Poster Session
JST‐CREST “Opt Bio” / WPI‐IIIS Joint Symposium
Deciphering the Brain through “Opt Bio” Tools

Data Blitz by Poster Presenters

13:20 – 13:50

Poster Presentation

13:50 – 14:30

Poster list#

Presenter

Title

1

Tsuyoshi Saito

Development of photocaged adenosine 2A receptor activator

2

Roy Koustav

Opto‐chemical control of sleep in the nucleus accumbens using a photocaged adenosine A2A receptor
allosteric modulator

3

Hikari Yashiki

Chemogenetic suppression of histamine receptor cells produces slow‐waves in mice

4

Iyo Koyanagi

Mechanisms of hippocampal adult‐born neurons for memory consolidation during sleep

5

Jiahui Yu

Adult‐born neuron activity in the establishment of fear generalization

6

Yuteng Wang

Synchronous young and matured neuron activity for memory consolidation

7

Pablo Vergara

CaliAli: A tool for inter‐session alignment of 1‐photon calcium imaging data allowing tracking neurons in
the non‐rigidly moving brain

8

Srinivasan Sakthivel

Transient recruitment of an adult‐born neuron ensemble for fear memory consolidation in REM sleep

9

Shingo Soya

Whole brain mapping and manipulation of activated neuronal populations by exhausted exercise

10

Saki Yamada

Capturing and manipulating neurons activated by exhausted exercise

11

Emi Hasegawa

Rapid eye movement sleep is initiated by dopamine signaling in the basolateral amygdala in mice

12

Sol Jeong

GABAergic neurons around Ventrolateral Periaqueductal Gray regulate Cataplexy in Narcolepsy model
mice

13

Yuki Saito

Visualizing input‐output architecture of orexin neurons with double‐color projection‐selective retrograde
tracing

14

Kseniia Prokofeva

Delineation of Neural Circuits of Galaninergic Neurons in the VLPO Implicated in Regulation of Sleep

15

Zhang Zhongwen

Mechanism by which the BNST→DpMe pathway induces arousal

16

Li RuthGastrin‐releasing Peptide‐producing Neurons in the Suprachiasmatic Nucleus play an Essential Role inRegulating Circadian Rhythm

17

Toru Takahashi

QIH induction using extremely sensitive optogenetics

18

Yoan Cherasse

Roles of monoamines and their regulatory systems in motivation and arousal

19

Katsuyasu Sakurai

The anatomical and functional understanding of neural dynamics of VTA dopaminergic neurons in female
sexual behavior

20

Ai Miyasaka

Dopamine dynamics in NAc‐vs control male sexual behaviors in mice

21

Hibiki Okamura

Analyses of the effects of prior social stress on brain activity during social interaction in mice using a novelsemi‐automated c‐Fos mapping program

22

Shinnosuke Yasugaki

Is increased REM sleep an adaptive response or an exacerbating factor in stress resilience?

23

Yoshifumi Arai

Characterization of the neuronal activity of a newly identified REM sleep‐regulating neuron using glass
pipette extracellular unit recording combined with optogenetics

24

Ami Kaneko

Analysis of the mechanism of REM sleep behavior disorder with focus on Parkinson’s disease

25

Ayako Imamura

Effects of chronic cell ablation in the preoptic area on sleep‐wake cycles

26

Yusuke Murakami

Toward label‐free molecular imaging of whole brain and brain cells using ultra‐broadband multiplex
CARS microspectroscopy

27

Olga Malyshevskaya

The role of hypothalamic supraoptic nucleus in maintaining wakefulness.

28

Javier Diaz Cisternas

Recovering EEG Generators from Their Collective Stochastic Interference

29

GoEun (Abby) Han

Dihydropyridine calcium blockers do not interefere with slow wave sleep

30

Satomi Okabe

The effects of olfactory stimulation during REM sleep on dream emotionality: a study focusing on
individual difference in olfactory perception

31

Tsuyoshi Nemoto

Recovery from systemic‐inflammation‐induced altered sleep is potentiated by sevoflurane preconditioning

32

Shigeru Chiba

Validation experiment using in‐home sleep electroencephalography and its clinical application

33

Neira Almanza Juan Carlos

Automatic assessment of sleep recordings from a portable IoT EEG device.

34

HarukaMetabolomic and pharmacologic analyses of brain substances associated with sleep pressure in mice

35

Tomohiro Kitazono

The novel intracellular signaling pathways for the regulation of homeostatic sleep need36

Cao Liqin

Natural history study of sleep disturbances in CDKL5 deficiency disorder mice

37

Hikari Yamamoto

OX2R‐selective orexin agonism is sufficient to suppress narcoleptic symptoms, cataplexy and wakefragmentation, without inducing drug‐seeking behavior in mouse models

38

Kaushik Mahesh Kumar

Pre‐narcoleptics are more prone to suvorexant‐induced cataplexy.

39

Mari Hondo

Orexin receptor antagonists ameliorate the symptoms of REM sleep behavior disorder in a novel mousemodel and in human patients

40

Kumar Deependra

Learning and memory deficit in adult dreamless mice41

Tomoyuki Fujiyama

NALCN in the forebrain and pons‐medulla regions have distinct roles in REM sleep regulation

42

Suzuki

Mahmoud Asmaa
Elhosainy Mohamed

Sleepy mouse as a model of idiopathic hypersomnia

43

Yuki TairaElectrophysiological analysis of ion channel mutations in mice with REM sleep abnormalities

44

Noriko Hotta

Sleep/wakefulness and body weight growth from infancy to adulthood in a hypersomnia model, Sleepy
mutant mouse.

45

Seoane Collazo Patricia

SIK3 in different hypothalamic areas mediates whole‐body energy balance

46

Kanako Iwasaki

Where does Sleepy mutation of SIK3 cause sleep phenotypes?

47

Fuyuki Asano

Sik3 regulates sleep need via glutamatergic neurons in cerebral cortex

48

Shinya Nakata

Molecular mechanisms for SIK3(Sleepy)‐mediated sleep/wake regulation

49

Minjeong Park

Sleep/wake behavior of mice lacking PKA phosphorylation site in SIK350

Kim Staci Jakyong

Loss of canonical Hdac4 signaling leads to disregulated NREMS

1

1 University of Tsukuba------------------------------------------------------------------

2 Hiroshima University

Development of photocaged adenosine 2A
receptor activator-----------------------------------------------------------------Photopharmacology may offer the possibility of curing
diseases and alleviating symptoms while preventing
uncontrolled drug activity, i.e., the drug is active only at
the times and places where it exerts its therapeutic effect.
Although chemical photo switches have been used
extensively in vitro, their use in vivo has been slow,
largely because of the difficulties in applying theseprobes in mammalian models. We revealed a prominentrole of indirect pathway neurons in the nucleus
accumbens (NAc) in sleep/wake regulation andproposed that the NAc links motivation and sleep. This
brain circuit may explain why we feel sleepy in theabsence of motivating stimuli, i.e., when we are bored.
Adenosine is a plausible candidate molecule foractivating NAc indirect pathway neurons to induceslow-wave sleep (SWS) because caffeine, the mostwidely consumed psychostimulant in the world,
produces its arousal effect in the NAc by blocking
adenosine A2A receptors (A2AR) on indirect pathway
neurons. However, the ability of adenosine incontrolling NAc indirect pathway neurons for sleep
induction remains to be elucidated. We recently reported
the first positive allosteric A2AR modulator, named
A2AR PAM, that evokes A2AR responses in the brain
and developed a visible-light photoactivatable
derivative of A2AR PAM (opto-A2AR PAM). OptoA2AR PAM showed remarkable water solubility (>10mM) and has an absorption maximum at 415 nm inaqueous solution. SWS was significantly increased for
5h in wild-type mice after intraperitoneal administrationof opto-A2AR PAM and stimulation with violet light(405 nm) for 1 h after drug treatment, whereas no effectwas observed in the absence of light exposure or in
A2AR knockout mice. By using opto-A2AR PAM, we
induced sleep for the first time in freely behaving miceby photopharmacologic allosteric A2AR modulation,suggesting that extracellular adenosine is involved in
the regulation of sleep in the NAc.
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Tsuyoshi Saitoh1, Shuji Ioka1, Mustafa Korkutata1,
Yohei Chitose2, Manabu Abe2, Michael Lazarus1,
Hiroshi Nagase1
1 University of Tsukuba
2 Hiroshima University-----------------------------------------------------------------Adenosine (Ad) is an endogenous somnogen, whichstrongly promotes sleep through the activation of the
adenosine 2A receptor (A2AR) in the nucleus
accumbens (NAc).1 Although the increased
accumulation of Ad during wakefulness leads to induce
sleep, the detailed temporal and spatial mechanisms of
adenosinergic control of the NAc have not been clarified.
Though photocaged A2AR activator has been expected
as a novel optical tool to control the single receptor
activity by light irradiation, the chemical tools based on
the known A2AR agonist have an issue for separation of
the endogenous and exogenous effects due to thebackground activity. We recently discovered a novel
positive allosteric modulator (PAM) YNT378.2
YNT378 enhanced the sensitivity of A2AR toendogenous Ad without showing agonist activity andinduced slow-wave sleep in mice. These findings led us
to develop the photocaged A2AR PAM (opto-YNT378)
to facilitate the optopharmacology study of A2AR.
The firth generation opto-YNT378, carrying 6nitroveratryl (Nv) group as a photolabile protecting
group, had problematic properties such as waterinsolubility, slow photoresponse, and short absorption
maximum. To improve the photochemical andphysicochemical properties of Nv group, we developed
a novel photolabile protecting group A400 based on 3aryl coumarine chromophore.3 The 2nd generation
opto-YNT378 bearing A400 exhibited high watersolubility, fast photoresponse, and an absorption
maximum at 420 nm. Moreover, the time-dependent
photoactivation of opto-YNT378 was observed in thecell-based assay.
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3------------------------------------------------------------------

Chemogenetic suppression of histamine
receptor cells produces slow-waves in mice2------------------------------------------------------------------

Opto-chemical control of sleep in the nucleus
accumbens using a photocaged adenosine A2A
receptor allosteric modulator

Hikari Yashiki1, Yo Oishi1, Michael Lazarus1
1 University of Tsukuba-----------------------------------------------------------------Sleep in mice is divided into 2 stages: non-rapid eye
movement (NREM) sleep and rapid eye movement(REM) sleep. Electroencephalogram (EEG) shows
typical waves during these phases. The delta-range
frequency (1–4 Hz) of EEG is dominant during NREM

Koustav Roy1, Shuji Ioka1, Mao Amezawa1, Yoan
Cherasse1, Hiroshi Nagase1, Masashi Yanagisawa1,
Kaspar Vogt1, Manabu Abe2, Tsuyoshi Saitoh1, Michael
Lazarus1

1

sleep, while theta-range frequency (4–8 Hz) is dominantduring REM sleep. Although these criteria are usedgenerally, the mechanism to generate these typicalwaves is not clear.
Antihistamines induce sleepiness/sleep in humans. So,
they have been used for sleep-promoting effect.
Antihistamines work as inverse agonists of histamine
H1 receptors (H1R, Gq-coupled receptors), causing
pharmacological effects opposite to those of agonist. At
present, the neuronal mechanisms of sleepiness/sleeppromoting effect of antihistamines are unknown and it
is also unclear whether suppression of H1R-expressing
cells affects on sleep/wake behaviors.
In this study, we examined the effect of neuronal
suppression in H1R-expressing cells on sleep/wakebehaviors in mice by a chemogenetic method. After thechemogenetic suppression, the EEG with highamplitude and low frequency was observed for a few
hours even during movement. These results suggest thatsuppression of H1R-expressing cells produces slow
waves in mice.
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rhythm with ABNs during REM sleep, we examinedhippocampal theta-phase specific ABN activity formemory consolidation by closed-loop optogenetic
manipulation. This study provides insights into how theunique cellular plasticity in the adult brain operates
synchronously with brain rhythm for memory
consolidation during sleep.
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5------------------------------------------------------------------

Adult-bornneuronactivityinestablishment of fear generalization

theJiahui Yu1, Pablo Vergara1, Toshie Naoi1, Yuteng Wang1,
Yoan Cherasse1, Takeshi Sakurai1, Masanori Sakaguchi1
1International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba-----------------------------------------------------------------Overgeneralization of fear in safe contexts is considereda hallmark of post-traumatic stress disorder. Traditional
behavior paradigm to elicit fear generalization requires
more than ten days, which prevents the understandingof how fear generalization establishes in parallel withmemory processing, including memory consolidation. It
is known that adult-born neurons (ABNs) continuously
generate in the hippocampus play key roles incontextual fear generalization. We have elucidated the
dynamic activity change of ABNs in REM sleep andtheir necessity in memory consolidation. However, theiractivity patterns responsible for fear generalization arecompletely unknown. In this study, we first constructeda robust behavior paradigm to analyze the process ofcontextual fear generalization. To reveal the temporaland spatial activity of ABNs, we employ calcium
imaging in freely moving mice. This study provides new
insights into understanding the mechanisms ofestablishing contextual fear generalization.
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4------------------------------------------------------------------

Mechanisms of hippocampal adult-bornneurons for memory consolidation during
sleep
Iyo Koyanagi1, 2, Yuteng Wang1, Sakthivel Srinivasan1,
Jiahui Yu1, Toshie Naoi1, Akinobu Ohba1, Deependra
Kumar1, Pei-Hsi Wu1, Kaspar Vogt1, Shinji Matsuda2,
Yoan Cherasse1, Takeshi Sakurai1, Masashi
Yanagisawa1, Michisuke Yuzaki4, Masanori Sakaguchi1
1International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba
2 Degree Programs in Comprehensive Human Sciences,
Graduate School of Comprehensive Human Sciences, Universityof Tsukuba
3 Department of Engineering Science, Graduate School ofInformatics and Engineering, The University of ElectroCommunications, Japan
4 Department of Physiology, Keio University School of Medicine,Japan.

6------------------------------------------------------------------

Synchronous young and matured neuron
activity for memory consolidation-----------------------------------------------------------------Sleep plays critical roles in memory consolidation, yet
the mechanisms are still unclear. Young adult-bornneurons (ABNs) in the hippocampal dentate gyrus (DG)
bestow unique plasticity to the memory circuit. We
found that ABNs exhibit sparse activity during REM
sleep, which is necessary for memory consolidation(Kumar and Koyanagi et al., Neuron, 2020). A
prominent synchronous neural activity (i.e., theta
rhythm) appears in the DG during REM sleep. Theta
rhythm coordinates both synaptic plasticity and memory
consolidation. To reveal the functions of the theta

Yuteng Wang1, Pablo Vergara1, Sakthivel Srinivasan1,
Jiahui Yu1, Toshie Naoi1, Yoan Cherasse1, Takeshi
Sakurai1, Masanori Sakaguchi1
1International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba-----------------------------------------------------------------Brain plasticity shifts from cellular to synaptic during
development. However, cellular plasticity remains
largely functional in the hippocampal dentate gyrus
2

(DG) throughout life; adult-born neurons (ABNs) arecontinuously generated in DG that play pivotal roles inmemory. Indeed, we found that the sparse activity of
ABNs during REM sleep is necessary for memory
consolidation (Kumar et al., Neuron, 2020). It suggests
synchronized ensemble activity between ABNs and thedevelopmentally-born matured neurons for memory
consolidation during sleep. However, the activity and its
functional significance remain completely unknown. To
tackle this issue, we have tested various methods
utilizing the Calcium-imaging and miniaturized
microscope we developed. Our study aims to pave a way
to uncover how cellular plasticity plays distinct roles inconcert with already existing circuits in the adult brain.
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8------------------------------------------------------------------

Transient recruitment of an adult-bornneuron ensemble for fear memory
consolidation in REM sleep
Sakthivel Srinivasan1, Iyo Koyanagi1, Yuteng Wang1,
Pablo Vergara1, Jiahui Yu1, Toshie Naoi1, Kaspar E.
Vogt1, Yoan Chérasse1, Takeshi Sakurai1, Keita Lio1,
Noriki Kutsumrura1, Hiroshi Nagase1, Masanori
Sakaguchi1
1International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba-----------------------------------------------------------------Memory replay during sleep is suggested to contributeto memory consolidation during sleep. The dentate
gyrus (DG) in the hippocampus encode contetual fear
memory trace. Moreover, we showed that the activity of
the adult-born DG neurons (ABNs) during REM sleep
is necessary for memory consolidation (Kumar andSrinivasan et al., Neuron, 2020). Therefore, we
examined whether re-activation of context eoncoding
ABNs during REM sleep is necessary for memory
consolidation. Our study reveals the coding mechanisms
critical for fear memory consolidation during sleep.
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7------------------------------------------------------------------

CaliAli: A tool for inter-session alignment of
1-photon calcium imaging data allowing
tracking neurons in the non-rigidly moving
brain
Pablo Vergara1, Sakthivel Srinivasan1, Toshie Naoi1,
Yoan Cherasse1, Takeshi Sakurai1, Masanori Sakaguchi1
1International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba-----------------------------------------------------------------Endoscopic calcium imaging allows monitoring theactivity of neurons across days and weeks. Current
algorithms to track neurons across sessions rely on thealignment of spatial footprints of neurons independently
extracted from each recording session. However,
because the brain is a non-rigid structure, movements
across sessions are often not expressed as a simple
translation transformation. In this scenario, the spatial
footprints of neurons are commonly not sufficient toalign imaging sessions, especially when neurons
overlap with each other, are sparsely distributed, orwhen their activities remap (i.e., neurons disappear insome recording sessions). To address this issue, wedeveloped CaliAli (Calcium imaging Inter-session
Alignment), a tool that utilizes blood vessels in addition
to spatial cues of neurons to align video sessions
automatically. Because CaliAli aligns video sessions
before neural extractions, it is possible to extract weakcalcium signals that would be otherwise undetected byindependent analysis of each recording session.
Moreover, CaliAli outperforms other tracking
approaches in conditions of high neural overlap orremapping of neuron activities and can be applied tohighly condensed populations such as the granule cells
in the dentate gyrus and also sparsely distributed
population such as the hippocampal adult-born neurons.
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9------------------------------------------------------------------

Whole brain mapping and manipulation ofactivated neuronal populations by exhausted
exercise
Shingo Soya1, Saki Yamada2, Takashi Matsui3, Takeshi
Sakurai1
1 Faculty of medicine, University of Tsukuba
2 Faculty of medical science, University of Tsukuba
3 Faculty of health and sports science, University of Tsukuba-----------------------------------------------------------------Exhausted exercise induces a variety of physiologicalresponses including increased core body temperature,
glycogen reduction, hypoglycemia, described with socalled “Fatigue”. However, neural mechanism that
integrates these physiological alterations has been
largely unknown. To depict the neural populations
which are active during exhausted exercise, we utilized
TRAP (Targeted Recombination in Active Populations)system. We crossed TRAP2-mice with Rosa26-CAGlsl-hM3Dq (hM4Di)-mCherry mice and used motordriven treadmill for capturing and manipulating neurons
activated in exhausted exercise in whole brain. We
found that exhausted exercise induced increased
expression of hM3Dq-mCherry in several brain regions
including the POA, DMH, PVN, PH, Pons, PAG.
3

Chemogenetic activation of the trapped neurons byintraperitoneal injection of CNO (Clozapine-N-Oxide)
showed reduction of body temperature and energy
expenditurewithimmobility.
Chemogenetic
inactivation of these neurons showed no change inenergy expenditure and body temperature. We foundsurface body temperature decreased during exhausted
exercise on treadmill. These results suggest that neurons
which are activated during exhausted exercise might
regulate surface body temperature to maintain core bodytemperature during exhausted exercise.
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11------------------------------------------------------------------

Rapid eye movement sleep is initiated bydopamine signaling in the basolateral
amygdala in miceEmi Hasegawa1, 2, Ai Miyasaka1, Katsuyasu Sakurai1,
Yoan Cherasse1, Yulong Li3, Takeshi Sakurai1, 2
1International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba
2 Faculty of Medicine, University of Tsukuba
3 State Key Laboratory of Membrane Biology, Peking University
School of Life Sciences10------------------------------------------------------------------

Capturing and manipulating
activated by exhausted exercise

neurons-----------------------------------------------------------------The sleep cycle alternates between REM (rapid eye
movement) and NREM (non-rapid movement) sleep,
which is a highly characteristic feature of sleep.
However, the mechanisms by which this cycle isgenerated have not been well-characterized. We foundthat a periodic transient increase of dopamine (DA)level in the basolateral amygdala (BLA) during nonrapid eye movement (NREM) sleep terminates NREM
sleep and initiates REM sleep. DA acts on dopamine
receptor D2 (Drd2)-expressing neurons in the BLA to
induce a transition from NREM to REM sleep. Thismechanism also plays a role in cataplectic attack, which
is a pathological intrusion of REM sleep into
wakefulness in narcoleptics. These results show acritical role of DA signaling in the amygdala in REM
sleep regulation and provide a neuronal basis of sleep
cycle generation.
------------------------------------------------------------------

Saki Yamada1, Shingo Soya2, Takashi Matsui3, Takeshi
Sakurai2
1 Faculty of medical science, University of Tsukuba
2 Faculty of medicine, University of Tsukuba
3 Faculty of health and sports science, University of Tsukuba-----------------------------------------------------------------Exhausted exercise induces a variety of physiologicalresponses including increased core body temperature,
glycogen reduction, hypoglycemia, described with socalled “Fatigue”. However, neural mechanism that
integrates these phenomena is largely unknown. First,
we utilized TRAP (Targeted Recombination in Active
Populations) system by using TRAP2-iCre mice which
express iCre under the c-fos promoter. We crossed
TRAP2-mice with Rosa26-CAG-lsl-hM3Dq-mCherry
and utilized motor-driven treadmill for capturing andmanipulating neurons activated in exhausted exercise inwhole brain. We found that exhausted exercise induces
increased expression of hM3Dq-mCherry in several
brain regions. Activation of these neurons byintraperitoneal injection of CNO (Clozapine-N-Oxide)
showed reduction of body temperature and energy
expenditure with increased immobility. To search thebrain regions which controls these phenomena, utilized
Cre-dependent AAV expressing hM3Dq-mCherry with
TRAP2-mice. We injected AAV into the AVPe, DMH,
PH regions to capture and manipulate neurons which areactivated during exhausted exercise. Chemogenetic
activation of AVPe neurons showed decreased bodytemperature and energy expenditure with immobility.
On the other hand, activation of DMH and PH neurons
showed increased energy expenditure and bodytemperature accompanied with tail vasodilation. Also,
we found body temperature decreases during exhausted
exercise on treadmill. These results suggest that neurons
which are activated during exhausted exercise showed
opposite effect on body temperature and energy
expenditure regulation.

12------------------------------------------------------------------GABAergic neurons in the ventrolateralperiaqueductal gray are implicated in
cataplexy of narcoleptic mice
Sol Jeong1, Shuntaro Uchida2, Shingo Soya1, Takeshi
Sakurai1
1 University of Tsukuba
2International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba-----------------------------------------------------------------The loss of orexinergic neurons cause narcolepsy, asleep disorder characterized by chronic sleepiness andcataplexy. Cataplexy is a sudden muscle atonia during
wakefulness, which is considered as the pathologicalintrusion of REM sleep into wakefulness. The midbrain
region, especially the ventrolateral periaqueductal
gray(vlPAG) is known to regulate REM sleep. However,4the role of vlPAG in cataplexy is poorly understood. We
identified vlPAG and several brain regions as the
upstream of glutamatergic neurons in sublaterodorsal
tegmental nucleus(SLD) projecting to ventromedial
medulla (VMM; GluSLD→VMM), which is known as
the common pathway of cataplexy and REM atonia(Uchida et al., 2021). Based on the previous research,
we injected Cre-dependent AAV expressing hM3DqmCherry into the vlPAG in vGAT-IRES-Cre mice.
Chemogenetic activation of vlPAG GABAergic neurons
decreased the amount of cataplexy in narcoleptic
orexin-ataxin3 mice. These observations suggest that
vlPAG GABAergic neurons suppressed cataplexy
through SLD, and a lack of orexin signaling might
induce the abnormal activity of vlPAG GABAergic
neurons leading to muscle atonia.
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14------------------------------------------------------------------

Delineation of Neural Circuits of Galaninergic
Neurons in the VLPO Implicated inRegulation of Sleep
Kseniia Prokofeva1, 2, Yuki C. Saito2, Arisa Hirano2, 3,
Takeshi Sakurai2, 3
1Graduate School of Comprehensive Human Sciences,
University of Tsukuba
2 International Institute for Integrative Sleep Medicine (WPI-IIIS),
University of Tsukuba
3 Faculty of Medicine, University of Tsukuba-----------------------------------------------------------------Sleep and wakefulness are regulated through dynamic
reciprocal interactions between sleep- and arousalinducing neuronal networks. In this intricated circuitry,
orexin neurons located in the lateral hypothalamus (LH)play a pivotal role in consolidation of wakefulness,while GABAergic and galaninergic (GAL) neurons in
the ventrolateral preoptic nucleus (VLPO) participate ininitiation and maintenance of sleep. Previous studies
showed that orexinergic neurons are innervated by
GABAergic neurons in the VLPO. However, anatomical
connection and functional interaction between orexin
neurons and galaninergic VLPO neurons has not yet
been examined. The aim of this study is to identify
sleep-implicated neural circuits comprising of thegalanin-producing neurons in the VLPO and orexinergic
neurons. We conducted monosynaptic retrograde rabiesmediated tracing from orexin neurons using newly
generated Orexin-iCre knockin mice and visualized
galanin (Gal) and vesicular GABA transporter (Vgat)
mRNAs in the VLPO inputs implementing FISH. We
found that Gal-positive VLPO neurons, mainly coexpressing Vgat, make direct synaptic inputs to orexin
neurons. Further, we conducted projection-specificrabies-mediated tracing from the GAL(VLPO→LH)
neurons and identified that they receive monosynaptic
inputs from various functionally diverse brain areas. We
are currently performing optogenetic stimulation of theaxons of galaninergic and GABAergic VLPO neurons
in the LH to assess contribution of the
GAL/GABA(VLPO→LH) pathways to regulation of
sleep. These findings uncover connectivity ofgalaninergic VLPO neurons and may shed light on therole of their circuits in governance of sleep.
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13------------------------------------------------------------------

Visualizing input-output architecture oforexin neurons with double-color projectionselective retrograde tracing
Yuki Saito1, Yoan Cherasse1, Yasutaka Niwa1, Takeshi
Sakurai1
1 University of Tsukuba-----------------------------------------------------------------Orexin-producing neurons (orexin neurons), located in
the lateral hypothalamic area (LHA), play a highly
important role in maintaining wakefulness. Orexin
neurons send widespread projections to nuclei
containing monoaminergic neurons such as VTA, LC,
TMN, and raphe nuclei, all of which containmonoaminergic neurons. We previously identified input
neurons that make direct synaptic contacts to orexin
neurons with modified rabies vector-based retrograde
tracing. That study showed orexin neurons received
input from various regions of the brain.
In this study, we used projection sites-specific rabiesmediated monosynaptic retrograde tracing to identify
the neuronal inputs to the orexin neurons withprojections to particular regions. In addition, we used
newly generated orexin-iCre KI mice to analyze theinput-output relationship of orexin neuronal circuits
using the modified multi-color simple-cTRIO method.
This new method allowed us to detect more than two
different input-output pathways in the same brain.
This study revealed that orexin neurons projecting toeach output region also send projections to all brain
regions we previously examined. However, we foundsome biased input and output architectures. Orexin(LHA→LC), Orexin (LHA→VTA), and Orexin (LHA→ DR) neurons have mostly overlapping but partly
distinct distributions of input neurons.
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15------------------------------------------------------------------

DpMe pathway induces arousal

Zhang Zhongwen1, 2, Yoan Cherasse2, Takeshi Sakurai25

molecular fluctuations in cycles of around 24 hours,
known as circadian rhythms, which are regulated by thecircadian master clock, or the hypothalamicsuprachiasmatic nucleus (SCN) in mammals. The SCN
contains distinct subtypes of neurons expressing
different neurotransmitters, such as arginine vasopressin(AVP), vasoactive intestinal peptide (VIP), and gastrinreleasing peptide (GRP). Studies assessing the role ofeach neurotransmitter have been conducted over the
past few decades, but little is known about the roles ofthe different neurons that express each peptide. In thisstudy, we focused on unraveling the role of gastrinreleasing peptide-producing neurons in regulating
circadian rhythm, as little research has been done toidentify the properties of these neurons. We used a
newly generated mouse line, Grp-iCre knock-in (KI)mice and Cre-dependent adeno associate virus (AAV) to
specifically manipulate SCN GRP-producing neurons.
We discovered that when we inhibited the SCN GRPproducing neurons with tetanus toxin light chain(TeNTLC), mice display attenuated behavioral
rhythmicity compared to control mice that express only
green fluorescent protein (GFP). When we examined theclock gene expression rhythm within the SCN of micewith inhibited SCN GRP-producing neurons, we
discovered that the oscillation amplitude of PER2
expression is drastically lower in these mice than incontrol mice. These results suggest that SCN GRPproducing neurons are crucial for sustaining SCN
molecular rhythmicity and in turn, is important for
regulating behavioral rhythm.
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1 Faculty of medicine, University of Tsukuba
2International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba-----------------------------------------------------------------Sleep disorders are known as one of the core symptoms
of depression, anxiety disorders and PTSD patients. We
previously demonstrated that the optogenetic
stimulation of GABAergic neurons in the Bed Nucleusof the Stria Terminalis (BNST) can trigger immediatetransition from nonrapid eye movement (NREM) sleep
to wakefulness in male mice. We confirmed dense
innervation from GABA-BNST neurons to the deep
mesencephalic nucleus (DpMe), one of the regions
implicated in promoting arousal. We demonstrated that
optogenetic stimulation of GLUT-DpMe neurons
resulted in immediate transition from NREM sleep towakefulness. We hypothesized that some GABA-DpMeinterneurons might inhibit GLUT-DpMe neurons andreceive inhibitory projections by GABA-BNST neurons.
We also performed retrograde neuronal circuit tracingwith a modified rabies virus to examine the neuronalconnectivity between BNST and DpMe. Combined within situ hybridization, we concluded that GABA-BNST
neurons do not make direct synaptic connections to
GLUT-DpMe neurons. However, we found that GABADpMe neurons receive direct synaptic input fromGABAergic and glutamatergic neurons in the DpMe as
well as GABA-BNST neurons. This research revealed
the neuronal circuits involving GABA-BNST and
GLUT-DpMe neurons that play an important role inquick switching from NREM sleep to wakefulness. We
hope that this research can help decipher the etiology ofsome stress or fear related insomnia symptoms observedin some psychiatric disorders, like post-traumatic stressdisorder (PTSD) and social anxiety disorder (SAD) in
the future.
------------------------------------------------------------------

17------------------------------------------------------------------

Induction of mouse hibernation-like stateusing high sensitive optogenetics
Tohru Takahashi1, Arisa Hirano1, Takeshi Sakurai1

16------------------------------------------------------------------

1 International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba

Gastrin-releasing Peptide-producing Neurons
in the Suprachiasmatic Nucleus play an
Essential Role in Regulating Circadian
Rhythm-----------------------------------------------------------------Although a wide variety of optogenetic tools has beendeveloped, a specific opsin that can be stimulated
continuously for a long time duration (e.g., 24 hours)
has not been reported yet. To create a new optogenetic
tool for long term photostimulation, we modified acertain opsin (here called HPN1) and applied it for thehibernation-like hypometabolic state named "Qneurons-induced hypometabolic state (QIH)" in mice,
which we recently established. Optical stimulation of
HPN1 expressed in Q neurons induced hypothermia fora long period by illuminating quite low-power light withhigh spatiotemporal resolution. The HPN1-mediated
QIH recapitulated some kinetics of physiological

Ruth Li1, 2, Yoan Cherasse2, Ran Inoue3, Hisashi Mori3,
Arisa Hirano2, 4, Takeshi Sakurai2, 4
1 Graduate School of Comprehensive Human Sciences, Doctoral

Program in Medical Sciences, University of Tsukuba
International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba
3 Department of Molecular Neuroscience, University of Toyama
4 Faculty of Medical Sciences, University of Tsukuba
2-----------------------------------------------------------------Most organisms exhibit behavioral, physiological, and6

Katsuyasu Sakurai1, Kai Yashiro1, Ai Miyasaka1, Liu
Qinghua2, Takeshi Sakurai1changes observed in the natural hibernation. The
extremely high-sensitive optogenetics will allow us formuch more localized light stimulation than has beenachieved so far. This could also enable proper
optogenetic manipulation in experiments that would be
affected by light or heat, such as research on circadianrhythms, thermoregulation.
------------------------------------------------------------------

1

International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba
2 National Institute of Biological Sciences (NIBS)-----------------------------------------------------------------The neural mechanisms of female sexual motivation
have been unclear. To understand the neural basis offemale sexual motivation, we focused on dopamine
which is recognized as a crucial neural substrate forsexual behavior in both male and female animals. Here,
we examined the neural activities of VTA dopaminergic
neurons in a female mouse during sexual behavior byusing in vivo imaging system, fiber photometry. We
discovered the dramatic surge of the neural activity of
VTA dopaminergic neurons in the female mouse
immediately after male mouse ejaculation. Currently,
we are trying to reveal the function or biologicalmeaning of this dramatic dopamine surge of the female
mouse at the timing of male mouse ejaculation by
applying neural circuit-specific manipulation methods
involving histological, optogenetic, and chemogenetic
strategies. We will introduce those data at the meeting.
------------------------------------------------------------------

18------------------------------------------------------------------

Roles of monoamines and their regulatory
systems in motivation and arousal
Yoan Cherasse1, Shingo Soya1, Yuki Cleo Saito1,
Hiroshi Ichinose2, Takeshi Sakurai1
1International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba
2 Tokyo Institute of Technology School of Life Science andTechnology-----------------------------------------------------------------The exact mechanisms by which monoamines (MA)such as dopamine, serotonin, histamine andnoradrenaline regulate human behaviours and how theirabnormalities result in several psychiatric disorders
have been incompletely understood. Our project aims at
producing a constructive understanding between
monoamines and psychiatric disorders, with aparticularly focus on the depression-related symptoms
of motivation and arousal.
We generated genetically modified mice expressing Cre
recombinase under the control of MA promoters with aconditional KO mouse for GTP cyclohydrolase 1(GCH1), the gene indispensable for production of MA.
We then evaluated psychiatric disorders in addition to
sleep and circadian rhythm.
Our experiments on serotonin deficient mice showed areduced wheel-running activity compared to control
groups. The noradrenergic deficient mice exhibited
unusual increase of electromyogram during NREM
sleep. Mice deficient in dopamine showed a drastic
increase of wakefulness during the dark phase compared
to control mice, accompanied by a massive reduction of
sleep and low anxiety. Finally, mice depleted in MA ofneurons projecting to the prefrontal cortex exhibited anabnormal response to fear conditioning. We also
analyzed effect of icv orexin on sleep/wakefulness
behavior in MA-deficient mice.
------------------------------------------------------------------

20------------------------------------------------------------------

Dopamine dynamics in NAc-vs control malesexual behaviors in mice
Ai Miyasaka1, Yuka Terakoshi1, Katsuyasu Sakurai1,
Takeshi Sakurai1, Qinghua Liu1, 2
1International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba
2 National Institute of Biological Sciences, Beijing-----------------------------------------------------------------In male mice, sexual behaviors consist of a series ofstereotypic steps, such as sniffing, mounting,intromission, and ejaculation. However, the neuralmechanisms by which these behaviors are regulated andcoordinated have remained unclear. By using GRABDA sensor to monitor dopamine (DA) signaling in the
nucleus accumbens (NAc), we found thatDA
dynamics in the NAc ventral shell region (NAc-vs)corresponded nicely to sequential steps of male sexual
behaviors: 1) DA release is increased at the initiation ofmounting; 2) DA level fluctuates rhythmically during
intromission; 3) DA level is decreased right before
ejaculation. To characterize the functional importanceof DA dynamics in NAc-vs, we optogeneticallymanipulated the NAc-vs projecting dopaminergic
neurons in the ventral tegmental area. Interestingly,
inhibiting the dopaminergic neurons at the onset ofsniffing or mounting stopped sexual behaviors.
Moreover, inhibition of the dopaminergic neurons

19------------------------------------------------------------------

The anatomical and functional understandingof neural dynamics of VTA dopaminergic
neurons in female sexual behavior
7during intromission induced ejaculation, whereas
activation during intromission prolonged ejaculation
latency. These results suggest that DA dynamics in
NAc-vs contribute critically to the regulation of sexual
behaviors in male mice.
------------------------------------------------------------------

activity during social interaction and provide a novel
method for efficient c-Fos mapping across various brain
regions.
------------------------------------------------------------------

22------------------------------------------------------------------

21

Is increased REM sleep an adaptive responseor an exacerbating factor in stress resilience?

------------------------------------------------------------------

Analyses of the effects of prior social stress on
brain activity during social interaction in miceusing a novel semi-automated c-Fos mapping
programShinnosuke Yasugaki1, 2, Mitsuaki Kashiwagi1, Mika
Kanuka1, Masashi Yanagisawa1, Yu Hayashi1, 3
1

1, 2

International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba
2 Doctoral Program in Biomedical Sciences, Graduate School ofComprehensive Human Sciences，University of Tsukuba
3 Human Health Sciences, Graduate School of Medicine, Kyoto
University

1, 3Hibiki Okamura , Shinnosuke Yasugaki , Haruka
Suzuki-Abe1, 4, Yoshifumi Arai1, 5, Katsuyasu Sakurai1,
Masashi Yanagisawa1, Hotaka Takizawa6, Yu Hayashi1,

7-----------------------------------------------------------------Our sleep consists of two stages: rapid-eye-movement(REM) sleep and non-REM (NREM) sleep. Currently,
the function of REM sleep is largely unknown. Here, we
focused on depression. Patients with depression almost
inevitably exhibit sleep disorders, and in particular,
abnormalities in REM sleep, most commonly increases
in REM sleep amounts, are frequently observed. Thereare controversies as to whether increased REM sleep
helps recovery from depression or rather contributes toworsening of the depression-related symptoms. As afirst step to address this in mice, we aimed to analyze
the effects of 10 days of social defeat stress on sleep inmice. Similar to patients with depression, stress
exposure in mice increased REM sleep. Moreover, theincrease in REM sleep that observed after acute stress
exposure tended to attenuate after chronic stress
exposure. Based on these results, we next investigatedthe effects of artificially increased REM sleep at specifictimings by chemogenetic activation of the REM sleeppromoting neurons recently identified in our laboratory.
Surprisingly, we found that repeated activation of thisneurons has antidepressant effects on a decrease insocial interests induced by chronic social defeat stress.
Now we are trying to reveal how artificially increased
REM sleep contributed to those behavioral phenotypes.
Our future work will provide new insights to address thecausal relations between stress resilience in mice andREM sleep.
------------------------------------------------------------------

1

International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba
2 Ph.D. Program in Humanics, School of Integrative and Global
Majors, University of Tsukuba
3 Doctoral Program in Biomedical Sciences, Graduate School ofComprehensive Human Sciences, University of Tsukuba
4 Research and Development Center for Precision Medicine,
University of Tsukuba
5 Master’s Program in Medical Science, Graduate School ofComprehensive Human Science, University of Tsukuba
6 Faculty of Engineering, Information and Systems, University ofTsukuba
7 Department of Human Health Science, Graduate School of
Medicine, Kyoto University-----------------------------------------------------------------Depression is a major psychiatric disorder worldwide.
Stressful social experience is a factor for depression.
However, the precise mechanism that links social stressto depression remains unclear. Mice exhibit depressionlike phenotypes following chronic stress. Social defeat
stress (SDS) is one of the methods to apply chronic
stress to mice. Here, we investigated how chronic SDS
affects BALB/c mice. While BALB/c mice are reported
to show higher stress susceptibility compared to thewidely used C57BL/6 mice, there are few studies thatapplied SDS to BALB/c mice. We found that BALB/cmice showed depression-like states including reducedsocial interest after chronic SDS and the effects
remained even after 2 weeks. Furthermore, we aimed toinvestigate the brain regions that show altered activity
following social interaction in mice that underwent SDS
by means of c-Fos mapping. To this end, we establisheda novel method to conduct whole-brain c-Fos mapping
based on semi-automated image processing, and thescreening revealed several brain regions that showed
different c-Fos expression levels in stressed micecompared to the control mice. These results contributeto understanding how prior social stress affects the brain23------------------------------------------------------------------

Characterization of the neuronal activity of a
newly identified REM sleep-regulatingneuron using glass pipette extracellular unit

8

recording combined with optogenetics

5

Department of Neurology, Faculty of Medicine, Academic
Research Division, Univ of Toyama
6 Department of Human Health Science, Graduate School of
Medicine, Kyoto University

Yoshifumi Arai1, 2, Mitsuaki Kashiwagi1, Mika Kanuka1,
Takaya Suganuma1, Kaeko Tanaka1, Masanori
Sakaguchi1, Masashi Yanagisawa1, Yoshimasa Koyama3,
Yu Hayashi1, 4-----------------------------------------------------------------During rapid eye movement (REM) sleep, the cerebral
cortex becomes activated and produces vivid dreams.
Yet, it normally does not lead to motor output owing to
expression of muscle atonia. However, patients with
REM sleep behavior disorder (RBD) exhibit impaired
muscle atonia during REM sleep and frequently act outof their dreams. For example, they talk loudly or exhibit
violent movements such as hitting and kicking. RBD
patients often wake up following such movements,
which can lead to reduced sleep quality.
A majority of RBD patients co-suffer fromsynucleinopathies including Parkinson’s disease anddementia with Lewy bodies or eventually develop these
diseases within 10-14 years. Thus, RBD is considered
as a prodromal of synucleinopathies. Here, we aimed tounderstand the mechanisms underlying the link between
RBD and synucleinopathies and establish an RBD
mouse model for future development of effective
treatment for RBD. A rare G51D α-synuclein mutation
leads to production of toxic α-synuclein fibril andpatients carrying this mutation show rapid progressionof Parkinson’s disease. We produced and injected G51D
α-synuclein fibrils into the brainstem pontine tegmental
area in mice, and observed effects on atonia during
REM sleep, behavior and motor symptoms.
------------------------------------------------------------------

1

International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba
2 Master's Program in Medical Sciences, Graduate School of
Human Comprehensive Sciences, University of Tsukuba
3 Department of Science and Technology, Fukushima University
4 Graduate School of Medicine, Kyoto University-----------------------------------------------------------------Human beings spend about one-third lifetime in sleep.
Mammalian sleep comprises non-rapid eye movement
sleep (NREM) sleep and rapid eye movement (REM)
sleep. Previous studies revealed that the brainstem plays
a crucial role in the regulation of REM sleep. However,
the mechanism of REM is still largely unknown. Glass
pipette extracellular unit recording, a conventional
electrophysiological technique, has been used tocharacterize neuronal activity in the sleep/wake cycle.
This method is robust in terms of temporal resolution
and can record even a small neuron. A new REM sleepregulating subtype neuron has been identified in thedorsal pons and the activity of this neuron in thesleep/wake cycle should be characterized. However, it
has been demanding to record a specific subtype neuron
and a neuron that fires at low frequency in the method.
To overcome this limitation, we combined the optotagging method with glass pipette extracellular unit
recording and made a cell-type-specific unit recording.
We found that some of the REM sleep-regulatingneurons showed increased activity during REM sleep
and these neurons may play important role in REM
sleep regulation.
------------------------------------------------------------------

25------------------------------------------------------------------

Effects of chronic cell ablation in the preoptic
area on sleep-wake cycles
Ayako Imamura1, Koichi Miyatake2, 3, Akiko Ihara2,
Sakiko Honjoh2

24------------------------------------------------------------------

1 Ph.D. Program in Humanics, University of Tsukuba

Analysis of the mechanism of REM sleep
behavior disorder with focus on Parkinson’s
disease2 International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba
3 JSPS Research Fellow-----------------------------------------------------------------Sleep and wakefulness are controlled by specific brain
regions. These regions have been identified by classic
electric stimulation of non-specific cell types andacute neural activity manipulation. However, despite
many experiments that show that acute manipulation ofthese regions has a temporary effect on the sleep-wakecycles, chronic inhibition of neural activity often has noeffect.
This is thought to be due to adaptation andcompensation by other brain regions. In this study, we
focused on the preoptic area (POA), which has been

Ami Kaneko1, 2, Shinnosuke Yasugaki1, 3, Hideki
Hayakawa4, Kensuke Ikenaka4, Cesar Aguirre4,
Masashi Yanagisawa1, Kousuke Baba4, 5, Hideki
Mochizuki4, Yu Hayashi1, 6
1International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba
2 Ph. D. Program in Humanics, SIGMA, University of Tsukuba
3 Doctor Program in Biomedical Science, Graduate School ofComprehensive Human Science, University of Tsukuba
4 Department of Neurology, Graduate School of Medicine, Osaka
University

9

reported to promote sleep . With conventional methods
such as electrolytic lesions, axons passing through theregion are also affected, so functional inhibition is likely
not brain region-specific. The effect of brain regionspecific cell ablation in the POA on sleep-wake cycles
is unclear. Then, we investigated the effect of brain
region-specific cell ablation in the POA on the sleepwake cycle. In this method, we injected taCasp3 AAV,
which overexpresses procaspase-3 in a Cre-dependent
manner and induces apoptosis, in the POA -specific toavoid the effects of passing axons and ablate the cells inthe brain region. As a result, the fragmentation of sleepwake cycles was observed, the frequency of transitions
from wakefulness to sleep increased, and the duration of
sleep decreased. This effect was sustained for more thanfour weeks, and no compensation by other brain regions
was observed. These results suggest that POA is
necessary for consolidated sleep-wake cycles.
------------------------------------------------------------------

imaging with single-cell resolution will allow acomprehensive analysis of molecules in the brain andwill lead to a better understanding of brain function andstructure.
------------------------------------------------------------------

27------------------------------------------------------------------The role of hypothalamic supraoptic nucleus
in maintaining wakefulness.
Malyshevskaya Olga 1
1International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba-----------------------------------------------------------------This project arisen from accidental finding thatphotostimulation of supraoptic (SO) nuclei resulted in
prolonged wake state. Based on previous in-situ
hybridization data we assumed that SO nucleus is
involved in cannabinoid convulsive effects andtherefore investigated the effect of optogenetic
activation of SO nucleus. Bilateral photostimulation of
SO nuclei did not induce electrographic seizures,
however,
we
discoveredthat
prolonged
photostimulation (4 hours) resulted in increased
wakefulness (time in wake) with complete absence ofsleep (NREM and REM) during stimulation, which was
highly significant and resulted in sleep reboundaccompanied by increased NREM sleep amount anddelta NREM amount immediately after the end of
stimulation. We have also examined the effect of 8 hrs
photostimulation, which resulted in normal wake
behavior absence of sleep for the first 4 hours andsignificant decrease of sleep for the rest ofphotostimualtion time. This suggests that SO nucleus isstrongly involved in inducing and possibly maintaining
arousal state. To our knowledge this data has not beenreported by anyone yet. We are planning to deeply
investigate how activation of SO nuclei produces
arousal and what mechanism underlie this phenomenon
and to anatomically dissect inputs and outputs of sucharousal effect.
------------------------------------------------------------------

26------------------------------------------------------------------

Towardlabel-free molecular imaging ofwhole brain and brain cells using ultrabroadband
multiplex
CARS
microspectroscopy
Yusuke Murakami 1
1International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba-----------------------------------------------------------------Mapping the distribution of chemical bonds throughout
brain tissue is inevitable for understanding brain
functions. We applied label-free ultra-broadband
multiplex CARS (MCARS) spectroscopic imaging
system to construct the whole brain chemical molecularmap. Our experimental setup enabled us to acquire the
MCARS spectrum with the spectral coverage ofapproximately 3000 cm^-1 from the macro (tissue level)
and micro (cellular level) scales. As a result, the
MCARS images of the whole brain slice at CH_2
stretching and CH_2 scissoring bands were assigned as
lipids, which mainly visualize the white matter. Next,
we visualized the hippocampus and cortex on microscale. The ratio of the vibrational band due to CH_3
stretching to that of OH stretching indicates that thewater content of these neurons is more abundant thanother surrounding regions. In the cortex, localized lipidrich areas were also observed. This probably
corresponds to the distribution of lipid-rich glial cells
such as astrocytes and oligodendrocytes, which are oneof the most abundant cells in the brain. These results
were suggesting that MCARS spectroscopic images canbe used to distinguish cell types in the brain. In
conclusion, the whole brain label-free molecular28------------------------------------------------------------------

Recovering EEG Generators from Their
Collective Stochastic Interference
Javier Diaz1, Go-Eun Han1, Olga Malyshevskaya1,
Xifang Hayashi1, Juan-Carlos Letelier2, Hiroyasu
Ando3, Masashi Yanagisawa1, Kaspar E. Vogt1
1

10International Institute for Integrative Sleep Medicine (WPI-

encephalogram (EEG) as oscillations in the delta (0.5-4Hz) range. Slow wave activity is to date the bestindicator for homeostatic sleep regulation; it isincreased after prolonged waking and slowly reduced
during NREM sleep. The precise mechanisms
underlying sleep homeostasis and the generation of slow
waves are unknown. Activity-dependent neuronalcalcium influx has been hypothesized to play animportant role in generating slow oscillations and might
be involved downstream signaling that mediates sleep
function. Dihydropyridine blockers of L-type voltage
gated calcium channels (VGCCs) are in wide clinical
use to treat hypertension and other cardiovasculardisorders and are readily blood-brain-barrier (BBB)
penetrant. We therefore wanted to investigate theirpotential effect on slow wave generation andhomeostatic NREM sleep regulation. In-vivo twophoton imaging of cortical neurons showed larger
spontaneous calcium transients in slow wave sleep
compared to waking. Application of the dihydropyridine
calcium blocker nicardipine significantly reduced
cortical calcium transients, without affecting slow wave
generation. Nicardipine also did not affect the slow
wave activity over 24h following application. Timespent in slow wave sleep and episode duration were also
not affected. We conclude that despite evidence thatneuronal calcium influx may be involved in NREM
sleep function, blocking calcium entry through L-type
VGCCs does not interfere with slow wave generation or
regulation.
------------------------------------------------------------------IIIS), University of Tsukuba
2 Departamento de Biología, Facultad de Ciencias, Universidad
de Chile
3 Advanced Institute for Materials Research (AIMR), Tohoku
University.

-----------------------------------------------------------------Mainstream descriptions of EEG rely on its analysis in
the frequency domain and basically explain theobserved EEG oscillations as emerging from neuronalsynchronization processes. Traditionally, theseoscillations are categorized into spectral bands whose
correlates with different brain states are well established.
Recently, however, some alternative views of EEG
emphasize its arrhythmic nature, focusing on global
properties of the spectrum as, for example, the observed
1/f spectrum. Following this line of thought, in thepresent study we modeled EEG as a stochastic process
emerging from the superposition of arrhythmic pulses
described by arbitrary functions. Even though this kindof superposition generates colored Gaussian noise, fromour simulations we discovered that it is still possible tostatistically recover the shape of the underlying pulse.
Applying these concepts to actual EEG, unique patterns
characterizing NREM sleep, REM sleep, quiet wake,and active wake can be identified. Remarkably, using
the presented methodology, all these behavioral states
can be determined directly from EEG, not requiring
EMG. In addition, although it is well-known atropine
induces EEG delta waves that can be confused with
NREM sleep EEG, our method shows a distinctive
pattern for the atropine effect, very different from thosepatterns associated with NREM sleep or any otherphysiologic state.
------------------------------------------------------------------

30------------------------------------------------------------------The effects of olfactory stimulation during
REM sleep on dream emotionality: a study
focusing on individual difference in olfactory
perception

29------------------------------------------------------------------

Dihydropyridine calcium blockers do notinterefere with slow wave sleep
1

2

Satomi Okabe1, 2, Takashi Abe1

1,3

GoEun Han , Sumire Matsumoto , Javier Diaz ,Robert W. Greene1,4, Kaspar E. Vogt1

1
International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba
2 JSPS Research Fellowship for Young Scientists

1 International Institute for Integrative Sleep Medicine (WPI-IIIS),
University of Tsukuba, Tsukuba, Japan
2 Frontier Research Institute for Interdisciplinary Sciences,
Tohoku University, Sendai, Japan
3 Departamento de Biologia, Facultad de Ciencias, Universidad
de Chile
4 Department of Psychiatry & Neuroscience Peter O’Donnell
Brain Institute, UT Southwestern Medical Center, Dallas, TX,
USA-----------------------------------------------------------------Odors are useful stimuli in sleep research because its
rarely cause arousal. In addition, odor has anotherunique characteristic that there are large individual
differences of perception.
In the present study, we examined how the effects ofpresentation of olfactory stimuli during REM sleep on
dreams differed according to individual differences inodor perception.
We focused on the preference at study 1, and thefamiliarity at study 2, the effects of odor were stronger-----------------------------------------------------------------Slow wave or non-rapid eye movement (NREM) sleep
is tightly homeostatically regulated and essential forsurvival. Slow waves are observed in the electro-

11when the subjects were preferring and familiar to theodor, and their dream became more negative.
However, no such effect was found when the subjective
intensity was focused on at study 3.
These results, preference and familiarity related andsubjective intensity did not relate to the effect that makedream more negative, were confirmed at the pooled
analysis of study 2 and 3.
In our presentation, we will present these results andwhy preferred and familiar odor induced negative dreamand subjectively intense odor did not.
------------------------------------------------------------------LPS group than in the LPS-only group.Conclusions:
Sevoflurane preconditioning promotes recovery fromaltered sleep induced by systemic inflammation.
Activation of PPTg/LDTg is considered a mechanism
underlying sleep reintegration. The recovery
phenomenon shows potential for clinical application incases of sleep disturbances induced by systemic
inflammation..
------------------------------------------------------------------

32------------------------------------------------------------------

Validation experiment using in-home sleep
electroencephalography and its clinicalapplication31------------------------------------------------------------------

Recoveryfromsystemic-inflammationinduced altered sleep is potentiated by
sevoflurane preconditioning

Shigeru Chiba1, 2, Yoko Suzuki1, Takashi Abe1, Hideaki
Kondo3, Takashi Kambayashi1, 2, Masashi Yanagisawa1

Tsuyoshi Nemoto1, Irukayama-Tomobe Yoko2, Yuki
Hirose3, Hiromu Tanaka1, Genki Takahashi1, Satoshi
Takahashi4, Masashi Yanagisawa1, Takashi Kanbayashi1

International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba
2 Ibaraki Prefectural Medical Center of Psychiatry
3 Shunkaikai Inoue Hospital1-----------------------------------------------------------------To construct a system that can evaluate sleep asaccurately as polymsomnografy (PSG), the gold
standard for sleep evaluation, we developed an in-home
EEG. We conducted a series of experiments to validate
the accuracy of the device.The first study included 50
healthy subjects and verified the validity of sleep
evaluation for in-home EEG using simultaneous
measurements of in-home EEG, PSG, and Fitbit Charge
3, an accelerometer with HRV analysis, in the subjects.
The mean ± standard deviation (SD) of the accuracy andkappa coefficient of the in-home EEG, in which thescoring result of the PSG for the correct answer, was
86.8% ± 3.8% and 0.80 ± 0.05, respectively. The lowest
accuracy and kappa coefficient were 77.4% and 0.65,
respectively. The accuracy in stage N1 was 59.5%,
which was low, but good accuracy was obtained in theother sleep stages. The mean ± SD of the accuracy andkappa coefficient of the Fitbit was 69.5 ± 8.3% and 0.50
± 0.14, respectively, and the accuracy and kappa
coefficient of the Fitbit sleep stage scoring was
significantly lower (P < 0.05) than those of the in-home
EEG. Another empirical study of sleep assessment
including 100 patients with sleep disorders of breathing(SDB) using in-home EEG, has just begun. A highproportion of patients have been diagnosed as
obstructive sleep apnea syndrome (OSAS), andsimultaneous measurement of their sleep using in-home
EEG and PSG showed that the accuracy of in-home
EEG depends largely on their severity of the disease. In
patients with 5-15 AHI (apnea and hypopnea index), theaccuracy was as high as that of healthy subjects, but in

1

International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba
2 Medical Mycology Research Center (MMRC), Chiba University
3 Department of Anesthesiology, University of Tsukuba Hospital
4 Department of Anesthesiology, Katsuta Hospital-----------------------------------------------------------------Background: Despite extensive evidence on the organ
protective effects of sevoflurane, its effect on disturbed
sleep remains unclear. We hypothesized that sevofluranepreconditioning positively impacts disturbed sleep
caused by systemic inflammation.Methods: A mousemodel of lipopolysaccharide (LPS)-induced systemic
inflammation was employed to investigate the effects ofsevoflurane on sleep recovery. We evaluated symptoms
recoverythroughelectroencephalography/electromyography(EEG/EMG) and histological studies. The mice were
exposed to 2% sevoflurane before and after peritoneal
injection of LPS. The EEG/EMG were recorded for 24
h after the procedure. Brain tissue was harvested after
the sevoflurane/LPS procedure and was immunostainedusing
individual
antibodies
againstcholineacetyltransferase (ChAT) and Fos. We quantitatively
analyzed the ChAT-positive and ChAT/Fos doublepositive cells in the pedunculopontine tegmental
nucleusandlaterodorsal
tegmental nucleus(PPTg/LDTg). Results: Mice preconditioned with
sevoflurane showed a significant recovery in rapid eye
movement (REM) sleep following the LPS challenge.
They also demonstrated shorter REM latency, indicating
an early recovery from LPS-altered sleep. We observed
more ChAT/Fos double-positive cells in the
PPTg/LDTg in the sevoflurane preconditioning plus

12

patients with over 30 AHI, who are considered as having
severe OSAS, the accuracy was below 70%.
------------------------------------------------------------------

2 Researchand Development Center for Precision Medicine,
University of Tsukuba
3 Life Science Research Center, Technology Research Laboratory,
Shimadzu Corporation
4 Department of Anatomy, Faculty of Medicine, Toho University
5 Department of Molecular Genetics, University of Texas
Southwestern Medical Center, Dallas, TX, USA
6 Life Science Center for Survival Dynamics, Tsukuba Advanced
Research Alliance, University of Tsukuba
7 R&D Center for Frontiers of Mirai in Policy and Technology(F-MIRAI), University of Tsukuba

33------------------------------------------------------------------

Automatic sleep stage and arousal assessmentof sleep recordings from a portable IoT EEG
deviceJuan Carlos Neira1, Leo Ota2, Kazumasa Horie2,
Masashi Yanagisawa3, Hiroyuki Kitagawa3-----------------------------------------------------------------Sleep need is accumulated during wakefulness anddissipated during sleep. Sleep deprivation (SD) has beenused as a method to investigate the molecular changes
under high sleep need. However, SD induces changes
not only reflecting increased sleep need but also
inevitable stresses and prolonged wake state itself. The
Sleepy mutant mice exhibit constitutively high sleep
need despite sleeping longer, and have been useful as amodel of high sleep need. Here we conducted a crosscomparison of brain metabolomic profiles between SD
versus ad lib slept mice, as well as Sleepy mutant versus
littermate wild-type mice. 203 metabolite were
quantified in total, of which 43 metabolites showedsignificant changes in SD, whereas 3 did in Sleepy
mutant mice. The large difference in the number ofdifferential metabolites highlighted limitations of SD as
methodology. The cross-comparison revealed that adecrease in betaine and an increase in imidazole
dipeptides (IDs) are associated with high sleep need inboth models. Furthermore, we found that central
injection of IDs increased subsequent NREM sleep time,suggesting the possibility that IDs may participate in theregulation of sleep in mice.
------------------------------------------------------------------

1 PhD program in Humanics, University of Tsukuba
2

Center for Computational Sciences (CCS), University ofTsukuba
3 International Institute for Integrative Sleep Medicine (WPI-IIIS),
University of Tsukuba-----------------------------------------------------------------Since its inception, EEG recording and scoring has
become a standard procedure for sleep research andsleep diagnosis. EEG has traditionally been recorded ina laboratory setting, as part of the Polysomnogram(PSG), but recently it is possible to record it in otherspaces thanks to the advent of portable EEG devices.
EEG is scored following the R&K or AASM guidelines,
an activity that requires the visual inspection of eachsleep epoch in a whole sleep recording, a very time
consuming process that is prone to error. With theinterest of speeding up this process, different machinelearning models have been proposed, but they are
limited to specific tasks, such as stage scoring or arousal
scoring but not both; therefore, limiting the possibility
to evaluate other sleep variables. For this reason, I am
proposing a model called SAAS U-Net, for thesimultaneous assessment of sleep stages, arousal eventsand other sleep variables using the recordings of a
portable IoT EEG device, with the purpose of improving
the early diagnosis of sleep disorders, benefitting theusers of this device and at the same time, contributing to
the promotion of a better quality of sleep.
------------------------------------------------------------------

35------------------------------------------------------------------The novel intracellular signaling pathways forthe regulation of homeostatic sleep need
Tomohiro Kitazono1, Noriko Hotta-Hirashima1, Kanako
Iwasaki1, Tomoyuki Fujiyama1, Hiromasa Funato1, 2,
Masashi Yanagisawa1, 2

34------------------------------------------------------------------

Metabolomic and pharmacologic analyses ofbrain substances associated with sleep
pressure in mice

1 International Institute for Integrative Sleep Medicine (WPI-IIIS),

University of Tsukuba
2 Department of Anatomy, Faculty of Medicine, Toho University
3 Department of Molecular Genetics, University of Texas
Southwestern Medical Center
4 Life Science Center for Survival Dynamics, Tsukuba Advanced
Research Alliance (TARA), University of Tsukuba

Haruka Suzuki-Abe1, 2, Kazuhiro Sonomura3, Shinya
Nakata1, Kazuya Miyanishi1, AsmaaMahmoud1, Noriko
Hotta-Hirashima1, Chika Miyoshi1, Taka-Aki Sato2,
Hiromasa Funato1, 4, Masashi Yanagisawa1, 5, 6, 7-----------------------------------------------------------------Sleep is an ubiquitously conserved behavior, andhomeostatic sleep need is the fundamental element forsleep regulation. However, the molecular/cellular basis

1

International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba13

of sleep need remains unknown. Our recent study
suggested that changes of sleep need can be translated
into cumulative phosphorylation of certain brain
proteins, and SIK3 kinase has an essential role in thisregulatory mechanisms. In this study, to reveal themolecular/cellular basis of sleep need, we aim to
identify upstream and downstream components of SIK3.
As a possible upstream regulator of SIK3, we focusedon LKB1 kinase. We generated postnatal neuronspecific LKB1 knockout (nKO) mice, and LKB1 nKO
mice showed weaker sleep pressure. Furthermore, thisphenotype is recovered by constitutively active
mutation of SIK3. These results suggest that LKB1
regulates sleep/wake behavior by acting upstream of
SIK3.
To identify the downstream signaling pathway of SIK3,
we conducted in vitro substrate screening of SIK3 bykinase-oriented substrate screening (KIOSS) method(Nishioka et al., 2015) and the pathway analysis. These
results suggest that Rho-GTPase signaling pathway may
function downstream of SIK3 in sleep/wake regulation.
------------------------------------------------------------------(EMG) recording. At both baseline and following sleep
deprivation, young and older Cdkl5 KO mice exhibited
significantly increased sleep latency, shorter sleep
episode duration and frequent transitions between sleep
and wakefulness compared with their littermate controls,
which resemble sleep disturbances observed in human
CDD patients. The results suggest that Cdkl5 KO mousemodel may be a valuable genetic model for studying
sleep disruptions in CDD patients.
------------------------------------------------------------------

37------------------------------------------------------------------

OX2R-selective orexin agonism is sufficient tosuppress narcoleptic symptoms, cataplexy
and wake fragmentation, without inducing
drug-seeking behavior in mouse models
Hikari Yamamoto1, Yasuyuki Nagumo1, Yukiko
Ishikawa1, Yoko Irukayama-Tomobe1, Tsuyoshi
Nemoto2, Hiromu Tanaka2, Genki Takahashi2, Akihisa
Tokuda1, Tsuyoshi Saitoh1, Hiroshi Nagase1, Hiromasa
Funato1, 3, Masashi Yanagisawa1

36------------------------------------------------------------------

1

International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba
2 Department of Anatomy, Faculty of Medicine, Toho University
3 School of medicine, University of Tsukuba

Natural history study of sleep disturbances in
CDKL5 deficiency disorder mice
Cao Liqin1, Tingting Lou1, Xin Zhang1, Jing Ma1, 2,
Zhiqiang Wang1, 2, Teruyuki Tanaka3, Hiromasa Funato1,
Qinghua Liu1, 4, Masashi Yanagisawa1-----------------------------------------------------------------Acquired loss of orexin neurons causes a chronic sleep
disorder narcolepsy-cataplexy. Narcoleptic symptoms
are characterized by cataplexy and sleep/wakefragmentation. Orexin receptor agonist is expected as amechanistic treatment of narcolepsy. However, it has
been unclear whether the activation of only OX2R, orboth OX1R and OX2R, is required to replace theendogenous orexin functions in the brain. To examine
whether the selective activation of OX2R is sufficient toameliorate the cataplexy and sleep/wake fragmentation,
we compared the therapeutic efficacy by peptidic orexin
in narcolepric model mice. Here we concluded that
OX2R selective orexin agonism is sufficient toameliorate both cataplexy and wake fragmentation
without reward or reinforcing effects. These findings
provide a proof-of-concept for a safer mechanistic
treatment of narcolepsy-cataplexy through OX2Rselective agonism.
------------------------------------------------------------------

1 International Institute of Integrative Sleep Medicine (WPI-IIIS),

University of Tsukuba
Center for Life Sciences, School of Life Science andTechnology, Harbin Institute of Technology
3 Graduate School of Medicine, The University of Tokyo
4 National Institute of Biological Sciences
2 HIT-----------------------------------------------------------------CDKL5 deficiency disorder (CDD) is an X-linked
severe neurodevelopmental disorder caused bypathogenic mutations in the Cyclin-dependent kinaselike 5 (CDKL5) gene. Patients with CDD display avariety of clinic symptoms including early-onset
seizures, intellectual disability, and autism spectrum
disorder. The majority (>86%) of patients have sleep
problems such as difficulty falling asleep, sleep
fragmentation, frequent night awakenings, nightscreaming and excessive daytime sleepiness. Sleep
disturbances impair neural plasticity and exacerbate
illness state in patients with autism and epilepsy.
However, little is known about the sleep phenotypes in
Cdkl5 mutant mice. To this end, we characterizedbaseline sleep and recovery sleep after sleep deprivation
in young and older Cdkl5 knock-out (KO) male mice
and
their
wildtype
littermatesusing
electroencephalography (EEG) and electromyography38------------------------------------------------------------------

Pre-narcoleptics are moresuvorexant-induced cataplexy

prone

to

Mahesh K. Kaushik 1, Kosuke Aritake1, Yoan Cherasse1,

14

Aya Imanishi2, Takeshi Kanbayashi1, Yoshihiro Urade1,
3, Masashi Yanagisawa1, 4, 5, 6

University, Tokyo
4 Cardiovascular Respiratory Sleep Medicine, Juntendo
University Graduate School of Medicine
5 Department of Cellular Neurobiology, Brain Research Institute,
Niigata University
6 R & D Center for Frontiers of MIRAI in Policy and Technology,
University of Tsukuba

1 International Institute for Integrative Sleep Medicine (WPI-IIIS),

University of Tsukuba
2 Akita University Graduate School of Medicine
3 Isotope Science Center, The University of Tokyo
4 Department of Molecular Genetics, University of Texas
Southwestern Medical Center at Dallas
5 Life Science Centre, Tsukuba Advanced Research Alliance,
University of Tsukuba
6 R & D Center for Frontiers of MIRAI in Policy and Technology,
University of Tsukuba-----------------------------------------------------------------Rapid eye movement (REM) sleep behavior disorder(RBD), a parasomnia characterized by the loss of REM
atonia, is associated with a high probability of later
developing α-synuclein diseases. Novel transgenic
models of RBD are useful for promoting the discoveryof new RBD therapeutics. Here we generated transgenic
mouse lines lacking the glycine receptor alpha1 subunitin cholinergic neurons (ChGlyR-KO) or in a subset ofsomatomotor neurons (MnGlyR-KO). Both ChGlyRKO and MnGlyR-KO mice displayed excessive bodyand limb movements during REM sleep, including
jerking, kicking, punching, and chewing behaviors that
resemble human RBD. This phenotype was ameliorated
by the administration of clonazepam, a benzodiazepine
often used clinically to treat RBD symptoms in human,
indicating that the ChGlyR-KO mouse is a good animalmodel for studying RBD. We also found the dual orexin
receptor antagonist DORA22 was effective for treating
the RBD phenotype of ChGlyR-KO mice. Further, inhuman RBD patients, the clinically available orexin
antagonist suvorexant significantly reduced the loss of
REM atonia. Our observations indicate that orexin
signal blockade could be a potential treatment for RBD
symptoms.
-----------------------------------------------------------------------------------------------------------------------------------Orexins/hypocretins are key neuropeptides responsible
for the regulation of the central arousal and reward
circuits. Suvorexant, a dual orexin receptor antagonist,
induces sleep in mice, dogs, and humans. Suvorexant
induces sleep by reducing the orexin synthesis. We
hypothesize that individuals with low brain orexin
levels (pre-narcoleptics), maybe at a higher risk ofcataplexy by suvorexant. Heterozygous orexin
knockout (Ox-KO(-/+)) mice could mimic a perfect prenarcoleptic condition. As expected, a biochemical
analysis showed that Ox-KO(-/+) mice had asignificantly low level of orexin peptide as compared towild-type mice. The behavioral analysis showed thatsuvorexant administration in Ox-KO(-/+) mice induce
narcolepsy as evident by the appearance of sleep onset
REM (SOREM) sleep and cataplexy (direct transition
from wake to REM sleep) in a dose-dependent manner.
Sleep/wake did not show major changes. Our datasuggest that Ox-KO(-/+) mice have insufficiently low
brain orexin to show behavioral narcolepsy. However,upon suvorexant administration brain orexin may
further decrease, which along with the inhibition of theorexin receptors, results in the appearance of narcoleptic
symptoms in Ox-KO(-/+) mice.
------------------------------------------------------------------

40------------------------------------------------------------------

Learning and memory deficit in adultdreamless mice
Deependra Kumar1, Tomoyuki Fujiyama1, Takeshi
Kanda1, Chika Miyoshi1, Miyo Kakizaki1, Masanori
Sakaguchi1, Hiromasa Funato1, Masashi Yanagisawa1

39------------------------------------------------------------------

Orexin receptor antagonists ameliorate thesymptoms of REM sleep behavior disorder ina novel mouse model and in human patients

1 International Institute for Integrative Sleep Medicine (WPI-IIIS),

University of Tsukuba-----------------------------------------------------------------Earlier REM sleep deprivation studies in rodents
showed severe consequences of loss of REM sleep onlearning and memory. However, finding of those studies
is likely to affect by stressful sleep deprivation
procedures that are difficult to bypass. To address thisissue, we used Dreamless mice, which have inherently
reduced REM sleep (Funato et al., 2016). Further, weused genetically encoded tools for neural circuit
dissection to relevant cellular populations at learningand memory stages in dreamless mice. We crossed
dreamless mice with TRAP2 (Targeted Recombination

Mari Hondo1, Takuro Endo2, Ayako Mochizuki3,
Masanori Dantsuji3, Takeshi Kanda1, Makito Sato1,
Fusae Kawana4, Yukiko Ishikawa1, Kazue Suenaga2,
Shiro Nakamura3, Manabu Abe5, Tomio Inoue3, Kenji
Sakimura5, Hiromasa Funato1, Takeshi Sakurai1,
Masashi Yanagisawa1
1 International Institute for Integrative Sleep Medicine (WPI-IIIS),

University of Tsukuba
2 Tokyo Sleep Medical Center, Sleep Clinic Chofu
3 Department of Oral Physiology, School of Dentistry Showa

15

in Active Population) and Ai27 (tdTomato) to label
activated (TRAPed) cells at memory encoding,consolidation, and retrieval stages. Adult dreamlesstriple transgenic mice (3X: Drl -TRAP2-Ai27) and theirlittermates double transgenic (2X: TRAP2-Ai27)
underwent a classical contextual fear conditioning
paradigm. Next day when mice were placed in samecontext, dreamless 3X mice showed a significantly low(half) freezing percentage compared to littermates 2X
mice. Histological examination of dreamless brain
obtained at learning stage showed an overall low
number of TRAPed cells in hippocampus, particularly
in Dentate Gyrus region, indicating low hippocampal
activation. Hippocampal activation is required forsuccessful memory formation and storage, which isprobably affected due to low REM sleep amount indreamless mice.
------------------------------------------------------------------

point mutation in wild-type mice through genome
editing confirmed that the mutation was responsible for
REMS abnormality, suggesting an important role of
NALCN in REMS regulation. To elucidate theresponsible brain regions and neuronal subtypes
through which NALCN regulates REMS, we generated
flox and FLEx (flip-excision) knock-in mice bearing
Cre-dependent loss-of-function and gain-of-function
Nalcn alleles, respectively. In Nalcn-FLEx mice, we
confirmed that the mice crossed with a systemic Creexpressing line Actb-iCre phenocopied the Dreamlessmice on electroencephalogram and electromyogram(EEG/EMG) analyses. In Nalcn-flox mice, we
confirmed a neuronal subtype-specific deletion of Nalcn
mRNA in adult brain tissues. Recently we observed that
NALCN has distinct roles in forebrain and ponsmedulla regions for REM sleep regulation, with Foxg1IRES-Cre or En1-Cre lines. Now we are analyzing thedetailed sleep phenotype of these two Nalcn geneticallymodified mice with sleep stage scoring.
------------------------------------------------------------------

41------------------------------------------------------------------NALCN in the forebrain and pons-medulla
regions have distinct roles in REM sleep
regulation

42------------------------------------------------------------------

Sleepy mouse as a model of idiopathic
hypersomnia

Tomoyuki Fujiyama1, Satomi Kanno1, Noriko
Hirashima Hotta1, Miyo Kakizaki1, Aya Ikkyu1, Yosuke
Tsuneoka2, Manabu Abe3, Ayako Mochizuki1, Patricia
Seoane-Collazo1, Takehiro Miyazaki1, Seiya Mizuno4,
Chika Miyoshi1, Takeshi Kanda1, Masahiko Watanabe5,
Kenji Sakimura3, Satoru Takahashi4, Hiromasa Funato1,
2, Masashi Yanagisawa1, 6, 7

Asmaa Elhosainy Mohamed Mahmoud 1, 2, Haruka
Suzuki-Abe1, Mahesh Kaushik1, Staci Jakyong Kim1,
Tsuyoshi Saitoh1, Yukiko Ishikawa1, Noriko HottaHirashima1, Chika Miyoshi1, Hiromasa Funato1,
Masashi Yanagisawa1, 3, 4

1 International Institute for Integrative Sleep Medicine (WPI-IIIS),

1 International Institute for Integrative Sleep Medicine (WPI-IIIS),

University of Tsukuba
2 Department of Anatomy, Toho University
3 Department of Cellular Neurobiology, Brain Research Institute,
Niigata University
4 Laboratory Animal Resource Center, University of Tsukuba
5 Department of Anatomy, Hokkaido University
6 Life Science Center for Survival Dynamics, Tsukuba Advanced
Research Alliance (TARA), University of Tsukuba, Tsukuba
7 Department of Molecular Genetics, University of Texas
Southwestern Medical Center

University of Tsukuba
2 Doctoral Program in Medical Sciences, University of Tsukuba
3 Life Science Centre for Survival Dynamics, Tsukuba Advanced
Research Alliance (TARA), University of Tsukuba
4 Department of Molecular Genetics, University of Texas
Southwestern Medical Center at Dallas-----------------------------------------------------------------Idiopathic hypersomnia (IH) is a neurologic disordercharacterized by excessive daytime sleepiness, longnight sleep, non-refreshing naps, and difficulty of
awakening after naps or after night sleep, which
presents as “sleep drunkenness.” Daytime sleep latency
is shortened in IH patients, as typically assessed bymultiplesleep
latency
test(MSLT). The
pathophysiology of IH is still unknown and there is no
approved drug for this disorder. The Sleepy mutant
mouse, a mouse pedigree with a splicing mutation in the
Sik3 protein kinase gene, has longer sleep time andincreased sleep need compared with wild-type mice.
Therefore, we aimed to validate the Sleepy mouse as amouse model for IH. We used a mouse version of MSLT
and investigated sleep drunkenness in Sleepy mice. In
addition, we aimed to evaluate the effectiveness of-----------------------------------------------------------------Although REM sleep (REMS) is ubiquitous in
mammals, the molecular/neural mechanism of REMS
regulation remains unknown. We performed a largescale screening of ENU-mutagenized mice in order to
identify genes regulating sleep/wake behavior, andestablished the Dreamless mutant pedigree exhibiting~50% reduction in 24-h REMS amount. We identified asingle nucleotide substitution specific to Dreamlessmutant mice within the exon 9 of the Nalcn gene. The
mutation leads to a single amino acid substitution(N315K) of the NALCN protein, a voltage-independent,
non-selective leak cation channel. Introducing the same16

orexin agonists in treating the sleepiness symptom of the
Sleepy mouse. Sleepy mice showed normal sleep latency
compared with wild-type mice in the light phase andshorter sleep latency for the first 3 trials of the MSLT in
the dark phase. They also showed reduced decay of
NREM
delta
densityduringwakefulness.
Intracerebroventricular injection of orexin-A promoted
wakefulness for 2 h and 3 h after injection in Sleepy
mice and wild-type mice, respectively. Moreover,Sleepy mice but not wild-type mice showed a sleep
rebound after the orexin-A-induced wakefulness.
Intraperitoneal injection of the small-molecule orexin
agonist YNT-185 promoted wakefulness for 2 h in
Sleepy mice. These results indicate the validity of the
Sleepy mutant mouse as a model for idiopathic
hypersomnia, and the effectiveness of orexin agonists intreating sleepiness due to causes other than orexin
deficiency, which makes them potential drugs to
improve sleepiness in sleep disorders other thannarcolepsy.
------------------------------------------------------------------

1 International Institute for Integrative Sleep Medicine (WPI-IIIS),

University of Tsukuba
2 Department of Anatomy, Faculty of Medicine, Toho University
3 Department of Molecular Genetics, University of Texas
Southwestern Medical Center
4 TARA, University of Tsukuba-----------------------------------------------------------------For technical reasons, few studies have been conducted
to examine sleep/wakefulness during infancy using
electroencephalogram
andelectromyogram(EEG/EMG). Therefore, the significance of sleep during
infancy remains enigmatic and there are many
unresolved issues regarding sleep during postnatal
development, such as the onset of sleep abnormalities
observed in adult mice. Here, we developed a method torecord EEG/EMG from P21 immediately after weaning
to P57 every 4 days. Using this system, we examinedwhen SIK3 Sleepy mutant mice (SLP) start to exhibit
the long sleep time observed in adulthood. In addition,
EEG/EMG recordings were performed on these SLP
mutant mice and their wild-type littermates at 10-13
weeks and 30-33 weeks of age. At P21, whensleep/wake behavior was immature, there was no
significant difference in NREMS time between SLP
mutant and wild-type littermates. Subsequently, the SLP
mice showed a constant increase in NREM sleep timefrom P25, while the wild type showed a slight decrease.
Furthermore, weekly weight measurements from 4 to 30
weeks of age confirmed that SLP mice begin to become
obese after the onset of hypersomnia.
------------------------------------------------------------------

43------------------------------------------------------------------

Electrophysiological analysis of ion channel
mutations in mice with REM sleep
abnormalities
Yuki Taira1
1 International Institute for Integrative Sleep Medicine (WPI-IIIS),

University of Tsukuba-----------------------------------------------------------------We will perform a functional analysis of a novel mousefamily with REM sleep abnormalities that was
discovered in a previous study by random mutagenesis
and EEG analysis.
The gene responsible for the sleep abnormalities in thismouse family has been identified as an ion channel, but
how the mutation changed the biophysical properties of
the ion channel as a result of the mutation is still
unknown. The purpose of this study is to characterize
the biophysical properties of this mutant ion channel
using the patch clamp method.
------------------------------------------------------------------

45------------------------------------------------------------------

SIK3 in different hypothalamic
mediates whole-body energy balance

areas

Patricia Seoane-Collazo1, 2, Masashi Yanagisawa2,
Hiromasa Funato1, 3
1 International Institute for Integrative Sleep Medicine (WPI-IIIS),
University of Tsukuba
2 Cimus, Santiago de Compostela University
3 Department of Anatomy, Faculty of Medicine, Toho University-----------------------------------------------------------------In addition to its role in determining sleep need, Saltinducible kinase 3 (Sik3) is likely to regulate energy
homeostasis since mice mutant for exon 13 of the Sik3
gene (Sleepy) display an obese phenotype. The main
objective of this project is to identify the role of SIK3 inthe central nervous system in the regulation of energybalance. To achieve this, neuron-specific mutant micelines for the Sleepy gene were generated, as well as viral
genetic approaches in Sik3-ex13 floxed mice were used
to identify the brain regions involved in SIK3 actions.
Our data showed that Vglut2-specific Sleepy mice also
show an obese phenotype, suggesting dependency of44------------------------------------------------------------------

Sleep/wakefulness and body weight growthfrom infancy to adulthood in a hypersomnia
model, Sleepy mutant mouse
Noriko Hotta-Hirashima1,Chika Miyoshi1, Aya Ikkyu1,
Miyo Kakizaki1, Satomi Kanno1, Kumi Ebihara1,
Hiromasa Funato1,2 , Masashi Yanagisawa1

17

glutamatergic signaling. SIK3 gain of function, by AAV
injection in Sik3-ex13 floxed mice, in the ventromedial
nucleus of the hypothalamus induced feedingindependent increase in body weight, associated withaltered glucose homeostasis. On the other hand, SIK3 inthe paraventricular nucleus of the hypothalamus caused
hyperphagia and reduced energy expenditure leading to
an obese phenotype. Overall, these data suggest a roleof SIK3 in the regulation of energy metabolism in a
nucleus-specific manner.
------------------------------------------------------------------------------------------------------------------------------------

47------------------------------------------------------------------

Sik3 regulates sleep need via glutamatergic
neurons in cerebral cortex
Fuyuki Asano1, Tomoyuki Fujiyama1, Chika Miyoshi1,
Tomohiro Kitazono1, Noriko Hotta-Hirashima1, Miyo
Kakizaki1, Aya Ikkyu1, Satomi Kanno1, Shinya Nakata1,
Nodoka Asama1, Seiya Mizuno2, Fumihiro Sugiyama2,
Satoru Takahashi1, 2, Hiromasa Funato1, 3, Masashi
Yanagisawa1, 4, 546------------------------------------------------------------------

Where does Sleepy mutation of SIK3 cause
sleep phenotypes?

1 International Institute for Integrative Sleep Medicine (WPI-IIIS),

University of Tsukuba
2 Transborder Medical Research Center, University of Tsukuba
3 Department of Anatomy, Faculty of Medicine, Toho University
4 Department of Molecular Genetics, University of Texas
Southwestern Medical Center
5 Life Science Center for Survival Dynamics, Tsukuba Advanced
Research Alliance, University of Tsukuba

Kanako Iwasaki1, Tomoyuki Fujiyama1, Shinya Nakata1,
Minjeong Park1, Chika Miyoshi1, Noriko HottaHirashima1, Aya Ikkyu1, Miyo Kakizaki1, Yukiko
Ishikawa1, Fumihiro Sugiyama2, Seiya Mizuno2,
Manabu Abe3, Kenji Sakimura3, Satoru Takahashi2,
Hiromasa Funato1, 4, Masashi Yanagisawa1, 5, 6-----------------------------------------------------------------Sleep is regulated by sleep need in a homeostatic
manner. We recently identified Sik3 as a gene regulating
sleep/wakefulness through forward-genetics approach.
The pedigree, Sleepy, which has a splicing mutation
Sik3(Slp) in Sik3 resulting in the skipping of exon 13,
exhibits increased sleep need and prolonged sleep time.
On the other hand, the SIK3 kinase activity is tightly
regulated by phosphorylation of T221 in the kinasedomain T-loop. Interestingly, the phosphorylation of
T221 is increased in wild-type mice after sleep
deprivation, indicating SIK3 kinase activity increases inmice with a higher sleep need. However, how SIK3
kinase activity is involved in sleep regulation remains
unknown.
Here, we report the functional analysis of SIK3 kinase
activity for sleep/wake regulation using mice expressing
a non-phosphorylatable T221A or phosphomimetic
T221E mutant of SIK3(WT) or SIK3(SLP), respectively.
Our EEG-EMG-based sleep analysis of those mutant
mice and biochemical assays of those mutant SIK3
proteins indicate that SIK3 kinase activity regulates
sleep need and is required to increase sleep amountfollowing another functional alternation such as proteinprotein interaction.
------------------------------------------------------------------

1 International Institute for Integrative Sleep Medicine (WPI-IIIS),

University of Tsukuba
2 Laboratory Animal Resource Center, University of Tsukuba
3 Department of Animal Model Development, Brain ResearchInstitute, Niigata University
4 Department of Anatomy, Faculty of Medicine, Toho University
5 Department of Molecular Genetics, University of Texas
Southwestern Medical Center
6 Life Science Center, Tsukuba Advanced Research Alliance,
University of Tsukuba-----------------------------------------------------------------Recently, we found a gain-of-function Sleepy (Slp)mutation in the Sik3 gene, which produces the mutant
SIK3(SLP) protein, increases sleep amount and nonREM sleep (NREMS) EEG delta density, an index of
sleep need. However, it remains to be elucidated where
SIK3(SLP) increase sleep amount and NREMS EEG
delta density, since SIK3 is expressed in various tissues.
Here, we investigated whether SIK3(SLP) in matureneurons is sufficient for the sleep phenotypes of Sleepy
mutant mice with Synapsin1CreERT2; Sik3Sleepy-flox
mice. Tamoxifen administrated Synapsin1CreERT2;
Sik3Sleepy-flox mice exhibited increased NREMS timeand NREMS EEG delta density. It suggested that SIK3
plays roles regulating sleep amounts and sleep need inmature neurons.
Furthermore, we explored neural populations which
increase NREMS time upon SIK3(SLP) expression with
AAV vectors. We found that SIK3(SLP) expression inmedial parts of hypothalamus increased NREMS, but
not NREMS EEG delta density. It implies that neural
populations that increase NREMS and NREMS EEG
delta power in Sleepy mutant mice are not identical.

48------------------------------------------------------------------

Molecular mechanisms for SIK3(Sleepy)mediated sleep/wake regulation
Shinya Nakata1, Haruna Abe-Komiya1, Tomoyuki
Fujiyama1, Chika Miyoshi1, Aya Ikkyu1, Fuyuki Asano1,
18

Seiya Mizuno2, Fumihiro Sugiyama2, Satoru Takahashi2,
Hiromasa Funato1, 3, Masashi Yanagisawa1

containing a well-conserved protein kinase A (PKA)phosphorylation site, serine 551. The skipping of exon
13 results in a deletion of 52 amino acids including S551.
Also, Sik3 S551A knock-in mice showed reduced total
wake time and increased sleep need. These results
suggest that the existence of S551, a PKA recognition
site, is crucial for the normal sleep/wake regulation andmaintenance of daily sleep need.
In SIK3, there are three PKA recognition site, threonine
469, serine 551 and serine 674. To examine whether thephosphorylation of T469 and S674 of SIK3 is requiredfor proper sleep/wake behavior, we generated mutant
mice in which SIK3 T469 and SIK3 S674 were
substituted by alanine through the CRISPR/Cas9
method. Sik3 T469A mice showed increased NREM
sleep time and NREM sleep delta power, an index for
sleep need. Sik3 S674A mice showed no changes in
NREM sleep time and NREM sleep delta power. Thesefindings indicate the PKA recognition sites of SIK3,
especially T469 and S551 are required for regulation of
sleep/wake behavior.
------------------------------------------------------------------

1 International Institute for Integrative Sleep Medicine (WPI-IIIS),

University of Tsukuba
2 Faculty of Medicine, University of Tsukuba
3 Department of Anatomy, Toho University-----------------------------------------------------------------Sleep is regulated by sleep need in a homeostatic
manner. We recently identified Sik3 as a gene regulating
sleep/wakefulness through forward-genetics approach.
The pedigree, Sleepy, which has a splicing mutation
Sik3(Slp) in Sik3 resulting in the skipping of exon 13,
exhibits increased sleep need and prolonged sleep time.
On the other hand, the SIK3 kinase activity is tightly
regulated by phosphorylation of T221 in the kinasedomain T-loop. Interestingly, the phosphorylation of
T221 is increased in wild-type mice after sleep
deprivation, indicating SIK3 kinase activity increases inmice with a higher sleep need. However, how SIK3
kinase activity is involved in sleep regulation remains
unknown.
Here, we report the functional analysis of SIK3 kinase
activity for sleep/wake regulation using mice expressing
a non-phosphorylatable T221A or phosphomimetic
T221E mutant of SIK3(WT) or SIK3(SLP), respectively.
Our EEG-EMG-based sleep analysis of those mutant
mice and biochemical assays of those mutant SIK3
proteins indicate that SIK3 kinase activity regulates
sleep need and is required to increase sleep amountfollowing another functional alternation such as proteinprotein interaction.
------------------------------------------------------------------

50------------------------------------------------------------------

Loss of canonical Hdac4 signaling leads todysregulated NREMS
Staci Jakyong Kim1

1 International Institute for Integrative Sleep Medicine (WPI-IIIS),

University of Tsukuba-----------------------------------------------------------------Sleep is observed universally across various species
from nematodes to mammals and the properly
maintained sleep is directly linked to the general wellbeing of the organism. Much advances has been madein the identification of the distinct groups of neuronal
population that are involved in the transitions between
sleep and wake states. These networks of neurons candrive vigilance stage switches and changes in neuronal
activity related to sleep and wakefulness. Despite thegrowing interest and findings in the maintenance ofsleep/wakefulness,theintracellular
regulatory
mechanism of the response to sleep need changes
remains largely unknown.
Our EEG/EMG-based forward genetics approach inmouse sleep/wake behavior has successfully identified
several sleep regulatory genes. We previously reported
a long-sleep pedigree carrying a gain-of-function
mutant allele, Sleepy, in the Sik3 gene and showed SIK3
as a central regulator of sleep homeostasis. Sik3-Sleepy
mice showed marked increase in daily total non-REM
sleep (NREMS) sleep, as well as inherent increase inslow-wave activity during NREMS. Our effort inelucidating the key component of sleep/wakemodulation has continued with discoveries of othersleep regulating genes. Sleepy2 is one of such mutant

49------------------------------------------------------------------

Sleep/wake behavior of mice lacking PKA
phosphorylation site in SIK3
Minjeong Park1, Takato Honda2, Chika Miyoshi1,
Tomoyuki Fujiyama1, Miyo Kakizaki1, Aya Ikkyu1,
Seiya Mizuno3, Satoru Takahashi3, Hiromasa Funato4,
Masashi Yanagisawa1
1 International Institute for Integrative Sleep Medicine (WPI-IIIS),

University of Tsukuba
2 Department of Brain and Cognitive Sciences, Massachusetts
Institute of Technology
3 Laboratory Animal Resource Center, University of Tsukuba
4 Department of Anatomy, Toho University School of Medicine-----------------------------------------------------------------We previously identified a kinase, SIK3, as an important
sleep regulator through screening of randomly
mutagenized mice. Mice that express mutant SIK3
lacking the 52 amino acids encoded by exon 13 showeda decrease in wake time and an increase in NREM sleep
time. SIK3 is an AMPK-family protein kinase19

pedigrees with a loss-of-function of HDAC4. The
heterozygous mutant mice showed increase in both
NREMS time and slow-wave activity during NREMS.
Importantly, HDAC4 is known as a primary target of
SIK3, where the phosphorylation by SIK3 of aconserved serine residue is essential in the nuclear-tocytoplasmic transfer of HDAC4. Indeed, mice with aphosphodeficient mutation in this residue of HDAC4
showed an opposite phenotype: these mice showed
increase in daily wake time and significant decrease in
the slow-wave activity during NREMS. Taken together,sleep/wake maintenance and proper response to sleep
need changes require canonical signaling of SIK3HDAC4.
For further understanding of specific neuronal network
where the SIK3 and HDAC4 may function, we
employed a neuronal type-specific manipulationengineered by Cre-loxP recombination. From thecomparison of various Cre reporter lines with Hdac4flox
or Sik3-Sleepyflox mice, we discovered the NREMS timeand slow-wave activity during NREMS are modulatedby distinct neuronal types. Our results show that SIK3HDAC4 mediated regulation of the amount and qualityof sleep are controlled by discrete neuronal network in
type- and region-specific manner.
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