acids were sequentially removed from the N-terminalside of CTD, and showed that mGluR6 surface localization was unaffected until the entire CTD
was deleted. We demonstrated that mGluR6 with CTD consisting of only a middle segment(851-856) was deficient in surface expression. These results suggest that a single histidine orlysine residue at the N or C terminus of CTD is sufficient for mGluR6 surface localization andreceptor function, whereas mGluR6 CTD may contain regulatory elements for intracellular(COI:No)
trafficking and signaling.

Single-Molecule Fluctuations and Conformational Changes of the
Human Transient Receptor Potential Vanilloid 1 (TRPV1) Channel
Recorded using Diffracted X-ray Tracking
Hirofumi Shimizu1，Takuya Kobayashi2，Masayuki Iwamoto3，Kentaro Kajiwara4，
2 1 Dept Integ Sys Phisiol, Univ
Nagomi Kurebayashi2，Haruo Ogawa5，Takashi Murayama（
Fukui Fac Med Sci, Japan, 2 Dept Cell Mol Pharm Juntendo Univ Grad Sch Med, Japan,

3 Dept Mol Neurosci, Univ Fukui Fac Med Sci, Japan, 4 SPring-8 /JASRI, Japan, 5 IQB, The

Univ of Tokyo, Japan）

The transient receptor potential vanilloid 1 (TRPV1) channel senses various stimuli, such as
vanilloid, heat, and pain. Recent progress in the structural analysis of the channel has revealed
an important clue for understanding the structure-function relationship. We applied diffracted
X-ray tracking to the human TRPV1 channel to reveal/investigate further the molecularmechanisms underlying the activations. The purified channels were fixed on the plate at theextracellular side, while the gold nanocrystals were attached to the other side as a probe.
The diffraction spots from the crystals were tracked on the plane of the 2D X-ray detector
at the synchrotron facility, which was then translated into the motions of the channels. We
have introduced a toroidal mirror and high-speed 2D X-ray detector at the BL28B2 beamline
at Spring-8, which can achieve high temporal (sub-millisecond) and spatial (~0.1º) resolutions
to track the motions. In the presence of capsaicin, the enhancement of the global fluctuations
and twisting motions along the axis of the ion permeation pathway were observed, which
was suppressed by its antagonist. In contrast, only small fluctuations were observed in theabsence of capsaicin. These results suggested that the observed large fluctuations and twisting
motions were involved in the gating motions of the TRPV1 channels. In this presentation, we
will discuss the detailed behavior of the TRPV1 channels with respect to activations, and the(COI:No)advantages of our method for revealing the dynamic properties of proteins.

2P-0152P-0181 1 Dept Physiol, Grad
Etsuko Minobe1，Jianjun Xu1，Masayuki Mori X.2，Masaki Kameyama（

1 1 Integrative Physiol, Grad Sch
Yoshifumi Okochi1，Hidekazu Tsutsui2，Yasushi Okamura（Calmodulin (CaM) performs critical regulation of Cav1.2 Ca2+ channels. Ca2+-free CaM preassociates dynamically with the channels, and subsequent Ca2+ influx drives conformationalchanges that induce the channel regulation. Several conformations of the channel-CaM complex
have been reported, but it remains to be controversial. We have previously reported that the
wildtype Cav1.2 channel requires CaM and ATP to induce its activity. The model, based on our
experiments, consists of two CaM-binding sites in the channel each for Ca2+-dependent facilitation and inactivation. In this study, we have investigated the conformation of Cav1.2 channel inthe inactivated state by recording the activity of carboxyl-terminal (CT) deleted channel liked
to CaM (CaM-linked channel) and amino-terminal (NT) deleted CaM-linked channel (delN-CaMlinked channel) in the inside-out mode of patch-clamp technique. We found that the CaM-linked
channel maintained the activity in the presence of ATP, and that CaM-dependent inactivation(CaMDI) could be isolated from Ca2+-dependent inactivation (CDI). The CaM-linked channel
showed both CDI and CaMDI, while the delN-CaM-linked channel showed only CaMDI but not
CDI. GST-pulldown assay was performed in different Ca2+ and CaM concentrations to explore
the interaction NT and CT peptide with CaM. The results suggested that the inactivationinduced by one CaM linked with the channel may require NT of the channel. The inactivationinduced by more than one CaMs may not require NT, but involve CT of the channel. Thus,
there might be two types of the conformation of Ca2+/CaM-dependent inactivation of Cav1.2
channel. (COI:No)

Recently, several ion channels functioning in endomembrane system have been identified. Since
one of these channels is a voltage-dependent ion channel, a possibility arose that membranepotential in endomembrane system could change. However, it is still lack the method measuring membrane potential change in this system. To challenge this problem, we set out theestablishment of a method measuring membrane potential in endomembrane system. We
chose phagosome, a vesicle separated from plasma membrane, formed when phagocytes eat
pathogens and dead cells. We have reported previously that we succeeded visualization ofphagosomal membrane potential in RAW264.7 macrophage cell line by using FRET-based
voltage probe Merm2 and phagosomal membrane potential hyperpolarizes with maturation ofphagosome, comparing to cell membrane. However, absolute value of membrane potential ofphagosomes remains unknown. In this study, we measured fluorescence intensity of Merm2 incell membrane of RAW cells by setting reference voltage using patch clamp technique thereby
calibrating fluorescence intensity of RAW cells. We found that cell membrane potential of
RAW cells was roughly estimated to hyperpolarize from -30 mV to -80 mV when these cells
were stimulated with ATP. We are now trying to estimate phagosomal membrane potential of
isolated phagosomes using the same method.
What is the biological significance of hyperpolarized phagosomal membrane? In general, hyperpolarization of membrane can facilitate Ca2+ flux by increase of driving force. Since phagosome
is known to supply Ca2+ to cytoplasm through ion channels, it is assumed that hyperpolarization in phagosomal membrane is required for facilitation of Ca2+ flux into cytoplasm. We aretrying to manipulate phagosomal membrane potential by optogenetic and pharmacological(COI:No)
techniques to understand significance of hyperpolarization in phagosomes. 

Ca2+ dependent inactivation of Cav1.2 channel induced by twomolecules of calmodulin
Sch Med ＆ Dent Sci, Kagoshima Univ, Japan, 2 Dept Chem, Sch Med, UOEH, Japan）

S118

Establishment of a method measuring membrane potential inphagosomes
Med, Osaka Univ, Japan, 2 Japan Advanced Institute of Science and Technology）

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

2P-0192P-022

Daiju Yamazaki（Div of Pharmacol, NIHS, Japan）

Seiya Miyazaki1，Motohiro Mizuguchi1，Tomoaki Ishida1，Tomoe Ueyama2，Takahiro Sogo2，
2 1 Dept. of Biomed. Sci., Col. of Life Sci., Ritsumeikan
Shu Nakao1, 2，Teruhisa Kawamura1, （Contractility assessment of engineered heart tissue using human iPS
cell-derived cardiomyocytes
A number of anticancer agents manifest cardiotoxicity including left ventricular dysfunction
and congestive heart failure as side effects. Doxorubicin (DOX) is among the most effective
and widely used anticancer agents in the clinic. Although DOX are applied to several typesof cancer, it causes a dose-related cardiotoxicity that can lead to heart failure in a subset ofpatients. In the research and development period at Pharmaceutical companies, if anticancer
agents are evaluated for side effects risk such as heart failure before clinical and established
for initial dosage, it is possible to reduce the risk in the clinical. However, there is no in vitro
contractility assay for detecting cardiotoxicity by anticancer agents.
Here, we produced engineered heart tissue (EHT) using human induced pluripotent stem
cell derived cardiomyocytes (hiPS-CMs) and developed contractility assay for evaluating
contractile dysfunction to DOX. First, we examined gene expression analysis for maturationof hiPS-CMs. 4-weeks cultured EHTs increased cardiomyocytes maturation marker including
MYH7, TNNI3 and KCNJ2 compared with unloaded and 1-week cultured EHTs. It was suggested that EHTs matured hiPS-CMs. Next, we investigated β-adrenergic receptor responses
with isoproterenol as an activator and propranolol as a blocker. Isoproterenol showed dosedependent positive chronotropic and inotropic action and propranolol showed dose-dependent
negative chronotropic and inotropic action. These responses were similar with human heart.
Finally, we chronically applied DOX to EHTs. Low dose of DOX (0.1 µM) increased beat rate
in a time-dependent manner. Middle and high dose (0.3 and 1 µM) of DOX increased beat rateuntil 48h and impaired contractility in a time- and dose-dependent manner. It is suggested thatEHTs may be useful tool for detecting cardiotoxicity by anticancer agents.
(COI:No)

Gene Expression Proﬁling of the Sinoatrial Node in Mice: the Effect ofEndurance Exercise
Univ., 2 Global Innov. Res. Org. Ritsumeikan Univ.）

Chronic endurance training causes slowed resting heart rate known as exercise-induced
bradycardia. Severe bradycardia leads to syncope, sudden cardiac death, and necessity ofpacemaker implantation. Our previous studies demonstrated that exercise-induced bradycardia is caused by dysfunction of the sinoatrial node (SAN), the primary pacemaking site of theheart, through ion channel remodeling. However, it remains elusive which type of stimuli
from exercise directly affect the expression and function of cardiac ion channels in SAN. We
here investigate exercise-induced changes in the gene expression profile of the SAN that may
cause resting bradycardia using a mouse model. Mice were subjected to high-intensity forced
swimming twice a day and 6 days per week for 4 weeks. The swimming sessions resulted insignificantly lower resting heart rate in mice than in sedentary mice. To address the global
gene expression profile in the SAN, we next performed RNA isolation from the SAN tissues
dissected based on the anatomical landmarks. Total RNA samples pooled from three individual
mouse SANs were preamplified and used to generate a cDNA library for RNA-sequencing.
Transcriptome analysis revealed downregulation of 4,306 genes in the trained SAN. Gene set
enrichment analysis showed that significantly reduced expression levels of genes enriched in
Ca2+ regulation-, ion channel-, impulse conduction-, autonomic receptor-related gene sets intrained SAN, suggesting dysfunction of the pacemaker activity. GO analysis demonstrated
that exercise-induced downregulation of lipid metabolic process and immune system process
was observed, while there were upregulated genes annotated in GO terms associated withmuscle contraction, hypertrophy, oxidative phosphorylation and mitochondrial function.
Although further functional analysis is required, our results suggest that chronic exercise
stimulates adoptive response of the myocardium to increased hemodynamic stress, and it also
downregulates cardiac ion channels and autonomic regulators through metabolic remodeling(COI:No)in the SAN, leading to bradycardia.

2P-0202P-023

Nobuhiro Watanabe1，Satoshi Sasaki1, 2，Kazuto Masamoto1, 2, 3，Harumi Hotta1

Tsubasa Izaki, kei Ishii, Ryota Asahara, Hidehiko Komine（AIST, AHFRC, Tsukuba, Japan）

Vascular gap junctions contribute to pial artery dilation response tosomatosensory stimulation in isoﬂurane-anesthetized rat cortex（ 1 Dept Auton Neurosci, Tokyo Metropol Inst Gerontol, Japan, 2 Grad Sch Info Eng, Univ
Electro-Commun, Japan, 3 CNBE, Univ Electro-Commun, Japan）
Somatosensory stimulation increases cerebral blood flow (CBF) in the representative regionsof the somatosensory cortex, accompanying with cerebral arterial dilation. Such stimulationinduced arterial dilation is believed to propagate through the vascular endothelium from thecerebral parenchyma to the cortical surface. Vascular gap junctions transmit vasodilation
signals in vitro, but its role in vascular regulation in vivo remains unclear. The present study
aimed to investigate whether vascular gap junctions contribute to the regulation of the pial andpenetrating arterial tones during somatosensory stimulation. Male Wistar rats were artificiallyventilated under isoflurane anesthesia. For somatosensory stimulation, the left forepaw waselectrically stimulated. Rhodamine-labeled Ficoll was intravenously administered to fluorescently label the vasculature. The artery in the forelimb area of the right cortex was imaged via
a cranial window using two-photon microscopy, and the diameter was measured. In separate
experiments, CBF was measured using a laser speckle flowmeter, and the somatosensoryevoked potentials were recorded in the right somatosensory cortex. To block vascular gap
junctions, carbenoxolone (CBX) was intravenously administered. The forepaw stimulation
increased the diameter of the pial artery by 7.0% and of the penetrating artery by 5.0% of the
pre-stimulus diameter, without changing the systemic arterial blood pressure. The stimulation
increased regional CBF by 24.1% of the pre-stimulus levels, and induced somatosensory-evoked
potentials. Following CBX administration, the pial artery dilation response was reduced to
3.2%, however the extent of penetrating artery dilation or of CBF increase was not affected.
Further, CBX did not alter on the resting level of the blood pressure or the magnitude ofsomatosensory-evoked potentials, indicating that CBX does not influence the neural functions
but presumably affects the cerebrovascular responses. These results suggest that vascular gap
junctions, possibly on the endothelium, contribute to pial artery dilation but not to penetratingartery dilation during somatosensory stimulation.
(COI:No)

The arterial baroreﬂex suppresses psychological distress evoked bysocial exclusion
Social exclusion (neglect or rejection by others) evokes psychological distress termed “socialpain”. A previous study reported that hypertension attenuated sensitivity to social pain. We
thus hypothesized that an afferent input of the arterial baroreflex attenuates social pain. To
test the hypothesis, we investigated the influence of the arterial baroreflex on social pain
evoked by experimentally-induced social exclusion in human. The arterial baroreflex was
induced by neck chamber technic (negative pressure (-30-20 mmHg) was applied within achamber wrapped around the neck). Experimentally-induced social exclusion was evoked by
the cyberball task. The cyberball task is a virtual ball tossing game among three players (onesubject and two computer players). Social exclusion was simulated by reducing the ratio ofthrows to a subject (<10%). Subjects receive a ball in the first half of the task, but there are only
throws among computer players in the second half of the task. Neck suction was applied at
the time when throws among computer players were performed in the second half of the task.
Heart rate was noninvasively measured three-lead electrocardiogram. Subjects played two
kind of the cyberball task: applying negative pressure during the task (the baroreflex condition)and not applying negative pressure (the control condition). After playing each condition, social
pain was assessed by the need-threat scale (self-reported scale for assessing social pain). In thebaroreflex condition, HR dropped in the second half of the cyberball task compared to in thefirst half of the task. Social pain score was lower in the baroreflex condition than in the control
condition (baroreflex: 14.47 ± 1.94, control: 15.75 ± 2.02, p<.05±). These findings suggest that
(COI:No)the arterial baroreflex suppress social pain evoked by social exclusion.

2P-021

2P-024

Takako Yokota, Toru Akaike, Susumu Minamisawa（ Dept. of Cell Physiology, The Jikei

2 1 Dept Cardio Physiol, Grad Sch Med,
Keiko Kaihara1，Keiji Naruse1，Gentaro Iribe（

Background: Oxygen sensitivity plays an important role in vascular tone. In ductus arteriosus(DA) closure, the increase in oxygen concentration inactivates Kv and KATP channels, whichleads to depolarization and enhances Ca2+ intake. In contrast, the reaction of pulmonary arteriolae (PA) to oxygen is opposite to DA, and high sensitivity to oxygen allows PA to dilate soonafter birth. The specific genes that contribute to this difference remain unknown, however.
Therefore, to identify the oxygen-sensing mechanism, we sought to clarify the differential gene
expression pattern in DA and PA before and after birth.
Aim: The aim of this study is to identify the genes in rat DA and PA that contribute tooxygen sensitivity.
Methods and Results: Using Agilent SurePrint G3 Rat GE 8X60K, V2 Microarrays (Agilent®),
we compared the gene expression profiling in DA and PA of Wistar rats before (on embryonicday 21: e21) and after birth (on day 2: d2). We found 19 genes in which the expression levels
increased in DA and decreased in PA after birth (d2DA/e21DA>2.0 and d2PA/e21PA<-2.0)
and 21 genes in which the expression levels were vice versa. Among these 40 genes thatexhibited an opposite expression pattern in DA and PA, 15 are already demonstrated toexpress in smooth muscles. We therefore considered these 15 genes to have a high probabilityof being involved in vessel closure as a reaction to the increase in oxygen concentration. In
addition, referencing a database about their expected oxygen sensitivity, the functional closure
mechanism and the epidemiological characteristics of PDA, we selected 5 genes (dgkg, scara5,
abcc8, nr4a1, kcne3) as important factors for DA and PA to develop oxygen sensitivity.
Conclusions: We identified five candidate genes that may contribute strongly to oxygen
sensitivity in rat DA and PA.(COI:No)

Reactive oxygen species (ROS) plays important physiological roles in the cardiovascular system.
We have previously reported that myocardial stretch increases ROS derived from NADPH
oxidase (NOX)2 to stimulate the sarcoplasmic reticulum (SR) to increase Ca2+ spark rate andincreases cellular contractility. However, behavior of NOX4-derived ROS in this response isunknown. In the present study, we investigated the role of NOX4 on acute stretch-induced
ROS, Ca2+ sparks and cellular mechanics.
Ventricular cells were enzymatically isolated from either 8～12 week old mice (WT) or NOX4
knock out (KO) mice hearts. Isolated mouse ventricular myocytes were exposed to 8～10%
axial stretch using computer-controlled piezo-manipulated carbon fibers, attached to both cellends. Net ROS production and Ca2+ sparks were studied using DCF and flou-4, respectively. In
single cell mechanics study, slopes of end-systolic force-length relation curves was measured
as an index of cellular contractility.
Genetic NOX4 deletion (NOX4 KO) or pharmacological NOX4 inhibition by GKT136901 abolished stretch-induced increase in ROS production and reduced cellular contractility. However,deletion or inhibition of NOX4 did not affect stretch-induced increase in Ca2+ spark rate. The
results suggest that role of NOX4-derived ROS in the response to myocardial stretch is different from that of NOX2.
(COI:No)

Candidate genes that contribute to oxygen sensitivity were identiﬁed
from rat ductus arteriosus and pulmonary arteriolae
University School of Medicine, Tokyo, Japan）

Effects of NADPH oxidase (NOX) 4 on single cell mechanics in mouse
ventricular cardiomyocytes
Okayama Univ, Japan, 2 Dept Physiol, Asahikawa Med Univ, Japan）

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S119

2P-025

2P-028Megumi Kato1，Mizuyo Kojima2，Kaori Yamashita1，Eiji Wada1，Yukiko Hayashi1

Yuichi Kuda1，Yasutaka Kurata1，Takayuki Ikeda2，Mamoru Tanida1，Tsumoto Kunichika1，
2 1 Dept Physiol 2, Sch Med, Kanazawa Med
Toshishige Shibamoto1，Hideto Yonekura（Anti-IL6 receptor antibody may worsen cardiomyopathy related tolaminopathy（ 1 Dept Pathophysiol, Tokyo Medical Univ, Japan, 2 Diversity Promotion Center, Tokyo
Medical University, Japan）
Purpose: Mutations in LMNA which encodes A-type lamins can cause several human diseases
including fatal cardiomyopathy with conduction defects. Activation of MAPK pathways wasreported to link Lmna-related cardiomyopathy. Interleukin-6 (IL-6) is a multi-functional cytokine
and known to promote fibrosis in heart. In addition, the IL-6 receptor antibody (MR16-1) was
reported to suppress inflammation after myocardial infarction, and improved cardiac remodeling in mice. In this study, we investigated the roles of IL-6 in Lmna-related cardiomyopathy
and the therapeutic
effects of MR16-1.
Methods: We used Lmna p.H222P knock-in mice (H222P) and C57BL/6J mice as control (WT).
We performed qPCR to check gene expression, western blotting to measure protein levels of
IL-6 signaling pathway, and histological analyses by Masson’s trichrome staining.
Results: mRNA of IL-6 was significantly increased in H222P heart. After MR16-1 treatment,
mRNA of IL-6 in H222P heart was decreased, whereas mRNA of collagen1α1, and TGFβ2
was increased. Furthermore MR16-1 enhanced phosphorylation of ERK1/2 and increased
fibrosis.
Conclusions: These results suggest bloking IL-6 receptor may worsen Lmna-related cardiomyopathy.
(COI:No)

2P-026

Experimental and theoretical study on mechanism of Ca2+ and Na+
permeation in CaV1.3 L-type calcium channels
1 1 Dept Physiol,
Futoshi Toyoda1，Akinori Noma2，Ding Wei-Guang1，Hiroshi Matsuura（

Ionic mechanisms of pacemaker activity in HL-1 mouse cardiomyocytes
Univ, Japan, 2 Dept Biochem 2, Sch Med, Kanazawa Med Univ, Japan）

Aim: HL-1 mouse atrial myocytes, often used for systematic electrophysiological studies, exhibit automaticity and thus would be useful for investigating ionic and dynamical mechanismsof pacemaker activity. Therefore, we determined the ionic mechanism of automaticity in HL-1
cells.
Methods: We measured membrane action potential, sarcolemmal ionic currents, and intracellular Ca2+ concentration changes in HL-1 cells by whole-cell patch clamp methods andfluorescence imaging of Ca2+ and membrane potential. Roles of the inward rectifier K+ channel
current (IK1), hyperpolarization-activated cation channel current (If)and Ca2+ releases from thesarcoplasmic reticulum (SR) in automaticity were determined by using Ba2+, ivabradine andcaffeine to cause IK1 block, If block and SR Ca2+ depletion, respectively.
Results: There exist both pacemaking and non-pacemaking cells. Larger IK1 was detected in
non-automatic cells by whole-cell patch clamp. By the patch clamp and fluorescence imaging
of membrane potential, automaticity was promoted by Ba-induced block of IK1. In the absenceof Ba2+, caffeine attenuated Ca2+ transient, slowed pacemaking, and abolished automaticity.
In contrast, Ba-induced automaticity did not disappear with co-administration of caffeine orivabradine.
Conclusions: The density of IK1 was considered to be an important factor that determines
whether pacemaker activity emerge or not. Our findings suggest that the mechanisms ofpacemaker activity in HL-1 cells vary depending on the IK1 density: SR Ca2+ release-mediatedpacemaking and membrane clock-dependent (If-independent) pacemaking occur at higher andlower IK1, respectively.
(COI:No)

2P-029Serotonin uptake via plasma membrane monoamine transporter during
ischemia-reperfusion in the heart
Takashi Sonobe, Tsuyoshi Akiyama, James Pearson（Dept Cardiac Physiol, Natl Cereb

Shiga Univ Med Sci, Japan, 2 Dept Bioinformatics, Col Life Sci, Ritsumeikan Univ）

Cardiovasc Ctr Res Inst, Suita, Japan）

We have reported that CaV1.3 L-type calcium channel (LTCC) is responsible for the generationof the sustained inward Na+ current (Ist) in cardiac pacemaker cells. However, it remains unclear how CaV1.3 could mediate both Ca2+ and Na+ currents. Here, the competitive permeationof Ca2+ and Na+ was examined in LTCCs in mouse sinoatrial node (SAN) cells and CaV1.3
channels in heterologous system. In SAN cells, L-type Ca2+ current (ICa, L) typically elicited at-10 mV was gradually decreased and mostly disappeared as the external Ca2+ concentration([Ca2+] o) was lowered from 1.8 to 0.01 mM. Further reduction in the [Ca2+] o (≤1 µM) conversely
induced an inward current, indicating a switch of conducting ion through LTCC from Ca2+ to
Na+. These responses are characteristic of an anomalous mole-fraction dependence of LTCCs
reported previously. On the other hand, the persistent inward current at -60 mV, assumed
to be Ist, was gradually increased by lowering [Ca2+] o, consistent with differential responses
between ICa, L and Ist to the external Ca2+ in our previous study. Reconstituted CaV1.3 channels
successfully recapitulated ICa, L at the physiological [Ca2+] o of 1.8 mM, which was typicallymanifested at -10 mV but also evident at -60 mV as a sustained inward current similar to Ist in
SAN cells. Importantly, CaV1.3 also reproduced the distinct response of ICa, L and Ist to lowering[Ca2+] o, suggesting both currents in SAN cells are attributable to the permeability propertyof CaV1.3 channels. Theoretical analysis using Almers ＆ McCleskey model well explained
the experimental observation of anomalous mole-fraction dependence of ICa, L as well as thegeneration of Ist through CaV1.3, but predicted the presence of a large Na+ conducting modewith higher Ca2+ sensitivity under extremely low [Ca2+] o condition. (COI:No)

Serotonin (5-HT) accumulates in the heart during ischemia-reperfusion. The accumulated 5-HT
is considered to be taken up into cells and then metabolized with producing 5-hydroxyindole
acetic acid (5-HIAA) and hydrogen peroxide, which worsens the myocardial injury.
Our previous results suggested the contribution of 5-HT transporters, which were resistant
to fluoxetine (selective serotonin reuptake inhibitor) to take up 5-HT from the myocardialinterstitial space during ischemia-reperfusion.
Therefore, the aim of this study was to investigate contribution of plasma membrane monoamine transporter (PMAT) to the 5-HT uptake in the heart.
By means of microdialysis technique to the heart of anesthetized rats, we monitored myocardial interstitial levels of 5-HT and 5-HIAA during coronary occlusion followed by reperfusion
period. We also examined the effects of local administration of a PMAT inhibitor, decynium-22
on the changes in 5-HT and 5-HIAA level.
Baseline dialysate 5-HT concentration significantly increased and 5-HIAA concentration
decreased by decynium-22. Dialysate 5-HT concentration further increased during the coronary occlusion and then decreased after the reperfusion, while 5-HIAA increased after thereperfusion. In the presence of decynium-22, the decrease in 5-HT and the increase in 5-HIAA
during the reperfusion period were significantly prevented. These results indicated that 5-HT
uptake/5-HIAA production was interrupted by the inhibition of PMAT.
In conclusion, PMAT regulates basal level of interstitial 5-HT and contributes to take up 5-HT
into cells during reperfusion after ischemia in the heart.
(COI:No)

2P-027

2P-0305 1 Dept
Yuma Sato1, 2，Tri Doan Huu1, 3，Masayuki Matsumoto4, 5，Tadachika Koganezawa1, （Hidefumi Waki1，Makoto Suzuki1，Keisuke Tomita2，Ko Yamanaka1，Kei Tsukioka1，
3 1Grad Sch Health ＆ Sports Sci, Juntendo Univ, Japan, 2 Faculty of Med
Sabine Gouraud（

Dopaminergic system is involved in cardiovascular responses inducedby lateral habenula activation in rats
Physiol, Fac Med, Univ Tsukuba, Ibaraki, Japan, 2 Master’s Program in Medical Sciences,
Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba,
Ibaraki, Japan, 3 Doctoral Program in Biomedical Sciences, Graduate School ofComprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan,
4 Department of Cognitive and Behavioral Neuroscience, Faculty of Medicine, University ofTsukuba, Tsukuba, Ibaraki, Japan, 5Transborder Medical Research Center, University ofTsukuba, Tsukuba, Ibaraki, Japan）

The habenula is a pair of small nuclei which is located above the thalamus and divided into themedial and lateral nuclei (LHb). It is known that exogenous stress events activate LHb neurons,
which in turn inhibit dopamine neurons in the midbrain. Although the stress events also induce
various autonomic physiological responses such as changes of blood pressure and heart rate,
little is known about whether and how the LHb regulates the cardiovascular responses to thestress event. To understand the roles of the LHb and dopaminergic system in the cardiovascular responses to stress events, here we investigated the effect of the electrical stimulation of the
LHb on blood pressure and heart rate with a dopaminergic receptor blocker. We used Wistar
male rats that were anesthetized by urethane (1 g/kg, i.p.). Arterial pressure was recorded
from the femoral artery. Heart rate was analyzed from R-R intervals of the electrocardiograph.
The LHb was electrically stimulated with a coaxial electrode (300 µA, 0.5 ms duration, 100 Hz,
for 10 s). The LHb stimulation-induced cardiovascular responses were observed before andafter administrating clozapine (1 mg/kg, i.v.), a non-selective dopamine receptor antagonist. As
a result, electrical stimulation of the LHb significantly increased the mean arterial pressure anddecreased heart rate. In the presence of the clozapine, these cardiovascular changes induced
by electrical stimulation to the LHb were significantly attenuated. These results suggest thatthe activation of LHb neurons produces the pressor response and bradycardia by affecting
the autonomic nervous system, and that the effect of the LHb activation on the stress-induced(COI:No)
cardiovascular responses is mediated by the dopaminergic system.

S120

Counteractive effects of daily exercise on stress-induced alteration of
NTS transcriptome in rats
Sci, Teikyo Univ of Sci, Japan, 3 Dept of Biol, Faculty of Sci, Ochanomizu Univ, Japan）

Background: We have reported that chronic restraint stress increased resting blood pressure(BP), whereas daily exercise exhibited anti-hypertensive effects. Since the nucleus tractus
solitarius (NTS) plays important roles in regulating BP, we hypothesized that the NTS is
involved in mechanisms underlying both stress-induced hypertension and exercise-dependentanti-hypertensive effects. In this study, we tested this hypothesis by transcriptome analysis at
the level of the NTS.
Method: Eighteen Wistar male rats (Four-week-old) were divided into three groups (n=6 each):control (Co); chronic restraint stress (ST); and ST + voluntary exercise (SE) groups. ST was
produced by one hour-immobilization per day for 3 weeks (5 days per week). After the end ofintervention period, PCR array (RT2 Profiler PCR Array) targeting genes related to neuronal
functions was used to screen for differentially expressed NTS genes among three groups.
Results and Discussion: Six genes in the NTS were identified as differentially expressed genes
between Co and ST groups, however only two genes were found as differentially expressed
between Co and SE groups, suggesting that exercise modifies stress-induced alteration of gene
expression profiles in the NTS. Such modified genes by exercise include dopamine receptor
D1 (Drd1) and neuropeptide Y receptor type 5 (Npy5r), both of them are known to modulate
neuronal functions.
Conclusion: These results suggest that altered gene expression of the NTS might be involvedin the mechanism underlying both stress-induced high BP and the preventive effect of exercise.
(COI:No)
Functional roles of identified genes in the NTS need to be elucidated.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

2P-0312P-034

Yoshiyuki Fukuba1，Marina Morimoto1，Saki Namura1，Kohei Miura1，Masako Endo Y.1，
1 1 Dept Exerc Sci Physiol, Pref Univ Hiroshima, Japan,
Anna Oue2，Hideaki Kashima（

Eri Tabuchi, Takeshi Sakaba, Yuki Hashimotodani（Lab Mol Syn Fun, Grad Sch Brain Sci,

Effects of warming or cooling of the forearm on brachial artery
endothelial function during leg cycling exercise in humans
2 Toyo Univ）

Analysis of short-term potentiation at the supramammillary nucleus to
the dentate granule cell synapses
Doshisha Univ, Japan）

It is well recognized that leg cycling exercise induces endothelial adaptation of the conduitartery in an inactive limb (i.e., brachial artery; BA) beyond the active lower limbs. Suchbeneficial effects on the endothelium may be because of exercise-induced elevated shear stress(SS) due to increased blood flow to the BA. Modulation of the SS profiles, such as the pattern ofantegrade and retrograde SS, is especially important for vascular adaptation. Warming of theforearm is often used as an experimental manipulation to modulate the SS profile in the BA,
thereby improving flow-mediated dilation (FMD) via shear-dependent mechanisms. Therefore,
the aim of this study was to examine the effect of modulating the SS profile using forearm
warming and cooling on the subsequent endothelial function in the BA during exercise. The 12
healthy young subjects immersed their right forearm into water at either 15 ℃ or 42 ℃ during
60-min leg cycling exercise at heart rate of 120–130 bpm. A control trial was the same exercise
without water immersion. The endothelial function in the right BA was evaluated by means of
FMD (%) before (baseline) and after the exercise. In addition, the shear rate (SR, an estimateof SS) profiles before, during, and after the exercise were simultaneously evaluated. Afterthe exercise, there was no significant change in the FMD the warm immersion and control
trials. However, in the cool immersion trial, the post-exercise FMD at 60 min decreased frombaseline significantly and was lower than the warm and control trials. Concomitant forearm
cooling during leg exercise attenuates post-exercise endothelial function and it is associatedwith modulation of shear patterns. It is concluded that the modulation of shear patterns in the
BA during exercise appears to be associated with subsequent endothelial function. [supported
by JSPS-KAKENHI (#17K01616)]
(COI:No)The supramammillary nucleus (SuM) of the hypothalamus has strong connections with the hippocampus and has been implicated in spatial learning, emotional behavior and sleep-wake state.
We recently found that SuM neurons make monosynaptic connections to granule cells (GCs)and interneurons in the dentate gyrus and co-release glutamate and GABA at these synapses.
While we previously revealed synaptic connections between the SuM and dentate gyrus, little
is known about synaptic plasticity at SuM-GC synapses. Here, we investigated whether SuMGC synapses can undergo short- or long-term plasticity. To this end, we performed whole-cellpatch-clamp recordings from GCs in acute hippocampal slices. To optogenetically activate SuM
afferents, we injected AAV carrying channelrhodopsin-2 into the SuM of VGluT2-Cre mice.
By light illumination, SuM-GC EPSCs were monitored in the presence of GABAA receptor
antagonist picrotoxin. We found that postsynaptic depolarization induced a robust potentiationof EPSCs, which lasted for 10-15 min. This depolarization-induced potentiation (DIP) is inputspecific, as DIP was not observed at perforant-path inputs, main excitatory inputs onto GCs.
We further found that postsynaptic loading of the Ca2+ chelator BAPTA, or bath applicationof nifedipine, a L-type Ca2+ channel blocker, abolished DIP, indicating that a postsynaptic Ca2+
influx by postsynaptic depolarization is required for induction of DIP. To test whether DIP isexpressed as presynaptic or postsynaptic mechanism, we monitored pharmacologically isolated
NMDA-EPSCs. If DIP is due to an enhancement in glutamate release, NMDA-EPSCs are
expected to show a similar potentiation. However, DIP was not observed in NMDA-EPSCs,
suggesting that DIP is postsynaptically expressed. Remarkably, DIP was not observed in
IPSCs mediated by GABA co-released from SuM terminals, suggesting that DIP changes
glutamate/GABA ratio at SuM-GC synapses. Taken together, our results indicate that SuM
inputs may have important roles for information processing in the dentate gyrus by temporalchanging the excitatory/inhibitory balance.
(COI:No)

2P-032

2P-035

Sayaka Niwa（ Dept LifeSci, Grad Sch LifeSci, Ritsumei Univ, Japan）

Suguru Kobayashi, Yuzuki Murao, Nana Komatsu, Hisayo Sadamoto（Kagawa SchlWe aim at developing the human ventricular cell (HuVEC) model (Himeno et al. 2015).
This study asked whether the force of contraction of the HuVEC model well reproduces
the intraventricular pressure to perform the pumping function of a ventricle. Toward thisend, we applied the Laplace equation to convert the developed tension of HuVEC model tothe ventricular pressure. The blood circulation (total volume = ~5800 ml) was composed byconnecting the Laplace ventricle to an afterload, a systemic vascular resistance, a preload, asinus volume and Laplace atrium. A common rate of shortening of the sarcomere length (dX/
dt in the NL model) was determined between the myofilament kinetics and the volume changeof the Laplace ventricle at each cycle of numerical integration of the model. The Bernoulli’s
equation was used for blood pressure with the total pressure E, lateral pressure P and the
dynamic pressure v with the blood density ρ.
E = P + (ρ× v2) / 2
When the HuVEC model was paced with an interval of 800 ms, the major feature of a cardiac
cycle was reproduced, such as the ejection fraction, intraventricular pressure, blood pressurein the afterload, and volume changes in each compartments. It was suggested that the HuVEC
model meets main requirements well for driving the blood circulation. Limitations of the model
will be discussed in this presentation.
(COI:No)

Synchronous oscillatory network is vital for cognitive functions of the brain of both vertebrates
and invertebrates. In the central nervous system of the terrestrial slugs, periodic oscillationis recorded from the surface of the laminar structure of procerebrum (PC) and its frequencychanges are suggested to encode the olfactory information and memory. We recently foundoscillatory activity is generated spontaneously in dispersed cell culture of PC neurons. Application of acetylcholinesterase inhibitor or nicotine increased the number of spontaneous activities
and furthermore, induced synchronous oscillatory activity. On the other hand, biogenic aminesor neuropeptides often changed the number of spontaneous activities without generation ofsynchronous oscillation on PC neurons. Previous results suggest that acetylcholine could befunction as a driving force on the synchronous oscillatory activity of the PC neuron network via
nicotinic acetylcholine receptors activation. In present study, differences between synchronousand asynchronous network were examined in cultured PC neuron (7-21 days). We found alower excitability and a higher sensibility to cholinergic activation in synchronous PC neurons.
PC neurons cultured after 10 days could induce synchronous oscillation, and included twogroup, (a) acetylcholine sensitive and (b) insensitive/driven by a.
(COI:No)

2P-0332P-036The cardiac cycle reconstructed using the human ventricular cell
(HuVEC) model

Controlling development of phase-2 early afterdepolarizations in humanventricular myocytes of lomg QT syndromes: A theoretical study using
bifurcation analyses of two mathematical models
Yasutaka Kurata1，Kunichika Tsumoto1，Ichiro Hisatome2，Yuichi Kuda1，Mamoru Tanida1
12（ Dept Physiol 2, Sch Med, Kanazawa Med Univ, Japan, Dept Regener Med Therapeut,
Grad Sch Med Sci, Tottori Univ, Japan）

Aim: Early afterdepolarization (EAD) causes ventricular arrhythmias in long QT syndrome(LQTS). The aim of this study was to provide a theoretical background for controlling EAD
formation in LQTS via slow-fast decomposition analysis based on bifurcation theory.
Methods: Assuming inhibition of delayed-rectifier K+ channel currents (slow component IKs for
LQT1 and rapid component IKr for LQT2), we used LQT1 and LQT2 versions of human ventricular myocyte models (Kurata et al, 2005; ten Tusscher-Panfilov, 2006), which can reproduce
EADs during β-adrenergic stimulation and bradycardia, respectively. Bifurcation diagrams
for a fast subsystem were constructed as functions of a slow variable, e.g., activation gating
variable of IKs (xs), Ca2+-dependent inactivation gating variable of L-type Ca2+ channel current
ICaL (fCa), or Ca2+ concentration in the sarcoplasmic reticulum (SR). Parameter-dependent shifts
in Hopf, saddle-node and period-doubling bifurcation points were determined as functions ofparameters such as maximum conductance of IKr (gKr), IKs (gKs) and ICaL (gCaL).
Results and Discussion: In the Kurata et al model, with increasing xs2 or decreasing fCa,
an equilibrium point (EP) at depolarized potential was destabilized via a Hopf bifurcation(HB); as gKr decreased, HB points shifted toward higher xs2 or lower fCa, leading to transient
trapping of full system trajectories around the stable EP, i.e., EAD formation. Decreasing
gCaL (or increasing gKr/gKs) shifted HB points toward prevention of trapping of full systemtrajectories (EAD formation). Accelerating IKs activation or inhibition of the transient outward
current (Ito) was also effective in preventing EADs. The ten Tusscher-Panfilov model further
reproduced spontaneous SR Ca2+ release-mediated EADs via destabilization of intracellular
Ca2+ concentrations; decreasing SR Ca2+ pumping rate was effective in preventing the SR
Ca2+ release-mediated EAD. This study demonstrates that bifurcation analysis is useful forsystematically investigating how EADs can be controlled by modifications of ionic channels(COI:No)and transporters. 

Cholinergic induction of network oscillations in invertebrate olfactory
neuron in vitro
Pharmaceut Sci, Tokushima Bunri Univ, Japan）

The endocannabinoid 2-arachidonoyl glycerol in dentate granule cells
suppresses kainate-induced seizures
1 1 Dept. Neurophysiol., Grad. Sch. Med.,
Yuki Sugaya1，Kenji Sakimura2，Masanobu Kano（
Univ. of Tokyo, Japan, 2 Dept. Anim. Model Dev., Brain Res. Inst., Niigata Univ., Japan）

The endocannabinoid 2-arachidonoyl glycerol (2-AG) produced by the activity of diacylglycerol
lipase α (DGLα) mediates retrograde suppression of synaptic transmission and prevents excessive excitability of neural circuits. In line with this notion, we have demonstrated previouslythat kainate-induced seizures are substantially more severe in global DGLα knockout micethan in wild-type littermates. However, it remains unclear which cell types are responsiblefor 2-AG production and ameliorating seizures, although many reports suggest involvementof the hippocampal dentate gyrus in seizures. Therefore, we examined possible roles of
2-AG produced in granule cells of the dentate gyrus in kainate-induced seizures. To obtain
conditional knockout mice with DGLα deletion specifically in dentate granule cells, we crossed
the pro-opiomelanocortin (POMC)-cre mice with the DGLα floxed mice. Immunofluorescencestaining showed that the POMC-cre positive offsprings (gcDGLα -/- mice) lacked DGLα indentate granule cells but exhibited intact DGLα expression in other hippocampal neurons.
In contrast, localizations of CB1 cannabinoid receptors in the dentate gyrus were identicalbetween the gcDGL -/- mice and their cre negative littermates (gcDGLα fl/fl mice). We
injected kainate (30 mg/kg, i.p.) to both genotypes and determined the latency to the onsetof generalized tonic clonic seizures in gcDGLα -/- and gcDGLα fl/fl mice. We found thatthe latency was significantly shorter and the mortality was significantly higher in gcDGLα-/- mice than in gcDGLα fl/fl mice. The latency of gcDGLα -/- mice was comparable to thatof global DGLα knockout mice. These results suggest that 2-AG produced in dentate granulecells ameliorates seizures and is crucial for survival during hyperexcitability. (COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S121

2P-037

Postnatal stimuli modulate cortico-hippocampal network dynamics
2 1 Dep. Histology, Jichi Medical
Yoshiaki Shinohara1，Shinnosuke Koketsu2，Takatoshi Ueki（

Univ., 2 Dep Integrative Anatomy, Med, Nagoya City Univ）

Phase-locked synchronization between the cerebral cortex and hippocampus has been
implicated to be important during working memory and spatial navigation. However, howanimal experience organizes cortico-hippocampal dynamics is largely unknown. To analyze
synchronized activity between the hippocampus and various cortical areas, we used transgenic
mice (G7NG817), which expresses the calcium indicator G-CaMP7 in astrocytes and the majority of excitatory neurons in the cortex. The mice allow us to observe the temporal dynamics
of cortical calcium in 25-100Hz transcranially. Environmental effects on cortico-hippocampal
dynamics were observed by comparing mice reared under two distinct conditions, either inenriched environment (ENR) or isolated condition (ISO). After 4 weeks rearing, we measured
cortical calcium dynamics and hippocampal local field potentials simultaneously.
We found that the cortical calcium dynamics was closely correlated to hippocampal EEG
status in urethane anaesthetized mice. Regardless of the rearing conditions, calcium activities
observed during theta and non-theta states showed distinct spatial and temporal patterns.
Cortical calcium level is higher in wide areas of the cortex during the theta state, and thetemporal calcium fluctuation is relatively mild. During non-theta states, basal calcium level islower, but larger cortical calcium elevations that spread over cortical areas co-occurred withhippocampal sharp wave and ripple (SWR) oscillations.
Next, we investigated spatio-temporal dynamics between hippocampal SWR events andcortical calcium using head-restrained un-anaesthetized mice monitoring animal sleep/awake
states. In both states, calcium activities preceded hippocampal SWR events in vision-related
cortical areas. During awake SWRs, wide areas of cortical calcium elevation co-occurredwith hippocampal SWRs, while sleep SWRs showed delayed frontal cortical activation after
SWRs. Interestingly, ENR mice exhibited sparser activity patterns. Our data indicate that thecortico-hippocampal dynamics show distinct patterns depending on animal states, but postnatal(COI:No)
experience also play a role for modulating the network dynamics.

2P-040Orexin receptor activation induces a novel slow afterhyperpolarizationthat results from the calcium-dependent closure of cation channels inserotonergic dorsal raphe neurons
2 1 Dept of Neurophysiol,
Masaru Ishibashi1，Atsuo Fukuda1，Christopher Leonard S（

Hamamatsu Univ School of Med, Japan, 2 Dept of Physiol, New York Medical College, USA）

Serotonergic (5-HT) dorsal raphe (DR) neurons regulate numerous brain functions including
sleepwake states, circadian phase, reward and mood. Moreover, orexin receptor signaling at
5-HT DR neurons appears critical in the sleep disorder narcolepsy, which emerges following
the loss of orexin signaling. We recently reported that in addition to producing a slow depolarization, orexin-A enhances the post-spike afterhyperpolarization (oeAHP), which alters spikeencoding by increasing spike frequency adaptation. Mechanistically we found that the oeAHP
involved two distinct components that required Ca2+ influx. The first was of medium-duration(tau ~ 0.5s) and involved apamin-sensitive SK Ca2+-activated K+ channels. The second was oflonger duration (tau ~ 5s), was apamin-insensitive (termed the ai-oeAHP) and appeared similar
to a slow AHP (sAHP). In this study we used whole-cell patch clamp recordings and Ca2+
imaging in mouse brain slices to investigate the mechanisms and function of this ai-oeAHP.
we found that the ai-oeAHP was not attenuated by a cesium-based patch solution as expected
for a K+ currents, but rather was blocked by substituting NMDG for Na+ in the ACSF orby application of flufenamic acid (FFA), both of which attenuated the orexin-induced inward
current. Moreover, we found that the increase in baseline membrane conductance produced
by orexin-A was reduced during the ai-oeAHP suggesting that the ai-oeAHP was mediated bya transient, Ca2+-dependent closure of the cation channels activated by orexin. These results
suggest that ai-oeAHP is a novel type of Ca2+-dependent sAHP that is conditionally expressedfollowing orexin-activation of non-selective cation channels. (COI:No)

2P-0382P-041

Daisuke Ikutame1，Masamichi Shinoda2，Kentaro Urata1，Shintaro Fujiwara1，
2 1 Dept Complete Denture Prosthodont, Nihon Univ Sch
Toshimitsu Iinuma1，Koichi Iwata（（Neurobiology Research Unit, OIST, Japan）

Age-related changes in the M1/M2 polarization of microglia intrigeminal spinal subnucleus caudalis following intra-oral injury
Dent, Japan, 2 Dept Physiol, Nihon Univ Sch Dent, Japan）

Pain perception in the orofacial region is known to change during aging. It is reported that the
microglial activation in the trigeminal spinal subnucleus caudalis (Vc) is involved in orofacial
pain hypersensitivity following intra-oral injury in young adult animals, but it is unclear how
the aging affects the pain hypersensitivity enhanced by microglial activation following orofacial
tissue injury. Here, we examined that aging-related changes in the M1/M2 polarization of
microglia in Vc following intra-oral injury in mice.
Senescence-accelerated mouse prone 8 (SAMP8) and senescence-accelerated mouse resistant
1 (SAMR1) as control were used in this study. Palatal mucosal incision (Length: 3mm, Depth:1 mm) was performed under the deep anesthesia. The mechanical withdrawal threshold(MHWT) in the incised site was measured for 21 days following intra-oral tissue injury. Furthermore, expression pattern of CD11c-immunoreactive (IR) cells (affective microglia (M1)) andCD163-IR cells (protective microglia (M2)), and TNFalpha-IR M1 and M2 in Vc was analyzed
immunohistochemically on days 3 and 11 following intra-oral tissue injury.
The decrease of MHWT following intra-oral tissue injury is significantly enhanced in SAMP8.
M1 activation was enhanced and the number of TNFalpha-IR M1 and M2 was increased on
day 3 following intra-oral tissue injury in SAMP8 compared with SAMR1. On day 11, M1 andM2 activation was observed in both groups, whereas M2 activation was attenuated in SAMP8.
Moreover, the increase in the number of TNFalpha-IR M1 was more extensive than that of
TNFalpha-IR M2.
The present findings suggest that mechanical allodynia following intra-oral tissue injury waspotentiated and sustained in SAMP8 due to the enhancement of TNFalpha signaling following
M1 activation and M2 attenuation in Vc.(COI:No)

Difference of cholinergic modulation on corticostriatal synaptic plasticity
between direct and indirect spiny projection neurons
Atsushi Tamura, Julie Chouinard, Kiyoto Kurima, Yumiko Akamine, Jeffery Wickens R.
The cholinergic interneurons (CINs) of the striatum are crucial for behavioral flexibility. CINsof the dorsomedial striatum (DMS) play a role in strategy switching. However, how CINs
modulate the neural circuitry underlying strategy switching is unclear. The glutamatergic
afferents from the cerebral cortex to the striatum display activity-dependent plasticity in thecorticostriatal synapses, and may be involved in certain types of learning. One hypothesis isthat strategy switching may be realized by a modulatory effect of CINs on corticostriatal
plasticity. Here, we investigated the effect of CINs on activity-dependent plasticity in thecorticostriatal synapses. To control tonic firing of CINs, adeno-associated virus (AAV) encoding
halorhodopsin (NpHR) was injected into DMS of ChAT-cre mice. AAV injected mice expressed
NpHR in CINs and we can optogenetically inactivate CINs firing. We made whole-cell exvivo slice recordings from spiny projection neurons (SPNs), which are the output neurons of
the striatum, and recorded EPSPs induced by electrical stimulation of the corpus callosum.
Activity dependent synaptic plasticity was induced by high-frequency stimulation under the
Mg-free conditions. This conditioning stimulus combined with optogenetic inactivation of CINs
during HFS induced long-term potentiation in some SPNs. However, other group of SPNs
showed long-term depression in response to the same conditioning stimuli. To identify the cell
type showing opposite plsticity, we observed HFS-induced LTP on either direct or indirect
SPNs.These recordings revealed that pause in CINs faculitate LTP on dSPNs and disrupte
LTP on iSPNs. This result suggest that pause in CINs work on synaptic plasticity in different(COI:No)
manner between dSPNs and iSPNs.

2P-039

2P-042Masatoshi Ando, Masamichi Shinoda, Koichi Iwata（ Dept Physiol, Sch Dent, Nihon Univ,

Dongyu Liu1，Tomokazu Ohshiro1，Kazuyuki Fujihara2，Yuchio Yanagawa 2，
1 1 Dept Physiol, Grad Sch Med, Tohoku Univ, Japan, 2 Dept Gen Behav
Hajime Mushiake（

Oxytocin attenuates orofacial mechanical allodynia following infraorbital
nerve injury
Japan）

Oxytocin (OXT) signaling is known to attenuate nociceptive neuronal hyperexcitability whichis responsible for pain hypersensitivity. In this study, we examined the effect of oxytocin application into the nerve-injured site on orofacial mechanical hypersensitivity following infraorbital
nerve injury (IONI) in rats.
A one-third thickness of the infraorbital nerve was tightly ligated with 6-0 silk under deep
anesthesia, MedGel® containing OXT (20 µl, 50 mg/ml in 0.01M PBS) is placed directly into
the ION-injured site. The head-withdrawal threshold to mechanical stimulation (MHWT) of thewhisker pad skin was measured using von Frey filament following IONI. Trigeminal ganglion(TG) neurons innervating the whisker pad skin were identified employing retrograde labelingwith 3% Fluoro-Gold (FG). FG-labeled OXT receptor-positive, transient receptor potential vanilloid 1 (TRPV1)-positive, and TRPV4-positive TG neurons were examined immunohistochemically on day 5 following IONI.
The MHWT was significantly decreased from day 1 onward following IONI. OXT application
into the nerve-injured site attenuated the decrease of the MHWT from day 5 onwards. On day
5 following IONI, OXT receptors were expressed in uninjured ION and FG-labeled TG neurons
Furthermore, the number of FG-labeled TRPV1-positive and TRPV4-positive TG neurons wasincreased on day 5 following IONI, and its increments were significantly depressed by the
OXT application into the ION-injured site. In addition, the suppression of the increased numberof FG-labeled TRPV1-positive and TRPV4-positive TG neurons was significantly inhibited byconcomitant OXT receptor antagonist (Atosiban, 20 µl, 100 µM) application.
These findings suggest that OXT application into the ION-injured site inhibits the increases in
TRPV1 and TRPV4 expression in TG neurons innervating the whisker pad skin, resulting in
the attenuation of orofacial mechanical allodynia following IONI.
(COI:No)

S122

Neurological analysis of the GAD67 knockout rats
Neuro, Grad Sch Med, Gunma Univ, Japan）

An inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) is synthesized by two
isozymes, glutamate decarboxylase (GAD) 65 and GAD67. GAD65 is localized in the synapses
and responsible for synaptic GABA production. Its deficiency causes spontaneous epilepsy
in both mice and rats, indicating indispensable roles of the protein in the central nervous
system. On the other hand, GAD67 is a cytoplasmic protein and GAD67 knockout mice areborn with severe cleft palate and all of them die at birth, which hampered further detailed
analysis. Thus, it remains unknown whether deficiency of GAD67 causes any abnormal brain
function in animals.
Recently we generated GAD67 knockout rats by a gene-editing method and found that incontrast to GAD67 knockout mice, GAD67 knockout rats do not exhibit cleft palate and someof them can survive to adulthood. Therefore, these GAD67 knockout rats brought us an opportunity to examine the function of GAD67 in vivo. The GAD67 knockout rats exhibited no
drastic convolutions or myoclonus as in the case of the GAD65 knockout rats. To examine if
the GAD67 knockout rats exhibit any abnormal brain activity, we recorded the EEG fromthe animals. A hand-made headset with eight wire electrodes was chronically implanted overthe epidural space of the homozygous GAD67 knock-out rats. Heterozygous or wild typeanimals for the gene locus were used as control. Basic time-series analysis of the raw EEG
revealed rather normal frequency distributions of the slow sleep waves and the awake fast
waves, comparable to those of the control animals. However, a series of short slow waves (3～6 seconds) were observed intermittently in the EEG from the GAD67 knockout rats while
awake. We are currently investigating whether these paroxysmal waves reflect any petit-mal(COI:No)seizure, by monitoring their behaviors when these paroxysmal waves occur.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

2P-043

2P-046

Tenpei Akita, Atsuo Fukuda（ Dept Neurophysiol, Hamamatsu Univ Sch Med, Japan）

1 1 Dept Integr
Yoshihiko Wakazono1，Ryosuke Midorikawa1，Shogo Oka2，Kogo Takamiya（

Two cation current components in GABAergic interneuron precursors in
the medial ganglionic eminence of the embryonic brain
Proliferation and migration of developing cells are accompanied by cell volume changes, which
are achieved by the net ion flux and the resulting passive water movement across the cell
membrane. The opening of several types of ion channels and the electrochemical gradientsof permeating ions can produce the net ion flux. The precursors of GABAergic interneurons
in the cerebral neocortex are born primarily in a region in the ventral telencephalon of theembryonic brain, called the medial ganglionic eminence (MGE), and they migrate toward theneocortex during prenatal and perinatal periods. The types of ion channels involved in thedevelopment of interneuron precursors have not yet been identified. By whole-cell patch
clamping of acutely isolated interneuron precursors in MGE, we found two cation current
components under normal conditions. One component was activated at the membrane voltage
level more than －30 mV and exhibited steep outward rectification that was suppressed by
the removal of K+ from the patch pipette solution. The tail current generated at －60 mV
after depolarizing voltage steps was, however, inward and abolished by external Na+ removal.
The reversal potential of the tail current was around －30 mV. Thus, this voltage-gated current component must be generated by an ion channel highly permeable to K+ with a small
significant permeability to Na+. Another component was a small Na+ leak current with linear
voltage dependence. These two cation current components might cooperatively regulate the(COI:No)development of interneuron precursors.

2P-044

Concentration dependent inhibition of evoked EPSPs produced byammonium in rat hippocampal CA1 neurons
Naomitsu Kameyama, Eiichiro Tanaka, Yoshinaka Murai, Kiyoshi Kikuchi（Dept physiol, Sch
of Med, Kurume Univ, Japan）

Hyperammonemia suppresses the central nervous system, e.g., it can induce comas. Why
hyperammonemia induces impairments of consciousness has not yet been clarified. On theother hand, a high concentration (1–5 mM) of NH4Cl has been found to inhibit excitatory
postsynaptic potentials (EPSPs) in in vivo or in vitro animal model experiments, although themechanism responsible for the inhibition of EPSPs by NH4Cl is still under discussion. We
investigate details of the inhibition of EPEPs by NH4Cl using intracellular and extracellular
recordings made from the hippocampal CA1 and CA3 neurons of male Wistar rats. Fast EPSPs
were elicited via the focal stimulation of Schaffer collaterals through monopolar electrodes.
The AMPA type fast EPSPs initially increased in amplitude by 5 mM NH4Cl superfusion, thenafter suppressed and disappeared. Application of various concentration of NH4Cl induced the
suppression of the AMPA type fast EPSPs with concentration dependent manner. Applicationof 5mM glutamate induced a membrane depolarization, which is increased in amplitude in thepresence of 5mM NH4Cl. On the other hand, a Na+ spike elicited in a CA3 neuron suppressedby the application of 5mM NH4Cl. A Ca2+-dependent spike was elicited in a CA3 neuron in
the presence of 1 µM tetrodotoxin / 20mM tetraethyl ammonium. The Ca2+-dependent spike
initially prolonged its duration by application of 5 mM NH4Cl, and then shortened.
These results suggest that sustained suppression of fast EPSP may be due to presynaptic
inhibition. Mechanism of the presynaptic inhibition may include, at least, inhibition of the Na+
spike generation or conduction in the excitatory nerve, and of the influx of the Ca2+ into
the excitatory nerve terminals. Impairment of consciousness due to hyperammonemia may
cause by the reduction of excitatory inputs to the central nervous system due to presynaptic
inhibition.
(COI:No)A crucial role of N-glycosylation of homomeric GluA1 AMPA receptor in
LTP induction
Physiol, Fac Med, Univ Miyazaki, Japan, 2 Dept Biol Chem, Human Health Sci, Grad Sch
Med, Kyoto Univ, Japan）

The intracellular molecular mechanisms underlying the regulation of the AMPA receptor havebeen dramatically elucidated in the past few decades. In contrast, the functional regulation of
the extracellular domain remains unclear. Here, we focused on N-glycosylation of the AMPA
receptor in the extracellular domain and tried to clarify their functions by combining molecularbiological and electrophysiological techniques.
We previously reported that 401 asparagine residues (N401), putative N-glycosylation site, in
GluA1 subunit might be a responsive site for drastic changes of glutamate responses of AMPA
receptor from desensitization to re-sensitization, and that long-term potentiation (LTP) induction of hippocampal slices prepared from N401Q GluA1-expressing lentivirus vector-injected
GluA1 KO mice was impossible to maintain its potentiation.
In the present study, we tried to clarify molecular mechanisms underlying the defect of LTP
maintenance in N401Q GluA1 mutant. Analysis of miniature EPSC (mEPSC) revealed that thefrequency, amplitude and rise times showed no significant change, and that the decay time
became significantly faster than that of wild-type GluA1. In addition, when expressed N401Q
GluA1 in primary cultured neurons prepared from embryonic GluA1 KO mice brain, theglutamate response did not always show the re-sensitization. These are not consistent withresults in N401Q GluA1-expressing HEK cells. To explain this discrepancy, we next examined
the involvement of GluA2 subunit, which is possible to form heteromers with GluA1 subunit.
Glutamate response in N401Q GluA1-expressing HEK cells reverted the re-sensitization to
the desensitization by co-expression with GluA2 subunit. These results suggest that the resensitization in N401Q GluA1 homomers might influence to synaptic currents during the LTP
induction period, not in the resting state.
(COI:No)

2P-047Long-term survival and proper axonal elongation of grafted
hypothalamic Neurons from Mouse ES Cells
Miho Kawata1，Yu Kodani1，Yoko Kaneko S.1，Akira Nakashima2，Hiroshi Nagasaki1（ 1 Dept Physiol, Sch Med, Fujita Health Univ, Japan, 2 Dept Physiol chem, Sch Med, Fujita
Health Univ, Japan）
We have established the method to transplant the differentiated hypothalamic neurons frommouse embryonic stem cells (mESC) into the supraoptic nucleus (SON) of the hypothalamus. In
this study, we investigated whether grafted neurons could survive for a long time and functionin the host brain.
EB5, a mESC cell line, was induced to hypothalamic neurons by floating culture method. The
hypothalamic progenitors were purified using the cell-surface antigens. For differentiation,these cells were enzymatically dissociated and cultured on Matrigel-coated cover glass. Afterday 28, when cells developed to show hypothalamic nature, the cells were infected AAVCAG-tdTomato for labeling with red fluorescent protein. Then, the cells were dissociated andtransplanted to SON of the SCID/NOD mice. To examine the long-term survival and functionof the grafted cells, we collected brain samples at 1 month or 3 months after the grafting.
Graft tissue survived up to 3 months. The axons from tdTomato positive neurons were identified widely in hypothalamic area after 1 month. Three months after grafting, the tdTomato
positive axons showed beaded-shape, and some of them have reached to the neural lobe of
pituitary.
These results suggested the grafted hypothalamic neurons survived in the hypothalamus areaof immunnodeficient mice for long-term and followed the pattern of axon guidance as seen in
the host hypothalamic neurons.
(COI:No)

2P-045

2P-0481 1 Dept Physiol, Faculty Med, Osaka
Yoshihiro Egashira1，Akio Ojida2，Fumihito Ono（

Munehisa Maeshiba1, 2，Hiroshi Kajiya1, 3，Takashi Tsusumi2，Takashi Tsuzuki2，Koji Okabe3，
1 1 Res Cent for Regener Med, Fukuoka Dent Coll, Japan, 2 Dept Oral Rehabil,
Jun Ohno（

The identity of the synaptic vesicle pools underlying evoked andspontaneous synaptic transmission in larval zebraﬁsh neuromuscularjunction
Medical College, Japan, 2 Grad Sch Pharm Sci, Kyusyu Univ, Japan）

The information transfer at a synapse is mediated by the release of neurotransmitters stored
in the synaptic vesicles (SVs). All synapses show both evoked (action potential dependent) andspontaneous (action potential independent) forms of neurotransmitter release. Aside from themode of neurotransmitter release, neurotransmitter-filled SVs are generally categorized into
three distinct pools; a readily releasable pool (RRP), a reserve pool and a resting pool. The RRP
and reserve pool together form the recycling pool of SVs. Although the relationship between
evoked and spontaneous neurotransmitter release has been investigated using a variety ofmodel synapses, it remains controversial whether the SVs undergoing evoked and spontaneous
fusion belong to the same SV pool. Here we addressed this issue in the larval zebrafish neuromuscular junction by generating a novel transgenic fish in which a pH sensitive fluorescentprotein (super-ecliptic pHluorin; SEP) and Halo tag were fused in tandem to the luminal side ofa SV protein and expressed specifically in the motor neurons. Live imaging of SEP fluorescenceallowed us to monitor exo / endocytosis of SVs induced by repeated electrical stimulation,
whereas Halo tag-based labeling enabled us to separately tag SVs mobilized by evoked andspontaneous activities. Using this technique, we found that, in zebrafish neuromuscular junction, about 80% of SVs were mobilized by both high frequency electrical stimulation and highK+ depolarization stimulation, and thus constituted the recycling pool. In contrast, SVs fused
spontaneously comprised only 12% of total SVs. Sequential labeling of SVs with spontaneousand evoked activities indicated that the two SV populations overlapped completely. Therefore,
our data showed that the SVs undergoing spontaneous fusion originated from the identical SVpool responding to action potentials, and the population of the spontaneously-fusing SVs was(COI:No)
limited to a small proportion.

Hyperocclusion suppresses the cognitive activity via the expression ofamyloid beta
Fukuoka Dent Coll, Japan, 3 Dept Physiol Sci Mol Biol, Fukuoka Dent Coll, Japan）

Background: In recent years, severe periodontitis has been reported to suppress cognitive
ability. However, it is unclear the relationship between malocclusion and cognitive ability. The
purpose of this study was to clarify the effects of malocclusion on cognitive ability.
Materials and Methods: To create hyperocclusive state in jaw movement stainless wireattached to occlusal surface in upper right molar in 2-month-old and 12-month-old mice. We examined the behaviors analyses such as the 8-arm radial maze test and novel object recognition
test on a trial per day. The analyses were measured the total stay duration and error scores in
the 8-arm radial maze test and recognitive scores in novel object recognition test. Furthermore,
the hippocampus samples were collected on before (0) and after (1 and 4 weeks) hyperocclusion
loading in these mice and examined the change in expression of cognition related molecules.
Results and Discussion: The long-term cognitive ability and social cognitive ability transiently
suppressed on 1 week after hyperocclusion in 2 month-old mice, but not in 12 month-old mice.
The expressions of cognition suppressing molecules were also upregulated on 1 week after hyperocclusion using RT-qPCR and Western blotting. The malocclusion suppressed the cognitive
ability via the upregulation of suppressive molecules in the cognition, especially amyloid beta,
indicating that strict occlusion play an important factor in normal cognitive ability.  (COI:No)

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2P-049

2P-0522 1 Biophotonics
Sota Hiro1, 2, 3，Yoshiko Yamada2，Tomomi Nemoto1, 2, 3，Ryosuke Enoki1, （Taku Nakagawa1，Toshiharu Yasaka2，Noriyuki Nakashima1，Kensuke Oshita3，
1 1 Dept Physiol, Med, Kurume Univ, Japan, 2 Dept Immunol, Grad Sch
Makoto Takano（

Optical analysis of nuclear-cytoplasmic Ca2+ dynamics in the mastercircadian clock
Research Group, Exploratory Research Center on Life and Living Systems, National
Institutes of Natural Sciences, Japan, 2 Laboratory of Molecular and Cellular Biophysics,
Research Institute for Electronic Science, Hokkaido University, Japan, 3 Graduate School ofInformation Science and Technology, Hokkaido University, Japan）

The temporal order of the mammalian physiology and behavior is coordinated by the biologicalcircadian clock that is synchronized with the environmental light-dark cycle. The master clockof circadian rhythm is the suprachiasmatic nucleus (SCN) that is located in the hypothalamus.
The SCN is composed of ca. 20,000 neurons, and the individual neurons are thought to keep
24-hour rhythms by the transcriptional and posttranslational feedback loop (TTFL) involving
the clock genes and their protein products. The cytoplasmic circadian Ca2+ rhythm is thought
to regulate the TTFL (Enoki et al., Scientific Reports, 2017). On the other hand, the SCN
neurons exhibited circadian Ca2+ rhythms in the cytoplasm but not in the nucleus (Ikeda etal., Neuron, 2003). It is still a matter of discussion whether the cytoplasmic Ca2+ enters into
the nucleus or whether the cytoplasmic Ca2+ is regulated independently of the nuclear Ca2+.
In the present study, we simultaneously expressed two genetically-encoded Ca2+ probes,
GCaMP6s and jRGECO1a: The former expressed specifically in the nucleus of the SCN neurons and the latter in the cytoplasm by combining with nuclear localization signal or nuclear
exporting signal, respectively. Ca2+ dynamics were monitored at the single-cell level using a
Nipkow-disk confocal time-lapse imaging system. We found robust circadian Ca2+ rhythms in
the nucleus (nuclear circadian Ca2+ rhythms). Both nuclear and cytoplasmic circadian Ca2+
rhythms had identical topological patterns in the SCN network: the advanced phase in thedorsal region compared with the central and ventral regions. Moreover, nuclear circadian
Ca2+ rhythms had the same phase as cytoplasmic circadian Ca2+ rhythms. These results showthat spatiotemporally organized nuclear circadian Ca2+ rhythms might play a crucial role in atranscriptional regulatory mechanism in the SCN.
(COI:No)

2P-050Establishment and characterization of neuron-like PC12-derived celllines with hypersensitivity or hyposensitivity to temperature-regulated
repeated thermal stimulation (TRTS)
Tada-aki Kudo1，Kanako Tominami1，Satoshi Izumi1，Yohei Hayashi2, 3，Takuya Noguchi4，
5 1 Div Oral Physiol, Grad Sch Dent, Tohoku Univ, Japan, 2 CRCBR, IDAC,
Guang Hong（Tohoku Univ, Japan, 3 Grad Sch Life Sci, Tohoku Univ, Japan, 4 Lab Health Chemist, Grad
Sch Pharma Sci, Tohoku Univ, Japan, 5 Liaison Cent Innov Dent, Grad Sch Dent, Tohoku
Univ, Japan）

Neurite formation allows functional formation or restoration of the nervous system. Previously,
we developed a novel in vitro method for induction of neuronal cell differentiation in rat
neuron-like PC12 cells using temperature-controlled repeated thermal stimulation (TRTS) witha heating plate for temperature regulation of the culture medium. Based on sensitivity to
TRTS, the PC12 cells were divided into two types: hypersensitive to TRTS with elongation of
the neurites, and hyposensitive to TRTS for unknown reasons. To investigate the mechanismof hyposensitivity, this study aimed to establish two PC12-derived cell lines according tosensitivity to TRTS by a general method of subcloning during the course of neuronal celldifferentiation using a 96-well culture plate. To characterize these two models, the cell size,
growth rate, and elongation of the neurites were evaluated in the subcloned cells treated withneurite inducers, such as nerve growth factor (NGF) and bone morphogenic protein (BMP),
or TRTS. Two types of subcloned cell lines were obtained: PC12-P1F1, TRTS-hypersensitive
PC12 cell line and PC12-P1D10, TRTS-hyposensitive PC12 cell line. No significant differencesin the cell size and growth rate were observed among the parental PC12 cell line and thetwo subcloned cell lines. Elongation induction of the neurites by BMP or TRTS was higher
in PC12-P1F1 than in the parental PC12 cells, while no induction of neurite elongation wasobserved in PC12-P1D10. In contrast, NGF-induced neuritogenesis was observed in all the celllines. These results suggest that PC12 parental cell line shows differences in cell characteristics
related to TRTS-induced neuronal differentiation, and BMP signaling pathway is required for
TRTS-induced neurite outgrowth in PC12 cells. (COI:No)

2P-051

Cytotoxicity of nanomaterials and neuronal cell death
Yuji Kamikubo, Yoshie Hashimoto, Takashi Sakurai（Dept Pharmacol, Juntendo Univ Sch

Characterization of HCN4-immunoreactive neurons that localized atlaminae Ⅱ-Ⅲ in mouse spinal cordMed, Kagoshima Univ, Japan, 3 Dept Anesth, Med, Kurume Univ, Japan）

The hyperpolarization-activated, cyclic nucleotide-gated channels (HCN1～4) are drawing attention as a potential target of pain treatment; in the dorsal root ganglion, it has been reported
that HCN2 expressing neurons play the central role in the transmission of inflammatory andneuropathic pain. We have previously reported that HCN4 channels are expressed in the spinal
dorsal horn, using transgenic mouse in which the expression locus of HCN4 can be visualized
by chemical luminescence by firefly luciferase. However, little is known about the function of
HCN4-expressing neurons in the spinal dorsal horn. We therefore aimed to characterize theHCN4 expressing cells using the immunohistochemistry in the transgenic mouse expressing the artificial chromosome in which GFP was knocked into the genetic locus of vesicular
GABA transporter (VGAT). We found that the HCN4-immunoreactive cells showed laminar
distribution restricted to lamina II inner (IIi) and lamina III, and hardly co-localize with GFPsignal derived from VGAT. These findings suggested that most of HCN4-expressing neurons
were putatively excitatory interneurons. It has been reported that excitatory interneurons at
the border between lamina IIi and III receive the tactile inputs and simultaneously receive
the feed-forward-inhibition from the inhibitory interneurons. The failure of such feed-forwardinhibition reportedly underlie the neuropathic pain. Because HCN channels were thought toregulate the synaptic integration by suppressing the fluctuation of membrane potential, HCN4
expressed in the excitatory interneuron might participate in the modulation of such local(COI:No)
circuit.

2P-053The mechanism underlying central sodium sensing for mediating saltinduced hypertension
3 1 Division of Molecular
Kengo Nomura1，Takeshi Hiyama.Y1, 2，Masaharu Noda1, 2, （Neurobiology, National Institute for Basic Biology, 2 School of Life Science, SOKENDAI,

3 Research Center for Cell Biology, Institute of Innovative Research, Tokyo Institute ofTechnology）

Increases in sodium concentrations ([Na+]) in body fluids elevate blood pressure (BP) by enhancing sympathetic nerve activity (SNA). However, the mechanisms by which information on
increased [Na+] is translated to SNA have not yet been elucidated. We demonstrated that [Na+]increases in body fluids mediated sympathetic activation leading to BP elevations in wild-type
mice, but not in mice which genetically disrupts Nax, an atypical Na channel. We identified Nax
channels expressed in specific glial cells in the organum vasculosum lamina terminalis (OVLT)
as the sensors detecting increases in [Na+] in body fluids, and showed that OVLT neurons
projecting to the paraventricular nucleus (PVN) were activated via acid-sensing ion channel 1a(ASIC1a) by H+ ions exported from Nax-positive glial cells. These results provide an insight into
the neurogenic mechanisms responsible for salt-induced BP elevations.
(COI:No)

2P-054

Response properties to amplitude changes of sound envelope in theneurons of the anterior auditory ﬁelds

Med, Japan）

Sohei Chimoto（Dept NeuroPhysiol, Div of Med, Univ of Yamanashi, Japan）

With the development of nanoscience, nanomaterials with various sizes, chemical elements,
shapes, and surface modifications have been produced for industries and consumers. Althoughbulk materials have constant features regardless of their size, nanomaterials possess novelsize-dependent physical and chemical properties and have been used in a broad range of processes and products including paints, foods, cosmetics, clothes, polishing of fine structures, andpharmaceuticals. Nanomaterials including nanoparticles (NPs) of titanium dioxide, aluminumoxide, zinc oxide, silicon dioxide (silica) are well studied and have been applied in various fields.
Because silica nanoparticles are inexpensive, innocuous, and easy to produce and functionalize,
they are applied as adsorbents, catalyst carriers, materials for bio-imaging, and drug-delivery
systems. Despite these benefits, there are concerns that exposure to nanoparticles can havedetrimental effects on certain types of tissues. Because NPs can cross the blood–brain barrier
and the blood–placental barrier, they may cause toxic effects such as immune responses,
hemolysis, and developmental abnormalities in the brain and developing embryos. Although
investigations of the toxicity of NPs to neurons are essential for medicinal use, few studies haveassessed the direct effects of NPs on cells derived from the central nervous system. In thisstudy, we showed that treatment with silica and metal oxide NPs caused oxidative stress andneural cell death. Furthermore, we found that these cytotoxicities was reduced by NPs surfacefunctionalization or protein coating, and pretreating cells with an antioxidant, suggesting that
contact-induced oxidative stress may be responsible for NPs-induced cell death. These data will
(COI:No)be valuable for utilizing NPs in biomedical applications.

Natural sounds such as human speech and animal species-specific sounds show a broad of timeenvelope changes in amplitude under acoustic environments. They contain a sound with aquick attack in amplitude (damped sounds) and a sound with a slow increase (ramped sounds).
It has been also examined that the temporal amplitude changes influence sound perception andidentification in human psychophysical experiments. In the previous studies we found that thehigher auditory cortices as well as the primary auditory cortex (A1) include velocity-sensitive
cells for the damped- and ramped-sounds and some neurons in the secondary auditory cortex(A2) and posterior auditory fields (PAF) had the sensitivities to direction of the amplitudechange. The neurons in anterior auditory fields (AAF) rostral to A1 show tonotopic responses
and are also responsive to various kinds of sounds such as noise busts, amplitude modulated
sounds, and natural sounds. However, little is known about their response properties during
the stimuli with amplitude change. In the present study we recorded single unit activities fromAAF of awake animals and examined response properties during ramped and damped sounds.
Spike trains responding to each sound were used to construct peristimulus time histogramsand the height of PSTH was transformed into the driven rate by subtracting the mean of
background firing rates. We found that most of AAF neurons showed sensitivities to the quickchange of stimulus envelope and a few neurons showed sensitivities to the slow amplitudechanges during both damped- and ramped-sounds. Most AAF neurons also show weak
selectivity for damped- or ramped-sounds by comparing between the peak response amplitude
during both sounds. These results suggest that the majority of AAF neurons are tuned to thehigher velocity of the envelope change but not to the direction of the change.
(COI:No)S124

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

2P-055

GABAergic inputs to ON and OFF starburst amacrine cells arecontrolled by different acetylcholine receptors
Mie Gangi, Makoto Kaneda（ Dept Physiol, Nippon Med Sch, Tokyo, Japan）
Starburst amacrine cells (SACs) are considered to play an important role for the formationof direction selectivity in the retina. They are classified into ON SACs and OFF SACs basedon their morphological properties. The ON SACs have cell bodies in the ganglion cell layer
and arborize their dendrites in sublamina b of the inner plexiform layer (IPL). The OFF
SACs have cell bodies in the inner nuclear layer and arborize their dendrites in sublamina aof the IPL. Because of the easy accessibility to the soma in the whole-mount preparation, theelectrophysiological properties of ON SACs were rigorously studied, and the electrophysiological properties of OFF SACs were believed to be same as those of ON SACs. In a seriesof experiments, we showed that the electrophysiological properties of OFF SAC were notpredictable from those of ON SACs. First, P2X2-purinergic signaling pathway is predominantly
used in OFF SAC. Second, glycinergic signaling pathway is predominantly used in ON SAC.
Further, we recently showed the possibility that the pathways of acetylcholine (ACh) synthesis
might be different between ON SAC and OFF SAC.
In the present study, we examined whether such a possible difference of ACh synthesis can
produce further difference in cholinergic signaling pathways in the mouse retina. Application of
ACh or carbachol (CCh) increased GABAergic IPSCs in both SACs. In ON SACs, a muscarinic
agonist increased IPSCs, while in OFF SACs, a nicotinic agonist increased IPSCs. These results
indicate that ON and OFF SACs receive GABAergic inhibitory inputs from different amacrine
cells expressing different subtypes of ACh receptors.
(COI:No)

2P-058

Electrical synapses of pyramidal cells in developing visual cortex andretinal ganglion cells can enhance excitatory synaptic outputs throughsynchronous neural spiking between these cells
2 1 Dept Physiol, Fujita Health Univ Sch Med, 2 Dept Pathol,
Soh Hidaka1，Chiyuki Kaneko（

Faclt Med Tech, Fujita Health Univ Sch Health Sci）

Electrical synapses are present in many types of visual cells expressing channel subunit, connexins (J. Neurosci., 2004; J. Integra Neurosci., 2008; Brain Res., 2012). Electrical current spreadthrough gap junctions between presynaptic neurons is expected to regulate chemical output
synapses from these neurons onto postsynaptic neurons. In our recent studies, physiologicalproperties of electrical synapses between α retinal ganglion cells have been characterized(J. Neurosci., 2004; J. Integra Neurosci., 2016). In the present study, we examined electricalsynapses of retinal ganglion cells and pyramidal cells in visual cortex of developing rats andprimate common marmosets. First, we investigated the localization of gap junctions between
these visual excitatory cells by immunocytochemical studies of connexins. Second, we analyzed
physiological properties of electrical synapses between these cells under dual whole-cell patch
clamp recordings. We then investigated relationship between electrical synapses and chemical
output synapses of these visual cells. Connexin-36 electrical synapses occur between these cells,
where depolarizing responses in the cells increased through cells’ electrical synapses. Synchronous injection of subthreshold currents (pulses) in two electrically-coupled cells increased (thelikelihood of Na+) neural spiking. These electrically-coupled visual cells generated synchronousneural spiking within 4.4msec under patch clamp recordings. These results suggest that visualcells’ chemical excitatory synaptic outputs onto postsynaptic cells appear to increase together
with synchronous neural spiking through electrical synapses between these gap-junctionally(COI:No)connected visual excitatory cells.

2P-0562P-0593 1 Sys Neurosci Sect, Dept ofKouji Takano1，Nana Hayashi-Morita1，Kenji Kansaku1, 2, （Yoshiyuki Tsuboi（Dept Physiol, Nihon Univ Sch of Dent, Japan）

Optimal degree of visual angle for the SSVEP-based brain-computerinterface stimuli
Rehab for Brain Funct, Res Instit of Natl Rehab Cent, 2 Dept of Physiol and Biol
Information, Dokkyo Medical University School of Medicine, 3 Cent for Neurosci and Biomed
Eng, The Univ of Electro-Communications）

Introduction: Our group has developed an EEG based steady-state visual evoked potential (SSVEP) brain-computer interface (BCI) system for amyotrophic lateral sclerosis (ALS) patients,
including completely locked-in syndrome (CLIS). We reported that a CLIS patient who cannot
control the direction of their eyes, was able to control the BCI system (Okahara, 2018). We haveexperienced that position of visual stimuli (LED flicker) was required to adjust in each patient.
In this study, we evaluated optimal degree of visual angle for the SSVEP-based BCI stimuli.
Methods: Ten subjects (5 females, age 31.1) participated in this study. The visual stimuli were
presented by LED. The LED was placed in the peripheral visual field at 5, 10, 15, 30, and45 degrees on the opposite side of the dominant eye. Participants were asked to gaze at a
central fixation point and attend to or ignore the LED (attention/ignore tasks). EEG signals
were measured from Oz, PO7, and PO8. We calculated the power spectrum density (PSD) andimaginary coherence (ImC) of the EEG data as the parameters to discriminant the subjects
attend or ignore the LED.
Results: In PSD analyses, over 70% accuracies of discrimination were observed at 5, 10, 15 degrees, and in ImC analyses, these were observed at 10, 45 degrees. The highest accuracy was
75% (7 subjects exceeded 70%) at 5 degrees in PSD analyses and 71.9% (6 subjects exceeded70%) at 45 degrees in ImC analyses.
Discussion: The results suggested that optimal degree of visual angle for the SSVEP-based(COI:No)BCI stimuli were dependent on the used analyzing methods (PSD or ImC). 

Effects on compound action potentials at dorsal root after the injectionof QX314 / Flagellin (Q/F) solution to the sciatic nerve in rats
QX-314 is a derivative of lidocaine and block the conduction of C-fibers by binding Na+ channel from the inside after entrance to the inside of nerve fiber through the TRPV1 channel
following the capsaicin application. Flagellin is a ligand of the Toll-like receptor 5. Recently,
it has been reported that Q/F solution selectively blocked Aβfibers in mice (Nature 2015).
We also found that thresholds for mechanical stimulation to foot were increased in rats withsciatic nerve injury (SNI: Spared Nerve Injury) at 40 min to 3 h after Q/F solution injection
to foot skin. Aim of this study is to clarify reduced inputs to dorsal spinal cord qualitatively
and quantitatively after injection of Q/F solution to the sciatic nerve trunk. Compound action
potentials were recorded with monopolar lead method from the dorsal roots of lumber 4-6
following the electrical stimulation of sciatic nerve at the level of the distal of the mid-thighunder gas anesthesia (2 % isoflurane with 2 l/min O2). We investigated effects of Q+F solution
on conductions of Group I to III after an injection of Q+F solution to sciatic nerve trunk at thelevel of hips. At first, the application of Q+F solution effectively blocked Group II fibers in doseand infusion-velocity dependent manner. But the solution also finally blocked other types offiber and response did not recover over 3 h. These results suggest that Q+F solution may berequired any contrivance for the application.
(COI:No)

2P-0572P-0601 1 Lab. Mol ＆ Dev Biology, Protein
Yuko Sugita1，Kenichiro Miura2, 3，Takahisa Furukawa（

Kotaro Honda1，Mitsutoshi Tominaga1，Fumiya Kusube1, 2，Fumiyuki Yamakura3，
5 1 Inst Env Gend Specif Med, Juntendo Univ, Japan, 2 Dep
Hisashi Naito4，Kenji Takamori1, （Photoreceptor ribbon synapse is a factor regulating light adaptation of
the visual information processing in miceInst, Osaka Univ, Japan, 2 Dept. Integ Brain Sci, Grad Sch. Med, Kyoto Univ, Japan,

3 National Center of Nerology and Psychiatry, Japan）

The ribbon synapse is a specialized synaptic structure which connects photoreceptor synapse
terminal with bipolar and horizontal cell. An extracellular matrix protein Pikachurin is essential for the proper formation of the ribbon synapse. Several lines of evidence suggest that
the Pikachurin deletion specifically influences the ON pathway; the localization of mGluR6
is restricted to the postsynaptic site of ON bipolar cells in the ribbon synapses of the outerplexiform layer.
To understand how influence of ON pathway contributes to visual information processing, we
examined optokinetic responses (OKRs) elicited by two-frame animations presented with aninter-stimulus interval (ISI) in Pikachurin null mutant mice that show improper formation of
the photoreceptor ribbon synapse.
The wild-type mice showed OKRs to two-frame-motion stimuli in the veridical direction in
the absence of an ISI. The OKRs decreased progressively as the ISI got longer and becamealmost zero when the ISI was 106.7ms. When the ISIs were 106.7 ms or longer, the OKRs weredirected in the opposite direction. The Pikachurin－/－ mice showed a different dependence on
ISIs from that of the wild-type mice. We simulated the dependences on ISIs of individual miceusing a computational model of visual motion detection (elaborated Reichardt motion detector)
to estimate the characteristics originated from the temporal filters embedded in the visualsystem of mice.
The frequency characteristics of temporal filters in Pikachurin－/－ mice showed no difference inthe optimal temporal frequency, but showed stronger adaptation to an exposure to a sustained
light signal than the wild-type mice. These results suggest that the ribbon synapse is a factor
regulating light adaptation of visual information processing.
(COI:No)

Investigation of the antipruritic mechanism of the kappa opioid receptor
agonist in the murine spinal dorsal horn
Bio Sci Tec, Fac Ind Sci Tec, Tokyo Univ Sci, Japan, 3 Fac Int Lib Art, Juntendo Univ,
Japan, 4 Inst Health Sports Sci Med, Juntendo Univ, Japan, 5 Dep Derm, Juntendo Univ
Urayasu Hosp, Japan）

The kappa opioid receptor (KOR) antagonist is used as a drug for intractable itch, whichis resistant to histamine receptor antagonists. However, little is known about specific sites
important to the antipruritic effects of the agonist. Because intractable itch is presumed to beabnormal in the neuronal pathway of spinal cord, our research aims to identify the action pointsof the KOR agonist in the spinal itch pathway.
To identify the anti-pruritic action of the agonist of kappa opioid receptor, in vivo electrophysiology, behavioral tests, and high-sensitive in situ hybridization (ISH) were conducted in murine
spinal dorsal horn, using C57BL/6J mice.
Behavioral tests showed that intrathecal injection of the KOR agonist reduced but not eliminated gastrin-releasing peptide (GRP)-evoked scratching behaviors. In vivo electrophysiologicalrecordings revealed that administration of the KOR agonist suppressed chloroquine-responsive
dorsal horn neurons in 15.8% (3/19) of mice. Only one of three-suppressed neuron responded
to GRP administration in the spinal cord. ISH in three sections of the spinal cord showed that
24.6% (154/777) were double positive for GRP and KOR, and 13.6% (68/567) for GRP receptor(GRPR) and KOR in total KOR+ cells. Most KOR+ cells were negative for GRP and GRPR.
Intrathecal injection of dynorphin-saporin did not decrease scratching bouts caused by GRP.
Unexpectedly, it tended to increased scratching bouts by the administration of GRP alone andthe combination of GRP and the KOR agonist.
In conclusion, the KOR agonist targets both GRP+ KOR+ and GRPR+ KOR + cells which arepresent in a 2:1 ratio, in the spinal dorsal horn. These findings suggest that GRP+ KOR- and
GRPR+ KOR- cells may function as interneurons in the spinal dorsal horn.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

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2P-061

2P-064Chengzhu Yin, Toshiyuki Ishii, Makoto Kaneda（ Dept. Physiol, Nippon Medical School,

Ryusuke Yoshida1，Keisuke Sanematsu2，Shingo Takai2，Shusuke Iwata2，
2 1 Dept Oral Physiol, Grad Sch Med, Dent and Pharma, Okayama
Noriatsu Shigemura（Contribution of bicarbonate permeability to the reversal potential of
GABA responses in bipolar cells of the mouse retina
Tokyo, Japan）

The decreases in taste recognition thresholds in humans by addition of
low concentration of salts

In the retina, the different polarity of surround responses between ON- and OFF-bipolar cells
is thought to be mediated by the differences of the reversal potentials of GABA responses.
In retinal bipolar cells, GABA responses are mediated by the GABAA and GABAC receptors.
In the present study, we examined whether the difference of the bicarbonate ion (HCO3－)
permeability to GABA receptors can generate the difference of reversal potentials between
the GABAA and GABAC receptors using patch clamp technique. We also examined whether
the anion permeability of GABAA and GABAC receptors reported in heterologous expression
system can be true in vivo. The reversal potentials of GABAA and GABAC receptors without
extracellular HCO3－ did not show significant difference (-4.9 ± 2.4 mV (n = 19) for GABAA and-5.1 ± 3.6 mV for GABAC (n = 18)). The reversal potentials of both GABAA and GABAC receptors with extracellular HCO3－ (24 mM) did not differ significantly but slightly shifted to thepositive side (-2.4 ± 2.6 mV (n = 19) for GABAA and -0.6 ± 4.1 mV (n = 18) for GABAC). The
permeability of anions such as I－, NO3－, HPO4－, CH3COO－ to GABAA and GABAC receptorsof bipolar cells were similar to the permeability reported the previous study in vitro. These
results rule out the possibility that the expression level of GABA receptor subtypes withdifferent HCO3－ permeability contributes to form the differences of the reversal potentials of
GABA responses between ON- and OFF-bipolar cells, although the permeability of HCO3－ canaffect the reversal potential of GABA responses. Our results also show that anion permeabilityof GABAA and GABAC receptors reported in vitro study can be applicable to assess the ionicmechanisms of GABAA and GABAC receptors in vivo.
(COI:No)

Univ, Japan, 2 Sect of Oral Neurosci, Grad Sch of Dental Sci, Kyushu Univ, Japan）

2P-062

2P-065

Teruaki Nasu1，Yoshiko Uchimura1，Asako Kubo2，Koutaro Yamada3，Kazue Mizumura1, 2

Aiko Moridera（ Dept Ergo, Grad Sch Med, UOEH, Kitakyushu, Japan）

Analysis of the peripheral mechanism of muscular mechanicalhyperalgesia induced by repeated cold stress in rats
（ 1Coll. Life Health Sci., Chubu Univ, Kasugai, Japan, 2 Dept Physiol. Nihon Univ, School of
Dentistry, 3 ZERIA Pharmaceutical Co. Ltd）
There are many patients suffering from chronic musculoskeletal pain, however, its mechanism
is not well understood so far. Previously we reported that rats exposed to repeated cold stress(RCS) showed long lasting muscular mechanical hyperalgesia (Nasu et al, 2010). Its spinal
mechanism has been partly clarified (Nasu et al, 2019), and we also showed RCS changed
mechanosensitivity of muscular nociceptors (Wakatsuki et al., 2015). Yet, it is not known what
sensitizes peripheral nociceptors in RCS. Purpose of the present study is to clarify factors thatsensitize muscular nociceptors.
We examined expression of mRNAs of mediators (NGF, GDNF, family, BDNF, NT-3, IL-1β,
IL - 6, TNF-α)by RT- PCR in extensor digitorum longus muscle of the control rats and rats
underwent RCS. We also measured pH in tibialis anterior (TA) muscle by a needle typepH electrode under 1.5% isoflurane anesthesia. Consequently, we pharmacologically examined
whether vesicular ATPase (V-ATPase) contributes to muscular pH change in RCS, andwhether muscular pH change contributes to muscular mechanical hyperalgesia.
RT-PCR revealed that expression of mRNA of any mediators examined were not changed by
RCS. We found RCS significantly lowered muscular pH. Subcutaneous injection of bafilomysin(25μg/Kg), a V-ATPase inhibitor, and APETX2, an ASIC3 antagonist, reversed muscularmechanical hyperalgesia. These results suggest that muscular acidification contributes topersistent muscular mechanical hyperalgesia by RCS.
As we previously reported that chondroitin sulfate (CS) decreased acid-induced facilitation of
mechanosensitive currents in dorsal root ganglion neurons (Kubo et al., 2013) and mechanical response of single muscular thin-fibers (Hotta et al, in press), we examined whether CS
reversed mechanical hyperalgesia after RCS. As expected CS attenuated the mechanicalmuscular hyperalgesia after RCS dose dependently. Collectively, these results suggest that
RCS induces acidification of the muscles and this acidification sensitizes muscular nociceptors,(COI:No)
resulting in mechanical hyperalgesia. 

2P-063

It is well known that sweet taste is enhanced by addition of NaCl, however physiological/
molecular mechanisms for sweet enhancement by addition of NaCl is not elucidated. Sweet
receptor forms heterodimeric complex of T1R2 and T1R3. Therefore, sweet enhancementby salt may be caused by modulation of T1R2/T1R3 activity by salt. To test this possibility,
the effects of addition of salt to tastants on taste recognition thresholds in humans wereanalyzed. Sweet recognition thresholds for sucrose, glucose and aspartame were significantly
lowered by addition of 3 mM NaCl, which was almost tasteless to participants. In contrast,
recognition thresholds for bitter (quinine), sour (HCl) and umami (monosodium glutamate +inosine-5’-monophosphate) were not affected by addition of 3 mM NaCl. Other salts such as
N-methyl-D-gluconate chloride (NMDG-Cl), KCl and NaHCO3 also lowered sweet recognition
threshold for sucrose and glucose but not for aspartame. These results suggest that the addition of salt selectively enhances sweet taste sensitivity in humans and that there are multiple
sweet receptor systems in humans.
(COI:No)

Effect of sleep deprivation on sleep homeostasis in secretin receptor
knockout mouse
Secretin is a peptide hormone released from the duodenum to stimulate the secretion ofdigestive juice by the pancreas. Secretin also functions as a neuropeptide hormone in the brain.
Recently, it is reported that secretin neurotransmission might be related to the pathophysiology of autism. As sleep disturbance is one of the symptoms of autism, we hypothesed that thesecretin neutorransmission might play a role in the sleep control. We used secretin receptor
knockout (SctR KO) and wild type mice to compare baseline sleep profile and homeostatic
response to the sleep deprivation (SD). The mice were surgically implanted a radio transmitter
into the peritoneal cavity for measuring ambulatory activity and body temperature, and theelectrodes for measuring electroencephalogram and electromyogram under the sevoflurane
anesthesia. All animals were put in the home cage for at least one week for recovery andthen transferred to the recording chamber. After a 24 hours of baseline recording, SD was
performed for four hours from the light onset and rebound responses were recorded foranother 24 h. The SctR KO mice showed a tendency to need more time to recover from the SD
compared to control group. This result suggests that secretin neurotransmission might have arole in the sleep control especially with sleep homeostasis.
(COI:No)

2P-066

The response to whisker stimulation in visual associated area of
monocular deprived mice in vivo

Massage-like stroking stimulation increases call rate of 50-kHz
ultrasonic vocalizations with various call subtypes in young adult rats

Akari Hashimoto1，Akiko Miyamoto2，Koichiro Haruwaka1，Daisuke Kato1，
1 1 Div System Neuroscience, Grad Sch Med, Kobe
Yoshihisa Tachibana1，Hiroaki Wake（

4 1Center Med Sci, Int Univ
Rie Shimoju1，Miyo Hori2，Hideshi Shibata3，Mieko Kurosawa1, （Univ, Japan, 2 Systems Neuroscience Group, AIST, Japan）

Information processing of sensory inputs in higher-order cortex are crucial to detect theexternal environment. Blind subjects compensate the disadvantage on it by recruiting andprocessing the other remaining senses in the visual associated area, which phenomenon is
known as ’’cross-modal plasticity’’. The previous studies using positron emission tomography(PET) showed that activity of visual cortex occurred in the blind people when reading Braille.
Whisker-dependent activation of visual cortex was demonstrated in the eye enucleated mice.
However, the functional changes and its mechanism in vivo have not been shown yet. In thisresearch, we try to unravel the effect of early monocular deprivation (MD) on the activationof the visual associated area with whisker stimulation. We first visualized the axonal projection from S1 (primary somatosensory cortex) to V2 (extrastriate cortex) by injecting adenoassociated virus coded eGFP with synapsin promotor (AAV1-syn-eGFP). Retrograde labellingof S1-V2 axons by Cholera Toxin Subunit B (Recombinant) Alexa FluorTM 488 Conjugate
also showed the existence of the projection. Then we hypothesized the activation triggeredby whisker stimulation in S1 can be transmitted and processed in V2 as well. To verify our
hypothesis, we combined in vivo two-photon imaging with the injection of AAV coding Ca2+
indicators (GCaMP6s/GCaMP6f) and visualized the activity of S1-V2 axons and neurons in V2
during whisker stimulation. We finally assumed that microglia are a key player for the synaptictransferring from V1-V2 connection to S1-V2 axon. To verify the microglial contribution, we
assessed morphology and gene expression of microglia, which might be a driving force tomodify the neuronal circuit with MD. This study will be an important clue to understand the(COI:No)
compensating ability of the cortex for the future therapeutic target.

S126

Health ＆ Welfare, Otawara, Japan, 2 Fund Advancement of Intl Sci, Tsukuba, Japan, 3 Lab
Vet Anat Ins Agric, Tokyo Univ Agric ＆ Tech, Tokyo, Japan, 4 Dept Pharm Sci, Intl Univ
Health ＆ Welfare, Otawara, Japan）

Rats emit 50-kHz ultrasonic vocalizations (USVs) associated with reward. We have previously
shown that massage-like stroking stimulation of the abdomen under vertical holding condition
vigorously induced 50-kHz USVs. 50-kHz USVs were categorized according to 14-subtype
scheme (Wright et al., 2010), and some of the subtypes are thought to be associated withspecific emotional state and emotion-related behaviors. This study sought to classify thesubtypes induced by stroking stimulation of the abdomen. In addition, the effects of stroking ofother cutaneous areas (back or head) on 50-kHz USVs were examined. The experimenter held
tightly the rat’s upper back skin with the left hand and stroked gently the skin (abdomen, backor head) with the right hand. USVs were recorded during stroking stimulation. Massage-likestroking stimulation of the back or head significantly increased 50-kHz USVs as stimulation of
the abdomen did. The total call numbers of 50-kHz USVs evoked by stroking of the abdomen,back or head during stimulation were not different. On the other hand, stroking stimulation
altered 50-kHz call subtype profile in a stimulus site dependent manner. In abdomen-stroked
group, “step down” and “composite” calls were more, while “flat” calls were less than back orhead-stroked group. Furthermore, we found a new subtype which has not been shown in the
14-scheme by Wright and coworkers. The new subtype was monotonous call in harmonics andwe defined it as “harmonic flat”. The “harmonic flat” was well observed in response to stroking
stimulation of all the skin areas tested. These results demonstrate that stroking stimulationof various skin areas generally evoked 50-kHz USVs; however, emotional state induced bystroking stimulation may be partly different depending on the stimulus site.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

2P-067

2P-070Constant light exposure leads to the down-regulation of clock genes
expression with circadian clock disorganization in pregnant beef cows

Fibroblast growth factor 5 (FGF5) plays a critical role in central
regulation of mouse behavior

3 1 Lab. Anim. Pro.
Tsuyoshi Otsuka1，Hiroki Mitsuishi2，Hiroki Onishi1，Masato Yayota2, （Kazuto Matsuda1，Kai Kimura1，Kazuaki Nakamura2，Tsuyoshi Hamada3, 4，Junko Imaki4，
1 1 Dept Ani Sci, Teikyo Univ Sci, Yamanashi, 2 Nat Cent Child HealthYasuhiko Kondo（

Mgmt., Fac. Appl. Biol. Sci., Gifu Univ., Japan, 2 Lab. Anim. feed. Nut., Fac. Appl. Biol.
Sci., Gifu Univ., Japan, 3 GeFAH., Gifu Univ., Japan）

Circadian rhythm is generated by transcriptional translational feedback loop system of clock
genes, which are regulated by photoperiod. In mammals, variation in circadian rhythm has
an affect on physiological function such as behavior, metabolism, and reproduction. Therefore,
understanding the circadian rhythms of clock genes contribute to improvement of production
and health in animals. It is well known that hair follicle cells are noninvasive, convenient,and useful biopsy sample for detecting the expression pattern of circadian clock in human.
However, whether the expression rhythms of clock genes are able to measure in cows using
hair follicle cells remains unclear. Here we demonstrate the expression rhythms of several
clock genes (Bmal1, Per1, and Cry1) were shifted by difference of day length and constantlight exposure caused a loss of circadian clock in hair follicle cells of non-pregnant beef cows.
Interestingly, the expression levels of several clock genes in pregnant beef cows fluctuate
by difference of day length. Moreover, pregnant beef cows show the reduction of most clock
genes expression levels with disrupted of circadian rhythms by constant light exposure. Thesefindings suggest that it is possible to detect the circadian clock in beef cows using hair follicle
cells, and light control may provide a critical impact on circadian rhythm of pregnant beef
(COI:No)cows.

2P-068

Spontaneous neuronal activity in the bed nucleus of the stria terminalis
across sleep-waking cycles in rats
Kazumi Takahashi, Satoshi Eifuku（Dept Systems neurosci, Med, Fukushima Med Univ,

Develop, Tokyo, 3 Fac Health Med Sport, Teikyo Heisei Univ, Chiba, 4 Dep Develop Anat,
Nat Def Med Coll, Saitama）

Fibroblast Growth factor 5 (FGF5) broadly expresses in the central nervous system. However,
our knowledge of the function is limited except for the blood brain barrier, and little has been
known on behavioral regulation. In this study, we first demonstrated behavioral characteristicof FGF5 null mutant male mice, compared to that of wild-type (WT) males. When exposing
to odors of sexually active males and estrous females, although both WT and mutant males
preferred female odor to male odors, mutant males showed significantly shorter investigationfor either odors than WT males. In mating tests, mutant males showed lower activities andless ejaculation. Furthermore, performance in Morris water maze was deteriorated in mutant
males compared to WT males. Since we found severe dysfunction in a variety of behavior inmutant mice, we reexamined FGF5 mRNA expression in the forebrain, indicating that FGF5
gene expresses broadly such as in the olfactory bulb (OB), the amygdala, the hippocampus,
and the hypothalamus. Immunohistochemistry (IHC) and in situ hybridization (ISH) studies
detected the signals in common in the hippocampus, the cerebral cortices, and the thalamic reticular nucleus. However, the signals were generally weak and able to confirm the PCR results
only partly. Furthermore, the inconsistency was found between IHC and ISH. Intense signals
in the granular layer of CA2, CA3 and dentate gyrus, but weak in CA1, of the hippocampus
were observed by ISH, while immunoreactive patchy particle (not cellular form) were found in
the hippocampus including CA1 by IHC. ISH detected weak signals in the OB, but IHC failed.
The current study demonstrates that FGF5 in the central nervous system is involved in neural
regulation of various behaviors, suggesting that FGF5 deficiency can be a good model for some
psychiatric disease in human.
(COI:No)

2P-071

Sexual Activity Correlates Olfactory Sensitivity in Male Rats
Yui Shimomi, Yasuhiko Kondo（Dept Ani Sci, Teikyo Univ Sci）

The bed nucleus of the stria terminalis (BNST), receiving input mainly from the amygdala, isinvolved in various physiological and behavioral responses to stress, fear and anxiety. It has
been also reported that BNST neurons play important roles in the generation of wakefulness
through their multiple projections to waking centers in the hypothalamus and brainstem, including the locus coeruleus, the parabrachial nuclei and others. However, the characteristics ofthe spontaneous activity of BNST neurons across sleep-waking cycles have not yet been fully
elucidated. In the present study, we recorded state-dependent changes of neuronal activity inthe posterior part of the BNST (BNSTp) through a wire-bundled tetrode along with corticalelectroencephalogram and neck muscle activity in freely moving rats. Most neurons recordedin the BNSTp showed tonic and frequent discharge during both wakefulness and paradoxical
sleep. During slow-wave sleep, these neurons showed phasic and clustered discharge, which
was highly synchronized among the neurons recorded simultaneously, and the timing of thedischarge clustering showed a correlation with the phase of cortical slow-wave rhythmicity.
These results suggest that a homogeneous neuronal population in the BNSTp may exert
strong influences on systems that generate cortical slow-wave activity during slow-wave sleep,in addition to the pathway of cortical activation during wakefulness and paradoxical sleep.
(COI:No)On screening the sexual activity of male rats, researchers became aware of the existence,
among naïve rats, of a subpopulation with little or no sexual behavior, even after several
mating sessions. The present study investigated whether such sexually sluggish (SS) males
show behavioral differences from normal copulatory (NC) males, other than sexual behavior.
Sexually naïve Long-Evans male rats were subjected to weekly olfactory preference tests,
which measured the time spent nose-poking sexually active males and sexually receptive
females for odor exploration in a three-chamber apparatus, each followed by copulation tests
with receptive females for 3 weeks. Depending on their sexual behavior performance, the rats
were assigned to NC males who showed ejaculation in these copulation tests and SS males
who showed very low sexual activity resulting in no ejaculation. The preference tests indicatethat although both NC and SS males significantly prefer receptive female odor to male odor, SS
males spent significantly less time nose-poking to investigate receptive females than NC males
did. Thereafter, the food finding test was performed after overnight fasting. In the test, all theNC males found the buried pellet within 5 min, whereas over 60% of the SS males failed tofind it. Furthermore, the males were tested for finding a buried bag containing soiled beddingcollected from estrous female cages. The bag was found by 80% of NC males, but only by 20%
of SS males. Our current results suggest that the difference between SS and NC male rats is
not only in sexual behavior but also extends to other functions such as olfaction to detect foods.
(COI:No)

2P-069

2P-072

Sachiko Chikahisa, Yoshitsugu Kondo, Tetsuya Shiuchi, Noriyuki Shimizu, Daisuke Tanioka,
Hiroyoshi Sei（Dept Ingeg Physiol, Biomed Sci, Tokushima Univ Grad Sch, Japan）

Kuniaki Satori1，Yu Takarada1，Masashi Arake3，Yuji Morimoto3，Toshiaki Ishizuka2，
1 1 Dept Engineering Science, Tokyo Denki Univ, Japan,
Hiroyuki Ohta2，Tatsuji Takahashi（

Japan）The role of PPAR-alpha and ketone bodies in the regulation of arousal
during food deprivation

Peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription
factor involved in the regulation of fatty acid metabolism. PPARα is known to regulate thesynthesis of ketone bodies via 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2). Ketone
bodies such as acetoacetic acid and β-hydroxybutyric acid are produced from fatty acids inhepatocytes and brain astrocytes and become the main fuel in many tissues during starvation.
We recently found that PPARα and ketone bodies are involved in the regulation of sleep
homeostasis. In this study, we investigated how sleep-wake changes during food deprivationin PPARα knockout (KO) mice. We evaluated sleep, locomotor activities, body temperature,arterial pressure and heart rate, under ad lib-fed conditions and 24-hour food deprivation.
Under ad lib-fed condition, KO mice showed enhanced sleepiness. Under food deprivation, theamount of wakefulness, locomotor activities, arterial pressure and heart rate were markedly
decreased in KO mice, compared with that of WT mice. In addition, a lower concentration ofplasma ketone bodies and decreased mRNA expression of Hmgcs2 in the liver and brain wereobserved in KO mice under food deprivation. These results suggest that PPARα and ketone
bodies play an important role in the maintenance of wakefulness during food deprivation in
mice.
(COI:No)How do mice balance the trade-off between exploration andexploitation?
2 Dept Pharmacology, NDMC, Japan, 3 Dept Physiology, NDMC, Japan）

How should an animal choose between exploiting a well-known resource or exploring new
options? This problem is widely known as the exploration-exploitation trade-off.
Reinforcement learning models play an important role in understanding the behavior andneural mechanisms of choice action in animals and humans, especially as for the adaptivebalancing of exploration and exploitation. Previous studies have shown that in the two- or
three-armed bandit problems, the action choice of rats are similar to a reinforcement learningmodel, the forgetting Q-learning algorithm operated under the softmax action selection policy(Ito and Doya 2009; Cinotti 2019). Our experiments have shown that the action choice of miceshows less exploration when the reward probabilities are high overall.
Thus we suspected that there is a factor that contributes to balancing between exploration andexploitation that is sensitive to the reward probabilities.
Therefore, we proposed models of value-update and policy.
Specifically, a model in which the value-update and the policy parameters are scaled using theestimated mice weight, the food intake within the last 12 hours, and the Q-value.
We also applied satisficing as a policy, which explores until an aspiration level is met, and thenthe greedy action selection is performed.
As a result of comparing 24 models, a model in which the learning rate of the Q-value isinversely proportional to the magnitude of the Q-value itself best explained the mice behavior.
The result indicates that mice put great value on unexpected rewards and preserve highly(COI:No)
expected choice values.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S127

2P-073

2P-0761 1 Dept Physiol, Grad
Yusuke Tsuno1，Takashi Maejima1，Emi Hasegawa2，Michihiro Mieda（

Mai Sawabe1，Yohei Yamashita1，kenji Hanamura2，Hiroyuki Yamazaki2，Tomoaki Shirao2，
1 1 Dept Life Science, Grad Sch of Life Science, Toyo univ, Japan, 2 Dept
Nobuhiko Kojima（

In vivo analysis of circadian rhythm of the suprachiasmatic nucleus in
AVP neuron-speciﬁc vesicular GABA transporter knock-out mice
Sch Med, Kanazawa Univ, Japan, 2 IIIS, U of Tsukuba）

Is drebrin required for metabotropic glutamate receptor-mediatedchanges in anxiety-related behavior and spine morphology?

The suprachiasmatic nucleus (SCN) is the circadian rhythm center of the mammals. Individual
SCN neurons have their intrinsic daily rhythms controlled by transcriptional-translational
feedback loops. However, how these individual circadian rhythms can synchronize is largely
unknown. GABAergic transmission may be a good candidate, but its contribution remains
controversial.
To elucidate the functional meaning of GABAergic transmission within the SCN, we usedconditional knock-out mice in which vesicular GABA transporter (VGAT) was specificallydeleted in AVP neurons (AVP-Vgat －/－). We found that AVP-Vgat －/－ mutant mice showed
lengthening and a splitting pattern of the activity time in behavioral circadian rhythm. By
recording miniature GABA-mediated postsynaptic potential (mGPSCs), the frequency ofsmall amplitude mGPSCs was decreased in both AVP and non-AVP neurons during thelight period, suggesting the decrease of GABA release from AVP neurons. To examine theclock gene expression in the SCN, we made an acute slice to observe the daily rhythm of
the clock gene, Per2, expression by using Per2:: Luc mice. We found the peak phase of theclock gene expression was delayed relative to the onset of activity period of behavior in
AVP-Vgat －/－mutant mice, indicating the phase relationship change between SCN molecular
clocks and the locomotor activity.
To examine the rhythmicity of the activity of AVP neurons in vivo, we used fiber photometry
to visualize calcium rhythm with a calcium indicator (jGCaMP7s) at the right SCN in AVP-Cre(control) and AVP-Vgat －/－ mutant mice. We found a possible change of phase relationshipbetween circadian calcium rhythm and locomotor activity in AVP-Vgat －/－ animals. These
results suggest that GABAergic transmission of AVP neurons may regulate the phase relationship among the SCN molecular clock, SCN neuronal activity rhythm, and locomotor activity
rhythm, and thereby control the timing of SCN output to behavior.
(COI:No)of Neurobio and Behav, Grad Sch of Med, Gunma Univ, Japan）

2P-0742P-077

Tetsu Hatsukano, Kazuhiro Sano, Sonoko Ogawa（ Faculty of Human Science, University of

Chihiro Sato1，Kunikazu Tanji2，Mana Kishimoto1，Shun Mori1，Shuhei Koeda1，
1 1 Dept of Occcupi Ther, Grad Sch Health Sci, Hirosaki
Misaki Mikami1，Junko Yamada（

Effects of reversible manipulation of estrogen receptor α expression in
the ventromedial nucleus of the hypothalamus on aggressive behavior
in male mice

Dendritic spine is dynamic structure and its morphology is related not only to synaptic plasticity of individual neurons, but also to higher functions of the brain. Actin cytoskeleton supports
the structure of spines and regulates dendritic spine remodeling in response to neuronal activity. Recent lines of evidence indicate that the group I metabotropic glutamate receptor (mGluR)
is involved in structural plasticity of dendritic spines. It is known that mGluR activation triggers long-term depression in synapses and dysregulation of mGluR signaling causes aberrant
spine morphology. These findings suggest that mGluR activity may link to actin cytoskeleton.
However, the intracellular molecular cascade that relays mGluR activity to actin cytoskeletal
dynamics is largely unknown. We have demonstrated that the actin-binding protein drebrin
plays a critical role in spine morphogenesis and plasticity. Drebrin possesses a binding motifof Homer that is known as a scaffold protein of mGluR. So, we make the hypothesis thatdrebrin mediates the interplay between mGluR and F-actin through the Homer-binding. In thisstudy, we examined the effects of the lack of drebrin on animal behavior, spine morphology
and protein interaction in dendritic spine using drebrin knockout (DXKO) mice. We foundthat these KO mice showed increased anxiety in the open field test. Histological investigation
with Golgi-Cox staining revealed that a significant decrease in spine density in hippocampal
CA1 regions of DXKO mice. Among the types of spines, stubby, thin and mushroom-typespines were decreased, and filopodia-type spines were conversely increased. These observations suggest that the lack of drebrin affect the maturation of dendritic spines. We have now
examined whether the lack of drebrin cancels out mGluR-mediated changes in animal behavior
and spine morphology. This study will uncover the molecular basis for the roles of mGluR on
anxiety-related disorders and structural plasticity. (COI:No)

The motor recovery and synaptic plasticity was affected by the exercise
in the hemorrhage model rat

Tsukuba, Japan）

Univ, Japan, 2 Dept of Neuropathology, Grad Sch Med, Hirosaki Univ, Japan）

Testosterone (T) facilitates male aggressive behavior by acting on estrogen receptor α (ERα)
after being aromatized to estradiol in the brain. Using virally mediated RNAi methods forsite-specific ERα knockdown, we have found that ERα in the ventromedial nucleus of the
hypothalamus (VMH) at the time of behavioral testing in adult is necessary for “activational
action” of T to induce aggression (Sano et al., Eur J Neurosci, 2013). However, it is not known
whether expression of ERα in the VMN is involved in possible “organizational action” of T
during pubertal period. To test this, it is necessary to be able to knockdown ERα expression during pubertal period but not at the time of testing in adult. In the present study, wedeveloped a new method to achieve reversible manipulation of ERα expression by inducing
gene knockdown with the tet-on system. We first injected inducible ERα knockdown vectors
in adult mice and confirmed that ERα expression in the VMH was suppressed under doxycycline (DOX)+ condition and recovered by terminating DOX treatment. We then knocked down
ERα in the VMH during only pubertal period by injecting the inducible ERα knockdown
vectors on postnatal day (PND) 21 and giving DOX containing food between PND 21 to 56.
When the mice were tested on PND 84 under DOX- condition, they showed equivalent levels of
aggressive behavior as mice treated with a control virus during pubertal period. On the other
hand, consistent with our previous findings, levels of aggressive behavior in mice injected with
the inducible ERα knockdown vectors on PND 70 were reduced under DOX+ condition but
restored afterward in DOX- condition. These results suggest that ERα in the VMH is required
for the activational action of T in adulthood but not involved in the organization action duringpubertal period for male aggression.
(COI:No)

Previously, we reported that the exercise promotes the motor recovery and the recovery ofvoluntary exercise (V-Ex) was earlier than forced exercise (F-Ex) in the intracerebral hemorrhage (ICH) rats. However, the factors to promote the motor recovery were unknown. The aimof this study is to clarify the mechanisms of recovery by the rehabilitation. We focused on theearly phase when there was significantly difference between the trained groups.
To clarify the synaptic function of the injury side, DiI was injected into the right striatum.
Whole cell patch clamp recordings were performed from DiI labeled cells in the layer V inmotor cortex (M1) using voltage -clamp mode (V hold = -70 mV). The amplitude and frequencyof miniature excitatory postsynaptic currents (mEPSCs) were recorded in the presence of
TTX at 32 ℃. The amplitude and frequency of mEPSCs were increased in both V-Ex and F-Ex
groups than the non-exercise group.
To assess the difference between the V-Ex and F-Ex group, the motivation and stress levels
with exercise were compared among three groups. The immunoreactivity of ΔFosB in
nucleus accumbens was used as a marker of activated neuron on the reword system, andcorticosterone concentrations were analyzed to assess the stress level. The expression of
ΔFosB was increased in the V-Ex group than the N-Ex group. The plasma corticosterone washigher in the F-Ex group than the other groups.
These data suggested that the motor recovery was due to the synaptic plasticity in M1 thatproject to striatum, and the synaptic functions might be affected by the motivation or stress.
It is known that the KCC2 level is decreased after brain injury in mature neuron. There might
be alterations in KCC2 expression in brain on the recovery. We need to determine the effect(COI:No)of the KCC2 for the neural recovery.

2P-0752P-078

Ryota Kato1，Kenjiro Sato2，Ryosuke Hitomi3，Kazuki Toyoda3，Nobuhiko Kojima1, 2Kazuaki Nishimura1, 2，Kazuhiko Baba1，Kenya Sanada1，Haruki Nishimura1，
Kentaro Tanaka1，Satomi Sonoda1，Mitsuhiro Yoshimura1, 3，Takashi Maruyama1，
1 1 Dept Physiol, UOEH, Japan, 2 Dept ObGyn, UOEH, Japan,
Kiyoshi Yoshino2，Yoichi Ueta（

Memory impairment and changes in brain gene expression on heatexposed mice（ 1Grad Sch of Life Sciences, Toyo Univ, Japan, 2 Res Ctr for Biomed Eng, Toyo Univ, 3 Schof Life Sciences, Toyo Univ, Japan）
Global warming increases the risk to health damage including brain dysfunction. It is reported that the serious heat stroke patient shows a symptom of the higher brain dysfunction.
However, the details of how heat stress affects cognitive functions are still unknown. In thisstudy we focused on memory function among cognitive functions, and aimed to elucidate themolecular basis underlying heat-induced memory impairment.
To evaluate the memory function of heat-exposed mice, we conducted the novel object recognition test. The heat exposed (1 hour at 45℃) immediately after the object presentation did
not affect the retention of the memory tested 24 hours after the heat exposure. By contrast,
the second day’s test showed a significant memory impairment in the heat-exposed mice.
However, the third day’s test, the heat-exposed mice showed normal retention of the previous
day’s memory. Thus, memory impairment was only detected first day after heat exposure,suggesting that heat-induced memory impairment seems to be transient.
To understand the molecular mechanism that caused this transient memory impairment, we
examined the effect of heat exposure on the expression of genes in the brain. PCR array
analysis showed that heat exposure caused changes in expression of various synaptic plasticityrelated genes in the brain. Thus, it is considered that the increased expression of these genes
by heat stress causes functional and structural changes in synapses and remodeling andreconstruction of neural circuits.
We also found that the changes in expression of apoptosis-related genes and the inflammatory
marker were observed 24 hours after heat exposure. Such pathological events may impact on
subsequent memory acquisition.
These molecular changes after heat stress may be involved in not only transient impairmentof memory, but also recovery of memory processes. Our studies may contribute to better(COI:No)
understanding of molecular basis for heat-induced cognitive deficit. 

S128

Possible relationship between estrogen-dependent hypothalamic
oxytocin synthesis and body fat accumulation in female rats

3 Dept Translational Health Science, Sch Med, Univ Bristol, UK ）Oxytocin (OXT) is produced in the supraoptic (SON) and paraventricular nuclei (PVN) of
the hypothalamus and is secreted into the systemic circulation from the posterior pituitary
gland (PP). Although OXT regulates labor and lactation, OXT is also related to feeding as
an anti-obesity peptide. However, the relationships between estrogen, hypothalamic OXT
synthesis and body weight, in particular fat mass are unclear in women. Our aim in thepresent study was to evaluate whether estrogen regulates hypothalamic OXT synthesis andbody fat accumulation in female rats that express the OXT-monomeric red fluorescent protein
1 (mRFP1) fusion gene.
The synthesis of OXT in the hypothalamus was evaluated by the intensity of the mRFP1
fluorescence in the SON, PVN and PP. The distribution of body fat accumulation was measured by microcomputed tomography (micro-CT). The fluorescence intensity of OXT-mRFP1
in the SON, PVN and PP were most strongly observed during the estrus stage of female
rats and decreased significantly in ovarectomized (OVX) rats. The subcutaneous fat mass
was significantly increased in OVX rats in comparison with sham-operated rats. Estrogen
replacement in OVX rats caused significant increases of the fluorescent intensities in the SON,PVN and PP in a dose-dependent manner. Depending on the dose of estrogen, subcutaneous
fat mass was reduced at low doses of estrogen replacement and visceral fat mass as well as
subcutaneous fat mass was reduced at high doses of estrogen replacement. These data suggestthat hypothalamic OXT synthesis and accumulation of body fat mass may be regulated by
estrogen level.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

2P-079

Cardiovascular response evoked by social defeat stress and projections
to the rostroventral medulla from the midbrain in rats
2 1 Department of
Mio Matsuyama1，Ena Yamamoto1，Yuliang Hu2，Jouji Horiuchi1, （Biomedical Engineering, Toyo University, Japan, 2 Dept Biomedical Engineering, Toyo
Univ, Japan）

2P-082

Does physical activity in daily life cause differences in circulatory
dynamics and autonomic nervous system activity between men andwomen?
Xinru Sun, Junko Hoshi, Hiromasa Tanno, Emi Kanno, Ryoko Maruyama（ Health Science,

Grad Sch Med, Tohoku Univ, Japan）

Acute psychological stressor evokes typical cardiovascular responses, such as increases inblood pressure (BP) and heart rate (HR) accompanied by a physical reaction. It has been shownthat the sympathetic vasomotor pathway of acute psychological stress is mediated via neurons
in the rostroventral medulla (RVM) indirectly from the dorsomedial hypothalamus which is apossible autonomic center for the stress response. However, it is still unknown detailed neuralprojections from the hypothalamus to the RVM. In the present study, we investigated that
direct projections to the RVM and distribution of c-Fos expressed neurons during social defeat
stress (SDS) in conscious rats. In order to verify neural projections to the RVM, we injected
a retrograde tracer, FluoroGold (FG) into the unilateral RVM in the rat. The FG injected rat
was allowed to recover for a week, and then was exposed to the SDS challenge. After the
SDS exposure, neuro-excitatory marker, c-Fos and FG were stained and observed throughout
the brain. We have already shown that the SDS challenge evoked pressor response andtachycardia in our previous study. The FG labeled neurons were observed from the pons to
the hypothalamus. The double-labeled (c-Fos and FG) neurons were distributed in the lateral/
ventrolateral periaqueductal grey matter (PAG) in the midbrain. Therefore, we microinjected
GABA antagonist, Bicuculline (Bic) to excite neurons distributed in the lateral/ventrolateral
PAG in some anesthetized rat preparations (urethane;1.3g/kg, iv). The Bic injection into the
PAG caused profound increases in BP and renal sympathetic activity. Taken together withall our results, it is suggested that neurons in the lateral/ventrolateral PAG contribute to thepressor response evoked by acute psychological stress, like the SDS, via sympatho-excitatory(COI:No)vasomotor pathway.

Physical activity (PA) provides health benefits: the PA profile (type, intensity, amount) is associated with enhanced health and QOL. We have used non-intervention methods to observe theassociation between PA in daily living, circulatory dynamics, and autonomic nervous system(ANS) activity in healthy young adults. We aimed to investigate how PA in daily life affects
ANS activity and circulatory dynamics in men and women.
Forty-five participants aged 20–30 years were recruited, and we measured the PA of dailyliving over 1-week using pedometer. Accordingly, the high PA group (≥28 METs/week)
comprised 7 men and 16 women and the low PA group (<28 METs/week) comprised 8 men
and 14 women. Height, weight, BMI, muscle mass, visceral fat rating, and basal metabolic rate
were measured using a body composition analyzer. We also measured blood pressure (BP)and heart rate variability (HRV) at rest and during the postural change from the supine to thesitting position. Spectral analysis of HRV was performed to evaluate low-frequency (LF) power,
high-frequency (HF) power, and LF/HF ratio to observe the changes in cardiac sympathetic
and parasympathetic nerve activities.
Significant differences were observed between the men groups that the high PA group interms of body muscle mass is higher. The women in the high PA group had significantly
higher leg muscle mass than those in the low PA group. During the postural change from all
positions, women groups of ANS activities were more sensitive than that in men groups. Thus,
we observed greater PA of daily living affects body muscle composition, and the impact on
ANS activities exhibited gender differences.
(COI:No)

2P-0802P-083

Akari Hori1，Yuka Ichinotsuka2，Tatsuya Kumaki2，Ena Yamamoto1，Mio Matsuyama1，
2 1 Department of Biomedical Engineering, Toyo University,
Jouji Horiuchi1, （Grad Sch Med, Tohoku Univ, Japan）

Distribution of c-Fos expressed neurons and the cardiovascular
reaction evoked by social defeat stress in serotonin-deﬁcient rats
2 Dept Biomedical Engineering, Toyo Univ, Japan）

Social defeat stress (SDS) that mimics stress caused by interpersonal relationship trouble, isa kind of psychological stress and causes us to have various autonomic responses such as
hypertension and tachycardia. In our previous study, central activation of 5-hydroxytryptamine
1A receptors suppressed the stress-induced cardiovascular response seen when stimulating
the dorsomedial hypothalamic area (DMH) in anesthetized rats. Also, we found that the SDS
increased blood pressure (BP) and heart rate (HR) and increased the neural excitability in the
DMH in conscious rats. Thus, it is suggested that central serotonin system plays an importantrole in the stress-induced cardiovascular response, but the detailed mechanism is not clear.
In this study, we investigated the effect of the SDS on the cardiovascular response and theexcitability of the DMH neurons in serotonin-deficient model rat (FH/Ham rat). Experimental
rats were implanted a telemetry probe to measure BP and HR during the SDS and thenthe rats’ brains were fixed and stained a neuro-excitatory marker, c-Fos in the DMH. The
initial change in BP caused by SDS in FH/Ham rats was the same as those from Wistar rats
(control). However, the pressor response in FH/Ham rats soon returned to pre-SDS levels,
whereas the pressor response persisted during the SDS exposure in the control group. The
immediate increase in HR after the SDS in FH/Ham rats was significantly suppressed, andthe tachycardia did not sustain during the SDS period. The number of c-Fos immunoreactive
neurons in the DMH significantly increased in FH/Ham rats compare to the control. Therefore,
the neural excitability of the DMH, which is one of stress centers, augmented in FH/Ham rats.
These results suggest that there is a discrepancy between DMH neural excitability and the(COI:No)
cardiovascular responses during SDS in serotonin-deficient rats.

Does listening to music affect HR, BP and autonomic nervous activity
in men and women differently?
Junko Hoshi, Xinru Sun, Hiromasa Tanno, Emi Kanno, Ryoko Maruyama（ Dept Nursing,
It has been reported that listening to music has beneficial effects on physical and mental health.
Moreover, it has been reported that autonomic cardiac control and auditory perception differs
between men and women. Despite these widespread claims, previous studies have showninconsistent results regarding physiological responses to music. We aimed to evaluate the gender differences in heart rate (HR), blood pressure (BP) and autonomic nervous system (ANS)
activity in healthy adults in response to listening to music. Twenty-five volunteers participatedin 3 tasks consisting of 3 experimental conditions. Including 10 min of rest and test-to-load on
sympathetic nervous activity, the conditions were listening to Mozart’s Piano Sonata for Two
Hands, D-major (K448) or silence as control for 8 min in randomized order. Electrocardiography(ECG) was continuously performed from start to end of each data collection point. There
were no significant differences between men and women in terms of changes in HR and BP.
However, significant decrease after listening to K448 in the ratio of low frequency (LF)/ highfrequency (HF) as an index of sympathetic nervous activity was observed only young women.
Conversely, natural logarithmized HF (logHF) as an index of parasympathetic nervous activity
in men significantly decreased after listening to K448. Our finding suggests that ANS activity
in response to music varies between men and women whereas this gender difference was not(COI:No)
noted in terms of HR and BP. 

2P-081

2P-084

Akihiro Fukushima, Kazuhiro Nakamura（ Dept Integrative Physiol, Nagoya Univ Grad Sch

Hiromi Ueda1，Mitsuya Shiraishi1，Hiroyuki Nakamori2，Takahiko Shiina3，Yasutake Shimizu3，
1 1 Dept. Basic Vet. Sci., Fac. Vet. Sci., Kagoshima Univ., Kagoshima,
Kiyotada Naitou（

Oxytocinergic Transmission from Paraventricular Hypothalamic
Nucleus to Rostral Medullary Raphe Stimulates Brown Adipose Tissue
Thermogenesis
Med, Japan）

Oxytocin (OXY), a neuropeptide synthesized in the paraventricular hypothalamic nucleus(PVH) contributes to many brain functions including maternal, social and sexual behaviors
and stress responses, which all involve autonomic responses. To investigate the roles of PVH
oxytocinergic neurons in the central regulation of autonomic functions, in this study, we first
performed neural tract tracing in rats from oxytocinergic PVH neurons to the rostral medullaryraphe region (rMR), which contains sympathetic premotor neurons controlling brown adipose
tissue (BAT) thermogenesis. A recombinant adeno-associated viral vector (OXY-Tet-palGFP)
was injected into the PVH to express palGFP under the oxytocin promoter. Confocal imaging
showed OXY-immunoreactive puncta in PVH-derived, palGFP-expressing fibers distributedin the rMR. palGFP-expressing fibers in the rMR were also closely associated with neurons
expressing vesicular glutamate transporter 3 (VGLUT3), a marker for BAT sympathetic
premotor neurons. Next, we co-injected another viral vector (TRE-ChIEF-mCherry) with the
OXY-Tet-palGFP vector into the PVH to express the engineered channelrhodopsin variant,
ChIEF, in OXY neurons. In whole-cell slice recordings of PVH neurons, GFP(+)/mCherry(+)
double-labeled neurons faithfully exhibited action potentials in response to 10-Hz blue-lightstimuli. In in vivo BAT sympathetic nerve recording, optogenetic stimulation of PVH-derived
OXY terminals in the rMR increased BAT sympathetic nerve activity and temperature, expired CO2, and heart rate, eliciting thermogenic and cardiac sympathetic responses. Moreover,prior nanoinjection of OXY into the rMR significantly potentiated BAT sympathetic nerve
activity and other sympathetic responses evoked by NMDA nanoinjection into the rMR. Thesefindings indicate that OXY transmission from the PVH to the rMR stimulates thermogenic andcardiac sympathetic outflows to BAT and the heart. This OXY pathway may be involved inemotion-related metabolic, thermal and cardiac responses.
(COI:No)

Analysis of the effect of intraspinal administration of alpha-melanocytestimulating hormone on colorectal motility
Japan, 2 Dept. Cell Physiol., Nagoya City Univ. Grad. Sch. Med. Sci., Nagoya, Japn, 3 Dept.
Basic Vet. Sci., Lab. Physiol., Unit. Grad. Sch. Vet. Sci., Gifu Univ., Gifu, Japan）

Colorectal motility is regulated by two defecation centers, one located in the brain and one in
the spinal cord. However, the precise mechanisms of central regulation of colorectal motility arestill unclear. An orexigenic peptide, ghrelin, reportedly affects the spinal defecation center andenhances colorectal motility. Previously, we showed that somatostatin, which has the opposite
effect to ghrelin in the hypothalamus, enhanced colorectal motility through acting on the spinal
defecation center. In this study, we focused on alpha-melanocyte-stimulating hormone (alphaMSH), another neuropeptide with an opposite effect to ghrelin in the hypothalamus. We aimed
to elucidate the action of alpha-MSH on the spinal defecation center and the consequent effecton colorectal motility in anesthetized rats. Colorectal motility was enhanced by intraspinal
administration of alpha-MSH at the L6-S1 level (the location of the spinal defecation center), but
not by intravenous injection. Intraspinal alpha-MSH still enhanced colorectal motility when thethoracic cord was transected at the T4 level. Prevention of parasympathetic outflow from thespinal cord by cutting the pelvic nerves, but not prevention of sympathetic outflow by cutting
the colonic nerves, abolished the effect of alpha-MSH. Our results demonstrate that intraspinalalpha-MSH activates the parasympathetic outflow to the colorectum by acting on the spinal
defecation center and enhances colorectal motility. The findings provide novel insight into thecentral regulation of colorectal motility. Considering the role of alpha-MSH in hypothalamic
appetite regulation, we suggest that this neuropeptide may link appetite modulation to the(COI:No)
central regulation system for defecation.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S129

2P-085

Generation and analysis of scn4aa and scn4ab double knock-out
zebraﬁsh

2P-088

Withdraw

Chifumi Terai, Souhei Sakata, Fumihito Ono（Dept Physiol, faculty Med, Osaka Med Col,
Japan）

Muscle excitation starts by activation of the acetylcholine receptor (AChR), followed by the action potential generation, opening of the dihydropyridine receptor (DHRP) and the elevation of
the intracellular calcium level. In contrast to this classical model, muscle contractions without
the action potential have been reported in the slow-twitch fibers of the zebrafish trunk muscle(Buckingham et al.) and the trunk muscle of juvenile ascidians (Ciona intestinalis) (Nishino etal.). In these muscle fibers, the depolarization directly contracts the muscle and enables animal
locomotion. To re-examine the significance of action potentials, we knocked out voltage-gatedsodium channels (Nav, scn) in zebrafish fast-twitch fibers. If the muscle fibers lacking the action
potential contract, the fish will swim normally. Alternatively, if the fibers lose their activity,
the fish will show abnormal swimming pattern as reported in Zempo et al. (In this meeting).
Initially, we performed RNA sequencing and found that the abundant read counts of scn4aa
and scn4ab in the zebrafish trunk muscle. In addition, much smaller counts of scn5lab (scn12ab)were also observed. The PCR experiment confirmed the gene expression of scn4aa and scn4ab,while scn5lab was absent. Depending on these data, we generated a double-knock out zebrafish
line - lacking scn4aa and scn4ab - using the CRISPR-Cas9 system. Unexpectedly, the double
knock-out fish exhibited swimming pattern similar to wild-type fish. This may imply thedispensability of the action potential. We are recording the sodium current from the knock-out
zebrafish. At the meeting, we will discuss the role of scn genes and the action potential in(COI:No)
swimming activity of zebrafish. 

2P-086Detection of muscle hardness after cramping
Akihiro Kaizu, Yoshiyuki Tsuboi（Dept Of Physiol, Sch Dent, Nihon Univ, Japan）
Background: Muscle clamps are painful involuntary contraction. Muscle clamps have a good
prognosis, but many people complain of pain and hardness even after muscle clamping. Stretch
is commonly used for pain relief. However, little is known about the cause of remaining painor hardness after clamping.
Purpose: In this study, we aimed to measure muscle properties after cramping.
Methods: Seven-week-old SD rats (average 239 g) were used. A wire electrode was connected
and attached to the rat tibial nerve under general anesthesia (2% isoflurane). Tetanic contraction was caused by applying electrical stimulation. The rat lower leg muscle clamping model
was obtained by performing electrical stimulation for 5 minutes at an intensity (frequency 50
Hz, duration 100 µs, 10 times the threshold) that causes complete tautness. Muscle properties
such as Tone (Hz), Stiffness (N/m) and Elasticity was measured by using Myotone Pro®. In
addition, in order to examine the effect of stretching, the lower leg muscles after cramping
were stretched for one minute, and same parameters were evaluated.
Results: Muscle Tone (Hz) and Stiffness(N/m) were increased significantly (P<0.05). Although
there was no significant difference, Elasticity was also increased after muscle cramping. These
indicators were improved by stretching treatment.
Discussion: This study suggests that remaining hardness after cramping may be caused bymuscle morphological changes. Because Muscle hardness was detected even in the absence ofelectrical stimulation. To consider the details, cytohistological study is necessary.
(COI:No)

2P-089

Role of cyclic AMP pathway on expression of MyHC Ⅱ and IL-6 mRNAs
in mouse myocytes
2 1 Deptf Nutr. Sci., Kansai Univ of Welf.
Junko Yamaji1，Reiko Hiroshima2，Yoshiaki Mori（
Sci., Japan, 2 Dept Rehab, Kansai Univ of Welf. Sci., Japan）

CGRP is a neuropeptide secreted from motor neuron at neuromuscular junctions and also
activates cyclic AMP (cAMP) pathway. However, the role of the cAMP pathway in expression of MyHCII mRNA in skeletal muscle remains unknown. Recently, we reported that IL-6
induced by calcineurin activation might increase MyHCIIb mRNA, but that MyHCIIb mRNAlevel was decreased by medium supplemented with CGRP in C2C12 cells. In this study, weinvestigated that the role of cAMP pathway and CGRP on expression of MyHCIIb and IL-6
mRNAs in C2C12 myocytes.
C2C12 cells were cultured by medium containing with or without agents at the beginningof differentiation. The levels of mRNA expression were measured by quantitative RT-PCR
method using Taqman probes.
Then our results are as follow:(1) The mRNA expression level of MyHCIIb was significantly upregulated by IL-6 induced bycalcineurin activatiors and was significantly attenuated by calcineurin inhibitor.
(2) The mRNA expression level of MyHCIIb was not affected by medium supplemented withforskolin, with 8-bromo cAMP and with clenbuterol.
(3) The IL-6 mRNA level was not affected by medium supplemented with CGRP and withforskolin.
These results suggested that calcineurin-mediated IL-6 production increases MyHCIIb mRNAbut that cAMP pathway is not essential for expression of IL-6 and MyHCIIb mRNAs in C2C12(COI:No)
cells.

2P-087

2P-090

Sachiko Ootsuka（ Dept Frontier Health Science, Grad Human Health Sciences, Tokyo1 1 Dept of Rehabil
Yoshiaki Mori1，Junko Yamaji2，Reiko Hiroshima1，Manabu Miyamoto（

1-Fluoro-2, 4dinitorobenzene delays the relaxation process of theskinned carotid artery
Metropolitan Univ, Japan）

Purpose: Phosphocreatine (PC) is known to accelerate Ca2+ removal induced relaxation
process in smooth muscle. To investigate whether the acceleration of the relaxation is due
to ATP regeneration through Lohmann reaction, we examined the effects of 1－Fluoro－2,
4dinitorobenzene (FDNB), a potent inhibitor of creatine kinase, on the relaxation process using beta escin skinned (cell membrane permeabilized) taenia cecum and carotid artery fromguinea－pig.
Materials and Methods: A small intact strip of carotid artery or taenia cecum from guinea－
pig was firstly skinned with beta escin and Ca ionophore A23187, then contracted with 10 µM
Ca2+. When the active force reached to a steady level, Ca2+ was quickly removed by applicationof ethylene glycol-bis(2-aminoethlylether)-N, N, N’, N’－tetraacetic acid (EGTA) in the presenceor absence of FDNB.
Results and Discussion: FDNB significantly slowed the relaxation process both in skinned
carotid artery and taenia cecum at 30μM and higher. Regression analysis of the relaxation
process suggested that FDNB accelerated reattachment and/or slowed subsequent detachment and/or slowed subsequent detachment of myosin with actin rather than the initial fastcross-bridge dissociation. Since the slowing effects of FDNB on the relaxation process wasobserved even in the absence of PC in the intracellular solution, FDNB might affect intrinsic
PC regenerating mechanisms and/or other Lohmann reaction independent mechanisms of the(COI:No)skinned smooth muscle.

S130

Activation CGRP-cAMP-dependent signal transduction pathways
upregulate MyHC I mRNA in C2C12 cells
Sci, Kansai Univ of Welf Sci, Kashiwara, Japan, 2 Dept of Nutr Sci, Kansai Univ of Welf
Sci, Kashiwara, Japan）

Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide which belongs to a family of
related peptides including calcitonin, amylin, and adrenomedullin. CGRP peptides are mainly
localized in sensory and central neurons and have been implicated in a variety of physiologicalprocesses. CGRP has also been identified in spinal motoneurons of several species and in thenerve terminals of the rodent neuromuscular junction. Our previous study using differentiated
C2C12 cells indicated that myosin heavy chain type I (MyHC I) mRNA expression level was
significantly increased by the application of CGRP, but myosin heavy chain type IIb (MyHC IIb)mRNA expression level was not increased by the application of it. C2C12 cell line appears to
express CGRP receptors coupled to adenylyl cyclase activity. Therefore, we examined the contribution of cAMP-dependent pathways on the upregulation of MyHC I mRNA levels in C2C12
cells. C2C12 cells were induced to differentiate to myotubes by medium exchange to D-MEM
containing 2%FBS. The cells were incubated in D-MEM containing 2%FBS with chemicalcompounds at the beginning of differentiation and removed after 24hr, and were maintainedin differentiation medium for 3 days. MyHC I and MyHC IIb mRNA expression levels were
measured by the real-time PCR method. MyHC I mRNA levels were significantly increasedby the administration of CGRP, although MyHC IIb mRNA levels were not affected by it.
Additionally, the effects of forskolin or 8-Br-cAMP on these mRNA levels were identical to thatof CGRP. However, MyHC I mRNA expression levels were not affected by the application ofcAMP response element binding protein (CREB) inhibitor, 665-15. These results suggest thatthe upregulation of MyHC I mRNA level by the activation of CGRP-cAMP-dependent signaltransduction pathways is not involved in cAMP/CREB-mediated transcriptional regulation in(COI:No)
C2C12 cells.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

2P-091

Elucidation of the pathogenic mechanisms in HSPB8 myopathy

2P-094

Withdraw

Yukika Kawabata-Sakata, Genri Kawahara, Yukiko Hayashi K.（Dept Pathophysiol, Tokyo
Medical Univ, Japan）

Heat shock protein B8 (HSPB8), a member of the small heat shock protein family, prevents
protein denaturation and aggregation, and plays a role in degrading abnormal proteins via
the chaperone-mediated selective autophagy (CASA) pathway in skeletal muscle. Previous
studies reported that mutations in HSPB8 cause hereditary motor neuropathy and myopathy.
Recently, two novel candidate mutations of HSPB8 were identified in families with proteinaggregated myopathy. However, the pathogenic mechanisms of HSPB8 myopathy remains
to be clarified. In this study, to elucidate the pathological mechanisms of HSPB8 myopathy,
we analyzed phenotypes and intracellular aggregates using zebrafish and cultured cellswith novel HSPB8 mutations. We carried out microinjection of wild-type or mutant human
HSPB8 mRNA in zebrafish embryos. Then their phenotypes at 5 days post-fertilization wereanalyzed. Overexpression of mutant HSPB8 mRNA resulted in morphological abnormalities
at higher rate compared to expressing wild-type HSPB8 mRNA-injected and uninjected fish.
Furthermore, it revealed that these abnormal fish had severe muscle degeneration and protein
aggregation. Aggregate formation was also observed in cultured cells. In addition, the aggregates contained CASA-related molecules along with mutant HSPB8. Our data suggest that
failure of the protein quality control system with HSPB8 may cause myopathy with protein(COI:No)aggregation.

2P-092Ca2+-dependent and –independent contractile responses to 1-ﬂuoro-2,
4-dinitrobenzene (FDNB) in the taenia caecum isolated from the guinea
pig
Yukisato Ishida1, 2，Richard Paul2，Satoko Mihashi1，Sachiko Ohtsuka1，Naoya Nakahara3，
1 1Grad Sch Human Health Sci, Tokyo Metropolitan Univ, Tokyo, Japan,
Masaru Watanabe（
2 Coll Med, Univ Cincinnati, Cincinnati, Ohio, USA, 3 Dept Mol Physiol, Jikei Univ Sch Med,Tokyo, Japan）

In the guinea pig taenia caecum, both cytosolic and mitochondrial cratine kinases are present(Ishida et al., 1991), and phosphocreatine (PCr) production is dependent on the presence ofoxygen (Ishida and Paul, 1990), indicating the compartmentation of PCr and ATP metabolism
in a smooth muscle cell (Ishida et al., 1994). Since FDNB inhibits creatine kinases, we here
investigate effects of FDNB on contractile responses and phosphagen contents of the taenia.
The presence of 0.4 mM FDNB gradually elicited a contractile response of the intact taenia
for more than 60 min with a peak tension approximately 15 min after the onset of contraction.
The depletion of Ca2+ from the medium with 0.2 mM EGTA inhibited the FDNB-induced peaktension by 60% of control, but did not inhibit the late phase of contraction. On the other hand,pre- or post-treatment with 10 µM verapamil did not affect the contractile response to FDNB.
In the skinned taenia with β-escin and A23187, 0.1 mM FDNB elicited a contractile responsein the medium contained with CTP as a nucleotide, but not with ATP and PCr. In the intact
taenia, the presence of 0.4 mM FDNB gradually reduced tissue ATP (by half at 15 min andnearly depleted at 60 min), but not PCr, and increased ADP and AMP contents, measured byisotachophoresis, indicating that FDNB inhibits creatine kinases in the intact taenia. These
results suggest that FDNB elicits two components of contraction in the intact taenia, where theearly phase of Ca2+ channel-insensitive contraction is dependent on the presence of external
Ca2+, but the late phase is independent. The early phase seems to be an active contraction, but
the late phase be a rigor- or latch-like contraction of the taenia due to the elevation of ADP in(COI:No)
the presence of FDNB.

2P-093

Amiloride and spirantol sensitivity for NaCl responses in the tastesensitive neurons in the rostral nucleus of the solitary tract in rats
Tatsuko Yokota, Katsunari Hiraba（ Dept Physiol, Sch Dent, Aich-Gakuin Univ, Japan）
In the present study, we investigated whether amiloride (epithelial Na+ channel blocker) andspirantol (salt-taste enhancer, a pungent extract from an aster family, Spilanthes acmella)affected low-threshold (LT) and/or high-threshold (HT) neurons for NaCl in the first-ordertaste relay, the rostral nucleus of the solitary tract (rNST) in the medulla. Here, we recorded
extracellular single unit activities in the rNST neurons using multi-barrel glass micropipettes
while under urethane anesthesia. Taste solutions were applied to the tongue and the oralcavity, and rinsed by distilled water (water-condition), amiloride and spirantol. Net responses
during taste stimulations were calculated by subtractions of averaged spikes during water applications. Amiloride significantly reduced the net responses for 0.1 ~ 0.8 M NaCl in 33% (10/30
neurons), and all of the amiloride-sensitive neurons were the LT neurons. Spirantol increased
the net responses for NaCl in 50% (6/12 neurons) by spirantol. The spike activities for the LT
neurons were frequently increased by spirantol solution for itself. The HT neurons which did
not respond to 0.1 and/or 0.2 M NaCl during water-condition responded to their low concentrations during spirantol administration. The enhancement of spirantol may be prominent in the
HT neurons with amiloride-insensitive. These results suggest that enhancement of sodium
taste by spirantol result from not only increased response magnitudes but also recruitment ofneurons. (COI:No)

2P-095

Oral discrimination of viscosity in sweet ﬂavor in humans and rats
2 1 Dept Physiol, Grad Sch Dent Med, Kyushu Dent Univ, Japan, 2 Dept
Yuichi Miyamura1, （Oral Radiol, Grad Sch Dent Med, Kyushu Dent Univ, Japan）

The unpalatability of the texture and/or flavor of an additive cause some methodologicalproblems in studies of texture perception. Here, we examined oral viscosity discrimination inhumans and rats in sweet flavor to improve unpalatability and additive flavor. In nine healthy
volunteers, the palatability and viscosity perception of xanthan gum (0-3%)-contained fluids
were evaluated by visual analogue scales. Apple sweet flavor greatly improved the palatabilityof fluids without impairment of viscosity perception. In animal experiments, carboxymethyl
cellulose (CMC: 0-3%)-contained saccharin fluids were fed to food-deprived rats. The intake ofviscous fluids was same as non-viscous fluid until 1% and slightly decreased in 3%. Next, we
performed conditioned avoidance test by viscous fluid without saccharin and LiCl or saline(vehicle) to evaluate viscosity discrimination in rats. Relative intake of the viscous saccharincontaining fluid against vehicle treatment was significantly lower than that of the non-viscoussaccharin-containing fluid in 1% CMC conditioning, but not in 0.3% conditioning. These results
suggest that viscosity can be discriminated in the oral cavity under improvements of theunpalatability of texture and/or flavors of an additive by adding sweet flavors. (COI:No)

2P-096

Nutrient sensing mechanisms in gustatory ganglion cells of rats
Takeshi Suwabe, Toshiaki Yasuo, Noritaka Sako（Dept Oral Physiol, Sch Dent, Asahi Univ,
Japan）

Purpose: Sensory function is influenced by nutritional balance. However, little is known
about mechanism for integration of sensory and nutritional informations. In this study, weinvestigated the existence of taste receptors for nutrient sensing in the rat geniculate ganglion
to determine possible mechanism for integration of nutrition and gustatory informations in the
peripheral nervous system.
Methods: Subjects were adult male (7-8 weeks old) rats. Reverse transcribed total RNA
extracts of geniculate ganglia from rats were tested by qRT-PCR for the presence of therenin-angiotensin system (angiotensinogen, renin and angiotensin-converting enzyme) andangiotensin II receptor (type 1a, type 1b and type 2 receptors) mRNAs. 10-μm-thick freefloating cryosections of geniculate ganglia were prepared and incubated with anti-angiotensin
II antibody, stained with Alexa Fluor 568-conjugated secondary antibody and Alexa Fluor
488-conjugated anti-NeuN (a neuronal cell maker) antibody.
Results: A salt taste receptor aENaC, a sweet taste receptor component T1R3, a taste-specific
G protein subunit a-gustducin and the downstream element Trpm5 mRNAs were detected
in geniculate ganglia of rats by qRT-PCR. Angiotensinogen, renin and angiotensin-converting
enzyme mRNAs were detected in the geniculate ganglia by qRT-PCR. Angiotensin-II-immunoreactivity was detected in the cytoplasm of NeuN (a general neuronal marker)-immunopositive
cells of the geniculate ganglia by immunohistochemistry. Angiotensin II type 1a, type 1b andtype 2 receptor mRNAs were detected in the geniculate ganglia by qRT-PCR.
Conclusions: A salt taste receptor and a sweet taste receptor component mRNAs wereexpressed in the geniculate ganglion suggesting the ability to sense fluctuations of sodium andglucose concentrations in body fluid. The renin-angiotensin system and angiotensin II receptor
mRNAs were co-expressed in the geniculate ganglion suggesting cellular communication via
angiotensin II signal within the ganglion.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S131

2P-097

Mechano-sensitive ion channel of rat squamous cell carcinoma
Motoki Ishizaki1，Haruna Toda1，Sadao Ohyama1，Wataru Ohfusa1，Asuka Higashikawa1，2 1 Dept Physiol, Tokyo Deantal
Maki Kimura1，Yoshiyuki Shibukawa1，Tatsuya Ichinohe（

Univ, Japan, 2 Dept Dental Anesthesiology, Tokyo Deantal Univ, Japan）

2P-100Mechanical hypersensitivity induced by adult-onset hypothyroidism due
to peripheral nerve hyperexcitability based on voltage-gated potassiumchannel downregulation in mice
Machiko Suda1，Yusuke Takatsuru2，Izuki Amano1，Shogo Haraguchi3，Noriyuki Koibuchi1

Some patients with oral cancer in early stage do not complain any pain sensation while painis one of the important symptoms of oral cancer. Recent study has suggested that cancer
cells are capable to release pain-inhibiting substances to the surrounding tissue to suppress
pain caused by increasing tissue pressure during tumor growth. In this study, we aimed toinvestigate the mechano-sensitivity of rat squamous cell carcinoma cells, and whether cancer
cells are capable to release diffusible pain inhibitors into surrounding environment or not.
Rat squamous cell carcinoma cells (SCC-158) were used. Intracellular free Ca2+ concentration([Ca2+] i) was measured by fura-2/AM. Direct mechanical stimulation to single SCC-158 cells
was performed, and [Ca2+] i changes were recorded from mechanically stimulated cells andtheir neighboring cells, to investigate the intercellular signal communication among cancer
cells. We utilized Gd3+ (non selective mechano-sensitive cation channel inhibitor) and GsMTX4(Piezo1 channel inhibitor), as mechanosentive channel antagonists.
During direct mechanical stimulation to SCC-158, we could observe transient [Ca2+] i increase.
Repeated mechanical stimuli to the SCC-158 induced repeated [Ca2+] i increases, but theincreases did not show desensitizing effects. Transient [Ca2+] i increases were also observedin neighboring cells to the stimulated SCC-158 cells. Application of Gd3+ inhibited mechanicalstimulation-induced Ca2+ increase in the 35% of stimulated SCC-158 cells (5 cells/14 cells).
Application of GsMTX4 inhibited mechanical stimulation-induced Ca2+ increase in the 33% ofstimulated SCC-158 cells (5 cells/15 cells).
We suggest that rat squamous cell carcinoma cells express mechano-sensitive cation channels,
and their activation may result in releasing intercellular transmitters from the cells. (COI:No)（ 1 Dept of Integrative Physiol, Grad Sch Med, Gunma Univ, Japan, 2 Dept of Psychiatry,
Johmoh Hospital, 3 Dept of Biochem, Showa Univ Sch of Med）

2P-098

2P-101

Withdraw

Thyroid hormone (TH) is crucially involved in the function of both the central and peripheral
nervous system. Approximately half of adult-onset hypothyroid patients suffer from sensory
symptoms including pain, which may be caused by the peripheral neuropathy. However, themechanisms causing the pain in hypothyroidism have not yet been clarified. We generated
adult-onset hypothyroid mouse model by administrating 50 ppm propylthiouracil (PTU) for
5 weeks. The mechanical hypersensitivity, examined by von Frey test, was observed
during PTU exposure and recovered after the termination of PTU treatment. Compound
action potential of the sciatic nerve was also analyzed. Threshold, conduction velocity andamplitude of Aδ- and C-fibers were measured by single pulse stimulation. The latency delay
and amplitude decrease during train pulse stimulation were also measured. No significant
change in thresholds, conduction velocities, or amplitudes by single pulse stimulation wasobserved in PTU administered group. However, the latency delay and amplitude decrease
in Aδ-fiber components by train pulse stimulation were less in PTU administered group 4
week after the PTU exposure, indicating relative hyperexcitability in nociceptive fibers. In
the C-fiber component, less conduction delay by the train pulse stimulations was observed in
PTU administered group. Voltage-gated potassium channel 1.1 and 1.2 protein levels decreased
significantly in the sciatic nerve of PTU administered group simultaneously. These results
indicate that adult-onset hypothyroidism in mice causes mechanical hypersensitivity due tohyperexcitability of peripheral nerves, and reduction of Kv1.1 and Kv1.2 may be involved in(COI:No)such alteration.

Bromocriptine administration to pregnant mice during late pregnancy
induces impaired expression of maternal behavior in offspring
Kwan Ee Oh1，Seika Sato1，Shinnosuke Masuda1，James Taku Sairenji1，Mitsue Miyazaki 2，
1 1 Dept of Integrative Physiology, Gunma
Noriaki Shimokawa1, 2，Noriyuki Koibuchi （University Graduate School of Medicine, Japan, 2 Dept of Nutrition, Takasaki University ofHeatlh and Welfare, Gunma, Japan）

Although maternal behavior is one of the indispensable behaviors for the preservation of
species, our understanding towards the development of maternal behavior is still limited. Using
genetically modified mice, we previously reported the possibility that maternal prolactin (PRL)
during prenatal period could be the critical factor for the development of maternal behavior
in the subsequent generation. However, it is yet unclear whether the change in maternal
PRL itself influences the development of maternal behavior. Here, using wild-type mice administered with bromocriptine, a D2-dopamine agonist, we show that PRL signaling duringlate pregnancy is important in the development of maternal behavior in offspring. We firstmeasured the plasma PRL concentration during late pregnancy. Antepartum PRL surge was
identified from gestational day 19 and lasted until parturition. Interestingly, we also identified asmall nocturnal surge of PRL occurring daily at late pregnancy. By inhibiting secretion of PRL
from the pituitary gland using bromocriptine, we managed to keep the plasma PRL concentration at low level throughout late pregnancy. For the evaluation of maternal behavior in thenext generation, we measured the survival rate of pups. Mice born to mother administeredwith bromocriptine showed a lower survival rate of pups, indicating impaired expression ofmaternal behavior. In contrast, when PRL was administered together with bromocriptine,
survival rate of pups showed no significant difference compared to the controls. Collectively,
these findings suggest that PRL signaling during late pregnancy contributes to the normal(COI:No)development of maternal behavior in offspring. 

2P-099Sexually dimorphic neural projections of calbindin-D28K neurons in the
medial preoptic area of mice
Masahiro Morishita1，Kota Ono1，Yousuke Tsuneoka2，Shuhei Horio3，Shigeki Kato4，
1 1 Div Life Sci, Grad Sch Sci and Engr, Saitama
Kazuto Kobayashi4，Shinji Tsukahara（

Univ, Japan, 2 Dept Med, Toho Univ, Japan, 3 Div Endocrinol and Metab, Natl Inst for
Physiol Sci, Japan, 4 Dept Mol Genet, Inst Biomed Sci, Fukushima Med Univ, Japan）

The medial preoptic area (MPA) of mice contains a sexually dimorphic nucleus composed ofneurons expressing calbindin-D28K (Calb) [thereafter termed the calbindin-sexually dimorphic
nucleus (CALB-SDN)]. The CALB-SDN exhibits a male-biased sex difference in the numberof Calb neurons, although the projection site of Calb neurons and its sex difference remain
unclear. To determine the projection site of Calb neurons and its sex difference, we conducted
an anterograde neurotracing study using an adeno-associated virus (AAV) vector that enables
Cre recombinase-expressing cells to express green fluorescent protein (GFP) and wheat
germ agglutinin (WGA). We injected the AAV vector into the MPA of transgenic mice that
expresses Cre recombinase in Calb-expressing cells (Calb-Cre mice) and observed the brain
sections of male and female Calb-Cre mice to find GFP-expressing fibers and WGA-expressing
cell bodies. As a result, we found both GFP-expressing fibers and WGA-expressing cell bodies in the ventral tegmental area (VTA), suggesting that the VTA is a projection site ofCalb neurons. Next, we performed a retrograde neurotracing study using an AAV vector
that infects neurons at nerve terminals and expresses tdTomato after Cre recombination.
We injected the AAV vector into the VTA of male and female Calb-Cre mice and counted
the number of tdTomato-expressing Calb neurons in the CALB-SDN of Calb-immunostained
brain sections. As the results, many tdTomato-expressing Calb neurons were found in themale CALB-SDN, but few tdTomato-expressing Calb neurons in the female CALB-SDN. Calb
neurons that did not express tdTomato were also observed in the CALB-SDN of both sexes,
but no sex difference was found in such neurons. These results suggest that the CALB-SDN
contains two types of Calb neurons, which are neurons serving as interneurons within the
CALB-SDN and neurons projecting to the VTA. The latter may be a significant component forforming a sexually dimorphic neural circuit. (COI:No)

S132

2P-102

Effects of chronic iodine excess on brain function
1 1 Dept ofIzuki Amano1，Yusuke Takatsuru2，Shogo Haraguchi3，Noriyuki Koibuchi（

Integrative Physiol Gunma Univ Grad Sch of Med, Gunma, Japan, 2 Department ofMedicine, Johmoh Hospital, Gunma, Japan, 3 Department of Biochemistry, Showa University
School of Medicine, Tokyo, Japan）
Iodine is one of the essential micronutrient which is required for the synthesis of thyroid hormones. Thus, iodine deficiency may result in the hypothyroidism. Iodine deficiency is one of themost common cause of preventable mental retardation and brain damage in the world. On theother hand, Japanese iodine intake exceeds that of most other countries, due to the significant
seaweed consumption such as kelp. Iodine tolerance among individual humans varies greatly,
and the excess iodine can cause both hyper- and hypo- thyroidism. Furthermore, the effect of
thyroid dysfunction due to iodine excess on brain function has not been clarified. In this study,
we generated a mouse models for chronic iodine excess and evaluated its effect on brain
development. C57BL/6 mice were treated with KIO3 37.4mg/l and 374mg/l through drinking
water. The visual discrimination test and the three-chambered social test were conducted at 10
weeks old. After the experiment was completed, thyroid hormones were measured and tissues
were collected. Excess iodine intake caused hypertrophy of thyroid follicles regardless of theadministered dose. Female mice administered highest dose showed a slight decrease in thyroid
hormone levels, whereas male mice showed a slight increase. In the behavioral analysis, female
mice showed an increase in learning ability. In summary, the high overdose of iodine affects
thyroid hormone levels, cognitive learning function, and social behavior. The gender differencein the consequence was also observed. From the above, it was clarified that the high doseexerts various changes, although the body is tolerated with excess iodine.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

2P-103

2P-106

Yoko Kaneko S.1，Yu Kodani1，Miho Kawata1，Akira Nakashima2，Hiroshi Nagasaki1

Ryo Hatano1，Mikiko Takayama2，Kotoku Kawaguchi2，Toshiyuki Fukutomi3，Toru Kimura3，
2 1 Dept Med Physiol, Grad
Hiroyuki Sakurai3，Eunyoung Lee1，Takashi Miki1，Shinji Asano（

Elucidation of the relationship between TNF receptor subtypes and thedevelopment of insulin resistance（ 1 Dept Physiol, Sch Med, Fujita Health Univ, Japan, 2 Dept Physiol Chem, Sch Med, Fujita
Health Univ, Japan）
Type2 diabetes is a multifactorial disease and develops by overlapping multiple genetic
predispositions and environmental factors. Among them, obesity by overeating, high-fat diet
(HFD) intake and insufficient exercise are related to it. Recently, it has become clear adipocytes
secrete various physiologically active substances called adipokines. TNF-α, one of them, isinvolved in the development of insulin resistance.
There are two receptor subtypes of TNF-α. In this study, mice lacking type1 TNF receptor(R1-KO), mice lacking type2 receptor (R2-KO), mice lacking the both (D-KO) and C57BL/6
mice (WT) were reared with normal chow or HFD. The amounts of ingested calories andbody weight were measured at every three days up to eight weeks. The concentration of
triglyceride, glucose and insulin in blood were measured with assay kit.
The weight-gain rate of R1-KO, R2-KO, D-KO and WT reared with HFD was 57.7%, 75.3%,
73.4% and 73.4%, respectively. R2-KO and D-KO gained significant weight even with normalchow and it was 13.2% and 19.6%, respectively. The blood glucose level increased about 1.5
times and the concentration of insulin increased more than 5 times in R2-KO, D-KO and WT
by HFD feeding. The responses to HFD of R1-KO were not significant in the experiments.
HOMA-IR index is calculated from insulin and glucose levels in fasting blood, and it is used as
an indicator of insulin resistance. The value was 35.2 in R2-KO, 40.0 in D-KO and 21.0 in WT
reared with HFD, indicating they showed severe insulin resistance. The index of D-KO withnormal chow was 4.3, indicating they were in wild insulin resistant state. Those data suggested
there was a difference in the development of insulin resistance depending on the TNF receptor(COI:No)
subtypes that transduce the different signals into cells.

Ezrin regulates multiple solutes reabsorption via the regulation ofseveral membrane protein localization in the proximal tubules
Sch Med, Chiba Univ, Japan, 2 Dept Mol Physiol, Col Pharm Sci, Ritsumeikan Univ, Japan,

3 Dept Pharmacol Toxicol, Kyorin Univ Grad Sch Med, Japan）

Ezrin is a member of ERM (ezrin-radixin-myosin) proteins, which commonly possess a binding
domain for membrane proteins and phosphoinositide, which is located at amino-terminal and abinding domain for actin cytoskeleton, which is located at carboxyl-terminal. In kidneys, intense
expression of ezrin is observed in proximal tubules, and it is postulated that ezrin plays important roles in tubular solute reabsorption via the regulation of apical membrane localizationof several transporters. We previously reported that ezrin knockdown (Vil2kd/kd) mice show
hypophosphatemia due to misvocalization of Sodium dependent phosphate transporters and its
scaffold protein, NHERF1. However, we haven’t investigated the influence of loss of ezrin on
the membrane localizations of other transporters and membrane proteins. Thus, we performed
comprehensive proteomic analysis of renal brush border membrane vesicles (BBMVs) of
proximal tubules from Wild type (WT) and Vil2kd/kd mice in this study. We prepared BBMVs
by MgCl2 precipitation method from renal cortex. We identified totally 1,412 proteins from six
mice. Scaffold proteins including NHERF1 and PDZK1 were significantly decreased in Vil2kd/
kd mice. Several transporters including Slc5a1, Slc5a11, Slc22a4 and Slc22a5 showed marginallysignificant reduction. We also found that BBMV localizations of several other solute transporters associated with the reabsorptions of amino acids, glucose, and organic anions were totally
decreased in Vil2kd/kd mice, suggesting that Vil2kd/kd mice exhibit the phenotype of Fanconi
syndrome, which is accompanied with massive urinary solute waste. Vil2kd/kd mice showeda defective endocytosis of FITC-labeled beta2-microglobulin similar with Fanconi syndromemodel mice, suggesting that ezrin plays important roles in the regulation of several membranetransporter localizations in the proximal tubules.
(COI:No)

2P-104

2P-1072 1 Deprt Biomed Sc, Coll Life Sc,
Hiroki Matsuda1，Fumihiro Kato1，Yukihiko Kubota（

Kenya Sanada1, 2，Hiromichi Ueno1, 2，Kazuhiko Baba1，Kentaro Tanaka1，Haruki Nishimura1，
Kazuaki Nishimura1，Satomi Sonoda1，Mitsuhiro Yoshimura1, 3，Takashi Maruyama1，
1 1 Dept Physiol, UOEH, Japan, 2 Department
Tetsu Miyamoto2，Yutaka Otsuji2，Yoichi Ueta（

Analysis of Cell Behaviors in Pancreatic Islet during Pancreatic β cellregeneration in larval zebraﬁsh
Ritsumeikan Univ, Japan, 2 Deprt Bioinformatics, Coll Life Sc, Ritsumeikan Univ, Japan）

Pancreatic β cells produce Insulin and play a central role for glucose homeostasis. Regenerative capacity of mammalian β cells is limited, so that loss and dysfunction of β cells causes
type 1 and type 2 diabetes, respectively. On the other hand, zebrafish have high regenerative
capacity of pancreatic islets, including β cell, throughout their entire life. Thus, zebrafish is an
attractive model for the study of β cell regeneration.
Previously several groups have reported about molecular mechanisms underlying pancreatic
β cell regeneration. However, it is still unclear from what cells regenerating β cell arise andwhen β cell regeneration is completed. Therefore, we analyzed behaviors of pancreatic β
cells, α cells and neurod1 expressing endocrine progenitor cells in the regenerating islet using
several transgenic lines, to address these two questions. Our results revealed 1) that pancreatic
β cell arose from neurod1 expressing cells during β cell regeneration and 2) that pancreatic(COI:No)β cell completely regenerated within 13 days after β cell ablation. 

Visualizing expression of hypothalamic arginine vasopressin change
during peritoneal dialysis by using transgenic ratsof Cardiovascular medicine and Nephrology, 3Translational Health Sciences, School of
Medicine, University of Bristol, UK ）Background: Peritoneal dialysis (PD) is a renal replacement therapy that stores hypertonic
substances in the abdominal cavity to drain excess water and waste products. In PD patients,
volume excess is a major clinical problem. Arginine vasopressin (AVP), which is synthesizedin the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus,
increases water reabsorption in the collecting duct. AVP synthesis and release in the hypothalamus are mainly controlled by plasma osmolality and blood volume. We hypothesizedthat intraperitoneal administration of hypertonic substances like PD solutions could affect thesynthesis of AVP by altering osmolality and blood volume.
Methods: First, after administration of 3% hypertonic saline (HTN) as a dialysis solution for ashort-time dwell or polyethylene glycol (PEG) as a dialysis solution for a long-time dwell, we
evaluated the AVP-enhanced green fluorescent protein (eGFP) fluorescent intensity in the
hypothalamus. The eGFP intensity is a quantitative indicator of AVP synthesis in transgenic
rats. Second, we then quantified the Fos-like immunoreactive (IR) cells in several brain regionsthat are known to be involved in maintaining fluid homeostasis by control of AVP synthesis
and/or which have interactions with the hypothalamus. Next, we evaluated the gene expressions of AVP, GFP and CRH in situ hybridization histochemistry.
Results: eGFP fluorescent intensities were significantly increased in the hypothalamus afteradministration of HTN and PEG. Immunohistochemistry for Fos revealed that several brain
areas were activated after the administration of HTN and PEG. The gene expressions of AVP,
GFP and CRH were revealed by in situ hybridization.
Conclusion: We visualized and semiquantitatively evaluated upregulation of AVP-eGFP
synthesis and neuronal activations during PD. Upregulation of the hypothalamic AVP might
be involved in the mechanism of fluid retention in PD patients.
(COI:No)

2P-105

2P-108

Takeshi Kato（Nutr Phyol, Grad Phar Sci, Josai Univ, Japan）

3 1 Neuro Rehab, HIRI, AIST, Japan,
Ryota Tamura1, 2，Nobuo Kunori1，Ichiro Takashima2, （It is very important to prevent the progression of renal failure in order to reduce medical
expenses for a super-aging society in Japan. Among the polyunsaturated fatty acids (PUFAs),arachidonic acid (ARA) is important for kidney development and function, while docosahexaenoic acid (DHA) is important for anti-inflammatory action. In this study, chronic renal failure
model rats were fed different PUFAs, and the effects on the progression of renal failureand changes in PUFA metabolites during that progression were examined. Sprague Dawley
male rats were randomly divided into 4 groups (Control, ARA, DHA, and ARA+DHA) andwere fed diets containing different compositions of PUFAs for each group, ad libitum. One
month later, 5/6 of the kidney was excised from each rat. After 4, 8, 12, and 16 weeks, bloodand urine samples were collected. Creatinine levels in plasma and urine were quantified, andcreatinine clearance (Ccr) and urinary albumin (U-ALB) excretion were determined. Further,
PUFA metabolites in plasma were quantified using Liquid chromatography tandem-mass
spectrometry. Ccr decreased after renal failure treatment but was not affected by PUFA
intake. U-ALB increased after renal failure treatment. The U-ALB levels in the DHA+ARA
group significantly decreased compared to the Control group. DHA metabolites, ProtectinD1,
and ResolvinD1, increased in the DHA and DHA + ARA diet groups. On the other hand, ARA
metabolites increased in the ARA group. The correlation between PUFAs and U-ALB was
examined, and a negative correlation was found between DHA and U-ALB in the renal failure
model rats after 4 weeks. These results suggest that the effects of the combined use of DHA
and ARA may slow the progression of renal failure. (COI:No)

Transcranial Direct Current Stimulation (tDCS) is a non-invasive neuromodulation techniquethat delivers a low direct electric current via electrodes placed on the skin of head. It has
recently attracted attention as one of the therapeutic techniques expected to be effective forvarious brain disorders. Previous several studies on animals and humans have reported that it
has an effect of changing cortical excitability directly under the stimulation electrode (Nitsche
and Paulus, 2000). However, it has not fully understood how tDCS affects neuronal activity of
distant brain regions that is anatomically connected to the stimulation site. In the present study,
in order to verify the effect of tDCS in the brain region away from the stimulation site, first, we
injected retrograde adeno-associated virus (AAV) vector genetically encoded a calcium indicator jGCaMP7f (pGP-AAV-syn-FLEX-jGCaMP7f-WPRE) into the primary motor cortex (M1) ofrats and identified the brain areas where GCaMP expressed by immunohistological analysis.
Furthermore, we chronically recorded the neuronal activity in these GCaMP-expressing brain
regions of behaving rat by using fiber photometry calcium imaging and analyzed the changes
in activity associated with DC stimulation of the M1. Preliminary results showed transientenhancement of calcium signaling associated with DC stimulation in multiple brain regions,such as motor thalamus and M2. This result suggests that DC stimulation applied to the
cerebral cortex may affect neural activity of surrounding cortex and subcortical areas which(COI:No)
has connected with injection site.

Change in polyunsaturated fatty acid metabolite proﬁles in plasma upon
progression of chronic renal failure

Effect of transcranial direct current stimulation to M1 on subcorticalneural activity
2 Grad Sch Comprehensive Human Sci, Tsukuba Univ, Japan, 3 HIRI, AIST, Japan）

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S133

2P-109

2P-112

Masashi Watanave, Nobutaka Takahashi, Ayumu Konno, Hirokazu Hirai（Dept Neurophysiol,

Indriani Dwi Wahyu（Div. System of Neurophysiology, NIPS, Japan）

Protein Kinase Cγ regulates the ʼgainʼ of excitatory signal during
propagation from Climbing Fiber-Purkinje Cell synapses to the cell body
Grad Sch Med, Gunma Univ, Japan）

Protein kinase Cγ (PKCγ) is expressed exclusively in neurons of the brain. In the cerebellar
cortex, only Purkinje cells (PCs) expressed PKCγ. PKCγ-deficient PCs show normal cerebellar long-term depression, but persistent innervation by multiple climbing fibers (CFs) and
impaired motor coordination. We found that viral vector-mediated re-expression of PKCγ in
PCs resulted in significant rescue of behavioral defects seen in PKCγ-deficient mice, suggesting that PKCγ had a pivotal role in adult PCs and thereby, regulating the cerebellar function.
To clarify a role of PKCγ in mature PCs, we compared the electrophysiological propertiesof PKCγ-null PCs with those of wild-type (WT) PCs. We found no difference in synaptictransmission from PF to PC and from interneuron to PC between WT and KO mice. In
contrast, we found significant decrease in CF-EPSC amplitudes and imput resistances (Ri) in
KO PCs, which was elicited in the presence of EGTA in the internal solution at holding current(Hc) of -10mV, but not at Hc of -70mV, suggesting the voltage dependency of CF-PC EPSC andRi modulation by PKCγ.
Intriguingly, CF-EPSC amplitudes and Ri were comparable between genotypes when using
the internal solution containing 10mM BAPTA, instead of EGTA, or the extracellular solution
containing 500µM TEA, suggesting that modulation of CF-EPSC amplitude and Ri by PKCγ
was Ca2+ and K+ dependent. Based on these results, we hypothesized that the modulation
on CF-EPSC was mediated through activation of large conductance potassium (BK) channels, which was activated by voltage-gated calcium channels. Namely, we assumed that BK
channel function was augmented in KO mouse PCs. Enhanced BK current in KO PCs could
cause shunting effect and attenuate the excitatory signal during the propagation from CF-PC
synapses to cell-body. To test this, we are planning to measure the BK current and show you(COI:No)the results.

Abnormal basal ganglia activity of L-dopa-induced dyskinesia model
mice
Parkinson’s disease (PD) is caused by progressive loss of dopaminergic (DA) neurons in thesubstantia nigra pars compacta in the basal ganglia (BG) and characterized by akinesia, rigidity,
tremor, and postural instability. Administration of dopamine precursor, L-dopa, can compensate for DA reduction and ameliorate PD symptoms. However, as the disease progresses
with long-term use of L-dopa, PD patients may develop uncontrollable movements known
as L-dopa-induced dyskinesia (LID). This study tried to elucidate the mechanism underlying
LID by recording neuronal activity in mice model of LID under awake state. LID mice weregenerated by treating 6-hydroxydopamine-lesioned PD mice chronically with an intraperitoneal
injection of L-dopa. The extracellular recording was performed in the external segment of theglobus pallidus (GPe), a relay station of BG, and substantia nigra pars reticulata (SNr), an output
nucleus of the BG. Spontaneous recording of GPe and SNr activity showed increase of burst
firings during PD, dyskinesia-off (24 hours after L-dopa treatment), and dyskinesia-on (after Ldopa treatment, when mice developed dyskinesia) in comparison to the control mice. In normal
mice, cortical stimulation induces a triphasic response both in the GPe and SNr, consisting of
early excitation, inhibition, and late excitation. PD state increased cortically evoked late excitation in GPe neurons, which later decreased during the dyskinesia-on state. In the SNr, corticallyevoked inhibition was decreased during PD state. During dyskinesia-on state, cortically evoked
inhibition was prolonged, while late excitation was reduced. Cortically evoked inhibition andlate excitation in the SNr are mediated by the direct and indirect pathways of the BG that
contribute to releasing and stopping movements, respectively. Therefore, the present result
suggests that increased facilitatory signal through the direct pathway and decreased stop
signal through the indirect pathway for movements lead to the emergence of LID symptoms.
(COI:No)

2P-110

2P-113

Noriko Isoo1，Naoyuki Murabe1，Hiroshi Kameda1，Satoshi Fukuda1，Miki Ikeda1，
1 1 Dept Physiol,
Sayaka Teramoto2, 3，Toshihiro Hayashi1，Shin Kwak 2, 3，Masaki Sakurai（

Hiroyuki Kida, Koh Sakai, Hiroyuki Ohtaku, Fumimaro Tsukamoto, Zin Han Thiri,
Dai Mitsushima（ Dept Physiol, Grad Sch Med, Yamaguchi Univ, Japan）

Isolation of spinal motoneurons of a deﬁned pool: diversity in gene
expressions of motoneurons
Sch Med, Teikyo Univ, Japan, 2 Grad Sch Med, Tokyo Univ, Japan, 3 Dept Mol
Neuropathogen, Tokyo Med Univ, Japan）

Motoneurons control various types of skeletal muscles for voluntary movement. Muscles
exhibit considerable diversity, e.g. distal forearm muscles producing fine movements andaxial muscles mainly involved in posture control. Motoneurons innervating a single muscle
constitute a motoneuron pool. Motoneuron pools are organized in functional ensembles inthe spinal cord, which could predict diversity of motoneurons in each pool. The molecularsignaling pathways underlying the diversity remain poorly understood. Here we attempted todevelop a method for isolation of motoneurons in a defined pool to investigate differences ingene expression. To determine the locations of the forearm and paraspinal pools, motoneurons
innervating forearm or paraspinal muscles were retrogradely labeled by cholera toxin subunit
B-conjugated Alexa Fluor 594. According to the distribution and morphological characteristics,
motoneurons in both pools stained with toluidine blue were isolated by laser captured microdissection. Cells identified as motoneurons exhibited immunoreactivity to ChAT. They expressed
substantial amounts of ChAT mRNA, but negligible amounts of Olig2 and GFAP mRNA.
These findings confirmed the collected cells as motoneurons. Whole genome microarray analysis of the total 26,597 gene expression changes was performed to identify the gene expression
profiling of the collected cells. The number of upregulated genes (ratio ≧ 2.0-fold, p-value<0.05) and downregulated genes (ratio ≦ 0.5, p-value <0.05) were 799 and 1,641, respectively
in motoneurons in the forearm pools compared with those in the paraspinal pools. Gene set
enrichment analysis revealed that expression amounts of gene set related to axonal transport
were reduced. Using our method, as many as 2,440 genes demonstrated significant differencesin expression between two pools, suggesting comparable diversity in motoneuron pools. Thismethod developed to isolate motoneurons of a defined pool would be expected to contribute
further to clarification of molecular mechanisms underlying diversity in motoneurons.(COI:No)

Motor training strengthens the GABAergic synapses onto corticospinal
tract neurons in the primary motor cortex

Nerve fibers in the corticospinal tract originate from pyramidal cells in layer V of the motor
cortex. To analyze motor learning-induced plasticity at layer V synapses in the primary motor
cortex (M1), we trained rats with a rotor rod test (10 sessions per day). The motor performance
was significantly improved at the final session of the 1st day of training. Then, rats were
further trained up to 2 days. First, we examined the role of local glutamatergic transmission
in the behavioral study. Compared with vehicle injected controls, bilateral pretreatment witheither CNQX or APV significantly impaired motor performance. Second, we took layer V
specific sample from the M1 to quantify the protein level of AMPA receptor GluA1 subunit.
In the synaptosome fraction, 2-days trained rats significantly increased the GluA1 subunit
level, suggesting long-term increase in the training-induced AMPA receptor delivery into theexcitatory synapses. Third, we made acute brain slices of the M1 to evaluate synaptic plasticity. Vertical fibers from layer II/III neurons to layer V neurons were stimulated to evaluate
the ratio of AMPA receptor-mediated current vs NMDA receptor-mediated current (AMPA/NMDA ratio). The ratio in 2-days trained rats increased the ratio up to 177 % of untrained
rats. Finally, we recorded miniature EPSC and IPSC to evaluate synaptic plasticity in layer
V neurons by retrograde tracer in C4 cervical spinal cord. Compared to untrained controls,
trained rats showed significantly higher miniature IPSC amplitude and frequency, suggesting
the training-induced strengthen at GABAA receptor mediated inhibitory synapses.
Thus, the motor-training induced dynamic change in the plasticity at layers V synapses in M1.
Together with the plasticity at layers II/III synapses (Kida et al, 2016), the plasticity at GABAA
receptor mediated synapses in the M1 circuit may contribute to reorganize the control system(COI:No)of pyramidal tract after the motor training. 

2P-111

2P-114

Kojun Tsuji, Yoshihide Satoh（Dept Physiol, Nippon Dent Univ Niigata, Japan）

Shinya Suzuki1, 2，Tsuyoshi Nakajima2，Shun Irie2，Ryohei Ariyasu2，Hiroyuki Ohtsuka2，
2 1 Dept Phys Ther, Sch Rehabil, Hlth Sci Univ
Tomoyoshi Komiyama3，Yukari Ohki（

Administration of dopamine receptor antagonists to the striatum
attenuates the swallowing reﬂex in the rat
Since dysphagia is frequently caused by hemorrhage of the striatum putamen and the Parkinson’s disease, there is no doubt that disorders of the basal ganglia are related to dysphagia.
However, the mechanism is not completely understood, and it is not known how the basal
ganglia and neural pathways are related to swallowing function. In this study, we investigatedthe effect of microinjection of dopamine receptor antagonists into the striatum on swallowing
reflex. These experiments were performed on rats anesthetized by urethane (1.3 g/kg, i.p.).
Electromyograms were recorded from the mylohyoid and thyrohyoid muscles to identify
swallowing event. The swallowing reflex was evoked by repetitive electrical stimulation (0.2
ms duration, 30 Hz) of the right superior laryngeal nerve (SLN). The dopamine receptor
antagonists were microinjected (2 µl, 90 seconds) into the central part of bilateral striatum.
D1 receptor antagonist Sch-23390 (5 µg/µl) alone, or Sch-23390 (2.5 µg/µl) together with D2
receptor antagonist Domperidone (2.5 µg/µl), or D2 receptor antagonist Sulpiride (20 µg/µl)
alone were used. Co-administration of Sch-23390 and domperidone to the striatum suppressed
SLN-evoked swallows. The onset latency of the first swallow was not significantly difference
between before and after administration of dopamine receptor antagonist. These results suggested that the direct or indirect pathways of the basal ganglia may be involved in inductionof the swallowing reflex.
(COI:No)

S134

Modulation of indirect corticospinal excitation prior to visually-guided
online adjustment of target reaching movement
Hokkaido, Japan, 2 Dept Integr Physiol, Sch Med, Kyorin Univ, Japan, 3 Div Hlth Phys
Educ, Fac Educ, Chiba Univ, Japan）

Cervical interneurons have been suggested to receive the motor commands for visually-guided
reaching (VGR) adjustment of the forelimb in cats. However, their roles in humans have notbeen elucidated. We investigated modulations of indirect corticospinal excitation while humansubjects executed VGR adjustment of the arm.
Healthy subjects, who all gave informed consents, were seated with recording of surface
electromyograms (EMG) from the right biceps brachii (BB) muscle. Subjects were asked toreach with the right arm toward the illuminated target in front of the subjects. The target
position was sometimes changed to the right or left by 10 cm just after movement initiation to
evoke VGR adjustment. Transcranial magnetic or electrical stimulation (TMS/TES) over thecontralateral motor cortex was applied at various timing to induce motor-evoked potentials(MEPs) in BB. In a spatial facilitation test, TMS and electrical stimulation of the ipsilateralulnar nerve (NERVE) were delivered separately or in combination. Inter-stimulus interval
for the combined stimulation (CS) was set at 10 ms (NERVE ahead) to ensure simultaneousconvergence of both inputs onto the cervical interneurons.
In BB, background EMG activities were enhanced in the left jump trials to make adjustments
to the new target. The MEP was also significantly enhanced from 70 ms after target jumpin the left jump trials, which started ~20 ms earlier than the onset of the background EMG
modulation. In contrast, the MEP was suppressed from 100 ms after right jump of the target.
The spatial facilitation by CS was observed in any trial, but it was stronger in the left jumpcompared to the no jump trial. The present findings suggest that the corticospinal excitationin the cervical interneurons is enhanced just prior to VGR adjustment, which may result inincreased descending commands to BB motoneurons.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

2P-115

2P-1182 1 Dept Reg Physiol, Dokkyo
Gen Takei1, 2，Darya Tourzani2，Bidur Paudel2，Pablo Visconti（

1 1 Dept Cellular andMasao Miyake1，Susumu Yoshie1，Satoru Kaneko2，Akihiro Hazama（

Reactive oxygen species (ROS) are necessary for capacitation but are
independent of cAMP/PKA phosphorylation pathways
Med Univ, Japan, 2VASCI, UMass, Amherst）

Mammalian sperm have to undergo capacitation, a collective name of biochemical and physiological changes that occurs within female reproductive tract, to achieve fertilization. Several
studies have reported that appropriate amount of ROS has a positive role on capacitation. It
has been reported that human sperm activate ROS generation during capacitation. However,molecular identity of ROS-generating enzyme and signaling pathways involved upstream ofcapacitation-activated ROS generation is still controversial. In addition, it has not been shown
whether ROS is necessary for fertilization.
In the present study, we investigated the association of known capacitation-associated pathways with ROS generation using mice sperm as a model.
First, we confirmed that ROS generation was up-regulated during capacitation. ROS generation was monitored by chemiluminescence using luminol. Bicarbonate ion and bovine serum
albumin in capacitating media act synergistically to up-regulate ROS generation. When
nicotinamide adenine dinucleotide phosphate (NADPH) was extracellularly added, mouse
sperm greatly enhanced ROS generation. Apocynin, an inhibitor of NADPH oxidase (Nox),
significantly inhibited ROS generation by mouse sperm. These results suggest that ROS are
generated by Nox.
Although it has been reported that ROS activates cAMP synthesis and tyrosine phosphorylation in human and bovine sperm, removal of ROS by catalase and superoxide dismutase had
no effect on phosphorylation of PKA substrates and tyrosine residues. On the other hand,inhibitors or activators of adenylate cyclase and PKA did not affect ROS generation. These
results indicate that ROS generation and capacitation-associated cAMP/PKA and tyrosine
phosphorylation pathways are regulated independently.
Finally, we investigated the effect of ROS on fertilization. The result showed that in vitro fertilization was completely blocked when sperm were preincubated with catalase to remove ROS.
Taken together, these results suggest that ROS are necessary for capacitation/fertilization,and that its generation is regulated independent of cAMP/PKA/ and tyrosine phosphorylation
signaling pathways.
(COI:No)

Oocyte selection by membrane potential measurement after freezethaw cycle
Integrative Physiol, Fukushima Med Univ Sch Med, Japan, 2 Ichikawa General Hospital,
Tokyo Dental Coll, Ichikawa, Japan）

Morphological inspection is the most commonly used technique to pick quality oocytes andembryos for artificial fertilization. To raise reproductive ratio, a new selection method fromnew point of view is needed, however, there are few techniques which is able to distinguishgood oocytes. The membrane potential reflects expression of ion channels and completeness ofcell membrane, it may evaluate oocyte quality. We previously showed that there was a wide
dispersion of membrane potential among eggs without morphological difference, using MII and4-cell stage eggs. It implied this technique could be applied for quality selection.
In this study, we analyzed the relationships between embryogenic outcome and membranepotential of mouse oocytes after freeze-thaw cycle. Oocytes were collected from hyperovulated
4-week old female mice. After freeze-thaw cycle, oocytes were used to measure membranepotential. And we measured membrane potential with voltage-sensitive fluorescent dye after
direct recording using single electrode, following insemination. Some of oocytes performed
good morphological characteristics, could reach blastocysts.But most of oocytes which performed near zero voltage stopped development. The near zero voltage oocytes are possible tobe scratched during conventional protocol. This method may be applicable to ignore damaged
oocytes. And the effect of mechanical damage on membrane potential will be discussed. All
animal experiments were planned toward institutional guidelines and reviewed by institutional(COI:No)
animal care and use committee.

2P-116

2P-1191 1 dept Physiol,
Hiromasa Satoh1，Mayu Hanaue1，Ken Takamatsu2，Naofumi Miwa（

Sho Aki1，Md Azadul Kabir Sarker1，Kazuaki Yoshioka1，Koji Kuno2，Yasuo Okamoto1，
1 1 Department of Physiology Kanazawa
Kazuhiro Ishimaru1，Noriko Takuwa3，Yoh Takuwa（

Suppressive action of dicalcin on female fertility via interaction withmouse cumulus cell-oocyte complex
Saitama Med Univ, Japan, 2 Dept Physiol, Toho Univ, Japan）

Class II PI3Ks a and b Are Required for Rho-Dependent Uterine
Smooth Muscle Contraction and Parturition in Mice

Fertilization is a well-coordinated and sequential process, beginning with taxon-specificsperm-binding to egg-coating structures. Although the principal cellular events at fertilization
have been eludiated, the molecular mechanisms whereby the fertilization is controlled remain
largely unknown. We discovered that Xenopus dicalcin, present in the frog egg envelope,
remarkably suppresses fertilization in vitro. This action was brought about by its binding toenvelope-constituent glycoproteins, and the dramatic changes in the orientation pattern ofthe envelope filaments and the viscoelasticity of the entire envelope. We have also found thatmouse dicalcin is present in the ciliary oviductal cells and the cumulus cells among cumulus
cell-oocyte complex prepared from the oviduct lumen following ovulation. In this study, we investigated the potential role of dicalcin on mouse in vitro and in vivo fertilization. Pretreatmentof the cumulus cells-oocyte complex with mouse dicalcin substantially reduced the efficiency ofin vitro fertilization as judged by the formation of two pronuclei, whereas pretreatment with
anti-dicalcin antibody increased the efficiency. Next, we examined the efficiency of in vitro
fertilization using the cumulus cells-oocyte complex prepared from dicalcin knock-out mice,and found that the cumulus cell-oocyte complex of dicalcin (-/-) female mice showed a higher
efficiency than those of dicalcin (+/-) and wild type. In addition, the number of born pups from
dicalcin (-/-) female mice was significantly greater than those of dicalcin (+/-) mice and wildtype
female, following the natural mating with wild type male. These results suggested that mouse
dicalcin inhibits fertilization efficiency by hampering sperm-egg interaction via its direct action
on cumulus cells among cumulus cell-oocyte complex. There are no conflicts of interest to
declare.(COI:No)

University School of Medicine, Japan, 2 Cancer Research Institute, Kanazawa University,
Japan, 3 Department of Health Science, Ishikawa Prefectural University, Japan）

2P-117

2P-120Effects of serotonin on sperm hyperactivation and IVF in mice
Masakatsu Fujinoki（Dept Physiol, Dokkyo Med Univ, Japan）
Capacitation is an essential process for mammalian sperm in order to be fertilized. Capacitatedsperm exhibits a specialized flagellar movement that refers to “HYPERACTIVATION”. In
addition, it is showed that ability of sperm hyperactivation is associated with success of IVF.
Previously, I reported that hyperactivation was enhanced by serotonin and was suppressedby GABA in hamster sperm (Fujinoki (2011) Reproduction, 142, 255-266; Fujinoki and Takei(2017) Journal of Reproduction and Development, 63, 67-74). However, I was not able to examine
effects of serotonin on in vitro fertilization (IVF) because it is very difficult to perform hamster
IVF. In the present study, I show that mouse sperm hyperactivation is regulated by serotonin.
Moreover, I also show effects of serotonin on IVF.
At first, I examined effects of serotonin on mouse sperm hyperactivation. Serotonin increased
hyperactivation through 5-HT2, 5-HT3, 5-HT4 and 5-HT7 receptors. In the next step, I examined
effects of serotonin on IVF. Serotonin, 5-HT2 receptor agonist, 5-HT4 receptor agonist and5-HT7 receptor agonist increased the success of IVF although 5-HT3 receptor agonist did notaffect it.
These findings show serotonin regulates fertilization through inducing of hyperactivation in
mice. In mouse, serotonin is released from cumulus cells and matures an oocyte. Therefore, it islikely that serotonin regulates an interaction between sperm and the oocyte in mice. (COI:No)

Class II phosphoinositide3-kinases (PI3Ks), PI3K-C2α andPI3K-C2β, are highly homologous
and distinct from class I and class III PI3Ks in catalytic products and domain structures. In
contrast to class I and class III PI3Ks, physiological roles ofPI3K-C2α and PI3K-C2β are notfully understood. Because we previously demonstrated that PI3K-C2α is involved in vascularsmooth muscle contraction, we studied the phenotypes of smooth muscle–specific knockout(KO) mice of PI3K-C2α and PI3K-C2β. The pup numbers born from single PI3K- C2α–KOand single PI3K-C2β–KO mothers were similar to those of control mothers, but those fromdouble KO(DKO)mothers were smaller compared with control mice. However, the number of
intrauterine fetuses in pregnant DKO mothers was similar to that in control mice. Both spontaneous and oxytocin-induced contraction of isolated uterine smooth muscle (USM) strips wasdiminished in DKO mice but not in either of the single KO mice, compared with control mice.
Furthermore, contraction of USM of DKO mice was less sensitive to a Rho kinase inhibitor.
Mechanistically, the extent of oxytocin-induced myosin light chain phosphorylation was greatly
reduced in USM from DKO mice compared with control mice. The oxytocin- induced rise in
the intracellular Ca2+ concentration in USM was similar in DKO and control mice. However,Rho activation in the intracellular compartment was substantially attenuated in DKO micecompared with control mice, as evaluated by fluorescence resonance energy transfer imaging
technique. These data indicate that both PI3K-C2α and PI3K-C2β are required for normal
USM contraction and parturition mainly through their involvement in Rho activation.(COI:No)

The role of cerebral vessels in the healthy life expectancy on
Alzheimerʼs disease
2 1 International University of Health and Welfare,
Yu Hasegawa1, 2，Shokei Kim-Mitsuyama（

School of Health Sciences at Fukuoka, Fukuoka, Japan, 2 Department of Pharmacology andMolecular Therapeutics Kumamoto University Graduate School of Medical Sciences）

Objective: Increasing evidences support the notion that vascular factors play a pivotal role in
the progression of Alzheimer’s disease (AD). We herein evaluated the role of vascular factors
in the pathogenesis of AD using 5XFAD mouse.
Methods and Results: 1) Continuous intracerebroventricular injection with angiotensin
II (AngII) was performed to 5XFAD and wild-type mice through 4 weeks. Although bothmice showed similar increased blood pressure, AngII significantly augmented accumulationof amyloid-β (Aβ) on cortical arteries, cognitive dysfunction, and sarcopenia in 5XFAD.
2) Continuous intracerebroventricular injection with angiotensin (1-7), which suppresses
AngII-induced vascular injuries, was performed to 5XFAD through 4 weeks. Angiotensin(1-7) improved cognitive dysfunction and the effect was associated with increased cerebral
vasoreactivity and decreased accumulation of Aβ on cortical arteries. 3) Brain Aβ was significantly reduced in wild-type mice with deletion of apoptosis signal-regulating kinase 1 (ASK1),a member of the MAPK kinase kinase kinase. In addition, increased cerebral vasoreactivity
was observed in the mice. On the other hand, there was no significant difference on cognitive
function, vasureactivity, or cytotoxic changes in aged 5XFAD with deletion of ASK1.
Conculsion: Those results suggest that additive vasotoxic effects on cerebral amyloid angiopathy deteriorate healthy life expectancy. However, healthy cerebral arteries might inhibit theaccumulation of Aβ and cognitive impairment in AD.(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S135

2P-121

The developmental change of gap junction in starburst amacrine cellsof the mouse retina
1 1 Dept Physiol, Nippon Med Sch,
Takuma Maruyama1，Toshiyuki Ishii2，Makoto Kaneda（
Tokyo, Japan, 2 Dept physiol, Nippon Med Sch, Tokyo, Japan）

In the retina, electrical synapses formed by gap junctions are used in two different ways. Gap
junctions between the homologous neurons, which are common electrical coupling in the adult
retina, are used for the summation of signals and the cancellation of background noise, while
gap junctions between heterologous neurons are used for the conservation of synaptic inputs.
In the previous study, we have shown that chemical synaptic inputs onto starburst amacrine
cells (SAC), an only cholinergic neuron in retina, are different between ON-type SAC (ON-SAC)
and OFF-type SAC (OFF-SAC). In this study, we examined how the gap junctions are used forsignal processing in ON- and OFF-SAC in the mouse retina using patch clamp technique. In
the whole mount preparation, membrane capacitance (Cm) of ON-SACs with intact dendrites
decreased after an application of meclofenamic acid (MFA), a gap junction blocker, at P10,
suggesting the presence of electrical coupling. Because of the difficulty to access OFF-SACs inwhole mount preparation, systematic comparison of Cm between ON- and OFF-SACs during
development was carried out in slice preparation, which might give a possible damage todendrites of SACs. In both ON- and OFF-SAC, Cm was relatively high until P10 and started
to decrease at P15. Reduction of Cm continued until P28. Finally, to evaluate the cell type connected with SACs through gap junction, we injected gap junction permeant tracer, neurobiotin,
to ON-SACs in whole-mount preparation. The filled ON-SACs coupled with other types ofneurons but not with neighboring ON-SACs at P10. These results suggest that both ON- and
OFF-SACs do not summate homologous signals after eye opening. Gap junctions in SACs andheterologous neurons in the early developmental stage might be used for the signal transfer
pathway of the retinal waves, which are important for neural circuit formation in the retina.
(COI:No)

2P-124

Appropriate combinations of herbal components of Japanese Kampo
medicines exert laxative actions on colonic epithelium cells throughactivation of K and Cl channels
1 1 Dept Physiol,
Tomohiro Numata1，Kaori Sato-Numata2，Yasunobu Okada3, 4，Ryuji Inoue（
Grad Sch Med, Fukuoka Univ, Japan, 2 JSPS, 3 Dept Physiol, Kyoto Pref Univ of Med,Japan, 4 NIPS, Japan）

Japanese Kampo medicines Junchoto and Mashiningan are mixtures of numerous herbal plant
extracts and empirically known to exert laxative actions by stimulating fluid secretion from thecolonic epithelium, thereby softening stools and promoting intestinal movement. However, it isunknown which and how the herbal components of these crude Kampo drugs are effective tostimulate ion effluxes causing colonic fluid secretion. Here, we selected four herbal componentsof Junchoto and Mashiningan, Mashinin (MSN), Kyonin (KYN), Tonin (TON), and Daio (DIO),
which are putatively laxatives, and examined their effects on the ion channel activity of humancolonic epithelial Caco-2 cells.
Patch-clamp analyses revealed that in both Caco-2 and CFTR-expressing HEK293T cells, MSN
activated whole-cell current characteristic of the cystic fibrosis transmembrane conductanceregulator (CFTR) chloride channel, which is sensitive to an adenylyl cyclase inhibitor and a
CFTR blocker whereas KYN, TON, and DIO activated whole-cell potassium currents, which issensitive to a K+ channel blocker. K+ currents activated by DIO, but not KYN and TON, were
eliminated by intracellular Ca2+ chelation with 5 mM BAPTA. In addition, single-channel currents activated in a cell-attached patch by KYN, TON or DIO exhibited a unitary conductanceof around 200 pS.
The coulter counter for electronic cell size measurement showed that MSN induced thesecretory volume decrease (SVD) sensitive to a CFTR blocker, whereas TON, KYN, and DIO
induced SVD sensitive to a K+ channel blocker.
In conclusion, MSN and TON, KYN, or DIO promote fluid secretion from colonic epithelial cellsby activating CFTR chloride channels and the large-conductance and voltage-activated K+(BK)
channels, respectively. Thus, Japanese Kampo medicines, Junchoto and Mashiningan, exertanti-constipation actions by inducing KCl efflux in colonic cells through the combined actions of
CFTR- and BK-stimulating herbal components.
(COI:No)

2P-122

2P-125

Keitaro Satoh1, 2，Kaori Satoh2，Yoshie Imaizumi-Ohashi2，Mika Yokoi-Hayakawa2，
2 1 Dept Pharmacol, Sch Dent, Asahi Univ, Japan,
Toshiyuki Fukada3, 4, 5，Yoshiteru Seo（

Moyuru Hayashi1, 2，Tomomi Watanabe-Asaka1, 2，Daisuke Maejima2，Yoshiko Kawai1, 2，
2 1 Dept Physiol, Med, TMPU, Japan, 2 Dept Medical-Health, Med, Shinshu
Toshio Ohhashi（

Postnatal development of the temporomandibular joint of the Sprague
Dawley rat measured by MR imaging
2 Dept Physiol, Sch Med, Dokkyo Med Univ, Japan, 3 Lab Mol Cell Physiol, Fac Pharma Sci,
Tokushima Bunri Univ, Japan, 4 Div Pathol, Sch Dent, Showa Univ, Japan, 5 Lab Integr
Genom, IMS, RIKEN, Japan）

Water intake-mediated secretion of ATP from myoﬁbroblast cells in rat
small intestine
Unv, Japan）

Temporomandibular joint (TMJ) growth studies previously focused on the condyle and its
cartilage, and largely paid less attention to the articular disc and temporal bone. In this study,
we employed magnetic resonance (MR) images to observe the temporal bone, mandibular
condyle, and articular disc growth as a single unit. The changes in the size and relative positionof each component were observed using 7-tesla MR images during the postnatal growth.
Growth of the mandibular condyle chondrocytes was assessed by immunohistochemistry using
the expression of the zinc transporter ZIP13. We obtained 3-dimensional T1-weighted (T1w) MR
images of the TMJ of Sprague Dawley rats at 4 – 78 days-old (P4 – P78) with a voxel resolutionof 65 µm. Two-dimensional T1w MR images were obtained after a subcutaneous injection of
the contrast reagent gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA). Sections of
the TMJ were incubated with an anti-ZIP13 antibody. The T1w MR images showed that themandibular condyle was located posterior to the temporal bone until P20, but afterwards, itchanged to a location underneath the temporal bone. In the Gd-DTPA enhanced images, thearticular disc could be identified as a region with a lower signal intensity. The number of
ZIP13-positive chondrocytes at P6 was larger than that at P24. These observations suggestthat the mandibular condyle with cartilage and disc grows in the posterior side of the mandibular fossa until P20. After the growth of the articular disc, joint cavity, and condyle cartilage, thecondyle fits to the fossa, and completes the functional unit of the jaw joint.
(COI:No)We demonstrated currently water intake increased mesenteric lymph flow and the total fluxof albumin, long-chain fatty acids, and ILC-3-dependent IL-22 in rats (Nagashio S et al., Am J
Physiol 316: G155-G165, 2019). In the present with animal, cells, and molecular experiments,
we attempted to examine the effects of shear stress stimulation produced by water intake
on the myofibroblast cells in rat small intestine walls and then evaluate pivotal roles of theshear stress stimulation in innate immunological and physiological meanings. Thus, the shear
stress stimulation around 1 dynes/cm2 on cultured myofibroblast cells isolated from rat
small intestines produced significantly release of ATP. In agreement with the finding, theimmunohistochemical expression of cell surface F1/F0 ATP synthase was marked observedon the cultured myofibroblast cells. In rat in vivo experiments, an intragastric administrationof distilled water caused significant increases of mesenteric lymph flow and the total flux ofalbumin and ILC-3-mediated IL-22, but no or little total flux of ATP in the mesenteric lymph.
In concomitant with the in vivo experiments, the immunohistochemical expression of a specific
ATP nucleotidase was confirmed on the submucosal cells including central lacteal canals inthe small intestine. ATP also induced specifically significant immunohistochemical expressionof podoplanin in the bottom region of submucosal interstitial tissues in the small intestine. In
addition, ATP produced a significant increase of IL-22 mRNA expression in the isolated ILC-3
from the submucosal tissues. In conclusion, water intake-mediated shear stress stimulation
produced a significant release of ATP from myofibroblast cells in rat small intestine, which
may play key roles in the IL-22-dependent innate immunology in the body.
(COI:No)

2P-123

2P-126

In vitro modeling of hypothalamic tanycyte development using mouseembryonic stem cells
Yu Kodani1，Yoko Kaneko S.1，Miho Kawata1，Akira Nakashima2，Hiroshi Nagasaki1（ 1 Dept Physiol, Fujita Health Univ Sch Med, Japan, 2 Dept Physiol Chem, Fujita Health
Univ Sch Med, Japan）
Tanycytes are specialized ependymal cells lining the floor and ventro-lateral walls of the thirdventricle and an important modulator of hypothalamic functions. Recent studies have identified
tanycytes as an adult neural stem cell (NSC) population that supplies new neurons to the hypothalamus and that releases exosomes showing anti-aging effects. Tanycytes are considered tobe remnants of radial glial cells (RGCs), a major embryonic NSC population, but the molecular
events underlying functional maturation remain unknown. We previously reported that mouseembryonic stem cell (mESC)-derived hypothalamic tissues generate tanycyte-like NSCs. Here,
we examined whether this mESC culture system can recapitulate the developmental transition
from RGCs to tanycytes. To visualize both cell types, we used a Rax::GFP knock-in mESC line
(Rax is a transcription factor specific to RGCs and tanycytes in the hypothalamus). Rax::GFP+
cells comprised around 50% of total cells during the early phase of neurogenesis from mESCs(～3 weeks) and then decreased to less than 10% during the late phase (～5 weeks). The
proliferation marker Ki-67 was expressed in 67.5% of Rax::GFP+ cells on day 14 but only in
1.6% on day 35, indicating that residual Rax+ cells after neurogenesis are mostly in a quiescentstate like mature tanycytes. We newly identified a cell surface antigen for native tanycytes
and it was expressed on Rax::GFP+ cells at rates of 28.2% and 94.9% after 14 and 35 days of
mESC differentiation, respectively. Along with the surface antigen expression, the morphologyof Rax::GFP+ cells was shifted from RGC-like to tanycyte-like one. Our results suggest that in
the mESC-derived hypothalamic tissue, Rax+ cells reproduce the RGC-to-tanycyte transition
process that occurs in the developing hypothalamus. Thus, this culture system provides auseful in vitro model for understanding the regulation of tanycyte development, maintenance,
and function.
(COI:No)

S136

Regulation of esophageal striated muscle motility via ATP-dependent
potassium channels
Takahiko Shiina1，Kazuhiro Horii1，Shouichiro Saito2，Sawa Onouchi2，Yasutake Shimizu1, 3（ 1 Dept Basic Vet Sci, Lab Physiol, Unit Grad Sch Vet Sci, Gifu Univ, Gifu, Japan, 2 Dept
Basic Vet Sci, Lab Anat, Unit Grad Sch Vet Sci, Gifu Univ, Gifu, Japan, 3 G-CHAIN, Gifu
Univ, Gifu, Japan）

The external muscle layer of the mammalian esophagus consists of striated muscle fibers andsmooth muscle fibers. In the present study, we clarified roles of ATP-dependent potassiumchannels (KATP channels) in motility of the striated muscle portion in the esophagus. A segmentof the esophagus was isolated from the rat and was placed in an organ bath. Mechanical responses of the esophagus were recorded using a force transducer. Electrical stimulation of thevagus nerve trunk with single pulses evoked monophasic (twitch-like) contractile responses in
the rat esophagus. The contractile responses were abolished by treatment with d-tubocurarine,
a blocker of nicotinic acetylcholine receptors. Application of glibenclamide, an antagonist of
KATP channels, increased amplitude of vagally mediated twitch contractions of the rat esophagus. On the other hand, minoxidil, an agonist of KATP channels, decreased amplitude of twitch
contractions. RT-PCR revealed the expression of subunits of KATP channels in esophageal
tissue. In addition, immunopositivity for subunits of KATP channels was observed in the striatedmuscle cells of the esophageal muscle layer. These findings indicate that KATP channels might
be involved in motor regulation of striated muscle in the rat esophagus. (COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

2P-127

Inhibition of glucose absorption by acetic acid in isolated small intestineof mice – Effects of Na+ concentration
Tomoo Homma1, 2，Saemi Sugiyama1，Kazuya Terashima2，Satoru Ishihara3，
4 1 Dept Biotech, Fac Tech, Maebashi Inst Tech, Japan, 2 Div Biotech,Shin-Ichiro Karaki（

Grad Sch Tech, Maebashi Inst Tech, Japan, 3 Gunma Agric Tech Center, Japan, 4 Lab
Physiol, Dept Env Life Sci, Univ Shizuoka, Japan）

Authors reported that cabbage vinegar (CV) inhibited glucose (Glu) absorption in isolated small
intestine of mice and the main factor of this inhibition by CV was acetic acid (AA). In order toclarify inhibitory mechanism of Glu absorption by CV, instead of CV, we have been studying
inhibitory mechanism of Glu absorption by AA. The present study investigated effects of Na+concentration on Glu and AA absorptions by using everted sac specimen of isolated small intestine of mice, because it was known that both Glu and AA were absorbed by Na+-dependenttransporters. Absorbed quantities of Glu and AA were measured after 20 min and 40 min
since Glu and/or AA applications. In the presence of AA, Glu absorption was inhibited, but
AA was absorbed. On the other hand, under the condition that Glu was continuously absorbed,
Glu absorption was inhibited in the middle after AA was applied. Absorbed quantity of AA
was larger than that of Glu under the low Na+ concentration (10 mM). At then, absorbed Na+
seems to be preferentially used for AA absorption, and Na+ for Glu absorption might become
short. For further analysis, stoichiometry and affinity of substrates ＆ Na+ of transporters for
Glu and AA should be considered.
(COI:No)

2P-130

Activation of airway ciliary beating by an [Ca2+] i increased via
nifedipine-sensitive Ca2+ channels stimulated by amboxol
Daichi Saito1，Kotoku Kawaguchi1，Shinji Asano1，Yoshinori Marunaka2，Takashi Nakahari2（ 1 Dept. Mol. Physiol., Coll. Pherm. Sci., Ritsumei Univ, Japan, 2 Res. Unit for Epithelial
Physiol., Res. Org. of Sci. and Tech., Ritsumeikan Univ., Japan）

The effects of a mucolytic agent, ambroxol (ABX), on ciliary beat frequency (CBF) and ciliary
bend distance (CBD, an index of amplitude) were examined in lung airway ciliary cells of
mice. Airway ciliary cells were isolated by an elastase treatment from lungs of mouse. Ciliary
beatings of airway ciliary cells were observed by the videomicroscopy equipped with highspeed camera (500 fps) and CBD and CBD were measured by a image analysis program using
a recorded images. ABX(10 µM) increased CBF and CBD by 30 %. ABX-stimulated increases
in CBF and CBD were partially inhibited by Ca2+-free solution and nifedipine. However, addition of BAPTA-AM (10 µM) completely inhibited increases in CBF and CBD stimulated by
ABX. Measurement of [Ca2+] i revealed that ABX stimulated a transient increase in [Ca2+] i ina Ca2+-free solution, suggesting ABX stimulates Ca2+-release from internal stores followed by
Ca2+ influx. The KCl solution, in which NaCl is replaced with KCl, alone stimulated increases in
CBF and CBD, which were inhibited by nifedipine, and ABX still increased CBF and CBD in
the KCl solution. These indicate that nifedipine-sensitive Ca2+ channels exist in airway ciliary
cells and were activated by ABX. Moreover, we are going to identify voltage-activated Ca2+
channels in airway ciliary cells of mice. The role of nifedipine-sensitive Ca2+ channels on airway
ciliary cells still remains uncertain, however, ABX appears to stimulate the nifedipine-sensitive
voltage activated Ca2+ channels leading to increase CBF and CBD in airway ciliary cells.
(COI:No)

2P-128

2P-131

Tetsuo Yamashita1，Takeshi Hashimoto1，Junsuke Igarashi1, 2，Hiroaki Kosaka1，
1 1 Dept Cardiovasc Physiol, Fac of Med, Kagawa Univ, Japan, 2 Dept Med
Katsuya Hirano（

Daisuke Kobayashi, Ziasmin Khatun, Natsumi Nishimura, Akihiro Hazama（ Dept Cell

N-terminal region of apoptosis-inducing factor facilitates dimer
formation by stabilizing the charge transfer complex
Eng. Fac of Heal Sci, Morinomiya Univ of Med Sci, Japan）

Apoptosis-inducing factor resides in mitochondria as AIFmit, which lacks a mitochondrial
targeting signal (1-53), and its release from the membrane is a prerequisite for induction of
apoptosis. AIFmit has been considered to be anchored to the membrane by an N-terminal
region. However, we have shown that both AIFmit and AIFsol, which further lacks a region of
54-101 in AIFmit, bind to the membrane via ionic bond and preferably in a dimer form. NADH
facilitates the membrane binding of AIFmit to a greater extent than that seen for AIFsol.
NADH-bound AIF is known to favor the dimer form. It is therefore hypothesized that the Nterminal region stabilizes NADH binding and facilitate dimer formation of AIFmit. The present
study addressed this hypothesis. AIFmit and AIFsol were expressed as recombinant proteinstagged with (His)6 in E. coli. Gel filtration assay revealed that both AIFmit and AIFsol exist
as monomers in the absence of NADH, and as homodimer in the presence of NADH. Underaerobic conditions, the addition of NADH to both AIFmit and AIFsol decreased the absorbanceat 451 nm, which reflect the reduced state of FAD, and increased the absorbance at 750
nm, which reflect the formation of charge transfer FADH2-NAD+ complex. The dissociation
constant of NADH for AIFsol was three times higher than seen with AIFmit, whereas the
NADH oxidase activity of AIFsol was significantly higher than that of AIFmit. These findings
indicate that the charge transfer complex in AIFsol is unstable. The N-terminal region of
AIFmit is suggested to prevent electron transfer from FAD to oxygen and stabilize the chargetransfer complex, thereby facilitating dimer formation and membrane binding.
(COI:No)

Cytoskeletal actin ﬁber elongation was perturbed by cesium application
in NIH/3T3 cells
Integrat Physiol, Grad Sch Med, Fukushima Med Univ, Japan）

Our laboratory previously showed that cesium inhibited the growth of the human cancer cells
by inhibition of the glycolytic pathway. We also investigated the effects of cesium on murine
fibroblast cells (NIH/3T3) proliferation and migration. Regarding microscopic observation of
migration assay, a shape of migrating cells seemed to be different between in the absence
and in the presence of cesium. We assumed that the morphologic difference was caused bycytoskeleton difference. In this study, a structure of cytoskeletal actin fiber was visualized with
AlexaFluor568-conjugated phalloidin. The NIH/3T3 cells treated with 10 mM cesium chloride
have a tendency to round shape, and its actin fibers were condensed at peripheral membrane
ruffle compared with control. Moreover, there were structures look like slender fingers ofmembrane, as well as filopodia in cesium chloride application cells. These results indicated that
perturbation of cytoskeletal actin fiber elongation was caused by cesium.
(COI:No)

2P-1292P-1322 1 mandom corp,
Masayuki Takaishi1，Maori Kono1，Hidefumi Ikeda1，Makoto Tominaga（
Osaka, Japan, 2 Exploratory Research Center on Life and Living Systems, National Institutesof Natural Sciences）

Kazuki Nanjo（Dept of Regen Med, Grad Sch of Med, Yokohama City Univ, Japan）

The cellular mechanisms of skin irritation through TRP channels
activation

TRPA1 or/and TRPV1 is known to be one of the factors to mediate inflammatory pain. Skin
preparation such as cosmetics cause continuous slight skin stimulation without injury to leadskin irritation. It is not clear how these TRP channels act as molecular sensors of skin irritation
through signaling pathway activated by intracellular calcium increases or thereby increasing the membrane potential. Therefore, we investigated TRPs-mediated signal transduction
pathways and transcription factors that lead to skin irritation.
In this study, we focus on secreted proteins through intercellular signaling system activatedby TRPA1 and TRPV1. We established HEK293T cell lines stably expressing high levels ofhuman TRPA1 (hTRPA1), human TRPV1 (hTRPV1) or MOCK-transfected cells (MOCK). We
selected HEK293T cells since these cells do not express endogenous TRPs including TRPA1
and TRPV1, and do not have crosstalk between TRPs, meaning that HEK293T cell line isconsidered to be appropriate for detecting only TRP channel-dependent reactions at highsensitivity. At first, we conducted micro array analysis to determine the relative expression
levels of genes among hTRPA1, hTRPV1 and MOCK treated with or without AITC (a TRPA1
agonist) or capsaicin (a TRPV1 agonist) and the relative signaling pathway. We picked TRPA1
or TRPV1-dependent signaling pathways and downstream releasing factors via activation of
the specific pathway. We especially focused on secreted protein as chemokines and cytokines
because some of them play an important role in the pathogenesis of skin irritation. We identified that some common chemokines and cytokines were involved in the activation of both
TRPA1 and TRPV1, and some of them were uniquely associated with the activation of either
TRPA1 or TRPV1.
In the future, we will investigate production of chemokines and cytokines in various time-points
following various agonist-exposure to clarify which is the most important factor for skin irritation though TRP channel activation.
(COI:No)

Perfusion culture improves functions of hiPSC-liver tissue with large
blood vessel
We previously developed a technology to generate the vascularized liver bud composed ofhuman induce pluripotent stem cell (hiPSC)-derived hepatic endoderm, endothelial cells andmesenchymal cells and proposed the novel cell therapeutics for acute liver failure (Takebe T
et al., Nature, 2013). To apply this technology to end stage liver failure such as liver cirrhosis,
it is necessary to reconstitute large liver tissue. As low oxygen and less nutrients inside of thelarge tissue cause apoptosis, it is necessary to generate large tissue with blood vessel to supply
oxygen and nutrients by perfusion through large blood vessel.
In this study, we generated hiPSC-liver tissue with large blood vessel (0.5-1 mm diameter) andexamined the effect of perfusion on liver tissue functions. At first, we tried to fuse hiPSC-livertissue and mouse aorta punctured by needle. hiPSC-liver tissue successfully fuses with aorta
and mouse endothelial cells were observed in human liver tissue under the stimulation with
VEGF. Next, large vessels from different origins (femoral artery and vein from mouse and rat,
and mouse aorta) were fused with hiPSC-liver tissue and compared their albumin secretion.
Among these samples, hiPSC-liver tissue with rat femoral artery showed the highest albumin
secretion. Finally, perfusion culture of hiPSC-liver tissue with large blood vessel was conducted
at a flow rate of 30 ul/min. As a result, hepatic clusters and albumin secretion were increasedin hiPSC-liver tissue with perfusion culture.
Taken together, novel culture technology to generate hiPSC-liver tissue with large blood vessel
was developed and perfusion culture through large blood vessel improved hiPSC-liver tissue
functions such as increase of hepatic clusters and albumin secretion. This technology could be
a substitute for organ transplantation in the future and contribute to solve donor shortage of(COI:No)the organ transplantation.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S137

2P-133

Building-up the primary culture system for ependymal ciliary cells fromnewborn mice
Yurie Namoto1，Yuki Ishii1，Tomonori Okazaki1，Shinji Asano1，Yoshinori Marunaka2，
2 1 Dept Mol Phisiol, Coll of Pharm Sci, Ritsumeikan Univ, Japan,
Takashi Nakahari（
2 Res Uni for Epithelial Phisiolgy, Res Org of Sci Tech, Ritsumeikan Univ, Japan）

2P-136Functional expression of ascorbate peroxidase derived from
Cyanidioschyzon merolae in mammalian cells
Suzuro Hitomi1，Shoichiro Kokabu2，Kenichiro Matsumoto3，Izumi Ujihara1，Kentaro Ono1

（ 1 Div Physiology, Kyushu Dental Univ, Kitakyushu, Japan, 2 Div Mol Signal Biochem,
Kyushu Dental Univ, Kitakyushu, Japan, 3 NIRS, Chiba, Japan）

The brain ventricle wall is covered with beating cilia. The ependymal ciliary beating is thoughtto drive cerebrospinal fluid flow, because its impairment induces a hydrocephalus. To study
ependymal ciliary beating, the stable ependymal ciliary cells in primary culture are required.
In the present study, we are exploring the culture condition to obtain the stable sheet of
ependymal ciliary cells in primary culture.
We tested two culture conditions for ependymal ciliary cells in primary culture. The wholebrain from a newborn mouse was cut into small pieces and then, they were digested bya trypsin treatment. The isolated cells were cultured with DMEM containing 10% FBS in
the flask for a week. After the first culture, cells were seeded on the transwell filter andcultured further 2 days. In the first culture method (culture A), cells were cultured with MEM
without FBS in apical and basolateral sides. In the second culture method (culture B), cells
were cultured with MEM without FBS in the apical side and with DMEM containing 10%
FBS in the basolateral side. Beating cilia of ependymal ciliary cells on the transwell filter wereobserved by a video microscope equipped with a high-speed camera (500 fps). The numberof ependymal cells growing on the transwell filter was much larger in culture B than culture
A. The ependymal ciliary beating is activated by an application of CO2/HCO3－-free solution inboth cultures, suggesting they are stimulated by an elevation of pH i.
In conclusion, ependymal ciliary beating was observed in the primary culture systems A and B.
The culture B seems to be better compared with the culture A to get ependymal ciliary cells
primary culture. Furthermore, the ciliary beating of ependymal ciliary cells in primary culture
may be stimulated by an elevation of pHi.
(COI:No)

Ascorbate peroxidase (APX) is an anti-oxidative enzyme limited to plants and photosynthetic
protists. The primitive red alga Cyanidioschyzon merolae (C. merolae) has high adaptive ability
to live in high-temperature and acidic environment. Probably, C.merolae-derived APX (cAPX)
has greater anti-oxidative effect than other plants living in normal environment. In the present
study, we evaluated the possibility of increasing anti-oxidative ability in cAPX-transfectedmammalian cells. The cAPX gene was introduced into the mouse embryo fibroblast cell line
10T1/2 cells by lipofection. Cell proliferation was not changed by cAPX transfection. cAPXexpressed cells were more tolerant to oxidative stress induced by application of H2O2 thanmock cells. Cell viabilities of the cAPX-expressed cells cultured in low pH (pH 3) was increased
compared with those of mock cells. Our present study demonstrated that APX gene from C.
merolae could be functionally expressed in mammalian cells. The cAPX-expressed cells will beavailable for cytotherapeutic approaches, such as cell transplantation for tissue regeneration.
(COI:No)

2P-134

2P-137

Hiroaki Miyazaki（Dept Life Sci, Fac Sci Eng, Setsunan Univ, Japan）

Akihiro Hazama, Kei Kakinouchi, Susumu Yoshie（Dept Cell Integrative Physiol. Sch Med,The molecular mechanism of intracellular Cl in tumor progression by
regulating JAK-STAT signaling cascades
A malignant tumor is a serious health problem because cancer cells can spread to distantparts of the body, so-called metastasis. For example, gastrointestinal cancer cells arising in
the gastrointestinal tract enter the bloodstream and spread to distant organs such as thelung. Since metastasis is the most common cause of death from cancer, this process is an
important therapeutic target. However, the molecular mechanisms of the metastasis are notfully understood. We previously clarified that the reduction of intracellular Cl－ concentrations([Cl－] i) inhibits the cell proliferation of gastric cancer MKN28 cells by diminishing the transition
rate from G1 to S cell cycle phase. If it is clarified that the intracellular chloride acts asa signal to regulate function of cancer cells, it may lead us to development of novel andunique therapeutic approaches. From this viewpoint mentioned above, in the present study, weinvestigated whether the intracellular Cl－ regulates cancer cell proliferation, cell migration andcell invasion abilities in human prostate and esophageal cancer cell lines. Our study indicatesthat the intracellular Cl－ is a key factor regulating JAK-STAT signaling cascades involved intumor proliferation, migration and invasion. These results strongly suggest that changes of(COI:No)[Cl-] i would play important roles in cancer migrations and invasion.

2P-135Differential regulation of cortical actin cytoskeleton by intracellular
calcium in mouse eggs

Cl channel regulates epithelial to mesenchymal transition in oral/headand neck squamous cell carcinoma
Fukushima Med Univ, Japan）

Background: Squamous cell carcinomas in the oral/head and neck region are particularly aggressive due to high incidence of recurrence and distant metastasis. However, the mechanismof epithelial to mesenchymal transition (EMT) and distant metastasis in the oral/head and neckregion has not been fully elucidated. It has been known that Cl－ channel controls cell volume
and several signaling pathways. Thus, Cl－ channel may be important trigger for morphologicalchange such as EMT. The aim of this study is to investigate the role of Cl－ channel on EMT
in OSC 20 cell line, which is a squamous cell carcinoma line of the oral/head and neck region.
Methods: OSC-20 cells were cultured with low serum medium containing TGFβ1, which
is a potent inducer of EMT, or Cl－ channel blocker (NPPB, DIDS, and 9AC) for 5 days. The
morphological change, gene expression, migration, and signaling pathway of OSC-20 cells
treated with TGFβ1 or Cl－ channel blocker were evaluated.
Results: The morphology of OSC-20 cells treated with TGFβ1 was not so changed. On theother hand, OSC-20 cells treated with Cl－ channel blocker showed typical morphology of mesenchymal cells. Furthermore, the expression levels of mesenchymal markers such as vimentin,
ZEB1, and snail in these cells were higher than that of untreated cells and TGFβ1-treatedcells. A large number of vimentin-positive cells were appeared in OSC-20 cells treated with Cl－
channel blocker. Additionally, these cells showed migration. Interestingly, Cl－ channel blocker
did not activate smad2 signaling pathway, which is a signaling pathway that causes EMT.
Conclusion: OSC-20 cells treated with Cl－ channel blocker showed mesenchymal phenotype
via non-smad2 signaling pathway. These results suggest that Cl－ channel is a key regulator of
EMT in the oral/head and neck region.
(COI:No)

2P-138

Toxicity evaluation of Turritopsis sp. secretory component
2 1 Dept
Masahiro Kondo1，Tomoyasu Hirano1，Kazunao Hobo2，Shin Kubota3，Yuri Mukai1, （Hideki Shirakawa, Shunta Arakawa, Takashi Yoshida（ Dept Eng Sci, Grad Sch Info Eng,Electr Bioinform, Sch Sci Tech, Meiji Univ, Japan, 2 Dept Electr, Grad Sch Sci Tech, Meiji
Univ, Japan, 3Turritopsis Res Inst Reg Biol, Shirahama, Japan）

In mouse eggs, actin filaments (F-actin) are localized in the cortex beneath the plasma membrane, and play various roles in the cellular events at fertilization, such as sperm incorporation,
cortical granule exocytosis, and polar body emission. In the present study, we investigated
the spatiotemporal changes in F-actin organization, in relation to repetitive rises in intracellular Ca2+ concentration, or Ca2+ oscillations, induced by sperm-borne egg-activating protein
PLCζ. The actin cap (AC), which is a thick cortical layer of F-actin near the chromosomes inmature eggs arrested at the metaphase of the second meiosis, rapidly degraded after a few
Ca2+ transients. Calpain, a Ca2+-dependent protease, may participate in this process, since thetreatment with calpain inhibitor delayed the onset of the AC. Interestingly, the timing of the
AC degradation coincided with the beginning of chromosome separation, suggesting that itis regulated coordinately with the metaphase-to-anaphase transition in meiotic cell cycle. In
contrast to the AC, the F-actin forming the bundles in and beneath microvilli distributed overthe cortical region other than the AC, increased transiently upon Ca2+ rises. The increase wassuppressed by inhibitors for actin polymerization, but not by CK-666 or SMIFH2, inhibitors
for actin nucleation mediated by Arp2/3 or formins, respectively. By examining the effects ofseveral inhibitors for kinases, it was suggested that calmodulin-dependent kinase II and Src
family tyrosine kinases are involved in the regulation of Ca2+-dependent increase in cortical Factin. In the presentation, the relationship between the Ca2+-dependent changes in the cortical
F-actin and those in PIP2 will also be discussed. (COI:No)

In a wide range of the fields centered on drug discovery and regenerative medicine, it isstrongly expected that the control of cell death and cell differentiation will be useful for treatment of intractable diseases. Therefore, to find the protein which is as an inducer of cell deathor cell differentiation of human cells is urgent. On the other hand, the loss of biodiversity
due to the uniform use of agricultural and fishery resources and the deterioration of the
agricultural and fishery resources production base have become major problems in Japan inrecent years, and the sustainable production infrastructure is also required for pharmaceuticaland medical development in the next-generation. Thus, Turritopsis sp. is focused on as acompletely metamorphic and sustainable organism that develops large-scale cell death and celldifferentiation including the reversal of the life cycle in this study.
Aiming at the construction of an artificial production base for Turritopsis sp., the laboratory
culture was tried in this study. As a result, it was found that mucus is secreted from vicinity oftentacles of medusa and polyps. It was also found that this mucus is toxic to the adult Artemia,
which is a feed for the Turritopsis sp., and plays a major role in preying by limiting the activityof Artemia. Furthermore, from the viewpoint of discovering medicinal and medical seeds, theeffects of Turritopsis sp. body fluids on HeLa cells were examined. The relationship with thesecretory components obtained was clarified in this study.
(COI:No)

Univ Electro-Comm, Japan）

S138

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

2P-139

2P-142

Kyoji Horie, Junko Yoshida（ Dept Physiol II, Nara Med Univ, Japan）

4 1 Dept Physiol, Sch Med, Aichi
Maki Sato1, 2，Dominika Kanikowska3，Toshiaki Shiomi（

Identifying the heterogeneity of ground state pluripotency in mouseembryonic stem cells and elucidating its regulatory mechanism
Mouse embryonic stem cells (ESCs) maintain their pluripotent states in serum-free medium
supplemented with Mek, Gsk3 inhibitors and LIF (2i/LIF), which is called a ground state
culture condition. Core pluripotency transcription factors (TFs) known to fluctuate underserum/LIF such as Nanog are homogeneously expressed under 2i/LIF, implicating this groundstate pluripotency is static in nature. However, recent reports of single-cell transcriptome
analyses revealed the existence of heterogeneously expressed gene modules in the groundstate, with the significance of this remains elusive. Here we report heterogeneity of groundstate ESCs correlating with distinct differentiation potencies. A gene trap vector using Venus
as a reporter was randomly inserted genome-wide and clones showing heterogeneous Venus
expression were isolated. By sorting Venus-positive and -negative ESCs in each clone and inducing differentiation, we identified a clone showing different differentiation potencies between
Venus-positive and -negative ESCs. In this clone, a noncoding gene with no known function wastrapped. When we generated chimeric mice, we observed higher chimerism in Venus-negative
ESCs compared to Venus-positive, indicating heterogeneity of ESC pluripotency in the groundstate. To elucidate the mechanism regulating this heterogeneity, we conducted single-cell
ATAC-seq and identified TF-binding motifs differentially accessible between Venus-positiveand -negative ESCs. We disrupted these TFs by CRISPR/Cas9 and identified TFs regulating
this heterogeneity. Our results show that the ground state pluripotency is not static in nature(COI:No)but is dynamically fluctuating.

2P-140Glutamatergic and GABAergic populations of prostaglandin EP3
receptor-expressing preoptic neurons are heat-responsive
Yoshiko Nakamura（ Dept Integrative Physiol, Nagoya Univ Grad Sch Med, Japan）
The thermoregulatory center in the preoptic area (POA) receives thermosensory inputs
from peripheral thermosensors to control body temperature. The POA also contains neurons
expressing the EP3 subtype of prostaglandin E receptors, which receives the pyrogenic
mediator, prostaglandin E2 to trigger fever. However it is unknown whether EP3 receptorexpressing neurons in the POA contribute to basal body temperature regulation. In this study,
we examined the responsiveness of EP3 receptor-expressing POA neurons to environmental
temperatures and the neurotransmitters contained by these neurons by performing histological analyses combining fluorescent immunohistochemistry and in situ hybridization in rats
exposed to 36, 24 or 4℃ for 2 hours. Analyzing confocal images showed that EP3 receptorimmunoreactive POA neurons consisted of a large population expressing the glutamatergic
marker, VGLUT2 mRNA and a smaller population expressing the GABAergic marker, GAD67
mRNA. Heat exposure (36℃) significantly increased expression of c-Fos, a marker for neuronal
activation, in both glutamatergic and GABAergic populations of EP3 receptor-expressing
POA neurons, compared with control exposure (24℃). In contrast, cold exposure (4℃) did
not increase c-Fos expression in EP3 receptor-expressing POA neurons. These observations
suggest that EP3 receptor-expressing POA neurons contribute to heat-defensive control ofbody temperature under hot environments. Further studies are required to elucidate the rolesof the glutamatergic and GABAergic populations of EP3 receptor-expressing POA neurons in
the central circuit mechanisms for thermoregulation and fever.
(COI:No)

Sleep duration and melatonin secretion in preschool children in different
regions in JapanMed Univ, Japan, 2 Institutional Research, Sch Med, Aichi Med Univ, Japan,

3 Pathophysiology, Sch Med, Poznan Med Univ, Poland, 4 Sleep Disorders Center, Aichi Med

Univ, Japan）

Adequate sleep duration is an essential factor for general health, including cardiovascular,
metabolic and mental health, immunologic function, developmental health, and human performance. Amount of sleep for children is decreasing in past decades. According to recommendations of the Academy of Sleep Medicine (2016) for the pediatric population (children 3 to 5 yearsof age), the amount of sleep should be 10 to 13 hours. Shortage of sleep durations induces theconsequences of the children growth problems due to inadequate hormonal secretion. Recently,
the use of smartphone and tablet has increased among children, and in childcare. Blue lightfrom smartphone and tablet has a disruptive effect for melatonin secretion and sleep quality
at night.
We investigated the melatonin secretion in preschool children in different regions in Japan.
Total number of twenty-six children of age 3-6 years old from two regions of urban and rural
areas participated in this study. Saliva samples for melatonin were collected three times a day(at 20:00 h, 7:00 h and 12:00 h). The smartphone usage and sleep duration for 2 weeks wereasked by questionnaire and sleep daily. In the results, smartphone usage were 58.3% of children
in urban and 35.7% of children in rural areas in this study. The sleep duration of children in age
5-6 years old was shorter in urban than in rural area. However, melatonin concentration was
not different between the two areas. These data suggested that children in urban area might
be exposed to blue light more than that in rural area, indicating the LED blue light at night(COI:No)should be limited duration particular in young children.

2P-143

Emotional behaviors of next-generation offspring mice would be
affected by paternal stress just before mating
Noriyuki Shimizu, Sachiko Chikahisa, Tetsuya Shiuchi, Daisuke Tanioka, Hiroyoshi Sei（Dept Integ Physiol, Inst of Biomed Sci, Grad Sch Univ of Tokushima, Japan）

Emotional behaviors including anxiety, depression and fear are influenced by psychologicalstress. However, these mechanisms are still unclear. In recent study, we found a possibility
that paternal psychological stress affects emotional behaviors in next generation. On the basisof this result, we hypothesized that paternal stress just before mating would affect a formationof offspring’s emotional behaviors.
To make a paternal stress, we used a dexamethasone (glucocorticoid receptor agonist). Male
C57/BL6 mice (aged 8-9 weeks) were subjected to i.p. injection of dexamethasone (200 µg/kg). Administration of dexamethasone was continued for 2 weeks, and then each male mouse
was mated with same aged virgin female for 2 days. Pregnancy was confirmed in vaginal
plug formation. When the offspring have reached 10 weeks of age, we performed a behavioral
analysis to evaluate emotional behaviors.
Prenatally dexamethasone-treated offspring mice showed enhancement of anxiety, depressionlike behavior and a strong fear response to electrical stimulation compared with vehicle-treatedcontrol mice. Furthermore, when paternal mice had recovery period for 2 weeks after last
administration of dexamethasone before mating, their offspring mice showed normal emotional
behavior, which was similar to vehicle-treated control mice.
Collectively, these results suggest a possibility that enhancement of paternal stress before
mating would affect a function of offspring’s emotional behaviors.
(COI:No)

2P-141

2P-144

Mitsugu Yoneda1，Yui Kikuchi1，Tsukika Noda1，Kyoko Nishikawa1，Honoka Hasegawa1，
Tomoaki Fujisaku1，Hayate Tanigami1，Masataka Nakagawa1，Takashi Yoneda2，
1 1 Rehab, Health Sci, Kanazawa Univ, Japan, 2 Inst Liberal Arts ＆Takako Ohno-Shosaku（

Shizuka Ikegame, Kenju Miki, Misa Yoshimoto（Dept Physiol, Nara Women’s Univ, Japan）

Effects of environmental enrichment on fecal corticosterone levels andreinforcement learning in mice

Sci, Kanazawa Univ, Japan）

Repeated exposure to stress induces depression-like behavior in mice. Environmental enrichment has been reported to increase BDNF (brain-derived neurotrophic factor) levels in thecortex and hippocampus, and reduce the effect of stress. Previously, we have reported thatorally-administered theobromine can increase BDNF levels and improve motor learning inthree-lever operant task in mice. In this study, we examined effects of environmental enrichment (EE) on fecal corticosterone levels and the lever-pressing behavior in mice. We used
the operant box containing three levers. The mice were trained to press one of active levers
for a food reward (1-lever task). The number of active levers was initially set to three, thendecreased to two, and finally to one, depending on the performance. In the next step, the mice
were trained to press three levers in a given sequence (3-lever task), and then in a reversedorder (reverse 3-lever task). Before starting operant task, mice were housed in large cages(25,000 cm3) supplemented with running wheels, tunnels, huts, and mazes (EE mice) or small
cages (2,200 cm3) (control mice) for 4 weeks. For corticosterone measurements, feces werecollected 1, 10, 20, and 26 days after mice arrived at our laboratory, and once a week after
starting operant task. Compared with control mice, EE mice showed a slightly lower fecal
corticosterone level, smaller number of sessions required for completion of one-lever task,
and some difference in win-stay and lose-shift behavior in the 1-lever task. Our data suggestthat housing conditions (EE or control) can influence corticosterone levels and reinforcement
learning in mice. (COI:No)

Recall of fear memories activates hypothalamic paraventricular
neuronal and sympathetic nerve activity in conscious rats
Recall of fear memories activates the sympathetic nervous system and exerts adverse effects
on cardiovascular function. The hypothalamic paraventricular nucleus (PNV) is involved in
the regulation of sympathetic nerve activity (SNA) and cardiovascular function, suggestingthat the PVN may be involved in SNA and cardiovascular responses when recalling fear
memories. However, the potential contribution of PVN neuronal activity (PVNNA) to the
SNA responses and cardiovascular function when recalling fear memories remains unclear.
The aim of the present study was to investigate functional relationships among PVNNA,
SNA, and cardiovascular function in response to recalling fear memories in rats. Male Wistar
rats were chronically instrumented with multiple electrodes (100-µm stainless steel wires) for
measurement of PVNNA, renal SNA (RSNA), lumber SNA (LSNA), and electroencephalogram,
electromyogram, and electrocardiogram data, and a catheter was used to measure arterialpressure (AP). In the fear conditioning trials, a tone (conditioned stimulus) paired with a briefelectrical shock (0.1 mA, 1 s) was administered to rats twice per day over 2 days. During thecourse of fear conditioning, rats showed freezing behavior in response to only the tone without
foot shock. Upon presentation of the tone, PVNNA, RSNA, LSNA, and AP immediately andsimultaneously increased, while the heart rate increased gradually. These data suggest thatrecalling fear memory evoked by a conditional stimulus could activate PVNNA, resulting insimultaneous increases in RSNA, LSNA, and AP, while PVNNA may not be directly involvedin the heart rate response in conscious rats.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S139

2P-145

2P-148

Kana Yaguchi, Yuzuka Masuda, Kenju Miki, Misa Yoshimoto（Dept autonomic, Nara women’s

4 1 Dept Physiol, Sch
Takuya Matsui1，Chihiro Ito2，Toshiyuki Shibata3，Masataka Itoigawa（

Effects of social defeat stress on hippocampal neuronal andsympathetic nerve activity in conscious rats

Comparative proteomics analysis reveals the effect of phulorotanninfrom Sargassum carpophyllum on activation mast cells

Univ, Japan）

The hippocampus has been implicated in depression symptoms. Chronic social defeat stressis associated with depression, an increase in anxiety-like behavior, and disorders of autonomic
function in rodents. It remains unclear how chronic social defeat stress influences hippocampal
and autonomic function. In the present study, we attempted to measure the time course ofchanges in hippocampal CA1 neuronal activity (CA1NA), renal sympathetic nerve activity(RSNA), and lumber sympathetic nerve activity (LSNA) during social fighting and social defeat
in conscious rats. Wistar rats were chronically implanted with a combined probe consistingof multiple electrodes for measurement of CA1NA, RSNA, LSNA, and electroencephalogram,cervical electromyogram, and diaphragm electromyogram data, and a catheter was used tomeasure arterial pressure (AP). Social defeat was induced by introduction of a Long-Evansrat, which is typically more aggressive and heavier than a Wistar rat, into the Wistar rat’s
home cage for 30 minutes. The intrusion of the Long-Evans rat sometimes resulted in fighting
between the Ling-Evans and Wistar rats. Wistar rats that were defeated displayed a submissive posture and then exhibited immobilization for serval minutes. Immediately after fighting,
CA1NA was suppressed abruptly and in a sustained manner during the immobilized behavior.
However, the AP, heart rate, RSNA, and LSNA were not suppressed during the immobilized
behavior. These findings showed that defeat stress suppressed hippocampal function in a
tonic manner, and RSNA, LSNA, AP, and heart rate responses were dissociated from theCA1NA response, suggesting that these peripheral parameters are not an accurate index of(COI:No)
hippocampal function during defeat stress. Med, Aichi Med Univ, Japan, 2 Faculty Pharm, Meijo Univ, Japan, 3 Grad Sch Bioresources,
Lab Marine Food Chem, Mie Univ, Japan, 4 Sch Sports and Health Sci, Tokai Gakuen Univ）

Mast cells play a crucial role in inflammation such as allergic and fibrotic lesions, and their regulation can lead to inhibition of such pathological conditions. Recently, functional food has garneredmuch attention as a reliever of inflammation. Previously, we reported the isolation of compounds
from plants such as Rutaceae, which is used as a fruit, flavoring agent, and traditional medicine,that could suppress the release of chemical mediators from mast cells. Sargassum carpophyllum
is an edible seaweed, commonly consumed in Japan, and known to contain phlorotannins, which
are phloroglucinol polymers with reported anti-inflammatory activities. In the current study, weinvestigated the anti-allergic effects of phlorotannin.
Rat mast cell lines (RBL-2H3) that were pre-treated with three different phlorotannins were
stimulated with dinitrophenol-human serum albumin (DNP-HSA). The anti-allergic effects of phlorotannin on intra-cellular Ca2+ levels, reactive oxygen species (ROS) levels, and β-hexosaminidase
release in activated mast cells were investigated by spectrofluorometry and enzyme assay. Additionally, to identify the varied intra-cellular proteins affected by phrolotannins, an iTARQ-based
comparative proteomics analysis was performed between phulorotannins-pre-treated cells andcells stimulated DNP-HSA alone using liquid chromatography-mass spectrometry.
Phlorotannin significantly and dose-dependently reduced β-hexosaminidase release (IC50 49 56 µM). At concentration of 40 µM, phlorotannin reduced the ROS levels by ~50% comparedwith those in cells stimulated with DMP-HSA alone and non-stimulated cells, although the intracellular ROS levels did not differ between the DNP-HSA-stimulated and non-stimulated cells. The
intra-cellular Ca2+ levels in phlorotannin-treated cells were marginally reduced after DNP-HSA
stimulation. The proteomics analysis revealed that phulorotannins treatment upregulated theexpression of 27 proteins and down-regulated that of 35 proteins compared with that in cells
stimulated with DNP-HSA alone.
These results demonstrate that phlorotannin reduces ROS levels and β-hexosaminidase release,making it potentially useful for attenuating immediate hypersensitivity.
(COI:No)

2P-146

2P-149Hikaru Fujii, Takashi Nishida, Saeko Kimura, Shigenobu Matsumura, Kazuo Inoue,
Kousaku Ohinata（ Div Food Sci and Biotech, Grad Sch Agr, Kyoto Univ, Japan）

1 1 Dept Neurophysiol, Hamamatsu
Miho Watanabe1，Kristopher Kahle T.2, 3，Atsuo Fukuda（
Univ Sch Med, Hamamatsu, Japan, 2 Dept Neurosurgery, Yale Sch Med, New Haven, CT,
3 Depts of Pediatrics and Cell. and Mol Physiol; Centers for Mendelian Genomics, Yale Sch
Med, New Haven, CT）

Antidepressant-like peptide derived from Rubisco, a major protein fromgreen leaves

Currently, many bioactive peptides have been identified from natural food proteins. We previously reported that bioactive peptides can act on the nervous system and alter emotional
behavior. Rubisco, the key enzyme for carbon dioxide fixation and photorespiration, is known
as the most abundant protein in the entire biosphere. We identified Rubisco anxiolytic-like
peptides (rALPs) from the digest of rubisco based on comprehensive peptide analysis andstructure-activity relationships [1]. In this study, we investigated the antidepressant-like effects
of rALPs.
Antidepressant-like effects were evaluated by the tail suspension test, which can be used toscreen antidepressants, using male mice. We also chemosynthesized and purified peptides by
reversed-phase HPLC. We found that a heptapeptide, named rALP-1(1-7), released from spinach Rubisco gastrointestinal proteases reduced the immobility time after oral administration(0.03 mg/kg). Thus, rALP-1(1-7) exhibits potent antidepressant-like effects that are comparable
or better than those of general known antidepressants after oral administration.
Next, we investigated the mechanism of the antidepressant-like effects of rALP-1(1-7). We
used an antagonist of the serotonin 5-HT1A receptor, which is associated with antidepressantlike behavior. The antidepressant-like activity of rALP-1(1-7) was inhibited by WAY100135,
a 5-HT1A antagonist. The anxiolytic-like effects of rALP-1(1-7) were inhibited by the sameantagonist, suggesting that 5-HT1A mediated both the antidepressant-like and anxiolytic-like
effects of rALP-1(1-7). At present, we are investigating whether other mediators function in
the antidepressant-like effects.
In conclusion, a green leaf protein-derived heptapeptide, rALP-1(1-7), exhibits antidepressantlike effects after oral administration by activating the 5-HT1A receptor.
[1] Kimura S et al., Biochem Biophys Res Commun. 2018;505(4):1050-1056

Developmentally regulated KCC2 phosphorylation is essential for
dynamic GABA-mediated inhibition and survival

Despite its importance for GABA inhibition and involvement in neurodevelopmental disease,
the regulatory mechanisms of the K+/Cl－ cotransporter KCC2 (encoded by SLC12A5) during maturation of the central nervous system are not entirely understood. Here, we applied
quantitative phosphoproteomics to systematically map sites of KCC2 phosphorylation during
CNS development in the mouse. KCC2 phosphorylation at Thr906 and Thr1007, which inhibits
KCC2 activity, underwent dephosphorylation in parallel with the GABA excitatory-inhibitory
sequence in vivo. Knockin mice expressing the homozygous phosphomimetic KCC2 mutations
T906E/T1007E (Kcc2E/E), which prevented the normal developmentally regulated dephosphorylation of these sites, exhibited early postnatal death from respiratory arrest and a marked
absence of cervical spinal neuron respiratory discharges. Kcc2E/E mice also displayed disrupted
lumbar spinal neuron locomotor rhythmogenesis and touch-evoked status epilepticus associated with markedly impaired KCC2-dependent Cl－ extrusion. On the other hand, knockin miceexpressing the homozygous dephosphorylation of KCC2 mutations T906A/T1007A (Kcc2A/A),
which prevented normal phosphorylation of these sites in early developmental period, exhibited reduced anxiety, deficit in social novelty recognition, and reduced startle response. These
data identify a previously unknown phosphorylation-dependent KCC2 regulatory mechanism
during CNS development that is essential for dynamic GABA-mediated inhibition and survival.
(COI:No)(COI:No)

2P-147

2P-150Fatma Zahra Sakhri1, 2，Naoki Adachi1，Zahia Kabouche2，Sakina Zerizer2，
1 1 Dept Physiol, Sch Med, Showa Univ, Japan, 2 Univ des freres
Masataka Sunagawa（Natsuko Iizuka1, 2，Yuri Masaoka1，Masahiro Ida3，Masaki Yoshida4，Akira Yoshikawa1，
Nobuyoshi Koiwa5，Satomi Kubota1, 2，Motoyasu Honma1，Yumika Kokudai1, 2，
1 1 Dept Physiol, Showa Univ, Sch Med,
Sayaka Wada1, 6，Kenjiro Ono2，Masahiko Izumizaki（

Evaluation of antioxidant property, cytotoxicity and toxicity of Cydonia
Oblonga fruit extract
Mentouri, Constantine 1, Lab d’obtention de substances therapeutiques, Constantine-Algeria）

The present investigation was carried out to evaluate the antioxidant activity of Cydonia
Oblonga fruit (CO), and its toxicity. For this purpose, hydroalcoholic extract of this fruit was
prepared (EtOH/H2O; 80/20) and the antioxidant activity was determined by the 2, 2-diphenyl1-picrylhydrazyl (DPPH) radical scavenging assay. In the acute toxicity test, female Wistar
rats received oral administration of CO as 2,000 mg and 6,000 mg/kg Bwt. Mortality, signs of
toxicity, body weights were assessed for 14 days. Blood samples were collected for hematological assessments as well as organs were weighed. To more fully evaluate the toxicity potentialof CO, in vitro cytotoxicity was determined in primary culture of rat cortical cells.
The CO extract had an inhibitory concentration IC50 value of (480.46 ± 3.46) µg/ml for the
DPPH scavenging activity. Neither mortality nor treatment related changes in their behaviour
and external appearance were observed, indicating that the LD50 value of CO is higher than
6,000 mg/kg Bwt. No significant differences were noticed in body and organ weights and inhematological parameters between the control and treated groups. The CO extract did notshow significant cytotoxic effect in the range from 0.1 to 5.0 mg/ml.
The present research allows to conclude that the CO extract can be used as an easily accessible source of natural antioxidants with a potential therapeutic role, which is relatively safe
from toxic effects.
(COI:No)

S140

Declining of olfactory ability and volumes of hippocampus subﬁelds inelderly subjects

Tokyo, Japan, 2 Dep Neulolo, Showa Univ, Univ, Sch Med, Tokyo, Japan, 3 National Hospital
Organization Mito Medical Center, 4 Dept Ophthal, Jikei Med Univ, Tokyo, Japan, 5 Dept of
Health and Sci, Univ Human Arts and Sci, Saitama, Japan, 6 Dept Ophthal, Showa Med
Univ, Tokyo, Japan）

It is now well recognized that impairments in olfaction often provide the first sign of neurodegenerative disorders such as Alzheimer’s disease (AD). In AD, pathological changes, including
accumulation of senile plaques and neurofibrillary tangles, occur first in the parahippocmapus(paraHI), which are key areas for olfaction. Previous our study showed that the decrease of olfactory ability was associated with small paraHI volume, especially that of the left hemispherein healthy elderly subjects. The paraHI is an important relay area projecting hippocampus toretrieval of the memory. In this study, we measured paraHI volume and each hippocampus
subfields volume (subiculum, dentate gyrus, CA1, CA3) to investigate how paraHI volume
associated with each subfield. All 18 elderly subjects underwent magnetic resonance imaging
to measure anatomical brain volume (Freesurfer, Version 6), and subjects were assessed using
tests of olfactory acuity and cognitive function. Multiple linear regression analysis revealed that
olfactory ability was associated with the left paraHI and left dentate gyrus. Subjects with lower
olfactory ability showed small volume of the paraHI and dentate gyrus. Volume of the paraHI
included entorhinal cortex which is the gateway to the dentate gyrus, and also project back to
the orbitofrontal cortex. Volume changes in entorhinal cortex and dentate gyrus may be due topathological changes, and that may be primarily associated with declining of olfactory function(COI:No)
before actual decrease of cognitive function in elderly subjects. The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

2P-151

Brain and autonomic nerve activity preceding spontaneous perceptual
switching of bistable apparent motion
Arata Ichiki, Tamami Nakano（Grad Sch Frontier Biosci, Osaka Univ, Japan）
The neural mechanisms involved in spontaneous perceptual switching of bistable stimuli
remain unclear. A previous study reported that the arousal level increases at perceptual
switching; however, it appears to be associated with the button response. Therefore, thisstudy examined changes in arousal state several seconds preceding spontaneous perceptual
switching. The autonomic nerve activity was measured based on pupil diameter, heart rate,and skin conductance under two conditions of bistable apparent motion: the rivalry condition inwhich perceptual images alternated endogenously and the replay condition in which perceptual
images alternated exogenously. Reduction in pupillary diameter was observed approximately 3
seconds before perceptual switching in the rivalry condition compared with that in the replay
condition, suggesting that the arousal level decreased before spontaneous perceptual switching.
Next, we examined brain activity related to this change in arousal state preceding spontaneous perceptual switching using an electroencephalograph. The theta and delta band powerin the left frontal region and the delta band power in the parieto-occipital region increased
significantly in the rivalry condition compared with that in the replay condition approximately
4 seconds before perceptual switching. Moreover, we observed that the more increase in thedelta power in the left frontal region, the more decrease in the pupillary diameter before
spontaneous perceptual switching. Our study suggests that increases in the power of the slow
frequency band with decreases in the arousal level are involved in triggering spontaneous
perceptual switching of bistable apparent motion.
(COI:No)

2P-154

Effects of voluntary wheel running on conditioned fear learning andextinction learning
Hiroaki Kitagawa, Keita Nakaji, Kenny Daun, Anri Kuroda, Natsu Koyama, Seiji Hitoshi（Dept Physiol, Shiga Univ of Med Sci, Japan）

Voluntary exercise or environmental enrichment have been shown to increase adult hippocampal neurogenesis and improve learning ability. The current experiment investigated the effects
of voluntary wheel running on not only conditioned fear learning but also extinction learning.
Method: C57BL/6 male mice were used for current experiment. The exercise mice werereared in the cage equipped with running wheel and shelter for four weeks, and non-exercise
control mice were reared in normal cage. Pavlovian classical fear conditioning has been widelyused as an experimental paradigm for investigating mechanisms underlying fear memory
formation and extinction. In fear conditioning, a neutral contextual and auditory-cue stimulus(conditioned stimulus; CS) is paired with an aversive foot-shock experience (unconditioned
stimulus; US). After three days of conditioning training, contextual test and cued test were
performed, and the effects of associative learning were evaluated as freezing response. In
extinction training, CS was loaded unpaired with US.
Results: At recent memory one day after conditional learning, freezing time in exercise
mice was significantly longer than non-exercise control mice. But there was no statisticalsignificance in remote memory four week after conditioning between exercise group and nonexercise group. On the other hand, freezing time in exercise group was significantly shorter
than non-exercise control mice at the memory after extinction learning. Taken together, thesefindings indicate that voluntary wheel running enhances both fear conditioning learning andextinction learning. We will discuss about exercise related-neurogenesis in subependymal zone
(SEZ) and subgranular zone in dentate gyrus (SGZ).
(COI:No)

2P-152

2P-1553 1 Sch Med, Kyoto Univ,
Yuki Tamaki1，Ryo Sasaki2，Norihiro Takakuwa2，Tadashi Isa2, （1 1 Dept Physiol, Facult
Atsushi Chiba1，Kazunori Morita2，Ken-ichi Ohio1，Masahiko Inase（

Causal role for integrating expected value in risky choice in macaqueventrolateral prefrontal cortex
Kyoto, Japan, 2 Dept Neurosci, Grad Sch Med, Kyoto Univ, Kyoto, Japan, 3 Institute for the
Advanced Study on Human Biology, Kyoto Univ, Kyoto, Japan）

Recent studies have shown that many brain areas represent positive and/or negative rewardrelated parameters like expected value (EV), reward probability, size and risk etc. However,
little is known about where and how those parameters are integrated to make a decision.
To answer this question, we investigated how macaque monkeys handle their risky choice
when they might get reward with high risk and high return or low risk and low returncondition. We defined the risks as the reward probabilities and the returns as the reward sizes.
We also compared this in the condition with different EVs. Two monkeys were required tochoose one of the two color targets presented simultaneously by a saccade. Each color target
was assigned a certain combination of EV and risk/return. We found that monkeys preferredrisky choice as default mode which is consistent with other previous studies (McCoy and Platt,
Nat Neurosci. 2005; Stopper et al., Neuron 2014). Interestingly, we also found that monkeys
preferred higher risk when EV was relatively small. These results suggest that monkeys madea choice by integrating risk and EV.
We next investigated the causal role of a variety of brain areas in the behavioral choices by
reversible inactivation with microinjection of a GABAA receptor agonist, muscimol. We identified and localized the target area using MRI image with injection of gadolinium in advance.
When muscimol was injected into the bilateral ventrolateral prefrontal cortex (vlPFC) whilemonkeys were performing the task, the sensitivity to risky choice was gradually weakened,
although the sensitivity to EV was nearly unchanged. Interestingly, the interaction between
risk and EV was also weakened. Our results suggest that the integration of risk/return and
EV might be accomplished in vlPFC. We will also discuss about the role of other brain areas(COI:No)
like OFC, ACC and VTA. 

Neuronal activity of the monkey prefrontal cortex in a duration
estimation and production task
Med, Kindai Univ, Osaka-Sayama, Japan, 2 Dept Physiol, Iwate Med Univ）

To investigate whether the same neuronal mechanism works for duration estimation of avisual stimulus and for time production in motor preparation, neuronal activity in prefrontal
cortex (PFC) of the monkey was examined during a duration estimation and production task. A
green square (C1) was presented on the center of the monitor for 0.8, 1.6, or 3.2 sec. Following a
1sec delay period, a red square (C2) was presented on the monitor and kept on until the end of
the trial. The monkey was trained to release the hold key and to press a target button during
the allowed press period that was indicated by C1 duration. When the C1 was presented for
0.8 sec, the subject needed to press the button between 3.2 and 4.8 sec after the start of the C2
presentation. When the C1 duration was 1.6 or 3.2 sec, the allowed press period was 1.6 to 3.2
sec, or 0.8 to 1.6 sec after the C2 onset, respectively. Response times between the C2 onset andthe hold key release were differentially distributed among the short, middle, and long C1 trials.
Of 297 recorded PFC neurons, 70 neurons showed the task-related activity that was probably
involved in interval timing. A group of PFC neurons exhibited gradually increasing activity
during the latter part of long C1 presentation. The activity may function as an accumulator
to estimate C1 duration. Another group of neurons showed build up activity increasing to thestart of the allowed press period. This activity may be related to duration production for the
button press preparation. These results suggest that different groups of PFC neurons are likely
to work for the duration estimation of visual stimulus and for the time production in motor
preparation.
(COI:No)

2P-153

2P-156

Mizuho Gotoh1, 2，Kazuaki Nagasaka1, 3，Mariko Nakata1，Ichiro Takashima1, 2，
1 1 AIST, Tsukuba, Japan, 2 Univ. of Tsukuba, Tsukuba, Japan, 3 Niigata
Shinya Yamamoto（

Makoto Wada1, 2，Masakazu Ide1，Kouji Takano3，Yoshitake Sano4，Yo Shinoda5，
7 1 Dev Disorders Sect, Dept Brain Rehab, Res Inst of
Teiichi Furuichi4，Kenji Kansaku3, 6, （Effects of cortical temperature on neural excitatory/inhibitory balance
Univ. of Health and Welfare, Niigata, Japan）

Changes in brain temperature affect various brain functions. However, little is known about
how temperature affects neural information processing when activities of individual inputs mediated by different neurotransmitters are integrated. Herein, we examined whether and how
local brain temperature affects post-synaptic potentials in anesthetized rats. Evoked potentials
triggered by electrical stimulation to the midbrain dopamine area (VTA/SNc) were recordedin the frontal cortex while local cortical temperature was manipulated. Peak amplitudes of
evoked potentials decreased/increased as temperature increased/decreased at >～16℃; thatis, local temperature and amplitude were negatively correlated. This was inconsistent with thetraditional view that brain cooling inactivates neural activity. We then found that glutamate
receptor antagonists (NBQX and (R)-CPP) reduced the amplitude while it remained negatively
correlated with temperature; dopamine receptor antagonists (SCH-23390 and raclopride) did
not affect the amplitude. Interestingly, GABA receptor antagonists (gabazine) increased theamplitude, which was positively correlated with temperature in a high concentration of gabazine. These results suggest that the excitatory (glutamatergic) and inhibitory (GABAergic)
input (E/I) balance is temperature-dependent, with inhibitory inputs dominating at higher temperatures and excitatory inputs dominating at lower temperatures. Previous studies showedthat brain temperature changes dynamically within ranges of >1℃, and even that 1℃ changes
can alter neural activity. Our results thus indicate that brain temperature potentially mediates
neural information processing through E/I balance adjustment.
(COI:No)

Correlational analysis of c-Fos expression during rubber tail task in mice
NRCD, 2 Dept Informatics, Shizuoka Univ, 3 Sys Neurosci Sect, Dept Brain Rehab, Res Instof NRCD, 4 Dept App Biol Sci, Fac Sci and Tech, Tokyo Univ of Sci, 5 Dept Environ Health,
Sch Pharm, Tokyo Univ of Pharm and Life Sci, 6 Cent for Neurosci and Biomed Eng, The
Univ of Electro-Communications, 7 Dept Physiol & Biol Info, Dokkyo Med Univ Sch of Med）
We previously found that mice responded as if their own tails were being touched when therubber tails were grasped after synchronous strokes to their tails and rubber tails (Rubber tailtask; Wada et al., 2016), and we also found that the response was weakened in the Caps2-KO
mice, autism spectrum disorder model mice (Wada et al., 2019). Moreover, we found that
c-Fos positive cell densities were significantly lower in the posterior parietal cortex (PPC) andprimary somatosensory cortex (S1) in the Caps2 KO mice, compared to WT mice that received
the same synchronous visuotactile stroking (Wada et al., 2019). In this study, we investigatedthe correlational relationship of c-Fos expression between brain regions during the task.
After data matrix of c-Fos positive cell density were combined among the Caps2 KO mice (n=8) and wild type (n= 6) mice. Correlations with seed regions where significant difference wasobserved between the groups were calculated at each data point.
When the S1 was set as the seed region, we found that c-Fos positive cell densities in the motor
cortex, PPC, auditory and visual areas were significantly correlated (P<0.05, uncorrected). In
contrast, when the PPC was set as the seed region, we found that c-Fos positive cell densities
in the insular cortex, motor cortex, S1, retrosplenial cortex and hippocampus were significantly
correlated (P<0.05, uncorrected). In addition, c-Fos positive cell densities in these regions weregenerally low in the KO mice.
The results suggest that functional connections among the sensory areas exist, while functional
connections between the posterior parietal cortex and limbic system existed during the synchronous visuotactile stimulation. And they were partly overlapped.
We speculate that the functional connections are related to the multisensory integrations and(COI:No)the change of body ownership after the integrations, respectively. 

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S141

2P-157

2P-160Tsuyoshi Setogawa1, 2，Takashi Mizuhiki1, 2，Narihisa Matsumoto3，Barry Richmond J.4，
2 1 Faculty of Medicine, Univ. of Tsukuba, 2 Grad Sch. of
Munetaka Shidara1, （Univ, Nara, Japan）

The role of monkey orbitofrontal cortex in reward value computation in
cost-beneﬁt-based decision-making
Comprehensive Human Sci. Univ. of Tsukuba, 3 Human Informat. Res. Inst., AIST, 4 Lab.
Neuropsychol., NIMH/NIH ）When faced with having to choose one from some alternatives, animals, including humans,
will normally choose more valuable options than less valuable ones. Previous studies havereported that neuronal activities in orbitofrontal cortex (OFC) are related to the subjective
values of offered options. Here we studied whether 1) neurons in OFC encode the differencein value between offered options, and 2) there is a causal link between OFC neuronal activity
and choice.
Two monkeys were trained to perform a reward schedule task which consists of 1, 2 or 4
trials of visual discrimination to earn 1, 2 or 4 drops of liquid reward. After learning this task, adecision-making schedule task in which two kinds of choice target (CT) were sequentially presented was introduced. The CT brightness and length indicated reward amount and required
number of trials, respectively. Then, these two CTs were simultaneously reappeared. The
monkey could choose one of them, and then the chosen reward schedule started.
We recorded 256 single neurons from OFC (Monkey P: 137, Monkey H: 119). For 56/256(21.9%) of the neurons, the neuronal firing during the second target presentation period wassignificantly correlated with the subtraction between the first CT value and the second CT
value, suggesting that these neuronal responses encode the difference in value between two
CTs. To examine the causal relationship between these neural activities and choice, we
injected muscimol into a small regions of OFC rich in neurons coding for choice-related values.
Inactivating this tissue led the monkeys to choose slower and more likely to choose the lessvaluable alternative, when the difference in value was small. These results suggest that OFC
neurons code for value information that could be used to guide choices, and these signals have
(COI:No)a direct influence on the choice.

Effect of systemic acyl ghrelin administration on thermoregulatory
behavior in rats in the cold
Yuki Uchida, Chinami Tsunekawa, Izumi Sato（Fac of Hum Life and Env, Nara Women’s
Introduction: Ghrelin is known to be a peptide hormone to increase appetite; however, recent
reports suggested another physiological effects. There are two types of ghrelin (acyl and desacyl ghrelin). Des-acyl ghrelin facilities thermoregulatory behavior in rats in the cold (Uchida
et al., 2018, Brain Res); however, the effect of acyl ghrelin on the response is unknown yet. We
investigated the effect of acyl ghrelin on thermoregulatory behavior in rats in the cold.
Methods: Male Wistar rats received an i.p. saline or 24μg acyl ghrelin injection, then exposed
to 27℃ or 15℃ for 2-h with continuous body temperature, tail skin temperature, and thermoregulatory behavior (tail-hiding behavior) measurements.
Results: At 15℃, body temperature and the duration of thermoregulatory behavior were notdifferent between the acyl ghrelin and control groups; however, tail skin temperature in theacyl ghrelin group was greater than that in control group.
Conclusion: These results indicated that acyl ghrelin might not affect thermoregulatory
behavior; however, it affected tail skin temperature in the cold.
(COI:No)

2P-158

2P-161

Kei Mochizuki, Akira Murata, Masahiko Inase（Dept Physiol, Facult Med, Kindai Univ,

Toshiaki Yasuo, Takeshi Suwabe, Noritaka Sako（Dept Oral Physiol, Asahi Univ Sch Dent,

It is known that human has a cognitive bias to underrate the temporal interval between one’s
own action and its consequence. For instance, we usually perceive good synchrony between
key presses and the appearance of characters on the computer even in the presence of inevitable mechanoelectrical delays. This bias in time perception (“intentional binding”) is thought
to arise from subject’s involuntary tendency to temporally associate (“bind”) own action to its
consequence, and believed to be a charactaristic nature of the sense of agency.
To investigate the neurophysiological mechanism of this biased time perception, we establisheda new behavioral paradigm to study it in non-human primates. The monkey was trained tojudge the delay between two auditory stimuli as short or long compared to a predeterminedboundary. Stimuli were presented either passively or as a result of the monkey’s spontaneous
button press, in order to assess how precedent voluntary action would influence the perceived
temporal delay between the stimuli. The delay was varied from 100 to 600 ms and changedin each trial.
As a result, the monkey judged longer delays as “short” when timing was spontaneously
initiated. This indicates that the monkey tended to perceive the elapsed time shorter in trials
with self-initiated action. Furthermore, among different sets of delay lengths tested on separate
experimental sessions, this tendency was observed only in a limited set and range of potential
time delays. This suggests that the increase of “short” response in self-initiated trials was nota mere byproduct of the difference of task demands, and that the attribution of outer eventto one’s own action is strictly sensitive to the absolute temporal discrepancy on the order oftens of milliseconds. Our new task paradigm enables future investigations of biological underpinnings of the sense of agency by neurophysiological experiments in non-human primates.
(COI:No)When annimals lack a required nutrient, they must detect it in their surrounding environment
in order to ingest it. Vitamins are also essential nutrients. Most must be obtained from thediet because they cannot be synthesized in adequate quantities by an animals. Otherwise,
the deficiency syndrome may occur. Vitamin C (VC) deficiency, called scurvy, causes bodyweight loss, anorexia, reduction of taste nerve responses (Yasuo et al., 2019), weakness, fatigue,
depression, impaired wound healing, dysbasia, dental caries, dysfunctional dentine formation,
gingivitis, and dry mouth. It is therefore important to understand how VC intake is controlled
in annimals.
To investigate the mechanisms of ingesting VC, we conducted behavioral experiments using
osteogenic disorder Shionogi/Shi Jcl- od/od (od/od) rats, which lack the ability to synthesize
VC, and their wild-type controls osteogenic disorder Shionogi/Shi Jcl- +/+ (+/+) rats.
In the previous study, we demonstrated that VC-deficient these rats exhibited an increased
consumption of VC solution and decreased consumption of water relative to normal rats in
48-hour two-bottle choice test.
However, it remains unclear whether VC deficiency-induced body weight loss is linked to VC
intake or not.
To determine whether body weight loss influences VC intake in VC-deficient od/od rats or not.
We found that the VC-deficient rats showed an increase in VC intake and a decrease in water
intake; in contrast, BWC rats showed a decrease in water intake but no change in VC intake.
These data suggest that the change in VC intake displayed by VC-deficient rats cannot be(COI:No)
accounted for by changes in body weight.

2P-159

2P-162

Akira Terao1，Takeshi Yoneshiro2，Ryuji Kaede2，Kazuki Nagaya2，Manami Saito2，
2 1 School of
Juria Aoyama2，Elfeky Mohamed2，Yuko Okamatsu-Ogura2，Kazuhiro Kimura（

Ming-Liang Lee, Chitoku Toda, Kazuhiro Kimura（ Dept Biochem, Grad Sch Vet Med,

Perceptual bias for elapsed time caused by a self-initiated action inprimates
Osaka-Sayama, Japan）

Brown adipose tissue is involved in antiobesity effects of Melinjo(Gnetum gnemon L.) seed extract in high fat diet-fed mice
Biological Sci, Tokai Univ, Japan, 2 Dept Biochem, Grad Sch Vet Med, Hokkaido Univ, Japan）

Dietary supplementation of Melinjo seed extract (MSE) has been proposed as an anti-obesity
agent. However, the mechanism in which MSE modurates energy balance is unclear. In thisstudy, we investivated the effects of dietary MSE on energy intake, physical activity, andbrown (BAT) and white adipose tissue (WAT) thermogenesis in high fat diet (HFD)-fed mice.
Twenty-four C57BL/6J male mice were provided with the different diets for 17 weeks: thenormal diet (ND), HFD, and HFD added with 1% MSE. Food intake, spontaneous locomotor
activitiy, body composition, hepatic trigriceride (TG) content, and blood parameters were
examined. Expression levels of thermogenic molecules and inflammation markers in BATand WAT were examined by using quantitative PCR and Western blotting analysis. HFD
increased BAT and WAT weights and adiposity index compared with ND; however, HFDinduced gains in fat pad weight were decreased by dietary MSE. Dietary MSE did not affectenergy intake or spontaneous locomotor activity, but significantly suppressed HFD-induced fat
accumulation, hyperglycemia, and hyperinsulinemia. Homeostasis model assessment of insulin
resistance score and hepatic TG content were both lower in the MSE-supplemented HFD-fed
group than in the HFD-fed group, indicating reduced insulin resistance and a less fatty liver.
Dietary MSE upregulated thermogenic uncoupling protein 1 (UCP1) and mitochondrial marker
cytochrome c oxidase subunit IV protein expression in BAT; this was closely associatedwith sirtuin 1 mRNA induction. Obesity is closely associated with chronic inflammation in
adipose tissue, accompanied by increased proinflammatory and decreased anti-inflammatory
cytokine levels. mRNAs of adipose inflammatory markers, such as monocyte chemotactic 1
and interleukin-1, were induced by HFD but suppressed by MSE. Supplemental MSE activates
BAT thermogenesis through the Sirt1-mediated mechanisms and improves obesity-associatedadipose inflammation, hepatic steatosis, and insulin resistance. The MSE regimen would beapplicable to the interventions aimed at activating BAT and preventing metabolic disorders.
(COI:Properly Declared)

S142

The inﬂuence of body weight loss caused by vitamin C deﬁciency on
the ingestive behavior in rats
Japan）

In vivo labelling glucose excited neurons in ventromedial hypothalamus
unveils its role in the regulation of systemic glucose metabolism
Hokkaido Univ, Japan）

Hypothalamus plays a critical role in regulating whole body glucose metabolism. Glucose
excited (GE) neurons, one of the glucose sensing neurons, are excited by hyperglycemia
and believed to reduce blood glucose level by arising glucose uptake of skeletal muscle andadipose tissue and inhibiting liver gluconeogenesis. However, due to a lack of biomarker for
GE neurons, it is still a challenge to label and study actual role of these neurons in vivo.
Here, we developed a method to label hypothalamic GE neurons in vivo by a technique called
targeted recombination in active populations (TRAP), in which the tamoxifen-dependent Cre
recombinase will be expressed by the neuronal activity-dependent promoter. We intraperitoneally injected large amount of glucose to activate GE neurons and injected 4-hydroxytamoxifen
to trigger expression of a reporter protein in activated neurons. The neurons activated by
glucose were therefore labeled and called TRAPed. The ex vivo calcium imaging experiments
revealed these TRAPed neurons in the hypothalamus had higher neuronal activity under
2.5mM of glucose and lower activity under 0.2 mM glucose. To interrogate the physiological
functions of the TRAPed neurons, we selectively ablated TRAPed neurons in the ventromedial
hypothalamus (VMH) by AAV-delivered caspase3. Mice with ablated TRAPed neurons are
glucose intolerant and insulin resistant compared to control. In line with this, chemogeneticalactivation of the VMH TRAPed neurons improved glucose metabolism. Thus, we successfully
demonstrated the novel and reliable method to selectively label and manipulate GE neurons(COI:No)and that the GE neurons in the VMH regulates systemic glucose metabolism. 

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

2P-163

2P-166

Manami Oya, Kazuhiro Nakamura（ Dept Integrative Physiol, Grad Sch Med, Nagoya Univ,
Japan）

Keiko Morimoto, Yuho Yamauchi, Yuri Mizukami, Haruka Nakayama, Kyoko Ueshima,
Akira Takamata（ Dept Environ Health, Hum Life Environ Sci, Nara Women’s Univ, Japan）

Obesity is often developed with age due to attenuation of energy expenditure including metabolic thermogenesis. However, the mechanism of age-dependent attenuation of whole-bodymetabolism is unknown. In this study, we focused on age-dependent alteration of the central
neural circuit controlling metabolic thermogenesis in brown adipose tissue (BAT). Our in vivo
physiological experiments revealed that skin cooling-induced BAT thermogenesis was attenuated in older (6 months old) male rats compared with younger (9 weeks old) ones. Neurons
in the dorsomedial hypothalamus (DMH) are known to mediate thermogenic sympathetic
outflow to BAT and express melanocortin-4 receptors (MC4Rs), which play essential roles in
the regulation of appetite and energy homeostasis for anti-obesity. Thus, we hypothesized thatmelanocortin signaling mediated by MC4Rs in the DMH is altered in older animals. Supporting
this hypothesis, nanoinjection of melanotan-2 (MT-2), an MC4R agonist, into the DMH induced
blunted BAT thermogenesis in older rats, compared to younger rats. We conducted qPCR
analysis and found that the MC4R mRNA level in the DMH was comparable between older andyounger rats. To elucidate the mechanism of the age-dependent attenuation of MC4R sensitivity to melanocortin signals, we produced an anti-MC4R antibody. Using immunohistochemistry,
we have successfully confirmed the specificity of the anti-MC4R antibody in cultured cells
and brain slices. We are currently studying the mechanism of age-dependent attenuation of
MC4R signaling by analyzing age-dependent alteration of the subcellular distribution of MC4R
(COI:No)
proteins with this antibody.

Energy intake varies across the menstrual cycle in premenopausal women. Recent evidencessuggest that oral detections of fatty acids released from dietary triacylglycerol is involved in
fat intake. We examined whether oral sensitivity to fatty acid changes depending on menstrual
cycle and affects fat intake in young healthy women. Subjects (n=21) underwent the experiments at 4 phases of menstrual, preovulatory, mid-luteal, and late luteal phases during themenstrual cycle. The oral sensitivity to fatty acid was examined using a three-alternative,
forced-choice methodology for oleic acid. For the fat preference test, soups containing 4 different concentrations of canola oil were used for free-choice of the most preferred oil concentration. Furthermore, buccal mucosa was sampled to analyze the mRNA levels of the fatty acid,and estrogen receptors. In addition, they ate the laboratory-prepared lunch consisting of breadwith butter and soup in the experimental day.
Oral detection threshold for oleic acid was significantly lower in the preovulatory phase thanthat in the menstrual phase or late luteal phase. However, there were no differences in fat
preference and fat intake from the lunch among these four phases. In contrast, linear mixedeffect model analysis showed negative correlation between the oral fatty acid sensitivity andthe fat intake. RT-PCR revealed that CD36 mRNA level in buccal mucosa changed dependingon the menstrual cycle.
This study suggests that the oral fatty acid sensitivity changes depending on the menstrual
cycle, and affects fat intake through oral CD36 mRNA level in young healthy women.(COI:No)

2P-164

2P-167

Kan Kato, Kazuhiro Kimura, Chitoku Toda（ Laboratory of Biochemistry, Department of
Veterinary Medicine, Hokkaido University.）

Magdeline Carrasco 1，Ryoko Higa1，Kenshiro Shikano1，Ryohei Umeda1，Masanori Inoue2, 3，
1 1 Dept Neurophysiol, Fac
Kyoko Kiyota 3，Toshikatsu Hanada2，Kenji Ihara3，Reiko Hanada（

Production of anti-melanocortin-4 receptor antibodies to elucidate themechanism of age-dependent obesity

Impaired systemic glucose metabolism is a delayed onset response ofsocial defeat stress in mice

Effects of oral fatty acid sensitivity depending on menstrual cycle on fat
intake in young women

Molecular mechanism of APPL2 on NAFLD/NASH pathogenesis inzebraﬁsh

Depression is a neuronal disorder which influences systemic glucose metabolism, and thus
known as a risk factor of the type 2 diabetes mellitus in human. In mice, the chronic social
defeat stress (CSDS) is used to promote depressive symptoms, such as the social avoidance
and anxiety. Moreover, CSDS increases food intake, promotes obesity and deteriorates glucose
tolerance and insulin sensitivity. However, the effect of CSDS on glucose metabolism is still
controversial. To clarify the effect of CSDS on systemic glucose metabolism, we examined
glucose tolerance test (GTT) in the middle of and after CSDS.
C57BL/6J mice were introduced to the cage housed male ICR mice for 10 minutes to have aphysical interaction and kept in the same cage but separated from ICR mice by a wire nettingfor 24 hours. The same physical and psychological stress were applied to the C57BL micefor 10 days (day1-10). Open field test was done at two days after the end of CSDS (day12) toevaluate the depression. Food intake, body weight and glucose tolerance test were measured
during and after the CSDS (day0-19).
Food intake and body weight were not changed in both CSDS and control non-CSDS micethroughout the experiment (day0-19), even though CSDS increased anxiety-like behavior in the
open-field test compared to control mice at day12. Blood glucose levels were also not different
between groups at day 10. However, the CSDS mice impaired glucose tolerance in GTT
compared to control at day17.
Our data suggest that the abnormality of glucose metabolism by CSDS appears after theremoval of the stress, which is not seen in the middle of the stress exposure. Thus, the CSDS
is an unique stress protocol that affects systemic glucose metabolism with a delayed onset.
(COI:No)

Med, Oita Univ, Japan, 2 Dept Cell Biol, Fac Med, Oita Univ, Japan, 3 Dept Pediat, Fac Med,
Oita Univ, Japan）

2P-165

2P-168

Shinya Aoyama1, 2，Shuichi Kojima2，Keisuke Sasaki2，Takeru Shimoda2，Kengo Takahashi2，
2 1 Dept Neuro Behavi,
Rina Hirooka2，Yu Tahara2，Kazuyuki Shinohara1，Shigenobu Shibata（Emi Nakamura-Maruyama, Naoyuki Himi, Kazuhiko Narita, Risa Kai, Naohiko Okabe,
Osamu Miyamoto（Dept Physiol 2, Kawasaki Med Sch, Japan）

Effects of day-time feeding on murine skeletal muscle growth andsynthesis
Grad Med Sci, Nagasaki Univ, Japan, 2 Faculty of Sci and Eng, Waseda Univ, Japan）

Muscle mass is controlled by the balance between muscle synthesis and degradation. Althoughnutrition is important for the maintenance of muscle mass and growth, the effects of feedingtime have remained unclear. In the present study, we aimed to evaluate the effects of day- ornight-time-restricted feeding on the muscle volume using unilateral hypertrophy mouse models.
The day- and night-time-restricted feeding was conducted from zeitgeber time 2 (ZT2) to ZT10
and ZT14 to ZT22, respectively. One week after time-restricted feeding, muscle hypertrophy(overloading) of the plantaris muscle was induced by unilateral surgical ablation of the distal
tendons of the gastrocnemius and soleus muscles. Three or seven days after induction ofmuscle hypertrophy, plantaris muscle were collected at ZT6. The increase in muscle weight
was significantly attenuated by the day-time-restricted feeding. The muscle protein synthesis,
which was measured using SUnSET, was significantly increased on day 3 after overloadingin the NRF group but not in the DRF group. However, on day 7 after overloading, the muscle
protein synthesis was increased in the overloaded muscles of both groups, and there wasno significant difference between the groups. Similar response was observed in the level ofphosphorylated S6 protein, but not total S6 protein. The phosphorylation of S6 was increasedby overloading, with the increase being less in the DRF group than in the NRF group. These
results suggest that day-time feeding attenuated muscle growth via the inhibition of muscle
synthesis. Feeding at an irregular time such as a late-night meal could be detrimental formuscle growth.
(COI:No)

Currently, obesity and its related comorbidities, such as fatty liver disease, are being considered as an important public health problem because they account for the major mortality
causes nowadays. In Oita University Hospital, a case of family members was found presenting
an obese phenotype. A transcriptome analysis was performed to evaluate which genes could
be related to this phenotype, then it was found a point mutation in the gene coding for theadaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 2 (APPL2).
This mutation presented a change of cytosine for thymine in the position 1383 (C1383T).
APPL2 and its homologous APPL1 are adaptor proteins that interact with the intracellularregion of adiponectin receptors and mediate the mechanisms of adiponectin effect in glucose
and fatty acids metabolism. Previous studies report a tight association between APPL2 andNon-Alcoholic Fatty Liver Disease (NAFLD), however, the molecular and physiological mechanisms are not yet elucidated. In this study, to reveal the pathophysiological role of APPL2 in
NAFLD/NASH and obesity; we have established an APPL2 mutant zebrafish (Danio rerio).
Elucidation of APPL2 molecular and physiological mechanisms in NAFLD/NASH and obesity
entails a deeper understanding of these pathologies and their development which could allow(COI:No)for potential diagnostic and therapeutic approaches.

Alteration of Ca2+ release via ryanodine receptors in the hippocampus
of depression-like model mice

Although depressive disorders are common diseases, their pathogenic mechanisms are notfully understood. We have previously reported the changes in the expression of ryanodine
receptors (RyRs) in the hippocampus using depression-like model mice. In this study, in order
to investigate the functional changes of RyRs in depressive condition, we examined the alteration of Ca2+ release via RyRs.
We used the depression-like model mice (C57BL/6J, male, 11-12w) subjected to water immersion with restraint stress for 2 weeks. The hippocampal slices (350 µm) of the mice were madeby using a vibratome, the fluorescence intensity changes of pyramidal cells in the dentate
gyrus were measured as the amount of Ca2+ release by introducing Ca2+ ion indicators (Oregon Green 488 dye). Similarly, Ca2+ release in the hippocampal slices of the mice which weretreated with electro convulsive shock (ECS) was also measured. Induction of Ca2+ release via
RyRs was performed by flushing 200μl of caffeine (40mg/ml) into the recording chamber (final
concentration about 4mM). Concomitantly, actions of caffeine except for RyRs agonist werelimited by bath application of selective A1 and A2A adenosine receptor competitive antagonist,
8-cyclopentyl-1, 3-dipropylxanthine (DPCPX) and SCH58261, respectively.
As a result, the number of cells that showed caffeine-induced Ca2+ release decreased in the
hippocampal dentate gyrus of the model mice, and the incidence of caffeine-induced oscillation
also decreased compared with normal mice. On the other hand, these values of the model mice
returned to the level of normal mice by ECS.
These results suggest that the ability of Ca2+ release via RyRs decreased at the depressive
condition and returned to the normal level according to the recovery from it. Ryanodine receptors might be a novel therapeutic target for depressive disorders. (COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S143

2P-169

2P-172

Naofumi Miwa, Hiromasa Satoh, Ryohei Saito, Mayu Hanaue（Dept.Physiol., Saitama Med.

Chihpin Yang, Kana Sugimoto, Yukie Murata, Rina Gono, Yuichiro Hirata, Yohei Miyashita,
Kentaro Nakama, Kazuma Higashisaka, Kazuo Harada, Ryuichi Katada, Hiroshi Matsumoto

Identiﬁcation of a novel inhibitory factor for metastasis of mouseovarian tumor cells
Univ., Japan）

Molecular mechanisms of Wischnewski spot development on gastricmucosa in fatal hypothermia: an experimental study in rats

We have previously characterized a novel egg-coating envelope protein that suppresses
fertilization in Xenopus lavis (Hanaue and Miwa Sci Rep 2017). That protein (named dicalcin)
binds to a glycoprotein, a constituent of the egg-coating envelope filament; regulates theorientation pattern of the filaments and the viscoelasticity of the entire envelope, thereby
suppressing sperm-egg interaction. Cancer metastasis is a complex series of cellular processes
involving glycoprotein and oligosaccharides. To contribute to the study of cancer metastasis,
we investigated the action of dicalcin on the metastasis of mouse ovarian tumor cells. Extrinsically applied dicalcin remarkably binds to the cell surface as well as the cytoplasm of OV2944
cells. Pretreatment of OV 2944 cells with extracellularly applied dicalcin inhibited in vitro
invasion using Matrigel chamber in a dose-dependent manner. It also suppressed the bindingof OV2944 cells to Matrigel significantly; however, it unaffected the cell viability assessed by
the MTT assay, which indicated that it suppresses in vitro invasion through its direct binding
to OV2944 cells. Time-lapse imaging analyses discovered that the extracellular presence of
dicalcin inhibited migration of OV2944 cells on the plastic plate. We next examined in vivo
survival of mice that were injected with OV2944 cells. Concurrent injection with dicalcinderived peptide significantly prolonged the survival days of the mice, indicating that dicalcin
inhibited metastasis of OV2944 cells in vivo. In summary, our novel results elucidated the
suppressive action of dicalcin on metastasis of mouse ovarian tumor cells through its binding toOV2944 cells. We believe that our present study will provide insight into molecular machineryof metastasis processes, and may lead to the development of potent bioactive compound that is
capable of inhibiting cancer metastasis. There are no conflicts of interest to declare. (COI:No)（Dept Leg Med, Grad Sch Med, Osaka Univ, Japan）

2P-170

2P-173

Effects of calcium-release activated calcium channel on signaltransduction of mechanical stimulation to mouse synovial cells cultured
in medium containing IL-6
1 1 Dept. of Orthopaedic Surgery, Nara Med.
Yu Okumura1, 2，Keiji Asada1, 3，Miyako Takaki（
Univ., Kashihara, Japan, 2 Dept. of Phys. Ther., Fac. of Human Sci., Osaka Univ. of Human
Sci., Japan, 3 Dept. of Rehab., Fac. of Health Sci., Suzuka Univ. of Med. Sci., Suzuka, Japan）

Numerous dark-brown-colored small spots called “Wischnewski spots” are often observed on
the gastric mucosa in fatal hypothermia, but the molecular mechanisms through which they
develop remain unclear. We hypothesized that hypothermia may activate the secretion of gastric acid and pepsin, leading to the development of the spots. To investigate this, we performedexperiments using organotypic rat gastric tissue slices culture. Stomachs were obtained frommale Wistar rats (8 weeks old) after fasting for 6 hours. Gastric slices of 1-2 mm were takenfrom stomach between cardia and pylorus. The organotypic gastric slices were cultured in
37℃ (control) or 32℃ (cold) incubators. Cold loading for 6 h lowered the extracellular pH in
the culture medium. The mRNA expression of gastrin, which regulates gastric acid secretion,
increased after cold loading for 3 h. Cold loading increased the expression of gastric H+, K+ATPase pumps in the apical canalicular membrane and resulted in dynamic morphologicalchanges in parietal cells. Cold loading resulted in an increased abundance of pepsin C protein
and an elevated mRNA expression of its precursor progastricsin. Collectively, our findings
clarified that cold stress induces acidification by activating gastric H+, K+-ATPase pumps andpromoting pepsin C release through inducing progastricsin expression on the gastric mucosa,
leading to tiny hemorrhages or erosions of the gastric mucosa that manifest as Wischnewski
spots in fatal hypothermia.
(COI:No)

Mechanism of exercise-related sudden death under high temperatureenvironment
Rina Gono（Dept Leg Med, Grad Sch Med, Osaka Univ, Japan）

Purpose: Rheumatoid arthritis (RA) is a chronic inflammatory disease mainly consisting of
inflammation of the joint synovium, and interleukin-6 (IL-6) is involved in these symptoms.
In addition, previous studies have reported that calcium-release activated calcium channel(CRAC) is involved in the inflammatory response of synovial tissue in RA. However, its
mechanism has not been clarified. In the present study, we focused on the contribution of
CRAC to the response of mechanical stimulation (MS) in mouse synovial cells cultured in
medium containing IL-6.
Methods: Mouse synovial cells cultured in a control medium or a medium containing IL-6 wereused in the experiment. Intracellular Ca2+ concentration ([Ca2+] i) of synovial cell was measured
with loaded Fluo-3 AM by adding twice MS by a glass micropipette.
Results: Each MS elicited immediately [Ca2+] i increasing response in the presence of extracellular Ca2+. When the CRAC inhibitor (YM-58483) was treated, the ratio of the second responseto the first one in synovial cells cultured in medium containing IL-6 was significantly smaller
than that cultured in control medium.
Conclusions: The result indicated that CRAC possibly was involved in [Ca2+] i increasing
response to MS in mouse synovial cells cultured in medium containing IL-6.
(COI:No)Tokyo Olympic will open at 2020 summer. Recently, The International Olympic Committeeannounced that it was planning to move the Olympic marathon and race walking events
to Sapporo, the north city of Japan, which will mean significantly lower temperatures forthe athletes during the Olympic Games. Exactly, in the Utstein Osaka Project, 0.7% of outof–hospital cardiac arrest (OHCA) of cardiac origin (222/31, 030) between 2005 and 2012 in
Osaka Prefecture was related to exercise. Exercise increases core body temperature, leading
to a possibility of heat stroke, one of important factors in the sudden death during exercise.
We have showed that heat stress alone induced alteration of electrical rhythms using the heatstroke rat model and rat cardiomyocytes. Therefore, we hypothesized that exercise-induced
heat stroke increases a possibility of onset of arrhythmia. Then, the aim of this study was toclarify the effect of exercise on onset of arrhythmia under high temperature environment. Male
Wistar rats were initially subjected to an incremental speed exercise to evaluate their exercise
capacity. On the following day, they were randomly divided into 4 experimental groups: (i)control, (ii) exercise, (iii) heat, and (iv) exercise and heat. Rats were exercised at 15 m/min
speed for 15-30 min under temperate (22 ℃) or hot (37-40 ℃) environment condition. After
exposure, the rectal temperatures and electrocardiograms were measured under anesthesia
with isoflurane, and mRNA expression of various genes in heart was detected by quantitative
real-time RT-PCR. The rectal temperatures and heart rates were higher in the exercise and/or heat groups than the control group. The exercise and/or heat stress significantly changed
mRNA expression of autonomic nervous system-related genes. Therefore, these finding suggested that exercise-induced sudden death in the condition with high atmosphere temperature(COI:No)
may be mediated by the change of autonomic nervous system mechanisms. 

2P-171

2P-174

Yukie Murata1，Kana Sugimoto1，Chihpin Yang1，Rina Gono1，Teiji Harada1，Yohei Miyashita1，
Kazuma Higashisaka1，Kazuo Harada1，Ryuichi Katada1，Junya Tanaka2，Hiroshi Matsumoto1

Keizo Hiraishi1, 2，Haiyan Xu3，Lin-Hai Kurahara1, 2，Heping Zhang3，Katsuya Hirano2，
1 1 Dept. Physiol., Sch. Med., Fukuoka univ., Japan, 2 Dept. Cardiovasc. Physiol.,Ryuji Inoue（

Role of activated microglia/macrophages in brain edema formation after
brain infarction（ 1 Dept Leg Med, Grad Sch Med, Osaka Univ, Japan, 2 Molecular and Cellular Physiology,
Grad Sch of Med, Ehime Univ, Japan）

Brain edema after brain infarction affects mobility and mortality. Aquaporin-4 (AQP4), a water
channel protein, is expressed mainly in astrocytes and have increased after stroke in animal
experiments. Therefore, AQP4 may be a therapeutic target in ischemic brain edema, but its
expression mechanism is unclear. On the other hand, they have been reported in the animal
stroke model that activated resident microglia (MG) and bone marrow-derived infiltrating macrophages (MPs) accumulate in the ischemic core and stimulate astrocytes in the ischemic coreand penumbra after brain infarction and that IL-1α, TNF, and C1q produced from activatedMG are involved in astrocytes activation. Therefore, we investigated the role of MG/MPs on
the expression of AQP4 in the transient middle cerebral artery occlusion (tMCAO) model rats
and in the primary rat cultured astrocytes.
Brains were isolated at 0.25-7 days postreperfusion (dpr) in the tMCAO model. AQP4 mRNAlevel significantly increased in the penumbra at 3-7 dpr and core at 7 dpr. The change in
AQP4 mRNA expression was well correlated with those of Iba1, IL-1α, TNF, and C1q mRNA.
Furthermore, the rats with high expression of AQP4 mRNA in the ischemic core at 7 dpr had
high expression of IL-1α, but TNF and C1q did not. In the primary rat cultured astrocytes,
MG/MPs-conditioned medium or IL-1α treatment increased AQP4 mRNA expression.
Addition of IL-1 receptor type I antagonist reduced the increase in AQP4 expression. MG“signature” genes such as P2ry12, Olfm13, Gpr34, and Pros1 were enriched in the ischemic
core of AQP4High/ IL-1αHigh rats when compared to AQP4Low/ IL-1αLow rats, while MPs“signature” genes such as Ccr2 and Mybl2 were expressed at low levels. These findings suggest that IL-1α produced by the activated resident MG induces the AQP4 expression in theastrocytes in the ischemic core and penumbra.
(COI:No)

S144

Effects of breast milk-derived probiotics on a mouse model withcolorectal cancer induced by AOM/DSS treatment
Fac. Med., Kagawa univ., Japan, 3 Key Laboratory of Dairy Biotechnology and Engineering,
Ministry of Education, Inner Mongolia Agricultural univ., P.R.China）

Background: In ulcerative colitis (UC), one of inflammatory bowel diseases (IBD), the risk ofcolorectal tumors increases due to repeated remission and relapse of inflammation. Whether
this tumorigenic process involves intestinal flora has not been investigated yet. It has beenreported that the longer period of breastfeeding is correlated with the lower risk of IBD
and inflammatory tumors. In this study, we therefore examined whether breast milk-derived
probiotics have an effect on inflammation-related colon polyp formation.
Method: A colorectal cancer model was prepared by treating 6-week-old C57BL/6NCrSlc
mice with a single i.p. injection of azoxymethane (AOM : 12 mg/kg BW) and subsequent 2
sets of 7-day administration of 2% dextran sulfate sodium (DSS) in drinking water with one
week intermission. Breast milk-derived probiotic Lactobacillus rhamnosus M9 (2 x109 cells/
day/mouse) was administered to these mice by gavage in 2 sets of 7-day administration with
one-week intermission.
Result and summary: Twenty weeks after AOM administration, AOM+DSS group exhibitedan increased number of polyps, accompanied by a significant weight loss, bowel shortening,
increased scores of pathological stool and colon pathology. These changes were significantly
suppressed by M9 treatment. In addition, M9 suppressed the increase of interleukin-6/pSTAT3 and the Akt/NF-κB signaling associated with tumorigenesis. The metagenomic
analysis indicated a marked increase in Bacteroides and Helicobacter hepaticus; a marked
decrease in Eubacterium plexicaudatum in the AOM + DSS group compared with the vehicle
group, and these changes were suppressed by M9 treatment.
These results suggest that breast milk-derived Lactobacillus rhamnosus M9 suppressed inflammatory tumorigenesis by improving the intestinal flora and inhibiting inflammatory signaling incolon.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

2P-175

2P-178

Toshiyuki Saito1, 2，Yui Tsuzuki2，Chisako Kajiwara2，Kurumi Yokoyama1，Minori Shibutani1

Hideki Yamauchi, Shigeru Takemori（Div Phys Fitness, Dept Mol Physiol, The Jikei Univ

Inﬂuence of repeated restraint stress on the brain microglia and gut
microbiota in the rat（ 1Grad Sch Life Sci, Kyoto Sangyo Univ, Japan, 2 Dept Anim Med Sci, Fac Life Sci, Kyoto
Sangyo Univ, Japan）

The hypothalamic-pituitary-adrenal (HPA) axis is the major neuroendocrine system in responseto physical and psychological stressors. It is well-known that repeated or chronic exposure to
stressors may disturb regulation of the HPA axis, leading to neural diseases such as anxiety
and depression. However, patho-physiological mechanism still remains unknown to cause theneuro-endocrine disturbance with the stress response. In this study, we examined a sign ofpro-inflammatory events in the brain and any changes in composition of gut microbiota during
repeated restraint stress. Male Wistar-Kyoto rats (180-220 g) repeatedly received the restraint(2 hours in a day, totally for 14 days). The level of the plasma corticosterone (CORT) increasedon the day 1 and 7, but decreased on the day 14. In the paraventricular nucleus (PVN), there
were observed the Iba-1 immuno-positive cells (microglia) with only several branches. Analysisof 16SrRNA in the feces (sampled on the day 14) shows tendency for decrease in the ratio for
Lactobacillus in Firmicutes by the restraint, though the CORT level declined to that before thestress exposure (day －1). The present repeated restraint may change the gut microbiota withdelayed onset. Further studies need to examine relation between microglial differentiation (proinflammatory or neuro-protective) in the PVN and change in composition of the gut microbiota
by the repeated restraint stress.
(COI:No)

Age-related difference in autophagic adaptation and the effect of
resistance exercise in rat soleus muscle atrophied with unloading
Sch Med, Tokyo, Japan）

Aim: We have reported that unloading selectively affected type I fibers in aged rats disrupting
myofibrils with a decrease in sarcomeric proteins, forming inclusion body, and accumulating
abnormal mitochondria. Here, we aimed to clarify age-related differences in autophagic adaptation and the effects of intermittent resistance-exercise (IRE) in unloaded muscles of rats. IRE
is known to ameliorate sarcopenia.
Methods: Young (4 months, n=21) and aged (2 years, n=21) F344 female rats were randomly
divided into control, unloading, and unloading + IRE groups. Rats of the unloading and unloading + IRE groups have their hindlimbs unloaded by tail-suspension. 10-min IRE was performed
in unloading + IRE group 3 times per day every 4 hours in the dark period for 3 weeks. Soleus
muscle was examined at the end of the intervention.
Results: Unloading-induced atrophy with a degenerative decrease in myofibrillar protein
concentration was more prominently observed in aged than in young rats. Fbx32, a muscle
specific ubiquitin ligase, increased along with an increase in ubiquitinated protein by unloading
in both aged and young rats. LC3-II, an autophagy marker protein, and mitochondrial calcium
uniporter (MCU), a key protein activating mitochondrial biogenesis and of signaling pathways
for muscle hypertrophy, increased with unloading in young rats but decreased in aged rats.
PGC1, playing similar roles to MCU, specifically decreased with unloading in aged than inyoung rats. Intermittent IRE ameliorated atrophy similarly in the rats of both ages, while thelevels of LC3-II, MCU, and PGC1 were still lower than the control level in aged, but not inyoung rats.
Conclusion: Autophagic adaptation and myogenic response were critically different with age
in rat soleus. These differences may be responsible for age-related muscle responsiveness to(COI:No)
unloading and training.

2P-176

2P-179Maki Yamaguchi1，Takuhiro Kawahara1，Kie Iida2，Toshiko Yamazawa1，Nobutake Akiyama3，
1 1 Dept Mol Physiol, Jikei Univ Sch Med, Japan, 2 Dept Immunology, Jikei
Shigeru Takemori（Ryo Ishiwata, Yuji Morimoto（Dept Physiol, National Defense Medical College, Japan）

Effect of homogenate extract from adult skeletal muscles on the
proliferation and differentiation of myoblasts
Univ Sch Med, Japan, 3 Dept Ophthalmology, Jikei Univ Sch Med, Japan）

Residing next to mature skeletal muscle fibers, satellite cells are known to serve as theprogenitor of myoblast when triggered by various stimuli including damage to the muscle
fibers in vivo. Matsuoka et al. (2008) reported that the homogenate extract of various typesof adult chicken skeletal muscles induced differentiation of primary culture of myoblast
prepared from chicken embryo to express myosin heavy chain isoforms of the source skeletal
muscle of the extract. Inspired from their work, we tested the effect of homogenate extractfrom adult mouse skeletal muscle on primary culture of satellite cells prepared from young
mice. Unexpectedly, the extract seemed to enhance proliferation of the satellite cells without
inducing obvious differentiation in the expression pattern of myosin heavy chain. However, its
effect on the proliferation of satellite cells was unclear because of cellular contaminant such as
fibroblast and adipogenetic cells. So we tested the effect of muscle extract on C2C12 myoblast.
The proliferation of C2C12 myoblast was evaluated by the cytotoxic assay method. It was
found that the extract enhanced the proliferation of C2C12 myoblast dose dependently. The
mechanism of function of the extract will be discussed.
(COI:No)

Delayed umbilical cord clamping alters blood leukocyte proﬁles ofneonatal rats
Background: Delayed umbilical cord clamping (DUC) at birth is suggested to provide clinicalbenefits such as prevention of neonatal anemia and reducing the risk of late-onset sepsis. While
it is reported that DUC increases blood supply and the hematocrit values of neonates, theeffects of DUC on blood leukocyte profiles are not known.
Methods and Results: Full term Wistar rat fetuses at embryonic day 21 (e21) were used in
the study. Fetuses were delivered by Caesarean section. In the control group, the placenta
and the umbilical cord were immediately removed, while in the DUC group, the placenta
and the umbilical cord were kept attached for 1h until the collection of peripheral blood. The
collected blood samples were removed of red blood cells and were analyzed by flow cytometry.
Compared with prenatal fetuses, granulocyte population in the blood was increased in thecontrol group (1h after birth) and was further higher in the DUC group (8.1±1.1%, 11.6±1.7%,
and 15.6±2.2%, respectively, n = 5-6, p <0.05).
Granulocyte-stimulating factor (G-CSF) is a cytokine mediating release of granulocytes intoblood stream. Quantitative RT-PCR revealed that mRNA expression of G-CSF in umbilicalvein was higher than that in umbilical artery (2.01±0.37-fold, n=6, p <0.05) and significantly
increased by embryonic day (e21 vs. e19, 10.9±2.03-fold, n=6, p <0.01). Immunohistochemistry
showed that G-CSF was predominantly expressed in umbilical vein endothelial cells in e21
rat fetuses.
Conclusion: DUC increased granulocyte population in rat neonatal peripheral blood. G-CSF
that were highly expressed in umbilical vein endothelium may play a role in this process.
(COI:No)

2P-177

2P-180Hajime Arikawa1，Toshio Matsuoka1，Teppei Takahashi2，Tomoyoshi Terada3，Seiichi Era4

Kiyotaka Okada1, 2，Takahiro Okamoto2，Yoshimasa Takafuji2，Masayoshi Ishida2，
2 1 Dept Arts Sciences, Kindai Univ Facul
Naoyuki Kawao2，Osamu Matsuo2，Hiroshi Kaji（

Changes in ventilatory and muscle oxygen dissociation states insubjects not accustomed to exercise with vocalization（ 1 Faculty of Sports and Health Sci, Chubu Gakuin Univ, Seki, Japan, 2 Dept Oral andMaxillofacial Surgery, Gero Hospital, Gero, Japan, 3United Graduate School of Drug
Discovery and Medical Information Sci, Gifu Univ, Gifu, Japan, 4 Clinical Laboratory
Center, Japanese Red Cross Gifu Hospital, Gifu, Japan）

Purpose: We had previously reported that vocalization during continuous upper-limb exercise
at 80%VO2peak by subjects accustomed to exercising with vocalization (such as kendo players)tended to increase the value of FetCO2 (=PaCO2) and to suppress the decrease of oxygen dissociation states in exercising muscles. However, vocalization during exercise also suppresses
ventilations, which may make it difficult for those who are not used to exercising with vocalization to continue voicing or exercising. This study aimed to clarify the state of ventilation andactive muscle oxygen saturation during continuous upper-limb exercise in persons who are notused to exercising with vocalization.
Methods: Eleven male subjects who were not used to exercising with vocalization participatedin this study. They performed continuous upper-limb exercises using a hand ergometer at
80%VO2peak for 3 min under two conditions: “with vocalization (Voc)” or “without vocalization(non-Voc)”. We measured respiratory variables (such as FetCO2), venous oxygenation index(VOI) of middle fingertip, and tissue saturation index (TSI%) of triceps brachii (using NIRS),
before and immediately after exercise, and blood lactate concentration ([Lac‒]) before and 5
min after exercise.
Results: Voc value showed no difference in ΔFetCO2 compared to non-Voc value, but ΔVOI
tended to decrease more, ΔTSI% tended to decrease less, and Δ[Lac‒] tended to increase
more (no significant difference in any of them). In addition, some subjects were unable tocontinue vocalizing during exercise.
Conclusions: We observed that blood CO2 concentration did not increase because of inaccurate
vocalization during exercise. Even in this situation, the decrease in oxygen saturation of active
muscles tended to be suppressed. From these results, we inferred that rhythmic vocalization
with upper-limb exercise increased the rate of anaerobic energy supply and suppressed the(COI:No)
increase in oxygen consumption in active muscles.

The roles of PAI-1 in the delayed bone repair induced by glucocorticoid
Med, japan, 2 Dept Physiol Regenerat Med, Kindai Univ Facul Med, japan）

Glucocorticoids (GCs) regulate numerous physiological processes in a wide range of tissues. GCs
have been widely used for the treatment of chronic inflammatory diseases, such as the inflammatory bowel diseases, collagen diseases and rheumatoid arthritis for the anti-inflammatory
effects. It is an important clinical task that GC treatment induces osteoporosis, and previous
studies suggest that chronic GC treatment induces the delayed bone repair; however, themechanisms by which GC induces the delayed bone repair have still remained unclear. Here, weinvestigated the roles of plasminogen activator inhibitor-1(PAI-1) in GC-induced effects on bone
repair after femoral bone injury using female mice with PAI-1 deficiency and their wild-type
counterparts. Dexamethasone (Dex) enhanced plasma PAI-1 levels and PAI-1 mRNA levels in
the adipose tissues and muscles of wild type mice. PAI-1 deficiency significantly blunted thedelayed bone repair induced by Dex in mice. Moreover, PAI-1 deficiency significantly blunted
Runx2 mRNA levels and number of alkaline phosphatase-positive cells suppressed by Dex as
well as Dex-induced osteoblast apoptosis at the damaged site 7 days after bone injury of mice.
On the other hand, PAI-1 deficiency did not affect the ratio of receptor activator of nuclear
factor κB ligand / osteoprotegerin suppressed by Dex at the damaged site 7 days after
bone injury of mice. Moreover, PAI-1 deficiency did not affect the adipogenic gene expression
enhanced by Dex at the damaged site 7 days after bone injury in mice. In conclusion, we first
showed that PAI-1 is involved in the delayed bone repair after bone injury induced by GC in
mice. PAI-1 might influence early stage osteoblast differentiation and apoptosis during the(COI:No)
osteoblastic restoration phase of bone repair process.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S145

2P-1812P-184

Mariko Nakamura1，Chisaki Tamayose1，Satoru Hamada2，Chiaki Kijima1，Nobuyuki Hyakuna2，
1 1 Dept of Mol Cell Physiol,
Masanori Sunagawa3，Koichi Nakanishi2，Masayuki Matsushita（

Akira Yoshikawa1，Yuri Masaoka1，Masaki Yoshida2，Nobuyoshi Koiwa3，Natsuko Iizuka1, 4，
1 1 Dept Physiol,
Satomi Kubota1, 4，Ryo Manabe1, 5，Masahiro Ida6，Masahiko Izumizaki（

Signiﬁcance of measuring the blood platelet-derived microparticles(PDMP) and GPIb after pediatric hematopoietic stem cell transplantation
Grad Sch Med, Univ Ryukyus, Japan, 2 Dept Child Health, Grad Sch Med, Univ Ryukyus,
Japan, 3 Grad Sch Nurs, Meio Univ, Japan）

Pediatric hematopoietic stemcell transplantation has develop transplant-related complications:mucosal damage associated with pretreatment of anticancer drugs and radiation, organ
damage, severe infections associated with immunodeficiency and GVHD. After more than 3months of transplantation, life-threatening complications such as DIC, TMA, SOS are caused
by vascular endothelial injury. Clinical parameters for early detection of such coagulation
disorders and their measurement are expected. In this study, we focused on PDMP levels andplatelet membrane GPIbα kinetics for the purpose of identifying early coagulation disorders
after transplantation. As a result, an increase in PDMP value was observed in 4 out of 10
transplant patients at 4 weeks after transplantation, and the decrease in GPIbα expression,
which was simultaneously measured by the FACS, was observed in 3 cases. Although PDMP
did not increase, only GPIbα decreased in 2 cases. In the 5 cases with decreased GPIbα
exhibited severe complications such as severe infection, severe GVHD, autoimmune hemolytic
anemia, and nephrotic syndrome. In the 4 cases with increased PDMP, because proteases
induced by complications (inflammation/infection) after pediatric allogeneic hematopoietic celltransplantation may act on the platelet membrane, these are released into the plasma fromthe platelet membrane. Since GPIbα has a binding site for thrombin and vWF, we performedin vitro experiments using GPIbα and thrombin. As a result, the released GPIbα was notdetected by the FACS when bound to thrombin, indicating that the decrease in plasma GPIbαin patients with post-transplant complications is related to thrombin production in vivo. Posttransplant patients are likely to cause the complications when GPIbα decreases and PDMP
increases. Early detection of plasma GPIbα decrease can detect early complications such as
hypercoagulation after transplantation. Therefore, early detection of the dynamics of GPIbα
and PDMP after transplantation is presumed to be useful for early treatment of complications(COI:No)after transplantation.

Resting-State fMRI Connectivity Analysis between Hippocampus andMotor area in COPD patientsShowa Univ, Sch Med, Tokyo, Japan, 2 Dept Ophthal, Jikei Med Univ, Tokyo, Japan, 3 Deptof Health and Sci, Univ Human Arts and Sci, Saitama, Japan, 4 Dep Neulolo, Showa Univ,
Univ, Sch Med, Tokyo, Japan, 5 Dept Respiratory, Showa Univ, Sch Med, Tokyo, Japan,
6 Natl Hosp Org, Mito med Ctr, Tokyo, Japan）

Chronic obstructive pulmonary disease (COPD) is a primary airway inflammatory diseasecharacterized by irreversible airflow limitation which results in hypoxemia and hypercapnia.
Meanwhile, it is also realized as a complex multi-component disorder. Cognitive impairment
has been found as one of the important extrapulmonary manifestation in patients with COPD.
In our previous study, we found that left hippocampus volume was smaller in COPD patientscompared with those of control group. Resting-state functional magnetic resonance imaging (rsfMRI) connectivity analysis is one of the valuable ways to observe functional changes in humanbrain, especially cognitive functions. In this study, we evaluated the functional connectivity (FC)between left hippocampal and other brain area in COPD patients.
20 COPD patients and age-matched 25 control subjects participated in the present study.
Clinical 3T (Siemens) was used. Four minutes and thirty seconds rs-fMRI was recorded withcardiac pulse wave and respiration. We conducted a region of interest (ROI)-to-ROI FC analysis.
The FC between the left hippocampus and other brain were calculated with CONN toolbox.
COPD patients had significant lower FC between the left hippocampus and the right sensorimotor area than controls. In addition, COPD patients also had significant lower FC between
left and right hippocampus.
This study showed COPD having restricted physical activity due to respiratory problem
accompanied with dyspnea, anxiety and sleep problem may affect hippocampus volume
and reduced FC between hippocampus and motor-related areas. Since physical activity is aprognostic factor in COPD, reduced FC between hippocampus and motor-related areas could(COI:No)be one of an index for severity for restricted activity and poor quality of life. 

2P-182

2P-1853 1 Dept Physiol, Fac Med, Univ
Minako Okazaki1, 2，Saori Uozu1，Tadachika Koganezawa1, （Tsukuba, Tsukuba, Ibaraki, Japan, 2 Mstr Prog Med Sci, Grad Sch Comp Human Sci, Univ
Tsukuba, Ibaraki, Japan, 3Transborder Med Res Ctr, Univ Tsukuba, Ibaraki, Japan）

Kana Mieno, Satoshi Hoshino, Kaoru Ide, Susumu Takahashi（Grad Sch of Brain Science,

Endogenous hydrogen sulﬁde is essential to maintain eupneic
respiration in the in situ arterially perfused preparation of rats

Hydrogen sulfide (H2S) is generally known as toxic gas but endogenously generated and has
physiological roles in our body including the brain. However, roles of endogenous H2S to generate respiratory pattern in the respiratory center is not understood. The aim of this study wasevaluation of functional roles of endogenous H2S in generating the respiration.
We performed in situ arterially perfused preparations of decerebrated rats and recorded thephrenic and vagus nerves activities. We administrated amino-oxyacetate hemihydrochloride(AOA: 10 mM) or hydroxylamine hydrochloride (HA: 2 mM) to inhibit H2S production via cystathionine β-synthase (CBS), DL-propargylglycine (PAG: 10 mM) to inhibit it via cystathionine
γ-lyase (CSE), and S-adenosyl-L-methionine chloride dihydrochloride (SAM: 60 µM) to activate
it via CBS. Further, we applied riluzole (5 µM) to block persistent sodium channels which
can suppress pacemaker cells-based activity. And then, we compared the central respiratory
pattern before and after administration of drugs.
By inhibiting the CBS with AOA or HA, the respiratory pattern was switched from threephases eupneia into gasping-like respiration. Respiratory frequency and amplitude of phrenic
and vagus nerves activities were significantly decreased by the inhibition. On the other hand,in the presence of both riluzole and AOA or HA, gasping was little observed. Neither inhibiting
CSE with PAG nor activating CBS with SAM affected to eupneic respiratory pattern.
These results suggested that CBS-produced H2S has vital roles to maintain neuronal network
to generate eupnea. The mechanism to generate respiratory pattern might be switched fromnetwork- to pacemaker cells-based systems depending upon H2S concentration.
(COI:No)

Neural activity of place cell in various mazes in the same experimentalroom
Doshisha Univ, Japan）

Various forms of maze such as the T-maze, plus maze, and radial maze are routinely used to
examine learning and memory in animals. Place cells form a cognitive map because they fire
only when an animal passes through a particular location. The firing pattern of hippocampal
place cells tends to change when an animal moves to another room or the shape of the roomchanges. However, conventional mazes cannot easily change shape in the same physical location. Thus, it is difficult to complementarily compare and examine the results of experiments
conducted in the various shape of the maze. Here, we developed a system that can reconfigure
the maze shape in the same room. To examine how changes in cognitive maps affect learningand memory, we constructed several different shaped mazes using this system and examined
whether it affects their running trajectory and the response of hippocampal CA1 place cell
activity. First, we changed the shape of the maze from square to cruciform. As a result, place
field location in the hippocampus was shifted. Next, we changed the shape of the maze fromthe cruciform back to the square, place field location returned to its original position. Thesefindings suggest that the change of the maze shape in the same room caused different place
cell representation. Besides, to examine hippocampal-dependent working memory, we trained
rats to perform the delayed spatial alternation task for 10 days. As a result, the performance
significantly improved with experience. Finally, we discuss the results of a preliminary experiment in which the shape of the figure 8 maze is scaled or zoomed in the vertical or horizontal
directions.
(COI:No)

2P-183

2P-1861 1 Dept Anat ＆Shigefumi Yokota1，Noriyuki Hama2，Kotaro Takeda3，Masashi Fujitani（

Keisuke Ota1，Yasuhiro Oisi1，Takayuki Suzuki1，Tsubasa Ito1, 2，Yoshiki Ito1, 3，Kenta Kobayashi4，
Midori Kobayashi1，Maya Odagawa1，Chie Matsubara1，Yoshinori Kuroiwa5，Masaru Horikoshi5，
Junya Matsushita6，Hiroyuki Hioki7，Masamichi Ohkura8，Junichi Nakai9，Masafumi Oizumi1, 3，
1 1Center for Brain
Atsushi Miyawaki1，Toru Aonishi1, 2，Takahiro Ode1, 10，Masanori Murayama（

The pathway transmitting hypoxia information from the solitary nucleus
to the hypothalamus
Neurosci, Shimane Univ Sch Med, Japan, 2 Dept Physiol, Shimane Univ sch med, Japan,

3 Fac Rehabili, Sch Healthcare, Fujita Hlth Univ, Japan）

Obstructive sleep apnea decreases arterial oxygen pressure and then facilitates breathing aswell as arousal from sleep. It is suggested that the parabrachial nucleus (PB) known as the
pontine respiratory group is a key region in arousal by hypoxia. However, the neural circuitson this response are not fully understood. Under hypoxia, glutamatergic neurons in the medial
part of the caudal solitary nucleus (cNTSm) are activated. Therefore in this study, we firstexamined the indirect pathway via the PB from glutamatergic neurons in the cNTSm to
the perifornical area in the hypothalamus (PeF) where orexinergic neurons located. By using
a combined methods of a genetically anterograde labeling of glutamatergic cNTSm axons
and retrograde tracing, we found a dense plexus of axons originating from the cNTSm in
the external lateral PB subnucleus (PBel) and additional distribution of many axons in thecentral lateral PB subnucleus (PBcl) and dorsal lateral PB subnucleus (PBdl). On the other hand,
PeF-projecting neurons were observed in the PBcl and PBdl and they were overlapped to the
distribution of cNTSm axons. We also found that CGRP-immunoreactive neurons, which areindicated as arousal-inducing neurons were embedded in the plexus of cNTSm axons in the
PBel. Next, we demonstrated that some PeF-projecting neurons in the PBcl and PBdl, as wellas many CGRP-immunoreactive neurons in the PBel, expressed Fos protein after exposure to
8% hypoxic condition, using a combination of immunohistochemistry and retrograde tracing.
These results suggest that the glutamatergic cNTSm neurons may exert excitatory influence
not only upon PB neurons projecting to the PeF but also upon CGRP neurons in the PBel to(COI:No)
induce arousal by hypoxia.

S146

In vivo calcium imaging with a single cell resolution using“cosmoscope”, a new wide-ﬁeld two-photon microscope

Science, RIKEN, Saitama, Japan, 2Tokyo Institute of Technology, Yokohama, Japan, 3Univ. ofTokyo, Tokyo, Japan, 4 National Institute for Physiological Sciences, Okazaki, Japan, 5 Nikon
Corporation, Yokohama, Japan, 6 Hamamatsu Photonics K.K., Iwata-shi, Japan, 7 Juntendo
University Graduate School of Medicine, Tokyo, Japan, 8 Kyushu University of Health and Welfare,
Miyazaki, Japan, 9Tohoku University, Miyagi, Japan, 10 FOV Corporation, Yokohama, Japan）
In neuroscience, in vivo functional imaging with a single-cell resolution over a wide field of view(FOV) is challenging. To accomplish this, we developed a wide-field two-photon microscope,
which we named “cosmoscope”, that equips a very large objective lens with low magnification
and a high numerical aperture. We evaluated the optical resolution and aberrations of this microscope by means of simulation. Eighty percent of the energy was contained within a radiusof 1.1 µm in all FOV. This result was almost equivalent to the performance at the diffraction
limit, indicating that this microscope possesses the high efficacy of two-photon excitation andhigh spatial resolution in all three axes across the entire field of view. To further evaluate thismicroscope, we performed in vivo calcium imaging of L2/3 and L5 cortical neurons expressing
GCaMP in an awake mouse. GCaMP fluorescence was observed in the cytoplasm, not in the
nucleus, providing confirmation that this microscope possesses single-cell resolution. We also
developed a low computational cost cell detection (LCCD) algorithm (Ito et al., bioRxiv 502153),
because the data size acquired by our microscope was so large that the previously reported
algorithms could not detect neurons within a practical period of time. LCCD enabled us to
extract more than 16,000 neural activities of L2/3 cortical neurons. Finally, we identified thefunctional map of the neurons. Whereas sensory stimulus-evoked activity was localized to the
responsible region, movement-related activity was globally distributed. Cosmoscope will open
the door to monitor a great multitude of single cortical neurons.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

2P-187

2P-1902 1Toyohashi Univof Tech, Dept of Applied Chem andRika Numano1，kohjiro Matsumoto（

Manabu Saito1，2，Masaru Ninomiya3，Shuji Hakoshima3，Kenji Tsuchiya4，Yui Sakamoto1，
Kazutaka Yoshida1，Yuri Matsubara1，Ai Terui1，Shinya Ueno2, 5，Kazuhiko Nakamura1, 2

The novel electroporation of a Water-in-oil Droplet was applied tocytomorphology modiﬁcation
Life Science,, 2 Nepa Gene Co., Ltd.）Electroporation is the most widely used transfection method for delivery of cell-impermeable
molecules into cells. We developed a novel gene transfection method, water-in-oil (W/O) droplet
electroporation, using dielectric oil and an aqueous droplet containing mammalian cells andtransgene DNA. When a liquid droplet suspended between a pair of electrodes in dielectric oil
is exposed to a DC electric field, the droplet moves between the pair of electrodes periodicallyand droplet deformation occurs under the intense DC electric field.
During electrostatic manipulation of the droplet, the local intense electric field and instantaneous short circuit facilitate gene transfection.
This method has several advantages over conventional transfection techniques, including co-transfection of multiple transgene DNAs into even as few as 103 cells, transfection
into differentiated neural cells. In addition, there have been improvements in W/O droplet
electroporationelectrodes for disposable 96-well plates making them suitable for concurrent
performance without thermal loading by a DC electric field. This technique will lead to the(COI:No)development of cell transfection methods for iPS cells and genome editiong.

Development of ASD screening algorithm in 5-year-old children using
eye-tracking device (Gazeﬁnder)（ 1 Dept Neuropsychiat, Grad Sch Med, Hisosaki Univ, Japan, 2 Res Center for Child Mental
Dev, Grad Sch Med, Hirosaki Univ, Japan, 3 JVCKENWOOD Corporation, 4 Center forChild Mental Dev, Grad Sch Med, Hamamatsu Med Univ, Japan, 5 Dept Neurophysiol, Grad
Sch Med, Hisosaki Univ, Japan）

Children with Autism Spectrum Disorder (ASD) have a characteristic eye movement due tosocial attention that depends on the bias of interest, compared to children with typical development (TD). It is necessary to detect early ASD for improving the symptoms, but it is not easy
to diagnose ASD in early childhood. The aim of this study was to develop a screening algorithm
by extracting gaze characteristics in pre-school 5-year-old ASD. 97 typical developmental
children (TD) and 42 ASD children without coexisting other developmental disorder were
just looking at 2-minute video with contents of face, preference, biological motion, and pointing.
Statistics analysis was a principal component analysis on fixation rate at 89 Area of Interest(AOI) using diagnostic information and high sensitivity AOI based on correlation coefficient /p value / effect size. Considering the effects of gender, the analysis was divided into boys andgirls. There was a significant difference between TD and ASD in specific AOI of both boys andgirls. The fixation rate of girls (AOC=0.89) showed 86% sensitivity and 83% specificity, and the
fixation rate of boys (AOC=0.73) showed 69% sensitivity and 69% specificity by a cut-off score.
In all children, AOC was 0.78, and when the provisional cut-off score of the fixation rate was
50.4%, the sensitivity was 75% and the specificity was 75.8%. This result was appropriate as ascreening algorithm.
(COI:No)

2P-188Analgesic effects of voluntary running performed before or after the
induction of inﬂammatory pain
Risa Yamauchi1, 2，Hideshi Ikemoto1，Naoki Adachi1，Takayuki Okumo1，Jun Oike1，
Yusuke Kunieda1，Hiroyuki Horikawa1, 2，Mana Tsukada1，Tadashi Hisamitsu1，
1 1 Dept Physiol, Showa Univ, Sch Med, Tokyo, Japan, 2 Faculty of
Masataka Sunagawa（Arts and Sciences at Fujiyoshida, Showa Univ, Yamanashi, Japan）

AIM: Some studies support the effectiveness of physical therapy for reducing pain and improving the physical function. These effects might differ depending on the modalities (e.g. forced or
voluntary running) and the timing of physical exercises. In the present study, we investigated
the influence of voluntary running before or after the induction of experimental inflammationon acute pain in rats.
Methods: Experiment 1. Male Wistar rats were divided into a control, a nonrunning before theinjection of formalin (pre-NOR), and a voluntary running before the injection (pre-VR) groups.
Inflammation was induced by injecting formalin (1%, 50 µl,) into the hindpaw. Rats in the
pre-VR group were given free access to a running wheel for a week. The total time spent in
pain-related behaviors was quantified for 60 min after the injection. Experiment 2. Rats weredivided into a control, a non-running after the formalin injection (post-NOR), and a voluntaryrunning after the formalin injection (post-VR) groups. Formalin was similarly injected, andthen rats in the post-VR group were given free access to a running wheel for 12 days. The
inflammatory sensitization was tested with the von Frey test. The expression of activatedmicroglia in the spinal cord was analyzed by Western blotting and immunofluorescent staining.
Microglia are thought to be involved in the prolongation and the chronicity of pain.
Results: Voluntary running before the induction of pain did not provide the analgesic effect(Exp. 1). However, running after the induction of pain significantly accelerated the recovery
from pain, and significantly inhibited the expression of activated microglia (Exp. 2).
Conclusions: These results suggest that physical therapy early after surgery may promote the(COI:No)
recovery from inflammatory pain by inhibiting microglial activation.

2P-189Preemptive analgesia provided by complementary approaches in acuteinﬂammatory pain model rats
Nachi Ebihara, Hideshi Ikemoto, Naoki Adachi, Tamehiro Ebihara, Kayoko Ebihara,
Jun Oike, Yusuke Kunieda, Yasunori Takayama, Tadashi Hisamitsu, Masataka Sunagawa（Dept Physiol, Showa Univ, Sch Med, Tokyo, Japan）

Preemptive analgesia is a strategy that is designed to prevent central sensitization andchronic postoperative pain. The present study aimed to elucidate the preemptive analgesia
of complementary approaches, such as transcutaneous electrical nerve stimulation (TENS),
electroacupuncture (EA), a Japanese herbal medicine Yokukansan (YKS), and a combination of
EA and YKS, using rats with formalin-induced acute inflammatory pain.
Male Wistar rats were divided into six groups: a control, a formalin-treated (For), a pretreatment of TENS+For (TENS), a pre-treatment of EA+For (EA), a pre-administration of
Yokukansan (7 days)+For (YKS), and a EA+YKS+For (EA+YKS) group. Rats were injectedsubcutaneously with formalin (50 µl, 1%) into the hindpaw. TENS and EA were delivered at afrequency of 4 Hz for 30 min before the injection of formalin. EA was applied at the Zusanli(ST-36) acupoint. YKS was mixed with powdered rodent chow at a concentration of 3% andfed to the YKS-treated rats. The total time spent in pain-related behaviors was quantified
for 60 min immediately after the formalin injections. We also observed the expression ofphosphorylated extracellular signal-regulated kinase (pERK), which has been used as a markerof neural activation, in the spinal dorsal horn by immuno-staining. As a result, the duration of
pain-related behavior was significantly increased following the injection of formalin; however,
the increase was significantly inhibited in the TENS (p<0.05), EA (p<0.05), and EA+YKS groups(p<0.01). The number of spinal pERK (+) cells was also significantly increased; however, thechange was significantly inhibited in the TENS, EA, and EA+YKS groups (each; p<0.01).
These results suggest that TENS, EA, and a combination of EA and the administration of
YKS produce preemptive analgesia and inhibit the phosphorylation of ERK in the spinal cord.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S147

Poster Presentations
Day 3（ March 19 , 13 : 10 〜 14 : 10 ）3P-001〜3P-018

Ion Channel・Receptor（ 3 ）3P-019〜3P-032OU

Heart・Circulation（ 3 ）3P-033〜3P-050

Neuron・Synapse（ 3 ）3P-051〜3P-062

Sensory Function（ 3 ）3P-063〜3P-072

Behavior Science・Biorhythm（ 3 ）3P-073〜3P-075

Neurochemistry（ 3 ）3P-076〜3P-081

Autonomic Nervous（ 3 ）3P-082〜3P-089

Muscle Physiology（ 3 ）3P-090〜3P-094

Oral Physiology（ 3 ）3P-095〜3P-099

Endocrinology（ 3 ）3P-100〜3P-102

Kidney・Urination（ 3 ）3P-103〜3P-108

Motor Function（ 3 ）3P-109〜3P-112

Development・Growth・Aging（ 3 ）3P-113〜3P-123

Cell Physiology・Molecular Physiology（ 3 ）3P-124〜3P-129

Environmental Physiology（ 3 ）3P-130〜3P-132

Drug Actions（ 3 ）3P-133〜3P-136

Membrane Transport

3P-137〜3P-146

CNS Function（ 3 ）3P-147〜3P-154OU

Nutrition・Metabolism・Thermoregulation（ 3 ）3P-155〜3P-162OU

Pathophysiology（ 3 ）3P-163〜3P-165

Physical Fitness・Sports Medicine（ 3 ）3P-166〜3P-168

Respiration（ 3 ）3P-169〜3P-170

Study Methodology（ 3 ）3P-171〜3P-175OU

Others（ 3 ）3P-001

3P-004

Toshiko Yamazawa1，Haruo Ogawa2，Takashi Murayama3，Maki Yamaguchi1，
Hideto Oyamada4，Junji Suzuki5，Nagomi Kurebayashi3，Kazunori Kanemaru6，
6 1 Dept Mol Physiol, Jikei Univ Sch
Katsuji Oguchi4，Takashi Sakurai3，Masamitsu Iino（

Akiko Nakashima, Makoto Takano, Noriyuki Nakashima（ Dept. Physiol., Kurume Univ. Sch.

Molecular dynamics simulation of mutant type 1 ryanodine receptor

Med, Japan, 2The Univ Tokyo, Japan, 3 Juntendo Univ Sch Med, Japan, 4 Showa Univ Sch
Med, Japan, 5 Univ California San Francisco, USA, 6 Nihon Univ Sch Med, Japan）

In skeletal muscle cells, membrane depolarization is translated into intracellular Ca2+ signals,
and type 1 ryanodine receptors (RyR1), located in the sarcoplasmic reticulum membrane, playa key role in intracellular Ca2+ release. Mutations in the RyR1 gene cause severe muscle
diseases, such as malignant hyperthermia (MH), which is a disorder of Ca2+-induced Ca2+
release via RyR1 in the skeletal muscle. Thus far, more than 300 mutations have been reported
in RyR1 in patients with MH, and most of those mutations have been found in three hotspot
regions of RyR1. However, due to a lack of comprehensive analysis of the structure–function
relationship of mutant RyR1, the mechanism remains largely unknown. Here, we combinedfunctional studies and molecular dynamics (MD) simulation of RyR1 bearing disease-associatedmutations at the N-terminal region. When expressed in HEK293 cells, the mutant RyR1 caused
abnormalities in Ca2+ homeostasis. MD simulations of wildtype (WT) and mutants were
performed using crystal structure of the NTD monomer of RyR1. In WT, we found thatthe B(R283)-A(D61)-C(R402)-A(E40)-C(S406) hydrogen bonds/salt bridges network (B-A-C-A-C
network) around R402. MD simulation of the mutant of R402 revealed that alterations ofhydrogen bonds/salt bridges between NTD, consisting of A, B and C domains. The importanceof R402 was verified by functional studies and MD simulations with Alanine mutants of E40
and D61 that form tight interaction with R402. Our results reveal the importance of interdomain interactions within NTD in the regulation of the RYR1 channel and gain insights into
(COI:No)the mechanism of MH caused by the mutations at the NTD.

Mouse ameloblasts express inwardly rectifying K+ channels
Med., Japan）

Enamel is a surface layer of teeth with extreme hardness comprising of mineralized structure.
The enamel layer is formed by the secreting activity of ameloblasts. In addition to secretingmachinery, ameloblasts are known to express ion transporters. Here, we further investigatedthe channel expression of ameloblasts. We first prepared the semi-intact preparation of mouseincisors at 300-µm thickness by frontal sectioning and performed a voltage-clamp patch clamp
recording. We detected inwardly-rectifying channel activities, which were enhanced by theelevation of K+ concentration in the recording pipette. In the whole-cell configuration, we
confirmed the inwardly-rectifying currents, which were abolished by the application of 5 mM
Ba2+ in the bath solution. Based on these features characteristic to inwardly rectifying K+(Kir) channels, we investigated the mRNA expression patterns of Kir channel families by
reverse-transcriptase PCR using the incisor tissues. As a result, we revealed that Kir 2, 4 and6 subtypes were potentially expressed in the mouse incisor ameloblasts.
These results indicate that the membrane potential of ameloblasts is subject to the characteristic activities of Kir channels, e.g., in response to the extracellular K+ concentration or the
intracellular pH changes.
(COI:No)

3P-002

3P-005

Yasunobu Okada1，Toshiaki Okada2，Kaori Sato-Numata3, 4，Tomohiro Numata4

Noriko Ishizuka, Naotaka Ikumi, Hisayoshi Hayashi（Laboratory of Physiology, School of

Reexamination of the roles of LRRC8 and TTYH in the molecular
identity of volume-sensitive outwardly rectifying anion channel VSOR
（ 1 Dept Physiol, Kyoto Prefectural Univ of Med, Kyoto, Japan, 2 Nat Inst Physiol Sci,
Okazaki, Japan, 3 JSPS, Tokyo, Japan, 4 Dept Physiol, Fukuoka Univ, Fukuoka, Japan）
Cell volume regulation (CVR) is fundamental to survival/death and functions of animal cells.
CVR after osmotic swelling, called regulatory volume decrease (RVD), is attained by parallel
activation of K+ channels and Cl－ channels. The most important Cl－ channel directly activatedby cell swelling and involved in RVD is the volume-sensitive outwardly rectifying anion channel VSOR (also called volume-regulated anion channel VRAC). Two membrane-spanning
protein families, LRRC8(A+C/D/E) and TTYH(1-3), were reported to be the molecular identityof VSOR in 2014 and 2019, respectively. In the present study, the roles of these two protein
families were reexamined in human epithelial cells by microarray assay and gene knockout/
knockdown experiments. Actually siRNA-mediated quadruple knockdown of LRRC8A, 8C,
8D, and 8E or triple knockdown of TTYH1, 2 and 3 largely, though not completely, inhibitedswelling-activated VSOR currents in HeLa cells. However, differential microarray studies
showed that expression levels of mRNAs for LRRC8A-E and TTYH1-3 were not markedly
different between VSOR-deficient KCP-4 cells and VSOR-rich parental KB cells as well as
between KCP-4 and other VSOR-rich human epithelial cell lines including HeLa, HEK293T and
Intestine 407. Furthermore, we produced a stable LRRC8A-deficient cell line, Clone-3, derived
from HeLa cells by the CRISPR-Cas9 knockout method and then examined VSOR activity
induced by reducing intracellular ionic strength under isotonic conditions (Low-IS) and by
applying a hypotonic challenge. In Clone-3 cells, both Low-IS-activated and swelling-activated
VSOR currents were still sizably observed, though considerably suppressed compared to thosein wild-type HeLa cells. Moreover, siRNA-mediated triple knockdown of TTYH1-3 failed toabolish both types of VSOR currents in Clone-3 cells. Taken together, it is likely that someother core molecule is still missing, and thus further studies are warranted for the molecular
identification of VSOR.
(COI:No)

Coupling mechanism for Na+/H+ exchanger and Cl /HCO3 exchanger
in the intestine
Food and Nutritional Sciences, University of Shizuoka）

Intestinal NaCl absorption is considered to work by coupling a Na+ transporter and a Cl－transporter. Although the Na+ transporter has been shown to be Na+/H+ exchanger isoform
3 (NHE3) throughout the intestine, the Cl－ transporter has a different form in each intestinal
segment, which are Cl－/HCO3－ exchanger slc26a6 in the small intestine and another Cl－/
HCO3－ exchanger slc26a3 in the large intestine. We have also shown that NHE3 changes
coupling partner to H+-coupled peptide transport (pepT1) when H+-coupled peptide absorption is activated. However, the molecular mechanism of each coupling mode remains to be
determined. To investigate this, we determined the involvement of slc26a3 in NaCl absorptionin each intestinal segment of mice. Ussing chambers were used to measure transepithelial
22Na+ and 36Cl－fluxes across the intestinal membrane. In addition, we investigated a mode ofcoupling between slc26a3 and NHE3 by using intestinal organoids. The activity of NHE3 wasdetermined as the rate of Na+-induced intracellular pH recovery after acid loading. In theUssing chamber experiments, the addition of NHE3 specific inhibitor S3226 to the luminal sideinduced simultaneous inhibition of net Cl－ flux in the middle colon and cecum. However, thecoupling ratio of NHE3 and slc26a3 is different between the middle colon and cecum. These
results suggested that the mode of coupling of Na+ and Cl－ absorption is different along colonicsegments. (COI:No)

3P-003

3P-006

Nobutake Hosoi1，Koji Shibasaki2，Teiichi Furuichi3，Hirokazu Hirai1，Tetsushi Sadakata4（ 1WPI-NanoLSI, Kanazawa Univ., 2 Infiniti, Kanazawa Univ., 3 Coll. of Sci. and Eng.,
Kanazawa Univ., 4 Sch. of Life Sci., Fudan Univ.）

Action potential ﬁring and Na currents in cerebellar Purkinje cells ofclass II ARF-deﬁcient ʼaction tremorʼ mice: class II ARF may function asa Nav1.6 localizer at the AIS（ 1 Dept Neurophysiol ＆ Neural Repair, Grad Sch Med, Gunma Univ, Japan, 2 Dept Mol Cell
Neurobiol, Grad Sch Med, Gunma Univ, Japan, 3 Dept App Biol Sci, Faculty Sci Tech,
Tokyo Univ of Sci, Japan, 4 Edu Res Support Center, Grad Sch Med, Gunma Univ, Japan）

ADP-ribosylation factors (ARFs) are a family of small GTPases comprising six members categorized into three classes: class I (ARF1, 2, and 3); class II (ARF4 and 5); and class III (ARF6).
Although class I and III ARFs are known to be the key regulators in vesicular membranetrafficking, the cellular function of class II ARFs remains unclear. Using double knockout
technique, we produced the class II ARF hypomorphic mouse line carrying the ARF4+/－/
ARF5－/－ genotype. It turned out that ARF4+/－/ARF5－/－ mice (class II ARF-deficient mice)show action tremor-like movement disorders. In the present study, we focused on the electricalproperties of cerebellar Purkinje cells (PCs) which play an important role in motor control.
Slice patch-clamp experiments revealed that class II ARF-deficient PCs cannot maintain highfrequency spike firing in response to larger current inputs. Spontaneous spike discharges
were also reduced significantly in class II ARF-deficient mice. To elucidate the underlying
mechanism, we examined voltage-gated Na currents in PCs. Although the voltage dependenceof activation and inactivation of the Na currents in class II ARF-deficient PCs were similar
to that in WT PCs, the mutant PCs showed a reduction not only in the typical fast transient
Na current, but also in the resurgent Na current which is related to Nav1.6 pore-forming
alpha subunit and contributes to repetitive high-frequency firing in PCs. Immunohistochemical
analysis revealed that class II ARF-deficient mice exhibit reduced Nav1.6 localization to the
PC axon initial segment (AIS) which is the action potential initiation site. On the other hand,outside-out patch recording from PCs and immunohistochemistry suggest no alteration involtage-dependent K channels between WT and class II ARF-deficient mice. We conclude that
(COI:No)class II ARF plays an important role in localizing Nav1.6 to the AIS of PCs.

S150

Dynamic structure of transient receptor potential vanilloid 1 (TRPV1)
cation channel observed by high-speed atomic force microscopy
Ayumi Sumino1，Daichi Mukai3，Zhao Yimeng4，Mikihiro Shibata1, 2，Motoyuki Hattori4
Transient receptor potential vanilloid 1 (TRPV1) ion channel is a capsaicin receptor thatinduces burning pain. It is also the thermosensitive channel that opens over 43 ℃, functioning
as thermosensor of the cells. Single-particle analysis using cryo-electron microscopy (Cryo-EM)
have revealed high-resolution structure of the TRPV1 channel. However, molecular mechanismof the heat sensing and dynamic structural change upon gating is still unknown. Here, we applied high-speed atomic force microscope (HS-AFM) for observation of the dynamic structureof TRPV1. TRPV1 channels were solubilized and purified with detergent, then reconstituted
into phospholipid bilayer on an AFM substrate. AFM image showed tetrameric structure likea windmill, whose diameter and protrusion from bilayer surface were about 15 nm and 3 nm,
respectively. The cryo-EM structure shows similar structure with view from cytoplasmic side,suggesting that the structure imaged by AFM corresponds to cytoplasmic ankyrin repeat
domains (ARD). The protruded height of the ARD is 2 nm shorter than the cryo-EM structure,
implying that the ARDs might lie down on the bilayer surface. We will discuss how ligand(COI:No)binding and heat change the structure of ARDs and its fluctuation.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

3P-007

CACNA1C-E1115K Mutation Associated with Overlap Phenotype ofLong-QT and Brugada Syndrome Disrupts Cav 1.2 Ion Selectivity inPatient-speciﬁc iPS Cell-derived Cardiomyocytes
Asami Kashiwa1，Takeru Makiyama1，Hirohiko Kohjitani1，Thomas Maurissen2，
Yimin Wuriyanghai1，Yuta Yamamoto1，Jingshan Gao1，Hai Huang1，Tomohiko Imamura1，
Takanori Aizawa1，Taisuke Ishikawa2，Seiko Ohno3，Futoshi Toyoda4，Seiich Sato5，
Kazuhiro Takahashi6，Knut Woltjen2，Minoru Horie7，Naomasa Makita3，Takeshi Kimura1（ 1 Dept Cardiol, Grad Sch Med, Kyoto Univ, Japan, 2 CiRA, Kyoto Uni, Japan, 3 NCVC,
Japan, 4 Dept Physiol, Med, Shiga Univ, Japan, 5 Okinawa Prefectural Nanbu Medical Center,6 Kizawa Memorial Hospital, 7 Center for Epidemiologic Research in Asia, Shiga Univ, Japan）
Background: A missense mutation, E1115K in CACNA1C, encoding L-type Ca2+ channels (LTCCs), is
located in the crucial site of ion selectivity (Nature. 1993;366(6451):158-61). Recently, the mutation wasreported to cause diverse arrhythmogenic phenotypes including long-QT syndrome (LQTS) and Brugada
syndrome.
Objective: The aim is to investigate the disease mechanism of CACNA1C-E1115K using an iPSC model.
Methods and Results: We generated iPSCs from a 12-year-old boy carrying heterozygous CACNA1CE1115K, whose ECG presented QT prolongation and a drug-induced Brugada ECG pattern. Patch-clamp
recording using differentiated cardiomyocytes (CMs) revealed that the peak ICaL densities of E1115K
iPSC-CMs were reduced compared to controls (4.9±0.8 vs. 9.6±1.0 pA/pF, p<0.001), which might
contribute to Brugada phenotype. Impaired Ca2+ selectivity was demonstrated by the negative-shifted
reversal potential and marked outward currents through LTCC carried by monovalent cations in E1115K
CMs. In action potential (AP) recordings using a dynamic clamp system injecting synthetic IK1, E1115K
CMs exhibited significantly longer AP durations (APD90 444.0±12.3 vs. 354.6±20.1mV, p<0.001). We
also evaluated drug effects on APDs using voltage-sensitive dye imaging. Nifedipine (10 nM) significantly
shortened APDs in E1115K cells than controls (APD reduction rate: -45.8 vs. -36.1%, p<0.05). Furthermore,mexiletine and GS967, which can block sodium currents, significantly shortened APDs in E1115K cells(-11.1 vs. 0.7, p<0.05; -21.9 vs. -4.2%, p<0.01). These results suggest that late sodium currents are enhancedin E1115K cells, and might have an important role in APD prolongation.
Conclusion: We demonstrated impaired ion selectivity in CACNA1C-E1115K iPSC-CMs which might beassociated with the patient’s clinical phenotypes. Late sodium current blockers might be candidates to(COI:No)
rescue QT prolongation in this mutation. 

3P-010Identiﬁcation of binding partners of the voltage-gated sodium channel
Nav1.1
Ikuo Ogiwara（ Dept Physiol, Nippon Med Sch, Japan）
Nav1.1 is a brain-type of voltage-gated sodium channel α subunit. Nav1.1 is predominantly expressed in parvalbumin-expressing (PV) cells, and plays critical roles in maintaining sustained
fast-spiking from these inhibitory neurons. Mice with conditional deletion of Nav1.1 in PV cells
display epileptic seizures and autism-like behaviors that resemble clinical features of human
Dravet syndrome with mutations of the Nav1.1-encoding gene SCN1A. While Nav1.1 is also
detectable in some excitatory cells, conditional deletion of Nav1.1 in excitatory cells exert a
protective effect on seizure symptom. Nav1.1 interacts with sodium channel β subunits andubiquitously-expressed calmodulin. We here screened candidate proteins that could bind to
Nav1.1 using immunoprecipitation with anti-Nav1.1 antibody and mouse brain extract, followedby tandem mass spectrometry. The list of proteins included sodium channel β subunits,
calmodulin and other proteins. We selected some Nav1.1-binding partner candidates, whichwere seemingly expressed in subsets of neurons on the basis of the Allen brain atlas database.
We evaluated that two candidates, namely, A and B, were co-immunoprecipitated with Nav1.1,
and vice versa, using mouse brain extract. The proteins A and B have been reported to belocalized at the axons and post-synapses of neurons. We finally found that the proteins A andB both bound to the intracellular loop I of Nav1.1 using heterologous expressing systems with
HEK293 cells. Our findings may suggest that these Nav1.1-binding partners may be involvedin axonal localization of Nav1.1.
(COI:No)

3P-0083P-0114 1 Dept Physiol Sci and Mol
Kunitoshi Uchida1, 2，Makoto Tominaga2, 3，Jun Yamazaki1, （Yoshimichi Murata, Taiki Okabe, Ruka Kobayashi, Yoshio Maruyama（ Dept Physiol, Grad

The electrophysiological analysis of TRPM5 channel
Biol, Fukuoka Dental Col, Japan, 2 Div Cell Signal, NIPS, Japan, 3Thermal Biol, ExCELLS,
Japan, 4 Lab Vet Pharmacol, NIhon Univ, Japan）

TRPM5 is a monovalent cation-permeable channel activated by intracellular Ca2+, and its

activity is enhanced by temperature increase from 15℃ to 35℃. This channel is restrictedly
expressed in taste cells, the pancreas, the brainstem and olfactory epithelium, and is thought
to be involved in controlling membrane potentials. In taste cells, TRPM5 is expressed in type 2
taste cells, and involved in the signal transduction in the downstream of sweet taste receptor.
In this study, we analyzed TRPM5 by using whole-cell patch-clamp recording. We found thatTRPM5 is activated and irreversibly inactivated upon heat stimulation. The mechanism of
temperature-dependent inactivation could be different from that of voltage-dependent inactivation. Next, we analyzed TRPM5 channel protein electrophysiologically by using planar lipid
bilayer (PLB) method, which is one of the reconstitution systems. Thermosensitive activationand inactivation of TRPM5 channel protein were also observed in PLB method, suggesting that
other molecules contained in cells could not be required for temperature dependent activation(COI:No)and inactivation of TRPM5.

3P-009Characterization of disease-associated CFTR-mutations identiﬁed inJapanese cystic ﬁbrosis patients
Yoshiro Sohma, Rio Kimishima, Shogo Iwai, Hikaru Sohma, Nao Kobayashi, Kanako Nakao（Div Mol Therapeutics, Grad Sch Pharm, IUHW, Japan）

Cystic Fibrosis (CF) is reported to be very rare among Asiansand previous reports suggested
that the profiles of CF-caused CFTR mutations found in Japanese CF patients are different
from Caucasians.
At present twenty-four Japanese CF patients have been definitely identified their diseaseassociated CFTR mutations. Twenty-two mutations were identified in the CFTR proteins. 11
out of 22 mutations seemed to be in the class II and 10 mutations in the class III or IV.
Among the Japanese CF mutations, a massively deleted mutation lacking the coding sequences
along three exons without frameshift (dele 16-17b mutation) has been found with the highest
frequency (13 alleles out of 28 CF alleles). CFTR protein derived from the dele 16-17b CFTR
gene is expected to lack 153 amino acids from Gly970 to Thr1122 (Δ(G970-T1122)-CFTR). Most
importantly two non-consanguineous CF patients with homozygous dele 16-17b mutation havealready been found out of all 29 Japanese CF patients, which suggests a small but significant
population with heterozygous dele 16-17b mutation in Japanese.
In this study, we attempted to characterize effects of the Japanese-specific mutations on CFTR
protein. (COI:No)

proton transport in endoplasmic reticulum membrane
Sch Med, Tohoku Univ, Japan）

Many studies have revealed that pH regulation of intracellular membranous compartments is
critical for their function. Each organelle has own pH regulation mechanisms and maintains
their own pH for carrying out the function, although details are still unclear. Luminal pH
of ER (pHERL) is thought to about 7.2, close to cytosolic pH. It looks like resulting of absentof active regulation of H+ transport, i.e. pHERL is always passively following the cytosolic
pH. Though, regulation of pHERL and its mechanisms are not well understood. We tried toreveal the pHERL regulation agents using genetically encoded ratiometric fluorescent pH
probe, mCherrySEpHluorin-ER in nuclear envelopes (NEs: containing peri-nuclear endoplasmic
reticulum membrane). pH imaging of NE lumen was revealed that luminal pH followed pHchange of cytosolic side, but the degree of pH change was diminished against the pH changeof outside. For investigating the mechanisms of this proton transport, we carried out intra- andextraluminal ion substitution experiments. (COI:No)

3P-012

Prostaglandin E2 receptor EP4 induced Ca2+ inﬂux from theextracellular space via Orai1 and regulates the cell migration in oral
cancer cells
Kohei Osawa1，Masanari Umemura1，Rina Nakakaji1, 2，Kenji Mitsudo2，Yoshihiro Ishikawa1（ 1 Dept CVRI, Grad Sch Med, Yokohama City Univ, Japan, 2 Dept OMS, Grad Sch Med,
Yokohama City Univ, Japan）

Introduction: The EP4 prostanoid receptors is one of the four receptor subtypes for Prostaglandin E2 (PGE2). EP4 may play an important role in cancer progression. Its inhibition is apotential strategy for cancer therapy. However, little information is available regarding thefunction and cellular signaling pathway of EP4 in oral cancer. In this study, we show that EP4
signal regulates cell migration and metastasis in oral cancer.
Material and Method: Human-derived tongue squamous cell carcinoma cell lines, HSC-3 andOSC-19 were used. Changes intracellular Ca2+ level were measured by Fura-2, a Ca2+-sensing
fluorescent dye. Migration was examined with the scratch assay. Western blot was also
performed. In order to ablate EP4 or Orai-1, shRNA was induced with lentiviral infection in
HSC-3. In animal experiments, we established lung metastasis model mice to evaluate metastatic ability. Immunoprecipitation for EP4, Orai1 and transient receptor potential canonical 1(TRPC1) were performed.
Result: The EP4 agonist (ONO-AE1-437), rapidly increased intracellular Ca2+. EP4 agonist
promoted cell migration and increased phosphorylation of ERK in HSC-3. In contrast, EP4knockdown significantly reduced the cell migration. EP4-Knockdown decreased lungmetastasis in mice. Orai1-knockdown also negated the EP4 agonist-induced Ca2+ elevation.
Immunoprecipitation showed that EP4 was colocalized and formed complexes to both Orai1
and TRPC1.
Conclusion: EP4 regulates intracellular Ca2+ elevation via Orai1 and TRPC1, resulting inpromoting cell migration of oral cancer. (COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S151

3P-013

3P-016

Asparagine-linked glycosylation as a key regulator of gating properties
in cardiac Nav1.5 channels

ATP- and voltage-dependent gating of P2X2 receptor analyzed byvoltage-clamp ﬂuorometry

Pu Wang, Yangong Liu, Mengyan Wei, Shinichiro Kume, Tatsuki Kurokawa,
Katsushige Ono（Deptment Physiopathology, Grad Sch Med, Oita Univ, Japan）

2 1 Div Biophys and Neurobiol, Natl Inst Physiol
Rizki Tsari Andriani1, 2，Yoshihiro Kubo1, （Voltage-gated sodium channels are composed of a pore-forming α subunit and auxiliary β
subunits. SCN5A gene encodes a pore-forming α subunit of the voltage-gated sodium channel
Nav1.5 which is known to play an important role in human cardiac myocytes. In the Nav1.5
channel, several potential glycosylation sites are postulated including the asparagine-linked(N-linked) glycosylation. Although the N-linked glycosylation is one of the common post-translational modifications, the detail of their functions on the Nav1.5 channel are not been understoodwell. We applied the whole-cell patch-clamp technique to analyze the effects of the N-linked
glycosylation inhibition on the human Nav1.5 channel expressed in HEK293 cells. In our study
we observed that the addition of tunicamycin, a specific and potent inhibitor of N-linked glycosylation, increased the Nav1.5 channel current amplitude when the cells were incubated for 24hours. Aslo a significantly shift of the steady-state inactivation curve to the hyperpolarization
direction was observed, whereas the activation curve was unaffected. Recovery from inactivation was prolonged by tunicamycin treatment, where the fast phase (τfast) was unaffected
and the slow phase (τslow) was prolonged. Decay of Nav1.5 channel current was unaffected bytunicamycin. When the ubiquitin-proteasome pathway was inhibited with a proteasome inhibitor MG132 [N-[(phenylmethoxy)carbonyl]-L-leucy-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide],tunicamycin could not modify Nav1.5 current any further. These findings suggest that maturation and degradation of Nav1.5 channel protein depends on N-glycosylation for the channel
kineties.
(COI:No)P2X2 is a ligand-gated ion channel activated by extracellular ATP. This receptor shows acomplex gating depending on both [ATP] and voltage, in spite of the absence of a canonicalvoltage sensor domain. It remains unknown how the structural rearrangements occur during
voltage-dependent gating. Thus, in the present study we aim to analyze the structural rearrangements of P2X2 receptor upon ATP- and voltage-dependent gating, by voltage-clamp
fluorometry (VCF). We used a fluorescent unnatural amino acid (fUAA) named Anap as afluorophore which can be directly incorporated into the channel protein in Xenopus oocytes.
In addition to that, to improve the VCF recording optical signal by decreasing the intrinsic
background fluorescent of oocytes, a small molecule kinase inhibitor named HG-9-91-01 (SIK
inhibitor) was applied. We then observed Anap fluorescence intensity changes associatedwith voltage changes at Ala337 and Ile341 in the 2nd transmembrane domain (TM2). Thechanges upon step pulse application from Ala337Anap and Ile341Anap showed a much fasterkinetic than the voltage-dependent current activation. Moreover, both changes showed a linear
voltage- dependent behavior. These changes might indicate a phenomenon related to electrochromic effect, implying that there is an electric field convergence at the position of Ala337 andIle341 during P2X2 receptor complex gating. Voltage-dependent fluorescence change at Ala337
was larger in the absence of ATP than in the presence of ATP, reflecting the ATP-dependentchange of the focused electric field. Mutagenesis studies at Ala337 in TM2 and its possible
counterpart, Phe44 in TM1, suggested that the interaction between Ala337 and Phe44 in the
open (ATP-bound) state is important for the complex gating of P2X2 receptor. It is possiblethat Phe44 swings into the focused electric field and that the interaction between Ala337 andPhe44 upon ATP binding, which contributes to the opening, might be under the control of(COI:No)
membrane voltage.

3P-0143P-017α-mannosidase I-dependent N-linked glycosylation modiﬁes distinct

Sci, Okazaki, Japan, 2 Dept Physiol Sci, SOKENDAI, Hayama, Japan）

gating properties of the hERG channel

Modulations of ion channel function by Sigma-1 receptor, a multimodal
membrane protein

Mengyan Wei, Yangong Liu, Pu Wang, Shinichiro Kume, Tatsuki Kurokawa, Katsushige Ono

2 1 Div. Biophys
Chang Liu1, 2，I-Shan Chen1, 2，Ruth Murrell-Lagnado3，Yoshihiro Kubo1, （（Dept Morbid Physiol, Grad Sch Med, Oita Univ, Japan）

Asparagine-linked (N-linked) glycosylation has emerged as an essential post-translational
modification to control the number of channels embedded in the plasma membrane as well as
to regulate their functional gating properties. The human ether-a-go-go related gene (hERG)
channel is known to form the major subunits of the rapidly activating delayed rectifier potassium current (IKr) in cardiomyocytes. hERG channel proteins are initially synthesized in theendoplasmic reticulum, where they undergo N-linked core glycosylation to form immaturechannel proteins. Then the immature proteins are trafficked to the Golgi apparatus, during
which they undergo the complex processes of glycosylation to become fully glycosylated
mature proteins. Although many studies have reported functions of N-linked glycosylation, theexact role of α-mannosidase I-dependent N-linked glycosylation processed in Golgi apparatus
on the hERG channel is not well understood. We analyzed the effects of N-linked glycosylation
inhibitions on the hERG channel under the whole-cell patch-clamp technique using the heterologous expression system in HEK293 cells. The whole inhibitions of N-linked glycosylation withtunicamycin, an inhibitor of the synthesis of dolichol-PP-GLcNAc, decreased hERG channel
current amplitude when the cells were incubated for 48 hour. However, kifunesine, a selective
inhibitor of class I α-mannosidase, did not reduce the current amplitude; kifunesine modified
the gating property of the hERG channel when the cells were incubated for 48 hour. Morespecifically, kifunesine reduced the tail current of hERG channel when pluse protocol was
designed to evaluate the reactivation of the channel. These results lead to the conclusion thatα-mannosidase I-dependent N-linked glycosylation in the Golgi apparatus plays a key role in(COI:No)
modifing the distinct gating property of the hERG channel.

3P-015N-glycosylation inhibition attenuates heart automaticity by deranging
T-type Ca2+current and HCN current
Yangong Liu, Pu Wang, Mengyan Wei, Shinichiro Kume, Tatsuki Kurokawa, Katsushige Ono

and Neurobiol, NIPS, Okazaki, Japan, 2 Physiol. Sci, SOKENDAI, Hayama, Japan, 3 Schoolof Life Sciences, University of Sussex, Brighton, UK ）Sigma-1 receptor (S1R) is a protein expressed mostly on the endoplasmic reticulum membrane.
It is implicated in many psychiatric and neurological disorders including drug addiction,
juvenile amyotrophic lateral sclerosis and Alzheimer’s disease. S1R has been reported todirectly or indirectly interact with ion channels such as Kv channel family members andKir2.1 channel, while the underlying mechanisms are unknown. To examine the influence of
S1R on various Kv and Kir channels, we performed experiments using Xenopus oocytes as anin vitro expression system and observed the following results. (1) By immunohistochemical
staining, we confirmed the S1R expression in the oocytes injected with S1R cRNA. (2) Byelectrophysiological recordings, we observed that coexpression of S1R suppresses the current
amplitude of Kir3.1/Kir3.2 in a S1R expression level-dependent manner. (3) We observed that
coexpression of S1R diminishes the current amplitude of Kv2.1 but not those of Kv1.1 and Kv1.3.
(4) Mutations of Ser583 or Ser586 in Kv2.1 to Ala are known to disrupt the clusters of Kv2.1 andto increase the total current amplitude. However, we observed that S1R still decreases the current amplitude of the mutants, suggesting that the effect of S1R on Kv2.1 is not relevant to thechannel clustering. Taken together, our present data suggest that S1R modulates the functionof Kir3.1/Kir3.2 and Kv2.1 channels, but not those of Kv1.1 and Kv1.3 channels. These results
further our understanding of the modulation mechanisms of ion channels by S1R.
(COI:No)

3P-018Asparagine-linked glycosylation modiﬁes voltage-dependent gating
properties of CaV3.1-T-type Ca2+ channel
Shinichiro Kume, Yangong Liu, Pu Wang, Tatsuki Kurokawa, Katsushige Ono（Dept Physiol, Grad Sch Med, Oita Univ, Japan）（Dept Patphysiol, Grad Sch Med, Oita Univ, Japan）

Asparagine-linked glycosylation (N-glycosylation) is an essential post-translational modification
for a large number of crucial proteins in numerous physiological processes, and 20 percent of
the congenital disorders of glycosylation cases have been reported including cardiac complications, such as arrhythmias, cardiomyopathies, and structural defects. To know its cardiac
functions, we studied the effects of N-glycosylation defect on rat hearts and cardiomyocytes
using tunicamycin. The synthesis of glycoprotein and inhibition of protein glycosylation bytunicamycin were examined in in vivo and in vitro administration. We observed that thedaily abdominal injection of tunicamycin (0.1 mg/100 g) caused a gradual slowing down of theheart rate and severe bradycardia in rats within days. Further electrophysiological study inneonatal rat cardiomyocytes revealed that tunicamycin application rendered an attenuationof cardiomyocytes automatic beating, as well as reduction of T-type Ca2+current and theHCN current, which implies that N-glycans deficiency may cause bradycardia. Moreover, thecalnexin and calreticulin chaperone system in the endoplasmic reticulum could be postulated asa mechanism for a well assignment of N-glycans existing in CaV3.1 channel, the dominant isoform of T-type Ca2+current channels in the adult mammals. In conclusion, pacemaker-related
ion channel currents, T-type Ca2+current and HCN current, may therefore play roles in thepathogenesis of congenital disorders of glycosylation in the heart.
(COI:No)

T-type channels are low-voltage-activated channels that play a role in the cardiovascularsystem particularly for pacemaker activity. Glycosylation is one of the most prevalent posttranslational modifcations in protein. Among various glycosylation types, the most common one
is asparagine-linked (N-linked) glycosylation. The aim of this study was to elucidate the rolesof N-linked glycosylation for the gating properties of the CaV3.1-T-type Ca2+ channel. N-linked
glycosylation synthesis inhibitor tunicamycin causes a reduction of CaV3.1-T-type Ca2+ channel
current (CaV3.1-ICa.T) when applied for 12 h or longer. Tunicamycin (24 h) signifcantly shifted
the activation curve to the depolarization potentials, whereas the steady-state inactivation
curve was unafected. Use-dependent inactivation of CaV3.1-ICa.T was accelerated, and recovery
from inactivation was prolonged by tunicamycin (24 h). CaV3.1-ICa.T was insensitive to a
glycosidase PNGase F when the channels were expressed on the plasma membrane. Thesefndings suggest that N-glycosylation contributes not only to the cell surface expression of
the CaV3.1-T-type Ca2+ channel but to the regulation of the gating properties of the channel
when the channel proteins were processed during the folding and trafcking steps in the cell.
(COI:No)S152

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

3P-019

Contradictory responses of aortic Beta and iliac-femoral Beta during
diltiazem administration in rabbits
Shin-ichiro Katsuda1，Yuko Horikoshi2，Yuko Fujikura3，Akihiro Hazama1，Tsuyoshi Shimizu4，
5 1 Dept Cellular ＆ Integrative Physiol, Fukushima Medical Univ. Sch Med, Japan,
Koji Shirai（
2 Dept of Lab Med, Fukushima Med Univ Sch Med, Fukushima, Japan, 36th-Year Med

Student, Fukushima Med Univ Sch Med, Fukushima, Japan, 4 Inst Shimizu Space Physiol,
Suwa Maternity Clinic, Simosuwa-machi, Japan, 5 Seijinkai Mihama Hospital, Chiba, Japan）

3P-022

Carvedilol inhibits the emergence of waves resembling abnormal
Q waves, and the spread of inﬂammation and ﬁbrosis induced byisoproterenol toward the epicardium
Takeshi Yamaguchi1，Ryuichiro Mukai1, 2，Chinatsu Hiraoka1, 3，Hideyuki Nakagawa4（ 1 Lab Anat ＆ Physiol, Fac Nurs, Shikoku Univ, Japan, 2 Kidai Univ Hosp, Osaka, Japan,
3 Kitajima Taoka Hosp, Tokushima, Japan, 4 Dept Pharmacol, Fac Nurs, Shikoku Univ, Japan）

Background and purpose:The regulatory mechanism of the stiffness of the elastic and muscular arteries during calcium channel blocker has not been clarified. By applying the theoryof cardio-ankle vascular index (CAVI), we have established new arterial stiffness indices inthe entire aorta (aBeta) and in the iliac and femoral arteries (ifBeta). The effect of diltiazem on
aBeta and ifBeta was clarified using rabbits.
Methods: Fourteen male rabbits aged 10-12 months were studied under pentobarbital anesthesia. Pulse waves at the origin of the aorta (oA), distal abdominal aorta (dA) and distal end ofleft femoral artery (fA) and flow waves at oA were simultaneously recorded before and after
the intravenous infusion of diltiazem at 50 µg/kg/min for 2 min via the ear vein. PWV in theentire aorta (aPWV), from dA to fA (ifPWV) and from oA to fA (afPWV) was determined by
the difference in the rising time of two pulse waves and distance of two pressure sensors. Beta
was determined as Beta=2ρ/ΔP×ln(SBP/DBP)×PWV2 (ρ: blood density, SBP, DBP and
ΔP: systolic, diastolic and pulse pressures).
Results: When diltiazem was administered, blood pressure and pulse rate decreased. aBeta
increased significantly while ifBeta decreased significantly. Aortic-femoral Beta (afBeta) did notshow significant change despite the infusion of diltiazem.
Conclusion: During diltiazem administration, contradictory responses of aBeta and ifBeta
were observed, suggesting that there exist co-relating regulatory mechanism in which elastic
artery of central site contracted responding to dilated peripheral muscular artery by diltiazem.
(COI:No)

Purpose: This study examined the involvement of carvedilol (Crv) in the emergence of abnormal Q waves and the spread of inflammation induced by isoproterenol (Iso).
Materials ＆ Methods: Male SD rats were administered 20 mg/kg of Crv orally for 1 week.
The control (Con) groups were administered DMSO. After 1 week, they were subcutaneously
injected with 50 mg/kg of Iso. The groups continued to orally receive Crv or DMSO, respectively, for 1 week. ECG was recorded using standard lead II before and after Iso injection. ECG
was recorded again after 1 or 4 weeks and rats were perfused with 4% paraformaldehyde.
The heart was embedded in paraffin, and cardiac tissues were stained by HE (hematoxylin andeosin) and Masson-Goldner stain.
Results: Both groups exhibited almost the same ECG waves at 5 min after Iso injection. After
4 weeks, Con groups had developed waves resembling abnormal Q waves, but Crv groups had
not. The amplitude of QR waves in the Con group was more than 20% of the R wave amplitude.
However, in the Crv group, that of QR waves was less than 20% of that of R waves. The QRS
intervals were prolonged after Iso injection in the Con groups, but not as prolonged in the Crv
groups. The subendocardial tissues of the heart were infiltrated with inflammatory cells 48
h after Iso injection. Inflammatory cells were found near the epicardium after 4 weeks in the
Con groups, but few cells were found in the Crv groups. On Masson-Golder staining, a positive
area in the epicardium was observed in the Con group, but it was smaller in the Crv group.
Conclusion: These results suggest that Crv can inhibit the emergence of waves resemblingabnormal Q waves and the spread of inflammation induced by isoproterenol toward the(COI:No)epicardium.

3P-0203P-023

Hirotsugu Tsuchimochi1，Takashi Sonobe1，Takahiro Higuchi2，James T Pearson1

1 1 Dept Bioinfo,
Takao Shimamoto1，Kunichika Tsumoto2，Yasutaka Kurata2，Akira Amano（Coll Life Scis, Ritsumeikan Univ, Japan, 2 Dept Physiol, Kanazawa Med Univ, Japan）

The Goto-Kakizaki (GK) rat, a type 2 diabetes model, does not develop diabetes during early
postnatal period, but as it develops, it exhibits fasted hyperglycemia and impaired glucose
tolerance due to decreased insulin secretion. The purpose of this study was to investigate
the effects of low-intensity endurance exercise on the development of diabetes in young GK
rats. Four-week-old male GK rats (n=5) were subjected to low intensity endurance exercise 5days a week for 8 weeks. At 12 weeks of age, an oral glucose tolerance test (OGTT), endurance exercise test, echocardiography, and left ventricular pressure-volume (P-V) relationshipanalysis were performed. Transcriptome analysis was performed using isolated left ventricular(LV) myocardium. A group of sedentary animals (n=7) were used as a sedentary control
group, and male Wistar rats were used as a strain comparison group. Compared to Wistar
rats, GK rats showed a mild increase in fasting blood glucose at 4 weeks of age. Endurance
training improved endurance capacity in both GK and Wistar rats, but abnormal glucose
tolerance in GK rats did not improve except for a slight decrease in blood glucose levels 30 to
90 minutes after OGTT. Echocardiography and P-V loop analysis showed no effect of exercise
training on heart morphology and function in 12-week-old GK rats. Comprehensive analysis
of LV myocardial mRNA expression showed that the change in mRNA expression level wasmuch greater based on strain differences than the effect of exercise training. Since GK rats
genetically develop diabetes, the effect of exercise training on the suppression of diabetes waslimited. In order to examine the effect of exercise training on the prevention of the onset of
diabetes, therefore, we conclude that it is necessary to investigate diabetes models that arecaused by environmental factors.
(COI:No)

Abnormalities of action potentials (APs) in cardiomyocytes and of its AP propagation lead to
cardiac arrhythmias. It is believed that early afterdepolarizations (EADs) which transiently
depolarizes during the AP repolarization phase triggers fatal arrhythmias such as Torsades dePointes (TdP). Although, generative mechanisms of EADs have been intensively investigatednumerous experimental and theoretical studies, the mechanism of EAD development-mediatedarrhythmogenesis in the ventricle is not fully understood. To examine how EADs that evoked
locally in ventricular tissue lead to arrhythmias, we constructed a mono-domain sheet model (6cm × 6 cm) consisting of 360,000 human ventricular myocardial units and performed computer
simulation of AP propagation. In the present study, we investigated how the numbers andlocations of myocardial units evoking EAD in the myocardial tissue related the arrhythmogenesis. When myocyte units evoking EAD were located at the center of myocardial sheet asa square area and about 80% units in the myocardial sheet occurred coordinately EADs, theEADs occurrence followed by spiral wave-like excitations, i.e., reentrant arrhythmias, were
triggered. Furthermore, we found that if EAD evoked myocyte units account for the off-centerof the myocardial sheet, the reentrant arrhythmias can be triggered with fewer EAD evokedunits (25% of the total myocyte units). These results suggested that not only the numbers of
myocytes evoking coordinately EADs but also the location causing EADs may be involved in
fatal arrhythmia onsets.
(COI:No)

3P-021

3P-024

Akira Hanashima1，Yoshihiro Ujihara2，Momoko Ohira1，Misaki Kimoto1，Aya Kodama1，
1 1 First Dept Physiol, Kawasaki Med Sch, Japan, 2 Dept
Ken Hashimoto1，Satoshi Mohri（

2 1 Lab Physiol, Dept Health Sci at Fukuoka, IUHW,
Sachio Morimoto1，Kengo Hayamizu（

Effect of endurance exercise training on the development of diabetic
cardiomyopathy in young Goto-Kakizaki rats
（ 1 Dept Cardiac Physiol, NCVC, Suita, Japan, 2 Julius-Maximilian’s University of Wurzburg）

Evolution of the coronary circulation hearts by shortening the elastic
regions of connectin
Elec Mech Eng, Nagoya Inst Technol, Japan）

Vertebrate hearts are classified into two types: coronary circulation hearts and sinusoidal
circulation hearts. Though the sinusoidal circulation hearts take oxygen and nutrients intospongiosa myocardium directly from heart lumen, the coronary circulation hearts take theminto compacta myocardium via blood vessels, which enables heart to act as a strong pumpin mammals and birds. The emergence of coronary circulation is thought to have promoted
the vertebrate evolutions and the mammal births. However, the appearance time and theappearance reason of the coronary circulation are unknown because the hearts did not remain
as fossils. The extensibility of myocardium, which regulates the pump function of heart, isdetermined by the passive tension of the elastic protein connectin. Therefore, we thought thatthese problems could be clarified by comparatively investigating the elastic region of connectin.
In this study, we focused on connectin in cartilaginous fish hearts. Our analysis for gene anddomain structures found that the elastic region of elephant shark connectin had shorter PEVK
segment and fewer numbers of Ig domains than those in connectins of mammals and birds.
However, the RT-PCR experiments elucidated that the most of these different components
were spliced out in elephant shark heart, and become similar domain structure to that in adult
heart of mammals and birds. Our microscopic observation found that the elephant shark hearts
had coronary artery and consisted of the compacta myocardium, and the sarcomere structures
and the connectin localizations were similar to those in mammals and birds. Therefore, it is possible that the coronary circulation of cartilaginous fishes appeared independently of mammals
and birds, and the uniquely evolved elastic region of cartilaginous fishes connectin had beenshortened by heart-specific splicing to make a similar compacta myocardium to mammals andbirds as a result of the convergent evolution.
(COI:No)

Coordinately early afterdepolarizations evoked in ventricular tissue
trigger reentrant arrhythmias:in silico study

Role of a functional SNP of the gene coding brain serotonin synthesis
rate-limiting enzyme Tph2 in dilated cardiomyopathy
Japan, 2 Dept Anesthesiology and Crit Care Med, Kyushu Univ Grad Sch Med）

Brain serotonin dysfunction is involved in depression, which has been shown to increase therisk of death in heart failure (HF) patients. Tryptophan hydroxylase 2 (TPH2) expressed
specifically in the central nervous system is involved in serotonin synthesis in the brain asa rate-limiting enzyme. Inbred mice have a functional single nucleotide polymorphism (SNP)
C1473G in Tph2, with the Tph2 activity being lower in BALB/c strain with G/G allele than in
C57BL/6 mice with C/C allele. In this study, we examined the role of this SNP in the diseasephenotype of knockin mice with a dilated cardiomyopathy (DCM)-causing mutation ΔK210
in cardiac troponin T (cTnT) by creating single-gene congenic strain with Tph2 1473C/C or
G/G allele. The ΔK210-cTnT DCM mice on C57BL/6 background frequently suffered fromsudden cardiac death (SCD) with no heart failure symptoms, whereas the ΔK210-cTnT DCM
mice on BALB/c background mostly died of congestive HF instead of SCD. Introductionof Tph2 1473G/G allele into C57BL/6 background DCM mice caused congestive HF death
while decreasing SCD and extending the life expectancy. On the other hand, introductionof Tph2 1473C/C allele into BALB/c background DCM mice caused SCD while decreasing
congestive HF death and shortening the life expectancy. These results strongly suggest thatbrain serotonin function plays an important role in the disease phenotype of DCM. (COI:No)

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S153

3P-025

Atrial arrhythmia induced by pilocarpine application on footpads offreely behaving miceShinichi Sato, Yosuke Okamoto, Kyoichi Ono（Dept Physiol, Grad Sch Med, Akita Univ,
Japan）

Purpose: Previously, we reported that pilocarpine could penetrate through footpads of mouseand induce arrhythmia noninvasively by using a new multi-dry-electrode plate electrocardiogram (MDEP)-sensor system. In the present study, we discuss the characteristics of arrhythmia induced noninvasively in freely behaving mice.
Methods: Electrocardiogram (ECG)s of freely-behaving C57BL/6J mice were individually
recorded for one h by the MDEP-sensor system following free walking in a cage on the paper
soaked with 2% pilocarpine solution for 10 min. Fifteen ECG signals and one piezoelectric
sensor signal from the MDEP-sensor were stored in a PC via a 16 channel A/D converter.
Results: Four mice out of seven mice examined showed arrhythmia from the beginning of ECG
recording immediately after the pilocarpine treatment. A sudden increase in R-R interval up
to approximately 160 – 180 % frequently appeared among the R-wave trains with a frequency
of around 600 bpm. The relative incidence of arrhythmia declined about 30 min after the startof recording. The pilocarpine-induced arrhythmia is likely to be classified as atrial arrhythmia
according to the observation of ECGs obtained by the MDEP-sensor system. In addition, some
ECG traces may suggest the reentrant activity in the atria.
Conclusion: Pilocarpine acts as a muscarinic receptor agonist, which stimulates the secretion ofsaliva and sweat and is generally used to treat dry mouth and glaucoma. In the present study,
we first demonstrate the usability of pilocarpine to induce arrhythmia in freely behaving mice.
Pilocarpine should permeate the footpad skin and vascular walls and should be delivered byblood flow to the vicinity of the heart. However, it is not clear yet the role of pilocarpine for the
induction of arrhythmia. Further study is needed to understand the mechanism of arrhythmia
induction by pilocarpine.
(COI:No)

3P-028Development of a new treatment for dilated cardiomyopathy by mutatedtroponin T replacement
Yuya Yamaguchi1，Jun Tanihata1，Susumu Minamisawa2，Shunsuke Baba2，Sachio Morimoto3（ 1 Division of Aerospace of Medicine, Department of Cell Physiology, The Jikei University
School of Medicine, 2 Department of Cell Physiology, The Jikei University School of
Medicine, 3 School of Health Science, International University of Health and Welfare）

Introduction: Dilated cardiomyopathy (DCM) is characterized by cardiac dilation and pump
failure. Fundamental therapy for DCM has not been established. Especially, DCM whichdevelops in young age has poor prognosis. Cardiac troponin T (TNNT2) amino acid mutation(ΔK210) knock-in mouse (TNNT2ΔK210/ΔK210) generated by Morimoto et al., has the similar
phenotypes as human juvenile DCM. A previous report showed that overexpression of mutant
TNNT2 in wild type mice caused DCM phenotype, suggesting that overexpression of normal
TNNT2 in TNNT2ΔK210/ΔK210 mice may improve DCM phenotypes.
Purpose: The aim of this study is to investigate whether replacing the mutant TNNT2 withnormal TNNT2 overexpression would improve the DCM phenotypes caused by the mutant
TNNT2.
Methods and Results: No difference was observed in the cardiac weight and function by echocardiography of human TNNT2 overexpression (Tg) mice compared to non-Tg mice. Next, wemated with Tg mice and TNNT2+/ΔK210 mice to generate Tg/TNNT2ΔK210/ΔK210 mice. The
life span of Tg/TNNT2ΔK210/ΔK210 mice was slightly extended compared to TNNT2ΔK210/ΔK210
mice. In the Tg/TNNT2+/ΔK210 mice, the cardiac weight was significantly lower compared with
TNNT2+/ΔK210 mice at 7 weeks of age. Moreover, the expression levels of ANP mRNA in Tg/
TNNT2+/ΔK210 mice were decreased compared with those of TNNT2+/ΔK210 mice at 7 weeks ofage. Echocardiographic examination revealed that left ventricular end-diastolic dimension tended
to be decreased and left ventricular fractional shortening was increased in Tg/TNNT2+/ΔK210
mice of 10-weeks-old compared to those of TNNT2+/ΔK210 mice.
Conclusion: These results suggest that DCM caused by mutant TNNT2 is partially improvedby overexpression of normal TNNT2. In future, we will examine whether normal TNNT2 genetransfer using virus vectors could improve the DCM phenotypes caused by mutant TNNT2.
(COI:No)

3P-026

3P-029Akira Kimura, Shingo Murakami（Dept. EECE, Faculty of Sci. ＆ Eng., Chuo Univ.）

Cheng-Kun Du1，Dong-Yun Zhan1，Sachio Morimoto2，Tsuyoshi Akiyama1，Mikiyasu Shirai1，
1 1 Natl. Cereb. Cardiovas. Ctr., Suita, Japan,, 2 Int Univ Health ＆ Welfare,James Pearson（

The simulation based prediction method for early afterdepolarization in
drug-induced arrhythmia
Drug-induced arrhythmia is initiated by early afterdepolarization (EAD) occurring under IKr
block and resulting prolongation of action potential duration (APD). Therefore, to predict druginduced arrhythmia caused by IKr blockers, action potential prolongation and IKr block havebeen used. However, although there are IKr blockers that prolonged the APD for the sameamount of time, some cause EAD and some do not. For example, amiodarone causes few EAD
and therefore is known to be a relatively safer drug. In contrast, terfenadine and bepridil cause
drug-induced arrhythmia and EAD much more than amiodarone. Based on the above, wehypothesized that one needs to consider other factors than IKr block to predict drug-induced
arrhythmia. In the present study, voltage-dependent property of ICaL was adopted as a factor
and we examined the effect of the voltage dependence on EAD, by using the mathematicalmodel of human ventricular. Firstly, we confirmed that EAD is initiated under bradycardia andaction potential prolongation in the simulation. Next, we confirmed that voltage-independent
ICaL block suppressed EAD. Finally, we used the three ICaL block models of amiodarone andterfenadine and bepridil. The results showed that the model of amiodarone suppressed EAD
but the others increased the occurrence of EAD. Analyzing the simulation results in detail,we found that the ICaL block being weak in hyperpolarization side by terfenadine and bepridil
contributed to the increase of EAD occurrence and that by amiodarone did to the decrease.
Therefore, when we predict drug-induced arrhythmia, not only APD prolongation but also
voltage-dependent property of ICaL block should be checked.
(COI:No)

3P-027Histamine Excites Neonatal Rat Intracardiac Ganglion Neurons Via
Activation of Non-Selective Cation Channels and Inhibition of M-type
K+ channels
3 1 Div Homeostatic Development, NIPs, 2 Sch Allied Health Sci, Kitasato Univ,
Aya Sato1, 2, （3 Dept Ped, Shiga Univ of Med Sci）

Coronary spasm is a potentially life-threatening medical emergency that has been infrequently observed in allergy-related incidents in both children and adults. Here, its underlying
mechanism is still poorly characterized although previous studies suggest that it is precipitatedby histamine receptor 1 (H1 receptor) activation. Given that cardiac functions are greatly
regulated by the autonomic nervous system and that intracardiac ganglia act as relay stations
for all parasympathetic input to the heart, we therefore investigated the effect of histamine on
neonatal rat intracardiac ganglion neurons in vitro. Under current-clamp conditions, applicationof 10 µM histamine depolarized the membrane potential and elicited accompanying repetitive action potential firing. This histamine response was almost completely abolished in thepresence of the H1 antagonist triprolidine (1 µM), but not the H2 antagonist ranitidine (3 µM)
or the H3 antagonist clobenpropit (3 µM). Furthermore, application of the H1 agonist 2-pyridylethylamine (30 µM) was able to mimic this histamine response. Under voltage-clamp conditions,histamine evoked inward currents when the membrane potential was held at -60 mV. The
removal of extracellular Ca2+ substantially increased current amplitude whilst the additionalremoval of extracellular Na+ completely abolished it. This is consistent with the involvementof cation channels that are non-selective but show a greater permeability to Na+ relative toCa2+. Separately, the amplitude of M-current deactivation, induced by a hyperpolarizing stepfrom a holding potential of -20 mV to -60 mV, was reduced similarly by the application of either
histamine or the M-current inhibitor XE-991. Notably however, XE-991 application had noeffect on the resting membrane potential. Taken together, these results indicate that histamine
facilitates the excitation of intracardiac ganglion neurons via distinct H1 receptor dependent
signaling pathways that ultimately activate non-selective cation channels and inhibit M-type(COI:No)K+ channels.

S154

Cardiac pathology in a knock-in mouse model for human hypertrophic
cardiomyopathy at early postnatal stages
Okawa, Japan）

Purpose: Hypertrophic cardiomyopathy (HCM) is the most common monogenic genetic
cardiac disease, with an estimated prevalence of 1:500 in the general population. However,
little is known about the disease progression of HCM in children. In previous studies, we foundthat S179F HCM knock-in mice had a higher mortality rate before weaning. Therefore, in thisstudy, we focused on disease progression of HCM during early postnatal stages.
Methods: Cardiac function was evaluated at 15 and 30 days old using echocardiography andpressure volume loop analyses. Protein levels were measured by western blotting assay.
Results: Echocardiography showed that LV internal diameter during end diastole and end
systole of the HCM mice were less than WT, and LV ejection fraction was greater than WT,
but LV wall thickness was not different between HCM and WT mice both at 15 and 30 days
old. Echocardiography also showed a decrease in E/A ratio and an increase in isovolumic
relaxation time at 15 and 30 days old HCM mice. In vivo cardiac catheter measurements
showed a decrease in LV dP/dtmin in 30 day old HCM mice. Myocardial disarray was observed
at 15 and 30 days old, but fibrosis was observed only in 30 day old HCM mice. Further, wefound decreases in protein levels of phosphorylated phospholamban at 15 and 30 days and in SR
Ca2+-ATPase of 30 day old HCM mice. There was however no change in proBNP expression
at both time points.
Conclusions: In this study, we found that there was LV diastolic dysfunction in HCM but noincreases in proBNP level and ventricular wall thickness that were both evident at 3 monthsof age. These suggest that targeted therapies for retarding disease progression in HCM would
be more beneficial when commenced early in postnatal life. (COI:No)

3P-030Role of TRPC channels on single cell mechanics in mousecardiomyocyte
1 1 Dept Physiol, Med,
Yohei Yamaguchi1，Gentaro Iribe1，Keiji Naruse2，Akira Takai（

Asahikawa Med Univ, Japan, 2 Dept Cardiovas Physiol, Grad Sch Med, Okayama Univ,
Japan）

An increase in preload induces a biphasic response in the heart. The short-term increase inpreload rapidly augments contractile force owing to the Frank-Starling mechanism (FSM) that
accelerates Ca2+ sensitivity of the contraction protein. A further long-term increase in preload
for several minutes to hours causes the increase in [Ca2+] i, leading to slow force response to
stretch (SFR), a further increase in the contractile force. The authors’ previous study identified the involvement of TRPC3 and TRPC6, known as mechanosensitive non-selective cation
channels, in SFR. However, the participation of TRPC3 and TRPC6 in FSM remains unclear.
To clarify the role of TRPC3 and TRPC6 in FSM, cardiomyocytes were enzymatically isolated
from either wild-type (WT), TRPC3 knockout (KO), or TRPC6 KO mice. Both cell ends were
held by two pairs of carbon fibers attached to both upper and bottom cell surfaces to apply
stretch to the cells. The cells were paced at 4 Hz and superfused in normal Tyrode solution at
37 ℃. Cell length and active/passive force calculated from carbon fiber bending was recordedin six different preload conditions. The effect of each genetic deletion on cellular contractility
and elastance was assessed by the slope of end-systolic force-length relation curve (ESFLR)
and end-diastolic force-length relation curve (EDFLR). The slope of ESFLR was significantly
steeper in TRPC6 KO mice than in WT mice, though it remained unchanged in TRPC3 KO
mice. Meanwhile, the slope of EDFLR was not significantly different between WT, TRPC3 KO,
and TRPC6 KO mice. These results suggest that TRPC6 regulates the contractile property
via modulating the increase in the contractile force controlled by FSM, while TRPC3 is not
involved in FSM.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

3P-0313P-034Hiromichi Takano, Hikaru Hashitani（Dept Cell Physiol, Grad Sch Med, Nagoya City Univ,

Mai Hatano1，Tomoyuki Miyazaki1，Waki Nakajima1，Tetsu Arisawa1，Yuuki Takada1，
Yoshinobu Ishiwata2，Masaki Sonoda3，Naoki Ikegaya3，Masaki Iwasaki4，Naoto Kunii5，
Yuichi Kimura6，Fumio Yamashita7，Masataka Taguri8，Hideaki Tani9，Nobuhiro Nagai9，
Teruki Koizumi9，Shinichiro Nakajima9，Makoto Higuchi10，Hiroyuki Uchida9，Takuya Takahashi1

Different EDH properties between rat gastroepiploic and mesentericarteryJapan）

Endothelium derived hyperpolarization (EDH) has more important role to regulate the vascular resistance than nitric oxide especially in the resistance arteries. The objective of thisstudy is to compare properties of the EDH among the blood vessels. 3rd or 4th branch of
mesenteric arteries (diameter 200 – 300 µm) and gastroepiploic arteries(diameter 100 - 200 µm)
were dissected from rat. These preparation were pinned down to the bottom of the chamber(0.5 mL volume) and superfused with warmed (35 – 36 ℃) Krebs solution. The membranepotential was measured using a conventional microelectrode technique. The electrode wasinserted from the adventitial side. Drugs were administered in the superfusate. All experiments were conducted in presence of Nω-nitro-L-arginine (100 µM). The resting membranepotential was similar in both blood vessels (-53 ± 5 mV in mesenteric artery vs -55 ± 3 mV
in gastroepiploic artery). Acetylcholine (ACh, 1 µM) produced an initial (10mV), followed by asecond phase (4 mV) of membrane hyperpolarization in both types of blood vessels. dicrofenac
Na (1 µM) inhibited the second phase of those hyperpolarization for both types of arteries.
1 µM TRAM-34, an intermediate conductance Ca2+ activated K+ channel inhibitor, inhibited theinitial phase of hyperpolarization for gastroepiploic artery only. In presence of 0.1 µM apamin,a small conductance Ca2+ activated K+ channel inhibitor,, ACh failed to produce the initial
phase of hyperpolarization for the mesenteric artery, while evoked smaller but significant
hyperpolarization for gastroepiploic artery. In presence of diclofenac, TRAM-34 plus apamin,
ACh produced no hyperpolarization for both types of blood vessels. These results suggest that
2 types of K+ channels to produce EDH have different contribution for the hyperpolarizationand that those contribution might be different between those types of blood beds.  (COI:No)

3P-032OU

Novel direct effect of SGLT inhibitor, Canagliﬂozin, on humanmyocardial redox state
Hidekazu Kondo1，Akoumianakis Ioannis2，Akawi Nadia2，Kotanidis Christos2，
Antonopoulos Alexios2，Channon Keith2，Casadei Barbara2，Antoniades Charalambos2（ 1 Department of Cardiology and Clinical Examination, Oita University, 2 Division of
Cardiovascular Medicine, University of Oxford）

Background: Recent clinical trials have demonstrated an effect of sodium glucose cotransporter 2 (SGLT2) inhibitors in improving cardiovascular outcomes of heart failure patients, but
the exact mechanisms remain unknown.
Aim: We investigated the direct effects of Canagliflozin, a non-selective SGLT1/SGLT2 inhibitor on myocardial redox signalling in humans.
Methods: In Study-1, RAA samples from 51 patients were used in an ex vivo system to study
the direct effects of canagliflozin on O2.- generation and understand its role in controlling theactivity of NADPH-oxidases and uncoupled nitric oxide synthase (NOS). To further study theunderlying mechanisms mediating our findings, we then used human cardiomyocytes (hCM) ina series of in vitro experiments (Study 2).
Results: Ex-vivo 1-hour incubation of human myocardium with Canagliflozin significantly
reduced basal and NADPH-oxidase-derived O2.- and improved nitric oxide synthase (NOS)
coupling as determined by the L-NAME inhibitable O2.-. The effects on NADPH-oxidases
were mediated by AMP kinase-mediated reduction of membrane translocation of p47phox, andsuppression of Rac1 GTP-activation and consequent reduction of membrane translocationof Rac1. Canagliflozin also reduced tetrahydropterin oxidation, a NOS co-factor essential for
maintenance of NOS coupling, resulting into improved NOS coupling. These findings werereplicated in hCM, where canagliflozin was shown to regulate the AMP/ATP ratio, that could
be upstream of AMPK activation.
Conclusions: We now demonstrate for the first time in humans that Canagliflozin suppresses
myocardial NADPH-oxidases activity and improves NOS coupling through an AMPK/Rac1(COI:No)-mediated pathway in the human myocardium.

Positron Emission Tomography Tracer for AMPA receptors
Characterizes Psychiatric Disorders in Human（ 1 Dept Physiol, Grad Sch Med, Yokohama City Univ, Japan, 2 Dept Radiol, Grad Sch Med,
Yokohama City Univ, Japan, 3 Dept Neurosurgery, Grad Sch Med, Yokohama City Univ,
Japan, 4 Dept Neurosurgery, National Center Hospital of Neurology and Psychiatry, Japan,
5 Dept Neurosurgery, Grad Sch Med, Tokyo Univ, Japan, 6 Faculty of Biology-Oriented Scienceand Technology, Kindai Univ, Japan, 7 Division of Ultrahigh-Field, Institute for Biomedical
Sciences, Iwate Medical University, Japan, 8 Dept Data Science, Yokohama City Univ, Japan,
9 Dept Neuropsychiatry, Grad Sch Med, Keio Univ, Japan, 10 Dept Functional Brain Imaging
Research, National Institutes for Quantum and Radiological Science and Technology, Japan）

The glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor(AMPAR)
plays central roles in neuronal functions. However, clinical translation of AMPAR knowledge islimited due to the inability to visualize AMPAR in the living human brain. Here we developed apositron emission tomography(PET) tracer for AMPARs, named [11C] K-2, and showed its specific binding to AMPARs. Logan graphical analysis in first-in-human PET study with healthy
participants revealed reversible binding of [11C] K-2. Further, [11C] K-2 revealed systemic
reduction of AMPARs in patients with depression, while patients with schizophrenia exhibited
focal decrease of AMPARs in parahippocampal and cingulate gyrus. These decreases weresignificantly correlated with their symptomatology scores in both disorders. Thus, [11C] K-2
could be a useful tool to study biological base of psychiatric disease, and expected to be a novel(COI:No)diagnostic drug in the clinical setting.

3P-035The mechanisms of synaptic imbalance in pathophysiological state ofneuronal circuit at the prefrontal cortex in 15q11-13 duplication autism
model mice
1 1 Dept Pharmacol, Nippon Med Sch,
Fumihito Saitow1，Toru Takumi2，Hidenori Suzuki（

Japan, 2 RIKEN Center for Brain Science）

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits
in social communication and interaction and repetitive behaviors. A chromosome 15q11-13
paternal duplication (15q dup) mouse corresponding to cytogenetically frequent CNV in ASD
have core symptoms of ASD-like behaviors. Recently, we found that 15q dup mice showed ahyposerotonergic state in the brain and a reduction in the neuronal activity of the dorsal raphe
nucleus (DRN) neurons. Also, we found that administration of the SSRI fluoxetine during theearly postnatal period normalized the serotonin (5-HT) level in adulthood. Together with theserotonin restoration, impairments of electrophysiological properties in the DRN and a social
behavior were ameliorated. 5-HT is a well-known modulator of behavioral, physiological, andemotional functions at forebrain region. In this study, we investigated the regulatory mechanisms of synaptic excitatory/inhibitory (E/I) balance at the neuronal circuit in the prefrontal
cortex (PFC). Using whole cell recording from layer V pyramidal neurons in acute adult brainslices of PFC (~3 month-aged), we found the E/I balance shifted toward the excitatory in 15q
dup mice, consistent with our previous study observed in the sensory cortex. The imbalance of
E/I enhanced LTP of glutamatergic synaptic transmission. This enhancement was mimickedby partial blocking of GABAA receptors suggesting that the neuronal circuits in the PFC of
15q dup mice were in a reduced state of GABAergic inhibition. We also found that excitatory
synaptic inputs and 5-HT2 receptor-mediated modulation of parvalbumin-positive fast-spiking
interneurons (FSINs) contributed to control of optimal E/I balance. Taken together, theseresults provide new insights into the cellular mechanisms underlying maintenance of optimal
E/I balance in the PFC. (COI:No)

3P-033

3P-036

Teruyuki Fukushima（ Dept Physiol ＆ Biol Inform, Dokkyo Med Univ, Tochigi, Japan）

Kouhei Kitano, Yuka Nakaya, Masayuki Kobayashi（Dept Pharmacol, Sch Dent, Nihon Univ,

Peripheral nerve injury-induced effect on BK channel modulation of
GABAergic transmission in the superﬁcial dorsal horn of mice
Large-conductance calcium-dependent potassium (BK) channels are found in tissues including
nerves and smooth and cardiac muscles. Previously, we reported that BK channels locatedin presynaptic terminals reduce excitatory synaptic transmission in the superficial dorsal
horn (SDH) and that functional downregulation of BK channels accompanies neuropathic pain
induced by peripheral nerve injury. Here, we clarified the physiological significance of BK
channels in the modulation of GABAergic synaptic transmission in the SDH, where nociceptive
information is processed.
Spontaneously occurring GABAergic miniature inhibitory postsynaptic currents (sIPSCs) wererecorded from GFP-negative neurons located in the SDH of spinal cord slices from glutamate
decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice. The effect of a BK
channel blocker, iberiotoxin (IbTX), on GABAergic mIPSCs was analyzed.
IbTX increased the frequency of GABAergic sIPSCs without affecting their amplitude in the
sham-operated mice, suggesting that BK channels attenuated GABAergic synaptic transmission via presynaptic mechanisms.
We conducted similar experiments using sciatic nerve-ligated (SNL) neuropathic mice toclarify the role of BK channels in nociceptive information processing modulated by GABAergictransmission.
Before the IbTX application, the frequency of GABAergic mIPSCs in the SNL mice was significantly more than that in the sham-operated mice. During the IbTX application, the frequency
in the SNL mice was similar to that in the sham operated mice. Those would suggest that
SNL may induce functional downregulation of BK channels which suppress GABA release.
In the neurons that did not respond to IbTX, the both frequencies of GABAergic sIPSCs before
and during the application of IbTX in the sham-operated mice were significantly more thanthose in the SNL mice, which imply that SNL may decrease GABAergic synaptic transmission
in IbTX-unresponsive neurons by unknown mechanism except for BK channels.
Our results may clarify the underlying mechanisms of the nociceptive actions of BK channels(COI:No)in the SDH.

An optogenetic approach to investigate functional proﬁles of projection
from the insular cortex to the nucleus accumbens
Japan）The nociceptive information in orofacial region is transmitted to the trigeminal spinal nucleus
of caudalis, parabrachial nucleus, sensory thalamus, and cerebral cortex. Nociception is considered to be finally processed in several cortical regions: the primary and secondary sensory
cortices, and limbic cortex including the anterior cingulate and insular cortices. Interestingly,
the secondary somatosensory and insular cortices (S2/IOR) neurons send their axons to the
nucleus accumbens (NAc), a center of pleasure, and therefore, this pathway is considered tomodulate orofacial pain. However, little information is available in terms of the S2/IOR→NAc
pathway including physiological and pharmacological properties. We hypothesized that acetylcholine that control neuronal activities in the NAc modulates synaptic transmission from
S2/IOR neurons to NAc neurons, and the cholinergic modulation affects on limbic response
to nociceptive stimulation in orofacial region. To explore this hypothesis, we used a whole-cellpatch-clamp recording technique in combination with optogenetics. By injecting AAV5hSyn-ChR2(H134R)-mCherry into S2/IOR, S2/IOR neurons expressed ChR2 one month after
injection. Activation of ChR2 by blue light stimulation induced excitatory postsynaptic currents(EPSCs). Tetrodotoxin (TTX) application completely diminished EPSCs and application of
4-aminopyridine with TTX recovered EPSCs, suggesting that these EPSCs were mediatedvia monosynaptic connections. The glutamate receptor antagonist, DNQX, diminished EPSCs,
indicating that S2/IOR→NAc connections are mediated by AMPA receptors. Carbachol, acholinergic agonist, suppressed EPSCs, and this effect was blocked by atropine, a muscarin
receptor antagonist. Thus, cholinergic effect on the S2/IOR→NAc connection is suppressive
via musucarine receptors. This muscarinic effect might suppress limbic responses to oral pain.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S155

3P-037

Inhibitory local connections of parvalbumin-expressing neurons in the
rat globus pallidus
Tetsuya Higashiyama, Fuyuki Karube, Yasuharu Hirai, Fumino Fujiyama（ Lab Neural

3P-040Microglia mediate peripheral nerve injury-induced plasticity to thethalamusYoshifumi Ueta, Sachie Sekino, Yoko Katayama, Mariko Miyata（ Dept Physiol, Sch Med,

Circuitry, Grad Sch Brain Sci, Doshisha Univ, Japan）

Tokyo Women’s Med Univ, Japan）

The globus pallidus (GP) is known as a relay nucleus of the indirect pathway of the basal
ganglia (BG) circuit. The GP plays an important role for the control of action. The GABAergic
GP neurons can be classified into two subtypes on projection patterns. One type is a prototypicneuron that projects to the subthalamic nucleus and BG output nuclei. Another type is anarkypallidal neuron that solely projects to the striatum. Previous morphological and in vitro
electrophysiological studies showed that GP neurons had local GABAergic axon collaterals.
However, it is unclarified whether a strength and probability of the intra-GP connection dependon the subtypes and whether the connection affects the activity of each subtypes. To address
this question, we examined local synaptic inputs from parvalbumin (PV) expressing GP neurons(PV-GP neurons) to the two subtypes with in vitro whole cell recording. PV-GP neurons are themost numerous population of prototypic neurons which account for 70% of GP neurons. For
selective stimulation of PV-GP neurons, we combined Cre-dependent adeno-associated virus
expressing channelrhodopsin-2 and PV-Cre rats in which PV containing neurons express Cre
recombinase. We found that the photoactivation of PV-GP neurons elicited the optogeneticallyevoked inhibitory postsynaptic currents (oIPSCs) in both subtypes of post-synaptic GP neurons
in voltage clamp recording. The oIPSCs were mediated by GABAA receptors. The connection
probability and oIPSC amplitude were not significantly different between the subtypes ofpost-synaptic GP neurons. During 10Hz of the optical stimulations, PV-GP neurons suppressed
the firing rate of post-synaptic GP neurons in current clamp recording. These results indicatedthat the inhibitory local connections of PV-GP neurons might modulate inhibitory outputs
from prototypic neurons to downstream BG nuclei, and disinhibit the striatum via inhibition of(COI:No)arkypallidal neurons.

In the central nervous system, peripheral nerve injury reorganizes somatotopic representation.
We have previously reported in mice that the whisker deafferentation induced by transection of the infraorbital nerve disrupts topographic projections from the brainstem to thethalamic ventral posteromedial nucleus (VPM) by increasing non-whisker-information carrying’ectopic’ axons in VPM. However a mechanism mediating peripheral nerve damages to thiscentral reorganization is still unknown. Here we show the role of pathway- and region-specificmicroglial activation in the principal trigeminal nucleus (Pr5), a whisker sensory-recipient
brainstem region. Systemic or local manipulation of microglia reveals that reactive microglia
in Pr5 are necessary and sufficient for inducing ectopic axons in VPM. Microglia associatewith deafferentation-induced Pr5 neuronal hyperexcitability. Inactivation of Pr5 neurons can
suppress deafferentation-induced increase of ectopic axons in VPM in spite of microglial
activation in Pr5. Moreover, microglial activity is necessary for deafferentation-induced ectopic
hypersensitivity to lower jaw mechanical stimulation. Thus, brainstem microglial activity mediates nerve injury-induced plasticity to non-injured thalamic circuits and somatosensory-related
behavior.
(COI:No)

3P-0383P-041

Tomoko Oshima-Takago1, 2，Hirokazu Sakamoto 1，Shigeyuki Namiki1，Kenzo Hirose1，
2 1 Dept. of Pharmacology, Grad. Sch. of Medicine,
Masao Tachibana1, 2, 3，Hideki Takago1, （（Dept Physiol, Grad Sch Med, Yamaguchi Univ, Japan）

Optical measurement of glutamate release from multiple ribbon-type
synapses at the terminal of goldﬁsh retinal bipolar cell
The Univ. of Tokyo, Tokyo, Japan, 2 Dept. of Rehabilitation for Sensory Functions, ResearchInst., Nat’l Rehabilitation Ctr. for Persons with Disabilities, Saitama, Japan, 3 Ctr. for Systems
Vision Science, Organization of Science and Technology, Ritsumeikan Univ., Shiga, Japan）
Glutamate release at ribbon-type synapses in the retinal, cochlear and vestibular organs is
optimized for transmitting continuous sensory signals with graded potential changes. At
these synapses, presynaptic Ca2+ signals that trigger glutamate release show heterogeneity
within and across active zones (AZs). However, it is yet to be investigated whether glutamate
release from AZs at ribbon synapses shows heterogeneity. Furthermore, these cells exhibit
kinetically separate components of glutamate release: fast and slow components of release.
Besides, accumulating evidence from total internal reflection fluorescence (TIRF) microscopic
and/or electrophysiological studies indicates that ribbon-associated and ribbon-free AZs at theterminal of retinal bipolar cell might underlie fast and slow components of release, respectively.
This outlier hypothesis remains to be further tested ideally by visualizing the dynamics ofglutamate release at multiple AZs. Using a retinal bipolar cell (BC) specific enhanced glutamate
optical sensor (BC-eEOS), which has been optimized from the original eEOS, we here report
the establishment of optical measurement of glutamate signals from individual release sites at
the terminal of goldfish retinal bipolar cell. This novel imaging technique using the BC-eEOS
allowed us to find heterogenous nature of the fast and slow components of glutamate signals
at the ribbons, which were visualized with 5-TMRIA-conjugated CtBP-binding peptide. First,
the BC-eEOS fluorescence intensity for the fast component was variable across individual
ribbons. Second, the slow component was not observed at all the ribbons. Rather, sites forthe slow component appeared to be spatially confined. Third, the fast component occurred
predominantly at ribbon-associated sites, whereas slow component appeared to occur at both
ribbon-associated and ribbon-free sites. Taken these together, the novel imaging method using
BC-eEOS might enable us to analyze the fast and slow components of glutamate releaseat multiple ribbon-associated as well as ribbon-free sites in the retinal bipolar cell terminal.
(COI:No)

3P-039Mechanical stimulation-induced intercellular communication among
trigeminal ganglion neurons
Tatsuhiko Yazaki1, 2，Sadao Ohyama2，Wataru Ofusa2，Haruna Toda2，Hidetaka Kuroda3，
1 1 Dept Dent
Asuka Higashikawa2，Maki Kimura2，Yoshiyuki Shibukawa2，Tatsuya Ichinohe（
Anesthesiol, Tokyo Dent Coll, Japan, 2 Dept Physiol, Tokyo Dent Coll, 3 Dept Crit Care Med
Dent, Kanagawa Dent Univ）

Recently, paracrine communication between neurons and non-neuronal cells has been welldescribed. There has been a little report on the intercellular communication between neurons,
however. Although we have previously examined intercellular communication between trigeminal ganglion (TG) neurons by patch-clamp recordings, we could not record any responses
from nearby TG neurons to the stimulated ones. To examine possible intercellularcommnication via metabotropic receptors, we aimed to clarify whether the diffusible factors
or substances are capable to be released from the neurons by direct mechanical stimulation
to the single neurons or not using intracellular Ca2+ imaging. We dissected TG from neonatal
Wistar rats (7 days old) under pentobarbital sodium anesthesia (50 mg/kg with isoflurane). We
then acutely isolated TG cells, and the cells were primary cultured (5% CO2, 95% air, 37 ℃).
The cells ware loaded with fura-2, and the intracellular free Ca2+ concentration ([Ca2+] i) wasrecorded from the fluorescence (510 nm) intensity ratio at excitation wavelengths of 340 nm
and 380 nm. Direct mechanical stimulation to the TG cells increased [Ca2+] i. In addition, notonly directly stimulated neurons, but also the neurons which are located nearby stimulated
neurons showed an increase in [Ca2+] i. The results suggested that neurons that received
mechanical stimulation are capable to release diffusible intercellular transmitters to establish(COI:No)
inter-neuronal communication via metabotropic receptor activation. 

S156

Contextual learning requires phosphorylation at Ser408-409 of GABAA
receptor β3 subunit
Yuya Sakimoto, Ryota Sotomaru, Thein-Oo Paw-Min, Makoto Goshima, Dai Mitsushima
Although contextual learning requires plasticity at both AMPA receptor-mediated excitatory
and GABAA receptor-mediated inhibitory synapses in CA1 neurons, detailed mechanism of
the learning-induced plasticity at GABAA receptor-mediated synapses has been unknown.
We recently reported learning-induced increase in postsynaptic number of GABAA receptor channel (Sakimoto et al, Cerebral Cortex 2019), and phosphorylation of intracellular loop
(Ser408-409) of GABAA receptor β3 subunit (Sakimoto et al., FASEB J 2019). To further
examine the causal relationship among the Ser408-409 phosphorylation, synaptic plasticity,and the learning, we used cell permeable HIV tag peptide and synthesized with the novel
peptide-based phosphorylation inhibitor targeting sites at Ser408-409 (Tat pep β3-SS). Under
the freely-moving condition, we bilaterally microinjected the Tat pep β3-SS or site-specificmutated control (Tat pep β3-AA) into the CA1 region, and used them for the following behavioral test battery: contextual learning (IA task), emotion (open field task), perception (visualtask) and social behaviors (pairing test). Tat pep β3-SS but not Tat pep β3-AA impaired
the performance of retrieval test in IA task, while the effect was not observed in any otherbehavioral tests. Ex vivo whole cell patch clamp analysis further revealed that unilateral Tat
pep β3-SS microinjection clearly blocked the learning-induced increase in the postsynaptic
GABAA receptor-mediated Cl- current in CA1 pyramidal neurons.
These results suggest a causal relationship among the Ser408-409 phosphorylation, GABAA
receptor-mediated synaptic plasticity, and the learning. Understanding the functional role ofSer408-409 phosphorylation of the subunit might be beneficial for the drug discovery anddevelopment for multiple cognitive disorders.
(COI:No)

3P-042

Reduced glutamate uptake in cerebellar Purkinje cells
in Atp1a3 heterozygous knockout mice: glial compensation and its
impacts on long-term depression
2 1 Dept Physiol, Sch Med, Int Natl
Keiko Ikeda1, 2，ShinIchiro Satake3, 4，Kiyoshi Kawakami（
Univ Health Welfare, Japan, 2 Div Cell Biol, Center for Mo Med, Jichi Med Univ, Japan,
3 Natl Inst Physiol Sci, Okazaki, Japan, 4 Grad Univ for Advanced Studies, Fundamental
Neurosci, Japan）

Excitatory amino acid transporters (EAATs) are responsible for cellular uptake of neurotransmitter glutamate using the electrochemical gradients of sodium and potassium ions acrossthe cell membrane. Na, K-ATPase (Sodium pump) plays a key role in the maintenance ofthe gradient. Mutations in the human Sodium pump alpha3 subunit gene, ATP1A3, havebeen identified as the cause for rapid-onset dystonia with Parkinsonism (RDP) and alternating hemiplegia of childhood (AHC). The alpha3 subunit protein is abundantly expressed in
Purkinje cells (PCs), the sole output neuron of the cerebellar cortex. The pathophysiology ofthese disorders remains unknown. Here we focus whether EAATs relate to these sodium
pump neurological disorders. We examined the EAAT activity in the cerebellum in Atp1a3
heterozygous knock out mice (Atp1a3+/－). We found a remarkable reduction of the glutamate
uptake-coupled currents mediated by the EAAT4 subtype in PCs of Atp1a3+/－ comparedwith those of wild type litter-mates. On the contrary, the amplitude of EAAT currents in theastrocyte Bergmann glia (BG) was profoundly higher in Atp1a3+/－. Consistently, the protein
levels of the glia-specific EAAT1 subtype in the cerebellum were increased. Furthermore, in
Atp1a3+/－, long-term depression (LTD, postsynaptic origin) was diminished at the excitatory
synapses from parallel fibers (PFs) to PCs. The impaired LTD was rescued by application of
the EAAT1 inhibitor UCPH102 and the mGluR1 agonist DHPG. Taken together, it is suggested
that the enhanced glutamate uptake by BG submerged the weakened neuronal EAAT activity
in Atp1a3+/－, thereby attenuating the extracellular diffusion of the glutamate spilled out of the
PF-PC synaptic clefts and reducing the activation of the perisynaptically distributed mGluR1s
on PC dendritic spines. It is quite likely that these events interfere with LTD. Our findings
would provide underlying mechanisms for the onset of dystonia symptoms in RDP and AHC.

(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

3P-043

3P-046

Minori Fukuda1，Masako Ikutomo2，Ken Muramatsu3，Naomi Oshiro1，Masatoshi Niwa1

Israt Jahan, Ryota Adachi, Hiroshi Kuba（ Department of Cell Physiology, Graduate School ofMorphological study of motor neurons innervating the lumbar trunk
muscles in rats
（ 1 Department of Occupational Therapy, Kyorin University, Tokyo, Japan, 2 Department of
Rehabilitation, Tokyo Health Science University, Tokyo, Japan, 3 Department of Physical
Therapy, Kyorin University, Tokyo, Japan）
The lumbar trunk region has no ribs, and is thought to play an important role in maintaining
the trunk in an upright position and performing anteroposterior and lateral bending. In thisstudy, we aimed to clarify the localization and morphological feature of motor neurons innervating the lumbar trunk muscles in the first and second lumbar segments.
The retrograde labeling with fluorescently labeled dextran of motoneurons was performed onanterior branches(AB) of the spinal nerves that control the ventral side of the trunk muscles i.e.
abdominal muscles, and posterior branches(PB) that controls the dorsal side i.e. back muscles
from the first and second lumbar spinal cord in rats.
The anterior branch motor neurons were located in ventrolateral portion of anterior horn, theposterior branch motor neurons were located in ventromedial portion and there was no sitewhere both were mixed. AB motor neurons were located over the range of stained segments
and PB motor neurons on the rostral half of the stained segment. The average number andsize of the AB motor neurons were 141 and 839 µm2, they of the PB motor neurons were 82
and 819 µm2 respectively. The size distribution of AB motor neurons was unimodal while the
PB motor neurons was bimodal.
Previous studies have established that size distribution of hindlimb motor neurons show
bimodal distribution and the smaller group is composed mainly of gamma motor neurons
while the larger group is composed mainly of alpha motor neurons. Hence, difference of size
distribution may reflect difference of gamma motor neuron content in AB motor nuclei and PB
motor nuclei. The abdominal muscle and back muscle may have different modes of control byalpha and gamma motor neurons.
(COI:No)

Activity-dependent differentiation of axon initial segment in avian
cochlea nucleus
Medicine, Nagoya University）

The axon initial segment (AIS) is accumulated with voltage-gated sodium channels andinvolved in generation of action potentials. We previously showed in avian cochlear nucleus(nucleus magnocellularis, NM) that length of the AIS is determined in a tonotopic-regionspecific manner; it is shorter in neurons with high characteristic frequency (CF) (~20 µm)
than those with low CF (~25 µm), which plays a critical role in adjusting the output within the
nucleus. However, the mechanism underlying this differentiation is not well understood. We
addressed this issue in organotypic cultures of chicken brainstem, in which NM neurons aretotally isolated from afferent input. At 14 days in culture, corresponding to the period after
maturation, the AIS in high-CF neurons was shorter (~24 µm) than that in low-CF neurons(~30 µm). However, this difference disappeared when the cultures were incubated for 3-7 days
with TTX and DNQX, due to an elongation of the AIS in high-CF neurons (~28 µm), suggestingthat spontaneous activity shortened the AIS specifically in high-CF neurons, which caused
the difference of AIS length between the neurons. In support, the AIS length was reduced inhigh-CF neurons, when spontaneous activity was augmented by either blocking Kv1 channels
with dendrotoxin-I (~21 µm) or lowering [K+] o with a chelator, sodium polystyrene sulfonate(~18 µm). Moreover, when we depolarized the membrane via elevation of [K+] o (10.6 mM), theAIS (~20 µm) was shortened in high-CF neurons, but not in low-CF neurons. These results
indicated that the activity-dependent shortening of the AIS occurs more efficiently in higherCF neurons, which underlies the tonotopic differentiation of the AIS in NM.(COI:No)

3P-0443P-047

Tomohiko Irie（Div Pharmacol, NIHS, Kanagawa, Japan）

Kohgaku Eguchi, Peter Koppensteiner, Ryuichi Shigemoto（Shigemoto group, Institute of

Loose coupling between SK and P/Q-type Ca2+ channels in cartwheelcells of the dorsal cochlear nucleus
Small-conductance Ca2+-activated K+ (SK) and largeconductance voltage- and Ca2+-activated
K+ (BK) channels are Ca2+-activated K+ channels that control action potential firing in diverse
neurons in the brain. In cartwheel cells of the dorsal cochlear nucleus, blockade of either
channel type leads to excessive production of spike bursts. In the same cells, P/Q-type Ca2+
channels in plasma membrane and ryanodine receptors in endoplasmic reticulum supply Ca2+
to BK channels through Ca2+ nanodomain signaling. In this study, voltage-clamp experiments
were performed in cartwheel cells in mouse brain slices to examine the Ca2+ signaling pathways underlying activation of SK channels. As with BK channels, SK channels required theactivity of P/Q-type Ca2+ channels. However, this signaling occurred across Ca2+ micro- rather
than nanodomain distances and was ndependent of Ca2+ release from endoplasmic reticulum.
These differential modes of activation may lead to distinct time courses of the two K+ currents
and therefore control excitability of auditory neurons across different timescales.
(COI:No)

3P-045Cell-cycle Length of Medial Ganglionic Eminence Progenitors
Determines Interneuron Fate
Yong-Chun Yu, Min Wang, Yinghui Fu, Dan Shen（Institutes of Brain Science, State Key

Laboratory of Medical Neurobiology and Collaborative Innovation Center for Brain Science,
Fudan University, Shanghai 200032, China.）

Cortical interneurons represent a highly diverse population of cells with important roles in
network function. How interneurons originating in the ganglionic eminences (GEs) become
specified during development is an outstanding question. Although genetic mutations that
disrupt the cell cycle of interneuron progenitors alter interneuron fate determination, the exact
link between them remains poorly understood. Here, using a triple thymidine analog method
to label dividing progenitors in vivo, we demonstrate that cell-cycle length of interneuron
progenitors influences the fate determination of major subgroups of cortical interneurons.
We found that short cell-cycle progenitors mainly locate in the middle region of the medial
ganglionic eminence (MGE), whereas relatively long cell-cycle progenitors preferentially distribute in the ventral and dorsal regions of the MGE. A combination of cell transplantationand fate mapping studies further reveal that short cell-cycle progenitors mainly give rise tosomatostatin-expressing interneurons, whereas relatively long cell-cycle progenitors generate
predominantly parvalbumin-expressing interneurons. These results suggest that cell-cycle
length of progenitors plays a pivotal role in the fate determination of MGE-derived interneuron
subtypes.
(COI:No)

Advantages of acute brain slices prepared at physiological temperaturefor investigating synaptic functions
Science and Technology Austria, Am Campus 1, 3400 Klosterneuburg, Austria）

Acute brain slice preparation has been developed as a powerful experimental model for investigating the characteristics of synaptic functions in brain. The acute slice preparation is readily
accessible for electrophysiological and optical recordings and retains the cytoarchitecture andsynaptic circuits in vivo except for the long-range projections. In general, to prepare acuteslices, the dissected brain from an animal is immersed into the ice-cold cutting solution, andthen sliced at ice-cold temperature (CT) to avoid the neuronal excitotoxicity during cutting.
The acute slice preparation at CT, however, causes molecular and architectural changesof cellular components. Chilling of hippocampal slices induces disassembly of microtubules
and eliminates dendritic spines. Re-warming of the hippocampal slices revives microtubule
structures, but excessively reconstructs the dendritic spines resulting in higher density ofsynapses than that in intact brain (Kirov et al., 2004).
Recently, a method of acute slice preparation at physiological temperature (PT) has been developed to improve quality of cerebellar slices in aged rodents. In the warm-cut cerebellar slices,
Purkinje cells (PCs) survived better without altered intrinsic electrophysiological featuresof the cells (Huang and Uusisaari, 2013). However, it hasn’t examined whether the synaptic
features are better preserved in the warm-cut acute slices compared to the cold-cut slices.
In this study, we investigated ultrastructural and electrophysiological features of synapses
in mouse acute cerebellar slices prepared at CT and PT. In the slices prepared at CT, wefound significant synaptic vesicle rearrangement and decrease in synaptic proteins, both ofwhich were not detected in slices prepared at PT. Consistent with these structural findings,
slices prepared at PT showed higher release probability and higher detectability of long-term
depression after motor learning compared with slices prepared at CT. These results indicate
substantial advantages of the slice preparation at PT for investigating synaptic functions indifferent physiological conditions.
(COI:No)

3P-048WNK3 kinase maintains basal excitability by regulating inward
rectiﬁcation and intracellular chloride in layer V pyramidal neurons ofmouse medial prefrontal cortex
Adya Saran Sinha1，Tianying Wang1，Yasushi Hosoi1，Eisei Sohara2，Tenpei Akita1，
1 1 Department of Neurophysiology, Hamamatsu University
Shinichi Uchida2，Atsuo Fukuda（
School of Medicine, Hamamatsu, Japan, 2 Department of Nephrology, Graduate School of
Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan）

WNK kinases with their downstream phosphorylation cascades SPAK/OSR1 are critical for
their regulation of cation chloride cotransporters namely NKCC1 and KCC2 in determining neuronal responses to GABA as either excitatory or inhibitory based on the status of
intracellular chloride [Cl‒] i levels. Among the WNK family (WNK1-WNK4), WNK3 are highly
expressed in brain. WNK1 is observed to be more active in immature neurons and WNK3 is
proposed to regulate activity of WNK1 in mature neurons. Increased expression of WNK3 hasbeen reported in pathological conditions of schizophrenia and temporal lobe epilepsy. Using
genetically modified WNK3KO mice and WT litter-mates, we performed in-vitro patch clamp
electrophysiology to understand the role of WNK3 kinase on basal and activity dependent
regulation of excitability and [Cl‒] i levels in layer V pyramidal neurons from the medial
prefrontal cortex (mPFC) between postnatal day (P)21-27. We found that loss of WNK3 inmature neurons significantly reduced intrinsic excitability, as evidenced by hyperpolarized
resting membrane potential (RMP), decrease in input resistance thereby increasing the rheobase current for AP generation. Voltage-clamp recordings revealed the enhanced inwardly
rectifying potassium (IRK) conductance caused the hyperpolarized RMP. Indeed, introductionof the active form of WNK3 kinase into the knockout neurons suppressed the enhancement
indicating a phosphorylation dependent mechanism. In addition, gramicidin perforated patch
clamp recordings from layer V pyramidal neurons exhibited a depolarized GABA response incomparison to WT neurons indicating higher [Cl‒] i levels. We also investigated by western blot
analysis the downstream signaling cascade of WNK1, SPAK/OSR1and their phosphorylation
status critical for regulation of NKCC1 and KCC2 function. Evaluation of synaptic currents
also showed increased frequency of miniature inhibitory post synaptic currents (mIPSCs).
Together, these observations indicate an important role of WNK3 in regulation of neuronal(COI:No)
excitability of mature pyramidal neurons.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S157

3P-049

Proximodistal heterogeneity in learning-promoted pathway-speciﬁcplasticity at dorsal CA1 synapses
2 1 Department of
Paw-Min-Thein-Oo1，Yuya Sakimoto1，Hiroyuki Kida1，Dai Mitsushima1, （3P-052Estimation of stimulating electrodes in retinal prosthesis by recurrent
neural network
Tomomitsu Miyoshi1，Tomoyuki Naito2，Ryota Imai3，Takeshi Morimoto4，Takashi Fujikado3

Physiology, Yamaguchi University Graduate School of Medicine, Yamaguchi, 755-8505,
Japan, 2The Research Institute for Time Studies, Yamaguchi University, Yamaguchi,
753-8511, Japan）（ 1 Dept Integrat Physiol, Grad Sch Med, Osaka Univ, Japan, 2 Lab Cogni Behav Neurosci,
Grad Sch Med, Osaka Univ, Japan, 3 Grad Sch Frontier Biosci, Osaka Univ, Japan, 4 Dept
Advanced Vis Neurosci, Grad Sch Med, Osaka Univ, Japan）

Contextual learning requires the delivery of AMPA receptors to CA1 synapses in the dorsal
hippocampus. However, proximodistal heterogeneity of pathway-specific plasticity remains
unclear. Here, we examined the proximodistal heterogeneity in learning-induced plasticity at
the CA1 synapses with inputs from the entorhinal cortex layer III (ECIII) or from CA3. We
subjected male rats to an inhibitory avoidance task and prepared acute hippocampal slices forwhole-cell patch clamp experiments, where we stimulated ECIII-CA1 or CA3-CA1 input fibers
to analyze evoked excitatory postsynaptic currents (EPSCs). Compared to untrained controls,
trained rats exhibited higher AMPA/NMDA current ratios at proximal and intermediate,but not distal CA3-CA1 synapses, which suggested that region-specific plasticity occurred
after learning. Moreover, trained rats exhibited higher AMPA/NMDA current ratios atintermediate and distal, but not proximal ECIII-CA1 synapses. These findings suggested thepresence of proximodistal heterogeneity in pathway-specific postsynaptic plasticity. Regarding
presynaptic plasticity, training slightly, but significantly increased the paired-pulse ratios ofproximal and intermediate, but not distal CA3-CA1 synapses. Moreover, trained rats exhibited
higher paired pulse ratios at intermediate and distal, but not proximal ECIII-CA1 synapses,
which suggested region-specific presynaptic plasticity. Finally, learning was clearly prevented
by the bilateral microinjection of a plasticity blocker in the proximal or intermediate, but notdistal CA1 subfields, which suggested functional heterogeneity along the proximodistal axis.
Understanding region- and pathway-specific plasticity at dorsal CA1 synapses could aid incontrolling encoded memory.
(COI:No)We have been developed a novel retinal prosthetic system, Suprachoroidal Transretinal
Stimulation (STS) for photoreceptor degenerating diseases. In the STS, stimulating electrodearray is implanted in sclera so as to avoid damaging neural retina. To evaluate the prosthetic
response in central nervous system, previously we investigated the response properties ofsingle-unit activity in cat lateral geniculate nucleus. Here, we recorded prosthetic electricalevoked response of visual cortex with electrocorticogram (ECoG) electrodes, and investigatedwhether Long short-term memory (LSTM) network can recognize the position of the stimulating electrode from the ECoG response or not.
The STS array with 9 active electrodes was implanted into cat eye under general anesthesia
in acute experiment. The size of single electrode in the array was 0.5 mm in diameter and 0.3
mm in height, which was the same as the device for clinical trials. The stimulation parameter
was 500 uA amplitude, 0.5 ms/phase duration, biphasic, 1 Hz frequency. The prosthetic evoked
response was recorded from cortical area17/18 surface by ECoG array with 16 electrodes.
Simple LSTM+3-layered fully connected layers network was trained with data of 250ms ECoG
waveforms (from 50ms before trigger 200ms after trigger). 80% of full data was used fortraining and then, this trained LSTM classified the rest of waveforms. The accuracy of theclassification was 0.63 (significantly higher than the chance level). This result shows that LSTM
can discriminate the electrode position by ECoG with online single waveform.
(COI:Properly Declared)

3P-0503P-053

Han-Thiri-Zin，Yuya Sakimoto，Dai Mitsushima（ Graduate School of Medicine, Yamaguchi

Rumi Nakayama-Naono1，Hideki Funahashi2，Yu Miyahara 2，Keiju Kamijo1，Yusuke Ishida1，
2 1 Dept of Anatomy and Cell Biol, Fac of Med, TMPU, Japan, 2 Dept
Toshikazu Nishimori（Contextual learning promotes plasticity at the inhibitory synapses onto
the granule cells in the dentate gyrus
University）

Granule cells in the dentate gyrus of the hippocampus are thought to be essential to memory
function by decorrelating overlapping input patterns (pattern separation). To examine thelearning-dependent synaptic plasticity of granule cells, we subjected male rats to an inhibitory
avoidance (IA) task and prepared acute hippocampal slices for voltage clamp analysis in trained
and untrained animals. After addition of tetrodotoxin, we recorded the spontaneous miniature
responses induced by a single synaptic vesicle of glutamate or GABA. Both miniature EPSCs(mEPSC at -60 mV) and miniature IPSCs (mIPSC at 0 mV) were sequentially recorded from thesame granule cells. Compared with untrained control, IA-trained rats significantly increased
mIPSC frequency, suggesting learning-induced plasticity at GABAA receptor–mediatedinhibitory synapses. In contrast, IA training did not affect mEPSC amplitude and frequency.
Moreover, specific stimulation of excitatory fibers from mossy cells, medial performant
pathways, or lateral performant pathways failed to show significant changes in AMPA vsNMDA current responses, suggesting that the learning may not promote plasticity at AMPA
receptor–mediated excitatory synapses. Since contextual learning strengthened both inhibitoryand excitatory synapses onto the CA1 pyramidal neurons, learning-promoted plasticity seems
to be cell type–dependent.

Anti-inﬂammatory and anti-nociceptive effect of human gene-derived
peptidesPsychiatry, Fac of Med, Univ of Miyazaki, Japan）

Keywords: AMPA receptor, GABAA receptor, glutamic acid, GABA, synaptic plasticity(COI:No)

Endokinin peptides encoded by human TAC4 gene are a new member of tachykinin peptide
family. Our previous study indicated that some of these peptides has a crucial role in nociceptive processing in the spinal cord. Indeed, endokinin peptides consist of four peptides, endokinin
A, B, C and D, and endokinin A and B (EKA/B) elicits excitatory effects, while the functionof endokinin C and D (EKC/D) remains uncertain. The pretreatment with EKC/D attenuatedinduction of nocicceptive-related behaviors by substance P (SP) indicating that EKC/D has anantagonistic role for the neurokinin-1 receptor (NK1R) that is the receptor of SP. This functional
difference may contribute to that amino acids at the carboxyl-terminal of EKA/B or EKC/D
are leucine and methionine, respectively. Since it is necessary to improve the anti-nociceptive
effect of EKC/D, we synthesized EKC/D-derived peptides (D-EKC/Ds) having some of D-typeamino acids. Then, to clarify whether the inhibitory effect of EKC/D is altered by replacementwith D-type amino acids (D-EKC/Ds), the effect of these peptides was evaluated by SP-induced
nociceptive behavior, and by the inflammatory nociceptive behavior. Pretreatment with DEKC/Ds were long-lasting attenuated on SP-induced nociceptive behavior and inflammation
which compared with EKC/D, and these effects were dependent on the number and position of
D-type amino acid.These results indicate that, D-EKC/Ds were not only anti-nociceptive effectbut also anti-inflammatory effect and some of D-type amino acids may have a crucial role of(COI:No)
these effects.

3P-051

3P-054

Takumi Akagi, Atsushi Shimohata, Ikuo Ogiwara, Makoto Kaneda（ Dept Physiol, Nippon

Akimasa Tashiro, Yuji Morimoto（Dept Physiol, Natl.Defense Med Col.）

Membrane expression of mGluR6 transfected cultured retinal bipolarcells
Med Sch, Japan）

Metabotropic glutamate receptor 6 (mGluR6) is expressed at the dendritic tip of retinal ONbipolar cells (ON-BC), and plays critical roles in the initial step of visual information processing.
The localization of mGluR6 in ON-BC is restricted to the postsynaptic compartment at thedendritic tips where it mediates G-protein-dependent signaling cascades of ’light on-response’
depending on glutamate release from photoreceptors. While the spatial restriction of mGluR6
localization is thought to be crucial for the precise visual signal transduction, the mechanismsof mGluR6 synaptic targeting remain elusive. In our previous study, we suggested that intracellular C-terminal domain (CTD) of mGluR6 may contain regulatory elements to control themembrane surface localization of mGluR6 in the heterologous expression system (HEK293,
primary hippocampal neuron). In this study, to elucidate the molecular mechanisms of polarized distribution of mGluR6 in retinal ON-BC, we transfected the plasmid expression vector
consist of cDNA encoding rat mGluR6 with V5 epitope tag located at the N-terminal end to
primary cultured mouse retinal ON-BC, and examined localization of the expressed mGluR6.
At DIV 7-10, cells with bidirectional neurites showed positive immunoreactivity for PKCα,a rod bipolar cell marker. In these PKCα-positive cells, we observed membrane surface
expression of V5 tagged mGluR6. However, we could not observe the polarized localizationof mGluR6. These observation suggest that mGluR6 possesses the ability to express itself
to membrane surface of the primary cultured retinal bipolar cells, whereas an important
additional mechanism is required for the polarized distribution in the cultured bipolar cells.
(COI:No)

S158

Involvement of melanopsin on the photic excitation of neurons in the
medullary dorsal horn and lacrimation
Bright light can cause ocular discomfort and evokes protective reflexes such as blinking. Previously we reported that bright light activates trigeminal nerve activity through an intraocularmechanism driven by a luminance-responsive circuit (non-image visual functions) and increased
parasympathetic outflow to the eye. Melanopsin-containing intrinsically photosensitive retinal
ganglion cells (ipRGCs) are photoreceptors that mediate non-image visual functions.
To determine if melanopsin activation was necessary for light-evoked Vc/C1 excitation,
melanopsin antagonist (Opsinamides) was injected systemically while recording light-evoked
Vc/C1 unit activity. Under isoflurane anesthesia, units were recorded in laminae I-II at Vc/
C1 under low ambient light. All units received convergent mechanical input from the ocularsurface and facial skin. Units with a cutaneous receptive field (RF) were tested for responses
to pinch stimuli. Light stimulation (300W/m2, 30s) was delivered from a thermal-neutral fiber
optic source positioned 5 cm from the ocular surface at 20 min intervals. Light and mechanicalevoked unit activity was recorded before and after 10 min after Opsinamides (1mg/kg, iv).
Systemic administration of Opsinamides inhibited the light evoked unit activity. While, mechanical input from the convergent receptive field was not inhibited. Systemic administrationof Opsinamides prevented light evoked increases in tear volume. These findings confirm thatmelanopsin plays a pivotal role in mediating light-evoked neural excitation at the Vc/C1 regionand ocular-specific function such as reflex lacrimation. Activation of melanopsin may cause(COI:No)
discomfort called dazzling sensation after intense light.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

3P-055

Role of olfactory tubercle in the weaning process of neonatal mice
Masahiro Yamaguchi, Yasutaka Chikuda（ Dept Physiol, Kochi Med Sch, Kochi Univ, Japan）
There is a close relationship between feeding behavior and olfaction. Weaning of neonates isa typical example of feeding behavior development and learning, which proceeds between 2
and 4 weeks of age in mice. In the olfactory cortical regions, olfactory tubercle (OT) is thought
to contribute to the odor-guided behavior learning, where the anteromedial domain drives
attractive behavior and the lateral domain aversive behavior. Given that the OT develops
structurally and functionally during the weaning period, we examined the role of the OT in the
neonatal feeding behavior. By using transgenic mice expressing Cre recombinase in striatal
neurons and viruses expressing toxins in a Cre-dependent manner, principal neurons in the
OT domains were ablated during neonatal period. Cell ablation in the anteromedial OT domain
impaired the ability of neonates to find food pellets hidden beneath the bedding, and prolonged
their preference to lactating mother over food pellets. Ablation in the lateral OT domain did
not induce these impairments. These results indicated a crucial role of the anteromedial OT(COI:No)domain in the physiological weaning process.

3P-056

Activation mechanism of the olfactory tubercle in the odor-guided
attracted behavior learning in mice
Yuriko Koga, Kazuko Yokoi, Masahiro Yamaguchi（Dept Physiol, Kochi Sch Med, Kochi

Univ, Japan）

Odors induce various motivated behaviors, and most of the odor-guided behaviors are acquired
through learning process. When mice are presented with a cue odor and food reward at thesame time, they become attracted to the cue odor. On the other hand, when they are presented
with the cue odor and noxious stimuli, they become aversive to the cue odor. Among various
areas of the olfactory cortex, the olfactory tubercle (OT) is a crucial area to link odor cueswith motivated behaviors. The anteromedial domain of the OT is activated when mice learn totake attractive behavior to the odor, while the lateral domain is activated when then learn totake aversive behavior. However, the plastic mechanism of the neural circuits of how specificdomains of the OT become activated by odor-guided behavior learning remains elusive.
The OT receives inputs from various brain areas. In this study, we focused on the plasticchange of synaptic inputs from the piriform cortex (PC), the largest area of the olfactorycortex, to the OT. By using optogenetics in mice, synaptic inputs from the PC to OT domains
were activated and associated with food reward. Then the mice learned to take food-seeking
behavior by the photostimulation. We are analyzing the synaptic terminals of the photostimulated synaptic inputs to the OT domains histologically in the brain sections, to see whether thesynaptic inputs in the anteromedial and lateral domains are biased by the attractive behavior(COI:No)
learning.

3P-058Developmental changes in spike sequences of L4, L2/3 neurons in
response to whisker stimulations in the rat barrel cortex
2 1 Dept Mol. Neurosci, Grad Sch Med, Osaka Univ,
Fumitaka Kimura1，Hiroshi Tamura（

Japan, 2 Grad Sch Frontier Bioscience, Osaka Univ）

Spike timing-dependent plasticity (STDP) at L4-L2/3 cells plays a crucial role in deprivationinduced plasticity of sensory cortical maps. This is because sensory input produces L4 cellfiring followed by L2/3 cell firing (L4-preceding-L2/3 firing) in the corresponding corticalcolumn, which causes strengthening of this synapses under Hebbian STDP rule, while sensory
deprivation produces sudden reversal of firings, or L2/3-preceding-L4 firing in the deprived
column, which causes weakening of this synapses. Previously we found that parvalbumincontaining GABA neurons in L4 produce fast feedforward suppression on L4 and L2/3, whichplays an important role in producing L4-preceding-L2/3 firing. However, such fast feedforward
suppression completes around P13-P15 during development. In addition, we also found that
L4-L2/3 STDP shows developmental switch from long-term potentiation only STDP (all-LTP
STDP) to Hebbian STDP with LTP and LTD after P15 (Itami, 2012). Thus, we hypothesizedthat the switch of STDP and the completion of fast feedforward GABA circuit together initiate
the critical period of map plasticity (Kimura, 2019). This hypothesis predicts that L4-precedingL2/3 firing following sensory inputs occurs only after around P15. Present study was initiatedto examine this prediction. For this purpose, we performed extracellular recording of corticalcell activity in response to whisker deflection with 32 ch electrodes covering from whole layersof rat barrel cortex under urethane anesthesia from P10 to P20. Cross-correlation analysis(COI:No)seem to indicate that our prediction was generally correct.

3P-059Auditory responses of the rat globus pallidus neuron subtypes
Yasuharu Hirai, Fumino Fujiyama（ Lab Neural Circuit, Grad Sch Brain Sci, Doshisha Univ,
Japan）The globus pallidus (referring the external segment of that in primates) is the one of the basal
ganglia nuclei, which receives inhibitory inputs from “indirect pathway” striatal projection
neurons and makes GABAergic innervations to almost all nuclei of the basal ganglia. According to the classical scheme, the activation of indirect pathway suppresses the thalamocorticalinformation transfer, or “movement” in the case of motor function, whereas the activationof direct pathway induces opposite effect on it. However, this go/no-go effect is still under
the debate, and also it is unclear how the direct/indirect pathway framework relates to thesensory processing in the basal ganglia. To understand how the globus pallidus handles thesensory inputs as a part of the indirect pathway, we recorded the unit activity of urethaneanesthetized rat globus pallidus neurons during the acoustic stimulation. Tonal receptive fields
were investigated with relation to the location of neurons within the nucleus, to the brain
state (slow-wave sleep or cortical activation states), and to the neuron subtypes which can beclassified by electrophysiological character, molecular expression, and axon projection target.
We found that the most of neurons within the caudal part of globus pallidus responded to thesound mainly excitatory. It contradicts to the scheme but corresponds to the recent findings
showing the existence of direct excitatory inputs from cortical pyramidal cells. The neurons
showing high spontaneous firing responded to the sound even during slow-wave sleep states
whereas other neurons were only during cortical activation states. These findings indicatedthat globus pallidus neurons in sensory region of basal ganglia responded to the cortical activity
more than striatal activity, and there might be at least two processing neuron types; one ishaving high spontaneous activity and always responded to the sensory input and another isfunctional only during awaking.
(COI:No)

3P-0573P-060Noriyuki Nakashima1，Kie Nakashima2，Akiko Nakashima1，Akiko Taura3，Harunori Ohmori4，
1 1 Dept. Physiol., Kurume Univ. Sch. Med., Japan, 2 Grad. Sch. Biostudies,
Makoto Takano（

4 1 Dept ofToshihiro Kawase1, 2, 3，Toshimitsu Takahashi1，Soichiro Fujiki1，Kenji Kansaku1, （Olfactory marker protein buffers cAMP by direct binding to avoid
depolarization-induced silencing of olfactory receptor neurons
Kyoto Univ., 3 Dept. Med. Engineering, Facult. Health Sci., Aino Univ., 4 Dept. Physiol., Sch.
Med., Kanazawa Med. Univ.）

Sensory cells respond to external stimuli with resilient firing properties. Olfactory receptor
neurons (ORNs) use odour- induced intracellular cAMP surge to gate cyclic nucleotide-gatednonselective cation (CNG) channels in the limited ciliary space. The prolonged exposure tocAMP causes adaptation of CNG channels and attenuates neural responses. On the other hand,the odour-source searching behaviour of animals requires ORNs to be sensitive to the odour
while approaching the targets. How ORNs accommodate these conflicting aspects of cAMP
responses remains unknown. Here, we show that the cAMP-buffering machinery swiftly
bypasses surplus cAMP during signal transduction to maintain the sensitivity of ORNs. We
discovered that the cytosolic olfactory marker protein (OMP) directly captured cAMP, whichtransiently desensitized CNG channel activity and prevented sustained membrane depolarization upon the application of sensory stimuli. Under repetitive stimulation, OMP－/－ ORNs
were immediately silenced after burst firing due to sustained depolarization and inactivatedfiring machinery. Consequently, OM－/－ mice showed serious impairment during odour-source
searching tasks. Therefore, cAMP buffering by OMP transiently desensitizes CNG channels
by uncoupling the signal transduction, but maintains resilient sensory responses in ORNs.
(COI:No)

Relationship between proprioceptive drifts and sense of ownershipduring robot hand illusion of elbow movements
Physiol ＆ Biol Inf, Dokkyo Med Univ Sch of Med, Mibu, Japan, 2 Inst Biomater ＆ Bioeng,
Tokyo Med Dent Univ, Tokyo, Japan, 3 Inst Innov Res, Tokyo Inst of Tech, Yokohama,Japan, 4 Cent Neurosci ＆ Biomed Eng, Univ of Electro-Communications, Chofu, Japan）
Understanding how we consciously experience our bodies is a fundamental issue in neuroscience. In former literatures, researchers have reported that the rubber hand illusion is accompanied by a change of perceived position of the hand towards the rubber hand, which is
known as a proprioceptive drift, and some studies showed that the proprioceptive drift wascorrelated with the sense of ownership (SO) in moving rubber hand illusion, which used arubber hand mechanically connected to participants’ own hands (Kalckert and Ehrsson, 2014).
In this study, we report that the same phenomena were observed in a robotic arm that moved
synchronously with the participants’ elbows.
Seven able-bodied participants were recruited. The robotic arm, using myoelectric control with
one degree of freedom (elbow flexion and extension), consisted of a prosthetic glove and an
actuator. The joint positions of the robotic arm were controlled continuously by means of theparticipant’s muscular activity on the elbow flexor and extensor. The participants took partin the in-phase and out-of-phase movement conditions for 10 min each. Before and after each
10-min experiment, the participants pointed to indicate the sensed position of the right index
finger with their left index finger with their eyes closed. We measured the position pointed
to, and the proprioceptive drift in a vertical plane was then calculated by subtracting the two
position measurements from each other. The participants answered a questionnaire to assess
SO immediately after each experiment.
In the in-phase movement, the proprioceptive drifts were significantly correlated with theratings of SO (Spearman: r = 0.86, p = 0.024), and in the out-of-phase movement, these were notsignificantly correlated. The results are consistent with the former studies of moving rubber
hand illusion.
The preliminary results suggest that the robotic arm will contribute to our understanding of(COI:No)
our experience on our bodies.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S159

3P-061

Spatiotemporal structure of spontaneous activity in the marmoset
visual cortex
Teppei Matsui1, 2，Takayuki Hashimoto1，Tomonari Murakami1，Masato Uemura1，
3 1 Dept. Physiol, Grad Scho Med, Univ
Kouhei Kikuta1，Toshiki Kato1，Kenichi Ohki1, （Tokyo, Japan, 2 JST-PRESTO, 3 IRCN ）Spontaneous neuronal activity in the resting-state is robustly observed throughout the human brain using functional magnetic resonance imaging and has been used widely to assess
functional network structures at the scale of the whole-brain (Fox and Raichle, 2007). At themesoscale, previous optical imaging in the visual cortex of cats and ferrets reported that spatial
patterns of spontaneous cortical activity closely resemble spatial patterns of iso-orientation
columns (Kenet et al., 2003; Smith et al., 2018) suggesting that spontaneous activity containmesoscale network information. However, due to the lack of imaging technique applicableto primates, precise mesoscale structures of spontaneous neuronal activity in primates still
remain largely unknown. Recently, genetically encoded calcium indicator applicable to common
marmosets has been developed (Sadakane et al., 2013; Uemura et al., Society for Neuroscience Abstract, 2018). In this study, we utilized this technique and conducted widefield andtwo-photon calcium imaging of spontaneous cortical activity in a large field-of-view (6.5 x
6.5mm) spanning the primary (V1) and secondary visual cortex (V2) of the marmoset monkey.
Wide-field imaging of spontaneous activity in the marmoset V1 and V2 indeed revealed richspatiotemporal structures at various spatial scales. At a large spatial scale, wave-like spontaneous activity propagating across the cortex was readily observed. Importantly, at a smaller
spatial scale, columnar patterns resembling orientation maps were frequently observed andwere often embedded within the wave-like activity. Cellular scale imaging with two-photon
microscopy confirmed that the columnar spontaneous activity reflected spontaneously active
clusters of neurons. Spike triggered average of spontaneous activity (Tsodykes et al., 1999)
further showed significant overlap between the column-like spontaneously activity and isoorientation columns. These results suggest that the analysis of spatiotemporal patterns of
spontaneous activity provide useful information about the modular organization of primate(COI:No)
neocortex.

3P-064

Male Elevated Gonadotropin Attracts Sexually Active Male Rats
Himeka Hayashi, Yui Shimomi, Kazuto Matsuda, Ryoko Kumagai, Yasuhiko Kondo（Dept Animal Sci, Teikyo Univ of Sci, Japan）

When simultaneously presenting odors of gonadally intact and of castrated males, sexuallymature male rats show significantly longer exploration to castrated male odor than that ofgonadally intact males. Our previous study suggested that this incentive to normal males is
produced by elevated level of gonadotropin caused by disinhibition due to a lack of testosterone
following castration. To further examine this hypothesis, we tested the effect of gonadotropin
treatment in hypophysectomized castrates on the preference of sexually active males; hypophysectomy abolishes circulating luteinizing hormone (LH) and follicle stimulating hormone(FSH), so that the attractiveness of castrated males may decline.Then, we prepared 3 groupsof stimulus males (n = 3, each), two of them subjected to hypophysectomy (Hx), and the one
received castration and sham-Hx surgery (Sham). They were kept for a few weeks to be stablein hormonal levels before the testing. Six sexually vigorous Long-Evans males were selected
as probe males through the mating sessions with hormone-primed receptive females and olfactory preference tests between odors of intact males and receptive females. Two hours before
the tests, 3 Hx males were i.m. injected with 50IU human chorionic gonadotropin (HxhCG) and3 Hx males and Sham males were injected with saline (HxSal and Sham, respectively). Probemales were tested for olfactory preference with 3 stimulus pairs, Sham vs. HxSal, Sham vs.
HxhCG, and HxSal vs. HxhCG. Consequently, probe males showed significantly longer timespent exploration to HxhCG male odor than that of Sham or HxSal males, while the preference
between Sham and HxSal male odors was equivalent. The current results clearly demonstratethat heighten circulating gonadotropin in males produces the attractiveness to same-sex, sexually active male rats.
(COI:No)

3P-062

3P-065

Mutsuo Taniguchi, Yoshihiro Murata, Masahiro Yamaguchi, Hideto Kaba（ Dept Physiol,

Shota Miyazaki1，Yu Tahara2，Christopher S. Colwell2，Gene D. Block 2，Wataru Nakamura3，
2 1 Laboratory of Animal Physiology, School of Agriculture, Meiji
Takahiro J. Nakamura1, （A mechanism for the inhibition of GABAergic transmission byvasopressin V1a receptors at the reciprocal synapse in the mouseaccessory olfactory bulb
Kochi Med School, Kochi Univ, Japan）

Central vasopressin (AVP) facilitates social recognition and modulates numerous complex
social behaviors in mammals. Vasopressin neurons were reported to exist in the accessory
olfactory bulb (AOB). The AOB has been demonstrated to be a critical site for mating-induced
mate recognition in female mice. The effect of AVP, however, on the synaptic transmission
between dendrites in the AOB of female mice is largely unknown.
To address this issue, we previously measured synaptic currents (IPSCs) from mitral cells in
the AOB. We have demonstrated that AVP significantly reduced the IPSCs through AVP V1areceptors. The reciprocal transmission, however, contains both glutamatergic transmission
from mitral to granule cells and GABAergic transmission from granule to mitral cells. Thus, it
is unclear whether AVP acts on the excitatory and/or the inhibitory transmissions.
In the present study, in order to conduct further investigation on the role of V1a receptor in the
GABAergic trasmission, AOB slices were prepared from 23- to 35-day-old Balb/c mice. Using
the patch-clamp technique in whole-cell configuration (holding potential, –70 mV), the current
responses of mitral cells were recorded in the presence of antagonists for glutamatergictransmission, CNQX (10 µM) and AP5 (50 µM). An extracellular application of vasopressin did
not affect the magnitude of the response of mitral cells to GABA (10 µM and 100 µM), whereas
it slightly suppressed voltage-activated Ca2+ currents in the granule cells. The present results
suggest that AVP can modulate the synaptic transmission from granule to mitral cells through(COI:No)a presynaptic mechanism.

3P-063Pontine serotonergic system regulates body movement and respiratory
rhythm coordination
2 1 Dept Physiol, Hyogo
Hirotaka Ooka1，Chiaki Uchida1，Akiko Arata1，Youko Tsukamoto（Coll med, Japan, 2 Dept Food ＆ Nutr, Hagoromo Univ.Intl. Studies, Sakai, Japan）

Locomotor-respiratory coupling, phase-locking between breathing and stepping rhythms, occurs in many vertebrates (Daley MA et al. 2013). In the neural circuit level, respiratory rhythm
is belonging to the medullary rhythm generator and body movement is belonging to the spinal
rhythm generator. The group of serotonergic neurons are strongly activated during rhythmic
activities and modulates the rhythm generator like respiratory and/or body movement. Parabrachial nucleus (PBN) is known about conjunctive system for sensory-motor co-ordination.
However, the relationship between respiratory rhythm and body movement in the pons levelhas not been well investigated. In this study, we analyzed the relationship between respiratory
rhythm and body movement in the pons using rat pons-medulla-spinal cord preparation; andexamined the effect of serotonin on their co-ordination. Serotonin enhanced the respiratory
rhythm-body movement synchronization with pons preparation, but not synchronized to bothrhythms in the pons removal. In addition, we examined the distribution of optical signals in
the pons triggered by body movement. We found the optical signals in the dorsal pons (probably PBN). When we add the 5-HT1A blocker to the preparation with pons, respiratory-bodymovement correlation weakened; therefore, the respiratory-body movement coupling wasmediated by 5-HT1A receptor. According to immunochemistry approach, 5-HT1A receptors
located in lateral PBN which was projected by optical imaging. Moreover, we examined thesensory-motor relationship between dorsal root stimulation in the C8 and respiratory rhythm
entrainment. C8 dorsal root stimulation connected body movement to respiratory rhythm.
These results suggested that the signal of body movement projected to the dorsal pons, andthat serotonin activated the synchronization between respiratory rhythm and body movement(COI:No)through 5-HT1A receptor in the dorsal pons.

S160

In Vivo Monitoring Reveals Two Different Oscillators in
Methamphetamine Treated Mice
University, Japan, 2 Department of Psychiatry and Biobehavioral Sciences, University ofCalifornia Los Angeles, USA, 3 Department of Oral Chrono-Physiology, Graduate School of
Biomedical Sciences, Nagasaki University, Japan）

The central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus,
generates circadian rhythms responsible for organizing the timing of most behaviors andphysiological events in mammals. However, there are other unidentified circadian oscillators.
For example, chronic treatment of methamphetamine (MAP) induces locomotor activity
rhythm in a circadian oscillation, independent of the SCN and driven by a pacemaker outside
the SCN. However, it remains unknown whether MAP affects SCN rhythmicity in mice that
display MAP-induced circadian locomotor activity rhythm (MCLA). To explore the effect of
MAP, we employed in vivo real-time monitoring of multi-unit neural activity (MUA) in freely
moving mice.
Three-month-old, male C57BL/6J mice were treated with 0.005% MAP dissolved in drinking
water until they displayed MCLA. An MUA electrode was chronically implanted into the
SCN or striatum, which is associated with motor functions. After recovery, we simultaneously
recorded wheel running activity (WR) and MUA.
In the SCN, control mice showed diurnal and circadian rhythms of MUA which was antiphasewith the rhythm of WR. Conversely, mice that displayed MCLA also showed robust MUArhythms in light-dark and constant dark conditions, while WR gradually overlapped with the
MUA. In addition, MUA in the striatum showed robust rhythms parallel to WR under all
conditions.
These results indicate that MAP does not strongly affect the neural activity rhythm in the
SCN, and that MAP-induced circadian oscillator and the SCN are completely independent of(COI:No)
each other. 

3P-066

Factors affecting social hierarchy and glucose metabolism relationship
Momoyo Ibukuro, Rikako Ukichi, Yukari Takahashi, Yae Sugimura K., Fusao Kato（Dept
Neurosci, Jikei Univ Sch Med, Japan）

Besides being regulated by the peripheral mechanisms, the blood glucose level is underpotent influence of the central nervous system. For example, epidemiological studies suggest
frequency of type 2 diabetes depends on socioeconomic status of patients (Rabi et al., 2006). As
the first step toward understanding neuro-behavioral factors affecting the glucose metabolism,
we evaluated the rank of individual mouse using the tube test (Wang et al, 2011) and comparedwith the glucose tolerance. The rank order of each of four mice in the same cage was evaluatedrepeatedly for 1-2 weeks and blood glucose level after glucose injection was measured at theend of the sessions. We analyzed influences of the following three elements: (1) diet, (2) sex and(3) systemic inflammation. (1) Effects of diet. We conducted the tube test in 10 sets of four mice
fed with low-fat (5 sets) or high-fat diet (the rest). The diet did not essentially affect the generalproperties of the formed social hierarchy. (2) Influence of sex. We compared the hierarchy
formation between male and female mice. While the stability of hierarchy structure was stablein both male and female, glucose metabolism depended on individual rank in male mice but
not in female mice, suggesting that the relationship between the social rank and glucose
metabolism is sex-dependent. (3) Impact of systemic inflammation on hierarchy. We examined
how lipopolysaccharide injection (0.1-1 mg/kg, i.p.) affects the rank order and found its impact
was limited with the present experimental conditions. Overall, these results suggest that the
glucose metabolism is under influence of neuronal and behavioral factors, which would shedlight on the novel approaches for improving glucose metabolism in diabetes patients. (COI:No)

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

3P-067

Novel function of NMU/NMS system in formation of the fear memory
Akinobu Soda, Ryoko Higa, Kenshiro Shikano, Reiko Hanada（ Dept Neurophysiol, Fac Med,

Oita Univ, Japan）

Neuromedin U (NMU) and Neuromedin S (NMS) are neuropeptides with various physiological
functions such as behavior and regulation of circadian rhythm. To investigate novel function of
NMU/NMS system related to higher brain functions, we have established NMU/NMS double
KO mice (dKO) and examined a series of behavior tests. In our preliminary experiment, dKO
showed increasing of anxiety in feared conditioned test. In this study, we performed the passive
avoidance test and analyzed the neuronal activation with c-Fos expression level. In the passive
avoidance test, dKO showed statistical difference in fear memory formation compared withwild type mice (WT) in 1 day after electric foot-shock (EFS). The extinction of fear memoryin 7 days and 28 days after EFS was shown in WT, however not in dKO. Furthermore, dKO
had shown higher concentration of serum corticosterone level than WT. These data suggestthat dKO were enhanced fear memory consolidation and reduced extinction of fear memory.
Additionally, in 1 day after EFS, the number of c-Fos positive cells in the lateral amygdala
nucleus significantly increased in dKO than WT. This indicates the lateral amygdala nucleus
tightly contributes the fear memory formation in initial state. These results indicate that the(COI:No)NMU/NMS system is closely involved in the formation of the fear memory.

3P-070Establishment of screening method for sleep/wakefulness in micethrough forward genetics
Chika Miyoshi1，Noriko Hotta-Hirashima1，Satomi Kanno1，Aya Ikkyu1，Miyo Kakizaki1，
Mana Yamada1，Staci Kim J1，Shigeharu Wakana2, 3，Masashi Yanagisawa1, 4, 5，
6 1WPI-IIIS, University of Tsukuba, Tsukuba, Japan, 2 RIKEN
Hiromasa Funato1, （BioResource Center, Tsukuba, Japan, 3 Institute of Biomedical Research and Innovation,
Kobe, Japan, 4University of Texas Southwestern Medical Center, Dallas, TX, 5TARA,
University of Tsukuba, Tsukuba, Japan., 6 Department of Anatomy, Faculty of Medicine,
Toho University, Tokyo, Japan）

The network of genes and molecules that govern sleep and wakefulness remains largely
unknown. Forward genetics is a powerful approach in elucidating biological phenomena thatcannot be predicted from the function of known genes. We conducted a large-scale screening
in mice using the EEG/EMG-based sleep/wake monitoring. The search has succeeded in
identifying several sleep regulatory genes.
Among the established mouse pedigree, theSleepy mutant family shows a significant reduction
in total wake time. Sleepy mice have mutation in the Salt-inducible kinase 3 (Sik3) gene which
encodes a member of the AMP-activated kinase-related kinase. In addition to the hypersomnia,
Sleepy has a tendency to become obese. This mouse pedigree will be a model animal not only
for studying hypersomnia but also the obesity which are considered to be costly conditions forthe society.
(COI:No)

3P-0683P-071

Akihiko Ushigome1，Makoto Takahashi2，Shinichiro Takahashi2，Junichi Tanaka2, 3

Yoichi Minami, Tomoko Yoshikawa, Mamoru Nagano, Satoshi Koinuma, Keisuke Ikegami,
Atsuko Fujioka, Keiichi Furukawa, Yasufumi Shigeyoshi（Dept Anatomy, Faculty Med,

Involvement of glutamatergic inputs from the subfornical organ to themedian preoptic nucleus in the water ingestion induced by angiotensin
II in rats
（ 1 Dept Judo Physical Therapy, Fac Human Care, Teikyo Heisei Univ, Tokyo, Japan, 2 Dept
Disability Sci, Grad Sch Edu, Naruto Univ Edu, Tokushima, Japan, 3 Lab Neurosci, Naruto
Univ Edu, Tokushima, Japan）
The present study was conducted to examine the role of glutamatergic systems in the medianpreoptic nucleus (MnPO) in the water ingestion induced by administration of angiotensin
II (ANG II) in the subfornical organ (SFO) in the rat. Microdialysis data demonstrated thatmicroinjection of ANG II (10－10 M) into the SFO significantly increased the release of glutamate(Glu) in the MnPO in the rats under the condition that water is available for drinking and therats under the condition that water is not available for drinking. The amount of initial maximal
increases in the Glu levels elicited by the ANG II injection was quite similar in drinking and non
drinking rats, whereas the duration of the response was much longer in non drinking than indrinking rats. The amount of water ingestion in 20 min immediately after the ANG II injection
was significantly enhanced by previous injections of N-methyl-D-asparatate (NMDA, 10μM)
into the MnPO, while the ANG II-induced water ingestion was attenuated by pretreatment
with the NMDA antagonist dizocilpine (MK-801, 10 µM). The amount of water intake elicitedby the ANG II injection into the SFO was enhanced by previous injections of either the
non-NMDA agonist kainic acid (KA, 50 µM) or quisqualic acid (QA, 50 µM) into the MnPO. On
the contrary, the ANG II-induced drinking response was diminished by pretreatment with
the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX, 10 µM) in the MnPO.
These results suggest that the glutamatergic neural pathways to the MnPO may transmit the
information for eliciting drinking in response to ANG II acting at the SFO and the ANG IIinduced dipsogenic response may be mediated through both NMDA and non-NMDA receptor
mechanisms in the MnPO.
(COI:No)

3P-069Neurons in the medulla related to the regulation of sleep/wake cycles
and autonomic nervous system

Generation of a genetically modiﬁed rat overexpressing BMAL1
dominant negative form
Kindai Univ, Japan）

The circadian clock is the endogenous oscillator ticking about a day. At molecular level, thecircadian clock is composed by a set of clock genes such as Per2, Cry1 and Bmal1. Genetical
disruption of clock genes results in abnormal circadian rhythm. Because of technical advantages, mice are mainly used to generate molecular clock disfunction models. Of note, circadianperiod of mice behavioral rhythm is shorter than 24 hours, whereas that of humans and rats
are longer than 24 hours. In this study, we focused on rats for the phenotype similarity tohumans’ circadian clock systems. We overexpressed BMAL1 dominant negative (DN) form
which lacks C terminus CRY1 interaction domain specifically in neurons by using a mouseprion promoter (MoPrnP.XhoI). The transgene was injected to fertilized eggs collected fromtransgenic rats having destabilized firefly luciferase reporter gene driven by Per2 promoter(Per2-dLuc). We observed smaller amplitude of bioluminescence rhythm in BMAL1 DN transgenic rats using cultured SCN and rapid entrainment after an abrupt shift of the LD cycle.
These findings suggest that the attenuation of the circadian rhythm brings rapid elimination of(COI:No)jet lag.

3P-072

Withdraw

Yoshifumi Arai, Hayato Iwata, Tatsuya Suzuki, Kaname Mochizuki, Yoshimasa Koyama（Faculty of Symbiotic Systems Science, Fukushima Univ, Japan）

The medulla plays important roles in the promotion of rapid eye movement (REM) sleep
and muscle atonia during REM sleep and contains the cardiovascular center, that regulates
vasomotion and respiration. During REM sleep, rapid increases in blood pressure (BP) andheart rate (HR) are observed due to the turbulence of the autonomic nervous system. However,it remains to be known how the neurons in the medulla regulate sleep/wake cycles and BP
fluctuation during REM sleep.
We recorded single neuronal activity and BP in head-restrained and unanaesthetised rats
during sleep/wake cycles. Of 115 neurons recorded, 75.7% (87/115) showed increased activity
during REM sleep, during REM sleep and waking, or during Slow wave sleep and REM sleep,
which we term PS active neurons.
32.2 % of PS active neurons (28/87) started increasing their activity before the REM sleep
onset. They are mainly located from central to the caudal parts of the medulla. Among them,
the neurons showing tonic firing during REM sleep were distributed in the ventral parts, while
the phasic firing neurons were found in the dorsal parts.
Of 87 PS active neurons, 32.2 % (28) showed firing in close relation with BP fluctuation during
REM sleep, and 75 % of them (21/28) increased their firing rate before the BP fluctuation. The
time preceding to the start of the blood pressure increase was longer in the neurons located in(COI:No)the rostral medulla compared to those in the caudal medulla.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S161

3P-073

Microglia eliminate synapses by phagocytosis in mature brain; its
physiological roles
Junya Tanaka（ Dept Mol Cell Physiol, Grad Sch Med, Ehime Univ, Japan）
Microglia in the normal mature brain have long been recognized as resting cells that do notplay anything until any kinds of pathologic events occur. However, currently it is well known
that microglia constantly move their fine processes even in the physiological condition while
monitoring, probably, the activities of synapses. In the developing brains, microglia take partin the construction of neural network by eliminating inactive synapses. In this paper, we willshow the data on the involvement of microglial synaptic elimination in physiological functions.
1) Sleep and microglia. Decrease in synaptic density or activities during sleep have long been
noted, whereas the mechanisms underlying the synaptic loss have not been known. We haverecently found that microglia phagocytose synapses during sleep that binds eat-me signals
such as complements or MFG-E8. The eat-me signal molecules should have bound to phosphatidylserine, therefore, eliminated synapses were apoptotic inactive ones. 2) Parkinsons diseasemodel and microglia. Microglia may be involved in compensation of dopaminergic neuron loss
by eliminating glutamatergic synapses from subthalamic nuclei in the 6-OHDA-induced rat
Parkinsons disease model. Here, hyperactive synapses should have been eliminated. 3) Less
active microglia in the attention deficit/hyperactivity disorder (ADHD) model rats. Recently,
we have found that Lister hooded rats (LHRs), an inbred rat strain having black spots in
the head and the back, display ADHD-like behaviors. Microglia in the frontal cortex of LHR
with smaller somata expressed CD11b less significantly than those of Wistar rats. Synapsin I
immunoreactivity was decreased in the prelimbic region in the frontal cortex compared to thatin the Wistar rat brains. All these findings suggest that microglia may modulate various kinds(COI:No)of behaviors by eliminating synapses by phagocytosis.

3P-076

Localization of imidazoline 1 and α2-adrenergic receptors in newborn
rat brainstem
Yukari Nagakura, Ryoji Ide, Chikako Saiki, Yoichiro Kitajima, Nana Hashizume, Toshio Imai

（Dept Physiol, Sch Life Dent at Tokyo, Nippon Dent Univ, Japan）

Introduction: Dexmedetomidine (DEX), a α2-adrenergic receptor (α2-AR), has been used asa sedative and is also known to suppress blood pressure and heart rate through its stimulant
effect on imidazoline 1 receptor (I1R), as well as on α2-AR. However, compared to α2-AR,
little is known about the distribution of I1R in the area related to cardiorespiratory centersin the brainstem. Therefore, in this study we performed immunohistochemistry of newborn
rat brainstem to detect I1R and α2-AR-positive neurons in the ponto-medullary area to gain afurther understanding of the effect of DEX on the cardiorespiratory regulation mechanism inthe central nervous system.
Materials and methods: Newborn Wistar rats (n = 7, 8-11 g) were deeply anesthetized and aftertermination of breathing brainstem were dissected out. The dissected tissues were fixed in 4%
paraformaldehyde, rinsed in phosphate-buffered saline containing sucrose, placed in compound,and stored at -80 ℃. Frozen serial sections (10 µm thick) were cut with a cryostat. The sections
were incubated in a blocking solution and with primary and secondary antibodies. Immunofluorescence was visualized by using a spectral confocal microscope. As primary antibodies,
we used anti-NISCH and anti-α2A-AR antibodies to identify I1R and α2-AR, respectively, andanti-tyrosine hydroxylase (TH) antibody to identify both caudal ventrolateral medulla (CVLM)
and rostral ventrolateral medulla (RVLM), in which the basic cardiorespiratory centers arelocated. In CVLM and RVLM, clusters of TH-positive cells exist in the regions corresponding
to C1 and A1 areas, respectively.
Results: NISCH-positive neurons were detectable in brainstem, including CVLM, RVLM, andthe area close to locus coeruleus (LC) of caudal pons. In addition, among the NISCH-positive
neurons some neurons seemed to be co-expressed with α2A-AR.
Conclusion: Present results suggest a possibility that DEX influences central cardiorespiratory regulation mechanism by activating both I1R and α2-AR in the ponto-medullary area.
(COI:No)

3P-0743P-077

Kazuya Miyanishi, Minori Watanabe, Rina Uchida, Emamussalehin Choudhury, Hajime Yano,
Junya Tanaka（ Dept Mol Cell Physiol, Grad Sch Med, Ehime Univ, Japan）

Univ, Japan）

Parkinson’s disease (PD) is one of the frequent neurodegenerative diseases causing bradykinesia, tremor, muscle rigidity and postural instability. Although progressive dopaminergic(DArgic) neuron loss in the substantia nigra is a main pathology of PD, motor deficits arethought not to become obvious until most DArgic neurons are lost, probably due to compensatory mechanisms that overcome the reduction of DA level in the striatum. Even in animal PD
models, the compensatory mechanisms make it difficult to detect motor dysfunctions whenmost DArgic neurons are functional. In this study, we performed various behavioral tests(apomorphine-induced rotation, cylinder, forepaw adjusting steps (FAS), beam walking, rota-rod,
and open-field test), using 6-hydroxydopamine (OHDA) and lipopolysaccharide (LPS)-induced
hemi-PD model rats with various striatal DA levels, to find the best way to predict the DAlevel from earlier disease stages. Reduction in the striatal DA levels in the 6-OHDA-model was
severe, while LPS-model was less significant. Among the behavioral tests, data from cylinderand FAS tests, which evaluate forelimb motor function, best correlated with decline of the DAlevel. The beam walking and apomorphine tests showed less significant correlation than thecylinder and FAS tests. Open-field and rota-rod tests were not detectable. Expressional levels
of mRNA encoding tyrosine hydroxylase (TH), a marker of DArgic neurons, correlated wellwith the DA level. Metabotropic glutamate receptor 4 mRNA expression correlated with thestriatal DA level and may be related to compensatory mechanisms. These results suggest that
forelimb movements, or hands and forearms in clinical settings, rather than movement of the
body or large joints should evaluate motor impairments of PD. The combination of cylinderand FAS tests may be the best way to evaluate the rat PD models, in which many functional(COI:No)
DArgic neurons.

Humans and animals can judge whether the current state is favorable or unfavorable, andselect actions accordingly. To generate actions appropriate for the current state, autonomic
cardiovascular tuning to supply energy to active skeletal muscles is important. However, theexact neuronal mechanisms of dynamic adjustments of autonomic cardiovascular responses indifferent states are still unclear. In this study, we hypothesized that the amygdala plays a rolein autonomic cardiovascular tuning for adaptive behavioral control. We recorded the bloodpressure and heart rate of rats during appetitive and aversive classical conditioning tasks inwhich they learned varying associations between conditioned stimuli (CS) and unconditioned
stimuli (US) in three types of blocks: (1) in appetitive (REW) blocks, one tone cue (CS+, 10kHz) preceded reward (US+, 5% sucrose) and another (CS-, 4 kHz) preceded non-reward (US-);(2) in aversive (AVE) blocks, the CS+ preceded an air puff (US+) and the CS- preceded no
air puff (US-); (3) in neutral (NA) blocks, both CS tones preceded nothing. NA blocks werealternately deployed between REW and AVE blocks. In the REW blocks, blood pressure
gradually increased during the CS-US interval, followed by a vigorous pressor response to
the US+. In the AVE blocks, blood pressure showed phasic depressive responses to both
CS+ and US+. The blood pressure response to the CS+ in the REW blocks was significantly
higher than that in the NA blocks. The heart rate response to the CS+ in the AVE blocks
was significantly lower than that in the NA blocks. Furthermore, bilateral inactivation of thecentral nucleus of the amygdala by microinjection of the GABAA receptor agonist muscimol
significantly decreased the pressor responses during the REW blocks. These results suggestthat the central nucleus of the amygdala may contribute to pressor response tuning evoked byemotional stimuli.
(COI:No)

3P-075

3P-078Behavioral tests predicting striatal dopamine level in a rat hemiParkinsonʼs disease model

Activation mechanism of heat shock factor 1 induced after zebraﬁsh
optic nerve injury
Kayo Sugitani（Div Health Sci, Grad Sch Med Sci, Kanazawa Univ, Japan）
Generally, fish central nervous system (CNS) neurons can regenerate their axons even after
nerve transection, whereas mammalian CNS neurons cannot. We have identified many nerve
regeneration associated genes (RAGs) using the zebrafish optic nerve regeneration model.
Heat shock factor 1 (HSF1) is one of the RAGs, which acts as a key regulatory molecule
under the various acute stress conditions. Previous studies showed that HSF1 mRNA andphosphorylated-HSF1 protein rapidly started to increase in retinal ganglion cells within 30 min
after optic nerve injury. However, the rapid activation mechanism of HSF1 has not been clearly
elucidated. In breast cancer cells, activation of HSF1 is reportedly controlled by 17β-estradiol(E2). RT-PCR analysis of HSF1 and brain specific aromatase B, as a biomarker of E2 exposure,showed very similar temporal expression pattern in retina and optic nerve after nerve injury.
Here, we investigated the correlation of the levels of HSF1 and aromatase B during zebrafish
optic nerve regeneration. (COI:No)

S162

Switching of autonomic cardiovascular regulation to emotional stimuli
by the central nucleus of the amygdala in rats
Ko Yamanaka, Hidefumi Waki（Dept Physiol, Grad Sch Health and Sports Sci, Juntendo

Effects of pharyngeal mechanical stimulation on skeletal muscle blood
ﬂow and blood pressure
Yudai Tanaka1，Nobuhiro Watanabe2，Harue Suzuki2，Koji Fukui1，Harumi Hotta2（ 1 Sys Eng ＆ Sci, Grad Sch Eng ＆ Sci, Shibaura Inst of Technol, Saitama, Japan, 2 Dept
Auton Neurosci, Tokyo Metropol Inst Gerontol, Tokyo, Japan）
It has been shown that a mechanical stimulation to pharynx induced a reflex increase inthyroxine and calcitonin secretion from thyroid gland by excitation of thyroid parasympathetic
efferent nerve. In this study, we aimed to clarify whether the pharyngeal stimulation affects
muscle blood flow, by modulating muscle sympathetic nerve activity. For this purpose, we
compared the changes in muscle blood flow and mean arterial pressure (MAP) before and after
cutting lumbar sympathetic trunks (LSTs). In the experiments, we used anesthetized malerats. We inserted a catheter into the common carotid artery and measured MAP. Rats wereartificially ventilated via tracheal cannula. To measure muscle blood flow, we exposed soleusof left hindlimb and set a probe of the laser Doppler flowmeter on the muscle. The pharynx
was mechanically stimulated by a small balloon every 10 seconds for a period of 1-5 minutes.
LSTs were cut at a part of lumbar segments (L3-L5). When LSTs were intact, blood flow and
MAP increased during pharyngeal stimulation. The percentage increase in blood flow during
stimulation was 0-10% of the control value before stimulation. In the case of MAP, increase was
10-20%. It could be assumed that when vascular diameter of muscle is constant, the percentage
increase in blood flow should be same as that in MAP. However, we found response of bloodflow was smaller than that of MAP. Therefore, before cutting LSTs, we considered bloodvessels in muscle constrict during stimulation. After cutting LSTs, both increases in bloodflow and MAP were changed to the same extent (0-10%). The results indicate that reaction ofblood vessels induced by pharyngeal stimulation disappeared by cutting LSTs. Therefore, we
concluded that pharyngeal mechanical stimulation altered skeletal muscle blood flow and blood(COI:No)pressure by modulating muscle sympathetic nerve activity. 

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

3P-079

3P-082

Yoshihiro Mitoh1，Tadasu Sato2，Takehiro Yajima2，Masako Fujita1，Motoi Kobashi1，
1 1 Dept Oral Physiol, Okayama Univ Grad Sch Med
Hiroyuki Ichikawa2，Ryusuke Yoshida（

Takao Shioya（ Dept Physiol, Fac Med, Saga Univ, Japan）

Effects of appetite-boosting peptides on the superior salivatory nucleus
neurons in rats
Dent Pharm Sci, 2 Div Oral Craniofac Anat, Tohoku Univ Grad Sch Dent）

The superior salivatory nucleus (SSN) is the primary parasympathetic center for the submandibular and sublingual salivary glands. Since copious salivary secretion is observed during
feeding in various animals, it is possible that appetite-boosting peptides induce the salivation
by increasing SSN neuronal excitability, in addition to facilitating feeding behavior. In thisstudy, we mainly electrophysiologically investigated effects of some appetite-boosting peptides
on SSN neurons. Whole-cell patch-clamp recordings were performed from neonatal rat SSN
neurons retrogradely labeled with a fluorescent tracer. The membrane currents and potentials
were analyzed in bath-application of the peptides. Among peptides tested, only orexins (OXs;
OXA and OXB) affected SSN neurons. In current-clamp mode, application of OXs at resting
membrane potential induced depolarizations and often produced action potentials. OXA generated larger depolarization than OXB. In voltage-clamp mode, OXA generated inward currents
at -70 mV and the currents were completely inhibited by antagonists for OX1R and OX2R ofthe receptor subtypes, consistent with immunohistochemical data. The frequency of miniature
excitatory postsynaptic currents scarcely increased, suggesting that the neurons are mainly
activated via postsynaptic OX receptors. When a long depolarizing current pulse was given
at -90 mV (activation of A-currents), SSN neurons were classified into two types from thefiring pattern. We discussed the relationship between the responsiveness of two types of SSN
neurons to OXA and their target organs.
(COI:No)

Generation of EAD in heart cells involves reverse E-C coupling andreverse electrotonic conduction along T-tubules
Early after depolarization (EAD) is an abberant cardiac afterpotential that underlies thedevelopment of life-threatening ventricular arrhythmias. It is believed that the development
of EAD is caused by the reactivation of L-type Ca2+ current during the period of the action
potential plateau; however the cellular mechanisms that underlie the development of EAD
is still controversial. One favorable alternative is the depolarizing reverse-mode operation of
the Na+/Ca2+ exchanger, which is activated by aberrant Ca2+ release from the sarcoplasmic
retuculum in the process of reverse E-C coupling. Since EADs develop preferentially in damaged heart cells with abnormal Ca2+-signaling, here I studied the causal link between thedevelopment of EADs and aberrant intracellular Ca2+ level ([Ca2+] i) dynamics in mouse heartcells, using nystatin “superforated-patch” technique and Ca2+ imaging by fluo-3 AM. My results
show: 1) The generation of EADs was preceded by the development of depolarizing membranepotential (Vm) fluctuation. 2) The depolarizing Vm fluctuation occured concurrently with a local
brief [Ca2+] i elevation, and the Vm fluctuation was eliminated when Ni2+ was used to block the
Na+/Ca2+ exchanger. 3) The generation of the Vm fluctuation and EADs were suppressed after
the T-tubule system of the cell was detubulated. 4) Abbreviating the T-tubules’ length constantby increasing extracellular K+ level suppressed the development of the Vm fluctuation and
EADs accordingly. Taken together, I conclude that EADs are caused by the depolarizing Vm
fluctuation, which is induced locally in the T-tubule membrane by aberrant [Ca2+] i elevationand is conducted back electrotonically along the T-tubules. Hence, the membrane potential
waveform of the T-tubule membrane might be totally different from that of the surface plasma
membrane. (COI:No)

3P-0803P-083

Kentaro Taniguchi1, 2，Shizuka Nakajima1，Yuka Nomura1，Yuuki Kudo1，Naoya Okumura2，
2 1 Nagahama Inst, Bio-sci and Tech, 2 College Life andNaoya Jinno2，Akito Shimouchi（

1 1Grad Sch Health Sci, Tokyo Met
Masaru Watanabe1，Naoya Nakahara 2，Yukisato Ishida（
Univ, Japan, 2 Dept Mol Physiol, Jikei Univ Sci Med, Japan）

Saliva chromogranin A is associated with night sleep efﬁciency
evaluated by heart rate variabilities in young adults
Heal Sci, Chubu Univ）

Chromogranin A (CgA) is an acidic secretory protein belonging to the granin family, and saliva
CgA is related to the psychological stress. Heart rate variability (HRV) is one of the major
methods to obtain health information including sleep efficiency with non-invasive method. To
examine the relationships between CgA and sleep efficiency, we enrolled 20 young adults. HRV
and physical acceleration were obtained every minute for 24 hours during free moving. Saliva
was obtained before and after night sleep. The ratio of low frequency (LF)/high frequency(HF) and HFnu were used as HRV indices. Subjects with low saliva CgA after waking hadsignificantly lower modulation between sleep and after waking of LF/HF and HFnu. We
considered that lower levels of saliva CgA after wake-up suggested lower efficiency of night(COI:No)
sleep.

Modulation of myosin II function causes thin ﬁlaments disarrangement
through cross-bridge independent pathway in skinned smooth muscle

In the skinned (cell membrane permeabilized) guinea pig taenia caecum, blebbistatin, a selective inhibitor of skeletal and smooth muscle myosin II (Limouze et al., 2004, Katayama etal., 2006), suppressed both myosin light chain phosphorylation-dependent- and -independent
contraction, and also disrupted contractile filament organization (Watanabe et al., 2010). To
clarify the mechanisms of the smooth muscle filaments disorganization by blebbistatin, weinvestigated lattice like organization and periodic repeat of myosin and actin molecules ofbeta escin skinned taenia cecum using X-ray diffraction technique (Watanabe et al., 1993,
and Watanabe et al., 2009). The X-ray diffraction experiments were made at BL-6A in the
Photon Factory, the Institute of Materials Structure Science, Tsukuba, using the small-angle
X-ray camera. Diffraction patterns were recorded on GE healthcare imaging plates with a
specimen-to-plate distance of 220 cm. The size of the beam on the specimen was 1.0×0.5
mm. The exposure time was three times of 180 s. In the resting state, blebbistain weakened
14.4 nm meridional reflection from the thick filament as previously reported (Watanabe etal., 2009). Also, blebbistatin extended an equatorial reflection at a Bragg spacing of 11.4 nm
originated from lattice like arrangements of the thin filaments, but had little effects on thethird, fifth and higher order meridional reflections from collagen. In the absence of nucleotides
in the intracellular solution, blebbistatin did not show the extension of the 11.4 nm equatorialreflections. These results suggest that disruption of thick filaments by blebbistatin treatment
causes thin filaments disarrangement through cross-bridge independent pathways in skinned
taenia cecum, since blebbistatin is known to affect myosin nucleotide binding but dose not have
(COI:No)
direct effects on actin. 

3P-081

3P-084

Yuichirou Kimoto, Mamoru Tanida, Yasutaka Kurata（ Dept Physiol 2, Kanazawa Med Univ,

Naoya Nakahara（ Dept. Mol. Physiol., Jikei Univ. Sch. Med., Japan）

Effects of gastro-intestinal osmotic stimulation on afferent vagal nerve
outﬂows in the stomach, intestine and liver in rats
Japan）

Previously, it has shown that duodenal stimulation by hypertonic NaCl caused thermogenesis
and increase in metabolic rate (Osaka et la., Journal of Physiol. 2004). Vagal afferent pathways
in the gastro-intestinal organs connect to the brain for introducing various information in
the gastro-intestinal organs, but it has not been cleared whether osmotic stimulation affects
afferent vagal nerve activities in the stomach and intestine. Thus, we examined effects ofinjection of hypertonic NaCl solution into the stomach or duodenum on afferent vagal nerve
outflows in anesthetized mice.
We measured the afferent signals of the vagal nerve in the stomach branch, celiac branch andhepatic branch, and the efferent signals of the renal sympathetic nerve in urethane andαchloralose-anesthetized mice. The hypertonic solution (1M, 10ml/kg) was administered into
the stomach, intestine or portal vein. In addition, vagotomized mice by deletion of afferent
vagal branch were used to investigate role of vagal afferent pathways. We firstly showedthat hypertonic solution injection into the duodenum but not the stomach dose dependently
activated the afferent vagal nerve activity of the celiac branch, and it lowered blood pressure
30 min after injection. In addition, it stimulated the afferent vagal nerve activity of the hepatic
branch. In the vagotomized mice, stimulatory response of efferent renal sympathetic nerve to
intra-duodenal injection of NaCl solution was attenuated. These lines of evidence suggest that
osmotic stimulation in the intestine affects the vagal afferents and activates efferent discharge(COI:No)of the renal sympathetic nerve in mice. Actin ﬁlaments render considerable heat capacity to skeletal muscle
sarcomere
In biological tissue, crowding proteins considerably interfere with hydrogen bonding andmolecular motion of neighboring water molecules to form so called bound-water as well as
hypermobile water. Energetic consideration clearly indicates that functional proteins including
myoproteins utilize entropic free-energy derived from restriction and liberation of a cluster ofwater molecules to drive biophysical reactions such as muscle contraction. One of the methods
to detect the interaction between proteins and water is differential scanning calorimetry (DSC),
which records heat capacity change with temperature. We analyzed skeletal muscle fibers ofsartorius muscle from Rana Catesbeiana to analyze interaction of myoproteins with watermolecules. Demembranated fibers at rigor condition were used to avoid osmotic movement ofwater molecules across the cell membrane with freezing. With gradual increase in temperature(1℃/min), latent heat absorption peaks were observed at -24, -21, 0, 46, 65℃. The peaks at 46℃
and 65℃ would represent irreversible denaturation of myosin and actin, respectively, because
selective removal of myosin and actin decreased the corresponding peak. On the other hand,the -24℃ peak was largely affected by actin removal, and the -21℃ peak was affected by theremoval of actin as well as myosin. Integrated heat capacity in the range from -80 to +20℃ wassignificantly decreased by denaturation of actin. The decrease in the integrated heat capacitywith actin removal amounted to 65% on a per-myosin basis. These results indicate that actin
filaments render considerable heat capacity to skeletal muscle sarcomere.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S163

3P-085

Inhibition of actin polymerization accelerates relaxation process in beta
escin skinned smooth muscle
Satoko Mihashi（Human Health Sci, Grad. Sch, Tokyo Metropolitan Univ, Japan）

3P-088

Effect of TRPC knockout on pupil diameter adjustment
Toshiyuki Kaneko, Akira Takai（Dept Physiol, Asahikawa Med Univ, Hokkaido, Japan）

It is known that cytochalasin D, a fugal metabolite, and latrunculin B, a sponge toxin, are known
to inhibit actin polymerization, and also to suppress smooth muscle contraction. We found thatboth cytochalasin D and latrunculin B inhibited the maximal Ca ion induced force at around 1
micro M, but enhanced submaximal force development induced by lower Ca ion concentrations(S-13, Watanabe and Mihashi), indicating that inhibition of actin polymerization enhances Ca
ion sensitivity for the force. To investigate regulatory mechanisms of the thin filament liked
Ca ion dependent pathway in detail, we examined the cytochalasin D and latrunculin B effects
on Ca ion removal induced relaxation process in skinned (cell membrane permeabilized) taenia
cecum and carotid artery from guinea pig. When 10 micro M Ca ion was removed from theintracellular solution, the contractile force was decayed bi-exponentially with initial short timelag as previously reported (Yoshino et al., 2005, Hashimoto et al., 2008). Both cytochalasin
D and latrunculin B accelerated the relaxation process. The acceleration effect of the actin
polymerization inhibitors was observed even in the absence of nucleotide in the solution. On
the other hand, according to our preliminary results, myosin light chain inhibition with ML-7,
a kinase inhibitor, might attenuate the accelerating effects of cytochalasin and latrunculin B on
relaxation, indicating that modulation of actin function directly affect myosin light chain kinase(COI:No)
activity.

Since vision is an important sensory organ for mammals, mechanisms to properly capture
visual information are developed very well. The iris has a role of adjusting the size of the pupil
to adjust the amount of light incident on the retina, the pupillary sphincter contained therein
is only involved in the miosis and is dominated by parasympathetic nerves. On the other
hand, the pupil dilator muscles involved in mydriasis are dominated by sympathetic nerves,and the pupil diameter is adjusted antagonistically by these intraocular smooth muscles. To
maintain the light intensity properly, the iris needs to rapidly change the diameter of the pupil(rapid phase) and maintain the pupil for a long time (sustained phase). Extracellular calcium
influx is required for intraocular smooth muscle, but there are some molecular entities andmechanism of action, but details are unknown. So far, we have shown that calcium influx insustained phase is mediated by two nonselective cation channels (NSCC) with different unit
conductance by experiments in bovine ciliary muscle. Expression of TRPC1, TRPC3, TRPC4,
TRPC6, Orai1, etc. has been confirmed as a molecule candidate, but it is difficult to apply geneknockdown to bovine material, details of the relationship with NSCC have been clarified absent.
Therefore, experiments were carried out using TRPC 3 and TRPC 6 knockout mice and double
knockout mice based on these, as experimental materials, using mice which are relatively easy
to genetically modify. As a result, the pupil diameter during strong light stimulation decreased
significantly with the TRPC3 knockout but did not change with weak light stimulation. These
results suggest that TRPC3 may have some effect on the contraction of the pupil dilator(COI:No)muscles.

3P-0863P-089Contractile properties of rat epididymal duct
3 1 Dept Cell Physiol,
Retsu Mitsui1，Hikaru Hashitani1，Lang Richard J.2，van Helden Dirk F. （Differences between DOMS and muscle contusion in acuteinﬂammation

Nagoya City Univ Grad Sch Med Sci, Japan, 2 Sch Biomed Sci, Monash University,
Australia, 3 Sch Biomed Sci and Pharmacy, The University of Newcastle, Australia）

Kazuyoshi Hisatsune（Dept Musculoskeletal Functional Research and Regeneration, Grad

Background: The contractility of smooth muscle in the epididymal duct plays a fundamentalrole in transport of sperm from the testis to the vas deferens. Here, we examined spontaneousand sympathetic nerve-mediated contractions of the rat epididymal duct.
Methods: Short segments from two caudal regions (diameter: 200 µm and 400 µm) wereisolated, and both ends were loosely tied with threads. Changes in the longitudinal distance
between the threads were monitored with a video camera, and analysed using edge-tracking
software.
Results: Small ducts (200 µm in diameter) exhibited rhythmic phasic contractions that were
suppressed by yohimbine (1 µM), an α2-adrenergic receptor antagonist. Rhythmic contractions
were abolished by CPA (10 µM, sarcoplasmic reticulum (SR) Ca2+-ATPase inhibitor), 2-APB(100 µM, IP3 receptor inhibitor), niflumic acid (100 µM, Ca2+-activated Cl－ channel blocker)
or lowering extracellular Cl－ concentration (12.4 mM). Contraction frequency was decreasedby nifedipine (1 µM, L-type Ca2+ channel blocker) or ML218 (1 µM, T-type Ca2+ channel
blocker). Larger ducts 300-500 µm in diameter remained quiescent, but clonidine (100 nM),an α2-adrenergic receptor agonist, induced rhythmic contractions in a manner sensitive toyohimbine. In the larger ducts, electrical field stimulation (EFS; 50 µs duration, 2 Hz for 1 min,
5 min interval) induced a sustained contraction superimposed with rhythmic contractions.
Prazosin (1 µM), an α1-adrenoceptor antagonist, or PPADS (10 µM), a purinoceptor antagonist,
suppressed the sustained component, while yohimbine (1 µM) prevented the superimposed
rhythmic contractions. Depletion of sympathetic neurotransmitters (10 µM guanethidine)largely diminished the EFS-induced contraction.
Conclusion: 1) Sympathetic nerve-derived noradrenaline acts on α2-adrenoceptor to enhanceor induce rhythmic contractions. 2) Rhythmic contractions are likely to arise from SR Ca2+
release and depolarisation through resultant opening of Ca2+-activated Cl－－ channels. 3)
EFS-induced contraction consists of α1-adrenoceptor- and purinoceptor-mediated sustained
contraction and α2-adrenoceptor-mediated superimposed rhythmic contractions.
(COI:No)

Delayed onset muscle soreness (DOMS) and muscle contusion (MC) are often observed as
skeletal muscle injury for athletes, optical treatments could be different at each injury andthe progression. The purpose of the present study was to investigate histological and physiological differences between DOMS and MC in acute inflammation. Male SD rats were divided
into the DOMS group induced by lengthening contraction and MC group drop-mass method,respectively. The transverse sections at 48 h after injury were stained with hematoxylin andeosin for histological observation. Additionally, the calf circumference, pain sensitivity, andmotor function were evaluated at 24 and 48 h after injury. Necrotic areas with inflation ofmononuclear phagocyte were wider in the MC group than DOMS groups. Pain sensitivitiesof the DOMS group were higher at 48 h than 24 h after injury. Conversely, the sensitivitiesof the MC group were higher at 24 h than 48 h after injury. There were no differences in
the calf circumferences of the DOMS group between the intact and injured limbs at 24 and48h after injury. The circumferences of the MC group were longer in the injured limb than
the intact limb at 24 and 48h after injury. Downhill-walking speeds were almost the same
between the DOMS and MC groups at 24 and 48h after injury. These results suggest that
acute inflammation is occurred both in DOMS and MC, but the severity is remarkable in
MC. The change in pain sensitivity by 48h after injury was different between the injuries, notnecessarily correspond to the sign of swelling and motor function. Further research including
measurement of the physiological signs correlated with pain sensitivity is needed to clear
differences between DOMS and MC.(COI:No)

3P-087

3P-0902 1 Radiation
Tsuyoshi Fukuzawa1, 2，Ippei Yamato2，Shuichi Soeda2，Tetsuro Tamaki（

Mayumi Matsunaga1, 2，Maki Kimura2，Haruna Toda2，Sadao Oyama2，Wataru Ofusa2，
1 1 Dept. Dent. Anesthesiol.,
Asuka Higashikawa2，Yoshiyuki Shibukawa2，Tatsuya Ichinohe（

Quantitative evaluation of the decreased capacity of skeletal muscle
hypertrophy and the ratio of the bone marrow cell transplantation (BMT)
after the various total body irradiation (TBI)

Sch Biomedical and Health Sci, Hiroshima Univ, Japan.）Mechanical stimulation-induced intracellular cAMP- and Ca2+-signalingin human odontoblast

Therapy, Tokai Univ, Japan, 2 Muscle Physiol ＆ Cell Biol Unit, Tokai Univ. Japan）

Tokyo Dent. Coll., 2 Dept. Physiol., Tokyo Dent. Coll.）

The capacity of skeletal muscle hypertrophy was quantified after the various intensity of the
TBI in comparison of normal mice.The rate of BMT was also examined and compared.A single
TBI of 9.0(n=24), 5.0(n=6) and 2.5(n=5)Gy was delivered to C57BL/6mice, and the bone marrowstromal cells, which was obtained from GFP-Tg mouse, were injected from the tail vein(1x106
cells).Five weeks after the TBI, the compensatory hypertrophy was induced in the rightplantaris(PLT)muscle by the surgical ablation(SA) of the synergistic muscles(gastrocnemius
and soleus), and contralateral left-side was preserved as a control.Five weeks after the SA,
blood and muscle samples were obtained, and the rate of BMT in the blood(GFP-chimaerism)
and the ratio of muscle hypertrophy/contralateral-side was determined.The mean GFPchimaerism were 93±4.9% in9Gy, 88±7.8% in5Gy, and 0.5±0.1% in2.5Gy group following the
TBI intensity, and reducing ratio between 9Gy and 5Gy was small, but that was very large
between 5Gy and 2.5Gy with almost no replacement of bone marrow cells occurred.In contrast,
the muscle hypertrophy ratio was decreased following inverse correlation to the TBI intensity
as 2.3±15 in 9Gy, 35.6±11.5 in 5Gy and 44.5±12.3 in 2.5Gy groups. In this respect, we havefound that the normal mouse SA model always showed 71.4±5.6% hypertrophy, which bringout the almost maximum capacity of skeletal muscle.Therefore, when the value of normalmouse sets out the 100%, the TBI9Gy reduced 97% the capacity of muscle hypertrophy, 5Gy
reduced 50%, and 2.5Gy reduced 45%.Therefore, we concluded that the influence of TBI isbasically different from the bone marrow and skeletal muscle. (COI:No)We previously reported that mechanical stimulation evoked Ca2+ influx from extracellular medium through mechanosensitive transient receptor potential (TRP) channels in acutely isolated
rat odontoblasts. In addition, mechanosensitive piezo 1 channels are involved in mechanosensory processes in rat odontoblast-neuron complex. The mechanosensitive Ca2+ signaling inodontoblasts participates in reactionary dentin formation, and/or sensory transduction for thedentinal pain. We previously elucidated cyclic AMP (cAMP)-mediated crosstalk between TRP
vanilloid subfamily member 1 (TRPV1) channels and cannabinoid 1 (CB1) receptors in odontoblasts. CB1 receptor activation stimulates adenylyl cyclase, resulting in facilitation of cAMP
production. The increase in intracellular cAMP then activates TRPV1 channels, resulting in
Ca2+ influx. These findings suggested that the increase in intracellular cAMP concentration
mediates Ca2+ signaling elicited by mechanosensitive ionic channel activation, and intracellularcAMP might play key roles in dentin formation and/or sensory transduction. However, thedetailed intracellular cAMP signaling pathway, the role and the participation of cAMP on
the cellular function and the involvement of cAMP in mechanosensitive Ca2+ signaling inodontoblasts remain unclear. In the present study, we measured intracellular cAMP and Ca2+
concentrations following mechanical stimulation applied to human odontoblast (HOB) cells.
Intracellular cAMP level and intracellular free calcium concentration ([Ca2+] i) was recorded byfluorescence from mNeon Green-based cAMP sensor and fura-2, respectively. In the presenceof extracellular Ca2+, mechanical stimulation increased [Ca2+] i in both mechanically stimulated
HOB cells, and neighboring HOB cells to the stimulated ones. The increases were not desensitized by repeated mechanical stimuli. Mechanical stimulation decreased intracellular cAMPlevel in HOB cells, but did not induce any desensitizing effects on the intracellular cAMPlevel. These results indicated that mechanical stimulation elicited intracellular Ca2+ signalingin HOB cells, and the signal established intercellular odontoblast-odontoblast communication.
The results also suggested that mechanical stimulation inhibited intracellular cAMP signaling(COI:No)in odontoblasts.

S164

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

3P-091

3P-094Hiroshi Inoue, Seiji Goda（ Dept Physiol, Osaka Dent Univ, Japan）

Ratna Ramadhani, Kohei Mito, Toshiya Sato, Hisayoshi Ishii（Division of Physiology,

Inﬂuence S-PRG ﬁller eluate on secretion of MMP-1 and MMP-3 in
TNF-α stimulated human gingival ﬁbroblasts
The S-PRG filler is known to have a high cariostatic effect material. In this study, we examinedthe effect of S-PRG filler eluate on MMP-1 and MMP-3 secretion of human gingival fibroblasts(HGF). S-PRG filler eluate was added to HGF and we detected MMP-1 and MMP-3 secreted
into the culture supernatant by immunoblotting. As a result, secretion of MMP-1 and MMP-3
were enhanced by S-PRG filler eluate. We assessed the effects of S-PRG filler eluate induced
phosphorylation of p38, ERK 1/2 and JNK in HGF by immunoblotting. Results revealed that
phosphorylation of p38 and ERK 1/2 occurred within 1 minute. However, phosphorylationof JNK was not enhanced by S-PRG filler eluate. Secretion of MMP-1 and MMP-3 increasedby S-PRG filler eluate was slightly decreased by p38 inhibitor, but was markedly decreased
by JNK inhibitor and ERK inhibitor. Secretion of MMP-1 and MMP-3 significantly increased
by TNF-α stimulation was suppressed by S-PRG filter eluate. These results suggested that
phosphorylation of p38, ERK 1/2 and JNK may be involved in the secretion of MMP-1 and
MMP-3 by S-PRG filler eluate. Furthermore, it was suggested that S-PRG filler eluate may besuppressed the enhancement of MMP-1 and MMP-3 secretion by TNF-α stimulated HGF.

(COI:No)

Different Effects Between Trigeminal Sensory and Vagal Visceral Input
on Saivary Glands Blood Flow
Department of Oral Biology, School of Dentistry, Health Sciences University of Hokkaido）

Adequate blood supply to the three major salivary glands, submandibular (SMG), sublingual(SLG), and parotid (PG), is important for saliva production because the fluid in saliva originatesfrom blood capillaries and the interstitial fluid. Salivary gland hemodynamics is regulated by
the sensory and autonomic nervous system via several cranial nerves, including the trigeminal
and vagus nerves. Trigeminal sensory input is essential for regulating hemodynamics andsecretion of salivary gland in the orofacial area (Sato ＆ Ishii, 2015). Although afferent electricalstimulation of the vagus nerve has been reported to induce salivation in anesthetized rodent(Ueda et al., 2016), the effect of vagal-visceral input on hemodynamics in salivary glands remains unclear. Therefore, the purpose of this study was to evaluate and compare the different
effects of trigeminal sensory and vagal-visceral inputs on blood flow in salivary glands in deeply
urethane-anesthetized rats.
The left AVN, LN, and CVN were stimulated by bipolar electrodes. The cervical sympathetic
trunk (CST) on both side and the CVN on the right side were cut before the stimulation of theleft abdominal vagus nerve (AVN). The left CVN and lingual nerve (LN) were stimulated after
cutting the CST and CVN. The hemodynamics of the salivary glands (SMG, SLG, and PG) wererecorded using a laser speckle imaging blood flow meter. Systemic arterial blood pressure(SABP) was recorded from the femoral catheter using a Statham pressure transducer.
Our results indicated that LN stimulation elicited an increase in both SABP and blood flow in
the SMG, SLG, and PG. Furthermore, CVN and AVN stimulation induced an increase in SABP;
however, blood flow increase in the three salivary glands was lower than that observed after
LN stimulation. In conclusion, trigeminal sensory input rather than vagal-visceral input appears
to be involved in the regulation of blood flow in the salivary glands.
(COI:No)

3P-092

3P-095Mako Naniwa1, 2，Suzuro Hitomi1，Izumi Ujihara1，Takemi Shikayama1, 3，Kazunari Matsuda4，
1 1 Div Physiol, Kyushu Dent Univ, Japan, 2 Div of Oral Health Sci, Kyushu
Kentaro Ono（

1 1 Dept Integr
Hiroyuki Yajima1，Sumiyasu Ishii1，Wataru Miyazaki2，Noriyuki Koibuchi（

Effect of steroid-containing ointment in a rat oral ulcerative mucositis
model
Dent Univ, Japan, 3 Div of Periodontol, Kyushu Dent Univ, Japan, 4 Daiichi Sankyo Healthcare
Co. Ltd., Japan）

Oral ulcerative mucositis (OUM) is a common oral disease and causes pain during meals
and speaking. Ointment, which is composed of base and medicinal ingredient such as steroid,is frequently applied to the OUM region for healing. Since application of ointment on theoral cavity in experimental animals is technically difficult, little study have demonstrated theeffects of topical treatment on OUM pain. In this study, we investigated the effect of steroidcontaining ointment, which has high residence in the oral cavity, on OUM. First, we compared
the physical and sensory properties of various ointment bases (vaseline, plastibase, trafulointment [TO] base and traful ointment pro-quick [TOPQ] base). TO and TOPQ have highadhesiveness, hardness and viscosity in measurement by a rheometer. TO and TOPQ showed
higher adhesiveness and residue than vaseline and plastibase in human sensory evaluation.
Next, we investigated the effect of triamcinolone-containing TO(TO+Tmc) on OUM region
and OUM-induced pain. In rats, OUM was developed in the inferior labial fornix region bysoaking in 50% acetic acid under anesthesia. Ointment was applied twice a day. On day 2,
prolonged facial grooming behavior (a sign of spontaneous pain) was shortened by TO+Tmc
application, compared with non-treatment group. The head withdrawal threshold to von Freystimulation in the OUM region was significantly increased by TO+Tmc application. Moreover,mRNA level of the glucocorticoid receptor target gene GILZ was increased, and TNF-α and
COX2 mRNA levels were decreased. These results indicate that steroid in the highly-residual
ointments are effective for pain relief on OUM model rats.
(COI:Properly Declared)

3P-093Difference between somatosensory and gustatory input onparasympathetic increase in blood ﬂow in rat three major salivary
glandsToshiya Sato, Ratna Ramadhani, Kohei Mito, Hisayoshi Ishii（Div. of Physiol., Dept. of Oral
Biol., Sch. of Dent., Health Sci. Univ. Hokkaido）

Salivary gland hemodynamics is generally considered to be important for secretion of salivary
fluid. We have previously demonstrated that stimulation of trigeminal sensory nerves induces
rapid increases in blood flow in three major salivary glands mediated by parasympathetic
nerves, as well as salivation. This supports the importance of parasympathetic nerve activation
linked with orofacial sensory input in not only salivary secretion but also blood flow in salivary
glands. Relative secretion of the major salivary gland varies depending on the type of stimulus
such as mechanical stimulus or chemical stimulation of the sense of taste. Thus, it is assumed
that the glandular hemodynamics are also regulated in harmony with regulation of salivation, however, regulatory details remain unclear. To clarify this relationship between type of
sensory input and parasympathetic increase in blood flow among three glands, we analyzed
the glandular hemodynamics during electrical stimulation of the inferior alveolar nerve (IAN;
somatosensory input) or lingual nerve (LN; somatosensory and gustatory input) with 20 V atvarious frequencies (1-40 Hz) for 20 s in urethane-anesthetized rats. IAN or LN stimulationinduced frequency-dependent blood flow increases in three glands, and the increases weresignificantly inhibited by the intravenous administration of hexamethonium, autonomic ganglion blockade. The increase evoked by IAN stimulation was the highest in the parotid gland,
whereas that evoked by the LN stimulation was the highest in the submandibular gland.
Therefore, our results indicate that the parasympathetic increase in blood flow in parotid
gland is involved in somatosensory input, while that in submandibular gland is activated withgustatory input, and suggest that these differences would be related in the differences inrelative secretion rate of three salivary glands.
(COI:No)

Transcriptional kinetics altered by thyroid hormone during mouse
cerebellar development
Physiol, Grad Sch Med, Gunma Univ, Japan, 2 Dept Biosci Lab Med, Grad Sch Health Sci,
Hirosaki Univ, Japan）

Background: Thyroid hormone (TH) plays essential roles in the development of the cerebellumby regulating transcription of target genes. TH binds to TH receptor (TR) located in the cell
nucleus and stimulates transcription through TH response element (TRE). The expressionof many genes is temporary and spatially regulated by TH during cerebellar development.
However, the mode of transcriptional regulation by TR may vary among target genes. In theliver, altered duration of TH exposure resulted in distinct gene expression profiles. To examine
the mechanism of transcriptional regulation by TH in cerebellar development, gene expression
profile induced by various TH exposure duration was studied.
Methods: Anti-thyroid drug propylthiouracil (250 ppm in drinking water) was administered
to C57BL/6J mice from the gestation day 14 to postnatal day (P) 7 to generate perinatal
hypothyroid mice. To study the effect of continuous TH exposure, TH was subcutaneously
administered to hypothyroid pups from P2 to P7 (6 days group). To study the gene expression
profiles induced by single TH administration, TH was injected on P7 and mice were sacrificed
either 6 (6 hours group) or 24 hours (24 hours group) after injection. Cerebellar samples
were collected to extract RNA and subject to microarray analysis. Microarray results wereconfirmed after injection by RT-qPCR.
Results: Compared with hypothyroid mice, TH injected groups induced an alteration of mRNAlevels (upregulation, 1295 genes; downregulation, 1332 genes). Only 7.6% of the genes wereoverlapped in three groups among positively regulated genes, suggesting differential regulationof transcription stimulation in an exposure time-dependent manner of TH. In contrast, 57.2% of
the genes were common in the negatively regulated genes.
Conclusion: TH distinctively regulates transcription of target genes depending on exposure(COI:No)schedule in mouse developing cerebellum. 

3P-096

Acute mono-arthritis activates the neurohypophysial system andhypothalamo-pituitary adrenal axis in rats
Haruki Nishimura1，Makoto Kawasaki1，Takanori Matsuura1，Hitoshi Suzuki1，
Yasuhito Motojima1，Kazuhiko Baba1，Hideo Ohnishi1，Yoshiaki Yamanaka1，
Teruaki Fujitani1，Mitsuhiro Yoshimura2，Takashi Maruyama2，Hiromichi Ueno2，
Satomi Sonoda2，Kazuaki Nishimura2，Kentarou Tanaka2，Kenya Sanada2，Tatsushi Onaka3，
1 1 Dept Ortho, UOEH, Japan, 2 Dept Physiol, UOEH, Japan,
Yoichi Ueta2，Akinori Sakai（
3 Dept Physiol, Jichi Med Univ, Japan）

Various types of acute/chronic nociceptive stimuli cause neuroendocrine responses such asactivation of the hypothalamo-pituitary adrenal (HPA) axis and hypothalamo-neurohypophysial(oxytocin [OXT] and arginine vasopressin [AVP]) system. Chronic multiple-arthritis activates
the OXT/AVP system as well as the HPA axis, but the effects of acute mono-arthritis on the
OXT/AVP system and HPA axis in the same animals have not been evaluated simultaneously. In the present study, we used an acute mono-arthritic model induced by intra-articular
injection of carrageenan in a single knee joint of adult male Wistar rats. Acute mono-arthritis
was confirmed by a significant increase in knee diameter in the carrageenan-injected knee andsignificant decrease in mechanical nociceptive threshold in the ipsilateral hind paw. Immunohistochemical analysis revealed that the number of Fos-immunoreactive (ir) cells in the ipsilaterallamina I-II of the dorsal horn was significantly increased, and the percentage of OXT-ir and
AVP-ir neurons expressing Fos-ir in both sides of the supraoptic (SON) and paraventricular
nuclei (PVN) was increased in acute mono-arthritic rats. In situ hybridization histochemistry
revealed that levels of OXT mRNA and AVP hnRNA in the SON and PVN, CRH mRNA in
the PVN, and proopiomelanocortin mRNA in the anterior pituitary were also significantly
increased in acute mono-arthritic rats. Further, plasma OXT, AVP, and corticosterone levels
were significantly increased in acute mono-arthritic rats. These results suggest that acutemono-arthritis activates ipsilateral nociceptive afferent pathways at the spinal level and causes
simultaneous and integrative activation of both the OXT/AVP system and HPA axis, with adistinct pattern of upregulated gene expression compared to that of chronic multiple-arthritis.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S165

3P-097

Circadian rhythm of PVN neurons regulates glucose tolerance
1 1 Dept Physiol,
Toshiya Yamazaki1, 2，Rika Kita1，Boyang Zhang1，Masanori Nakata（

Wakayama Med Univ, Japan, 2 Dept Clinical Acupuncture, Kansai Univ of Health Sciences,
Japan）
Physiological functions, including feeding, body temperature, and hormone release, show
circadian patterns. Furthermore, impairment of the circadian patterns in these functions isthought to be causally implicated in diverse diseases including obesity and diabetes. Especiallythe paraventricular nucleus (PVN) in the hypothalamus integrates afferent signals from the
hypothalamus including SCN, the region of master clock, and plays a role in the regulationof energy metabolism. In this study, we aimed to clarify the function of circadian rhythm of
PVN on glucose metabolism. We generated and analyzed PVN-preferential Bmal1 KO mice.
KO mice resulted in impairment of glucose tolerance without affecting feeding and energy
expenditure. Interestingly, KO mice also exhibited decreases in insulin secretion without
insulin resistance. On the other hand, insulin release from isolated islets and increases incytosolic Ca2+ concentration of isolated pancreatic β-cell were maintained in KO mice as wellas Cre mice. These results suggested that the circadian rhythm in PVN neurons maintainneuronal regulation of insulin secretion.These results demonstrate that the circadian rhythmof PVN neurons drives neuronal regulation of insulin secretion, which is required for glucose(COI:No)
homeostasis.

3P-098

Identiﬁcation of allopregnanolone-biosynthesizing cells in adrenal grandand developmental change in GABA signaling machinery in adrenal
medullary cells
1 1 Dept Cell and
Keita Harada1，Hidetada Matsuoka1，Yuchio Yanagawa2，Masumi Inoue（

Systems Physiol, UOEH, Fukuoka, Japan, 2 Dept Genetic and Behavioral Neurosci, Gunma
Univ Grad Sch Med, Maebashi, Japan）
GABA is assumed to function as an autocrine or paracrine factor in adrenal medullary (AM)cells. We have reported that GAD67, an isoform of GABA synthesizing enzyme, and α3containing GABAA receptors are expressed in rat AM cells and allopregnanolone, a neuroactive steroid which is known to be synthesized in the adrenal gland, produces an increase in
GABAA receptor channel activity in guinea-pig AM cells. However, it is not clear how thisGABA signaling is established in the adrenal medulla. Before the birth, AM cells are notinnervated by the preganglionic sympathetic nerve fibers and do not form a compact massat the center of the adrenal gland. Thus, GABA signaling machinery might be establishedduring the development of the adrenal medulla after the birth, possibly under the influenceof the sympathetic innervation and/or adrenal cortical steroids. The present experiment
was undertaken to identify the cells where allopregnanolone is synthesized and investigate
whether GABA signaling in AM cells develop after the birth or not. 3α-hydroxysteroid dehydrogenase (3αHSD) and 5α-reductase, enzymes involved in allopregnanolone biosynthesis,
were immunohistochemically detected in mouse adrenal cortical (AC) cells, but not AM cells,and 3αHSD-like immunoreactivity (IR) was co-localized with calreticulin-like in AC cells, amarker protein of the endoplasmic reticulum. To investigate a developmental change in GABA
synthesis, GFP-GAD67 knock-in mice were used. GFP-expressing cells were located in theadrenal medulla, but not in the adrenal cortex. The number of GFP-expressing AM cells in
five-week old mice were much larger than that in one-week old mice. On the other hand,
GABAA receptor channel activities in newborn hamster AM cells did not differ from thosein adult AM cells. These results indicate that allopregnanolone is synthesized in AC cells andexpression of GAD67 and GABAA receptors in AM cells is differently regulated.
(COI:No)

3P-099The gastrin-releasing peptide system in the medial preoptic area
controls male sexual activity in rats
Sho Maejima1，Rei Nomura1，Keiko Takanami1, 2，Tatsuya Sakamoto1，Hirotaka Sakamoto1

3P-100

Renal tissue kallikrein may be involved in the regulatory Ca transport
along the kidney distal nephron
Yukiko Yasuoka1，Tomomi Oshima1，Yuichi Sato2，Noriko Takahashi1，Hiroshi Nonoguchi3，
4 1 Dept Physiol, Sch of Med, Kitasato Univ. Sagamihara, Japan,
Katsumasa Kawahara（
2 Dept of Mol. Diagnostics, Kitasato U. Sch. of Allied Health Sci., Sagamihara, Japan,

3 Internal Med., Kitasato U. KMC Hospital, Kitamoto, Japan, 4 Dept of Health and Nutrition,

Sendai Shirayuri Women’s College, Sendai, Japan）

Tissue kallikrein (TK), a serine protease in the distal nephron including the collecting ducts(CDs) and Henles loops (HLs) (Chen LM et al, 1995), may be a key to regulate the renal calcium(Ca) reabsorption (Picard N et al, 2005). To understand the molecular basis of the regulatory
Ca transport, a segmental and functional expression of TK, TRPV5 (luminal Ca transporter),
CaBP28K, (Calbindin D28K: cytosolic Ca buffering protein) was semi-quantitatively estimated inconjunction with blood and urine analysis.
Methods: Male mice (10 weeks) were treated with either 1% Ca-citrate diet (control) or low(0.05%)-Ca diet (LCa) for 1 week. Expression and localization of the TK, TRPV5, and CaBP28K
mRNAs and proteins were estimated by using in situ hybridization and immunohistochemistry.
Results: Plasma pH and Ca2+ concentration were unchanged in mice with LCa (pH7.38 and4.4 mg/dl (LCa) vs. pH7.36 and 4.5 mg/dl (control)), since the urinary excretion of Ca and NH3/
NH4+ was contrarily decreased and increased, respectively, from 186.0 (control) to 60.3 µg/day (LCa) and 120.7 (control) and 1101.6 µg/day (LCa) (P<0.005, n=8-9). Importantly, expression
levels of TRPV5 and CaBP28K proteins, co-localized in the distal convoluted tubule (DCT) andthe early connecting tubule (CNT), increased similarly during LCa. More importantly, TK
expression dramatically increased in the luminal membrane of the cortical and medullary
thick ascending limb of the HLs (CTAL and MTAL), the upper stream of the DCT and CNT.
Conclusion: Segment-specific increases of the TK, TRPV5, and CaBP28K expressions during
low Ca diet may be co-operatively involved in regulation of the renal Ca reabsorption along the(COI:No)
distal nephron.

3P-101

Thermal stimulation to lower back and rump skin improves voiding
efﬁciency in urethane anesthetized rats
Kaori Iimura, Nobuhiro Watanabe, Harumi Hotta（ Dept Auton Neurosci, Tokyo Metropol
Inst Gerontol, Japan）

Aim: The aim of the present study is to clarify whether somatosensory stimulation can
improve the voiding efficiency in underactive bladder.
Methods: We used female Fisher rats anesthetized with urethane. The cystometrogram wasrecorded via a bladder catheter inserted from bladder dome. Saline was continuously infused
via the catheter until the rat spontaneously voided. The voiding efficiency was calculated
based on infusion volume and voiding volume (the percentage of the voided volume relative
to the infused volume). Somatosensory thermal stimulation was applied to the lower backand rump skin using a Peltier thermode from start saline infusion to voiding. During stimulation, the temperature of the thermode was changed between 35℃ and 25℃ with a speed of
1-2℃/ second, repeatedly every 10 seconds. We compared voiding efficiency without and withthermal stimulation.
Results: Before applying thermal stimulation, voiding efficiency was approximately 24%.
During thermal stimulation, voiding efficiency significantly increased to approximately 40%.
There was 16% increase in voiding efficiency during thermal stimulation compared to control
condition before stimulation. The maximum pressure of bladder contraction was not different
between before and during stimulation. On the cystometrogram, we observed high frequency
oscillations (HFOs) during voiding, which reflecting repetitive contractions of the external
urethral sphincter. The duration of HFOs increased twofold from 1.1 ± 0.5 sec (mean ± SD)
before stimulation to 2.4 ± 1.0 sec during stimulation.
Conclusion: The results suggest that thermal stimulation to the lower back and rump skin
improved voiding efficiency by prolonging the duration of urethral sphincter contractions andextending the urine output time.
(COI:No)

3P-102

Effects of Docosahexaenoic acid- and Arachidonic acid-containing dieton Renal function of 5/6 nephrectomy rats
Hiroki Muramatsu, Kenji Sugibayashi, Masanori Katakura（ Nutr Phyol, Grad Phar Sci, Josai（ 1Ushimado Marine Institute, Fac Sch, Okayama Univ, Japan, 2 Natl Inst Genetics）

Univ, Japan）

In male rats, gastrin-releasing peptide (GRP) neurons in the spinal cord control male sexualfunction such as erection and ejaculation, and project axons into the brain. Central GRP is
known as a modulator of many physiological functions including itch, circadian rhythms, foodintake, fear memory consolidation. However, the role of GRP in male sexual behavior remains
to be uncovered. In this study, we investigated the involvement of central GRP neuron system
in regulatory mechanism of male sexual behavior. We generated a transgenic (Tg) rat that
expresses a human diphtheria toxin (DT) receptor and a monomeric red fluorescent protein(mRFP) depending on GRPR gene promoter in GRP receptor (GRPR)-expressing neuron. In
the morphological analysis using this Tg rat, we found that GRPR-expressing neurons are
distributed in several nuclei involved in male sexual functions such as the medial preoptic
area (mPOA; a regulatory center of male sexual behavior), bed nucleus of the stria terminalis,
ventromedial nucleus of the hypothalamus, and medial amygdala. A subpopulation of GRPRexpressing neurons exhibits a male-biased sexual dimorphism in the mPOA, and c-Fos expression in these neurons was significantly increased following to ejaculation. Then we examined
the lesion effect of GRPR-expressing neurons on sexual behavior by administration of DT into
the mPOA of Tg rats. Sexual activity of DT-administered males was significantly attenuatedin compared with before DT-administration. Subsequently, GRPR agonists and antagonists
were injected locally into the mPOA and male sexual activity was studied to verify the role ofendogenous GRP. Pharmacological stimulation of GRPR by agonists did not show any effects onmale sexual activity, whereas the inhibition of GRPR by antagonists significantly reduced malesexual activity. These results suggest that the GRP system in the mPOA plays an important(COI:No)role in male sexual activity in rats.

The kidneys are responsible for excretion, maintenance of pH, adjustment of blood pressure,and regulation of body fluid volume. The structure of the kidneys is highly complex, and it isvery difficult to recover after injury. Therefore, it is important to prevent the kidneys frombeing damaged. Arachidonic acid (ARA), an ω6 polyunsaturated fatty acid (PUFA), is involvedin the development and maintenance of renal functions. Docosahexaenoic acid (DHA), an ω3
PUFA, produces anti-inflammatory effects. In this study, we assessed the effects of consumption of different types of PUFAs on the development and progression of chronic kidney disease(CKD) using 5/6 nephrectomized rats. Male Sprague Dawley rats were randomly divided into
four groups, which were fed ad libitum, either control, ARA, DHA, or ARA+DHA-containing
diets for 4 weeks; then, 5/6 of each subject’s kidney was removed. Four weeks after surgery,rats were housed in individual metabolic cages for 24 hours and urine was collected. Then,
plasma and kidneys were collected for biochemical and histological analysis. Urinary albumin
excretion was increased in the control CKD group compared to the control sham group, but
it was attenuated by feeding the DHA diet. ARA and DHA levels in red blood cell wereincreased in groups.
fed diets containing ARA and DHA compared to the control CKD group. Peroxynitrite levelsin the kidney decreased in ARA, DHA, and ARA+DHA groups compared to the control CKD
group. Peroxynitrite levels in the kidneys had significantly negative correlation with creatinine
clearance. Lipid peroxide levels in the kidneys were not increased in any group. In contrast, inthe plasma, lipid peroxide levels increased in the control CKD group, the levels were attenuated by feeding the ARA+DHA diet. These results suggest that DHA, or a combination of
ARA and DHA, inhibits the progression of early stage of CKD.
(COI:No)S166

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

3P-103

How do the basal ganglia control thalamocortical activity?
3 1 Div Syst
Satomi Chiken1, 2，Hiromi Sano1, 2，Kenta Kobayashi2, 3，Atsushi Nambu1, 2, （Neurophysiol, Natl Inst Physiol Sci, Okazaki, Japan, 2 Dept Physiol Sci, SOKENDAI,
Okazaki, Japan, 3 Sec Viral Vector Devel, Natl Inst Physiol Sci, Okazaki, Japan）

The basal ganglia (BG) play a crucial role in controlling voluntary movements, and their
dysfunction causes severe motor disturbance, such as Parkinson’s disease and dystonia. The
BG receive information from a wide area of the cerebral cortices. The internal segment of theglobus pallidus (GPi), one of the output nuclei of the BG, sends GABAergic projections to thethalamocortical neurons and governs the original cortices. Thus, to elucidate roles of the BG
in control of voluntary movements, it is essential to understand how BG outputs control thalamocortical activity. In the present study, we identified thalamocortical neurons by electricalstimulation of the forelimb regions of the primary motor cortex and supplementary motor area,
and then examined their responses to electrical stimulation of the GPi in macaque monkeys.
Single-pulse stimulation induced a biphasic response composed of short-latency inhibition andfollowing excitation. Repetitive stimulation evoked a train of biphasic responses. Local injectionof GABA-A receptor antagonist into the vicinity of recorded thalamocortical neurons abolished
not only inhibition but also following excitation without changes in spontaneous firing rates,suggesting that the excitation is caused by a post-inhibitory rebound mechanism. Next, we
expressed halorhodopsin in the axon terminals of GPi-thalamic projections by injecting an
adeno-associated virus vector into the GPi, recorded thalamocortical activity using an optrodeduring performance of a hand reaching task, and selectively blocked GPi-thalamic inputs byilluminating yellow light in the vicinity of recorded neurons. Inhibitory responses evoked by
GPi-stimulation were successfully abolished by the optical stimulation. Task-related firing
increase was enhanced by blockade of GPi-thalamic inputs in the half of recorded thalamocortical neurons, but diminished in the other half. These results suggest that GPi-thalamic inputs
modulate thalamocortical activity through both GABAergic inhibition and following rebound(COI:No)
excitation and contribute to controlling voluntary movements.

3P-104

Interhemispheric inhibition during motor imagery of dominant or
non-dominant ﬁnger movement in humans: A transcranial magnetic
stimulation study
1 1 Dept Hum Health Sci, Grad Sch Med,Nan Liang1, 2，Amiri Matsumoto1，Hajime Ueda（

Kyoto Univ, Japan, 2 Dept Integr Physiol, Grad Sch Biomed Health Sci, Hiroshima Univ,
Japan）

3P-106

Activation of human spinal locomotor circuitry using transvertebral
magnetic stimulation
2 1College of Sports
Kazutake Kawai1，Toshiki Tazoe2，Kazuyuki Kanosue3，Yukio Nishimura（

Sciences, Nihon Univ, Japan, 2Tokyo Metropolitan Institute of Medical Science, Japan,

3 Faculty of Sport Sciences, Waseda Univ, Japan）

Rhythmic walking-like alternus movements in bilateral legs can be induced by non-invasivetransvertebral magnetic stimulation of the human lumbar spinal cord, indicating the existing lumbar locomotor center in mans. This magnetic stimulation would be a novel neurorehabilitation for gait disturbance due to spinal cord injury or stroke. It is important to know
the stimulus parameters that activates the spinal locomotor center, however the relationshipbetween stimulus intensity and induced gait has not yet been systematically investigated. The
purpose of this study was to find the stimulation intensity necessary to drive the spinal locomotor center effectively, and to verify the mechanism by which the neural elements are recruited.
Ten healthy adults’ participants were recruited. Transvertebral magnetic stimulation wasapplied at the lumbar spine level with a stimulation intensity from 10% to 70% maximum of themagnetic stimulator output with an increment of 10%. Most subjects showed no movements
in legs at 10% stimulation intensity. Low intensity stimulation tended to induce a hopping-likemovement in which both legs moved in phase. When the stimulus intensity was increased,
the induced movements changed to a walking-like movement in which the left and right legs
moved in anti-phases. In addition, as the stimulus intensity increased, the toe trajectory lengthof the induced walking-like movement tended to increase. The threshold required to induce
walking-like movement was significantly higher than that of hopping-like movement, and 40%
to 60% of the magnetic stimulator is required to drive the spinal locomotor center for walking
in mans.
(COI:No)

3P-107

Chemogenetics to decipher the functional role of the subthalamic
nucleus in macaque monkeys
3 1 Div of
Taku Hasegawa1，Satomi Chiken1, 2，Kenta Kobayashi2, 3，Atsushi Nambu1, 2, （System Neurophysiol, Natl Inst for Physiol Sci, Okazaki, Japan, 2 Dept of Physiol Sci,
SOKENDAI, Okazaki, Japan, 3 Sec of Viral Vector Development, Natl Inst for Physiol Sci,
Okazaki, Japan）

We have previously reported that interhemispheric inhibition (IHI) from the contralateral toipsilateral motor cortex (M1) increased during voluntary and imagined movements (Liang etal. 2014). By a pure central command of motor imagery, whether the extents to IHI differ
between dominant and non-dominant hands remains unclear. By using transcranial magnetic
stimulation (TMS) techniques, we examined that the excitability changes in the contralateraland ipsilateral M1 as well as IHI from the contralateral to ipsilateral M1 during motor imageryof dominant or non-dominant finger movement. The right-handed healthy subjects were asked
to perform imagery of the index finger abduction with their maximum force in the dominant
or non-dominant hand. Single pulse TMS was applied over the contralateral or ipsilateral
M1. Then, a conditioning TMS with an intensity ranging from 0.8 to 1.4 times the resting
motor threshold (rMT) was applied over the contralateral M1, and the test TMS with anintensity of 1.1 to 1.2 times rMT was applied over the ipsilateral M1. The motor evoked
potential (MEP) was recorded from the first dorsal interosseous (FDI) muscles. MEP amplitude
significantly increased in both FDI muscles during either dominant or non-dominant imagery.
The increased MEP in the left FDI muscle was negatively correlated with that in the right
FDI muscle during imagery of the right hand, while there was no correlation in the increased
MEPs between the muscles during imagery of the left hand. The conditioned MEP size waspositively correlated with that in the contralateral homonymous muscle during right hand
imagery, but with no correlation in the case of the left hand imagery. These results suggestthat IHI from the contralateral to ipsilateral M1 modulated depending on the contralateral M1
excitability during motor imagery of the dominant hand but not with that of the non-dominanthand.
(COI:No)The subthalamic nucleus (STN) is considered to play an essential role in motor control as
evidenced by effects of its lesion; the STN lesion induces hemiballismus, involuntary ballistic
movements of the limbs, and alleviates the motor symptoms in parkinsonian patients. However, it is not clear how the STN influences the output of the basal ganglia (BG) and controls the
movements. The STN sends glutamatergic axons to the internal segment of the globus pallidus(GPi), the output nucleus of the BG, as well as to the external segment of the globus pallidus(GPe), which in turn sends GABAergic axons back to the STN. We suppressed the STN
activity with the DREADD (Designer Receptors Exclusively Activated by Designer Drugs)
system and analyzed the activity change in the GPi and GPe.
An adeno-associated virus vector expressing the inhibitory DREADD receptor, hM4Di, wasinfused to the motor area in the STN of three Japanese monkeys (Macaca fuscata). Afterthe receptor expression (>3 weeks), abnormal involuntary movements were induced in thecontralateral forelimb by the systemic administration of a DREADD ligand.
The single unit activity in the forelimb motor areas of the GPe and GPi was recorded during
a reaching task and the following changes were observed after the STN suppression: 1) the
decrease in the baseline firing rates in the GPe, 2) the increase of the variability in spike timings
both in the GPe and GPi, and 3) the enhanced movement-related activity in the GPe and GPi.
Our results suggest that the STN controls movements not only through changing the firing
rates but also through adjusting the spike variability; the STN may play a role to stabilize thefiring timings of the GPe and GPi neurons through the excitatory innervation.
(COI:No)

3P-105

3P-1081 1 Dept Physiol,
Takashi Suzuki1，Kazunori Morita1，Masahiko Inase2，Katsumi Nakajima（Sch. Med, Iwate Med Univ, Yahaba, Japan, 2 Dept Physiol, Facult Med, Kindai Univ,
Osaka-Sayama, Japan）

Tatsuro Kumada1，Akira Yoshikawa2，Saho Morishita3, 4，Kazuya Hokamura5，
3 1 Fac Health Med Sci, Tokoha Univ, Japan,
Masahiko Izumizaki2，Kazuo Umemura（

Postural transformation during treadmill walking in Japanese monkeys:
kinematic and EMG analysis

Japanese monkeys can walk on a treadmill and volitionally transform its trunk posture fromhorizontal (quadrupedal gait) to vertical (bipedal gait) without interruption of stepping movement. To investigate behavioral processes of the postural transformation during locomotion,
we analyzed kinematics and EMG activity of the trunk and limbs in two monkeys. Lateraland back views of the walking animal were videotaped using two high-speed cameras. EMG
activity was recorded via chronically implanted Teflon-coated tungsten wires. Postural transformation was initiated by touchdown of either hindlimb (trigger limb, TL) during quadrupedal
gait and was accomplished within 1-2 step cycles. In this period, the body axis in the sagittal
plane increased from 5º to 70º.
In the frontal plane, the mediolateral hip position during quadrupedal gait located between
stance positions of the left and right hindlimbs and the head position between those of forelimbs. Just before the transformation, the hip and head positions shifted toward the TL side(the first postural adjustment). In the following righting-up period, these positions conversely
shifted to the opposite TL support side and then moved back to the TL side during subsequent
stance phase of the hindlimb contralateral to TL. Antigravity muscles in the hindlimb wereactive during stance phase and bilateral back muscles were coactive around touchdownof each hindlimb for quadrupedal gait. During righting up, phasic activity of these muscles
increased and burst activity of the left and right back muscles occurred alternately. Suchmediolateral trunk sway (the second postural adjustment) and activity patterns of antigravity
muscles observed during postural transformation continued into subsequent, stable bipedal
locomotion. Our results identified two distinct postural adjustments associated with righting-up
behavior during locomotion. They contribute to secure dynamic equilibrium during posturaltransformation and subsequent bipedal locomotion in the same manner as anticipatory postural(COI:No)adjustment precedes voluntary movements to maintain equilibrium.

Detection of motor defects in hindlimbs during locomotion in rats withfocal cerebral infarction by 3D kinematical analysis
2 Dept Physiol, Showa Univ, Japan, 3 Dept Pharmacol Hamamatsu Univ Sch Med, Japan,

4 Fac Health Promo Sci, Tokoha Univ, Japan, 5 Dept Med Edu, Hamamatsu Univ Sch Med,Japan）

Precise evaluation of motor outputs is required for unmasking the roles of local neuronal
circuits on controlling motor behavior and the long term effects of rehabilitation on motor
recovery after cerebral infarction. However, conventional behavior analysis in rodents is suitable for screening of animals with more severe defects, but not sufficient to access these issues.
Three-dimensional (3D) kinetic analysis allow us to quantify the motor behavior in spatiotemporal manner and detects even small changes in motor outputs. To elucidate the possibilityof this analysis, we developed rats with focal motor cortex infarction by photochemicallyinduced thrombosis (PIT) method, and have evaluated the movement of hindlimbs of theserats during locomotion on treadmill by using the KinemaTracer system (KISSEI COMTEC)
with some modifications. Rats with focal motor infarction did not exhibited apparent changes
in general gait parameters (gait cycle, stance phase, swing phase, step length and step width),but significant changes in trajectories of several hindlimb joints between pre- and post-1 day
operated rats. Interestingly, the deficits were more prominent in distal segments compared
to proximal segments. These results suggest that analysis of joint movements by 3D kinetic
analysis could evaluate the mild defects in motor behavior in ischemic model in rat. (COI:No)

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S167

3P-109

Effects of PPAR gamma agonist on senescence related gene
expressions in visceral adipose tissue of aged mice
Masaki Kimura1, 2，Ryo Shibakawa1，Yoshihiro Matsumoto1，Risako Ishii1，Natsumi Hirano1，
1 1 Dept Pharmacotherapeutics, Fuclt Pharmacy, Keio
Yoshimasa Saito1，Hidetsugu Saito（

Univ, Japan, 2 Lab Metabolic Med, Mater Med Res Ins, Univ of Queensland, Australia）

3P-112

Effects of cigarette smoke extract on endothelial cells-From theviewpoint of DNA damage and cellular senescence
Mari Ishida1，Chiemi Sakai1，Keitarou Ueda1，Masao Yoshizumi1，Takafumi Ishida2（ 1 Dept Cardiovasc Physiol and Med, Hiroshima Univ, Japan, 2 Dept Cardiovasc Med,
Fukushima Med Univ, Japan）

Background and Purpose: Senescence related markers are reported to be increased in white
adipose tissue of obese animals. In our previous study, PPAR gamma agonist seemed toameliorate these senescence related changes. The purpose of this study was to investigate
the effects of PPAR gamma agonist (pioglitazone) on senescence related gene expressions invisceral adipose tissue of aged mice.
Animal and method: 88-week-old male C57BL/6 mice were assigned to control (OC) or
pioglitazone ingestion (OP) group. The OP group took 12-week dietary ingestion of normal dietwith 0.02 % (w/w) pioglitazone. At the end of experimental period, abdominal fat tissues werecollected for the analysis of adipocyte cellularity and senescence related gene expressions.
Result and Discussion: There were no differences in both body weight and total food intake
between OC and OP group. Pioglitazone ingestion seemed to make some influences on visceraladipose tissue, those were indicated by the increase in plasma adiponectin concentration andthe decrease in fat cell diameter and MEST mRNA expression in epididymal fat. However,
we could not find enough changes in senescence related gene expressions in this visceral fat.
These results suggested that the further investigation about the age of mice and the volume
and duration of pioglitazone ingestion are needed to evaluate the effects of PPAR gamma
agonist on visceral adipose tissue of aged mice.
(COI:No)

Objective: Smoking exerts a detrimental effect to many organ systems and is responsiblefor illnesses such as atherosclerosis, cancer and chronic obstructive pulmonary disease. We
reported that DNA damage was increased in smokers’ blood cells compared to non-smokers.
Recently, it has been reported that DNA damage is involved in cellular senescence and thepathogenesis of atherosclerosis. In this study, we investigated whether cigarette smoke extract(CSE) exerts DNA damage and cell senescence in endothelial cells and the physiological influence of DNA damage.
Methods and Results: The influence of CSE extracted from tobacco smoke was examined inhuman umbilical vein endothelial cells (HUVEC). CSE was added to HUVEC and DNA damage
formation was quantified by fluorescence immunostaining; DNA single strand breaks (SSBs)
with RPA 2 and double-strand breaks (DSBs) with phosphorylated Histone H2AX as indices.
SSBs increased at 24 hours after CSE stimulation, and DSBs increased significantly at 72 hours
after stimulation. Continuous stimulation with CSE for 7 days resulted in an accumulationof cytosolic DNA and accelerated cellular senescence quantified by senescence-associatedβ-galactosidase activity. The mRNA expression of inflammatory cytokines, such as IL-6 andIL-1α was increased and prolonged by continuous CSE stimulation.
Conclusions: CSE was shown to induce DNA damage, accumulation of cytosolic DNA, cellularsenescence and inflammation of vascular endothelial cells. (COI:No)

3P-110

3P-113

Naoki Adachi1, 2，Misako Ando2，Fatma Zahra Sakhri 1，Hideshi Ikemoto1，Yusuke Ohashi1，
1 1 Dept Physiol,
Mana Tsukada1，Yasunori Takayama1，Yohei Hirai2，Masataka Sunagawa（Honing Huang（Appl Bio Chem, Grad Sch Agri, Kindai Univ, Japan）

Possible Mechanisms of Astrocyte Senescence in a Hepatic
Encephalopathy Model

Coriandrum sativum inhibits migration and invasion of cancer cellthrough suppressions of MMP-2 and u-PA expression

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome attributed to acute and chronic
liver dysfunction. The major symptoms of HE are mild cognitive impairment, stupor and coma
as well as motor disturbances such as ataxia and asterixis. Increased blood concentrationof ammonia is considered as one of the main causes of the symptoms. Interestingly, recent
studies suggest that ammonia-induced premature senescence in astrocytes may contribute todevelop the symptoms.
The aim of this study was to clarify the molecular mechanisms underlying the ammoniainduced astrocyte senescence, DNA damage levels, expression/activation of the cell cycle
regulators and sirtuin 1 (SIRT1), and senescence-associated secretory phenotype (SASP) using
cultured rat cortical astrocytes after ammonia exposure.
Ammonia (5 mM NH4Cl) exposure caused cellular senescence in astrocytes through upregulating cell cycle regulating factors such as p21 and p53. SASP was also observed as increased
IL-6 expression. When anti-senescence effects of resveratrol and nicotinamide mononucleotide(NMN) against the astrocyte senescence were determined, NMN showed significant suppression in these senescence-associated markers.
These results suggest a possible involvement of the astrocyte senescence in the pathophysiology of HE, and NMN could have a beneficial effect to ameliorate the neuropsychiatric symptoms in the patients with HE.(COI:No)

Coriandrum sativum, an annual herb and a member of Apiaceae family which is wildly used
as a spice worldwide. In our previous study, the effects of Coriandrum sativum on cancer cells
were investigated by using human hepatocellular carcinoma cell line (HepG2), human colorectal
adenocarcinoma cell line (Caco2) and mouse melanoma cell line (B16-F10) in vitro. It has been
demonstrated that the extract of Coriandrum sativum suppressed matrix metalloproteinase 2(MMP-2) activity and urokinase-type plasminogen activator (u-PA) activity in the conditioned
medium and inhibited migration and invasion of cancer cells. Since MMP-2 and u-PA wereinvolved in degradation of extracellular matrix, it was thought that the suppression of MMP-2
and u-PA activities induced the inhibition of migration and invasion of cancer cells. In thepresent study, we investigated the effects of Coriandrum sativum on expressions of MMP-2
or u-PA mRNA and antigen level. Furthermore, the alteration of signal transduction in B16F10 cells induced by Coriandrum sativum was studied. After lyophilization of Coriandrum
sativum for 24-48 hours, the extract of Coriandrum sativum was prepared with methanol. The
expression of MMP-2 or u-PA mRNA was quantified by RT-qPCR. MMP-2 or u-PA antigen
in the conditioned medium and the alteration of signal transduction in B16-F10 cells were
examined by western blot. Compared to control, incubation of B16-F10 cells with the extractof Coriandrum sativum significantly suppressed the expression of MMP-2 or u-PA mRNA anddecreased MMP-2 or u-PA antigen in the conditioned medium. The extract of Coriandrum
sativum significantly impaired phosphorylation of Erk and IKB, and translocation of NF-kB
into nucleus of B16-F10 cells. Based on these findings, it was confirmed that the extract ofCoriandrum sativum inhibited migration and invasion of cancer cells through suppression of(COI:No)
MMP-2 and u-PA expressions.

3P-111

3P-1142 1 Hyogo Univ of Health Sci of
Riku Arizono1, 2，You Ueda1, 2，Akira Tamaki1，Akiko Arata（

5 1GradTomomi Nakamura1，Toshinori Yasuzawa2, 3，Akira Mima4，Shigeru Ueshima1, 3, （Sch Agri, Kindai Univ., 2 Fac Health Sci, Kio Univ., 3 Fac Agri, Kindai Univ., 4 Dept
5Nephrol, Osaka Med Coll., Antiaging Ctr, Kindai Univ.）

Body movement analysis of human fetus has been studying using ultrasonic tomographic
imaging. However, that of rat fetus has not been much studying and there was no data of therat fetal movement. We created the equipment for analysis of rat fetal movement using theultrasonic tomographic imaging under non-anesthesia, and we studied the comparison of rat
fetal movement and human baby movement. In this study, we observed the fetal movement of
the total observation time (10 minutes) by using ultrasonic tomographic imaging system in theembryonic day15 (E15), E17, E19, E21 of Wistar rat fetus. We recognized three stages of fetal
movements; the body movement, the twitching movement like a reflex and the respiratory
movement. All of fetal movement was depressed by anesthetic drug application (GABA: midazolam + a2-adrenaline agonist: medetomidine) + kappa-receptor agonist: butorphanol). We also
examined the effects of each anesthetic drug on the fetal movement. Butorphanol significantly
depressed the body movement in E21, and midazolam and medetomidine depressed it inE17-19. However, the twitching movement was partially blocked by butorphanol in E19 andit was largely blocked by midazolam and medetomidine in E19-21. These results suggested
that 1) we recognized three stages of fetal movements in late pregnant term; 2) the effectsof between k-opioid receptor and both GABA and a2-adrenergic receptors might control thedifferent mechanisms in the fetal development. (COI:No)

Recently, obesity increases by the changes of lifestyle. The adipocyte hypertrophy causes
obesity, and it is involved in the development of diabetes, dyslipidemia and hypertension. Thesediseases are thought to advance arteriosclerosis. It is reported that Endothelial-to-Mesenchymal
Transition (EndMT) is involved in the development of arteriosclerosis. EndMT is the process
in which endothelial cells are transformed into mesenchymal cells, and is induced by TGF-β,
TNF-α or hyperglycemia. On the other hands, it is reported that eicosapentaenoic acid (EPA),
which is the n-3 polyunsaturated fatty acids, possesses the vasoprotection ability. In this study,
we investigated the effect of substances secreted by the progress of adipocyte hypertrophy
on EndMT of vascular endothelial cells (VEC) and further examined the influence of EPA on
it. 3T3-L1 preadipocytes were differentiated into adipocytes by changing the medium every
third day, and the culture medium was collected at each timing of medium-change. The lipid in
adipocyte was confirmed by Oil Red O staining and it was accumulated depending on differentiation. VEC were cultured with Dulbecco’s Modified Eagle Medium (DMEM). After reaching
to subconfluence, DMEM was changed to 3T3-L1 culture medium. In comparsion with control,
VEC cultured with 3T3-L1 culture medium from day 8 of differentiation significantly increased
ability of migration and the expression of SM22α(marker protein for mesenchymal cells),
decreased the expression of CD31 (marker protein for endothelial cells) and up-regulated Erk/
Snail signaling pathway. The addition of EPA into 3T3-L1 culture medium suppressed EndMT
and up-regulation of Erk/Snail signaling pathway induced by 3T3-L1 culture medium. Basedon these findings, it was suggested that substances secreted into the culture medium by
the progress of adipocyte hypertrophy cause EndMT of VEC, and EPA suppresses EndMT
through Erk/Snail signaling pathway. (COI:No)Sch Med, Showa Univ, 2 Dept Biomed Chemi, Sch Sci and Tech, Kwansei Gakuin Univ）

The effects of anesthetic drug on the rat fetal movement pattern
before and after in the non-anesthesia pregnant rat in the ultrasonictomographic method
Rehabilitation, Physical Ther for Int Disorders, Kobe, Japan, 2 Dept. of Physiol, Hyogo Collof Med, Nishinomiya, Japan）

S168

Eicosapentaenoic acid suppresses Endothelial-to-Mesenchymal
Transition of vascular endothelial cells induced by substances secretedby the progress of adipocyte hypertrophy

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

3P-115

Cesium ion suppresses ﬁbroblast migration in an applied electric ﬁeld
Tomohiro Kawamata, Shingo Tsuji, Daisuke Kobayashi, Akihiro Hazama（ Dept Cell
Integrative Physiol, Sch Med, Fukushima Med Univ, Japan）

3P-118Inﬂation of ex-vivo Rat Lung in Negative Pressure Chamber Induced
ATP Release in Alveoli and Surrounding Blood Capillary
1 1 MechanoBio, Grad
Kishio Furuya1，Ju Jing Tan2，Ryszard Grygorczyk 2，Masahiro Sokabe（

Fibroblasts located in the dermis play a key role of the wound healing and the overgrowthof fibroblast causes keloid formation. Endogenous electric fields (EFs), which are generated
by epithelial cells, are important to decide the fibroblast movement in the wound site. The
previous experiments by our group have shown that cesium ion suppresses cell proliferation
temporarily and this finding has a potential for clinical application. However, the effect of
cesium ion on the fibroblast movement in an applied EF has not bee reported. In this study,
we investigated the difference in fibroblast migration in the presence/absence of cesium ion
in an applied EF. The movement of murine NIH/3T3 fibroblast cells was tracked by thecomputer system with/without 3mM CsCl under 200 mV/mm EF. We observed that cesium
ion suppressed cell migration as compared to control medium. Moreover, fibroblasts acquired
the migratory ability again after washout of cesium ion. Further studies are needed in orderto unveil the detail mechanism under the inhibition of fibroblast migration by cesium ion.

(COI:No)Sch Med, Nagoya Univ, Japan, 2 CRCHUM, Univ Montreal, Canada）

3P-116

3P-119Kuya Yamamoto1，Kohki Ide2，Yuki Ichi2，Kazunao Hobo2，Shin Kubota3，Yuri Mukai1

Mitsutoshi Kurosaka, Yuji Ogura, Kazuhisa Kohda, Toshiya Funabashi（Department of

Cell death induction of human cancer cells by Mastigias papua ﬂuid
components（ 1 Dept Electr Bioinform, Sch Sci Tech, Meiji Univ, Japan, 2 Dept Electr, Grad Sch Sci
Tech, Meiji Univ, Japan, 3Turritopsis Res Inst Reg Biol, Shirahama, Japan）
Mastigias papua is a metamorphic animal that undergoes a major change in shape during its
growth, including a fertilized ovum, planula, polyp, strobila, ephyra, or medusa. In the process
of morphological changes, cell death and cell differentiation are occurring in the jellyfish body.
Therefore, the possibility is thought that there are components that show the same effect onhuman cells that are eukaryote as well as jellyfish. This study aims to discover the factorsthat induce cell death in HeLa cells derived from human cervical cancer and to analyze theirfunctions using the jellyfish extract as a sample.
As the result of the investigation what morphological changes occur when the jellyfish extract
was added to HeLa cells, it was found that cell death occurred. In addition, as a result that thecell membrane destruction was examined by trypan blue staining by cytotoxicity test, the cell
viability was decreased depending on the total protein concentration in the jellyfish body fluid.
Furthermore, the morphological differences in dead cells were observed between high andlow concentrations of jellyfish body fluid. Examining the relationship with cell death of HeLa
cells by the three types of jellyfish body fluids, extracted from umbrella, umbrella margin andoral arm, the strong activity of inducing cell death was found especially in oral arm extract. In
the future, the cell death inducer contained in the extract of the jellyfish oral arm should be
identified and clarify the mechanism of cell death.
(COI:No)

3P-117

Acute death of tumor cells induced by quinacrine with blue light
Hiromi Hiruma（ Dept Physiol, Kitasato Univ Sch Med, Japan）
Acridine orange, a weakly basic fluorescent dye, has been used as a photosensitizer for antitumor photodynamic therapy. We previously clarified that illumination of blue light on acridine
orange-loaded malignant melanoma cells showed intracellular vesicle disruption resulting incell death. This phenomenon is the underlying principle for acridine orange photodynamic
therapy. The present study searched the alternative photosensitizer to acridine orange, which
may lead to the extension of choice of photosensitizers. Of weakly basic dyes, only quinacrine
but not daunorubicin (daunomycin, an anti-neoplastic agent) showed a similar effect to acridine
orange when blue light was illuminated. Other fluorescent dyes Lucifer yellow, Congo red andruthenium red had no effect. When quinacrine was loaded in osteosarcoma cells, quinacrine
remained in intracellular vesicles. During illumination with blue light, the successive disruption of vesicles was observed as a flash of fluorescence, and shortly after that, blebs wereformed on the plasma membrane. These cells were died within 5 min. Vesicle disruption andcell death were inhibited by pretreatment with the H+-ATPase inhibitor bafilomycin and bysinglet oxygen scavengers, suggesting that vesicle acidification by H+-ATPase is needed forquinacrine accumulation in the vesicles and the generation of singlet oxygen causes vesicle
disruption and the following cell death. Therefore, quinacrine, which has already been used for
medicine for malaria and prion disease, may be a candidate of photosensitizer for anti-tumor
photodynamic therapy.
(COI:No)

Extracellular ATP and other nucleotides are important autocrine/paracrine mediators that
regulate diverse processes critical for physiological and pathological functions in the lung,including mucociliary clearance, surfactant secretion, ventilator induced lung injury (VILI) andchronic obstructive pulmonary disease (COPD). Cellular ATP release is mostly mechanosensitive; however, the impact of physical stimuli on ATP release during breathing has never beentested in intact lungs in real time. In this study, we investigated inflation-induced ATP releasein rat lungs ex vivo by real-time luciferin-luciferase (LL) bioluminescence imaging coupled withsimultaneous infrared tissue imaging under a macro-view microscope. We also developed anegative pressure chamber, which mimics the thoracic cavity, to apply physiological inflation
to the lung. With LL solution introduced into air spaces, brief inflation of the lung with –10 to–20 cm H2O) induced a transient ATP release in air-inflated alveoli and it remained spatiallyrestricted to single alveolar sacs or their clusters (70 to 260 µm). ATP release was stimulus
dependent: strong inflation evoked large ATP release that terminated upon alveoli deflation
while cyclic inflation produced cyclic ATP release. With LL introduced into blood vessels,
inflation induced transient ATP release in blood capillary in wide area of lung surface. The
response appeared diffuse but consisted of many small patch-like responses the size of alveolar
sacs. Findings suggest that inflation induces ATP release in both alveoli and the surrounding
blood capillary network; the functional units of ATP release presumably consist of alveolar sacs
(COI:No)or their clusters.

STAT6 negatively regulates differentiation and fusion of mouse
myoblasts
Physiology, St. Marianna University School of Medicine）

Purpose: Myoblast fusion is an essential for muscle fiber formation to accomplish skeletal
muscle development for regeneration and hypertrophy. However, the cellular signaling mechanisms of the myoblast fusion are not well known. It has been suggested that Interleukin-4(IL-4) plays a role in the process of myoblast fusion. Since IL-4 function is mediated by Signal
Transducers and Activator of Transcription 6 (STAT6) in many cell types, we hypothesizethat STAT6 is implicated in myoblast fusion as well. The aim of the present study was toclarify the role of STAT6 in the fusion of cultured mouse myoblasts.
Methods: Myoblasts were isolated from the lower limb of C57BL/6 mice, and expanded in
growth medium. The cells were transfected with short hairpin RNA (shRNA) for STAT6
or scrambled as a control for knockdown of STAT6. In separate experiment, the cells were
infected with either adenoviral (Ad)-STAT6 or Ad-empty as a control for overexpression of
STAT6. After those treatments, the cells were cultured with differentiation medium for 48 h
to induce myoblast fusion. The cells were fixed and immunocytochemically stained to examine
the differentiation index (percentage of the number of nuclei in MyHC+ cells to the total number of nuclei), fusion index (percentage of nuclei inside the myotube) and myotube diameter.
The cells were also processed to analyze the expression of myogenin and phosphorylation(phospho-) of p70S6K by Western blotting.
Results: The differentiation index, fusion index and myotube diameter were significantly
increased in STAT6 knockdown cells compared with the control cells. The expression of
myogenin and phospho-p70S6K in STAT6 knockdown cells were significantly higher thanthe control cells. Conversely, the differentiation index, fusion index, myotube diameter andphospho-p70S6K were significantly decreased in STAT6 overexpression cells compared withthe control cells.
Conclusion: STAT6 is a negative regulator for myoblast differentiation and fusion. (COI:No)

3P-120Effects of cell wall short-chain carbohydrate on cellular signaling
pathway in angiogenesis in melanoma mice model
3 1 Division of
Bhornprom Yoysungnoen1，Suthiluk Patumraj2，Rapepun Wititsuwannakul（

Physiology, Department of Preclinical Science, Faculty of Medicine, Thammasat University,
Rangsit Campus, Pathumthani 12120, Thailand, 2 Department of Physiology, Faculty of
Medicine, Chulalongkorn University, Bangkok 10330, Thailand, 3 Department ofBiochemistry, Prince of Songkla University, Hat-Yai, Songkla 90110, Thailand）

Cell wall short-chain carbohydrate (CW-SCC) is a short chain polysaccharide found in the plant
cell wall. CW-SCC has been showed an anti-angiogenic property; however, molecular mechanisms of CW-SCC against angiogenesis in malignant melanoma have yet not been reported.
Therefore, the purpose of this study was to determine the possible mechanisms of CW-SCC
against angiogenesis on VEGF signaling pathway in mice melanoma model. B16F1 melanoma
cells were injected into a tail vein of C57BL male mice. One day after injection, either vehicle
or CW-SCC (60 mg/kg) was orally administered for 14 consecutive days. After 2 weeks, themetastasized organs (lungs) were dissected out for further histological studies. The microvascular density (MVD) was evaluated by measuring the CD31 expression. Expressions of VEGF,
VEGFR-2, p-ERK1/2 and p-Akt were detected by immunohistochemistry. The results showedthat MVD in B16F1+vehicle group was markedly increased; however, it was significantly
decreased by treatment with CW-SCC. The expressions of VEGF, VEGFR-2, p-ERK1/2 andp-Akt were overexpressed in B16F1+vehicle group. Interestingly, they were attenuated whenmice were treated with CW-SCC. In conclusion, CW-SCC exhibited anti-angiogenic activity inthe melanoma mice model. It might be mediated by downregulation of VEGF-VEGFR-2 andsignaling molecules (p-ERK1/2 and p-AKT) expression. (COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S169

3P-121

Molecular mechanism of mitochondrial tRNA modiﬁcation enzyme
Mtu1 in reversible infantile mitochondrial disorder
Raja Norazireen Raja Ahmad1，Fan-Yan Wei2，Takeshi Chujo1，Kazuhito Tomizawa1（ 1 Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University,
2 Department of Metabolism and Physiology, Institute of Development, Aging and Cancer,
Tohoku University）
Mitochondrial transfer RNA (mt-tRNA) contains a variety of chemical modifications thatare introduced post-transcriptionally and essential for efficient protein translation as well as
energy metabolism in mitochondria. Among 22 subtypes of tRNAs encoded in mammalian
mitochondria, tRNALys, tRNAGln, and tRNAGlu undergo a unique 2-thiouridine modification(tm5s2U) at its “wobble position” (U34) of the anticodon. The TRMU nuclear gene-encoded enzyme, mitochondrial tRNA-specific 2-thiouridylase 1 (Mtu1) mediated this modification allowing
precise cognate codon recognition and ensuring accurate protein translation in mitochondria.
It is known that the loss of 2-thiolation modification are associated with the development ofreversible infantile liver failure (RILF) disease. As a number of pathogenic mutations werereported to be found in RILF patients with varying degrees of fatality and reversibility, wesought to elucidate the mechanism of Mtu1 role in reversible infantile mitochondria disorder.
We first generated Mtu1 KO cells and found that it abolished the 2-thiouridine formation in
mt-tRNAs. Loss of Mtu1 enzyme expression also impaired mitochondrial protein translationand complex I and IV of OXPHOS protein expression. Analysis on Mtu1 KO cells with expression of its equivalent pathogenic mutations reveals varying degrees of 2-thiolation modification
recovery and expression of OXPHOS complex proteins. It was found that the regulation of
Mtu1 enzyme function varies with each pathogenic mutation with some to be more critical tomitochondria function than others. These observations may provide a clue to the reversibility(COI:No)of mitochondrial disease in some patients.

3P-124Prenatal exposure to bisphenol A induces over-adaptation to predator
odor stress in rats
Tetsuya Fujimoto（Dept Physiol, Osaka Dent Univ, Japan）
Bisphenol A (BPA) is one of the major environmental endocrine disruptors. We have been
studying the effects of predator odor stress on behaviors. This time we examined whether it
can be applied to evaluate the effects of BPA. In this study, behavioral tests were conducted at
20 days of age in offspring after fetal exposure to BPA (1.5 mg/kg/day). Spontaneous behaviors(rearing, ambulation, grooming, and freezing) in the straight alley type field (6×44cm, 15cm
wall height) during 3 minutes were examined. Next, the same behavioral test was performedin the presence of a predator odor and compared to the non-odor session. There was no
significant difference in any parameters between the BPA and control groups in the non-odor.
When there was a predator odor, the data were fluctuated compared to the non-odor session,
but there was a difference in the fluctuation manner between the BPA and control groups.
There is no difference in the fluctuation manner of the rearing and the ambulation in bothgroups, in which, the scores were decreased by the odor. However, in freezing, the score wasincreased due to odor in the control group, while there was no change in the BPA group. In
grooming, there was no change in the control group but it was increased in the BPA group.
In addition, blood stress hormone levels were measured in both no-odor and odor sessions
and it was increased due to odor in both the BPA and control groups. In both groups, thehypothalamus-pituitary-adrenal system was activated by odor, but inhibition of immobility andincreased grooming were observed in the BPA group. These results suggest that BPA induces
a kind of the “over-adaptation” to odor stress.
(COI:No)

3P-122

3P-1251 1 Department ofSheng-Lan Shi1，Fan-Yan Wei2，Takeshi Chujo1，Kazuhito Tomizawa（2 1 Natl Inst Occup Safety Health,Ken Tokizawa1，Hirofumi Tsuchimoto2，Toru Shimuta（

Molecular basis of extracellular transport of chemically modiﬁed
nucleosides in human cells
Molecular Physiology, Faculty of Life Sciences, Kumamoto University, 2 Department ofMetabolism and Physiology, Institute of Development, Aging and Cancer, Tohoku
University）

RNA contains a wide variety of chemical modifications that are critical for maintaining
fundamental RNA functions such as structural integrity, intracellular localization, and decoding
efficiency. Deficits in RNA modification have been implicated in diverse diseases including
type 2 diabetes, myopathy, and live failure. These results demonstrate that RNA modifications
are indispensable for homeostatic regulation of human physiology as well as pathogenesis ofvarious diseases.
Interestingly, unlike reversible chemical modifications in protein and DNA, most of RNA
modifications are irreversible due to the complexity of RNA modifications and the lack of “demodifying” genes in genome. Therefore, even RNA is metabolized to single nucleoside, thesechemical modifications are stably attached to the nucleoside. The unique property of RNA
modifications thus leads to a fundamental question: what is the ultimate fate of these chemically modified nucleosides? Our study suggested modified nucleosides are actively transported
to extracellular space and transport of modified nucleosides are mediated by equilibrative(COI:No)
nucleoside transporters (ENTs).

Development of core temperature estimation system using patch-type
heat-ﬂux sensors on the chest
Japan, 2 Murata Manuf Co, Japan）

Industrial workers are at risk from heat illness whenever they work for a prolonged timein hot and humid conditions. Wearable technology is now being adopted, but a system that
accurately measures core temperature using wearable devices has yet to be reported. We
propose a new model based on a dual-heat-flux method that predicts core temperature using
data from patch-type sensors on the chest.
We performed experiments that compared our predicted temperatures (Tpre, using a revisedalgorithm from the dual-heat-flux method), with the actual temperatures in both esophageal(Teso) and rectal (Trec) sites during exercise in three heat conditions. Thirty-two volunteers
walked for 60 min at 4–5 km/h at 30℃, 35℃, or 40℃.
In the 40℃ condition, Teso, Trec, and Tpre increased from 37.2±0.2℃, 36.9±0.2℃, and
37.3±0.2℃ to 38.2±0.3℃, 37.9±0.3℃, and 38.0±0.2℃ (mean±standard deviation), respectively,
during exercise. The difference between Tpre and Teso was –0.10±0.15℃ and that between
Tpre and Trec was 0.02±0.19℃, using data sampled at 5-min intervals during exercise. In the
35℃ condition, the difference between Tpre and Teso was –0.06±0.17℃ and that between
Tpre and Trec was 0.04±0.14℃. In the 30℃ condition, the differences were –0.13±0.24℃(Tpre–Teso) and 0.06±0.25℃ (Tpre–Trec). Body mass, fat percentage, and sex did not affect
the Tpre algorithm, but skin temperature changes during exercise yielded errors.
The error ranges for our system are slightly superior to those in previous studies involving
noninvasive core temperature measurements. Our system uses simple wearable devices andcan provide real-time, subject-specific, and accurate body core temperature estimates underheat stress conditions.
(COI:No)

3P-123

3P-126Teruaki Yamaguchi, Hajime Yano, Junya Tanaka（ Department of Molecular and Cellular

Linh Pham T.1, 2，Ko Yamanaka5，Yasunori Miyamoto1, 3, 4，Hidefumi Waki5，
3 1Grad Sch Humanities and Sciences, Ochanomizu Univ, Tokyo, Japan,
Sabine Gouraud2, （Exploration of the role of glutamine metabolism in pro-inﬂammatory
reaction of microglia
Physiology, Ehime University Medical School）

Excessive pro-inflammatory reaction of microglia exacerbates neurodegenerative diseases including Parkinson’s disease. Therefore, controlling the excessive pro-inflammatory reaction canbe a new therapeutic target for suppressing the exacerbation of those disease. We found thatglutamine metabolism has the potential to play fundamental role in microglial pro-inflammatory
reaction. We employed mouse microglia cell line BV2 for in vitro experiments. BV2 exposed toa pro-inflammatory stimulus, Lipopolysaccharide (LPS), showed pro-inflammatory reaction including the synthesis of nitric oxide and the increase in mRNA expression of pro-inflammatory
cytokines. However, BV2 exposed to LPS with medium containing no glutamine exhibited
prominently attenuated pro-inflammatory reaction. In general, the intracellularly incorporated
glutamine is primarily converted to glutamic acid by glutaminase (GLS1), and is further supplied to various metabolic pathways. We observed the temporal upregulation of intracellularglutamic acid concentration accompanies to LPS stimuli in the presence of glutamine, whilealmost no change with low level in the concentration in the absence of glutamine. Moreover,
GLS1 inhibitor can suppress the pro-inflammatory reaction of BV2 induced by LPS. In addition,
we obtained the result suggest that glutathione, a metabolite of glutamine metabolism, can be
involved in pro-inflammatory reaction of BV2. We would like to discuss the role of glutamine(COI:No)
metabolism in pro-inflammatory reaction of microglia.

S170

Effect of plastic nanoparticles exposure on cardiovascular regulation:
focus on the inﬂammatory condition in NTS
2 Program for Leading Graduate School, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo,

Japan, 3 Department of Biology, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo, Japan,

4 Institute for Human Life Innovation, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo,

Japan, 5 Department of Physiology, Graduate School of Health and Sports Science, Juntendo
University, Inzai-city, Chiba, Japan）
A growing body of evidence shows that plastic nanoparticles (NPs) are omnipresent in theenvironment and food chain. According to recent studies, NPs can cross biological membranes
and blood-brain barrier, alter the gene expression of inflammatory molecules in cells, and trigger neurotoxic effects. How ever, the impact of NPs on mammalian health is not well studied.
We recently identified an association between the abnormal expression of pro-inflammatory
molecules in the nucleus of solitary tract (NTS), a central site for autonomic cardiovascularregulation, and the blood pressure (BP) levels. In this study, we investigated the effect of
NPs on inflammatory condition in the NTS and cardiovascular parameters. 50nm diameter
polystyrene nanoparticles (PSNPs) diluted in water (1ng/nl) or vehicle were microinjected into
the NTS of male Wistar rats (100nl bilaterally, n=5). The NTS tissues were collected 48 hours
after the injection for total RNA extraction and quantitative RT-PCR experiments. In parallel,other sets of male Wistar rats (4 weeks-old, n=6 per group) were exposed to PSNPs diluted
in water (1mg/100g body) or vehicle by oral gavage 3 times per week for one month. Weight,
BP and heart rate were regularly measured. Interestingly, the transcripts for inflammatory
molecules Chemokine (C-C motif) ligand 5 (CCL5)and Tumor Necrosis Factorα (TNFα) were
found significantly increased in the NTS of rats treated with PSNPs compared to control
rats treated with vehicle. Moreover, the BP of rats exposed to PSNPs was found significantly
lower than the one of control rats. Our results show that orally ingested PSNPs might affect(COI:No)
cardiovascular centers involved in BP regulation.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

3P-127

Effects of metabolic cage housing on oxytocin expression in rats
Hirofumi Hashimoto, Yoshiteru Seo（Dep. Regul. Physiol, Dokkyo Med. Univ.）
We usually used metabolic cage to house laboratory rats in some behavior studies. Metabolic
cages usually have grid flooring. Some previous studies showed that rats have some stress inhousing metabolic cages with grid flooring, as a result, their food intake and body weight gain
was decrease. We examined the effects of metabolic cage housing on central oxytocin (OXT)
on food intake in rats, on fluorescence intensity of OXT-monomeric red fluorescent protein
1 (mRFP1) in OXT- mRFP1 transgenic rats. The food intake had decreased for 4 days andrecovered until 7 days after housing metabolic cages. The mRFP1 fluorescence intensity wassignificantly decreased in the supraoptic nucleus, the paraventricular nucleus, and posterior
pituitary after 4 days after housing metabolic cages. These results suggested that central OXT
may be involved in anorexia induced by stress in metabolic cages in rats.
(COI:No)

3P-130Quantitative electrophysiological monitoring of anti-allergic effects ofantihistamines in rat peritoneal mast cells
Itsuro Kazama（ Nursing, Miyagi Univ, Japan）
Several food constituents, such as catechin in green tea, polyphenol in red wine, nobiletin inorange peel and lactobacilli in yogurt, are known to exert anti-allergic effects. However, theseeffects have not been examined quantitatively and their precise mechanisms are unknown.
Among second-generation antihistamine drugs, olopatadine exerts relatively stronger antiallergic effects. In addition to antagonizing histamine H1 receptors, olopatadine exerts mast cellstabilizing effects. Exocytotic process in mast cells can be detected electrophysiologically by
the changes in membrane capacitance (Cm). Therefore, to quantitatively determine the mastcell stabilizing properties by olopatadine, we examined its effects on the exocytotic processof mast cells. Applying the whole-cell patch-clamp technique in rat peritoneal mast cells, wemonitored the drug-induced changes in Cm during exocytosis. By the confocal imaging of
the lucifer yellow dye, we also examined the effects of olopatadine on the plasma membrane
deformation. Relatively higher concentrations of this drug (100 µM or 1 mM) almost completely
suppressed the GTP-γ-S-induced increase in the Cm. In addition, the drug totally washed off
the dye trapped within the cell surface. Electron microscopy revealed that olopatadine generated inward bending of the mast cell membrane, which counteracted the plasma membrane
deformation in degranulating mast cells. The results provided electrophysiological evidence
that anti-allergic drugs, such as olopatadine, exerted mast cell stabilizing properties. This approach would be useful to quantitatively determine the anti-allergic properties of certain drugs
or food constituents.
(COI:No)

3P-128

3P-1312 1Grad Sch Human Sciences,
Issei Kato1，Yuta Masuda1，Shuri Marui2，Kei Nagashima（
Waseda Univ., Japan, 2 Faculty of Human Sciences, Waseda Univ., Japan）

Waki Nakajima, Megumi Hara, Mai Hatano, Tomoyuki Miyazaki, Tetsu Arisawa,
Susumu Jitsuki, Takuya Takahashi（Dept Physiol, Sch Med, Yokohama Univ, Japan）

Aim: Assessment of thermal load during exercise is important in preventing hyperthermic
injury. However, the methods have not been well established. The aim of the present study
was to test the hypotheses that heart rates(HR) during resting periods of intermittent exercise
reflect thermal condition of body in any environmental conditions.
Methods: Young healthy male participants(n=12; age, 23.7 ± 2.4; bw, 64.34 ± 8.86 kg) had two
trials in hot and humid, and the control environments(HH and CON trials, respectively; HH,
ambient temperature(Ta) of 35℃ with 65% relative humidity(Rh); and CON, Ta of 25℃ with
30% Rh). Each participant conducted 5-set 6~8-min graded treadmill exercise(4~10 km/h; 3ºslope) with 2-min rest in between. Rectal temperature (Trec), skin temperature at 4 sites(chest,
upper arm, thigh, and lower leg; Tchest, Tarm, Tthigh and Tleg respectively) and HR were continuously measured. Mean body temperature(Tb) was calculated as 0.7*Trec+0.3*(0.3*(Tchest+Tthigh)+0.2*(Tarm+Tleg)). The minimum value of HR during each resting period was evaluated(HRmin).
Results: Both HR and Tb were greater in the HH trial than in the CON trial. HRmin wascorrelated with Tb at the time point in both HH and CON trials(r=0.83 and P<0.001; and r=0.79
and P<0.001, respectively). Regression equations for the HRmin and Tb was similar in both HH
and CON trials.
Conclusion: In the present study, HRmin reflects Tb. Moreover, the relationship between thetwo values was similar in both HH and CON trials. The present study may suggest that, during
intermittent exercise, HRmin are determined only by the extent of thermal strain, which often
differs among the thermoregulatory capacity of individuals.
(COI:No)AMPA receptor is one of the most important molecules controlling the neuronal activities.
Dysfunction of AMPA receptors is believed to underlie some of psychiatric disorders. Recent
studies have clarified that AMPA receptors expression is decreased in post-mortem brainof patients with major depressive disorder (Gibbons et al., 2012, Duric et al., 2013). Positive
allosteric modulators (PAMs) enhancing AMPA receptors function have been developed
toward patients with depression but the effects of PAMs on depression seem to be limited.
Here we show that we success in developing a new type of PAMs targeting AMPA receptors
improving the depressive phenotype of rodent depression model.
Firstly using LC-MS/MS screening method, we identified the compound, K-1, showing hightransitional potency through Blood-Brain Barrier (BBB). Secondly, we synthesized anothercompound, K-2, showing much higher transitional potency compared to K-1. Thirdly, we also
found that K-2 is metabolized immediately in vivo. To improve this character, we newlysynthesized another compound, K-4, hydrolysis-resistant form of K-2. Electrophysiologicalexperiments revealed that these thress compounds increased AMPA receptors current in
the CA3-CA1 synapses.
Previously we found the expression of AMPA receptors deceased in rodent depression model,
Wistar Kyoto (WKY) rat. To elucidate whether these compounds are effective on improving
depressive phenotype of WKY rat, we performed forced swim test (FST) to examine antidepressive effect of these compounds. Interestingly, K-2 and K-4 reduced immobility time in
FST compared to vehicle and K-1. Surprisingly K-4 reduced immobility time in FST comparedto K-2.
Taken together, K-4 may be the promising compound as a new type of anti-depressant drugs.
(COI:No)

3P-1293P-132Duong Duc Pham1，Seung Ku Lee2，Chol Shin2, 3，Nan Hee Kim4，John A. Eisman5，
1 1 Department of
Jacqueline R. Center5，Tuan V. Nguyen5, 6, 7, 8，Chae Hun Leem（Michiya Sugimori（Dept Integrative Neurosci, Grad Sch Med Pharm, Univ Toyama, Japan）

Relationship between minimum heart rates and body temperatureduring 2-min resting period of intermittent exercise

Koreans do not have higher percent body fat than Australians for agiven body mass index: a population-based comparison
Physiology, Ulsan College of Medicine, 2 Institute of Human Genomic Study, Korea

University Ansan Hospital , 3 Division of Pulmonary, Sleep, and Critical Care Medicine,
Department of Internal Medicine, Korea University, 4 Division of Endocrinology, Sleep,
Korea University Ansan Hospital, 5 Bone and Muscle Research Group & Faculty of Applied
Sciences, Ton Duc Thang University, Vietnam, 6 University of Technology Sydney (UTS),
Sydney, Australia, 7 Bone Biology Division, Garvan Institute of Medical Research, Sydney,
Australia, 8 St Vincent Clinical School, UNSW Australia, Sydney, Australia）
Many argue that body mass index (BMI) cutoff for obesity should be lower in Asians due
to evidence of a higher percent body fat (PBF) at a given BMI compared to the whites. We
compared the body composition between 1211 Korean and 1006 Australian men and women
aged 60 and higher enrolled in two population-based cohort studies. A whole-body scan using
a dual-energy X-ray absorptiometry (LUNAR Prodigy) was used to estimate lean mass, fat
mass, and PBF in both studies. Multivariate regression adjusted for age and BMI showedthat PBF was equal and lower in Koreans versus Australian in men and women, respectively(mean difference and 95% CI: -0.54 (-1.22 to 0.14) and -2.13 (-2.61 to -1.65) for men and women).
Propensity score pair-matching analysis for age and BMI (208 pairs and 423 pairs in men andwomen, respectively) revealed that Koreans had a compatible and around 2% lower PBF
than Australians, in men and women, respectively. No age and BMI interaction effects were
found. These findings do not support the need for lowering the threshold of BMI-based obesity
definition in Asians, at least for the Korean population.
(COI:No)

Development of new AMPA receptor positive allosteric modulator using
depression model rat

The combined efﬁcacy of OTS964 and temozolomide for reducing thesize of power-law coded heterogeneous glioma stem cell populations
Glioblastoma resists chemotherapy then recurs as a fatal space-occupying lesion. To improve
the prognosis, the issues of chemoresistance and tumor size should be addressed. Glioma
stem cell (GSC) populations, a heterogeneous power-law coded population in glioblastoma, arebelieved to be responsible for the recurrence and progressive expansion of tumors. Thus, we
propose a therapeutic strategy of reducing the initial size and controlling the regrowth of GSC
populations which directly facilitates initial and long-term control of glioblastoma recurrence.
In this study, we administered an anti-glioma/GSC drug temozolomide (TMZ) and OTS964, an
inhibitor for T-Lak cell originated protein kinase, in combination (T＆O), investigating whether
together they efficiently and substantially shrink the initial size of power- law coded GSC
populations and slow the long-term re-growth of drug-resistant GSC populations. We employeda detailed quantitative approach using clonal glioma sphere (GS) cultures, measuring spheresurvivability and changes to growth during the self- renewal. T＆O eliminated self-renewing
GS clones and suppressed their growth. We also addressed whether T＆O reduced the sizeof self-renewed GS populations. T＆O quickly reduced the size of GS populations via efficientelimination of GS clones. The growth of the surviving T＆O-resistant GS populations wascontinuously disturbed, leading to substantial long-term shrinkage of the self-renewed GS
populations. Thus, T＆O reduced the initial size of GS populations and suppressed their later
regrowth. A combination therapy of TMZ and OTS964 would represent a novel therapeuticparadigm with the potential for long-term control of glioblastoma recurrence via immediate(COI:No)and sustained shrinkage of power-law coded heterogeneous GSC populations.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S171

3P-133

3P-1363 1 Dept Physiol, Kitasato Univ Sch Med,
Hiroyasu Hatakeyama1, 2, 3，Makoto Kanzaki（

Naotaka Ikumi（Lab of Physiol, Grad Sch of Intg Pharma and Nutri and Sci, Univ ofWe previously provided critical quantitative information regarding intracellular trafficking of
membrane cargo proteins including the insulin-responsive glucose transporter GLUT4 and thetransferrin receptor (TfR). For example, statistic comparison of GLUT4 and TfR behaviors
with our intracellular trafficking nanometry based on single molecule imaging of quantum dot(QD) fluorescent nanocrystals clearly documented the unique regulatory system for GLUT4.
Specifically, we showed insulin-responsive liberation from its static states. We also identified
acute heterotypic endomembrane fusion of very small static GLUT4-containing vesicles witha subset of TfR-containing endosomes as being an initial process in the insulin-responsive
GLUT4 translocation by employing conventional cell-biological technique. These two distinct
principle-based imaging techniques, focused on elucidating the same biological phenomena,raise a simple question regarding the relationship between GLUT4 liberation and heterotypicendomembrane fusion. To directly answer this question, we here performed simultaneous
imaging analysis of these two phenomena within a cell. We first labeled myc-GLUT4-mCherrywith the QD nanocrystals and the fusion sensor simultaneously, and the labeled molecules
were then allowed to reyclce back to their stationary compartments, followed by TfR labeling.
The broad absorption spectrum and the large Stokes shift of QD allowed us to fully visualize
the two distinct fluorescent molecules simultaneously. We observed obvious increases in bothfusion sensor intensity and QD movement after several minutes of insulin stimulation andfound that the onset of the increases was much more rapid for the fusion sensor than for
QD movement. This indicates that heterotypic endomembrane fusion of GLUT4-vesicles with
TfR-containing endosomes precedes GLUT4 liberation from its static status in response to(COI:No)
insulin.

The primary function of intestinal epithelia is selective nutrient absorption and concomitantly,
blocking noxious substances from entering the internal compartment of living organisms. The
intestinal barrier is formed by a polarized monolayer of epithelial cells, which are connectedby intercellular junctions. The paracellular barrier is the tight junction (TJ), a complex locatedin the apicolateral membrane of epithelial cells, which consists of a number of tetraspan TJ
proteins, namely from the claudin family, and interconnects the apicolateral membranes ofneighboring cells like zippers. In addition to this bicellular TJ structure (bTJ), a tricellular
TJ (tTJ) is formed at the meeting point of three cells where three bTJ strands converge.
It extends far more basolaterally than the bTJ, forming a vertically orientated triple pairstrands structure with a “central tube”. This central tube is thought to be a weak point of theparacellular barrier. Angulin-1 is a member of the angulin protein family, which is exclusively
expressed at tTJ. The first hint that angulin-1 is involved in barrier function came when,in angulin-1 knockdown studies, a break-down of the barrier occurred. However, whether
angulin-1 is actually involved in the intestinal barrier has not been studied. To this end, weinvestigated the impact of deficiency of angulin-1 on the function of the small intestine byusing intestinal-specific angulin-1 deficient (angulin-1 cKO) mice. We measured unidirectional
22Na+ and 3H-Mannitol fluxes in isolated small intestine by using Ussing chambers. There wasno discernable change in 3H-Mannitol fluxes. However, transcellular and paracellular 22Na+
unidirectional flux were decreased in angulin-1 cKO mice. Na+-dependent nutrient absorption
activity was also decrease in angulin-1 cKO mice. Together, these results suggest that thetTJ protein angulin-1 may regulate bTJ ion permeability and transcellular nutrient absorption
mechanisms.
(COI:No)

3P-134

3P-1372 1 Lab Physiol, Dept Env Life Sci, Univ
Shin-Ichiro Karaki1，Kota Tsukamoto1，Ikuo Kimura（

Daisuke Ishii1, 2，Hironobu Osaki3，Arito Yozu1，Kiyoshige Ishibashi4，Kenta Kawamura5，
1 1Center for Medical Sciences, IPUHS, Japan, 2 Dept Cogni
Mariko Miyata3，Yutaka Kohno（

Simultaneous imaging analysis of insulin-responsive liberation andheterotypic fusion in GLUT4 trafﬁcking
Japan, 2 FRIS, Tohoku Univ, Japan, 3 Grad Sch Biomed Eng, Tohoku Univ, Japan）

Short-chain fatty acid-evoked transepithelial ion transport in the miceintestine
Shizuoka, Japan, 2 Dept Appl Biol Sci, Grad Sch Agri, Tokyo Univ Agri Tech, Japan）

Short-chain fatty acids (SCFAs) produced by intestinal bacterial fermentation are known not
only to be absorbed as nutrients, but also to stimulate large intestinal mucosa inducing fluid
secretion and smooth muscle contraction. The effects of SCFAs as stimulants had previously been investigated by utilizing rat distal colon, but there had no direct evidence of theidentification of SCFA receptor involving the SCFA-induced fluid secretion. Therefore, in thepresent study, we attempted to investigate the SCFA-evoked epithelial ion transport in wild
type (WT)-, FFA2 (GPR43)-KO and FFA3 (GPR41)-KO mice. In WT mice, mucosa-submucosa
tissue preparations of duodenum, jejunum, terminal ileum, cecum, proximal colon, middle
colon, distal colon and rectum were mounted on Ussing chambers, and short-circuit current(Isc) were measured as an index of net electrogenic transepithelial ion transport. The mucosal
addition of propionate (10－3 M) to each segment of the intestine evoked a phasic increasein Isc, and the effect was evoked the most potent in the cecal preparations. Therefore,
we investigated the effect of SCFAs on cecal preparations in detail. As a result, mucosal
addition of propionate (>10－5 M) concentration-dependently evoked a phasic increase in Isc,
and achieved the maximum at 3×10－3 M, but acetate and butyrate little evoked the responseeven at 3×10－3 M. In addition, the mucosal propionate-evoked response was attenuated byserosal addition of atropine (10－5 M), but not by the serosal addition of a neural blockade,tetrodotoxin (10－6 M). In FFA2-KO mice cecum, propionate (10－3 M) evoked the same response
as WT mice, but in FFA3-KO mice, propionate (10－3 M) little evoked the response. These
results suggest that the propionate-evoked ion transport in the mice cecum are mediated via
activation of FFA3 receptors in the apical site of the epithelium and released acetylcholine into
the basolateral site.
(COI:No)

Tricellular tight junction protein angulin-1 regulates bicellular tightjunction permeability and transcellular nutrient absorption mechanisms
Shizuoka, Shizuoka, Japan）

Role of the right frontal orienting ﬁeld in visuospatial attention
Behav Physiol, Grad Sch Med, Chiba Univ, Japan, 3 Dept Physiol (Neurophysiol), Sch Med,Tokyo Women’s Medical Univ, Japan, 4 Dept Physical Therapy, IPUHS Hospital, Japan,
5 Dept Physical Therapy, IPUHS, Japan）

Hemispatial neglect is a disorder of higher brain function that occurs after stroke and leads
to decreased attention and response to the opposite side of the damaged hemisphere. Because
patients with this condition often have motor paralysis and other symptoms owing to thepresence of several brain lesions, it is difficult to evaluate the recovery mechanism and training
effect of unilateral spatial neglect. Therefore, an effective training approach has not yet been
established. In this study, we created a mouse model of unilateral spatial neglect for thedevelopment of effective therapies under controlled conditions. Mice (C57/BL6J, 9 weeks old)
were intraperitoneally administered rose bengal. The right frontal orienting field (FOF) was
irradiated with an LED to produce a focal cerebral infarction. After creating the cerebral
infarction, unilateral spatial neglect was evaluated for six consecutive days. Mice in the control
group were intraperitoneally administered rose bengal after LED irradiation. The major findings were as follows: (1) Control group did not show unilateral spatial neglect; (2) Mice withcerebral infarction showed symptoms similar to those of unilateral spatial neglect; and (3)The symptoms did not recover 6 days after cerebral infarction. These findings suggest that
cerebral infraction of the FOF induces symptoms similar to those of unilateral spatial neglect.
(COI:No)

3P-135

3P-1381 1 Lab Physiol, Dept Env Life Sci, Univ
Kota Tsukamoto1，Ikuo Kimura2，Shin-Ichiro Karaki（

Daisuke Kato1，Hiroaki Wake1，Philip Lee2，Yoshihisa Tachibana3，Riho Ono3，
Shouta Sugio3，Yukio Tsuji3，Yasuyo Tanaka4，Yasuhiro Tanaka4，Yoshito Masamizu4，
Riichiro Hira8，Andrew Moorhouse9，Nobuaki Tamamaki6，Kazuhiro Ikenaka7，
Noriyuki Matsukawa5，Douglas Fields2，Junichi Nabekura7，Masanori Matsuzaki4

Short-chain fatty acid-evoked transepithelial ion transport in the miceterminal ileum
Shizuoka, Japan, 2 Dept appl Biol Sci, Grad Sch Agri, Tokyo Univ Agri Tech, Japan）

Short-chain fatty acids (SCFAs) defined as 2-6 carbon fatty acids, mainly acetate, propionate,
and butyrate, are produced from dietary fiber by bacterial fermentation in the intestine. They
not only are absorbed as nutrient, but also stimulate intestinal mucosa inducing a variety ofphysiological responses. The reflux of SCFAs from cecum to terminal ileum passing throughthe ileocecal valve may induce a fluid secretion in the terminal ileum, but the SCFA-induced ion
transport in small intestine is not fully understood. We therefore investigated the acetate- andpropionate-induced ion transport in the mice terminal ileum by the Ussing chamber technique.
Transepithelial potential difference was clamped 0mV, and short-circuit current (Isc) were
continuously measured as an index of net electrogenic transepithelial ion transport. In themice terminal ileum, mucosal treatment of acetate and propionate concentration-dependently
evoked phasic (achieved a peak within 1 min) and broad (achieved a peak within 2 – 10 min)increases in Isc. These effects were insensitive for tetrodotoxin, atropine and piroxicam. In theother presentation by us, we showed that the propionate-evoked increase in Isc was completely
abolished in the SCFA receptor FFA3 (GPR41)-KO mice cecum, but present study showed
that, even in the FFA3-KO mice terminal ileum, propionate and acetate evoked the same
increases in Isc. Whereas in FFA2 (GPR43)-KO mice, the propionate-evoked phasic increasein Isc was significantly reduced and the acetate-evoked phasic increase in Isc was completely
abolished. These results suggest that the effect of SCFAs in the mice terminal ileum was(COI:No)different from colon and further investigation of that should be performed.

S172

Illumination of abnormal neural activities caused by myelin impairment
suggests possible contribution to learning deﬁcits（ 1 Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate Schoolof Medicine, 2 National Institutes of Health, 3 Kobe University Graduate School of Medicine,
4The University of Tokyo, 5 Nagoya City University, 6 Kumamoto University, 7 National
Institute for Physiological Sciences, 8 National Institute for Basic Biology, 9The University of
New South Wales）
Myelin is sheath that forms around axons, regulating the speed of electrical impulses andefficiently transmitting them among the neurons. Myelinated bundles act as cables to connect
distant brain regions. Impaired myelin regulation or impairment of myelin itself is frequently
associated with deficits in learning and cognition in neurological and psychiatric disorders.
However, it has not been revealed what perturbation of neural activity induced by myelin
impairment causes learning deficits. Here, we measured neural activity in the motor cortex
during motor learning in transgenic mice with a subtle impairment of their myelin. This deficit
in myelin impaired motor learning, and was accompanied by a decrease in the amplitude of
movement-related activity and an increase in the frequency of spontaneous activity. Thalamocortical axons showed variability in axonal conduction with a large spread in the timing of
postsynaptic cortical responses. Repetitive pairing of forelimb movements with optogenetic
stimulation of thalamocortical axon terminals restored motor learning. Thus, myelin regulation helps to maintain the synchrony of cortical spike-time arrivals through long-range axons,facilitating the propagation of the information required for learning. Our results revealed thepathological neuronal circuit activity with impaired myelin and suggest the possibility thatpairing of non-invasive brain stimulation with relevant behaviors may ameliorate cognitive and(COI:No)
behavioral abnormalities in diseases with impaired myelination.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

3P-139

3P-142

Yoshiaki Arai（Dept Eiect ＆ Com, Nagano National Col, Japan）

Kensaku Nomoto1，Madoka Nakamura1，Hanako Murayama1，Kazunari Nagai1，
Mayuko Miyakoda1，Keisuke Hayashi1，Miki Sugihara1，Kazutaka Mogi1，Tsuyoshi Koide2，
1 1Companion Animal Research Lab, Sch Vet, Azabu Univ, Japan,
Takefumi Kikusui（

VEP in response to simple diagrams by EEG
Our first visual brain cortex consist of column architecture and reflect correct image which
suited the retina. And at the study of using fMRI, it is proved the original image is reconstructed by the activity of retina. But at the study of useing electroencephalography(EEG), thatis impossible now. If it would be possible, application would spread.
In this experiment, the purpose was to think the relations between an shape of visual evoked
potential(VEP) and original simple image which retina was stimulated by. 13 parsons ofvoluntary healthy aduits of 20 years old level cooperated.
9 subjects data that measured comparatively at low noise were analyzed. Each subjects
watched simple images a circle, a triangle, a square, a cross, a star shape and an nothing forstimulation. The obtained data was calculated 20 times of addition average for the response
form of VEP.
As a results of that the response of VEP stimulated by any images were bigger amplitude
than VEP stimulated by nothing image. Furthermore, differences were seen at form and sizeof the electric potential and might be correspondence of the image with the original image by(COI:No)
checking correration together.

Anterior cingulate cortex regulates the expression of observational fear

2 National Institute of Genetics, Japan）

Observational fear is the ability to exhibit fear-related behavior such as freezing and escaping when observing fear of a conspecific. Although previous studies have shown that theanterior cingulate cortex (ACC) plays an important role in observational fear, the underlying
neural mechanisms are not well understood. To address this issue, we performed behavioral,
neurophysiological, and chemogenetic experiments while animals performed the observational
fear paradigm, in which one mouse (termed as an observer) observed fear-related behavior
exhibited by a conspecific receiving footshocks (termed as a demonstrator).
First, we examined observational fear behavior in three mouse strains: MSM, C57BL/6J, andthe hybrid between MSM and C57BL/6J (MSMB6F1). Because MSMB6F1 strain showedadequate observational fear and moderate irritability compared to MSM and C57BL/6J strains,
we used MSMB6F1 mice in the following experiments.
Next, we recorded the neural activity of the ACC neurons during observational fear by using
fiber photometry. We found that the ACC neurons of the observer were activated when thedemonstrator received footshocks. We did not see significant activation associated with freezing behavior of the observer. This suggests that ACC activities are involved in the perceptionof fear of a conspecific, but not in the execution of freezing behavior.
Finally, we examined the necessity of ACC for the expression of observational fear. The ACC
neurons were inactivated in either non-specific or projection-specific manners by using the
DREADD system. Our preliminary results showed that chemogenetic inhibition of the ACCperiaqueductal gray (PAG) pathway decreased freezing behavior of the observer, suggestingthat this pathway plays an essential role in the expression of observational fear.
In conclusion, our results indicate that the ACC is necessary for converting perception of fearof a conspecific to the expression of observational fear, possibly through its projection to the
PAG.
(COI:No)

3P-140

3P-143

Yuri Masaoka1，Satomi Kubota2，Haruko Sugiyama3，Masaki Yoshida4，Akira Yoshikawa1，
Nobuyoshi Koiwa5，Motoyasu Honma1，Ryuta Kinnno2，Natsuko Iizuka1, 2，Masahiro Ida6，
1 1 Dept Physiol, Showa Univ School of Med, Japan,
Kenjiro Ono2，Masahiko Izumizaki（

Naoko Okada1，Masaki Okubo1，Yoko Machida1，Yutaka Komura2，Takashi Tominaga3，
1 1Grad Sch of Sci and Tech, Meiji Univ, Japan, 2 Grad Sch of Human andRiichi Kajiwara（

Parahippocampus volume changes provide an early indication of
declining of olfactory ability and cognitive function in elderly subjects

2 Dept of Neurol, Showa Univ School of Med, Japan, 3 Sensory Sci Res, Kao Corporation,

4 Dept of Ophthalmol, Jikei Univ, Japan, 5 Dept of Health Sci, Univ Human Art and Sci,

6 National Hospital Organization Mito Medical Center）

The aim of this study was to investigate the relationship between olfactory recognition andmorphological changes in olfactory brain regions including the amygdala, hippocampus, rectus,
parahippocampus, orbitofrontal cortex and medial frontal cortex in 27 elderly subjects. The
specific aim of the study was to determine which brain areas are associated with the initialdecline of olfaction in elderly subjects, which occurs before the onset of dementia. All subjects
underwent magnetic resonance imaging to measure anatomical brain volume, and subjects
were assessed using tests of olfactory test and Montreal Cognitive Assessment (MoCA). The
main finding of this study was that the decrease of olfactory ability was associated withsmall left parahippocampal. In addition, the group of subjects with lower left parahippocampal
volume showed lower cognitive scores. The parahippocampus has an important role in context
processing from the hippocampus and amygdala, relaying memory retrieval and emotional
reaction to the conscious awareness of the context organized in the orbitofrontal cortex. It is
possible that volume changes in the parahippocampus due to pathological changes contribute(COI:No)to the decline of olfactory recognition.

3P-141The effects of acute isometric handgrip exercise on cognitive function
Takuro Washio1，Kazuya Suzuki1，Shotaro Saito1，Kazuki Tamiya1，Hironori Watanabe1，
1 1Toyo University, 2 The University of ElectroSoichi Ando2，Shigehiko Ogoh（

The effects of muscimol injections into the nucleus accumbens on thewaiting behavior and brain slice analysis of the network activity
Environmental Studies, 3 Lab for Neural Circuit Systems, Inst of Neurosci, Tokushima
Bunri Univ）

The nucleus accumbens (NAc), an essential structure for guiding action selection and cost/benefit decision making, possesses many GABAergic medium spiny neurons (MSNs). The
MSNs are innervated by the dopaminergic neurons in the reward system as well as theexcitatory neurons in the corticolimbic circuitry. Their activity is modulated by the fast-spiking
GABAergic (and cholinergic) interneurons in the NAc. Thus, the GABAergic transmission in
the NAc should affect various actions such as impulsive-control behavior.
In the present study, we designed the two-choice task using a modified M-shaped-maze forinvestigating the waiting behavior for delayed reward in rats and investigated the involvementof the GABAergic transmission in the NAc. In the center lane of M-shaped-maze, the rat has
to wait for the LED-cue turned on before going into the correct lane where a sound stimulus
is presented. The waiting period, i.e., “delay for the reward” was presented randomly (0, 5, 10,
15, 20, 25 seconds). As the delay increased, the error rate of waiting behavior for future reward
was also increased and seemed to correlate with the delay period used at a previous trial.
The intra-NAc infusion of a GABAA receptor agonist fluorophore-conjugated muscimol (FCM)
affected the cue-selection behavior required waiting a period.
To confirm how the NAc neurons are activated and affected by drugs acting on GABA
receptors, we performed local field potential recordings and voltage-sensitive dye imaging inhorizontal brain slices containing bilateral NAc. We recorded the neural responses following
the electrical stimulation to the anterior commissure or the white matter at the rostral/
dorsal border of the NAc. The synaptic responses recorded from NAc-core show paired-pulse
facilitation, and the response was enhanced by GABAA receptor antagonist gabazine (1µM).
While, focal application of the FCM diminished the response in the NAc.
(COI:No)

3P-144A possible coding for experience: super bursts, ripple-like events, andsynaptic diversity
Junko Ishikawa, Takuto Tomokage, Dai Mitsushima（ Dept Neurosci, Grad Sch, Yamaguchi

Communications）

Univ, Japan）

There is strong epidemiology evidence for a positive association between hypertension or
age-related decline in cerebral blood flow (CBF) and cognitive impairment. However, therelationship between exercise mediated change in hemodynamics and cognitive function
remains unclear. A recent study reported that isometric handgrip exercise (IHG) training
reduces resting arterial blood pressure (ABP) and improves cognitive function in treatedhypertensive patients, indicating that IHG-induced reduction in resting ABP may be associatedwith improvement in cognitive function. We hypothesized that an acute bout of IHG improvescognitive function, and this improvement is associated with CBF and ABP responses. Fourteen
healthy subjects performed a cognitive task (go/no go task) before and immediately after
exercise protocol, which consisted of four sets of 2-min unilateral IHG at 25% of maximum
voluntary contraction. Mean arterial blood pressure (MAP) and middle cerebral artery blood
velocity (MCAv) were measured continuously throughout the experiment. MAP and MCAv
significantly increased during IHG (P<0.05) and returned to the resting baseline level after
the exercise protocol. The exercise protocol did not change the number of error trials in thego/no-go task but decreased its reaction time (RT) (P = 0.02), indicating that an acute bout of
IHG improved cognitive function. Interestingly, a difference in RT between before and after
exercise protocol was correlated with change in MAP during IHG (r = 0.59, P = 0.03), but
not that of MCAv and recovery of the hemodynamic response. These results indicate thatthe recovery of the hemodynamic response is not involved in enhanced cognitive function. In
contrast, since abnormal exercise pressor response affects cognitive function after IHG, ABP
monitoring during IHG may be important to ensure that blood pressure does not become
excessively elevated and to effectively improve cognitive function as well.
(COI:No)The hippocampal CA1 is necessary to maintain experienced episodic memory in many
species including humans. To monitor the temporal dynamics of processing, male rats wererecorded multiple-unit firings of CA1 neurons in habituated home cage and experienced either
4 episodes for 10 min: restraint stress, social interaction with female or male, or observationof a novel object. Before the experience, the neurons mostly exhibited sporadic firings withsome synchronized (≈ 50 ms) ripple-like firing events. After the onset of episode exposure, therestraint or social interaction with other rats induced spontaneous high-frequent firings (superbursts) intermittently, while the object observation induced the events inconsistently. Then,minutes after the initiation of episode, CA1 neurons frequently exhibited ripple-like firings
with less-firing silent periods. The number of ripple-like events depended on the experienced
episode and correlated with the total duration of super bursts. Experience clearly diversified
multiple features of individual ripple-like events with experience-specific manner, sustained formore than 40 min in the home cage.
Ex vivo patch clamp analysis further revealed the experience-promoted synaptic plasticity.
Compared with inexperience controls, the episodes with female, male, and restraint celldependently increased AMPA- or GABAA receptor-mediated postsynaptic currents, while thecontact with novel object increased GABAergic currents only. Since multivariate ANOVA in
multi-dimensional virtual space revealed experience-specific super bursts, and following ripplelike events and the synaptic plasticity, we hypothesized that the experience-specific superbursts and following synaptic plasticity are responsible to create the experience-specific ripplelike events and memory. It is possible to decipher encrypted experience by the deep learning of
the orchestrated ripple-like firings and synaptic plasticity in multiple CA1 pyramidal neurons.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S173

3P-145

AI analysis for episode-speciﬁc ripple-like ﬁrings of hippocampal CA1
neurons

3P-148

Physiological function of Neuromedin B in beige adipocyte
Ryoko Higa1，Ikuko Morisaki2，Kenshiro Shikano1，Toshikatsu Hanada2，Reiko Hanada1

Ryo Sato, Junko Ishikawa, Takuto Tomokage, Dai Mitsushima（ Dept Neurosci, Grad Sch
Med, Yamaguchi Univ, Japan）（ 1 Dept Neurophysiol, Fac Med, Oita Univ, Japan, 2 Dept Cell Biol, Fac Med, Oita Univ,
Japan）

Although the multiple CA1 neurons frequently forms synchronized (≈ 50 ms) ripple-like firing
events, the role of the events is still unclear. Here we recorded multiple-unit firings of CA1
neurons from freely-moving male rats in habituated home cage, and they experienced eitherof 4 episodes for 10 min: restraint stress, social interaction with female or male, or observationof a novel object. We subsequently recorded the firings for more than 30 min in their home
cage. The episodic experiences not only increased the number of events, but also changed
the 4 features of ripple-like events such as duration, amplitude, arc length, and number ofpeaks. Moreover, multivariate analysis of variance in the 4-dimensional virtual space revealed
significant episode-specificity of individual ripple-like events.
Using artificial intelligence (AI) system, we further analyzed thousands-pair of Euclidean
distance among the individual ripple-like events to measure the similarity. The diversityof ripple-like events was low before the experiences, but most ripple-like events exhibited
relatively long distance from other ripple-like events. Although the diversity of the events washigh in 20 to 30 min after the experience of restraint stress, many pairs of the events exhibitedclose Euclidean distance showing multiple clusters of similar ripple-like events. The diversified
ripple-like events and the clustered similarity might contain definitive information to represent(COI:No)
experienced episode. 

Beige adipocytes are thermogenic adipocytes distinct from classical brown adipocytes. It has
a key role in regulation of systematic energy homeostasis in mammals. Beige adipocytes werereported increasing the energy expenditure and improving metabolic abnormalities such as
obesity and insulin resistance. To reveal the molecular mechanism of beige adipocytes contributes to understand in the detail of energy metabolism. However, it is not fully understood.
Then, to assess differentiation and activation of beige adipocytes, we generated white andbeige adipocyte cell lines derived from the inguinal subcutaneous white adipose tissues of asingle C57BL/6J mouse. Comparing with the characteristics between white and beige adipocytes revealed that beige adipocytes are less adipogenesis than white adipocytes. To elucidate
which molecules could regulate beige adipocytes adipogenesis, we performed shRNA library
screening. Then, it was found Neuromedin B (NMB) as a candidate gene that suppresses beige
adipocyte differentiation. NMB is one of bombesin like peptides and known to be involved inregulation of energy homeostasis. However, there is no report on its relationship with beige
adipocytes. In this study, we generated NMB gene-deficient mice using the CRISPR-Cas9
system in order to investigate the function of NMB in beige adipocytes. Here, we will present
some data of NMB gene-deficient mice and their control mice in analysis of energy metabolism(COI:No)and beige adipogenesis.

3P-146

3P-149Alpha oscillations are related to accumulation of relevant information in
the parietal cortex
Yuki Suda, Takanori Uka（ Dept Integrative Physiol, Grad Sch Med, Univ Yamanashi, Japan）
Flexible decision making is accomplished by the integration of appropriate information depending on context. Although the spiking activity recorded in area LIP of the parietal cortex is
known to be related to the accumulation process for relevant information, little is known
concerning how the oscillatory alpha-band activity, known to be related to discrimination
performance, relates to the accumulation process. To address this issue, we investigated firing
rate and alpha oscillations of LIP neurons while monkeys performed a switching task.
We trained two macaque monkeys to flexibly switch between discriminating motion direction(upward vs downward) or stereoscopic depth (near vs far) in a random dot stereogram. Task
difficulty was varied by manipulating the motion coherence and the binocular correlation of
the visual stimulus. While the monkeys performed this task, we recorded local field potential(LFP) and single unit activity from isolated LIP neurons. We calculated the event-related
spectrum perturbations (ERSP) from LFP, and computed the time course of the alpha-band(8-14Hz) activity.
We confirmed the typical strength-dependent increase in firing rates for both motion coherence and binocular correlation following visual stimulus onset, and the convergence of firing
rates just before the saccade. On the other hand, the alpha-band activity gradually decreased
after stimulus onset irrespective of stimulus direction, and the decrement slope was dependent
on the absolute value of stimulus strength for relevant but not irrelevant features. These activities converged with respect to each choice around the time of saccade, suggesting that alphaoscillations are related to the evidence accumulation process in addition to spiking activity for(COI:No)
flexible decision formation.

Menstrual cycle inﬂuence on breast skin temperature measured during
sleep
Shuri Marui, Kaho Ito, Kei Nagashima（ Body Temp. Fluid Lab., Fac. Human Sciences

Waseda Univ., Japan）

Background: Body temperature at a rest (i.e., basal body temperature, BBT) is fluctuated inabout a month by changes in menstrual cycle in women of reproductive age. It is important todetermine the site of measuring the BBT more easily compared to the sublingual temperature.
The purpose of this study was to investigate whether breast skin temperature could be usedin healthy women to detect the menstrual cycle.
Methods: Data were collected from ten healthy young women. The subjects were nonsmokers,
were not taking oral contraception, and had regular menstrual cycles. The lower left breast
skin temperature was measured every 15 minutes during sleep using a temperature sensor attached to clothing and sublingual temperature was measured in the morning using a 10-second
digital thermometer. Both the breast skin and sublingual temperatures were measured every
day throughout three periods.
Results: The average temperature of the lower left breast until two hours before waking up(Tchest) was lower than the sublingual temperature among all subjects. Tchest was significantly
associated with the sublingual temperature among the subjects maintaining regular sleepwake cycles. Tchest showed a biphasic pattern similar to the sublingual temperature.
Conclusion: The menstrual cycle affects the breast skin temperature during sleep. It wassuggested that the BBT could be estimated from the temperature of the lower left breast.
(COI:No)

3P-147

3P-150

Tamami Ueda（ Nutr Phyol, Grad Phar Sci. Josai Univ, Japan）

Yasutake Shimizu1, 2, 3，Kanako Nomura1，Hiroki Shimaoka2，Yuuki Horii2，Kazuhiro Horii2，
2 1 Lab Vet Physiol, Fac Appl Biol Sci, Gifu Univ, Japan, 2 Dept Basic Vet
Takahiko Shiina1, （Effect of the genotype of elongation of very long chain fatty acids
protein 5 (ELOVL5) on the psychological state of Japanese elders
The number of patients suffering from mood disorder, a psychiatric condition, is increasing.
Systemic inflammation is now recognized as a major etiological factor for this condition. Creactive protein (CRP), a marker of inflammation, could also serve as a marker for mooddisorder. The expression of elongation of very long chain fatty acids proteins 5 (ELOVL5),
enzymes known to be involved in the synthesis of polyunsaturated fatty acids (PUFAs), hasbeen reported to reduce the risk of the disorder. Thus, the present study investigated the correlation between the Self-rating Depression Scale (SDS), apathy scale, or CRP and PUFA levelsin the red blood cells (RBCs). Furthermore, effect of the rs2397142 (CC versus CG+GG) alleleof ELOVL5 on enzyme activity, fatty acid (FA) synthesis, and psychiatric stabilization werealso investigated. In total, 853 samples from the Shimane chore study were assigned includedin this study. FA profiles of RBC membranes were analyzed by gas chromatography. Genomic
DNA was extracted from leukocytes using a standard phenol/chloroform method. SNPs in
ELOVL5 was genotyped using the TaqMan assay. The level of emotional disturbance in thesubjects was assessed using the SDS, apathy scale, and CRP were used. When the SDS score
was higher than 40, the subject was considered to have a tendency for emotional disturbance.
If the apathy scale was higher than 16, the subject was considered to have a tendency of low
motivation. CRP level was found to correlate with SDS score significantly. Although there wasno significant difference in the SDS score between the CC and CG+GG genotype groups, the
CRP levels tended to be higher in the latter. PUFA levels did not differ significantly based on
the rs2397142 genotype.
Collectively, the findings suggest that ELOVL5 rs2397142 exerts a very limited effect on the(COI:No)
psychological state of Japanese elders.

S174

Characterization of cold intolerance and daily torpor in the house musk
shrew (Suncus murinus)
Sci, Lab Physiol, Unit Grad Sch Vet Sci, Gifu Univ, Gifu, Japan, 3 G-CHAIN, Gifu Univ,
Japan）The house musk shrew (Suncus murinus: suncus) is cold intolerant because the animal is induced to hypothermia and immobilized in the cold environment. However, precise mechanismof cold intolerance in the suncus is still unclear. Therefore, the aim of the present study was
to characterize cold intolerance in the suncus. Both sexes of suncus were used in this study.
The suncus were bred in the cold room at 4℃, and then they were induced to hypothermia
and immobilized within 3 days. This result shows that the animals are cold intolerant. For
inducing habituation to cold environment, temperature in the breeding room was decreased
gradually. After habituation to cold environment, the suncus kept body temperature in thecold room at 4℃ for longer time. In addition, the size of brawn adipose tissue was increased.
These results indicate that cold intolerance of the suncus might be derived from insufficiencyof thermogenesis. On the other hand, we also examined property of daily torpor in the suncus.
(COI:No)So, we will report the results. The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

3P-151

3P-154OU

Jussiaea Valente Bariuan, Yuko Okamatsu-Ogura, Ayumi Tsubota, Kazuhiro Kimura

Teppei Ueda, Ryoko Higa, Kenshiro Shikano, Reiko Hanada（ Department ofCold-induced activation of BAT thermogenesis increases circulating
miR-122 level possibly through the secretion from muscle（Laboratory of Biochemistry, Faculty of Veterinary Medicine, Hokkaido University, Sapporo
060-0818, Japan）
Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis and required forbody temperature maintenance in cold environments. Since BAT enhances energy expenditure, it is considered as an anti-obesity target. BAT activity in human is negatively associatedwith circulating microRNA-122 (cir-miR-122) level, which is suggested to ordinarily come fromthe liver. To elucidate the relation of BAT function with cir-miR-122, we compared miR-122
levels between C57BL6/J mice exposed to cold environment (10±2℃) for 4 hours and placedat normal room temperature (23±2℃). Cold exposure significantly increased the expressionsof Uncoupling protein 1 (Ucp1), a key mitochondrial protein for thermogenesis, indicating theactivation of BAT. Cir-miR-122 level was significantly increased after cold exposure, but not
at room temperature. Cold exposure caused no change in miR-122 and its precursor levels in
the liver. In contrast, cold exposure significantly decreased miR-122 expression in the muscle,
but not in BAT, suggesting that increased cir-miR-122 level was due to the enhancement of its
secretion from the muscle. To examine whether BAT thermogenesis was a prerequisite forincreased cir-miR-122 and decreased miR-122 expression in the muscle, effect of cold exposure
was examined in UCP1-KO mice, lacking BAT thermogenic function. While the expressionsof thermogenesis-related genes in BAT, except for that of Ucp1, was significantly increased
after cold exposure, no significant changes were observed in cir-miR-122 and muscle miR-122
expression in UCP1-KO mice. These results suggest that cold-induced activation of BAT
thermogenesis increased cir-miR-122 through the secretion of miR-122 from muscle, althoughfurther study is required to find the missing link between BAT thermogenesis and miR secretion from the muscle.
(COI:No)

Physiological function of GRP in energy metabolism regulation
mechanisms
Neurophysiology, Faculty of Medicine, Oita University.）

Metabolic syndrome involves a large number of diseases and is a risk factor for obesity-relateddiseases. The number of those patients are increasing but there is no effective drug. In recent
years, beige adipocytes were discovered as a therapeutic target because of their ability to
produce heat and enhance energy metabolism like brown adipocytes. More recently, they
have been reported to improve glucose tolerance. However, there are still many unclear themechanism about differentiation and activation of beige adipocytes. To reveal the mechanismof beige adipocytes differentiation and activation, we established white and beige preadipocyte
cell lines from subcutaneous adipose tissue of mouse and compared mRNA expression levels
by RNA-seq. Then, we found a remarkable increasing of gastrin-releasing peptide (GRP) in
mature beige adipocyte activated by Forskolin. GRP, a bioactive peptide, is known to be
involved in systemic metabolism and central nervous system control. GRP receptor-deficient
mice decreased insulin secretion during elevated blood glucose periods. However, the relationship between beige adipocytes and GRP is unknown. In this study, we generated GRP geneknockout mice (GRP KO) using the CRISPR-Cas9 system to investigate the function of GRP
in beige adipocyte. We will present the data of GRP KO mice and wild type mice in analysisof body weight gain, food intake, energy metabolism, locomotor activity and glucose tolerance(COI:No)
under a normal diet or a high fat diet.

3P-152

3P-155JeongBeom Lee1，Lee Hye-Jin1，JeongHo Kim1, 2，Tae-Hwan Park 2，Young-Ki Min1

Aya Yamamura1，Nayeem Md Junayed 1，Hiroyuki Muramatsu2，Susumu Suzuki3，
Masato Watanabe2，Kogenta Nakamura2，Kazuhiro Yoshikawa3，Motohiko Sato1

Decreased thermal sweating of central sudomotor mechanism intropical Africans compared to temperate Koreans（ 1 Department of Physiology, College of Medicine, Soonchunhyang University, 31,
Suncheonhyang 6-gil, Dongnam-gu, Cheonan-si, 31151, Republic of Korea, 2 A student at theCollege of Medicine, Soonchunhyang University, 31, Suncheonhyang 6-gil, Dongnam-gu,
Cheonan-si, 31151, Republic of Korea）
Tropical natives sweat less and preserve more body fluid than temperate natives, toleratingheat stress. However, the mechanisms involved in such sweating reduction has not been fully
elucidated. We examined sudomotor responses of tropical natives (Africans, n=36 males) andtemperate natives (Koreans, n=41 males) subjects during hot water (43℃) leg immersion(central sudomotor response). Correlations between mean body temperature, basal metabolic
rate (BMR) and sweat rate were also examined.
All procedures were done in an automated climate chamber. Local skin temperatures and
BMR were measured and mean body temperature was calculated. Sweating activities which
include evaporative loss rate, sweat onset time, sweat rate, sweat volume and whole body
sweat loss volume were examined.
In the heat load test, Africans showed lower mean body and local skin temperatures than
Koreans before and after heating. Before and after heating, BMR declined significantly in Africans, while that of Koreans declined less. Local sweat onset time increased more in Africans
than in Koreans. Local evaporative loss rate, local sweat volume, local sweat rate, and wholebody sweat loss volume reduced in Africans than in Koreans. There were positive associationsof mean body temperature and resting BMR with mean sweat rate.
In conclusion, we observed the larger reduction of sudomotor activity in tropical Africans thanin temperate Koreans, which was associated with their lower mean body temperature and(COI:No)
lower BMR.

Pathophysiological role of VEGF receptor 3 on tumorigenesis of
prostate cancer（ 1 Dept Physiol, Aichi Med Univ, Japan, 2 Department of Urology, Aichi Medical University,
3 Division of Research Support, Research Creation Support Center, Aichi Medical University）
Prostate cancer is one of the most common and lethal malignancies in men worldwide. Although the androgen receptors (ARs) contribute to the progression of prostate cancer, otherhormones may be also involved in it. Vascular endothelial growth factors (VEGFs) play a
critical role in several cancer events including angiogenesis and tumorigenesis. However, thepathophysiological role of VEGF signaling in prostate cancer remains to be elucidated. In thepresent study, the involvement of VEGF receptors and their downstream pathways on cellproliferation, migration, and tumor development was examined using human primary prostateepithelial cell line (PrEC) and prostate cancer cell lines (LN-CaP and PC-3). Among three
VEGF receptors, the protein expression of VEGFR3 in PC-3 cells was much higher than otherprostate cells. PC-3 cells is a metastatic and highly migratory prostate cancer cells and thecell growth is androgen-independent. In PC-3 cells, VEGF-C facilitated the phosphorylationof VEGFR3 in a time-dependent manner and the stimulatory effect was mostly blocked bya selective inhibitor of VEGFR3, MAZ-51. MAZ-51 also inhibited the phosphorylation of Akt.
Importantly, the treatment with MAZ-51 dose-dependently reduced the viability, proliferation,and migration of PC-3 cells. Furthermore, MAZ-51 blocked the tumor growth of PC-3 cells
implanted into nude mice. These results strongly suggest that the activity of VEGFR3 involvedin cell proliferation, migration, and development of prostate cancer. Therefore, VEGFR3 may
be a novel therapeutic target for prostate cancer.
(COI:No)

3P-153

3P-1561 1 Department of
Hye-Jin Lee1, Jeong-Beom Lee1, Tae-Hwan Park 2 , Young-Ki Min（

Hisaki Hayashi1，Takuya Matsui1，Junayed Nayeem M.1，Rie Takahashi1，Aya Yamamura1，
1 1 Dept Physiol, Sch Med, Aichi Med Univ, Japan,
Mitsushi Ikemoto2，Motohiko Sato（

Evaluation of the quantitative sudomotor axon reﬂex test in healthyhumanPhysiology, College of Medicine, Soonchunhyang University, 31, Suncheonhyang 6-gil,
Dongnam-gu, Cheonan-si, 31151, Republic of Korea, 2 A student at the College of Medicine,
Soonchunhyang University, 31, Suncheonhyang 6-gil, Dongnam-gu, Cheonan-si, 31151,
Republic of Korea）
The purpose of this study was to quantitatively assess the difference in sudomotor function
between healthy males and females by measuring skin surface area and activated sweat glanddensity (ASGD). The quantitative sudomotor axon reflex test (QSART), a method for evaluating
autonomic nervous system activity, was used for quantification. In QSART, the sweat glands
are activated directly or indirectly by the subcutaneous application of neurotransmitters, suchas 10% acetylcholine, through iontophoresis (2 mA * 5 min). This series of mechanisms is called
the sudomotor axon reflex. Blue-black pigmented spots were counted in 0.5 cm×0.5 cm areas
under a microscope in triplicate, and average sweat gland density (count/cm2) was calculated.
After recording age, height, weight and several measurements of the forearm, QSART was
performed on 201 (116 males, 85 females) healthy subjects aged 21 to 78 years to measure
ASGD. The result of independent sample t-test showed higher ASGD in women. The bodysurface area and the surface area of the forearms were higher in men but the number ofactivated sweat glands was not significantly different according to sex. The activated sweat
gland counts of the body and forearms were analyzed through linear regression by age formales and females. The results demonstrate that an attenuation of sudomotor function occurs
with aging in both sexes. Moreover, the findings showed a progressive increase in onset timeand a decrease in sweat rates, SGD and SGO with increasing age in both sexes.
(COI:No)

Roles of oxidative stress induced extracellular vesicle derived frommesenchymal stem cell
2 AIST）

To date, mesenchymal stem cells (MSC) including bone marrow derived stem cells have been
intensively investigated as a cell–based therapy and regenerative medicine. Extracellular
vesicle, exosome, derived from MSC has been reported to have anti-oxidant potential, however,roles of exosome in age related macular degeneration, induced by oxidative stress in eyes,
has not been reported yet. Purpose of this study is to analyze the effect of bone marrow
MSC derived exosome on recovery from oxidative stress induced cellular damage in eyes.
First, exosome from bone marrow derived MSC was isolated by combination of filtrationand sucrose gradient ultracentrifuge method, and the quality was evaluated. Particle size
analyzer showed average diameter of exosome was 91.0 nm. Immunocytochemistry and flow
cytometry indicated 100% of cultured endothelial cells incorporated exosome labeled with
ExoGrow fluorescent labeling reagent. Transwell assay demonstrated that exosome stimulated
cell migration activity in cultured endothelial cells. These results indicated exosome isolated
here was biochemically intact and physiologically active. Since it is known that various stressstimulation change exosome profile of MSC, therefore, exosomal protein expression profile andits change induced by moderate reactive oxidative species are being analyzed with LC-MS/MS(COI:No)system, and will be discussed.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S175

3P-157

3P-160OU

Ammar Boudaka, Zakariya Al-Amri, Ismail Al-Qanobi, Nawaf Al-Mushifri, Hajar BaOmar,
Intisar Al-Lawati（Department of Physiology, College of Medicine and Health Sciences,

Hiroyuki Yatsuka1, 3，Kazumasa Hada1，Ryouhei Umeda2，Kenichi Kimoto3，Reiko Hanada2，
1 1 Department of Cell Biology, Oita University
Toshiaki Kubota3，Toshikatsu Hanada（

The Effect of Gum Arabic on the Diabetogenic Effect of Streptozotocin
in Mice
Sultan Qaboos University, Muscat, Sultanate of Oman）

Background: Diabetes mellitus is becoming increasingly common worldwide. Plant-based
therapies have been shown to have anti-diabetic properties. Gum Arabic (GA) is a prebiotic
agent that possesses antioxidant and anti-inflammatory properties. The effect of GA on the
diabetogenic effect of streptozotocin (STZ) is rather unknown. Therefore, the purpose of thecurrent study is to study whether GA plays any prophylactic or therapeutic roles in a mousemodel of streptozotocin-induced hyperglycemia.
Methods: Male C57Bl/6 mice (8 weeks) were randomly divided into five groups: control (received plane water), GA (in drinking water), STZ-injected, GA-STZ (GA for 2 weeks before STZ
injection) and STZ-GA (GA after STZ injection) groups. Body weight and fasting blood sugar
were measured in all groups. Then hyperglycemia was induced by a single intraperitoneal
injection of STZ into the respective groups. Post-STZ injection, body weight and fasting blood
sugar were recorded weekly for 4 weeks. At the end of the experiment, plasma insulin wasdetermined and pancreatic tissue sections were stained histomorphometric analysis.
Results: STZ and STZ-GA mice exhibited a significant weight loss and hyperglycemia over thecourse of 4 weeks compared to the control, GA and GA-STZ groups. Plasma insulin levels weresignificantly lower in STZ and STZ-GA compared with the other groups. Light microscopic assessment revealed an extensive loss of pancreatic islets in STZ and STZ-GA mice and dramatic
decrease in the size of the remaining islets. Immunofluorescence studies showed a decrease in
the number of insulin-positive beta cells in STZ and STZ-GA mice while the glucagon-positivealpha cells accounted for a major proportion of residual islet cell mass.
Conclusion: GA seems to play a prophylactic role against the streptozotocin-damaging effecton beta cells, which makes this dietary fiber as a novel pharmaceutical target with prophylactic
potential in diabetes mellitus.
Keywords: diabetes, STZ, hyperglycemia, insulin, Gum Arabic.
(COI:No)

Creation of neurodegenerative disease model and elucidation ofpathogenesis due to abnormal RNA metabolism
Faculty of Medicine, 2 Department of Neurophysiology, Oita University Faculty of Medicine,

3 Department of Ophthalmology, Oita University Faculty of Medicine）

The RNA exosome is an evolutionarily conserved ribonuclease complex that is critical forboth the processing and degradation of a variety of RNAs. Exosome component 2 (EXOSC2)
is a component of the exosome complex. Recently, homozygous or compound heterozygous
mutations in the EXOSC2 gene was identified in German families. The patients show neuronal
disorders, such as retinitis pigmentosa, progressive hearing loss, and mild intellectual disability.
Mutations.However, the relevance of the EXOSC2 gene mutation in the neurological disease
was entirely unknown. In this study, we aim to elucidate the pathophysiological mechanism ofRNAexosome related diseases. To investigate the in vivo function of EXOSC2, we generated
an exosc2 knockout zebrafish model by CRISPR/Cas9 system. The exosc2 knockout zebrafish
showed an abnormal phenotype in neuronal development, meaning that the phenotypic resemblance between human patients and zebrafish mutants. Furthermore, we showed senescencein exosc2 knockout zebrafish by histochemical assay for SA-β-gal activity. Considering theproperties of RNA exosomes, which contribute to degrading various types of RNA, it is conceivable that the accumulation of abnormal RNA molecules in cells is the trigger for neuronaldiseases and senescence. We are now under the investigation of global RNA analysis using the(COI:No)zebrafish model.

3P-158

3P-161OU

Hajime Yano, Tomoyoshi Sanada2，Islam Afsana1，Reina Tanimoto1，Teppei Ueda2，
1 1 Department of Molecular and Cellular Physiology,
Naohito Hato2，Junya Tanaka（

Ikuko Morisaki1，Yuji Arai2，Hiroshi Shiraishi1，Reiko Hanada3，Takashi Kobayashi4，
1 1 Department of Cell Biology, Oita University Faculty of Medicine,
Toshikatsu Hanada（

Roles of LOXL2 in exosomal fraction on lymph node metastasis of headand neck squamous cell carcinoma
Graduate School of Ehime University Medical School, 2 Department of Otorhinolaryngology,
Head and Neck Surgery, Ehime University Medical School）
The secretory enzyme lysyl oxidase like 2 (LOXL2) is assumed to contribute to tumor progression through participation in cellular events including remodeling extracellular matrix
and epithelial-mesenchymal transition. In the previous study, we identified elevated gene
expression of LOXL2 in human head and neck squamous cell carcinoma (HNSCC) lymph
node metastases. Here we assessed the significance of LOXL2 in the metastasis and of liquidbiopsies for detecting HNSCCs and their risk of the metastasis.
LOXL2 protein expression was assessed in human tongue HNSCC tissues by immunohistochemistry as well as in the serum from three patients by immunoblotting. Serum samples
were further fractionated in exosomes and supernatant by ultracentrifugation, which were
subjected to immunoblot and in vitro LOX activity analyses. Then, exosomal LOXL2 levels
from 38 serum samples from HNSCC patients and seven healthy volunteers were measured
using polymer sedimentation exosome preparation and subjected to statistical analysis.
Immunoblot analyses revealed that LOXL2 was present in serum exosomal fractions fromthree volunteers without anamnesis as well as three patients. Mean LOXL2 expression wasapproximately 3-fold higher in patients. Immunohistochemical LOXL2 staining was detected in
HNSCC cells in addition to non-cancerous lipid tissues and some muscles in a human tongue
HNSCC sample. Further measurements of exosomal LOXL2 by ELISA showed higher LOXL2
levels in patients.
Elevated serum exosomal LOXL2 levels can be an indicator of HNSCC. A follow-up clinicalstudy will be required to determine the clinical utility of using LOXL2 to diagnose HNSCC
and/or determine the risk of metastasis.
(COI:No)

Establishment and Functional Analysis of Pontocerebellar Hypoplasia
Type 10 mouse model
2 National Cerebral and Cardiovascular Center, 3 Department of Neurophysiology, Oita
University Faculty of Medicine, 4 Department of Infectious Disease Control, Oita University
Faculty of Medicine）

Pontocerebellar Hypoplasia is one of the hereditary neurodegenerative diseases, and thedisease has been reported up to 10 types so far. Our previous report found that CLP1 works
as RNA kinase which phosphorylate 5’ hydroxyl group RNA functions. Recently, it has beenreported that Pontocerebellar Hypoplasia type 10 is caused by the CLP1 mutation (R140H).
CLP1 is one of the components of transfer RNA (tRNA) splicing endonuclease complex (TSEN
complex) which removes intron from the pre-tRNA. We established and analyzed Clp1 kinasedead mouse model. We found the accumulation of abnormal mature tRNA fragments induced
apoptosis of neuron cell death. The region of R140H is different from the region of kinasedomain, so we established R140H model mouse using CRISPR/Cas9 system. R140H model
mouse displays milder neuron paralysis than kinase-dead model mouse. Future investigation
will aim to perform phenotypic and molecular analysis of the influence of abnormal maturetRNA fragments in motor neurons.
(COI:No)

3P-159OU

3P-162OU

Masanori Inoue1, 2，Kazumasa Hada1，Hiroshi Shiraishi1，Tohru Ishitani3，Masaki Matsumoto4，
1 1 Department of Cell Biology, Oita University Faculty of
Kenji Ihara2，Toshikatsu Hanada（

Masahito Shide1，Ryohei Umeda1，Kazumasa Hada2，Kenshiro Shikano1，Ryoko Higa1，
1 1 Dept
Hirotaro Urushibata2，Hiroshi Shiraishi2，Toshikatsu Hanada2，Reiko Hanada（tRNA processing defects link to p53-dependent neurodegenerative
disease
Medicine, 2 Department of Pediatrics, Oita University Faculty of Medicine, 3 Integrated

Signaling Systems, Department of Molecular Medicine, Institute for Molecular ＆ Cellular
Regulation, Gunma University, 4 Department of Molecular and Cellular Biology and Divisionof Proteomics, Medical Institute of Bioregulation, Kyushu University）
Fragments of transfer RNA (tRNA), derived either from pre-tRNA or mature tRNA, havebeen discovered to play an essential role in the pathogenesis of various disorders such asneurodegenerative disease. CLP1 is an RNA kinase involved in tRNA biogenesis, and mutations in its encoding gene are responsible for pontocerebellar hypoplasia type-10. Mutationof the CLP1 gene results in the accumulation of tRNA fragments of several different kinds.
These tRNA fragments are expected to be associated with the disease pathogenesis. However,
it is still unclear which of the tRNA fragments arising from the CLP1 gene mutation has thegreatest impact on the onset of neuronal disease. We found that 5’ tRNA fragments derived
from tyrosine pre-tRNA (5’ Tyr-tRFs) caused p53-dependent neuronal cell death predominantly
more than other types of tRNA fragment. Human neuroblastoma cells transfected with 5’ TyrtRFs were susceptible to cell death and the neuronal cell death induced by 5’ Tyr-tRFs wasprevented by p53 gene deletion. As an in vivo model, 5’ Tyr-tRFs caused severe developmental
abnormalities and induced neuronal cell death through p53 activation. These results provide
a new conceptual framework in neurodegenerative diseases for how the accumulation of 5’
Tyr-tRFs can affect neurons, and might help explain fundamental molecular principles of(COI:No)
neurodegenerative diseases. 

S176

Deﬁciency of VRK1 causes microcephaly and alters social interactions
in zebraﬁsh
Neurophysiol, Fac Med, Oita Univ, Japan, 2 Dept Cell Biol, Fac Med, Oita Univ, Japan）

Vaccinia-related kinase 1 (VRK1) is one of a serine/threonine kinase. It has been reported
that VRK1 gene mutation in human causes pontocerebellar hypoplasia and cerebral dysplasia.
However, the mechanisms of brain diseases caused by dysfunction of VRK1 are unclear. In thisstudy, to investigate the physiological functions of VRK1 and its involvement in pontocerebellar
hypoplasia, we measured body and brain sizes and performed a series of behavioral analyses
using VRK1 knockout zebrafish (VRK1 KO). We used CRISPR/Cas9 system to generate VRK1
KO. We established VRK1 KO which has 5 bp deletion causing a frameshift mutation. Morphological analysis showed that both body lengths and weights in VRK1 KO were smaller thancontrol zebrafish (CNT). In addition, microcephaly was observed in the VRK1 KO compared to
CNT. We measured the diameter of forebrain, midbrain, and cerebellum using HE stains in thebrain slices, and those of VRK1 KO was shorter than CNT in each brain region. The novel tankdiving test showed the decrease of locomotor activity and anxiety-like behavior in VRK1 KO.
The social interaction test suggested that VRK1 KO has a strong interest in novel zebrafish.
Taken together, we showed that VRK1 KO became microcephaly with growth retardation andincreased interest in novel zebrafish.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

3P-163

3P-1661 1 Institute of Humanities and Social
Kihachiro Fukada1，Hidehiko Kushi2，Terue Takashina（

Noriyuki Hama1，Shigefumi Yokota2，Masashi Fujitani1, 2，Yasumasa Okada3，
4 1 Department of Neural and Muscular Physiology,
Naohiro Koshiya4，Hidehiko Koizumi（

The effects of overweight on a2-plasmin inhibitor / plasmin complex
after strenuous exercise
Sciences, Nihon University, Tokyo, Japan, 2 College of Humanities and Social Sciences,
Nihon University, Tokyo, Japan）

Introduction: Some studies have reported an increase in fibrinolytic activity after acute strenuous exercise. Conversely, fibrinolytic activity is inhibited in overweight person. In this study,
our aim was to evaluate whether being overweight affects fibrinolytic activity after acutestrenuous exercise. Being overweight was defined using body mass index (BMI) as a measureof the degree of obesity.
Subjects and Methods: Twelve healthy young men aged 19 to 23 years old who engaged indaily exercise participated in this study. Seven of these men were categorized in the BMI<25group, and five were in the BMI>25 group. Venous blood samples were collected from thesubjects pre- and post-performance of the Cooper test. This test involved running as far as
possible within a 12-minute period. a2-plasmin inhibitor / plasmin complex (PIC, as a marker offibrinolytic activity) levels were measured using the collected blood samples.
Results: The PIC levels increased significantly in the BMI<25 group (pre: 0.5 ± 0.02 ug/mL,
post: 1.9 ± 0.3 ug/mL, P<0.05), but these were not significantly increased in the BMI>25 group(pre: 0.5 ± 0.08 ug/mL, post: 1.0 ± 0.1 ug/mL, p>0.05).
Conclusions: Using BMI as an index for evaluation, this study showed that fibrinolytic activity
is inhibited in overweight young men after acute strenuous exercise.
(COI:No)

Decrease in the expiratory duration induced by the photostimulation of
inhibitory neurons within the lateral solitary nucleus during inspiration
Shimane University School of Medicine, Shimane, Japan, 2 Dept of Anatomy, Shimane Univ.
Sch of Medicine, Japan, 3 Clinical Research Center, Murayama Medical Center, Japan,
4 Cellular and Systems Neurobiology Section, NINDS, NIH, USA）
Respiratory rhythm is generated by the central pattern generator (CPG) that is composed ofinterneurons within the ventral respiratory column and modulated by a variety of visceralinformation, such as blood gases and lung volume. The information from lung stretch receptor inputs to the respiratory CPG via GABA/glycinergic neurons within the lateral solitary
nucleus (lateral NTS). This inhibitory input is thought to cause the transition from inspiration to
expiration. However, it remains unknown how these inhibitory neurons affect the respiratory
rhythm. Using optogenetic approaches, we previously showed that the photostimulation oflateral NTS inhibitory neurons during the inspiratory phase terminated the ongoing inspiration and decreased the duration of following expiration, but the photostimulation during the
expiratory phase could not perturb the respiratory pattern. In this study, to examine therole of lateral NTS inhibitory neurons in the determination of respiratory phases, we applied
the photostimulation at the different timing within the inspiratory phase (0, 100 or 200 ms
after the onset of inspiration). The photostimulation applied at 0 or 100 ms after the onset
decreased the duration of ongoing inspiratory and following expiratory phases. In contrast, thephotostimulation applied 200 ms after the onset could not perturb the ongoing inspiration but
decrease the duration of the following expiration. These results suggested that the decreasein expiratory duration induced by activation of inhibitory lateral NTS neurons was not causedby the termination of inspiratory phase and inhibitory lateral NTS controls the expiratory(COI:No)duration.

3P-164

3P-167

3 1GradYuma Hara1，Linlang Quan1，Kei Hatano1，Yoshinori Ohtsuka2，Takahiro Yunoki（

Sayumi Kotani, Tien Lin Shih, Hiroshi Onimaru, Masahiko Izumizaki（Department of

We investigated the effect of inspiratory muscle fatigue on central fatigue and corticospinal
excitability in a leg muscle. Seven healthy male subjects performed a 30-min inspiratory resistive breathing (IRB) task (breathing frequency: 50 breaths/min, tidal volume: 2.0 times theresting value, inspiratory resistance: 40cmH2O). Vastus lateralis (VL) responses to transcranial
magnetic stimulation (TMS) of the motor cortex [motor evoked potential (MEP) and corticalsilent period (CSP)] and electrical stimulation of the femoral nerve [maximal M-wave (Mmax)]
were recorded during 5-s isometric knee extension at the intensity of 20% of maximal voluntary contraction (MVC). Voluntary activation (VA) of the VL was assessed via TMS during
5-s MVC. The VL responses, maximal inspiratory pressure (PImax), and respiratory effort
sensation were measured 5 min before the start of the IRB task (baseline) and between 10
and 15 min (middle phase) and between 25 and 30 min (end phase) after the start of the IRB
task. In addition, blood lactate concentration was measured before (baseline) and after (endphase) the IRB task. Although there was no significant difference in PImax among the threemeasurement phases, respiratory effort sensation and blood lactate concentration increased
significantly from baseline to the end phase of the IRB task (P<0.05). The CSP was significantly
longer in the end phase than in baseline and the middle phase (P<0.05), while MEP, Mmax, and
VA remained unchanged from baseline to the end of the IRB task. These results suggest thatdevelopment of inspiratory muscle fatigue or increased respiratory effort sensation increases
intracortical inhibition in the leg motor cortex.
(COI:No)

Monocarboxylate transporter (MCT) plays an important role in providing metabolic support toneurons by functioning as the principal transporters for lactate in the nervous system; lactatethat is produced by astrocytes is transported to neurons through the so-called astrocyteneuron lactate shuttle. It is unknown how the lactate shuttle system contributes to maintaining
respiratory neuron activity. Alpha-cyano-4-hydroxycinnamate (4-CIN) is known as a MCT-2
inhibitor. In the present study, we examined the effects of 4-CIN on respiratory activity inthe brainstem-spinal cord preparation from newborn rat (P0-P3). Bath application of 4-CIN(5 mM) depressed the respiratory rhythm accompanied by a decrease of the C4 amplitude.
After washout, the respiratory rhythm and C4 amplitude recovered. The respiratory activity
depressed by 4-CIN was partially recovered by bath-application of L-lactate (1 mM) or an
ATP-sensitive potassium channel blocker, glibenclamide. Local application of 4-CIN to spinal
cord or the medulla decreased C4 amplitude or the rhythm, respectively. Burst activities on
preinspiratory and inspiratory neurons in the medulla were also depressed. In the presence of
TTX, furthermore, some respiratory neurons were hyperpolarized during 4-CIN application.
In some respiratory neurons in which miniature-like EPSPs or IPSPs were detected in thepresence of TTX, these synaptic potentials were inhibited by 4-CIN with partial recovery
after washout. We concluded that 4-CIN might depress transmitter releases via action on
the presynaptic terminal as well as the postsynaptic membrane of respiratory neurons. Our
results suggest that the lactate shuttle is important in maintaining respiratory neuron activity.
(COI:Properly Declared)

3P-165

3P-1683 1 Inst. of Health and Sports ＆ Med.,
Yung-Li Hung1，Minenori Ishido2，Shuichi Machida（

Yoshihiko Oke1，Fumikazu Miwakeichi2, 3，Yoshitaka Oku1，Johannes Hirrlinger4, 5，
7 1 Dept Physiol, Hyogo Col of Med, Hyogo, Japan, 2 Dept Stats Modeling,
Swen Hulsmann6, （Continuous inspiratory resistive breathing increases motor cortex
inhibition in a lower limb muscle
Sch Educ, Hokkaido Univ, Japan, 2 Dept Sports and Human Studies, Sapporo Int Univ,
Japan, 3 Fac Educ, Hokkaido Univ, Japan）

The effects of estrogen on muscle hypertrophy and muscular waterhomeostasis in skeletal muscle following resistance exercise training
Juntendo Univ., Chiba, Japan, 2 Div. of Human Sci., Faculty of Engineering, Osaka Inst. of
Tech., Osaka, Japan, 3 Dept. of Health and Sports Sci., Juntendo Univ., Chiba, Japan）
Compared to older men, older women exhibit more frailty and loss of skeletal muscle mass. It isthought that the lack of estrogen after menopause may crucially contribute to reduced muscle
mass and strength in older women. The increase of extracellular water in the skeletal muscle
is associated with reduced estrogen levels. The extracellular water to intracellular water ratio
is increased and associated with muscle weakness in older women. Estrogen is considered
to regulate muscular water; however, the mechanism by which estrogen regulates muscularwater is unknown. Water channel aquaporin-4 (AQP4) and ion channel Na-K-Cl cotransporter
1 (NKCC1) regulate water homeostasis in the skeletal muscle, thus regulating its physiologic
functions, such as hypertrophy and atrophy. We investigated the effects of estrogen on AQP4
and NKCC1 in the skeletal muscle by using estrogen-deficient animals following resistance
exercise training. Female adult rats (10 weeks old) were divided into the following 6 groups:
sham sedentary, sham climbing training, ovariectomy sedentary, ovariectomy climbing training, ovariectomy plus estrogen treatment sedentary, and ovariectomy plus estrogen treatment
climbing training groups. The estrogen deficiency is caused by the ovariectomy. After 8 weeks
of climbing training, the weight of the flexor halluces longus (FHL) muscles were significantly
increased in the sham climbing training group but not in in the ovariectomy climbing training
group. On the contrary, ovariectomy plus estrogen treatment resulted in exercise-induced
muscle hypertrophy. AQP4 and NKCC1 protein expressions tended to be decreased after
ovariectomy, and the estrogen treatment reversed this decrease of AQP4 and NKCC1 inovariectomized animals. Nevertheless, these differences regarding protein expression among
the groups were not significant. Therefore, these data suggest that estrogen regulated
exercise-induced muscle hypertrophy but did not affect the expressions of AQP4 and NKCC1(COI:No)in the skeletal muscle.

The role of monocarboxylate transporters in maintaining respiratoryneuron activity
Physiology, Showa University School of Medicine, Tokyo）

Early postnatal development of inspiratory neuron-type in the preBötzinger complex of miceInst Stats Math, Tokyo, Japan, 3 Dept Stats Sci, Sch of Multidisciplinary Sci, Grad Univ Adv
Stud, Tokyo, Japan, 4 Carl-Ludwig-Inst for Physiol, Univ of Leipzig, Leipzig, Germany,
5 Dept of Neurogene, Max Planck Inst of Exp Med, Germany., 6 Clinic for Anesthesiol,
Gettingen Univ, Gettingen, Germany, 7 DFG Res CNMPB, Gettingen, Germany）

Spontaneous inspiratory rhythm is generated in the pre-Bötzinger complex (preBötC), one of
the kernel of the respiratory center in the brainstem. As components of the neuronal network
for rhythm generation, characteristics of single inspiratory neuron and functions of cell-types in
the preBötC have been well investigated. Next, the neuronal network structure of inspiratoryneurons should be addressed to understand the mechanism of rhythm generation. However,even ratios of inspiratory neuron-types to the total number of inspiratory neurons in the
network remain unrevealed. The ratios might change after birth. From late embryonic stage
to birth, the ratio of glycinergic and/or GABAergic neurons to the total number of inhibitory
neurons were shown to change in the ventrolateral medulla (Rahman et al., 2015). Furthermore, GABAA-mediated modulation of respiratory rhythm was changed after P3 (Ritter andZhang, 2000). Here, we estimated the ratios of inspiratory neuron-types to the total number ofinspiratory neurons in the preBötC during early postnatal days using double-transgenic miceexpressing EGFP in glycine transporter 2 -positive neurons and tdTomato in glutamic aciddecarboxylase 65 -positive neurons. We prepared rhythmic slices including preBötC from themice between postnatal day 1-10 and set the bin width to 2 days for the analysis. Rhythmic
bursting activities and neuronal activities could be recorded using local field potential (LFP)
and calcium imaging, respectively. Inspiratory neurons were detected as ROIs of radius 4
pixels (3.9 µm) in which the neuronal calcium signal had high maximum cross-correlation
coefficients to the LFP by screening and were double-checked by visually confirming thecalcium signal. Then, we classified inspiratory neurons into four types based on the expressionof the fluorescence proteins. Finally, we evaluated early postnatal development of inspiratory(COI:No)
neuron-types in the preBötC by comparison of the ratios at respective bins.

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S177

3P-169

3P-172

Yuri Hayashi1，Hiroki Takanari2，Eiji Hase2，Takeo Minamikawa2，Takeshi Yasui2，
3 1 Sch Med, Tokushima Univ, Japan, 2 pLED,
Mayuko Shimizu3，Koichi Tuneyama（

4 1 Sch Med,
Meiko Kanazawa1，Satoru Hashimoto2，Takanori Inoue3，Hiroki Takanari（

Quantitative assessment of liver ﬁbrosis using SHG
Tokushima Univ, Japan, 3 Dept Patho Lab Med, Tokushima Univ, Japan）

Assessment of dermatitis using Raman spectroscopy
Tokushima Univ, Japan, 2 RIEM, Nagoya Univ, Japan, 3 Sch Sci Tech, Oita Univ, Japan,
4 pLED, Tokushima Univ, Japan）

Introduction: Tissue fibrosis relates to various pathological conditions and impairs physiological function of the organs. Assessment of tissue fibrosis is mainly dependent on tissue staining.
However, it is difficult to get detailed information of fibrosis. It is known that the light withtwice frequency of irradiated light is generated when collagen is irradiated with an ultrashortpulse laser, which is called second harmonic generation (SHG). In the present study, we propose
to assess fibrosis in detail by a novel optical method, SHG.
Methods ＆ Results: Liver tissue were obtained from mice with steatohepatitis induced byhigh-fat feeding (SH) and mice with liver cirrhosis induced by intraperitoneal injection ofcarbon tetrachloride (LC), and sliced to 5-µm thickness. We have customized SHG microscope
equipped with a femtosecond pulse laser with a wavelength of 810 nm. The laser power was
adjusted to 20 mW on the stage, and Galvano mirror was set to scan a 162µm square range
on the x-y plane. The samples were exposed to the laser for 10 seconds and 256 pixels square
images were obtained. The intensity of SHG signal and fiber orientation based on SHG images
were analyzed by Image J software. There was no significant difference in the integrated
intensity of SHG between SH and LC. However, the fibrosis pattern was different between thetwo models; linear fibrosis in SH, and dotted fibrosis in LC. The dispersion of fiber orientation
was significantly large in LC (27.6¬¬±4.5, n=6) when compared to SH (12.8±3.1, n=7, p=0.018
vs. LC), suggesting that fibers are randomly constructed in LC.
Conclusion: We succeeded in detecting liver fibrosis using SHG in different pathological liverdisease models. The SHG microscopy could be a powerful tool for qualitative and quantitative(COI:No)
assessment of fibrotic tissue.

Background: Diagnosis of dermatitis mainly depends on subjectivejudgments such as doctor’svisual inspection and patient’s symptoms, and it is difficult to make an objective assessment.
When the materialis irradiated by a laser, Raman scattered light with a differentwavelengthfrom the laser is generated. Physical properties can beknown by analyzing the wavelengthtransition from the laser to theRaman scattered light. We propose to apply Raman spectroscopy tothe evaluation of dermatitis.
Methods: Dermatitis model mice in which ear skin was inflamed byPMA application wereprepared, and ear tissues were collected 6, 12, and 24 hours after PMA application. The tissue
without PMAapplication was used as normal control. The tissue was placed on thestage ofRaman microscope, and Raman scattered light generated bya 785 nm laser was recorded. The
Raman spectra were comparedbetween normal and inflamed skin. The analysis was conductedmainly on the Raman spectra newly generated in inflamed tissue.Pathological specimens
with HE staining were prepared after Ramanspectroscopy, scored for vasodilation and edema,and compared withthe score based on Raman spectra.
Results: In the inflamed skin, a new bimodal spectrum was frequentlyobserved at around 1,600
cm-1 that completely matched with Ramanspectrum of the blood, suggesting the spectrum
reflected vasodilationin the tissue. A broad spectrum was also observed at around 3,000cm-1
in inflammation model that was consistent with Raman spectrumof water, suggesting thesignal reflected interstitial edema. Thesespectra increased by time after PMA application
and reached maximum at 12 hours. Such changes in Raman spectra over timecoincided withchanges in histological vasodilation and edema.
Conclusion: We succeeded in detecting Raman spectra, which maydue to vasodilation andedema caused by inflammation, and thespectral and histological changes over time were
(COI:No)
consistent.

3P-170

3P-173OU

Samyoung Kim1，Sm. Ahasanul Hamid1，Mieko Imayasu1，Tomoyuki Yoshida 2，
1 1 JAIST, Japan, 2 Toyama University, Japan）
Hidekazu Tsutsui （Hiroyuki Fujinami1, 2，Hiroshi Shiraishi1，Toshitaka Shin2，Hiromitu Mimata2，
1 1 Department of Cell Biology, Faculty of Medicine, Oita University,
Toshikatsu Hanada（

Toward molecule-speciﬁc formation of neuron-microelectrode junctions

The microelectrode techniques have long enabled us to investigate the physiological properties of electrically active cells and networks. Although these techniques generally allow hightemporal resolution recordings, they intrinsically lack cell-type specificity. Target cells mustbe identified by means of other indirect criteria such as marker gene expression and cellularmorphology, which is not well-compatible with parallel recordings from highly heterogeneous
cellular networks.
By combining the robust bioactivities of synapse organizing molecules with microfabrication
techniques, we have set out to establish a means enabling the molecule-specific formationof neuron-microelectrode junctions. Following contact of neurons with a microelectrodeimmobilized with natural synapse organizing molecule, presynaptic terminals were successfully induced onto the electrode. Also, we have generated some sets of prototype engineered
synapse organizing molecules that do not cross-react with each other. It is expected that suchmolecular tools are exploited to develop the micro- or nano-electrode techniques which permit
selective recording from genetically specified cells. (COI:No)

3P-171

Summer training camps decreases cell-mediated immunity
1 1 Institute of Humanities and Social
Terue Takashina1，Hidehiko Kushi2，Kihachiro Fukada（

Sciences, Nihon University, Tokyo, Japan, 2 College of Humanities and Sciences, Nihon
University, Tokyo, Japan）

Introduction: The prevalence of upper respiratory infection has been reported to increase
due to excessive exercise. Although the effects of short-term exercise load on immune function have been studied, a few reports have evaluated the effects of prolonged exercise load.
Therefore, we focused on a rugby club training camp, in order to examine the effects ofprolonged high-intensity exercise load on cell-mediated immunity.
Methods: The subjects were 10 male university students who were members of rugby
football clubs (Age : 19.4 ± 0.2 year, Height : 177.0 ± 2.0 cm, Weight : 90.6 ± 2.8 kg). Blood
samples were collected from the subjects before and after a 25-day training camp. The countof T helper 1 (Th1) and natural killer cell (NK cell), natural killer cell activity (NK activity),
noradrenaline were measured.
Results: Th1 showed a significantly low value after the camp in comparison with before thecamp (pre : 20.5 ± 1.0 %, post : 16.8 ± 0.8 %, p <0.01). NK cell (pre : 22.1 ± 1.9 %, post : 16.1
± 1.2 %, p <0.01) and NK activity (pre : 52.7 ± 3.3 %, post : 40.8 ± 2.8 %, p <0.01) showeda significantly low value after the camp in comparison with before the camp. Noradrenaline
showed no significant difference (pre : 393.5 ± 36.5 pg/mL, post : 482.4 ± 38.3 pg/mL, p =0.057).
Conclutions: The results of this study demonstrate that summer training camps decreases(COI:No)
cell-mediated immunity.

S178

RNA kinase links prostate cancer progression
2 Department of Urology, Faculty of Medicine, Oita University）

In recent years, the relationship between abnormal RNA metabolism and various diseases
such as cancer and neurodegenerative diseases is becoming clear. In mammals, CLP1 and
NOL9 are known as RNA kinases that directly phosphorylate RNA. CLP1 is involved in transfer RNA (tRNA) maturation, and NOL9 is involved in ribosomal RNA (rRNA) maturation, bothof which have been shown to be involved in RNA metabolism. CLP1 has been reported to be agene fusion with cancer-related gene (AF10) in acute monoblastic leukemia. On the other hand,
NOL9 forms a complex with LAS1L, LAS1L has been shown to be more highly expressedin castration resistant prostate cancer compared to normal prostate cancer. However, themolecular mechanism of RNA kinase in the development and progression of cancer has notbeen clarified yet. Therefore, we analyzed the involvement of RNA kinase in cancer development and progression using a prostate cancer model. As a result, PC3 had higher RNA kinase
activity than LNCaP, and also higher cell growth, colony formation activity. Overexpression of
CLP1 in LNCaP enhanced RNA kinase activity and cell proliferation. CLP1 reduced 5’ tyrosinetRNA fragments accumulation in LNCaP. These results suggest that the RNA kinase activity(COI:No)of CLP1 is associated with cancer cell proliferation ability.

3P-174OU

Physiological 18F-FDG uptake in the anal canal in adults: evaluation
with PET/CT
Yankel Sena, Shunro Matsumoto, Christopher Silman, Kenichiro Otsuka, Takashige Kiyota（Departments of Radiology, Oita University Faculty of Medicine, Oita, Japan）

Objectives: To determine the physiologic uptake of 18F-fluorodeoxyglucose (FDG) in the anal
canal in adults using positron emission tomography fused with computed tomography (PET/CT).
Materials and methods: We conducted a retrospective study for 18F-FDG PET/CT imaging
on patients without symptoms and pathology in the anal region from January 2015 to August
2019. After excluding one patient with Crohn’s disease, one patient with hemorrhoid and twopatients with history of rectal cancer, a final total of 201 patients were included in this study.
FDG uptake were quantitatively and qualitatively evaluated on both early and delayed phases.
Quantitative method was calculated using maximum standardized uptake value (SUVmax)
and was compared to liver SUVmax and distal rectum SUVmax. Qualitative method was
performed with comparison to the background uptake such as perianal surrounding muscles.
Results: The mean of anal canal SUVmax on the early phase was 2.26 (range 1.00-6.30) andthat on the delayed phase was 2.52 (range 1.00-8.80). Early and delayed anal to liver SUVmax
ratio was 0.74 (range 0.24-2.25) and 0.81 (range 0.23-2.32). Furthermore, early and delayed anal
to rectal SUVmax ratio was 0.87 (range 0.30-1.89) and 0.90 (range 0.30-1.27), respectively. The
qualitative method found 22 (10.9%) patients with positive FDG uptake.
Conclusions: The physiologic 18-FDG uptake in the anal canal in adults can be occasionallyhigh in a visual manner. Measuring the anal canal SUVmax and comparing it to rectal uptake(COI:No)might help to differentiate between normal and pathological uptakes. The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

3P-175OU

TRAF6 in T cells exacerbates the severity of experimental autoimmune
encephalomyelitis by up-regulating CCR6 expression
Naganori Kamiyama, Benjawan Saechue, Astri Dewayani, Thanyakorn Chalalai,
Shinya Hidano, Nozomi Sachi, Sotaro Ozaka, Shinpei Ariki, Yasuhiro Soga, Mizuki Goto,
Takashi Kobayashi（Department of Infectious Disease Control, Faculty of Medicine, Oita

University, Oita, Japan）

Multiple sclerosis (MS) is a progressive neurological disease that affects the central nervous
system (CNS). Although many studies have implicated Th17 cells and their downstream
pathways in the pathogenesis of CNS autoimmunity, the pathogenic mechanism is not yet
clear. Experimental autoimmune encephalomyelitis (EAE) is the most commonly used for therodent model of MS. It has been reported that IL-17 produced by Th17 cells is crucial for thedevelopment of both EAE and MS.
TRAF6 transmits the intracellular signal from TLR, thereby regulates innate immune
responses. It has been reported that T cell-specific TRAF6-deficient (CD4-Cre; TRAF6fl/fl)
mice resulted in the increased number of Th17 cell suggesting TRAF6 function in the adaptive immunity. Thus, we sought to better understand TRAF6 function in the development
of EAE. We found that CD4-Cre; TRAF6fl/fl mice were highly resistant to EAE due to thedown-regulation of chemokine receptor CCR6 on Th17 cells. Recently, it has been revealed that
CCL20, the only ligand of CCR6, produced by astrocytes regulates Th17 cell trafficking into
the brain via CCR6 /CCL20 pathway in EAE mice. We confirmed that adoptively transferred
TRAF6-deficient T cells did not induce EAE. The number of infiltrated Th17 cells and thepathological impact of demyelination in the brain were significantly reduced in the mutant
mice suggesting that the migration ability of TRAF6-deficient Th17 cells into the CNS was
impaired. In vitro experiments showed that TRAF6 regulated the promoter activity of CCR6
gene. Taken together, CCR6 in Th17 cells up-regulates by TRAF6 leading to the enhanced
migration of auto reactive T cells into the CNS in EAE mice.
(COI:No)The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S179

Author Index

AUTHOR INDEXAUTHOR INDEX( Boldface indicates presenting author. )A
A LOWRY, Christopher
ABE, Chikara

O02-5
PS04-02
S15-01
1P-077

ABE, Kazuhiro

S13-04

ABE, Manabu

1P-031ABRIEL, Hugues

O11-2
O11-4

ADACHI, Naoki

1P-1032P-005
2P-147
2P-1882P-1893P-110

AL-LAWATI, Intisar

3P-157

AL-MUSHIFRI, Nawaf

3P-157

AL-QANOBI, Ismail

3P-157AMANO, Akira

PS06-04
PS22-02/
AP-4
PS22-04
O20-4
1P-018
1P-019
1P-079
3P-023AMANO, Izuki

S17-01
O02-2
O04-1
2P-100
2P-102ADACHI, Ryota

3P-046ADACHI-AKAHANE, Satomi

O07-5
O12-4

AMANO, Taiju

PS25-01

ANDO, Joji

PS09-02

AFSANA, Islam

3P-158

ANDO, Masatoshi

2P-039AGETSUMA, Masakazu

PS19-05
O17-1

ANDO, Misako

3P-110

ANDO, Soichi

3P-141

AOI, Kosuke

O08-2

AOKI, Ozora

1P-092AONISHI, Toru

2P-186

AONUMA, Kazutaka

1P-023

AOSHIMA, Megumi

AGEYAMA, Naohide

1P-121

AHMED, Rezwana

O02-1

AIZAWA, Hidenori

1P-003AIZAWA, Takanori

3P-007AKAGI, Takumi

2P-0032P-0143P-051AKAIKE, Toru

PS18-01
O17-4
2P-021ASAHARA, Ryota

PS05-01
1P-0142P-023

ASAI, Satoshi

PS24-03

ASANO, Ryotaro

O21-2

ASANO, Shinji

O01-1
O07-1
O09-5
2P-106
2P-1302P-133

ASHIHARA, Eishi

S03-02

ASHIHARA, Takashi

PS22-04

ASHIMORI, Atsushige

O02-1

B
BABA, Kazuhiko

O12-3
1P-076
1P-077
1P-1172P-078
2P-107
3P-096

BABA, Shunsuke

O03-2
O17-4
3P-028

1P-067

BAILEY, Tom G.

S06-03

O16-3

BANDO, Yogetsu

1P-124AOYAMA, Hiroki

1P-125BAOMAR, Hajar

3P-157

AOYAMA, Juria

2P-159BARBARA, Casadei

3P-032OU

2P-165BARIUAN, Jussiaea Valente

3P-151
O06-2

AOTANI, Daisuke

AOYAMA, Shinya

AKAJI, Sakiko

S03-01

ARAI, Yoshiaki

3P-139BARTLETT D., John

AKAMATSU, Motoyuki

1P-023

ARAI, Yoshifumi

3P-069BESSHO, Yasumasa

O02-1

AKAMATSU, Tsubasa

1P-031

ARAI, Yoshiyuki

PS19-05

BLOCK, Gene D.

3P-065

AKAMINE, Yumiko2P-041

ARAI, Yuji

3P-161OUBOCHIMOTO, Hiroki

AKI, Sho

PS23-01
O18-1
2P-119ARAKAWA, Shunta

2P-135

O03-2
O18-4

ARAKE, Masashi

2P-072BOUDAKA, Ammar

3P-157

ARATA, Akiko

1P-043
3P-063
3P-111BRĘBOROWICZ, Andrzej

O16-5

AKIE, Taichi

1P-072

AKITA, Tenpei

3P-048AKITA, Tenpei

2P-043

ARIKAWA, Hajime

2P-177AKIYAMA, Kanae

1P-114

ARIKI, Shinpei

3P-175OUAKIYAMA, Nobutake

2P-176

ARISAWA, Tetsu

AKIYAMA, Tsuyoshi

O21-1
2P-029
3P-0293P-034
3P-131

ARIYANI, Winda

S17-01

ARIYASU, Ryohei

2P-114

ARIZONO, Riku

3P-111

ARULLAMPALAM, Prakash

O11-2

ARULLAMPALM, Prakash

O11-4

ASADA, Keiji

2P-170AKSORNTHONG, Sirion

O10-5

AKUTAGAWA, Masatake

1P-095

AL-AMRI, Zakariya

3P-157

ALEXIOS, Antonopoulos

3P-032OU

ALFULAIJ, Naghum

1P-002S182

C
CARRASCO, Magdeline

2P-167CENTER, Jacqueline R.

3P-129CHAKRABARTY, Basu

S16-01CHALALAI, Thanyakorn

3P-175OU

CHARALAMBOS, Antoniades

3P-032OU

CHAROENPHANDHU, Narattaphol O01-5

O10-5
CHE, I-Shan

1P-008

CHEN, I-Shan

S13-01
3P-017

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

AUTHOR INDEX
CHEN, Jian

O03-4

EBIHARA, Tamehiro

2P-189

FUJITA, Masanori

1P-099CHERIAN, Lijo

O11-4

EGASHIRA, Yoshihiro

2P-045

FUJITA, Mayu

1P-070CHI JUNG, Hung

1P-050

EGUCHI, Akifumi

S17-04

FUJITA, Takayuki

AP-9

CHIBA, Atsushi

2P-155

EGUCHI, Kohgaku

3P-047

FUJITANI, Masashi

CHIBA, Koki

PS17-04
O17-5

EIFUKU, Satoshi

2P-0682P-183
3P-166

EISMAN, John A.

3P-129

FUJITANI, Teruaki

CHIBA, Seiichi

S14-01ELDSTROM, Jodene

PS01-03

O12-3
3P-096CHIKAHISA, Sachiko2P-069
2P-143EMANI MUNASINGHE, Pujika

PS16-02

FUJITA-YOSHIGAKI, Junko

CHIKEN, Satomi

O15-4
O15-5

EMANUELI, Costanza

PS16-02

O06-1
1P-071

EMOTO, Takahiro

1P-095

FUJIWARA, Aki

1P-056ENDO Y., Masako

2P-031

FUJIWARA, Hiroki

O04-5

ENOKI, Ryosuke

2P-049FUJIWARA, Shintaro

2P-038ENOMOTO, Yasushi

O14-1

FUJIWARA, Yasuyuki

1P-037

CHIKEN, Satomi

PS07-01
3P-103
3P-107

FUJIWARA, Yuichiro

S13-03

FUJIYAMA, Fumino

3P-0373P-059FUKADA, Kihachiro

3P-163
3P-171CHIKUDA, Yasutaka

3P-055

ERA, Seiichi

2P-177

CHIMOTO, Sohei

2P-054ESSERS, Maria

O11-2

CHOUDHURY, Emamussalehin

3P-074

ETO, Kei

1P-012CHOUINARD, Julie

2P-041

ETO, Kei

AP-8

CHOWDHURY, Srikanta

1P-050

ETO, Masatoshi

O01-2

FUKADA, Toshiyuki

2P-122CHRISTOS, Kotanidis

3P-032OU

EZURE, Hiromitsu

O05-5

FUKUBA, Yoshiyuki

2P-031

CHU, Zewen

1P-093FUKUDA, Atsuo

CHUJO, Takeshi

3P-121
3P-122

COFFEY, Sean

PS16-02

2P-0402P-043
2P-149
3P-048COLWELL, Christopher S.

3P-065CONWAY-CAMPBELL, Becky

1P-048CONWAY-CAMPBELL, Becky L.

O02-5

COOK J., Douglas

S09-01COOMBES, Jeff S.

S06-03

CUI, Dan

O13-5

CUI, Xiaoke

PS29-05

CUI, Yuanyuan

PS16-03
O11-2

DF
FEDIDA, David

PS01-03

FIELDS, Douglas

3P-138FUKUDA, Keiichi

ML2

FLYNN, Benjamin

1P-048FUKUDA, Masafumi

1P-120

FRY, Christopher

S16-01FUKUDA, Minori

3P-043

FU, Ou

S07-02FUKUDA, Norio

FU, Yinghui

3P-045

FUJIHARA, Kazuyuki

2P-042

PS29-01
PS29-03
O17-3
1P-064

FUJII, Ai

PS10-03

FUKUDA, Satoshi

2P-110

FUJII, Hikaru

2P-146FUKUHARA, Shigetomo

S15-04

FUJII, Noritaka

1P-055FUKUI, Koji

3P-078

FUJII, Satoshi

O04-5

FUKUI, Takuya

1P-124

FUJII, Takuto

PS27-03
2P-007FUKUNAGA, Masaki

O05-1

FUKUNAGA, Ryo

O17-1
O17-2

DATE, Yukari

LS2-1

DAUN, Kenny

FUJII, Yukihiko1P-120

2P-154DAVIS, Philip

FUJIKADO, Takashi

3P-052

PS16-02

FUKUSHI, Isato

O14-3

DEROUICHE, Sandra

FUJIKI, Soichiro

3P-060

1P-010
1P-012

FUKUSHIMA, Akihiro

2P-081

FUJIKURA, Yuko

3P-019FUKUSHIMA, Atsushi

O16-4

FUJIMOTO, Chisato

S15-02

FUKUSHIMA, Teruyuki

3P-033

FUJIMOTO, Tetsuya

3P-124

FUKUSHIMA, Yutaka

1P-017

FUJIMURA, Atsushi

O08-3

FUKUTOMI, Toshiyuki

2P-106

FUJINAMI, Hiroyuki

3P-173OUFUKUZAWA, Miyabi

1P-096

FUJINOKI, Masakatsu

2P-117

FUKUZAWA, Tsuyoshi

FUJIOKA, Atsuko

O19-1
3P-071

1P-0663P-087

FUNABASHI, Toshiya

FUJIOKA, Hitomi

O16-4

O16-4
3P-119

FUJIOKA, Mari

1P-092

FUNAHASHI, Hideki

3P-053

FUJIOKA, Yoichiro

1P-092

FUNAHASHI, Makoto

1P-070

FUJISAKU, Tomoaki

2P-141

FUNATO, Hiromasa

FUJISAWA, Ken

1P-106

O03-5
3P-070FURUICHI, Teiichi

2P-1563P-003DEWAYANI, Astri

3P-175OU

DING, Wei-Guang

2P-013

DIRK F., van Helden

3P-086

DOAN HUU, Tri

1P-0202P-027

DU, Cheng-Kun

3P-029DUC PHAM, Duong

3P-129DWI WAHYU, Indriani

PS07-01
2P-112E
EBIHARA, Kayoko

2P-189

FUJITA, Fumitaka

O11-1

EBIHARA, Nachi

2P-189

FUJITA, Masako

3P-079The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S183

AUTHOR INDEXFURUKAWA, Keiichi

3P-071

HAMADA, Satoru

2P-181FURUKAWA, Takahisa

2P-057

HAMADA, Tsuyoshi

2P-070HASHIMOTO, Ken

1P-015
1P-0243P-021FURUKAWA, Tetsushi

PS17-03

HAMAMURA, Maho

1P-091FURUKAWA, Tomonori

1P-104

HAMID, Sm. Ahasanul

3P-170HASHIMOTO, Masaaki

1P-084HASHIMOTO, Naoki

FURUKAWA, Yasuo

1P-009HAN THIRI, Zin

2P-113

1P-131HASHIMOTO, Satoru

FURUTA, Miyako

O16-4

HANADA, ReikoFURUYA, Kishio

3P-118FURUYASHIKI, Tomoyuki

PS20-02

1P-053
2P-1673P-067
3P-148
3P-154OU
3P-160OU
3P-161OU
3P-162OU

1P-119
3P-172HASHIMOTO, Takayuki

3P-061HASHIMOTO, Takeshi

2P-128HASHIMOTO, Yoshie

2P-051HASHIMOTODANI, Yuki

2P-034HASHITANI, Hikaru

S16-04
1P-062
3P-031
3P-086HASHIUCHI, Emi

PS02-03

HASHIZUME, Nana

3P-076HASHIZUME, Toko

O19-2

HASYASHI, Norito

O05-3

HATAKEYAMA, Hiroyasu

3P-133

HATANAKA, Nobuhiko

O15-5

1P-015
1P-0243P-021HATANO, Kei

3P-164

HATANO, Mai

3P-034
3P-131HANAUE, Mayu

2P-116
2P-169HATANO, Ryo

O01-1
2P-106GGANGI, Mie

2P-0142P-055

GAO, Jingshan

3P-007

GO, Yasuhiro

PS19-04

GODA, Seiji

3P-091

GODA, Yuki

S10-02

GONO, Rina

1P-118
2P-171
2P-172
2P-173

HANADA, Toshikatsu

HANAMURA, Kenji

1P-053
2P-167
3P-148
3P-159OU
3P-160OU
3P-161OU
3P-162OU
3P-173OU
1P-0302P-076GOSHIMA, Makoto

3P-041

GOTO, Ai

PS17-04
O17-5

GOTO, Jun-Ichi

O04-5

GOTO, Katsumasa

1P-067

GOTO, Mizuki

3P-175OU

HAN-THIRI-ZIN

3P-050

HATO, Naohito

3P-158

GOTO, Tsuyoshi

PS04-03

HARA, Megumi

3P-131

HATORI, Yasuhiro

1P-023

GOTOH, Mizuho

2P-153

HARA, Yuma

3P-164

HATSUKANO, Tetsu

2P-074

GOTOH, Yukiko

PS10-01

HARADA, Hidemitsu

O06-2

HATTORI, Atsunori

GOURAUD, Sabine

2P-030
3P-126

HARADA, Kazuo

1P-118
2P-171O05-4
1P-083

HATTORI, Motoyuki

3P-006

GREEN, Daniel J.

S06-03

HARADA, Kazuo

2P-172HATTORI, Noriaki

O08-1

GRYGORCZYK, Ryszard

3P-118

HARADA, Keita

3P-098HATTORI, Satoko

1P-008GRZELAK, Joanna

O16-5

HARADA, Teiji

HAYAMA, Yohsuke

O21-5

GUANYI, Wang

1P-050

1P-118
2P-171

1P-056

S03-03

3P-024

GUO, Shi-yu

HARADA, Yoshinori

HAYAMIZU, Kengo

1P-096

PS12-03

O16-3

HARAGUCHI, Atsushi

HAYASHI K, Yukiko

GUO, Tingting

2P-091

GWAK, Jongseong

1P-023

HARAGUCHI, Shogo

HAYASHI K., Yukiko

1P-046
2P-100
2P-102

HAYASHI, Yukiko

2P-025

HAYASHI, Himeka

3P-064

HAYASHI, Hisaki

3P-156

HAYASHI, Hisayoshi

O19-4
3P-005

HAYASHI, Keisuke

3P-142

HAYASHI, Mikio

1P-122

HAYASHI, Moyuru

1P-013
2P-125H
HADA, Kazumasa

HAGIWARA, Arisa

1P-053
3P-159OU
3P-160OU
3P-162OU
1P-067HAGIWARA-NAGASAWA, Mihoko PS17-04

O17-5
HAIJIMA, Asahi

S17-01
O04-1

HAKOSHIMA, Shuji

2P-190HAKUNO, FumihikoS10-02

HAMA, Noriyuki

2P-183
3P-166S184

HANASHIMA, Akira

HARATA, Daiki

O09-5

HARUWAKA, Koichiro

2P-063

HASE, Eiji

3P-169HASEGAWA, Emi

2P-073

HASEGAWA, Hiroshi

PS28-01

HASEGAWA, Honoka

2P-141

HASEGAWA, Mana

1P-055

HASEGAWA, Taku

HAYASHI, Toshihiro

2P-110

O15-4
3P-107

HAYASHI, Yasushi

1P-106

HAYASHI, Yohei

2P-050HAYASHI, Yuri

3P-169HAYASHI-MORITA, Nana

2P-056

HASEGAWA, Yu

2P-120HASHIMOTO, Akari

2P-063HASHIMOTO, Hirofumi

3P-127HASHIMOTO, Hitoshi

PS19-05

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

AUTHOR INDEXHAZAMA, Akihiro

1P-086
1P-0881P-089
1P-094
2P-118
2P-131
2P-1373P-0193P-115HEMPSTOCK, Wendy

O19-4

HIDA, Hideki

O05-4
1P-083
1P-090HIDAKA, Soh

HIRAI, Yasuharu

3P-0373P-059

HOBO, Kazunao

2P-138
3P-116HIRAI, Yohei

3P-110

HOKAMURA, Kazuya

3P-108HIRAI, Yoshiyuki

1P-070

HOLROYD, Sean

O14-5

HIRAISHI, Keizo

PS16-03
S01-04/AP-7
S18-05
2P-174

HOMMA, Tomoo

2P-127HONDA, Kotaro

2P-060

HONDA, Takeshi

1P-024

HONG, Guang

2P-050

LCS8-1HONJO, Tasuku

PL1

S01-04/AP-7
S18-05
1P-073
2P-128
2P-174

HONJOH, Sakiko

PS11-03

HONKURA, Naoki

O18-5

HONKURA, Naoki

PS23-02

HONMA, Motoyasu

2P-150
3P-140

HORI, Akari

2P-080

HORI, Hirofumi

1P-125

HORI, Miyo

2P-066

HORI, Yuichi

O12-4

HORIE, Kyoji

2P-139HORIE, Minoru

3P-007

HORIE, Mion

1P-131HORIGANE, Shin-ichiro

O14-4

HORII, Kazuhiro

O12-2
2P-126
3P-150HORII, Yuuki

O12-2
3P-150HORIKAWA, Hiroyuki

2P-188HORIKOSHI, Masaru

2P-186HORIKOSHI, Yuko

3P-019HORIO, Shuhei

2P-099HORIUCHI, Joji

1P-061HORIUCHI, Jouji

2P-079
2P-080HORIUCHI, Takatoshi

1P-061

HORTON S., Jaime

1P-002HOSHI, Junko

2P-082
2P-083HOSHI, Kenji

1P-128HOSHINO, Satoshi

2P-185HOSOGI, Shigekuni

S03-02HOSOI, Nobutake

O04-1
3P-003

HIRAKAWA, Kazunori
HIRANO, Katsuya

2P-058HIDANO, Shinya

3P-175OU

HIGA, Ryoko

1P-053
2P-1673P-067
3P-148
3P-154OU
3P-162OUHIRANO, Natsumi

3P-109HIRANO, Tomoo

1P-034HIRANO, Tomoyasu

2P-138HIRANO-IWATA, Ayumi

S04-02HIGASHI, Ryuhei

1P-062HIRAOKA, Chinatsu

3P-022HIGASHIBATA, Akira

O19-2

HIRASHIMA, Masanori

PS23-03HIGASHIJIMA, Shin-ichi

PS12-01

HIRATA, Hiromi

PS12-02HIGASHIKAWA, Asuka

1P-0692P-001
2P-016
2P-097
3P-039
3P-090HIRATA, Yuichiro

1P-118
2P-172HIRAYAMA, Tomoko

1P-125HIRAZAWA, Ki-ichi

PS13-02/AP-3
1P-004

HIRO, Sota

2P-049HIRONO, Moritoshi

1P-029HIROOKA, Rina

1P-096

HIROOKA, Rina

2P-165HIROSE, Jun

O18-3

HIROSE, Kenzo

PS15-01
3P-0382P-089
2P-090HIGASHISAKA, Kazuma

2P-172HIGASHISAKA, Kazuma

1P-118
2P-171HIGASHIYAMA, Tetsuya

3P-037HIGUCHI, Makoto

3P-034HIGUCHI, Sayaka

1P-130HIGUCHI, Takahiro

3P-020

HIKIDA, Takatoshi

PS07-03
O15-3

HIROSHIMA, Reiko

S09-03/AP-1

HIRRLINGER, Johannes

3P-168HIRUMA, Hiromi

3P-117HISADA, Toshiaki

PS29-05

HISADOME, kazunari

1P-070HISAMITSU, Tadashi

1P-056
1P-103
2P-1882P-189HIKOSAKA, Okihide
HIMENO, Yukiko

HIMI, Naoyuki

PS06-04
PS22-02/
AP-4
O20-4
1P-018
1P-079O05-3
2P-168HOSOI, Yasushi

3P-048HISATOME, Ichiro

2P-033HOSOKAWA, Yutaka

1P-105

HINO, Hideitsu

O05-2

HIOKI, Hiroyuki

HISATSUNE, Kazuyoshi

3P-089

2P-186HOSONO, Takayoshi

1P-087

HIRA, Riichiro

HITOMI, Ryosuke

2P-075

3P-138HOSSAIN, Akram

1P-073HIRABA, Katsunari

HITOMI, Suzuro

2P-093

HOSSAIN, Md Emon

PS28-05

HIRABAYASHI, Yusuke

S05-05

1P-0692P-136
3P-092

HOTTA, Harumi

HIRAI, Hirokazu

O04-1
2P-1093P-003

PS04-04
2P-0203P-078
3P-101

HOTTA, Norio

S06-04

HOTTA-HIRASHIMA, Noriko

3P-070HITOSHI, Seiji

2P-154

HIYAMA. Y, Takeshi

2P-053

HO, Quynh Mai

S05-01The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S185

AUTHOR INDEXHU, Yaopeng

S01-04/AP-7
O11-2

IKEDA, Eiji

O13-5

INOUE, Kazuo

2P-146

IKEDA, Hidefumi

2P-129INOUE, Kenichi

PS19-04

IKEDA, Keiko

1P-127
3P-042INOUE, Makoto

PS26-02

INOUE, Masanori

2P-167
3P-159OU

HU, Yuliang

2P-079HUANG, Hai

3P-007

IKEDA, Masaaki

O19-3

HUANG, Honing

3P-113

IKEDA, Miki

2P-110INOUE, Masumi

3P-098

HULSMANN, Swen

3P-168IKEDA, Riko

2P-002INOUE, Ryuji

HUNG, Yung-Li

3P-165

IKEDA, Takayuki

2P-028HYAKUNA, Nobuyuki

2P-181IKEGAME, Shizuka

2P-144HYE-JIN, Lee

3P-152

IKEGAMI, Keisuke

IBUKURO, Momoyo

3P-066

O19-1
3P-071IKEGAYA, Naoki

3P-034

IKEHARA, Toshitaka

1P-095

IKEMOTO, Hideshi

1P-056
1P-1032P-005
2P-1882P-1893P-110

PS16-03
S01-04/AP-7
S18-05
EP1-1
O01-3
O11-2
2P-008
2P-124
2P-174INOUE, Shutaro

1P-031INOUE, Takanori

3P-172INOUE, Tsuyoshi

PS02-02

INOUE, Wataru

PS20-03

INOUE, Yuriko

O05-5

INTHIWONG, Khachanan

O10-4

INUI, Tadashi

PS07-02

INUI, Toshio

O09-5

INUKAI, Youko

O08-4

INUTSUKA, Ayumu

PS25-02

IOANNIS, Akoumianakis

3P-032OU

IRIBE, Gentaro

PS17-01
PS17-02/
AP-6
2P-0243P-030I
ICHI, Yuki

3P-116ICHIKAWA, Hiroyuki

3P-079ICHIKAWA, Jun

2P-008ICHIKAWA, Misaki

1P-061ICHIKI, Arata

2P-151ICHINOHE, Tatsuya

2P-097
3P-039
3P-090IKEMOTO, Mitsushi

3P-156

IKENAKA, Kazuhiro

3P-138

IKENAKA, Yoshinori

S17-04ICHINOSE, Fumito

S02-01

IKEUCHI, Takeshi

1P-097ICHINOSE, Mitsuyuki

1P-107

IKKA, Tsunakuni

PS21-03

ICHINOTSUKA, Yuka

2P-080IKKYU, Aya

3P-070ICHISE, Nobutoshi

O03-1
1P-081IKUMI, Naotaka

3P-005
3P-136

IDA, Masahiro

2P-150
2P-184
3P-140IKUTAME, Daisuke

2P-038IKUTOMO, Masako

3P-043

IMAI, Daiki

O02-3
O21-3

IDA, Takanori

S10-01

IDE, Kaoru

2P-185

IMAI, Ryota

3P-052

IRIE, Shun

2P-114

IDE, Kohki

3P-116

IMAI, Takeshi

O12-5

IRIE, Tomohiko3P-044

IDE, Masakazu

2P-156

IMAI, Toshio

IDE, Ryoji

1P-102
3P-076

1P-102
3P-076

ISA, Tadashi

O12-1
2P-152IMAIZUMI-OHASHI, Yoshie

1P-078
2P-122

ISEKI, Mioko

1P-097

IGARASHI, Hiroyuki

1P-127ISHIBASHI, Kiyoshige

3P-137

IGARASHI, Junsuke

1P-073
2P-128IMAKI, Junko

2P-070ISHIBASHI, Masaru

2P-040IMAMURA, Tomohiko

3P-007IHARA, Kenji

ISHIDA, Akimasa

2P-167
3P-159OUIMAYASU, Mieko

3P-170IHARA, Kensuke

PS17-03

INABA, Yuka

PS02-03

O05-4
1P-083
1P-090

O20-1

ISHIDA, Mari

3P-112

IIDA, Kie

INAGAKI, Masashi

2P-176INAGAKI, Nobuya

O16-3

ISHIDA, Masayoshi

IIDA, Kurumi

1P-100
1P-101INAGAKI, Ryo

1P-111

O13-1
2P-180INAGAKI, Shigenori

O12-5

ISHIDA, Takafumi

3P-112

INASE, Masahiko2P-155
2P-158
3P-105ISHIDA, Tomoaki

2P-022ISHIDA, Yukisato

2P-092
3P-083INOUE, Akio

O05-5

ISHIDA, Yusuke

3P-053INOUE, Akitoshi

O05-5

ISHIDO, Minenori

3P-165INOUE, Hana

O18-2
2P-010ISHIGAMI, Tomoaki

PS18-04

ISHIHARA, Satoru

2P-127

IIMURA, Kaori

PS04-04
3P-101

IINO, Masamitsu

SL53P-001IINUMA, Toshimitsu

2P-038IIZUKA, Makito

1P-126
1P-127IIZUKA, Natsuko

2P-150
2P-184
3P-140INOUE, Hiroshi

PS02-03

ISHIHARA, Takeshi

1P-025INOUE, Hiroshi

3P-091

ISHIHARA, Yo

O18-4

O19-3

ISHII, Daisuke

3P-137INOUE, Ikuo

S186

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

AUTHOR INDEXISHII, Hisayoshi

3P-093
3P-094

ITO, Tsubasa

2P-186

ITO, Yoshiki

2P-186ISHII, Kazuhiro

1P-097

KABA, Hideto

3P-062

ITO-FUJISHIRO, Yasuyo

1P-121ISHII, Kei

PS05-01
1P-014
1P-0232P-023

KABOUCHE, Zahia

2P-147

ITOH, Junko

1P-097

KAEDE, Ryuji

2P-159ITOIGAWA, Masataka

2P-148KAGITANI, Fusako

O10-1

IWAI, Shogo

3P-009KAGIYAMA, Naoko

PS21-01

ISHII, Ken J.

O11-1

IWAKI, Mitsuhiro

PS29-02

KAHLE T., Kristopher

2P-149ISHII, Kuniaki

PS22-02/
AP-4
PS27-02

IWAMOTO, Erika

S06-05

KAI, Risa

2P-168IWAMOTO, Masayuki

PS13-04
2P-017

KAI, Yuko

S12-02ISHII, Masaru

PS02-05

IWASAKI, Masaki

3P-034KAIDA, Kohei

O19-2

ISHII, Risako

3P-109IWASAKI, Shinichi

S15-02

KAIHARA, Keiko2P-024ISHII, Shuya

PS29-01

IWASAKI, Yusaku

PS04-05

KAITSUKA, Taku

O07-4

ISHII, Sumiyasu

O10-3
3P-095IWASE, Satoshi

O08-4

KAIZU, Akihiro

2P-086

IWATA, Hayato

3P-069

KAJI, Hiroshi

2P-0032P-0142P-061
2P-121

IWATA, Koichi

O09-2
2P-0382P-039

S15-03
O13-1
2P-180

KAJI, Toshiyuki

1P-037

KAJIKURI, Junko

PS27-01

KAJIOKA, Shunichi

O01-2
O01-4

KAJIWARA, Chisako

2P-175

KAJIWARA, Kentaro

2P-017KAJIWARA, Riichi

3P-143

KAJIYA, Hiroshi

O06-2
O18-3
2P-048ISHII, Toshiyuki

ISHII, Yuki

2P-133

ISHIKAWA, Junko

3P-144
3P-145ISHIKAWA, Taisuke

PS06-02
3P-007

ISHIKAWA, Yoshihiro

PS14-03
S11-02
S11-03AP-9
3P-012

IWATA, Ryo

O12-5

IWATA, Shusuke

2P-064IZAKI, Tsubasa

1P-0232P-023K

IZUHARA, Luna

1P-130IZUMI, Ayana

1P-100
1P-101IZUMI, Satoshi

2P-050IZUMI-NAKASEKO, Hiroko

O17-5

KAKINOUCHI, Kei

2P-137IZUMI-NAKASEKO, Hiroko

PS17-04

KAKINUMA, YoshihikoS12-02
O19-3

ISHIMARU, Kazuhiro

O20-3
2P-119IZUMIZAKI, Masahiko

ISHITANI, Tohru

2P-150
3P-108

3P-159OU

KAKITA, Akiyoshi

1P-120ISHIWATA, Ryo

2P-179IZUMIZAKI, Masahiko

KAKIZAKI, Miyo

3P-070ISHIWATA, Shinichi

PS29-01
PS29-03
O17-3

1P-126
1P-127
2P-184
3P-140
3P-167

KAMATA, Satomi

2P-016

KAMBAYASHI, Ryuichi

PS17-04
O17-5

ISHIWATA, Yoshinobu

3P-034

1P-056

KAMEDA, Hiroshi

2P-110ISHIZAKI, Motoki

2P-097KAMEYAMA, Masaki

2P-015ISHIZUKA, Mayumi

S17-04KAMEYAMA, Naomitsu

2P-044

ISHIZUKA, Noriko

O19-4
3P-005KAMIJO, Keiju

3P-053

KAMIKUBO, Yuji

2P-051

ISHIZUKA, Toshiaki

2P-072KAMINO, Maho

1P-091

ISLAM, Mohammed Moinul

S05-02

KAMITORI, Kazuyo

S13-03

ISOMURA, Yoshikazu

O06-3

KAMIYA, Haruyuki

S04-04

ISOO, Noriko

2P-110KAMIYAMA, Naganori

3P-175OUKANAI, Anthony

S16-01KANAYAMA, Kiichi

1P-121

KANAZAWA, Meiko3P-172KANDA, Takeshi

O05-2

KANDA, Yasunari

O17-5

KANEDA, Makoto

2P-0032P-0142P-0552P-061
2P-121
3P-051

ITO, Chihiro

2P-148

ITO, Hiroki

S16-03

ITO, Hiromu

1P-131

ITO, Kaho

3P-149

ITO, Koichi

S10-02

ITO, Masanori

O07-5
O12-4

ITO, Megumi

1P-024

ITO, Minako

SL6

ITO, Tomoyuki

O14-2

IZUNO, Takuji

J
J WILLIAMS, Michael

PS16-02

JAHAN, Israt

3P-046JENSEN, Lars Jørn

PS16-04

JIN, HuilingPS16-01

JINNO, Naoya

3P-080JINNO, Yuka

PS13-03

JITSUKI, Susumu

1P-035/
AP-2
3P-131JITSUKI-TAKAHASHI, Aoi

1P-035/AP-2JUCHACZ, Aldona

O16-5

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S187

AUTHOR INDEX
KANEHISA, Kota

O07-2

KANEKO S., Yoko

2P-047
2P-103
2P-123

KATO, Daisuke

2P-063
3P-138

KATO, Fumihiro

2P-104

KATO, Fusao

O06-3
3P-066KAWANABE, Akira

PS13-03

KAWAO, Naoyuki

S15-03
O13-1
2P-180KANEKO, Kenya

O04-5

KAWASAKI, Makoto

O12-3

KANEKO, Chiyuki

2P-058

KATO, Hisanori

S10-02KAWASAKI, Makoto

3P-096KANEKO, Noe

PS10-03

KATO, Ikuo

O09-5

KAWASE, Toshihiro

3P-060KANEKO, Satoru

2P-118

KATO, issei

KAWASHIMA, Kaito

O02-4

KANEKO, Shuji

PS24-04

O02-4
3P-128KAWATA, Miho

2P-047
2P-103
2P-123

KANEKO, Tatsuroh

1P-052

KATO, Kan

KANEKO, Toshiyuki

3P-088

KATO, Mami

KANEMARU, Kazunori

3P-001KANEMURA, Yohei

1P-129KANIKOWSKA, Dominika

O16-5
2P-142KANNO, Emi

2P-082
2P-083KANNO, Satomi

3P-070

KANO, Masanobu

1P-031
2P-036KANOSUE, Kazuyuki

3P-106KANSAKU, Kenji

2P-056
2P-156
3P-060

2P-164
1P-1032P-0052P-006KAZAMA, Itsuro3P-130KEITH, Channon

3P-032OU

KATO, Megumi

2P-025KENNY, Paul J.

SL3

KATO, Ryota

2P-075KERSHAW, Yvonne

1P-048

KATO, Shigeki

O06-3
2P-099KERSHAW, Yvonne M.

O02-5

KHAIRINISA A., Miski

S17-01KHATUN, Ziasmin

1P-094
2P-131

KATO, Shiro

1P-019

KATO, Takeshi

2P-105

KATO, Toshiki

3P-061KHONGSOMBAT, Onrawee

O10-4

KATO, Yuko

AP-9

KIDA, Hiroyuki

KATSUDA, Shin-ichiro

3P-0192P-113
3P-049

KATSUMATA-KATO, Osamu

O06-1
1P-071

KIDA, Satoshi

PS20-04

KIJIMA, Chiaki

2P-181KANZAKI, Makoto

3P-133KAWABATA, Masanori

O06-3

KIKUCHI, Kiyoshi

2P-044KARAKI, Shin-Ichiro

2P-127
3P-134
3P-135KAWABATA-SAKATA, Yukika

2P-091KIKUCHI, Yui

2P-141

KAWADA, Toru

O20-1
O21-1
O21-5

KIKUSUI, Takefumi

3P-142KIKUTA, Kohei

1P-028KIKUTA, Kouhei

3P-061KIKUTA, Mari

1P-037KIKUTA, Satomi

1P-111KIKUYAMA, Sakae

1P-046

KAREMAN, Eljamal

O01-2

KARUBE, Fuyuki

3P-037KAWAGOE, Kentaro

O01-2

KASAGI, Satoshi

PS03-03

KAWAGUCHI, Kotoku

KASAI, Atsushi

PS19-05

KASAI, Haruo

ML3
PS15-03

O01-1
2P-106
2P-130KAWAGUCHI, Kotoku

O07-1

KIM J, Staci

3P-070

O12-1

KAWAGUCHI, Noriaki

O14-4

KIM, JeongHo

3P-152

O11-2

KAWAGUCHI, Shin-ya

1P-034

KIM, Nan Hee

3P-129KASHIMA, Hideaki

2P-031

KAWAHARA, Genri

KIM, Samyoung

3P-170KASHIO, Makiko

O11-3

PS12-03
2P-091

KAWAHARA, Katsumasa

KIMISHIMA, Rio

3P-009

3P-100

KAWAHARA, Michinari

KIM-MITSUYAMA, Shokei

2P-120

1P-131KIMOTO, Kenichi

3P-160OU

KASAI, Masatoshi
KASCHITZA, Daniela Ross

KASHIWA, Asami

O20-4
3P-007KASHIWADANI, Hideki

1P-052

KAWAHARA, Takuhiro

2P-176KASHIWAGI, Sayaka

1P-092

KAWAI, Eriko

KIMOTO, Misaki

1P-015
3P-021

KASUYA, Go

PS13-01

O02-3
O21-3

KAWAI, Kazutake

KIMOTO, Yuichirou

3P-081

3P-106KAWAI, Takafumi

KIMURA, Akira

3P-026

O09-1
1P-005KIMURA, Fumitaka

3P-058KAWAI, Yoshiko

1P-013
2P-125KIMURA, Hayata

1P-131KIMURA, Ikuo

KAWAJI, HIdeya

PS10-03

3P-134
3P-135KAWAKAMI, Junko

1P-128KIMURA, Kai

2P-070KAWAKAMI, Kiyoshi

3P-042KIMURA, Kazuhiro

3P-151KIMURA, Kazuhiro

2P-159
2P-162
2P-164KATADA, Ryuichi

1P-118
2P-171
2P-172KATAGIRI, Chiaki

O07-4

KATAKURA, Akira

2P-001KATAKURA, Masanori

3P-102KATAOKA, Naoya

PS20-01
1P-051KATARE, Rajesh

PS16-02

KAWAKAMI, Tomohisa

1P-018KATAYAMA, Yoko

3P-040

KAWAMATA, Tomohiro

3P-115

KATO, Aoi

1P-081

KAWAMURA, Kenta

3P-137

KIMURA, kenichi

PS09-04

KAWAMURA, Teruhisa

2P-022KIMURA, Koudai

O19-2

S188

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

AUTHOR INDEXKIMURA, Maki

1P-0692P-001
2P-016
2P-097
3P-039
3P-090

KOBAYASHI, Shiori

1P-054

KOKETSU, Shinnosuke

2P-037KOBAYASHI, Suguru

2P-035

KOKUBO, Michifumi

O04-1

KOBAYASHI, Takashi

3P-161OU
3P-175OU

KOKUDAI, Yumika

2P-150

KOMATSU, Masatoshi

O13-3

KOBAYASHI, Takeshi

O03-1

KOMATSU, Nana

2P-035KIMURA, Masaki

3P-109KOBAYASHI, Takuya

KOMINE, Hidehiko

KIMURA, Saeko

2P-1462P-0102P-017

KIMURA, Takeshi

O20-4
3P-007KOBIRUMAKI, Fuyu

1P-064PS05-01
1P-0232P-023

KOMIYAMA, Tomoyoshi

2P-114

KOMURA, Yutaka

3P-143

KIMURA, Toru

2P-106

KIMURA, Yuichi

3P-034KINNNO, Ryuta

3P-140KINOSHITA, Kengo

S13-02KINOUCHI, Yohsuke

1P-095KISHI, Hiroko

S18-01
S18-05
O13-4
O13-5

KOBIRUMAKI-SHIMOZAWA, Fuyu
PS29-01

PS29-03

KOMURO, Issei

ML1

KONDO, Hidekazu

3P-032OU

KONDO, Hiroko X.

S13-02KONDO, Masahiro

2P-138KONDO, Satoru

1P-028KONDO, Yasuhiko1P-085
1P-1142P-0772P-0702P-071
3P-064KONDO, Yoshitsugu

2P-069KONDOH, Daisuke

O18-4

KODA, Shigeya

O20-4

KODAMA, Aya

1P-015
1P-0243P-021KODANI, Yu

KOEDA, Shuhei

2P-047
2P-103
2P-123KISHI, Yusuke

PS10-01KISHIDA, Kota

1P-019KISHIMOTO, Mana

2P-077

KOGA, Yuriko

3P-056KISHIMOTO, Toshifumi

O18-2

1P-049KOGAMI, Sayaka

KONISHI, Masato

KITA, Rika

KONISHI, Ryoji

1P-073

3P-097

1P-035/AP-2

KITAGAWA, Hiroaki

2P-154

KONNO, Ayumu

2P-109KITAGAWA, Hiromu

1P-113

KONNO, Kohtarou

1P-008KITAJIMA, Ken

O19-1

KONNO, Yoshihiro

PS27-02

KITAJIMA, Yoichiro

1P-102
3P-076

3P-119KONO, Jin

S16-02

O20-4
3P-007

KONO, Maori

2P-129KOPPENSTEINER, Peter

3P-047KOHNO, Yutaka

3P-137KORYBALSKA, Katarzyna

O16-5

KOIBUCHI, Noriyuki

S17-01
S17-02
O02-2
O04-1
O10-3
2P-100
2P-101
2P-102
3P-095KOSAKA, Hiroaki

2P-128KOSHIYA, Naohiro

3P-166KOTANI, Sayumi

3P-167KOYAMA, Hiroyuki

O16-3

KOYAMA, Natsu

2P-154KOYAMA, Yoshimasa

3P-069

KUBA, Hiroshi

PS08-03
O04-4
3P-046

KUBO, Asako

2P-062

KUBO, Yoshihiro

PS13-02/AP-3
S13-01
1P-004
1P-007
1P-0083P-016
3P-017

KUBOTA, Satomi

3P-140KUBOTA, Satomi

2P-150
2P-184

KUBOTA, Shin

2P-138
3P-116KOGANEZAWA, Tadachika

KOHDA, Kazuhisa
KOHJITANI, Hirohiko

1P-0202P-027
2P-182KITANO, Kouhei

3P-036KITAURA, Hiroki

1P-120KITAZAKI, Satoshi

1P-023

KITO, Hiroaki

PS27-01

KIYAMA, Yuko

2P-014KIYOTA, Kyoko

2P-167KIYOTA, Takashige

3P-174OU

KOBA, Satoshi

O10-2
1P-058KOBASHI, Motoi

3P-079KOIDE, Tsuyoshi

3P-142KOBAYASHI, Daisuke

1P-086
1P-094
2P-131
3P-115

KOIE, Hiroshi

1P-121

KOINUMA, Satoshi

3P-071

KOIWA, Nobuyoshi

2P-150
2P-184
3P-140KOIZUMI, Hidehiko3P-166KOIZUMI, Teruki

3P-034

KOJIMA, Akiko

1P-0172P-013

KOBAYASHI, Kazuto

KOBAYASHI, Kenta

O06-3
1P-127
2P-099

2P-186
3P-103
3P-107KOBAYASHI, Masayuki

3P-036

KOJIMA, Mizuyo

2P-025KOBAYASHI, Midori

2P-186

KOJIMA, Nobuhiko

KOBAYASHI, Nao

3P-009

2P-0752P-076KOBAYASHI, Ruka

3P-011

KOJIMA, Shuichi

2P-165KOBAYASHI, Sei

S18-01
S18-05
O13-4
O13-5

KOJIMA, Tomoyuki

S11-03

KOJITANI, Hirohiko

KUBOTA, Tetsuo

AP-9

PS06-04

KOKABU, Shoichiro

KUBOTA, Tomoya

PS01-01

2P-136

KUBOTA, Toshiaki

3P-160OU

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S189

AUTHOR INDEXKUBOTA, Yasuo

1P-073

KUBOTA, Yoshiyuki

PS19-03

KUBOTA, Yukihiko2P-104

KUDA, Yuichi

PS22-04
2P-0282P-033

KUROSAWA, Mieko

1P-059
2P-066

LIU, Yanqing

1P-093

LIU, Yan-qing

1P-056KUROSE, Masayuki

1P-055

LOMBER G., Stephen

S09-01

KUSAKARI, Yoichiro

S05-03O03-2
1P-022

LOUCH, William E.

PS29-03

LU, Qian

O13-4
O13-5

LYU, Bochao

S18-01
O13-4
O13-5

2P-050KUSANO, Akinaga

1P-131

KUDO, Yuuki

3P-080

KUSE, Yoshiki

O19-2

KUMADA, Nao

O10-2

KUSHI, Hidehiko

KUMADA, Tatsuro

3P-108

3P-163
3P-171KUMAGAI, Ryoko

3P-064

KUSUBE, Fumiya

2P-060

KUMAKI, Tatsuya

2P-080KUSUHARA, Koichi

1P-117

KUMAMOTO, Yasuaki

S03-02
S03-04

KUSUMOTO-YOSHIDA, Ikue

S07-03

KUSUNOKI, Susumu

O19-1

KUWAHARA, Atsukazu

O09-5

KUWAHARA, Yuko

O09-5

KUWAKI, Tomoyuki

S07-03
1P-052

KUDO, Tada-aki

KUME, Shinichiro

3P-013
3P-0143P-015
3P-018M
MA, Jihao

S07-03

MACHIDA, Shuichi

3P-165

MACHIDA, Yoko

3P-143

MACHINO, Takeshi

1P-023

MACPHERSON, Tom

PS07-03

MAEJIMA, Daisuke

2P-125KUNICHIKA, Tsumoto

2P-028KUWAYAMA, Naohiro

PS10-01

MAEJIMA, Sho

3P-099KUNIEDA, Yusuke

2P-1882P-189

KWAK, Shin

2P-110

MAEJIMA, Takashi

2P-073

MAESHIBA, Munehisa

2P-048KUNII, Naoto

3P-034KUNIMATSU, Jun

S09-03/AP-1

LAWRENCE CHEUNG, Dennis

AP-8

KUNISUE, Tatsuya

S17-04

LEE, Eunyoung

2P-106

KUNO, Koji

2P-119

LEE, Hye-Jin

3P-153

KUNORI, Nobuo

1P-044
2P-108

LEE, Jeong Hoon

S05-01

LEE, JeongBeom

MAKI, Tomoko

O01-2

MAKITA, Naomasa

PS06-02
3P-007

MAKIYAMA, Takeru

PS06-03
O20-4
3P-007

3P-152

MANABE, Ryo

2P-184

3P-153

MANNING, Patrick

PS16-02
S12-02

L

KURACHI, Yoshihisa

PS22-04

KURAHARA H., Lin

PS16-03

KURAHARA, Lin-Hai

LEE, Ming-Liang

2P-162

MANO, Asuka

2P-174

3P-138

O05-5

S01-04/AP-7
S18-05

LEE, Philip

MANOME, Yoshinobu

KURAHARA, Rin

LEE, Seung Ku

3P-129MARUI, Shuri

LEEM, Chae Hun

S05-01
3P-129O02-4
3P-128
3P-149LEONARD S, Christopher

2P-040MARUNAKA, Yoshinori

LI, Jia

PS29-03

LI, Meihua

O20-1
O21-1

S03-01
S03-02
EP1-2
O09-5
2P-1302P-133

KURASAWA, Kentaro

S11-03

KURATA, Yasutaka

PS22-04
2P-0282P-033
3P-023
3P-081

LEE, Jeong-Beom

LI, Nan

2P-0173P-001

O13-4
O13-5

MARUO, Syunta

1P-087

LI, Tianbang

2P-012MARUYAMA, Akiko

1P-124KURIHARA, Fumiya

O19-4

LI, Yulong

SL4

MARUYAMA, Emi

O05-3

KURIHARA, Kinji

1P-068LIANG, Nan

2P-041

2P-082
2P-083

KURISAWA, Yutarou

1P-107

PS05-01
1P-014
3P-104MARUYAMA, Ryoko

KURIMA, Kiyoto

MARUYAMA, Satoshi

1P-099KURISU, Ryoko

O09-2

LIGHTMAN, Stafford

1P-048MARUYAMA, Takashi

LIGHTMAN, Stafford L

O02-5

LIN, Tzer-Bin

O21-4

LIU, Chang

3P-017

LIU, Dongyu

2P-042

LIU, Libin

O09-4

LIU, Yangong

3P-013
3P-0143P-015
3P-018

LCS7-1
1P-075
1P-076
1P-077
1P-1172P-078
2P-107
3P-096MARUYAMA, Takuma

2P-014
2P-121MARUYAMA, Yoshio

3P-011KUREBAYASHI, Nagomi

KURODA, Anri

2P-154

KURODA, Hidetaka

3P-039KUROIWA, Yoshinori

2P-186

KUROKAWA, Tatsuki

1P-002
3P-013
3P-0143P-015
3P-018KUROSAKA, Mitsutoshi

S190

3P-119The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

AUTHOR INDEXMARUYAMAI, Toru

LCS4-1

MATSUNO, Naoto

O18-4

MIMURA, Koki

PS19-04

MASAMIZU, Yoshito

PS19-01
3P-138

MATSUO, Megumi

O19-2

MIN, Young-Ki

MATSUO, Osamu

2P-180

3P-152
3P-153

MASAMOTO, Kazuto

2P-020

MATSUOKA, Hidetada

3P-098MINAMI, Yoichi

3P-071MASAOKA, Yuri

2P-150
2P-184
3P-140

MATSUOKA, Satoshi

S05-02
1P-021

MINAMIKAWA, Takeo

3P-169MINAMISAWA, Susumu

PS18-01
O03-2
O13-2
O17-4
1P-022
2P-021
3P-028MINOBE, Etsuko

2P-015MINOKOSHI, Yasuhiko

O16-3

MINOURA, Taro

1P-060

MITA, Kenichiro

O09-3

MITANI, Shohei

1P-011
1P-130

MASUBUCHI, Satoru

O11-3
O19-1

MASUDA, Shinnosuke

2P-101

MASUDA, Takahito

1P-085MASUDA, Tomoyuki

1P-097MASUDA, Yuta

PS28-04
O02-4
3P-128MASUDA, Yuzuka

2P-145MASUMOTO, Gen

S13-02MASUZAKI, Hiroaki

O16-3

MATSUBARA, Chie

2P-186

MATSUBARA, Takanori

O14-4

MATSUBARA, Yuri

2P-190

MATSUDA, Hiroki

2P-104

MATSUDA, Kazunari

3P-092

MATSUDA, Kazuto

2P-0703P-064

MATSUI, Sho

S07-04

MATSUI, Takuya

2P-148
3P-156

MATSUI, Teppei

3P-061

MATSUKAWA, Kanji

PS05-01
1P-014

MATSUOKA, Toshio

2P-177

MATSUSHIMA, Aya

O06-2

MATSUSHIMA, Ayami

S17-03

MATSUSHITA, Junya

2P-186

MATSUSHITA, Masayuki

O07-4
2P-181

MATSUURA, Hiroshi

O17-1
O17-2
1P-0172P-0132P-026

MATSUURA, Takanori

O12-3
3P-096

MITO, Kohei

MATSUYAMA, Mio

1P-061
2P-079
2P-080

3P-093
3P-094

MITOH, Yoshihiro3P-079MITSUDO, Kenji

3P-012

MATSUZAKA, Yoshiya

1P-128

MITSUI, Retsu

MATSUZAKI, Kentaro

PS28-05

S16-043P-086

MATSUZAKI, Masanori

PS19-01
3P-138

MITSUISHI, Hiroki

2P-067

MITSUSHIMA, Dai

2P-113
3P-041
3P-049
3P-050
3P-144
3P-145

MAURISSEN, Thomas

3P-007MD JUNAYED, Nayeem

3P-155

ME, Choudhury

O07-2

MECK, Warren

S09-05

MI, Xinya

1P-0172P-013

MIURA, Kenichiro

2P-057

MIURA, Kohei

2P-031MIAZUKAWA, Hazuki

S17-04

MIWA, Naofumi

MIDORIKAWA, Mitsuharu

1P-033

2P-116
2P-169MIDORIKAWA, Ryosuke

2P-046MIWAKEICHI, Fumikazu

3P-168
S08-04

MATSUKAWA, Noriyuki

3P-138

MATSUKI, Yuka

S11-05

MATSUMOTO, Akio

PS17-04
O17-5

MATSUMOTO, Amiri

3P-104

MIEDA, Michihiro

2P-073

MIYAGAWA, Shinichi

1P-090MIENO, Kana

2P-185MIYAGI, Etsuko

S11-03

S01-03
2P-092
3P-085MIYAHARA, Yu

3P-053MIYAKAWA, Tsuyoshi

1P-008MIYAKE, Masao

1P-086
1P-0892P-118MIYAKODA, Mayuko

3P-142MIYAMOTO, Akiko

2P-063MIYAMOTO, Manabu

2P-090MIYAMOTO, Osamu

O05-3
2P-168MIYAMOTO, Takeshi

O18-3

MIYAMOTO, Tetsu

1P-075
2P-107MIYAMOTO, Yasunori

3P-126MIYAMURA, Yuichi

2P-095MIYANISHI, Kazuya

S12-01
O07-2
3P-074

MATSUMOTO, Hidetoshi
MATSUMOTO, Hiroshi

1P-118
2P-171
2P-172

MATSUMOTO, Jumpei

PS19-04
1P-109

MATSUMOTO, Kenichiro

2P-136

MATSUMOTO, kohjiro

2P-187

MATSUMOTO, Mami

O16-3

MATSUMOTO, Masaki

3P-159OU

MATSUMOTO, Masayuki

1P-0202P-027

MATSUMOTO, Narihisa

2P-157

MATSUMOTO, Shunro

3P-174OU

MATSUMOTO, Yoshihiro

3P-109

MATSUMURA, Shigenobu

2P-146

MATSUNAGA, Masahiro

1P-046

MATSUNAGA, Mayumi

3P-090

MATSUNAGA, Yoshihiro

O11-5

MIHASHI, Satoko

MIKAMI, Kanta

O07-2

MIKAMI, Misaki

1P-085
1P-1142P-077MIKAMI, Tamaki

1P-114

MIKAMI, Yoshihiro

1P-126MIKAMI, Yoshinori

O07-5
O12-4

MIKI, Kenju

2P-144
2P-145

MIKI, Takafumi

O11-5

MIKI, Takashi

2P-106MIKOSHIBA, Katsuhiko

O04-5

MIMA, Akira

3P-114

MIMATA, Hiromitu

3P-173OU

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S191

AUTHOR INDEXMIYASHITA, Yohei

MIYATA, Mariko

1P-118
2P-171
2P-172
EP3-2
1P-033
1P-036
3P-040
3P-137MIYAWAKI, Atsushi

2P-186MIYAZAKI, Aiko

1P-040MIYAZAKI, Hiroaki

2P-134MIYAZAKI, Mitsue

2P-101MIYAZAKI, Seiya

2P-022MIYAZAKI, Shota

MORI, Shihomi

O06-2

MURAKAMI, Akikazu

1P-030

MORI, Shun

2P-077

MURAKAMI, Shingo

3P-026

MORI, Yasuo

O11-5
1P-002

MURAKAMI, Tomonari

3P-061

MURAKOSHI, Hideji

1P-090

MORI, Yoshiaki

2P-089
2P-090

MURAMATSU, Hiroki

3P-102

MORI, Yume

1P-100
1P-101

MURAMATSU, Hiroyuki

3P-155

MURAMATSU, Ken

3P-043

MURAMOTO, Kazuyo

S12-02MURAO, Yuzuki

2P-035

MURATA, Akira

2P-158MURATA, Koshi

PS03-02

MORIDERA, Aiko

2P-065MORIGUCHI, Takashi

1P-013

MORIMOTO, Keiko

1P-100
1P-101
2P-166

MURATA, Soichiro

O19-2

3P-065

MORIMOTO, Marina

2P-031

MURATA, Yoshihiro

MIYAZAKI, Takehiro

O05-2

MORIMOTO, Sachio

1P-041
3P-062MIYAZAKI, Tomoyuki

3P-034
3P-131

3P-0243P-029MURATA, Yoshimichi

3P-011

MORIMOTO, Sachio

3P-028MURATA, Yukie

MORIMOTO, Takeshi

3P-052

MORIMOTO, Yuicihi

PS15-03

1P-118
2P-171
2P-172

MORIMOTO, Yuji

2P-072
2P-179
3P-054

MURAYAMA, Hanako

3P-142

MURAYAMA, Masanori

2P-186

MURAYAMA, Takashi

MORISAKI, Ikuko

3P-148
3P-161OU

2P-0102P-0173P-001MORISHITA, Hirofumi

1P-049MURRELL-LAGNADO, Ruth

3P-017MORISHITA, Masahiro

2P-099MUSHIAKE, Hajime

2P-042MORISHITA, Saho

3P-108MORITA, Emiko

O02-3
O21-3
1P-125MIYAZAKI, Wataru

S17-01
S17-02
O02-2
O04-1
3P-095MIYOSHI, Chika

3P-070MIYOSHI, Tomomitsu

3P-052MIZOGUCHI, Hiroyuki

PS07-03

MIZOGUCHI, Naoko

S12-02MIZOGUCHI, Toshihide

1P-069MIZUGUCHI, Motohiro

2P-022MIZUHIKI, Takashi

2P-157MIZUKAMI, Yuri

2P-166MIZUMOTO, Ryo

2P-010MIZUMURA, Kazue

1P-042
2P-062MIZUNO, Masaki

S06-04

MIZUSAWA, Kanta

PS03-03

MIZUTANI, Kohta

O15-3

MORITA, Hironobu

MORITA, Kazunori

PS04-02
PS29-04
S15-01
S15-03
1P-077

2P-155
3P-105MIZUTANI, Natsuki

1P-006

MORITA, Masato

1P-034MOCHIZUKI, Kaname

3P-069MORITA, Tomoka

MOCHIZUKI, Kei

2P-158MOCHIZUKI, Naoki

S15-04

S18-01
O13-4
O13-5

MOGI, Kazutaka

3P-142

MOHAMED, Elfeky

2P-159MOHRI, Satoshi

1P-015
1P-0243P-021

NABEKURA, Junichi

PS19-05
AP-8
O17-1
1P-012
3P-138NABESHIMA, Syota

2P-007

NADIA, Akawi

3P-032OUNAGAI, Kazunari

3P-142NAGAI, Nobuhiro

3P-034NAGAI, Sayoko

2P-001

NAGAI, Takeharu

PS19-05
1P-003

MOTEGI, Shunichi

O08-1

NAGAKURA, Yukari

MOTOJIMA, Yasuhito

1P-123
3P-096

1P-102
3P-076NAGANO, Mamoru

O19-1
3P-071NAGASAKA, Kazuaki

PS05-03
2P-153NAGASAKI, Hiroshi

2P-047
2P-103
2P-123NAGASAWA, Yoshinobu

PS22-02/
AP-4

NAGASHIMA, Kei

PS28-04
EP2-1
O02-4
3P-128
3P-149NAGATA, Haruka

S10-02

MUANGKRAM, Yuttamol

1P-063

MUKAI, Daichi

3P-006MOMIYAMA, Toshihiko

O04-2

MUKAI, Noriko

1P-131MOMOSE-SATO, Yoko

1P-026
1P-027

MUKAI, Ryuichiro

3P-022

MUKAI, Yasutaka

MOMOTANI, Ko

S18-02

O15-3
1P-003MONTANI, Jean-Pierre

O20-5

MUKAI, Yuri

MOORHOUSE, Andrew

3P-138

2P-138
3P-116

MOORHOUSE, Andrew John

AP-8

MUKOHDA, Masashi

S18-04MORI X., Masayuki

O01-3
2P-015

MUNESUE, Yoshiko

1P-121

MURABE, Naoyuki

2P-110

MORI, Miyu

1P-131

MURAI, Yoshinaka

2P-044

S192

N

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

AUTHOR INDEXNAGATA, Osamu

S11-05NAKAMURA, Kei-ichiro

1P-062

NAMIKI, Shigeyuki

3P-038NAGATA, Sayaka

S10-05NAKAMURA, Kogenta

3P-155NAMOTO, Yurie

2P-133

NAGAYA, Kazuki

2P-159NAKAMURA, Madoka

3P-142

NAMURA, Saki

2P-031

NAING YE, Aung

PS22-02/
AP-4

NAKAMURA, Mariko

2P-181NANIWA, Mako

3P-092NAKAMURA, Masanori

1P-024NANJO, Kazuki

2P-132NAITO T., Atsuhiko

O17-5

NAKAMURA, Nozomu

O05-1

NARITA, Hirotaka

PS13-03

NAITO, Hisashi

2P-060

NAKAMURA, Ryosuke

1P-086

NARITA, Kazuhiko2P-168NAITO, Narumi

PS22-04

NAKAMURA, Takahiro J.

3P-065

NARUSE, Keiji

NAITO, Tomoyuki

3P-052NAKAMURA, Takashi

S11-03NAITO, Yuki

S14-02NAKAMURA, Tomomi

3P-114

NAITOU, Kiyotada

2P-084NAKAMURA, Wataru

NAKADA, Tomoaki

1P-046

PS26-01
3P-065

PS17-02/
AP-6
2P-0243P-030NARUSE, Keiji

O03-4

NAKADA, Tsutomu

O13-3

NAKAMURA, Yoshiko

2P-140

NASU, Teruaki

2P-062

NAKAGAWA, Atsushi

PS13-03

NAKAMURA, Yukihiro

S04-01

NATSUME, Rie

1P-008NAKAGAWA, Hideyuki

3P-022NAKAMURA-MARUYAMA, Emi 2P-168

NAYEEM M., Junayed

3P-156NAKAGAWA, Masataka

2P-141NAKAMURA-NISHITANI., Tomoe O16 -1

NEGORO, Hiromitsu

S16-02

NAKAGAWA, Taku

2P-052NAKANISHI, Koichi

2P-181NEMOTO, Takahiro

S10-03

NAKAHARA, Naoya

2P-092
3P-083
3P-084NAKANISHI, Miho

1P-039NEMOTO, Tomomi

2P-049NAKANISHI, Tomohiro

1P-064

NEPAL, Prabha

1P-092NAKANO, Takanari

O19-3

NGO, Jennifer

NAKANO, Takashi

O14-4
O15-3

PS16-01
S11-01NGUYEN, Tuan V.

3P-129NI, Tengyang

1P-093NIHONMATSU, Akira

1P-080NIIMI, Naoko

PS18-03
O19-5

NIIYAMA, Tetsushi

O08-2

NIIZEKI, Kyuichi

1P-132NIKAIDO, Yoshikazu

1P-104

NIN, Fumiaki

PS08-01

NINOIMYA, Yuzo

PS26-03

NINOMIYA, Ayane

O04-1

NINOMIYA, Masaru

2P-190NISHI, Hiroki

S10-02

NAKAHARA, Shiro

PS22-03

NAKAHARI, Takashi

O07-1
2P-1302P-133

NAKAHASHI, Mutsumi

1P-095NAKAHATA, Yasukazu

O02-1

NAKAI, Junichi

2P-186NAKAJI, Keita

2P-154NAKAJIMA, Katsumi

3P-105NAKAJIMA, Kazunori

PS10-04

NAKAJIMA, Shinichiro

3P-034NAKAJIMA, Shizuka

3P-080NAKAJIMA, Tsuyoshi

2P-114NAKAJIMA, Waki

3P-034
3P-131NAKAJO, Koichi

PS01-02
2P-011NAKANO, Tamami

2P-151NAKAO, Akito

O11-5

NAKAO, Kanako

3P-009NAKAO, Kazuwa

O16-3

NAKAO, Shu

O16-1
2P-022NAKASHIMA, Ai

PS11-01

NAKASHIMA, Akiko

3P-0043P-057NAKASHIMA, Akira

2P-047
2P-103
2P-123NAKASHIMA, Kie

3P-057NISHIDA, Daisuke

1P-069NAKASHIMA, Noriyuki

2P-052
3P-0043P-057NISHIDA, Iori

1P-131NISHIDA, Motohiro

O03-3/AP-5
O17-1

NAKAKAJI, Rina

3P-012NAKAMA, Kentaro

2P-172NAKATA, Mariko

2P-153

S10-04NAKATA, Masanori

3P-097NISHIDA, Takashi

2P-146NAKAMORI, Hiroyuki

2P-084NAKAYA, Yuka

3P-036NISHIJO, Hisao

NAKAMURA, Hiroaki

O06-5

NAKAYAMA, Haruka

2P-166NAKAMURA, Katsuki

PS21-02

NAKAYAMA, Hisako

1P-036

PS19-04
1P-042
1P-109NAKAMURA, Kayo

AP-8

NAKAYAMA, Masanori

PS23-04

NISHIKAWA, Kyoko

2P-141NAKAMURA, Kazuaki

2P-070NAKAYAMA, Shinsuke

NISHIKAWA, Takuya

NAKAMURA, Kazuhiko1P-085
1P-114
2P-190

O01-2
O01-4

O21-1
O21-5

NAKAYAMA, Shunya

1P-121NISHIMARU, Hiroshi

1P-109

3P-053NISHIMORI, Toshikazu

3P-053NALAYAMA, Shouta

S17-04NISHIMOTO, Rei

1P-012NAMBU, Atsushi

PS07-01
O15-4
O15-5
1P-111
3P-103
3P-107NISHIMURA, Akiyuki

O03-3/
AP-5

NAKAMACHI, Tomoya

NAKAMURA, Kazuhiro

NAKAMURA, Kazuomi

PS20-01
PS28-03
1P-051
1P-0602P-081
2P-163

1P-112NAKAYAMA-NAONO, Rumi

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S193

AUTHOR INDEXNISHIMURA, Haruki

NISHIMURA, Kazuaki

O12-3
1P-076
1P-077
1P-1172P-078
2P-107
3P-096

1P-076
1P-077
1P-1172P-078
3P-096NISHIMURA, Kazuaki

2P-107NISHIMURA, Natsumi

1P-094
2P-131NISHIMURA, Yuhei

PS24-02NISHIMURA, Yukio

3P-106NISHIOKA, Naritomo

O03-2
1P-022NISHIYAMA, Kazuhiro

O03-3/AP-5

NISHIZAWA, Kazuhisa

PS13-03

NISHIZAWA, Manami

PS13-03

O
O18-4

OBARA, Yutaro

PS27-02

OHTANI, Naoko

O06-5

PS29-04

OHTSUKA, Hiroyuki

2P-114

OCHI, Hiroki

S15-04OHTSUKA, Naruhito

O05-5

ODAGAWA, Maya

2P-186OHTSUKA, Sachiko

2P-092

2P-186OHTSUKA, Yoshinori

3P-164OHTSUKI, Gen

O04-3

OHTUBO, Yoshitaka

1P-040

OHYA, Susumu

PS27-01

OHYAMA, Sadao

1P-069
2P-097
3P-039

OBATA, Koji

ODE, Takahiro
OFUSA, Wataru

1P-0692P-016
3P-039
3P-090

OGATA, Yoshiki

1P-104

OGAWA, Haruo

2P-0173P-001OIKAWA, Shino

S12-02

OGAWA, Shino

1P-083

OIKE, Jun

OGAWA, Sonoko

2P-074

2P-1882P-189

OGAWA, Yoshihiro

PS02-01

OIKI, Shigetoshi

OGINO, Yoshihiro

S11-05

PS01-04
O09-3

OGIWARA, Ikuo

2P-0143P-0103P-051OISHI, Yumiko

LS2-2

OISI, Yasuhiro

2P-186OIZUMI, Masafumi

2P-186

1P-079NIWA, Masatoshi

3P-043

OGOH, Shigehiko

NIWA, Sayaka

2P-032

NODA, Mami

O08-2

NODA, Masaharu

2P-053

NODA, Tsukika

2P-141NOGAMI, Akihiko1P-023

NOGUCHI, Jun

PS15-03

NOGUCHI, Takuya

2P-050

NOMA, Akinori

SL2
PS06-04
PS22-02/
AP-4
O20-4
1P-079
2P-026

O21-2

OJIDA, Akio

2P-045

S06-02
S06-03
3P-141

OKA, Shogo

2P-046

OKABE, Akihito

1P-054

OKABE, Koji

O06-2
O18-3
2P-048

OGUCHI, Katsuji

3P-001

OGURA, Yuji

3P-119OH, Kwan Ee

2P-101

OKABE, Naohiko

2P-168OHASHI, Yusuke

3P-110

OKABE, Taiki3P-011

OHBA, Yusuke

1P-092

OKADA, Fumihiro

O11-1

OHFUSA, Wataru

2P-097

OKADA, Hiroyuki

O18-3

OHHASHI, Toshio

2P-125

OKADA, Jun-ichi

PS29-05

OHINATA, Kousaku

2P-146

OKADA, Kiyotaka

OHIO, Ken-ichi

2P-155

O13-1
2P-180OHIRA, Momoko

1P-015
3P-021

OKADA, Naoko

3P-143

OKADA, Ryo

O01-3

OHKI, Kenichi

1P-0283P-061

OKADA, Toshiaki

3P-002

OKADA, Yasumasa

O14-3
3P-166

OKADA, Yasunobu

1P-017
1P-074
2P-1243P-002

OKADA, Yosuke

1P-076

OKAMATSU-OGURA, Yuko

3P-151

OKAMATSU-OGURA, Yuko

2P-159OKAMOTO, Fujio

O06-2
O18-3

OKAMOTO, Keiichiro

1P-055

OKAMOTO, Motoi

1P-025

OKAMOTO, Satoshi

O19-2

OKAMOTO, Takahiro

2P-180OKAMOTO, Yasuo

O20-3
2P-119NOMIYAMA, Kei

S17-04NOMOTO, Kensaku

3P-142

NOMURA, Kanako

OHKI, Yukari

3P-150

2P-114

NOMURA, Kengo

1P-039
2P-053

OHKURA, Masamichi

2P-186OHMORI, Harunori

3P-057

NOMURA, Rei

3P-099OHMOTO, Makoto

S07-01NOMURA, Yuka

3P-080

OHMURA, Sayo

O01-3

NONOGUCHI, Hiroshi

3P-100OHNISHI, Hideo

3P-096NONOMURA, Satoshi

O06-3

OHNO, Jun

2P-048NOROTA, Ikuo

PS27-02

OHNO, Seiko

NOZAWA, Hana

1P-059PS22-05
3P-007NUMAGA-TOMITA, Takuro

2P-009OHNO-SHOSAKU, Takako

2P-141

NUMANO, Rika

2P-187OHNUKI, Yoshiki

PS18-02
O20-2

NUMATA, Tomohiro

1P-074
2P-1243P-002OHSHIMA, Daisuke

O07-5
O12-4

S194

2P-113

OBARA, Hiromichi

NISHIZUKA, Taiki

PS17-04
O17-5

PS10-03OHTAKU, Hiroyuki

OGO, Takeshi

NUNOI, Yoshio

OHTAKA-MARUYAMA, Chiaki

OHSHIRO, Tomokazu

2P-042

OHTA, Hiroyuki

2P-072The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

AUTHOR INDEXOKAMOTO, Yosuke

OKAMURA, Yasushi

PS06-04
PS22-02/
AP-4
3P-025
PS13-03
PS14-01
O09-1
1P-005
1P-0062P-018

ONO, Kota

2P-099PAUL, Richard

2P-092

ONO, Kyoichi

PS06-04
PS22-02/
AP-4
3P-025

PAW-MIN, Thein-Oo

3P-041

PAW-MIN-THEIN-OO

3P-049PEARSON, James

PS16-01
S11-01
O16-1
O21-1
2P-029
3P-029PEARSON, James T

3P-020PEARSON, James T.

O21-2

PENNINGER, Josef M

PL2

ONO, Munenori

O15-1

ONO, Riho

3P-138

ONO, Taketoshi

1P-109ONOUCHI, Sawa

2P-126

OKANO, Kie

1P-054

OOFUSA, Wataru

2P-001

OKAZAKI, Kazunobu

O14-1

OOKA, Hirotaka

OKAZAKI, Kazunobu

O02-3
O21-3

1P-043
3P-063OOTSUKA, Sachiko2P-087

PHAM T., Linh

3P-126

OKAZAKI, Minako

2P-182

2P-001POLAT K., Onur

O01-3

POLYAKOVA, Zlata

O15-5

PUPPULIN, Leonardo

S03-02OOYAMA, Sadao

OKAZAKI, Tomonori

O07-1
2P-133

ORTUSTE QUIROGA P., Huascar Pedro

OKE, Yoshihiko3P-168ORZECHOWSKA, Zofia

O16-5

OKOCHI, Yoshifumi

2P-018OSAKI, Hironobu

3P-137

OKU, Yoshitaka

O05-1
3P-168OSANAI, Makoto

1P-111OKUBO, Masaki

3P-143OSATO, Ayako

1P-085

OKUMO, Takayuki

1P-103
2P-188OSAWA, Kohei

3P-012OSHIMA, Tomomi

3P-100OKUMURA, Kazuki

1P-018OSHIMA-TAKAGO, Tomoko

3P-038

RAI, Dilip

2P-014

OKUMURA, Naoya

3P-080OSHIRO, Naomi

3P-043RAJA AHMAD, Raja Norazireen

3P-121

OKUMURA, Satoshi

PS18-02
O20-2

OSHITA, Kensuke

2P-052RAMADHANI, Ratna

OTA, Akemi

O02-3

3P-093
3P-094

OKUMURA, Yu

2P-170

OTA, Keisuke

2P-186RAVSHAN Z, Sabirov

1P-017

OKUTANI, Fumino

1P-041OTANI, Lila

S10-02RAWAL, Shruti

PS16-02OMATA, Daisuke

1P-054OTSUGURO, Ken-ichi

1P-091REYES-PRIETO, Nidia M.

O02-5

OMATA, Yasunori

O18-3

OTSUJI, Yutaka

3P-086

OMATSU-KANBE, Mariko

O17-1
O17-2

1P-075
2P-107

RICHARD J., LangRICHMOND J., Barry

2P-157OTSUKA, Kenichiro

3P-174OU

RIOS, Alain

O06-3

1P-086OTSUKA, Tsuyoshi

2P-067RODRAT, Mayuree

O01-5
O11-4

OMORI, Koichi

1P-067

Q
QUAN, Linlang

3P-164ROMORI, Toshiaki

S04-03OTSUKA-SAITO, Kaori

LCS3-2ROUGIER, Jean-Sébastien

ONAKA, Tatsushi

PS25-02
1P-117
3P-096

OTSURU, Naofumi

PS05-04

RUEDIGER, Stefanie

S06-03OUCHI, Kanae

1P-089

RUTKOWSKI, Rafał

O16-5

O-UCHI, Jin

S05-04

1P-127
3P-167

OUE, Anna

2P-031

OYA, Manami

2P-163OYAMA, Kotaro

PS29-01

OYAMA, Sadao

2P-016
3P-090ONIMARU, Hiroshi
ONISHI, Hideaki

PS05-04

ONISHI, Hiroki

2P-067

ONO, Daisuke

1P-050

ONO, Fumihito

PS12-04
1P-0652P-011
2P-0452P-085

ONO, Katsushige

ONO, Kenjiro
ONO, Kentaro

3P-013
3P-0143P-015
3P-018
2P-150
3P-140
1P-0692P-136
3P-092OYAMADA, Hideto

3P-001OZAKA, Sotaro

3P-175OUPSSACHI, Nozomi

3P-175OUSACHU, Riga

1P-109SADAKATA, Tetsushi

3P-003

SADAMOTO, Hisayo

2P-035SADATO, Norihiro

PS21-04

SADOSHIMA, Junichi

1P-016SAECHUE, Benjawan

3P-175OUSAGASAKI, Shota

1P-131

PAI, Chungyu

1P-121SAHARA, Naruhiko

1P-097PANUPINTHU, Nattapon

O10-5

SAHO, Kosuke

PARK, Tae-Hwan

3P-152
3P-153

O02-3
O21-3

SAIGOH, Kazumasa

O19-1

PATUMRAJ, Suthiluk

3P-120SAIKI, Akiko

O06-3

PAUDEL, Bidur

2P-115SAIKI, Chikako

PAUDEL, Sarad

1P-092

1P-102
3P-076The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S195

AUTHOR INDEXSAIRENJI, James Taku

2P-101

SAKOORI, Kazuto

1P-031

SAITO, Daichi

2P-130

SAKU, Keita

S11-04

SAITO, Hidetsugu

3P-109SAKUMA, Miho

1P-131

SAITO, Junichi

S11-03

SAITO, Kaori

O11-1

SAITO, Manabu

1P-085
1P-114
2P-190

SATO, Motohiko3P-155
3P-156

SATO, Nana

1P-102

SATO, Rina

1P-018SAKURAI, Hiroyuki

PS27-04
2P-106

SATO, Ryo

3P-145SAKURAI, Katsuyasu

S09-05

SATO, Seiich

3P-007SAKURAI, Masaki

2P-110

SATO, Seika

2P-101SAKURAI, Takashi

2P-051
3P-001

SATO, Shingo

S15-04

SATO, Shinichi

3P-025

SAITO, Manami

2P-159SAITO, Reiko

1P-117SAKURAI, Takeshi

S02-02

SATO, Shuhei

1P-096

SAITO, Ryohei

2P-169SANADA, Kenya

3P-096

SATO, Tadasu

3P-079SAITO, Shotaro

3P-141

SANADA, Kenya

SATO, Tatsuya

SAITO, Shouichiro

2P-126

O03-1
1P-081

SAITO, Toshiro

1P-016

SATO, Toshiya

SAITO, Toshiyuki

2P-175

3P-093
3P-094

SAITO, Yoshimasa

3P-109

1P-075
1P-076
1P-077
1P-1172P-078
2P-107

SATO, Yuichi

3P-100

SAITOH, Tadashi

1P-132

SANADA, Tomoyoshi

3P-158

SATO, Yuma

SAITOW, Fumihito

3P-035SANECHIKA, Akari

1P-019

1P-0202P-027SAKABA, Takeshi

PS15-02
2P-034SANEFUJI, Jun

1P-070

SATOH, Aya O.

1P-092SANEMATSU, Keisuke

PS26-03
2P-064

SATOH, Hiromasa

2P-116
2P-169SATOH, Kaori

2P-122

SATOH, Keitaro

2P-122

SATOH, Yoshihide

1P-082
1P-124
2P-111SATO-HASHIMOTO, Miho

S02-03

SATO-NUMATA, Kaori

1P-074
2P-1243P-002SAKAGAMI, Nami

1P-125SAKAI, Akinori

O12-3
1P-123
3P-096SANGO, Kazunori

PS18-03
O19-5

SANKAI, Tadashi

1P-121SAKAI, Chiemi

3P-112

SANO, Hideto

O18-5

SAKAI, Hideki

PS27-03
2P-007

SANO, Hiromi

PS07-01
3P-103SAKAI, Kenji

S14-01

SANO, Kazuhiro

2P-074SAKAI, Koh

2P-113

SANO, Yoshitake

2P-156SAKAI, KumikoS18-02SARAN SINHA, Adya

3P-048SAKAI, KumikoS14-01SATORI, Kuniaki

2P-072

SARKER, Md Azadul Kabir

2P-119SAKAI, Yutaka

O06-3

SATOU, Chihiro

O19-1

SASAGURI, Toshiyuki

O01-2

SAKAMOTO, Hirokazu

3P-038SATOU, Motohiko

O08-4

SASAJIMA, Hitoshi

1P-092SAKAMOTO, Hirotaka

PS25-04
3P-099SAWABE, Mai

2P-076

SASAKI, Keisuke

2P-165SAWADA, Yasuhiro

S15-04

SASAKI, Ryo

2P-152

SAWAGASHIRA, Ryo

1P-108SASAKI, Satoshi

2P-020

SAWAMOTO, Kazunobu

O16-3

SASAKI, Tsutomu

PS25-03

SCOTT H., Stephen

S09-01SASANO, Tetsuo

PS17-03

SEGAWA, Banri

1P-030SASAOKA, Toshikuni

1P-111

SEI, Hiroyoshi

SATAKE, ShinIchiro

3P-042

2P-069
2P-143SATAKE, Shota

1P-115

SEKI, Akiko

PS06-02

SATO, Aya

3P-027

SEKI, Hideaki

SATO, Chihiro

1P-085
1P-1142P-077

1P-072
1P-115
1P-116SEKINO, Sachie

3P-040

SATO, Issei

PS19-05

SENA, Yankel

3P-174OU

SATO, Izumi

2P-160

SEO, Eriko

1P-078

SATO, Jun

EP2-3

SEO, Yoshiteru

SATO, Katsushige

1P-026
1P-027

1P-078
2P-122
3P-127

SATO, Kenjiro

2P-075SETOGAWA, Tsuyoshi

2P-157

SHEN, Dan

3P-045

SHEN, Qin

PS10-02

SHI, Sheng-Lan

3P-122SAKAMOTO, Issei

O05-3

SAKAMOTO, Momoko

1P-037SAKAMOTO, Takeshi

1P-131SAKAMOTO, Tatsuya

3P-099SAKAMOTO, Yui

2P-190

SAKATA, Souhei

PS13-03
1P-0652P-085SAKHRI, Fatma Zahra

2P-147
3P-110

SAKIMOTO, Yuya

3P-041
3P-049
3P-050

SAKIMURA, Kenji

1P-008
1P-031

SAKIMURA, Kenji

2P-036

SAKO, Hidenori

LCS6-1

SAKO, Noritaka
SAKO, Noritaka

2P-096

SATO, Maki

2P-161
SATO, Makoto

S196

O16-5
2P-142
PS27-02

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

AUTHOR INDEXSHIBAKAWA, Ryo

3P-109SHIMIZU, Mayuko

3P-169SHIRAI, Nanae

O14-1

SHIBAMOTO, Toshishige

2P-028SHIMIZU, Noriyuki

SHIRAISHI, Hiroshi

SHIBANO, Kiwamu

1P-041

2P-069
2P-143SHIBASAKI, Koji

3P-003SHIMIZU, Takahiro

SHIBASAKI, Manabu

PS28-02

PS27-03
2P-007SHIBATA, Hideshi

1P-059
2P-066SHIMIZU, Takeshi

SHIRAISHI, Koichi

O05-5

SHIBATA, Mikihiro

3P-006

O05-4
1P-083
1P-090

1P-053
3P-159OU
3P-161OU
3P-162OU
3P-173OUSHIRAISHI, Mitsuya

2P-084SHIBATA, Shigeki

S06-01SHIMIZU, Tsuyoshi

3P-019SHIRAISHI, Takuya

1P-002SHIBATA, Shigenobu

EP1-3
1P-096
2P-165SHIMIZU, Yasutake

O12-2
2P-084
2P-126
3P-150SHIRAISHI, Yasunaga

1P-099SHIRAKAWA, Hideki

2P-135SHIRAO, Tomoaki

1P-0302P-076SHIROI, Rie

1P-100
1P-101SHIROTA, Matsuyuki

S13-02SHIUCHI, Tetsuya

PS03-04
2P-069
2P-143

SHUTO, Tsuyoshi

S14-03

SILMAN, Christopher

3P-174OU

SMALL-HOWARD L., Andrea

1P-002

SODA, Akinobu

3P-067SHIBATA, Tomohiro

PS19-04

SHIBATA, Toshiyuki

2P-148SHIBUKAWA, Yoshiyuki

1P-0692P-001
2P-016
2P-097
3P-039
3P-090SHIBUTANI, Minori

2P-175

SHICHITA, Takashi

PS02-04

SHIDARA, Munetaka

2P-157SHIDE, Masahito

3P-162OUSHIDO, Osamu

PS28-05

SHIGEMI, Kenji

S11-05SHIGEMOTO, Ryuichi
SHIGEMURA, Noriatsu

3P-047
PS26-03
EP3-1
2P-064

SHIGEYOSHI, Yasufumi

O19-1
3P-071

SHIH, Tien Lin

3P-167SHIINA, Takahiko

O12-2
2P-084
2P-126
3P-150SHIKANO, Kenshiro

PS03-01
1P-053
2P-1673P-067
3P-148
3P-154OU
3P-162OUSHIMODA, Kakeru

O03-3/AP-5
O17-1

SHIMODA, Takeru

2P-165SHIMOHATA, Atsushi

2P-0143P-051SHIMOJU, Rie

1P-059
2P-066SHIMOKAWA, Noriaki

2P-101SHIMOMI, Yui

2P-071
3P-064SHIMOMURA, Takushi

PS13-02/AP-3
1P-004

SOEDA, Shuichi

1P-0663P-087

SOGA, Yasuhiro

3P-175OUSHIMOUCHI, Akito

3P-080

SOGO, Takahiro

2P-022SHIMOYAMA, Shuji

1P-104

SOHARA, Eisei

3P-048SHIMOZAWA, Togo

PS29-03

SOHMA, Hikaru

3P-009SHIMUTA, Toru

3P-125

SOHMA, Yoshiro

3P-009

SHIN, Chol

3P-129SOKABE, Masahiro

3P-118

SHIN, Masashi

O06-2
O18-3

SOKABE, Takaaki

S08-02

SHIN, Toshitaka

SOMA, Shogo

O06-3

3P-173OUSHINBARA, Hisashi

SONOBE, Takashi

PS09-03

SHINJO, Satoko

O17-4

SHINODA, Masamichi

PS26-04
O09-2
2P-0382P-039PS16-01
S11-01
O21-1
2P-029
3P-020

SONODA, Masaki

3P-034

SONODA, Satomi

1P-076
1P-077
1P-1172P-078
2P-107
3P-096

SOTOMARU, Ryota

3P-041STOKES J., Alexander

1P-002

SU, Shaoyi

1P-070

SUDA, Machiko2P-100

SUDA, Yuki

3P-146

SUDO, Shunichi

O18-3

SUEHIRO, Yuji

1P-130SUEMITSU, Shunsuke

1P-025SUGAMA, Shuei

S12-04SUGAMA, Shuei

S12-02SHINODA, Yo

1P-037
2P-156SHINODA, Yoshikazu

O06-4

SHINOHARA, Kazuyuki

O02-1
2P-165SHIKAYAMA, Takemi

3P-092SHIMA, Yuta

1P-051SHIMA, Yuta

PS20-01

SHINOHARA, Yoshiaki

2P-037SHIMADA, Masahiko

O09-2

SHINOTSUKA, Takanori

PS19-01

SHIMAMOTO, Takao

PS22-04
3P-023SHINTANI A., Seine

O17-3

SHIOMI, Toshiaki

2P-142

O12-2
3P-150SHIOYA, Takao

3P-082SHIRAI, Kanon

2P-007SHIRAI, Koji

3P-019SHIRAI, Manabu

S11-01SHIRAI, Mikiyasu

O21-2
3P-029SHIMAOKA, Hiroki
SHIMIZU O., Chigusa
SHIMIZU, Chigusa

O07-4

1P-054SHIMIZU, Daisuke

PS03-03

SHIMIZU, Hirofumi

2P-017

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S197

AUTHOR INDEXSUGANAMI, Takayoshi

PS02-01SUZUKI, Honoka

1P-031

TAKAHASHI, Kentaro

PS17-03SUGAWARA, Yuka

1P-097SUZUKI, Junji

3P-001

TAKAHASHI, Makoto

SUGAYA, Yuki

2P-036SUZUKI, Kazuya

3P-141

1P-106
3P-068SUGIBAYASHI, Kenji

3P-102SUZUKI, Makoto

2P-030

TAKAHASHI, Mayu

O06-4

SUGIHARA, Miki

3P-142SUZUKI, Michiaki

S09-02

TAKAHASHI, Mutsumi

1P-124SUZUKI, Nao

1P-061

TAKAHASHI, Naohiko

SUZUKI, Shinya

2P-114LCS5-1
LCS9-1SUZUKI, Susumu

3P-155

TAKAHASHI, Nobutaka

2P-109SUZUKI, Takashi

3P-105

TAKAHASHI, Noriko

SUZUKI, Takayuki

2P-186

PS15-03
3P-100SUZUKI, Tatsuya

3P-069TAKAHASHI, Rie

3P-156

TAKAHASHI, Shinichiro

1P-106
3P-068SUGIMACHI, Masaru

O20-1
O21-1
O21-5

SUGIMORI, Michiya

3P-132SUGIMOTO, Kana

1P-118
2P-171
2P-172SUGIMOTO, Shunichi

1P-005SUZUKI, Yoshiro

O11-1

SUGIMOTO, Syunji

1P-105SUZUKI, Yuko

O18-5

TAKAHASHI, Shin-Ichiro

S10-02SUGIMURA K., Yae

O06-3
3P-066SUZUKI, Yuta

O02-3
O21-3

TAKAHASHI, Shu

1P-126

TAKAHASHI, Susumu

2P-185

TAKAHASHI, Takuya

1P-035/AP-2
3P-034
3P-131SUGIO, Shouta

3P-138SUGITA, Masaaki

1P-124SUGITA, Shukei

1P-024SUGITA, Yuko

2P-057SUGITANI, Kayo

3P-075SUGIURA, Akihiro

1P-098SUGIURA, Seiryo

PS29-05

SUGIYAMA, Atsushi

PS17-04
O17-5

T
TABIRA, Kazuyuki

1P-125TABUCHI, Eri

2P-034TACHIBANA, Masao

TAKAHASHI, Tatsuji

2P-072

3P-038TACHIBANA, Yoshihisa

TAKAHASHI, Teppei

2P-177

PS07-04
2P-063
3P-138
S09-04

TAKAHASHI, Toshimitsu

3P-060

TAKAHASHI, Tsutomu

1P-037

TAKAHASHI, Yu

1P-025

O19-2

TAKAHASHI, Yukari

O06-3
3P-066

TAKAI, Akira

PS17-02/AP-6
S01-01
3P-0303P-088TAKAI, Jun

1P-013

TAKAI, Shingo

2P-064

TAKAISHI, Masayuki

O11-1
2P-129TAKAKI, Miyako

PS29-04

TAKAKI, Miyako

2P-170

TAKAKU, Shizuka

PS18-03
O19-5

TAKAKUWA, Norihiro

2P-152

TAKAMATA, Akira

1P-100
1P-101
2P-166

TAKAMATSU, Ken

2P-116

TAKAMIYA, Kogo

2P-046

TAKAMORI, Kenji

2P-060

TAKAMURA, Yusaku

1P-109TAKANAMI, Keiko3P-099TAKANARI, Hiroki

1P-119TAKANARI, Hiroki

1P-129
3P-169
3P-172SUGIYAMA, Haruko

3P-140TADOKORO, Tomomi

SUGIYAMA, Saemi

2P-127TAEPAVARAPRUK, Pornnarin

O10-4

SUGO, Yoshinobu

S11-03TAGASHIRA, Iori

1P-009SUITA, Kenji

PS18-02
O20-2

TAGUCHI, Toru

1P-042TAGURI, Masataka

3P-034SUMIKAMA, Takashi

O09-3

TAHARA, Taiki

1P-079SUMINO, Ayumi

3P-006TAHARA, Yu

1P-047
2P-165
3P-065

SUN, Xinru

2P-082
2P-083SUNAGAWA A., Genshiro

S02-05SUNAGAWA, Masanori

2P-181SUNAGAWA, Masataka

PS09-01
1P-056
1P-093
1P-1032P-0052P-006
2P-147
2P-1882P-1893P-110SUWABE, Takeshi

2P-096
2P-161

TAJIRI, Naoki

O05-4
1P-083
1P-090

TAKADA, Naoki

O06-5

TAKADA, Yuuki

3P-034

TAKAFUJI, Yoshimasa

O13-1
2P-180TAKAGI, Daichi

PS22-02/
AP-4

TAKAGO, Hideki

PS08-02
3P-038TAKAHASHI, Aki

PS11-02

TAKAHASHI, Akira

1P-095SUZUKI, Akane

1P-013

TAKAHASHI, Akiyoshi

PS03-03

SUZUKI, Ayaka

O01-3

TAKAHASHI, Hirokazu

PS08-04

SUZUKI, Etsuko

O04-2

TAKAHASHI, Hiroki

2P-009SUZUKI, Harue

PS04-04
3P-078TAKAHASHI, Kazuhiro

3P-007

TAKAHASHI, Kazumi

2P-068SUZUKI, Hidenori

3P-035

TAKAHASHI, Ken

O03-4

TAKANO, Haruko

S15-04SUZUKI, Hitoshi

O12-3
3P-096

TAKAHASHI, Kengo

1P-096
2P-165TAKANO, Hiromichi

3P-031

S198

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

AUTHOR INDEXTAKANO, Kouji
TAKANO, Makoto

2P-056
2P-156
1P-062
2P-052
3P-0043P-057

TAKANO, Yu

S13-02

TAKARADA, Yu

2P-072

TAKASE, Minoru

1P-046

TAKASHIMA, Ichiro

1P-044
2P-108
2P-153

TAKASHINA, Terue

3P-163
3P-171

TAKUMI, Toru

3P-035TANAKA, Yoshiya

1P-076TAKUWA, Noriko

PS23-01
O18-1
O20-3
2P-119TANAKA, Yudai

3P-078TANDAI-HIRUMA, Megumi

1P-099TANI, Hideaki

3P-034TANIDA, Mamoru

PS04-01
PS22-04
2P-0282P-033
3P-081TANIGAMI, Hayate

2P-141TANIGUCHI, Hideki

O19-2

TANIGUCHI, Junichi

1P-079TANIGUCHI, Kentaro

3P-080TANIGUCHI, Mutsuo

3P-062TANIGUCHI, Takamichi

1P-059TANIHATA, Jun

O03-2
O13-2
3P-028TAKUWA, Yoh

PS23-01
O18-1
O20-3
2P-119TAMAKI, Akira

3P-111TAMAKI, Tetsuro1P-0663P-087TAMAKI, Yuki

2P-152TAMAMAKI, Nobuaki

3P-138TAKASUGI, Masaki

O06-5

TAMAOKA, Akira

1P-097TAKATA, Maki

1P-073

TAMAYOSE, Chisaki

2P-181TAKATSURU, Yusuke

O08-1
2P-102TAMIYA, Kazuki

3P-141

TAMURA, Atsushi

2P-041TAKATSURU, Yusuke

2P-100TAMURA, Hiroshi

3P-058TAKAYAMA, Chitoshi

O07-4
1P-054TANIHIRA, Hiroki

1P-111

TAMURA, Konomi

1P-096TANIMOTO, Reina

3P-158TAMURA, Ryota

2P-108TANIOKA, Daisuke

TAN, Ju Jing

3P-1182P-069
2P-143

TANAKA F., Kenji

1P-003

TANJI, Kunikazu

2P-077TANAKA H., Yasuyo

PS19-01

TANNO, Hiromasa

TANAKA R., Yasuhiro

PS19-01
PS19-05

2P-082
2P-083TANOUE, Rumi

S17-04

TAKAYAMA, Mikiko

2P-106

TAKAYAMA, Yasunori

1P-056
1P-1032P-005
2P-1893P-110

TAKAYAMA, Yasunori

2P-006TANAKA, Eiichiro

2P-044TARUNO, Akiyuki

TAKAYANAGI, Hiroshi

SL1

TANAKA, Hideo

1P-038
1P-039TAKAYANAGI, Yuki

PS25-02

S03-02
S03-03TASHIRO, Akimasa

3P-054

TANAKA, Junichi

1P-106
3P-068TASHIRO, Michiko

O18-2

TATEYAMA, Michihro

1P-007

O07-2
O07-3
2P-171
3P-073
3P-074
3P-123
3P-158TATSUMI, Kohei

O13-1

TAURA, Akiko

3P-057TAZOE, Toshiki

3P-106

TEI, Yuto

O12-4

TERADA, Jiro

O14-3

TERADA, Tomoyoshi

2P-177TERAI, Chifumi

2P-085TERAMOTO, Sayaka

2P-110TERAO, Akira

2P-159TERASHIMA, Kazuya

2P-127TERASHIMA, Yoshinori

O03-1
1P-081

TAKEDA, Atsushi

S14-04

TAKEDA, Haruna

O07-2

TAKEDA, Ikuko

AP-8

TAKEDA, Kotaro

O14-3

TAKEDA, Kotaro

2P-183

TAKEDA, Ryosuke

O02-3

TAKEDA, Yukari

1P-021

TAKEDA, Yuriko

2P-011TAKEI, Gen

2P-115

TAKEI, Tomohiko

S09-01

TAKEMORI, Shigeru

2P-176
2P-178TANAKA, Junya

TANAKA, Kentaro

1P-076
1P-077
1P-1172P-078
2P-107TANAKA, Kentarou

3P-096TANAKA, Kunihiko1P-098－TANAKA, Masaki

1P-108TERUI, Ai

2P-190O14-4

TANAKA, Michiko

EP3-3

TAKENAKA, AsakoTERUI, Takako

1P-064

S10-02TANAKA, Miyako

PS02-01

TAKENAKA, Yasuhiro

TIYASATKULKOVIT, Wacharaporn

O19-3

TANAKA, Risa

1P-131TAKENOYA, Fumiko1P-084

TANAKA, Sakae

TAKEO, Shin

1P-022TANAKA, Tomohiro

TAKESHIMA, Hiroshi

PS27-03

TAKETO, Megumi

1P-032TAKEUCHI, Ayako

S05-02TAKEYA, Kosuke

S01-02TAKEYA, Mitsue

1P-062TAKEMOTO, Yumi

1P-057

TAKEMOTO-KIMURA, Sayaka

O10-5

O18-3

TODA, Chitoku

2P-164

O03-3/
AP-5

TODA, Chitoku

2P-162

TODA, Haruna

2P-016TANAKA, Tomohiro

EP2-2
O16-3

TODA, Haruna

TANAKA, Yasuhiro

3P-138TANAKA, Yasuyo

3P-138

1P-0692P-001
2P-097
3P-039
3P-090The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S199

AUTHOR INDEXTODA, Koji

S09-05TOHSE, Noritsugu

O03-1
1P-081TOKIZAWA, Ken

3P-125TOKUDA, Masaaki

1P-073

TOMIDA, Taichiro

O07-5
O12-4

TOMIDOKORO, Yasushi

1P-097TOMINAGA, Makoto

PS14-02
S08-01
LCS3-1
O11-1
O11-3
1P-010
1P-012
2P-1293P-008

TSUJI, Kojun

1P-082
1P-124
2P-111

TSUJI, Shingo

1P-088
3P-115

TSUJI, Yukio

3P-138

TSUJIMURA, Takanori

PS26-02

TSUKADA, Mana

1P-056
1P-1032P-0052P-006
2P-188
3P-110UCHIMURA, Yoshiko

2P-062

UEDA, Hajime

3P-104

UEDA, Hiromi

2P-084

UEDA, Keitarou

3P-112

UEDA, Shuhei

O14-4

UEDA, Tamami

3P-147

UEDA, Teppei

3P-154OU
3P-158

UEDA, Yoshitomo

O05-4

UEDA, You

3P-111

UEKI, Takatoshi

2P-037

UEMURA, Kazunori

O20-1
O21-5

TSUKAHARA, Narutoshi

1P-030TSUKAHARA, Shinji

2P-099

TSUKAMOTO, Fumimaro

UEMURA, Masato

3P-061

2P-113

TSUKAMOTO, Ikuko

UEMURA, Satoshi

1P-013

1P-073

TSUKAMOTO, Kota

UENO, Hiromichi

3P-134
3P-135

1P-075
1P-076
1P-077
1P-117
2P-107
3P-096

UENO, Hiroshi

1P-025

UENO, Shinya

1P-104
2P-190TOMINAGA, Mitsutoshi

2P-060TOMINAGA, Takashi

3P-143TOMINAMI, Kanako

2P-050TSUKAMOTO, Youko

3P-063

TOMITA, Keisuke

2P-030TSUKIOKA, Kei

TOMITA, Shinnosuke

1P-131

O15-2
2P-030TOMIZAWA, Kazuhiko

O07-4

TSUMOTO, Kunichika

TOMIZAWA, Kazuhito

O08-3
3P-121
3P-122

PS22-04
2P-033
3P-023

TSUNEKAWA, Chinami

2P-160

UENO, Yasuharu

O19-2

TSUNEMATSU, Tomomi

PS11-04

UESAKA, Naofumi

1P-031TSUNEOKA, Yayoi

1P-037

UESHIMA, Kyoko

2P-166

2P-099UESHIMA, Shigeru

3P-114

UETA, Yoichi

LS1-1
O12-3
1P-048
1P-074
1P-075
1P-076
1P-077
1P-117
1P-1232P-078
2P-107
3P-096

UETA, Yoshifumi

3P-040UEYAMA, Tomoe

2P-022

UEZUMI, Akiyoshi

O06-5

UJIHARA, Izumi

2P-136
3P-092

UJIHARA, Yoshihiro

1P-015
1P-0243P-021TOMOKAGE, Takuto

3P-144
3P-145TOMOYOSE, Ryuji

1P-054TONOMURA, Sotatsu

1P-043TSUNO, Yusuke

2P-073TOSHIMA, Hiroko

1P-072
1P-115
1P-116TSUNODA, Keisuke

1P-045

TSURUSHIMA, Hideo

1P-023

TSUSUMI, Takashi

2P-048TSUTSUI, Hidekazu

2P-018
3P-170TSUZUKI, Atsushi

1P-092TSUZUKI, Takashi

2P-048TSUZUKI, Yui

2P-175TUNEYAMA, Koichi

3P-169TURNER C., Helen

1P-002TOSHIMA, Yoshito

S17-01

TOURZANI, Darya

2P-115TOYODA, Fumiyo

1P-046TOYODA, Futoshi

2P-0263P-007TOYODA, Kazuki

2P-075TOYOSHIMA, Yuka

S10-02

TSAI, Ching-Yi

O08-5

TSARI ANDRIANI, Rizki

3P-016

TSUBOI, Yoshiyuki

2P-059
2P-086TSUNEOKA, Yousuke

U
UBAIDUS, Sobhan

1P-069TSUBOSHIMA, Katsuyuki

1P-042TSUBOTA, Ayumi

UCAR, Hasan

PS15-03

3P-151

TSUCHIMOCHI, Hirotsugu

UCHIDA, Chiaki

PS16-01
3P-020
S11-01
O16-1
O21-2

1P-043
3P-063UCHIDA, Hiroyuki

3P-034

UJISAWA, Tomoyo

1P-010UCHIDA, Kunitoshi

3P-008

UKA, Takanori

3P-146UCHIDA, Rina

3P-074

UKICHI, Rikako

3P-066UCHIDA, Sae

LS1-2
O10-1

UMAKOSHI, Akihiro

O07-3

UMEDA, Ryohei

1P-053
2P-167
3P-162OU

UMEDA, Ryouhei

3P-160OU

UMEMURA, Kazuo

3P-108

TSUCHIMOTO, Hirofumi

3P-125

TSUCHIYA, Kenji

2P-190TSUCHIYA, Koichiro

1P-095TSUDA, Masayuki

S12-02TSUITSUI, Kazuyoshi

1P-046

S200

UCHIDA, Shinichi

3P-048UCHIDA, Yuki

2P-160UCHIDA, Yutarou

O19-2

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

AUTHOR INDEXUMEMURA, Masanari

AP-9
3P-012WATANABE, Masaru

S01-03
2P-092
3P-083

YAJIMA, Hiroyuki

3P-095YAJIMA, Takehiro

3P-079YAKO, Hideji

PS18-03
O19-5

YAMADA, Junko

1P-085
1P-1142P-077UOZU, Saori

2P-182URAKUBO, Hidetoshi

PS19-02

WATANABE, Masato

3P-155URANO, Tetsumei

O18-5

WATANABE, Miho

2P-149URATA, Kentaro

2P-038WATANABE, Minori

3P-074

URUSHIBATA, Hirotaro

1P-053
3P-162OUWATANABE, Nobuhiro

YAMADA, Kota

S09-05USHIGOME, Akihiko1P-106
3P-0682P-0203P-078
3P-101YAMADA, Koutaro

2P-062WATANABE, Satoshi

PS15-03

YAMADA, Mana

3P-070

1P-042WATANABE, Takaki

1P-031YAMADA, Mitsuhiko

WATANABE, Tatsuo

O10-2
1P-058
1P-112

O13-3
2P-009YAMADA, Rei

O04-4

YAMADA, Yoshiko

2P-049YAMADA, Yuta

1P-037YAMAGAMI, Fumi

1P-023

YAMAGISHI, Mariko

1P-110YAMAGUCHI, Kimitaka

O07-5

YAMAGUCHI, Maki

2P-1763P-001YAMAGUCHI, Masahiro

1P-041
3P-055
3P-056
3P-062YAMAGUCHI, Osamu

LCS1-1YAMAGUCHI, Ran

S07-03

YAMAGUCHI, Soichiro

1P-091YAMAGUCHI, Takanori

LCS2-1YAMAGUCHI, Takeshi

3P-022YAMAGUCHI, Teruaki

3P-123

YAMAGUCHI, Tomofumi

PS05-02

YAMAGUCHI, Yohei

PS17-02/
AP-6
3P-030

UTA, DaisukeV
VAN VLIET, Bruce N.

O20-5

VAN-HOUT, Isabelle

PS16-02

VISCONTI, Pablo

2P-115W

WATANABE-ASAKA, Tomomi	1P-013

2P-125WATANAVE, Masashi

2P-109

WEI, Fanyan

O08-3

WEI, Fan-Yan

3P-121
3P-122

WEI, Mengyan

3P-013
3P-0143P-015

WEI-GUANG, Ding

2P-026WENHUA, Liu

1P-073

WESTHOFF, Maartje

PS01-03

WICKENS R., Jeffery

2P-041

WADA, Eiji

2P-025

WADA, Makoto

2P-156

WADA, Sayaka

2P-150

WADA, Tadashi

1P-084

WADA, Yoshikuni

PS10-01

WADDINGHAM, Mark

PS16-01
S11-01

WADDINGHAM, Mark T.

O21-2

WIERS ROZENBROEK, Paul

AP-8

WAKABAYASHI, Masaki

S11-01WILLIAMS, Tory

PS12-04

WAKABAYASHI, Shigeo

O16-1

WINDA, Ariyani

WAKAMATSU, Yuma

PS12-02

S17-02
O02-2

WAKANA, Shigeharu

3P-070WAKASHIN, Hidefumi

1P-078WAKAZONO, Yoshihiko2P-046

WAKE, Hiroaki

2P-063
3P-138

WAKE, Naoki

PS08-04

WAKI, Hidefumi

O15-2
2P-0303P-077
3P-126

WANG, Chen

O03-4

WANG, Haibo

1P-093

WANG, Min

3P-045

WANG, Pu

3P-013
3P-0143P-015
3P-018

WANG, Tianying

3P-048

WANG, Yibin

PS18-01

WASHIO, Takumi

PS29-05

WASHIO, Takuro

3P-141

WATABE, Teruaki

1P-128WATANABE, Hironori

3P-141WATANABE, Masahiko

1P-008WITITSUWANNAKUL, Rapepun

3P-120WITOWSKI, Janusz

O16-5

WOLTJEN, Knut

3P-007WONGDEE, Kannikar

O01-5

YAMAGUCHI, Yoshifumi

S02-04

WONGMASSANG, Woranan

O15-4

YAMAGUCHI, Yuya

3P-028WU, ZhouS12-03

YAMAJI, Junko

WURIYANGHAI, Yimin

O20-4
3P-0072P-089
2P-090YAMAKURA, Fumiyuki

2P-060

YAMAMOTO, Ena

1P-061
2P-079
2P-080X
XIE, Yu

1P-017XIONG, Ping Yu

YAMAMOTO, Hideaki

S04-02

O03-2

XU, Haiyan

YAMAMOTO, Kazutoshi

1P-046

2P-174

XU, Jianjun

YAMAMOTO, Kazuya

1P-1162P-015

XU, Minhui

YAMAMOTO, Kimiko

PS09-02

O13-4

YAMAMOTO, Kohei

S09-05

YAMAMOTO, Kuya

3P-116

YAMAMOTO, Maho

1P-018PS04-05
LCS10-1

YAMAMOTO, Noriyuki

1P-084

YAMAMOTO, Shinya

2P-153

YAGI, Takashi

O16-3

YAMAMOTO, Takahiro

O08-3

YAGISHITA, Sho

PS15-03

YAMAMOTO, Tomomi

1P-008YAGUCHI, Kana

2P-145

YAMAMOTO, Yasuhiro

PS12-04

Y
YADA, Toshihiko

The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S201

AUTHOR INDEXYAMAMOTO, Yuta
YAMAMURA, Aya

O20-4
3P-007YANAGAWA, Yuchio

1P-029
2P-042
3P-098

3P-155
3P-156
S18-05YANAGI-ISHIHARA, Keiko

2P-004YANAGIDA, Takayuki

O14-4

YAMAMURA, Kensuke

1P-055YANAGISAWA, Masashi

YAMAMURO, Kazuhiko1P-049O05-2
3P-070YAMANAKA, Akihiro

PS07-03
O14-4
O15-3
1P-003
1P-050YAMANAKA, Daisuke

S10-02YAMANAKA, Ko

O15-2
2P-0303P-077
3P-126

YANG, Chihpin

1P-118
2P-171
2P-172

YANG, Qunhui

S18-03

YANO, Hajime

O07-2
3P-074
3P-123
3P-158YONESHIRO, Takeshi

2P-159YONEZAWA, Tomoko

PS13-03

YOSHIDA, Aiko

1P-092YOSHIDA, Eiko

1P-037YOSHIDA, Junichi

O06-3

YOSHIDA, Junko

2P-139YOSHIDA, Kazutaka

2P-190YOSHIDA, Masahide

PS25-02

YOSHIDA, Masaki

2P-150
2P-184
3P-140YOSHIDA, Norio

S13-02YOSHIDA, Ryusuke

2P-064YOSHIDA, Ryusuke

3P-079YASAKA, Toshiharu

2P-052

YASODA, Akihiro

YOSHIDA, Sachine

O03-5

S15-04YASUI, Hiroyuki

YOSHIDA, Takashi

2P-135

S14-02YASUI, Takeshi

YOSHIDA, Tomoyuki

3P-170

3P-169YASUO, Toshiaki

YOSHIDA, Yuki

1P-131

2P-096
2P-161YOSHIE, Susumu

YASUOKA, Yukiko

3P-100YASUTOMI, Yasuhiro

1P-121YASUZAWA, Toshinori

3P-114

1P-086
1P-0881P-0892P-118
2P-137YOSHIKAWA, Akira

2P-150
2P-184
3P-108
3P-140YAMANAKA, Yoshiaki

3P-096YAMANE, Yui

1P-058YAMANISHI, Yoshihiro

PS24-01

YAMANOUE, Tomomi

1P-035/
AP-2

YAMASAKI, Takaya

1P-121YAMASHINA, Yoshihiro

1P-125YAMASHITA, Fumio

3P-034

YAMASHITA, KaoriYATAGAI, Saya

S09-052P-025YAMASHITA, Masayuki

YATSUKA, Hiroyuki

3P-160OU

1P-001YAMASHITA, Shuichiro

YAYOTA, Masato

2P-0672P-016YAMASHITA, Takayuki

YAZAKI, Tatsuhiko3P-039YOSHIKAWA, Kazuhiro

3P-155

O14-4
O15-3

YIMENG, Zhao

3P-006YOSHIKAWA, Tomoko

3P-071YAMASHITA, Tetsuo

2P-128

YIN, Chengzhu

2P-061YOSHIKAWA, Yutaka

S14-02YAMASHITA, Tomohiro

1P-067

YIN, Henry

S09-05YOSHIMARU, Kouichirou

O01-4

YAMASHITA, Yohei

2P-076

YODA, Takanori

S11-03YOSHIMOTO, Junichiro

YAMASOBA, Tatsuya

S15-02

YOGI, Wakako

1P-103

O14-4
O15-3

YAMATO, Ippei

3P-056YOSHIMOTO, Misa

1P-0663P-087YOKOI, Kazuko
YOKOI-HAYAKAWA, Mika

YOSHIMURA, Akihiko

SL6

YAMAUCHI, Hideki

2P-178

1P-078
2P-122

2P-144
2P-145

2P-188YOKOTA, Shigefumi

YAMAUCHI, Yuho

2P-166

O14-3
2P-183
3P-166YOSHIMURA, Mitsuhiro

YAMAUCHI, Risa
YAMAZA, Takayoshi

O01-4

YOKOTA, Takako

2P-021YAMAZAKI, Daiju

2P-019YOKOTA, Tatsuko

2P-093

YAMAZAKI, Hiroya

O03-1
1P-081YOKOYAMA, Hisayo

O02-3
O21-3

YAMAZAKI, Hiroyuki

1P-030YOKOYAMA, Kurumi

2P-175YAMAZAKI, Hiroyuki

2P-076YOKOYAMA, Megumi

YAMAZAKI, Jun

3P-008O06-1
1P-071O02-5
1P-048
1P-075
1P-076
1P-077
1P-117
1P-1232P-078
2P-107
3P-096YOSHIMURA, Takashi

S08-03

YAMAZAKI, Masatoshi

PS06-01

YOSHIMURA, Yuki

1P-112YAMAZAKI, Nanae

1P-070YOSHINA, Sawako

YAMAZAKI, Tomohiro

1P-011
1P-130

1P-096YAMAZAKI, Toshiya

YOSHINAGA, Daisuke

O20-4

3P-097YAMAZAKI, Yoko

YOSHINAGA, Masao

PS22-01

O09-2

YAMAZAKI, Yoshihiko2P-141

2P-078

O04-5

YONEDA, Mitsugu

YOSHINO, Kiyoshi

YAMAZAWA, Toshiko

2P-1763P-001YONEDA, Takashi

2P-141YOSHIOKA, Kazuaki

YONEKURA, Hideto

2P-028YAMAZOE, Masahiro

PS17-03

YONEMOCHI, Keita

O08-1

PS23-01
O18-1
O20-3
2P-119S202

YOKOYAMA, Shingo

1P-067YOKOYAMA, Utako

S11-02
S11-03
AP-9
O18-2
2P-010The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

AUTHOR INDEXYOSHITOMO, Miki

PS19-05

YOSHIZUMI, Masao

3P-112

YOUM, Jae Boum

S05-01YOYSUNGNOEN, Bhornprom

3P-120YOZU, Arito

3P-137

YU, Yong-Chun

3P-045YUNOKI, Takahiro

3P-164

YURA, Kei

PS10-03YUZAKI, Michisuke

PS15-04Z
ZAWADA, Agnieszka

O16-5

ZEMPO, Buntaro

PS12-04

ZERIZER, Sakina

2P-147

ZHAN, Dong-Yun

3P-029ZHANG, Boyang

3P-097ZHANG, Heping

2P-174

ZHANG, Min

S18-01
O13-4
O13-5

ZHANG, Ying

S18-01
S18-05
O13-4
O13-5

ZHAO, Zidong

1P-048ZHENG, Can

O20-1

ZHOU, Li

1P-008The 97th Annual Meeting of the Physiological Society of Japan, March 17 − 19, 2020, Beppu

S203

Continued from back cover

Lunchtime Sessions 
S61Lunchtime Sessions

Luncheon Seminars 
S61Award Presentations ( Formerly The Japanese Journal of Physiology )Official Journal of The Physiological Society of Japan

Aims and Scope:
The Journal of Physiological Sciences
publishes peer-reviewed original papers,
reviews, short communications, technicalnotes, and letters to the editor, based on
the principles and theories of modern
physiology and addressed to theinternational scientific community.
All fields of physiology are covered,encompassing molecular, cellular andsystems physiology. The emphasis is on
human and vertebrate physiology, but
comparative papers are also considered.
The process of obtaining results must be
ethically sound.
Fields covered:·	Adaptation and environment·	Autonomic nervous function·	Biophysics·	Cell sensors and signaling·	Central nervous system and brain
sciences·	Endocrinology and metabolism·	Excitable membranes and neural cell
physiology·	Exercise physiology·	Gastrointestinal and kidney physiology·	Heart and circulatory physiology·	Molecular and cellular physiology·	Muscle physiology·	Physiome/systems biology·	Respiration physiology·	Senses

Luncheon Seminars

Editorial Board of The Journal of Physiological Sciences
Chief Editor

Associate Editors

Harumi Hotta, Tokyo
Hironobu Morita, Gifu
Makoto Tominaga, Okazaki

Review Editor

Yasunobu Okada, Okazaki
Yasuo Sakuma, Tokyo
Satomi Adachi-Akahane, Tokyo
Wataru Aoi, Kyoto
Hsiao Chang Chan, Hong Kong
Ying-Shing Chan, Hong Kong
Douglas C. Eaton, Atlanta
Yong-Jian Geng, Houston
Katsumasa Goto, Toyohashi
Yukiko Hayashi, Tokyo
Akihiro Hazama, Fukushima
Masaki Kameyama, Kagoshima
Masanobu Kano, Tokyo
Katsumasa Kawahara, Sagamihara
Toru Kawata, Suita
Kyungjin Kim, Seoul
Yoshihiro Kubo, Okazaki
Manabu Kubokawa, Morioka
Yoshihisa Kurachi, Suita
Tomoyuki Kuwaki, Kagoshima
Weimin Li, Shanghai
Yoshinori Marunaka, Kyoto
Satoshi Matsuoka, Kyoto
Kenju Miki, Nara
Yasuhiko Minokoshi, Okazaki
Shohei Mitani, Tokyo
Kei Nagashima, Tokorozawa
Mitsuyuki Nakao, Sendai
Yasuhiro Nishida, Tokorozawa
Kazunori Nosaka, Joondalup

Hiroshi Nose, Matsumoto
Shigehiko Ogoh, Kawagoe
Yasushi Okamura, Suita
Satoshi Okumura, Yokohama
Yoshitaka Oku, Nishinomiya
Brian Oldfield, Clayton
Lawrence G. Palmer, New York
Mathieu Piché, Trois-Rivières
Eduardo Rios, Chicago
Hideki Sakai, Toyama
Ichiro Sakuma, Tokyo
Motohiko Sato, NagakuteGeert Schmid-Schönbein, San Diego
Manabu Shibasaki, Nara
Yasutake Shimizu, Gifu
Masahiro Sokabe, Nagoya
Hideaki Soya, TsukubaMark Stewart, Brooklyn
Eiji Takahashi, Saga
Miyako Takaki, Kashihara
Makoto Takano, Kurume
Shigeru Takemori, Tokyo
Akiyuki Taruno, Kyoto
Yoichi Ueta, Kitakyushu
Marcel A.G. van der Heyden, Utrecht
Paul A. Welling, Baltimore
Kazuhiro Yagita, Kyoto
Jianqun Yan, Xi’an

Organizers of the 97th Annual Meeting in Beppu
Katsushige Ono, Yufu
Reiko Hanada, Yufu
Osamu Tokumaru, Oita
Tatsuki Kurokawa, Yufu
Shinichiro Kume, Yufu

21st Promotion Award of the Physiological Society of Japan for Young Scientists
10th Hiroshi and Aya Irisawa Memorial Promotion Award for Young Physiologists
10th Aya Irisawa Memorial Promotion Award for Excellence by Women Physiologists
10th Hiroshi and Aya Irisawa Memorial Award for Excellent Papers in The Journal of Physiological Sciences
10th Hiroshi and Aya Irisawa Memorial Award for Excellent Papers on Research in Circulation in The Journal of Physiological Sciences

Oral Presentations 

Yoshihiro Ishikawa, Yokohama

Editors

S66
S66
S67
S67
S67

Ryoko Higa, Yufu
Kenshiro Shikano, Yufu
Miki Watanabe, Yufu
Natsuki Matsuno, Yufu

S70
S71
S72
S73
S74
S75
S76
S77
S78
S79
S80
S81
S82
S83
S84
S85
S86
S87
S88
S89
S90

Kidney・Urination・Digestion・Absorption
Environmental Physiology
Heart・Circulation 1
Neuron・Synapse
Behavior Science・Biorhythm・Neurochemistry
Oral Physiology・Motor Function
Cell Physiology・Molecular Physiology 1
Pathophysiology
Ion Channel・Receptor 1
Autonomic Nervous・Endocrinology
Ion Channel・Receptor 2
Sensory Function
Muscle Physiology
OthersCNS Function
Nutrition・Metabolism・Thermoregulation
Heart・Circulation 2
Cell Physiology・Molecular Physiology 2
Development・Growth・Aging
Heart・Circulation 3
Heart・Circulation 4

Poster Presentations 
S92
S94
S96
S98
S99
S100
S101
S102
S103
S104
S104
S105
S106
S106
S107
S108
S109
S110
S111
S112
S112
S113
S113
S116
S119
S121
S124
S126
S128
S129
S130
S131
S132
S133
S133
S135
S135

Ion Channel・Receptor (1)Heart・Circulation (1)Neuron・Synapse (1)Sensory Function (1)Behavior Science・Biorhythm (1)Neurochemistry (1)Autonomic Nervous (1)Muscle Physiology (1)Oral Physiology (1)Endocrinology (1)Kidney・Urination (1)Motor Function (1)Development・Growth・Aging (1)Cell Physiology・Molecular Physiology (1)Environmental Physiology (1)Drug Actions (1)CNS Function (1)Nutrition・Metabolism・Thermoregulation (1)Pathophysiology (1)Physical Fitness・Sports Medicine (1)Respiration (1)Study Methodology (1)Others (1)Ion Channel・Receptor (2)Heart・Circulation (2)Neuron・Synapse (2)Sensory Function (2)Behavior Science・Biorhythm (2)Neurochemistry (2)Autonomic Nervous (2)Muscle Physiology (2)Oral Physiology (2)Endocrinology (2)Kidney・Urination (2)Motor Function (2)Reproduction
Development・Growth・Aging (2)

S136
S137
S139
S140
S140
S142
S143
S145
S145
S146
S146
S147
S150
S153
S155
S158
S160
S162
S162
S163
S164
S165
S166
S167
S168
S168
S170
S171
S172
S172
S174
S175
S177
S177
S178
S178

Digestion・Absorption
Cell Physiology・Molecular Physiology (2)Environmental Physiology (2)Drug Actions (2)CNS Function (2)Nutrition・Metabolism・Thermoregulation (2)Pathophysiology (2)Physical Fitness・Sports Medicine (2)Blood
Respiration (2)Study Methodology (2)Others (2)Ion Channel・Receptor (3)Heart・Circulation (3)Neuron・Synapse (3)Sensory Function (3)Behavior Science・Biorhythm (3)Neurochemistry (3)Autonomic Nervous (3)Muscle Physiology (3)Oral Physiology (3)Endocrinology (3)Kidney・Urination (3)Motor Function (3)Development・Growth・Aging (3)Cell Physiology・Molecular Physiology (3)Environmental Physiology (3)Drug Actions (3)Membrane Transport
CNS Function (3)Nutrition・Metabolism・Thermoregulation (3)Pathophysiology (3)Physical Fitness・Sports Medicine (3)Respiration (3)Study Methodology (3)Others (3)Volume 70 · Supplement 1 · 2020

Volume 70 · Supplement 1 · 2020
The 97th Annual Meeting of the Physiological Society of Japan
Plenary Lectures 
S2

Plenary Lectures

Special Lectures 
S3Special Lectures

S6
S6
S7

The Sunao Tawara Memorial Lecture
The Makoto Arita Memorial Lecture
The Susumu Hagiwara Memorial Lecture

Planned Symposia 
S10
S11
S12
S13
S14
S15
S16
S17
S18
S19
S20
S21
S22
S23
S24
S25
S26
S27
S28
S29
S30
S31
S32
S33
S34
S35
S36
S37
S38

Japan-Canada Joint Symposium Cutting-edge approaches to the functioning mechanisms and pathophysiology of ion channels
Neuro-immune communication and homeostatic responses
Integration of sensory and metabolic signals by neuropeptide in hypothalamus (navigates metabolic state.)
The metabolic modulation by sensory input from peripheral organs
Brain function for physical therapy and its physiological mechanisms
Recent Advancement of the Multidisciplinary Approaches to the Pathophysiology Underlying Arrhythmia
The emerging roles of pallidal nuclei in the basal ganglia circuitry
Systems biology of hearing: from the inner ear to the brain
Scientific basis of the oriental medicine: mechanisms improving muscle blood flow at rest
Japan-China Joint Symposium Cutting edge of cell fate determination and migration in the developing cerebral cortex
Women scientists in the optogenetic field – the new fact has come to light using optical approaches
New frontiers of locomotion research using zebrafish as a model system
Combined approaches of cutting-edge techniques and structural information to reveal the elaborate dynamics of membrane proteins
Past, present and future of JPS
Leading-edge approach for regulated exocytosis in neural system
New Insights into Vascular Aging Mechanisms and Therapeutic Targets
Cardiac mechano-physiology: physiology and pathophysiology of the mechano-electrical coupling
Inter-organ communication: molecular mechanism and pathophysiology induced by its disruption
Progress and perspectives of neuroscience with data-driven intelligence
Neural Mechanism of Psychological Stress: Molecules, Circuits and Disorders
The ethics, laws, and guidelines for human and animal researches
Challenge for the unsolved mechanism of arrhythmias; from the view of physiologists and cardiologists
Angiology evolving into new research fields
What is ‘data-driven’ science
Mechanisms of experience- or metabolism-dependent behavioral changes
New insights of sensory and brain functions affecting feeding behaviors: circadian, swallowing, taste and pain
Pathophysiology of ion channels and transporters in tumor growth
Basic and applied researches to progress the knowledge and therapy for heat stroke
Recent advances in muscle physiology

Symposia 
S40
S41
S42
S43
S44
S45
S46
S47
S48
S49
S50
S51
S52
S53
S54
S55
S56
S57

Deeper insights into smooth muscle physiology using natural products
Warm-blooded cool animals: hibernation and torpor physiology
Frontiers of biophotonics in physiology: Ion environments and molecule behaviors in the nano-space detected by Raman spectrum
Neural mechanisms unveiled by combined in vitro and in silico approaches
Mitochondrial function and its roles in cellular physiology/pathophysiology
Crosstalk in modifiable and non-modifiable risk factors for cardiovascular disease
Molecular and Neural basis for appetite and food preference
Physiological importance of environmental temperatures in ectotherms
Reverse engineering the brain functions for the control of adaptive behaviors
A new world of physiology developed by peptide hormones
AMED support for medical R&D: From basics to practiceControl of endogenous regulatory responses in glial cells in neuroinflammation - From embryonic development to pathogenesis of brain diseases
New strategies for understanding functions and mechanisms of ion channels/transporters/pumps in the upcoming post-structure era
The new sunrise of zinc physiology and zinc pathophysiology
Aging-related changes in physiological functions induced by the space environmentState-of-the-art physiology in urinary continence
Current advance in endocrine disruptor research
History and Up-to-date stories on the mechanism of smooth muscle contraction in health and disease

Educational Programs 
S60

Educational ProgramsContinued insede back cover

Volume 70 · Supplement 1 · 2020 · pp 1–203

Memorial Lectures Proceedings of the 97th Annual Meeting of the Physiological Society of Japan
March 17–19, 2020, Beppu, Oita, Japan