Poster Session
The 11th Annual WPI-IIIS Symposium～Deciphering the Mysteries of Instinctive Behaviors～

Data Blitz by Poster Presenters

13:40 – 14:55

Poster Presentation

14:55 – 16:00

Poster list#

Presenter

Title

1

Yusuke Iino

Synaptic plasticity in prefrontal cortex regulates sleep

2

Ayako Imamura

Both ablation and activation of POA cause sleep fragmentation

3

Prokofeva Kseniia

Regulation of Sleep and Wakefulness by Neuronal Circuits linking Ventrolateral Preoptic Nucleus andLateral Hypothalamic Area

4

Zhang Zhongwen

GABAergic neurons in the BNST projecting to the DpMe induce immediate transition from NREM sleep toarousal5

Ruth Li

Gastrin Releasing Peptide (GRP)-producing Neurons in the Hypothalamic Suprachiasmatic Nucleus (SCN)Mediates Photic Entrainment

6

Tohru Takahashi

24 hours QIH induction by OPN4-based optogenetics

7

Ai Miyasaka

Male sexual behaviors are regulated by dopaminergic signaling in NAc in mice

8

Susumu Umezawa

Ventral tegmental area dopaminergic neurons mediate sleep reduction induced by ablation of thesurrounding GABAergic neurons

9

Koustav Roy

Optochemical control of slow-wave sleep in the nucleus accumbens by a photoactivatable allostericmodulator of adenosine A2A receptors

10

Rintaro Otani

Development of photocaged drugs targeting adenosine 2A receptor

11

Keita Kajino

Design and synthesis of novel κ opioid receptor agonists with bicyclo [2.2.2] octane skeleton

12

Kao Hashimoto

Analysis of orexinergic function in chronic pain-like states

13

Insung Park

Orexin receptor antagonist modulates sleeping energy metabolism without overt effects on sleep
architecture

14

Yue Cao

Does different-intensity exercise affect the rest-activity rhythms among older adults?

15

Saki Tsumoto

Automatic Sleep Analysis for Individuals with Disordered Sleep Patterns

16

Javier Diaz

Assessing Wakefulness Intensity as a Counterpart to Sleep Depth During the Sleep-Wake Cycle

17

Zhiwei Fan

Binaural beats at slow frequencies less than 1 Hz shorten the latency of slow-wave sleep

18

Kazuki Sato

Targeted memory reactivation during REM sleep selectively enhances brain responses to unpleasantimages19

Hibiki Okamura

Exploration of a novel mechanism of sleep regulation that depends on feeding condition

20

Ami Kaneko

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

21

Sakthivel Srinivasan

Transient recruitment of adult-born neurons for fear memory consolidation in REM sleep

22

Pablo Vergala

CaliAli: neuronal tracing utilizing vasculature in calcium imaging

23

Noriko Hotta-HirashimaSex difference in sleep/wakefulness from infancy to adulthood in a hypersomnia model, Sleepy mutant
mouse

24

Liqin Cao

Sleep disturbances in mice with a CDKL5 kinase-dead missense mutation, a novel mouse model ofneurodevelopmental disorder

25

Tomoyuki Fujiyama

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

26

Minjeong Park

Sleep/wake behavior of mice lacking PKA-phosphorylation sites of SIK3

27

Tomohiro Kitazono

Upstream and downstream pathways of SIK3 in the regulation of sleep and wakefulness

28

Shinya Nakata

Molecular mechanisms for sleep/wake regulation by SIK3 kinase signaling

29

Fuyuki Asano

SIK3-HDAC4 in the suprachiasmatic nucleus regulates the timing of arousal at the dark onset and circadianperiod in mice

30

Kanako Iwasaki

SIK3 regulates sleep amount through hypothalamic nuclei

1wake cycles and chronic inhibition of neural activity inthese areas has little effect. These may have been an
adaptation and compensation by other brain regions.
The preoptic area (POA) has long been recognized as
the sleep center first proposed by von Economo.
However, a recent optogenetic study found thatphotostimulation of POAGAL neurons at 10 Hz failed toincrease sleep and surprisingly increased wake. These
results call into question whether POA neurons promote
sleep.
In this study, we investigated the effects of brain regionspecific cell ablation and chemogenetic activation in the
POA on the sleep-wake cycle. In this ablation method,
we injected taCasp3 AAV, which overexpresses
procaspase-3 in a Cre-dependent manner and induces
apoptosis, in the POA-specific to avoid the effects ofpassing axons and ablate the cells in the brain region.
The cell ablation results showed the fragmentation of
the sleep-wake cycle and a decrease in sleep duration.
This effect was sustained for more than four weeks, andno compensation by other brain regions was observed.
Next, chemogenetic activation of POA by
intraperitoneal injection of CNO (Clozapine-N-Oxide)surprisingly observed the fragmentation of the sleepwake cycle. These results suggest that the POA may not
be simply a sleep-promoting region, as previously
reported.
------------------------------------------------------------------------------------------------------------------------------------

Synaptic plasticity in prefrontal cortex
regulates sleep
Yusuke Iino1, Takeshi Sawada1,2, Kensuke Yoshida1,2,3,
Chika Shimizu1, Motoki Juichi1, Masashi Yanagisawa1,
Takeshi Sakurai1, Haruo Kasai2, Shoi Shi1,2
1 University of Tsukuba
2 University of Tokyo
3 RIKEN, CBS-----------------------------------------------------------------The synaptic strength in the cerebral cortex has beenshown to fluctuate according to the sleep-wake cycle.
Several pieces of empirical evidence show that synapses
are primarily potentiated during wakefulness andsubsequently downregulated during sleep. However, thecausal relationship between synaptic potentiation andsleep has remained elusive. In the present study, wedeveloped novel molecular tools to induce structural
long-term potentiation and depression (sLTP and sLTD).
By utilizing a chemical inducer of dimerization, we
confirmed that specific effector molecules could beaccumulated in dendritic spines. Accumulation ofmolecule X or Y in dendritic spines resulted in sLTP or
sLTD, respectively, in dissociated cortical neurons. By
using the two photon imaging, we verified that thismanipulation induced sLTP in cortical neurons in vivo.
Using this tool, we found that sLTP in the excitatoryneurons of the medial prefrontal cortex, but not in theinhibitory neurons, causally increased non-rapid eye
movement (NREM) sleep in mice. Conversely, synaptic
potentiation in excitatory neurons in the hippocampus
decreased NREM sleep, suggesting a circuit-dependent
regulation of NREM sleep by synaptic potentiation.
This study provides evidence for a causal link between
synaptic potentiation and the regulation of NREM sleep.
------------------------------------------------------------------

3------------------------------------------------------------------

Regulation of Sleep and Wakefulness by
Neuronal Circuits linking Ventrolateral
Preoptic Nucleus and Lateral Hypothalamic
Area
Kseniia Prokofeva1, Yuki C. Saito1, Yasutaka Niwa1,
Seiya Mizuno1, Satoru Takahashi1, Arisa Hirano1,
Takeshi Sakurai12

1 University of Tsukuba, Tsukuba, Japan-----------------------------------------------------------------------------------------------------------------------------------Uncovering of connectivity and functional relationships
between the preoptic area (POA) and lateralhypothalamic area (LHA) is crucial for comprehensive
mechanistic understanding of regulation of sleep andwakefulness. GABA- and galanin (GAL)-producing
neurons in the ventrolateral preoptic nucleus (VLPO) of
the POA are known to participate in promotion of sleep,
while orexin (hypocretin)-producing neurons (orexin
neurons) in the LHA play a vital role in maintenance of
wakefulness. Using retrograde rabies virus-mediatedtracing combined with in situ hybridization, we foundthat vesicular GABA transporter (Vgat)- and galanin(Gal)-expressing neurons in the VLPO makemonosynaptic inputs to orexin neurons in the LHA.

Both ablation and activation of POA cause
sleep fragmentation
Ayako Imamura1, Koichi Miyatake2, Akiko Ihara2,
Sakiko Honjoh2
1 Ph.D. Program in Humanics, University of Tsukuba
2International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba-----------------------------------------------------------------Sleep and wakefulness are controlled by specific brain
regions. These regions have been identified by classicalelimination of brain regions or by acute manipulation ofneural activity. However, acute manipulation of theseregions often has only a temporary effect on the sleep1

wakefulness. Retrograde neuronal circuit tracing with amodified rabies virus vector demonstrates neuronalconnectivity between the BNST and DpMe. Last, wehypothesize that some GABAergic interneurons in the
DpMe (GABADpMe) might inhibit GLUTDpMe neurons
and receive inhibitory projections from GABABNST
neurons. In conclusion, we could decipher the neuronal
circuit responsible for the promotion of wakefulness
between the BNST and the DpMe. Our data could
provide precious insight to improve sleep disorders in
people with stress-related sleep disorder.
------------------------------------------------------------------

Notably, over half (56.3±8%) of the VLPO inputs were
Vgat- and Gal-double-positive. Next, we examined
input neurons of the GABA- and GAL-producing
neurons in the VLPO projecting to the LHA anddetermined that the POA and LHA contained the biggest
numbers of inputs. Among other upstream brain areas
were the nucleus accumbens, bed nucleus of stria
terminalis, and wake-promoting regions, such as the
tuberomammillary
nucleus.
Further,using
multidimensional scaling and Spearman correlation
analyses of input neurons, we suggested that the GABAand GAL-producing VLPO neurons projecting to the
LHA compose a common neuronal population. We next
found that optogenetic stimulation of the
VLPOGABA→LHA pathway induced short bouts of
wakefulness, rather than impacted sleep. This study
reveals multilevel connectivity of the VLPO and LHA
and reports a new function of the VLPOGABA→LHA
pathway in regulation of sleep and wakefulness.
------------------------------------------------------------------

5------------------------------------------------------------------

Gastrin Releasing Peptide (GRP)-producing
Neurons
intheHypothalamic
Suprachiasmatic Nucleus (SCN) Mediates
Photic Entrainment
Ruth Li1,2, Pablo Vergara2, Yoan Cherasse2, Ran Inoue3,
Hisashi Mori3, Arisa Hirano2,4, Takeshi Sakurai2,44------------------------------------------------------------------GABAergic neurons in the BNST projecting
to the DpMe induce immediate transition
from NREM sleep to arousal

1

University of Tsukuba, Graduate School of Comprehensive
Human Sciences, Doctoral Program in Medical Sciences
2 International Institute for Integrative Sleep Medicine (WPI-IIIS)
3 University of Toyama, Faculty of Medicine, Department of
Molecular Neuroscience
4 University of Tsukuba, Institute of Medicine

Zhang Zhongwen1,2, Yoan Cherasse2, Takeshi Sakurai2-----------------------------------------------------------------Most animals display fluctuations in physiologicalphenomena that are around 24 hours (circadianrhythms).
In
mammals,thehypothalamicsuprachiasmatic nucleus (SCN) is the master clock thatgenerates and regulates the circadian rhythms. SCN
contains different subtypes of neurons that produce
distinct neurotransmitters, such as vasoactive intestinal
peptide (VIP), arginine vasopressin (AVP), and gastrin
releasing peptide (GRP). Compared to the othersubtypes, the GRP-producing neurons are scarce in
numbers and the roles they play in circadian regulationis unclear. Therefore, we used newly generated
transgenic mice, the Grp-iCre knock-in (KI) mice, andvirus vectors coding target genes that are expressed Credependently, to specifically manipulate the SCN GRPproducing neurons. When we deleted the SCN GRPproducing neurons by specifically expressing caspase,an enzyme that causes cell death, in the GRP-producing
neurons, we discovered that the mice’s behavioral
rhythms were not significantly affected. But when we
subjected these mice to jetlag, where the light onset wasadvanced for 6 hours, we discovered that the SCN GRPproducing neurons-deleted mice displayed significantly
slower entrainment to the new light-dark cycle. We
concluded that although SCN GRP-producing neurons
may not be essential for circadian rhythms generation,
they may play an important role in mediating photic

1 Faculty of medicine, University of Tsukuba
2 International Institute for Integrative Sleep Medicine (WPI-IIIS),

University of Tsukuba-----------------------------------------------------------------Negative emotions like fear and anxiety can arise inthreatened individuals, resulting in hyper arousal andincreased blood flow, allowing them to escape andminimize risks. Growing evidence shows the effects ofsleep on the regulation of emotional brain activity. Sleep
disturbance is frequent in patients suffering frompsychiatric stress disorders, such as depression, social
anxiety disorder (SAD) or post-traumatic stress disorder(PTSD), who are unable to regulate their negative
emotions. The bed nucleus of stria terminalis (BNST) isa crucial region of interest regarding human stressrelated psychiatric disorders, including PTSD. Ourgroup previously demonstrated that the optogenetic
excitation of GABAergic neurons in the BNST(GABABNST) triggered an immediate transition from
NREM sleep to wakefulness. Here, we describe theneuronal pathway responsible for this transition. We
found dense innervation from GABABNST neurons to thedeep mesencephalic nucleus (DpMe), one of the arousal
regulating centers, mainly populated by excitatory
glutamatergic neurons (GLUTDpMe). We demonstratethat optogenetic stimulation of GLUTDpMe neurons
results in immediate transition from NREM sleep to2

entrainment.
------------------------------------------------------------------

1 WPI-IIIS, University of Tsukuba
2 State Key Laboratory of MembraneBiology, Peking University

School of Life Sciences, Beijing, China.
3 National Institute of Biological Sciences, Beijing, China.
4 Tsinghua Institute of Multidisciplinary Biomedical Research,
Tsinghua University, Beijing, China.

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

24 hours QIH induction by OPN4-based
optogenetics-----------------------------------------------------------------Male mice are known to exhibit sequential sexual
behaviours such as sniffing, mounting, intromission andejaculation. However, the neural mechanisms that
regulate and coordinate these behaviours have remained
elusive. Based on our observations of GRAB-DA
dynamics in the nucleus accumbens (NAc), we foundthat there are several subregions in the NAc.
Specifically, the ventral shell region (NAc-vs) shows
elevated DA levels during intromission, while themedial shell region (NAc-ms) displays diminished DAlevels during intromission. To identify the inputs tothese subregions of the NAc, we employed two-colour
cholera toxin B subunit (CTB)-mediated retrogradelabeling to trace neuronal projections. We found that theNAc-vs receives input from the antero-lateral aspect ofthe ventral tegmental area (alVTA), while the NAc-ms
receives innervation from the postero-medial aspect of
the VTA (pmVTA). To identify the neuronal activity of
the alVTA and pmVTA, we monitored the GCaMPdynamics of dopaminergic neurons in various midbrain
regions. We observed that GCaMP fluorescencerecorded from the alVTA were augmented during
intromission, while those from the pmVTA weredecreased during this phase. Thus, the neuronal activityof the VTA subregions projecting to the distinct
subregions of the NAc corresponded to the DA releasepattern. By utilizing optogenetic manipulation of the
dopaminergic terminals in the NAc, we found that the
behavioral expression pattern of sexual behavior in malemice was altered. These findings suggest that DA
dynamics within the NAc are critical for regulating
sexual behavior in male mice.

Tohru M. Takahashi1,2, Arisa Hirano1,2, Takeshi
Sakurai1,2
1 International Integrative Institute for Sleep medicine (WPI-IIIS),

University of Tsukuba, Tsukuba, Japan
2 Faculty of Medicine, University of Tsukuba, Tsukuba, Japan-----------------------------------------------------------------Optogenetics has been used for neuronalmanipulation at a high temporal resolution tounderstand the neuronal mechanisms of many
physiologies. Current tools have been reported
to be effective for short-term manipulation(seconds to minutes), although it has beenchallenging for continuous stimulation ofneurons on much longer time scale (hours todays) to induce long-term behavioral andphysiological responses such as hibernation. In
this study, we aimed to establish a method
allowing stable 24-hour neuronal manipulation
with high reproducibility by using OPN4
(melanopsin), which is a GPCR(Gq)-type
photosensor with high-photosensitivity. We
recently found that excitatory manipulations of
Qrfp-expressing neurons in the preoptic area ofthe hypothalamus (Q neurons) induced ahibernation-likehypothermic/hypometabolicstate (QIH) in mice. To control QIH with higher
time resolution, we developed an optogenetic
method using modified OPN4. The engineeredOPN4 stably and reproducibly induced QIH forat least 24 hours by illuminating low-power light(3 µW, 473 nm laser) with high temporal
resolution. The optogenetic method would
enable us to identify neural mechanisms
underlying long-term dormancy states such as
sleep, daily torpor and hibernation.
------------------------------------------------------------------

8------------------------------------------------------------------

Ventral tegmental area dopaminergic neurons
mediate sleep reduction induced by ablationof the surrounding GABAergic neurons
Susumu Umezawa1, Michael Lazarus1, Yo Oishi1

7------------------------------------------------------------------

1 University of Tsukuba

Male sexual behaviors are regulated bydopaminergic signaling in NAc in mice-----------------------------------------------------------------It is widely accepted that sleep is controlled by two
processes, a homeostatic process and a circadian
process. However, sleep is also modulated by external
stimuli such as motivational stimuli. Dopaminergic
neurons in the ventral tegmental area (VTA) not only
play a central role in processing motivational stimuli,

Ai Miyasaka1, Takeshi Kanda1, Yuka Terakoshi1,
Cherasse Yoan1, Yukiko Ishikawa1, Yulong Li2, Masashi
Yanagisawa1, Katsuyasu Sakurai1, Takeshi Sakurai1,
Qinghua Liu1,3,4

3

but also regulate sleep. Recently, GABAergic neurons
around the VTA were also revealed to regulate sleep.
Chronic lesions of GABAergic neurons around the VTA(ventromedial midbrain/pons area: VMP) cause severe
daily sleep loss, yet the mechanism was unclear. In thisstudy, we examined the role of VTA dopaminergic
neurons (VTADA) in the severe sleep loss caused bylesions of VMP GABAergic neurons (VMPGABA). To
this end, we ablated VTADA in mice with VMPGABA
lesions and measured their daily sleep. As a result,
VTADA lesions attenuated the daily sleep loss in the lightperiod in mice with VMPGABA lesions. Furthermore, theanalysis of sleep architecture suggested that theattenuation of the sleep loss occurred via the increased
sleep episode number. In conclusion, this study revealed
that VTADA mediate the severe daily sleep loss causedby chronic lesions of GABAergic neurons around the
VTA.
------------------------------------------------------------------

molecule for activating NAc indirect pathway neurons
that express adenosine A2A receptors (A2ARs) to trigger
SWS but direct proof is lacking. Here, we developed a
BBB-permeable, visible-light (λ > 400 nm)
photoactivatable positive allosteric modulator of A2ARs(A2AR PAM) and used it to optochemically induce SWS
in the NAc of freely behaving mice. By contrast,
optochemical activation of A2ARs has no effect on theventrolateral preoptic area, a sleep center where A2ARs
are thought to promote sleep. Our approach to A2AR
optopharmacology should help in the generation ofphotoactivatable compounds for virtually any druggabletarget.
------------------------------------------------------------------

9

Rintaro Otani1, Shuji Ioka1, Mustafa Korkutata1, Yohei
Chitose2, Manabu Abe2, Michael Lazarus1, Tuyoshi
Saitoh110------------------------------------------------------------------Development of photocaged drugs targeting
adenosine 2A receptor------------------------------------------------------------------

Optochemical control of slow-wave sleep inthe nucleus accumbens by a photoactivatable
allosteric modulator of adenosine A2A
receptors

1 University of Tsukuba
2 Hiroshima University-----------------------------------------------------------------Adenosine (Ad) is an endogenous sleep-inducing
molecule that strongly promotes sleep throughactivation of adenosine 2A receptors (A2AR) in nucleus
accumbens (NAc). Increased accumulation of Ad during
wakefulness induces sleep, but the spatiotemporalmechanism of adenosinergic regulation in NAc remains
unclear. Photocaged drugs are known as photopharmacological tools to control the activity of target
receptors by light irradiation, but photocaged drugs
using A2AR agonists have problems in differentiationfrom endogenous effects due to background activity.
Our group recently found a positive allosteric modulator(PAM) YNT378 that enhances the sensitivity of A2AR toendogenous Ad and reported that it induces slow-wave
sleep in mice. In this study, we designed and synthesized
novel photocaged A2AR PAMs based on the structure of
YNT378 to elucidate the spatiotemporal mechanism of
A2AR activity.
As the first generation photocaged YNT378 with a 6nitroberatryl (Nv) photoremovable protecting group(PPG) showed poor water solubility, slow photoreaction
rate, and short reaction wavelength, we designed a newwater-soluble PPG, A400, based on 3-arylcoumarin, andsynthesized A400-YNT378. A400-YNT378 showed agood water-solubility and rapid uncaging reaction at
420 nm. In this presentation, we will report the detailsof the photochemical and pharmacological properties ofthese photocaged drug.
------------------------------------------------------------------

Koustav Roy1, Ioka Shuji1, Rintaro Otani1, Mao
Amezawa1, Kaspar Vogt1, Manabu Abe2, Yoan
Cherasse1, Hiroshi Nagase1, Masashi Yanagisawa1,
Radhika Basheer3, Tsuyoshi Saitoh1,4*, Michael
Lazarus1,4*
1 International Institute for Integrative Sleep Medicine (WPI-IIIS),

University of Tsukuba, Tsukuba, Ibaraki, Japan
2 Department of Chemistry, Hiroshima University, HigashiHiroshima, Hiroshima, Japan
3 Veterans Administration Boston Healthcare System andHarvard Medical School Department of Psychiatry, West
Roxbury, Massachusetts, United States
4 Department of Biomedical Sciences, Faculty of Medicine,
University of Tsukuba, Tsukuba, Ibaraki, Japan-----------------------------------------------------------------Optopharmacology, an emerging approach inpharmacology that involves the activation or
deactivation of molecules with light for targeted drugdelivery, may ease symptoms, cure diseases, andimprove quality of life while preventing uncontrolled
drug action. In-vivo use of optopharmacology to renderneurons or glia photoresponsive without the need to relyon genetic engineering has been slow to develop mainly
because of the inaccessibility of the brain to lightirradiation and inadequate delivery of photoactivatablecompounds through the blood-brain barrier (BBB). The
nucleus accumbens (NAc) is a novel region for slowwave sleep (SWS) regulation by integrating
motivational stimuli. Adenosine is a possible candidate
4Kao Hashimoto1,2, Yasuyuki Nagumo2, Keita Iio2,3, Yuki
Saito1,2, Yukiko Ishikawa2, Noriki Kutsumura1,2,3,
Takeshi Sakurai1,2, Masashi Yanagisawa1,2, Tsuyoshi
Saitoh1,2, Hiroshi Nagase2

11------------------------------------------------------------------

Design and synthesis of novel κ opioid
receptor agonists with bicyclo [2.2.2] octane
skeleton

1 Graduate School of Comprehensive Human Sciences, Universityof Tsukuba
2 IIIS, University of Tsukuba
3 Graduate School of Pure and Applied Sciences, University ofTsukuba

Keita Kajino1,2, Masahiro Yata1,2, Yasuyuki Nagumo2,
Akihisa Tokuda2,3, Noriki Kutsumura1,2,3, Tsuyoshi
Saitoh2,3, Hiroshi Nagase1,2-----------------------------------------------------------------Orexins neurons modulate pain sensitivity. However, it
is unclear how orexin neurons process afferent pain
information and produce analgesic effects in the central
nervous system. In this study, to reveal this mechanism,
we analyzed the neural pathways of pain regulation byorexin neurons using mice with chronic inflammatory
pain induced by complete Freund’s adjuvant (CFA).
We first demonstrated that CFA markedly increases cfos-positive activated orexin neurons in the lateralhypothalamus of mice. To evaluate the effects ofactivated orexin neurons on pain, we activated orexin
neuronschemogeneticallyand
CFA-induced
hypersensitivity was suppressed. These effects wererecovered by intracerebroventricular administration oforexin 1 receptor (OX1R) antagonist. Next, to reveal thedownstream of orexinergic neurons regulating pain, we
focused on noradrenergic (NA) neurons in the locus
coeruleus (LC), which dominantly express OX1R andplay an important role in pain regulation. A retrograde
tracer injected into the LC was transported to c-fospositive orexin neurons. The analgesic effects bychemogenetic activation of orexin neurons weresignificantly suppressed by injection of NA receptor
antagonist into the spinal cord, which is innervated by
NA neurons in the LC. We also found that a novel
selective OX1R agonist, originally developed in house,
induced analgesic effects. These results suggest thatorexin neurons are activated by afferent pain
information and then suppress their transmission at the
spinal cord through the activation of OX1R on NA
neurons in the LC, and selective OX1R agonist may be
a new type of analgesics.
------------------------------------------------------------------

1 Degree Programs in Pure and Applied Sciences, University ofTsukuba
2 International Institute for Integrative Sleep Medicine (WPI-IIIS),
University of Tsukuba
3 Graduate School of Human Sciences, University of Tsukuba-----------------------------------------------------------------κ opioid receptor (KOR) agonists have been expected as
analgesics without serious side effects derived from theactivation of μ opioid receptors (MOR) such as
addiction, sleepiness, nausea, and vomiting. Nalfurafine,the first selective KOR agonist, succeeded in affording
strong analgesic and antipruritic effects without
addiction, while, during its clinical trials for
postoperative pain, nalfurafine showed a severe sedative
effect at the same dose as an antinociceptive effect.
Therefore, the development of new KOR agonists
without sedative effect has been required.
During the course of drug discovery research based on
active conformational analysis of nalfurafine, we
recently discovered that YNT-1612 with a bicyclo[2.2.2] octene scaffold, which enables the side chainorientation toward the active conformation of
nalfurafine, exhibits potent antinociceptive activity
without a sedative effect. In this study, to elucidate themechanism of side-effect segregation, we conducted thestructure-activity relationship (SAR) study between theorientation of amide side chains and analgesic/sedative
effects.
Bicyclo [2.2.2] octane derivatives YNT-1623 and YNT1624 having different amide side chain orientations
were designed, synthesized, and evaluated forpharmacological activity. The results showed that both
YNT-1623 and YNT-1624 exhibited strong analgesic
effects, whereas YNT-1624 showed no sedative effects,
while YNT-1623 was significantly more sedative. These
results provide important information indicating that theorientation of the amide side chain contributes to theseparation of sedative effects, which will help elucidate
the mechanism of side-effect isolation in the future.
------------------------------------------------------------------

13------------------------------------------------------------------

Orexin receptor antagonist modulates
sleeping energy metabolism without overt
effects on sleep architecture

12

Insung Park1, Rikako Yoshitake2, Kazuki Kioka2, Asuka
Ishihara3, Katsuhiko Yajima4, Takashi Kanbayashi1,
Toshio Kokubo1, Ichiyo Matsuzaki1, Masashi
Yanagisawa1, Kumpei Tokuyama1------------------------------------------------------------------

Analysis of orexinergic function in chronic
pain-like states5

Sleep disturbances and poor rest-activity rhythms might
reduce the quality of life for older adults. Whether
exercise can affect circadian rhythms remain unknown.
This randomized controlled trial investigated the effects
of a home-based aerobic exercise intervention for 12
weeks in the evening on 54 older adults with lower sleep
quality (PSQI ≧ 6 points). Participants wererandomly allocated to a low-intensity exercise group(60-70 steps per minute), a moderate-intensity exercisegroup (110-120 steps per minute), or a control group.
Each intensity exercise group was performed for 30-min
daily at home in the evening (18:00 to bedtime).
Outcomes included the nonparametric approach
actigraphy data of 24/7 (inter-daily stability, IS; intradaily variability, IV; relative amplitude, RA; nocturnal
activity, L5; daily activity, M10; L5 and M10 start
times; the midpoint of L5 and M10 start times).
There were no significant differences in the IS, IV, RA,
L5, and M10. The moderate-intensity exercise group
showed a delay in L5 start time (23.8, 24,3, and 24.4
hours; P for trends = 0.002), and the midpoint of L5 andM10 start times (4.1, 4.5, and 4.6 hours; P for trends =0.004) during the intervention. However, the lowintensity exercise group and control group did notchange during the intervention.
Moderate-intensity exercise in the evening leads todelay rest-activity rhythms among older adults withlower sleep quality. Thus, it is necessary to consider thetiming and intensity of exercise when suggesting
exercise therapy (i.e., combination with CBT-I) to older
adults with sleep disorders.
------------------------------------------------------------------

1International Institute for Integrative Sleep Medicine, Universityof Tsukuba
2 Faculty of Health and Sports Sciences, University of Tsukuba
3National Institute of Diabetes and Digestive and Kidney
Diseases
4 Faculty of Pharmaceutical Sciences, Josai University-----------------------------------------------------------------Suvorexant, approved orexin receptor antagonist,targets the orexin-mediated wake-promoting system andoffers an alternative mechanistic approach to treating
insomnia. There is a crosstalk between regulators ofenergy metabolism and control of sleep / wakefulness.
A molecule, such as orexin, acts on regulation of energy
metabolism and sleep. Sleeping energy metabolism isrelated to sleep architecture, and orexin receptor
antagonist may affect energy metabolism as well as
sleep. Effect of suvorexant on sleeping energy
metabolism in human is not known, while stimulating
effects of orexin system on energy expenditure wasreported in animal studies. However, effect ofsuvorexant on oxidized substrate is inconsistent among
the animal studies.
In this placebo-controlled, double-blind, crossoverintervention study, metabolic rate of 14 healthy young
male without sleep problems was measured using
whole-room indirect calorimetry, during which
polysomnographic recording of sleep was made. The
participants consumed suvorexant (20 mg) or placebo
10 minute before bedtime.
Fat oxidation was increased but protein catabolism was
suppressed without changes in energy expenditure bysuvorexant during sleep. Interestingly, sleep
architecture was not notably affected by suvorexant.
The only difference in sleep architecture between 2
conditions was observed in NREM stage 1. The present
study suggested that the effect of suvorexant on energy
metabolism was not mediated by major changes in sleep
architecture.
------------------------------------------------------------------

15------------------------------------------------------------------

Automatic Sleep Analysis for Individuals with
Disordered Sleep Patterns
Saki Tsumoto1, Kazumasa Horie2, Masaki Matsubara3,
Fusae Kawana4,5, Masashi Yanagisawa6, Atsuyuki
Morishima3 , Hiroyuki Kitagawa6

14------------------------------------------------------------------

Does different-intensity exercise affect therest-activity rhythms among older adults?

1 Ph.D. program in Humanics, University of Tsukuba
2 Center for Computational Sciences ,University of Tsukuba
3 Faculty of Library, Information and Media Science ,University

Cao1,

Yue
JaeheeOkura2,3,5

Lee1,

JaehoonSeol2,3,4,

Tomohiroof Tsukuba
4
Yumino Heart Clinic
5 Juntendo University Graduate School of Medicine
6 WPI-IIIS, University of Tsukuba

1 Doctoral Program in Physical Education, Health and Sports-----------------------------------------------------------------Sleep stage scoring is the basis of sleep testing. Scoredstages based on biological signals and other data areclassified into five stages and used for sleep diagnosis.
This scoring process used to be performed manually bytrained technicians, but due to the rapid development of
information technology in recent years, automatedscoring methods using machine learning has been

Sciences, Graduate School of Comprehensive Human Sciences,
University of Tsukuba.
2 Faculty of Health and Sport Sciences, University of Tsukuba.
3 International Institute for Integrative Sleep Medicine,
University of Tsukuba.
4 Japan Society for the Promotion of Science.
5 R&D Center for Tailor-Made QOL, University of Tsukuba.

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

studied actively. However, most of these methods havetargeted only to healthy people, and there has not been
sufficient progress for patients with sleep disorders.
This is one reason why automatic scoring method has
not been widely used in actual clinical practice. In thisstudy, we develop a sleep stage scoring method forpeople who have sleep problems. In our experiment, wetrained a classification model for sleep stage scoringwith a dataset that contains biological signals taken fromboth healthy people and patients with sleep-disorderedbreathing, one of the most common sleep disorders. We
found that the model's accuracy drastically dropped forthe duration where apnea and hypopnea events occur.
Then, we developed separate models dedicated toapnea/hypopnea duration and normal duration andapplied them in a hybrid way. The results suggest that itis effective to apply the hybrid model to improve overall
accuracy. We are planning to take a similar approach tosleep analysis for various sleep patterns in the future.
------------------------------------------------------------------------------------------------------------------------------------

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

Binaural beats at slow frequencies less than 1
Hz shorten the latency of slow-wave sleep
Zhiwei Fan1, 2, Yunyao Zhu1,3, Chihiro Suzuki1, Yoko
Suzuki1, Yumi Watanabe4, Junki Endo4, Takahiro
Watanabe4, Takashi Abe1
1 International Institute for Integrative Sleep Medicine (WPI-IIIS),

University of Tsukuba, Tsukuba, Japan
2 The Japan Society for the Promotion of Science (JSPS) Foreign
Researcher
3 Graduate School of Comprehensive Human Science, Universityof Tsukuba, Tsukuba, Japan
4 KYOCERA Corporation, Japan-----------------------------------------------------------------Binaural beats are an auditory phenomenon in which
listeners hear a single tone equal to the frequency
difference between two tones transmitted to two ears
separately and simultaneously. Previous studies
suggested binaural beats can entrain neural oscillations
and induce related behavioral states. A previous study
showed listening to a 1-Hz binaural beat during a nap
increased the duration of slow-wave sleep. However, theeffect of binaural beats in the range of slow oscillations( ＜ 1-Hz) on sleep is poorly understood. We
hypothesized the 0.25-Hz beats could entrain neuraloscillations and enhance slow-wave sleep by shortening
the latency or increasing the duration. Twelve healthy
participants (6 women, 25.3±2.6 years) were included inthis study, approved by the research ethics committee.
The experiment included four 90-minute afternoon nap
sessions consisting of a sham condition (without
acoustic stimulation) and three binaural beats conditions(0-Hz, 0.25-Hz, or 1-Hz) with a 250-Hz carrier tone.
Participants were not informed of their conditions. The
acoustic stimuli were delivered through earphones andcontinued for a 90-minute nap. Our results showed boththe N2- and N3- latencies of the 0.25-Hz binaural beats
condition were shorter than those in the sham condition.
This study shows the potential capability of 0.25-Hz
binaural beats for sleep induction in generally healthy
populations.
------------------------------------------------------------------

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

Assessing Wakefulness Intensity as acounterpart to Sleep Depth During the SleepWake Cycle
J. Diaz1, H. Funato1, M. Yanagisawa1, K. E. Vogt1
1 WPI-IIIS, University of Tsukuba-----------------------------------------------------------------NREM sleep is associated with low frequency activity,
which is conventionally assessed using EEG delta
power, whereas wakefulness is associated with higher
frequency activity, which is diffusely distributed overthe EEG beta and gamma bands. EEG spectral power isorganized according to a 1/f-like law, implying that thepower of the frequencies identifying wakefulness is
negligible in comparison to the power of the delta band.
As a result, while EEG spectral analysis provides clear,easy-to-measure information that can be directly
associated with the dynamics of sleep homeostasis,
there is no equivalent for wakefulness. However, novel
methods for extracting the arrhythmic components of
EEG provide robust features specific to wakefulness forthe first time. Using this information, a variableindicating the intensity of wakefulness can be defined,
which serves as a counterpart to delta power-measured
sleep depth. The dynamics of the wakefulness-intensity
variable during the sleep-wake cycle in WT mice andthe mouse sleep mutants Sleepy and Dreamless areinvestigated in this preliminary report. This variable
anticorrelates with delta power and is highly correlatedwith the likelihood of being awake strongly supporting
its interpretation as the opposite of sleep depth. Sleepy
and Dreamless have subnormal and supranormal levelsof the wakefulness-intensity variable respectively.

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

Targeted memory reactivation during REM
sleep selectively enhances brain responses tounpleasant images
Kazuki Sato1,2, Satomi Okabe2,3,4, Yoko Suzuki2,
Takashi Abe2

7

1 Graduate School of Comprehensive Human Science, University

5 Doc Prog in Biomed Sci, Grad Sch of Comprehensive Humanof Tsukuba
2 International Institute for Integrative Sleep Medicine (WPI-IIIS),
University of Tsukuba
3 Department of Sleep-Wake Disorders, National Center of
Neurology and Psychiatry
4 Japan Society for Promotion of Science

Sci, Univ. of Tsukuba
6 Dept of Human Health Sci, Grad Sch of Medicine, Kyoto Univ-----------------------------------------------------------------Our sleep comprises two states: rapid eye movement(REM) sleep and non-REM (NREM) sleep. The
physiological function of REM sleep is poorly
understood. Recent human studies showed that low
amount of REM sleep is associated with a high risk ofdementia (Pase et al., 2017) and increased mortality(Leary et al., 2020). Moreover, in mice, the cerebral
blood flow increases during REM sleep (Tsai et al.,2021), suggesting that nutrition supply and theclearance of waste products occur actively during REM
sleep. Although the causal relationships are unclear,these studies imply that REM sleep is important for
maintenance of our health. However, currently it isdifficult to reliably increase the REM sleep amount in
humans. Here, we unexpectedly found that a certain
type of feed can increase the REM sleep amount in mice.
Switching to a normal feed reversed the REM sleep
amount to normal levels. We also found that this feed
condition-dependent change in sleep can be enhancedby the perturbation of certain neuronal circuits. Now, weare trying to elucidate the underlying mechanisms. This
study contributes to understanding a novel regulatory
system of sleep. Moreover, it might provide clues fordeveloping methods to increase REM sleep amount in
humans.
-----------------------------------------------------------------------------------------------------------------------------------Reactivation of emotional memories during REM sleep
has been hypothesized to lead to memory consolidation
and a decrease in emotional charge. However, previous
studies relying on subjective measures or remaining
under the influence of circadian rhythms have provided
conflicting evidence for this hypothesis. To address thisissue, the present study used the targeted memory
reactivation (TMR) procedure to reactivate specificmemories during sleep that are associated with a sensory
stimulus during wakefulness. Additionally, this study
used the late positive potential (LPP), a component of
event-related brain potentials, time-locked to thepresentation of affective images. Sixteen healthy adults
participated in the study. EEGs were recorded while
viewing unpleasant, neutral, or pleasant images (oldimages) with an odor stimulus. During subsequent REM
sleep, the same odor was presented in the TMR
condition, while an odorless stimulus was presented in
the control condition. Upon awakening, they performed
the same task as before sleep adding new images to test
memory for the images. They also rated their mood
before and after sleep. The results showed that TMR
increased the LPP amplitude after sleep for unpleasant
old images, but there were no changes in the LPP for
unpleasant new images as well as neutral or pleasantimages. In addition, there was a positive correlation
between LPP amplitude for unpleasant old images andmemory performance. Furthermore, negative mood was
reduced after sleep in the TMR condition. These results
suggest that TMR during REM sleep selectively
enhances attention to previously encountered
unpleasant stimuli while ameliorating negative affect.
------------------------------------------------------------------

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

Analysis of the mechanism of REM sleep withfocus on Parkinson’s disease
Ami Kaneko1,2, Shinnosuke Yasugaki1,3, Hideki
Hayakawa4, Kensuke Ikenaka4, Cesar Aguirre4,
Masashi Yanagisawa1, Kousuke Baba4,5, Hideki
Mochizuki4, Yu Hayashi1,6,7
1 WPI-IIIS, Univ of Tsukuba

19

2 Ph. D. Prog in Humanics, SIGMA, Univ of Tsukuba------------------------------------------------------------------

3 Doc Prog in Biomed Sci, Grad Sch of Comprehensive Human

Exploration of a novel mechanism of sleep
regulation that depends on feeding condition

Sci, Univ of Tsukuba
4 Dept of Neurology, Grad Sch of Medicine, Osaka Univ
5
Dept of Neurology, Faculty of Medicine, Acad Res Division,
Univ of Toyama
6 Dept of Human Health Sci, Grad Sch of Medicine, Kyoto Univ
7 Dept of Biol Sci, Grad Sch of Science, Univ of Tokyo

Hibiki Okamura1,2, Shimpei Taniguchi1,3, Mitsuaki
Kashiwagi1,4, Shinnosuke Yasugaki1,5, Mika Kanuka1,
Masashi Yanagisawa1, and Yu Hayashi1,4,6-----------------------------------------------------------------During rapid eye movement (REM) sleep, the cerebral
cortex is activated and produces vivid dreams. Yet, it
usually does not lead to motor output owing to theexpression of muscle atonia mechanisms. However,patients with REM sleep behavior disorder (RBD)
exhibit impaired muscle atonia and frequently act out of

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

Univ. of Tsukuba
2 Ph. D. Prog in Humanics, SIGMA, Univ. of Tsukuba
3 Master’s Prog in Med Sci, Grad Sch of Comprehensive Human
Sci, Univ. of Tsukuba
4 Dept of Biol Sci, Grad Sch of Sci, Univ. of Tokyo8

their dreams during REM sleep. Patients often wake up
following such movements, which can lead to reduced
sleep quality and excessive sleepiness or fatigue during
the daytime. RBD is considered a prodrome ofsynucleinopathies; most RBD patients eventuallydevelop synucleinopathies including Parkinson’s
disease and dementia with Lewy bodies within 10-14
years. In synucleinopathies, α-synuclein accumulates
and causes nerve cell death, which leads to motor
symptoms such as tremors, bradykinesia, and rigidity as
well as non-motor symptoms including cognitive
impairments and depression.
Here, we aimed to understand the mechanisms
underlying the link between RBD and Parkinson’s
disease and establish an RBD mouse model for the
future development of effective treatment for RBD. A
rare G51D α-synuclein mutation is observed in familial
Parkinson’s disease. We produced and injected G51D αsynuclein fibrils into the brainstem pontine tegmental
area in mice and examined the effects on muscle atonia
during REM sleep, behavior, and motor symptoms
across several months. Consequently, mice injectedwith α-synuclein fibrils exhibited impaired muscle
atonia during REM sleep. Moreover, these mice
exhibited motor impairment and propagation ofphosphorylated α-synuclein to other brain areas. We
expect this study will provide important insight into
how RBD develops at the early stages of Parkinson’s
disease.
------------------------------------------------------------------

REM sleep is necessary for contextual fear memory
consolidation (Kumar et al., Neuron). However, themechanism is not clear. Therefore, we hypothesize that
re-activation of the ABNs, which represent the context
to be associated with fear, during REM sleep isnecessary for fear memory consolidation (Koyanagi etal., Neur Regen Res). In this study, we silenced the ABN
activities representing the context during retrieval
which did not impair the memory. However, silencing
the ABNs during REM sleep impaired the memory. We
also found a distinct dynamics of the ABN activity
during REM sleep. Taken altogether, we propose that
contextual fear memory consolidation relies on thetransient recruitment of ABNs representing a specificcontext.
------------------------------------------------------------------

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

CaliAli: neuronal tracing utilizing vasculature
in calcium imaging
Pablo Vergara1, Sakthivel Srinivasan1, Yuteng Wang1,2,
Toshie Naoi1, Yuki Sugaya3,4, Masanobu Kano3,4, andMasanori Sakaguchi1,2,5
1International Institute for Integrative Sleep Medicine (WPIIIIS), University of Tsukuba, Tsukuba, Ibaraki, Japan
2 Doctoral Program in Neuroscience, Degree Programs in
Comprehensive Human Sciences, Graduate School ofComprehensive Human Sciences, University of Tsukuba, Tsukuba,
Ibaraki, Japan
3 Department of Neurophysiology, Graduate School of Medicine,
The University of Tokyo, Tokyo 113-0033, Japan
4 International Research Center for Neurointelligence (WPIIRCN), The University of Tokyo Institutes for Advanced Study(UTIAS), Tokyo 113-0033, Japan
5 Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki,
Japan

21
------------------------------------------------------------------

Transient recruitment of adult-born neurons
for fear memory consolidation in REM sleep
Sakthivel Srinivasan1, Iyo Koyanagi1,2, Pablo Vergara1,
Yuteng Wang1,2, Jiahui Yu1,2, Toshie Naoi1, Kaspar E.
Vogt1, Yoan Chérasse1, Takeshi Sakurai1,2,3, Noriki
Kutsumrura1, Taro Tezuka4, Masanori Sakaguchi1,2,3-----------------------------------------------------------------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 individual recording sessions. However,when neurons are intermittently detectable acrosssessions and misalignments are non-rigid, spatial
footprints are commonly insufficient to correct
misalignments and reliably track neurons. These issues
are common in experiments involving unstable neuralrepresentation, such as global remapping or drifts ofneural activities occurring through extensive periods. To
address these issues, we developed CaliAli (Calcium
imaging Inter-session Alignment): A tool that utilizes
brain vasculature and spatial cues of neurons to track
neurons across sessions automatically. Brain
vasculature is used as a reference structure to track
intermittently detectable neurons and to correct

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

University of Tsukuba, Ibaraki, Japan
2 Doctoral Program in Neuroscience, Graduate School ofComprehensive Human Sciences, University of Tsukuba, Tsukuba,
Ibaraki, Japan
3
Faculty of Medicine, University of Tsukuba, Ibaraki, Japan
4 Faculty of Engineering, Information and Systems, University ofTsukuba, Ibaraki, Japan-----------------------------------------------------------------Memory replay during rapid-eye-movement (REM)
sleep is suggested to contribute to memory
consolidation (Skaggs & McNaughton, Science). The
dentate gyrus (DG) in the hippocampus hosts contextual
fear memory trace (Liu et al., Nature) and adultneurogenesis (Akers et al, Stem cells). Recently, we
provided evidence that the activity of ABNs during
9

Tanaka2, Qinghua Liu1, 3, Hiromasa Funato1,4, Masashi
Yanagisawa1

misalignments. CaliAli uses an alignment-beforeextraction approach, enabling the detection of weakcalcium signals that would be otherwise undetected byanalyzing isolated sessions. CaliAli outperforms stateof-the-art algorithms in conditions of high neuron
overlap, sparsity, intermittent neuron detectability, andvarying signal-to-noise ratios. This evidence suggeststhat CaliAli is a reliable tool for tracking neuralactivities.
------------------------------------------------------------------

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

University of Tsukuba, Japan
2 Graduate School of Medicine, The University of Tokyo
3 National Institute of Biological Sciences, China
4 Department of Anatomy, Toho University Graduate School of
Medicine-----------------------------------------------------------------Pathogenic variants in the Cyclin-dependent kinase like5 (CDKL5) gene cause CDKL5 deficiency disorder(CDD), a severe neurodevelopmental disordercharacterized by refractory early-onset epilepsy,
intellectual disability, motor defects, cortical visual
impairments, and autistic features. The majority (>86%)of patients have sleep problems such as difficulty falling
asleep, sleep fragmentation, frequent awakening fromsleep at night, night screaming and laughing, andexcessive daytime sleepiness, which greatly impact thequality of life of patients and their families. However,
sleep study using electroencephalography (EEG) andelectromyography (EMG) recordinginmousemodels with CDD is limited. We have generated a novel
mouse model of CDD, the Cdkl5 kinase-dead K42R
knock-in (Cdkl5 K42R KI) mouse, which carries amutation mimicking a human CDD lost-of-function
missense mutation. In this study we have for the firsttime comprehensively analyzed sleep disturbances inboth female and male Cdkl5 K42R KI mice using
EEG/EMG recording. The results show that Cdkl5
K42R KI micerecapitulate sleep disturbances observed in human CDD
patients. Our study provides insights into better
understanding of sleep disturbances in CDD and will
facilitate identifying novel therapeutic strategies to treat
sleep problems in CDD and neurodevelopmental
disorder patients.
------------------------------------------------------------------

23------------------------------------------------------------------

Sex differences in sleep/wakefulness from
infancy to adulthood in a hypersomnia model,
Sleepy mutant mouse
Noriko Hotta-Hirashima1, Chika Miyoshi1, Aya Ikkyu1,
Satomi Kanno1, Kumi Ebihara1, Hiromasa Funato1,2,Masashi Yanagisawa1
1 WPI-IIIS, Univ. of Tsukuba
2 Dept. of Anat., Fac. of Med., Toho Univ.
3 Dept. of Mol. Genet., Univ. of Texas Southwestern Med. Cent.,

Dallas, USA
4 TARA, Univ. of Tsukuba-----------------------------------------------------------------It is known that there is a sex difference in sleep-wakebehavior in adult mice. However, due to technicaldifficulties in recording of electroencephalogram andelectromyogram(EEG/EMG)-based
sleep-wakebehavior in infant mice, sex differences in thedevelopmental stages from infancy have not been
investigated. Therefore, we established a method torecord EEG/EMG every 4 days from postnatal day 21
immediately after weaning to postnatal day 57. Utilizing
this methodology, we examined sex differences in thedevelopment of the hypersomnia phenotype observed in
Sik3_sleepy mutant mice (sleepy). Furthermore, when
EEG/EMG measurements were performed on sleepy
and their wild-type littermates at 10–13 weeks and 30–
33 weeks of age, females matured in sleep-wakebehavior about 1 week earlier than that of male mice.
We also found that prolong non-REM sleep time,observed in sleepy mutant mice, is more prominent infemales than in males.
------------------------------------------------------------------

25------------------------------------------------------------------NALCN in the forebrain and pons-medulla
regions have distinct roles in REM sleep
regulation
Tomoyuki Fujiyama1, Satomi Kanno1, Kazuya
Miyanishi1, Noriko Hotta-Hirashima1, Satsuki
Hayakawa1, Hikari Yamamoto1, Kanako Iwasaki1,
Ayako Mochizuki1, Patricia Seoane-Collazo1, Takehiro
Miyazaki1, Staci J Kim1, Deependra Kumar1, Miyo
Kakizaki1, Aya Ikkyu1, Yukiko Ishikawa1, Manabu Abe3,
Kenji Sakimura3, Seiya Mizuno4, Satoru Takahashi4,
Chika Miyoshi1, Takeshi Kanda1, Yosuke Tsuneoka2,
Hiromasa Funato1, 2, Masashi Yanagisawa1, 5, 6

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

Sleep disturbances in mice with a CDKL5
kinase-dead missense mutation, a novel mousemodel of neurodevelopmental disorder
Liqin Cao1, Xin Zhang1, Tingting Lou1, Teruyuki

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

10

1 International Institute for Integrative Sleep Medicine,

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 Life Science Center for Survival Dynamics, Tsukuba Advanced
Research Alliance (TARA), University of Tsukuba, Tsukuba
6 Department of Molecular Genetics, University of Texas
Southwestern Medical Center

University of Tsukuba
2 Department of Brain and Cognitive Sciences, Massachusetts
Institute of Technology
3 Laboratory Animal Resource Center, University of Tsukuba
3 Department of Anatomy, Toho University School of Medicine
4 University of Texas, Southwestern Medical Center-----------------------------------------------------------------SIK3 was identified as an important sleep regulator
through a screening of randomly mutagenized mice.
Moreover, mice lacking serine 551, the PKA
phosphorylation site of SIK3, showed increased NREM
sleep time and sleep need. These results suggest that theexistence of PKA recognition sites, such as S551, is
crucial for the normal sleep/wake regulation andmaintenance of daily sleep need.
In addition to S551, there are two more PKA recognition
sites, threonine 469 and serine 674. To examine whether
the phosphorylation of T469 and S674 of SIK3 is also
required for proper sleep/wake behavior, we generated
mutant mice in which SIK3 T469 and SIK3 S674 were
substituted by alanine.
Similar to Sik3 S551A mice, Sik3 T469A mice also show
increased NREM sleep time and NREM sleep delta
power, an index for sleep need. In contrast, Sik3 S674A
mice show no change in NREM sleep time and NREM
sleep delta power. Both SIK3 T469A and SIK3 S551A
proteins showed reduced immunoreactivities for antiphospho-S551 substrate and anti-phospho-T469
substrate. It can be assumed that phosphorylation of
S551 and T469 may interact each other and synchronize.
Moreover, SIK3 T469A and SIK3 S551A proteins have
reduced binding to 14-3-3. Attenuation of the binding
between SIK3 and 14-3-3 is consistent with a longer
sleep phenotype.
This study indicated that the lacking PKA recognitionsite of SIK3, especially T469 and S551, is a gain offunction and increased NREM sleep time and NREM
sleep delta density. Also, SIK3 T469A and SIK3 S551A
reduced the binding with 14-3-3 and it is thought to havea significant effect on sleep/wake behavior.
-----------------------------------------------------------------------------------------------------------------------------------Although REM sleep (REMS) is ubiquitous in
mammals, the molecular/neural mechanism of REMS
regulation remains unknown. We previously establishedthe Dreamless mutant pedigree exhibiting abnormal
EEG power spectra and ~50% reduction in REMS
episode duration (Funato et al., Nature 2016). We
identified an SNP specific to Dreamless mutant 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 same 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 the responsible brain
regions and neuronal subtypes through which NALCN
regulates REMS, we generated flox and FLEx (flipexcision) knock-in mice bearing Cre-dependent loss-offunction and gain-of-function Nalcn alleles,
respectively. In Nalcn-FLEx mice, we confirmed thatthe mice crossed with systemic Cre-expressing lines
phenocopied the Dreamless mice on EEG/EMG
analyses. In Nalcn-flox mice, we confirmed a neuronalsubtype-specific deletion of Nalcn mRNA in adult brain
tissues. With Foxg1-IRES-Cre or Emx1-Cre lines, weobserved that both gain-of- and loss-of-function
mutants showed theta/delta dissonance during REMS.
With En1-Cre, we observed fragmentation of REMS. It
is suggested that NALCN in forebrain and pons-medulla
regions has distinct roles for REM sleep regulation, and
NALCN in excitatory cortical- and hippocampal neuron
plays a key role for the maintenance of theta/delta
oscillation during REMS.
------------------------------------------------------------------

27------------------------------------------------------------------

Upstream and downstream pathways of SIK3
in the regulation of sleep and wakefulness26------------------------------------------------------------------

Sleep/wake behavior of mice lacking PKAphosphorylation sites of SIK3

Tomohiro Kitazono1, Noriko Hotta-Hirashima1, Shinya
Nakata1, Kazuya Miyanishi1, Kanako Iwasaki1,
Tomoyuki Fujiyama1, Hiromasa Funato1, 2, Masashi
Yanagisawa1, 3, 4

Minjeong Park1, Takato Honda2, Chika Miyoshi1,
Tomoyuki Fujiyama1, Kanako Iwasaki1, Miyo
Kakizaki1, Aya Ikkyu1, Seiya Mizuno3, Satoru
Takahashi3, Hiromasa Funato1,4 and Masashi
Yanagisawa1,5

1 Univ of Tsukuba
2 Dept of Anat, Fac of Med, Toho Univ
3 Dept of Mol Genet, Univ of Texas Southwestern Med Cent,11

is under genetic control, the intracellular signaling
pathway regulating sleep/wakefulness remains
unknown. We recently identified Salt-inducible kinase 3(Sik3) as a sleep promoting gene. The gain-of-function
mutation (Slp) induces increased sleep need and amount.
On the other hand, sleep deprivation specificallyenhances the phosphorylation level at T221 in SIK3.
Phosphorylation level at T221 in the SIK3 kinasedomain modulates the kinase activity, suggesting that
SIK3 kinase activity regulates sleep need.
Here, to examine the role of SIK3 kinase activity insleep/wake regulation, we performed EEG/EMG-based
sleep/wake analysis of mice carrying a T221A or T221E
mutation in Sik3. Both Sik3T221A and Sik3T221E
mice did not show any significant differences in sleep
time. While Sik3T221A mice exhibited marked
reduction of delta density during NREM sleep, which isa marker for sleep need, Sik3T221E mice also showeda tendency to decrease the delta density, which isconsistent with SIK3(T221E) have a constitutive but
less kinase activity. Moreover, we investigated the roleof kinase activity of SLP mutant SIK3 in sleep/wakeregulation. Sik3T221A-Slp mice exhibited theabolishment of hypersomnia phenotype of Sik3Slp mice.
In contrast, Sik3T221E-Slp mice showed increased
sleep time compared with Sik3T221E mice. These
results suggest SIK3 kinase activity regulates sleep needand is crucial for the induction of increased sleep
amount.
------------------------------------------------------------------

Dallas, USA
4 TARA, Univ of Tsukuba-----------------------------------------------------------------Sleep behavior is conserved from vertebrates toinvertebrates, and is regulated by the accumulation anddissipation of homeostatic sleep need. Despite its
importance, the molecular and cellular basis of
homeostatic sleep need remains unclear. We haverecently revealed that salt-inducible kinase 3 (SIK3) inthe brain plays a key role in this process (Funato et al.,Nature, 2016, Kim et al., Nature, 2022). In this study,
we aim to further explore the molecular and cellularbasis of homeostatic sleep need by identifying upstream
and downstream components of SIK3.
As a potential upstream regulator of SIK3, we focusedon liver kinase B1 (LKB1). We generated postnatal
neuron-specific LKB1 knockout (nKO) mice, and foundthat LKB1 nKO resulted in a significant decrease in
EEG delta power during non-rapid eye movement sleep(NREMS), which is an indicator of sleep need.
Furthermore, when we induced a constitutively active
mutation of Sik3 (T221E) in LKB1 nKO mice, we
observed a rescue of weaker NREMS EEG delta power.
These results suggest that LKB1 regulates sleep/wakebehavior by acting upstream of SIK3.
Downstream components of SIK3 have not been wellinvestigated. To identify downstream signaling
pathways of SIK3, we employed substrate screening
and revealed that the RhoA-GTPase signaling pathway
is strongly related to phosphoproteins identified in thescreening. Furthermore, when expressing a
constitutively active form of RhoA in the whole brain,
NREMS EEG delta power during NREMS was
decreased, suggesting that RhoA is also involved insleep/wake regulation. Our studies provide novel insight
into sleep/wake regulation.
------------------------------------------------------------------

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

SIK3-HDAC4 in the suprachiasmatic nucleus
regulates the timing of arousal at the dark
onset and circadian period in mice
Fuyuki Asano1, Staci J Kim1, Tomoyuki Fujiyama1,
Chika Miyoshi1, Noriko Hotta-Hirashima1, Nodoka
Asama1, Kanako Iwasaki1, Miyo Kakizaki1, Seiya
Mizuno2, Michihiro Mieda3, Fumihiro Sugiyama2,
Satoru Takahashi2, Shoi Shi1, Arisa Hirano1,4, Hiromasa
Funato1,5, Masashi Yanagisawa1,6,7

28------------------------------------------------------------------

Molecular mechanisms for sleep/wakeregulation by SIK3 kinase signaling
Shinya Nakata1, Haruna Abe-Komiya1, Tomoyuki
Fujiyama1, Fuyuki Asano1, Chika Miyoshi1, Aya Ikkyu1,
Seiya Mizuno2, Fumihiro Sugiyama2, Satoru Takahashi2,
Hiromasa Funato1,3, Masashi Yanagisawa1

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

University of Tsukuba
2
Laboratory Animal Resource Center in Transborder Medical
Research Center, Institute of Medicine, University of Tsukuba
3 Department of Integrative Neurophysiology, Graduate School of
Medical Sciences, Kanazawa University
4 Faculty of Medicine, University of Tsukuba
5 Department of Anatomy, Toho University Graduate School of
Medicine
6 Department of Molecular Genetics, University of Texas
Southwestern Medical Center
7 Life Science Center for Survival Dynamics, Tsukuba Advanced
Research Alliance, University of Tsukuba

1 International Institute for Integrative sleep medicine, Universityof Tsukuba
2 Faculty of Medicine, University of Tsukuba
3 Department of Anatomy, Toho University-----------------------------------------------------------------Sleep amount within a day is tightly regulated by the
homeostatic sleep need accumulating during
wakefulness and dissipating during sleep. Althoughseveral studies suggests that the required sleep amount12-----------------------------------------------------------------Sleep is a fundamental behavior conserved fromvertebrates to invertebrates. Sleep/wakefulness is under
the control of “two processes”: homeostatic sleep needand circadian rhythm. Salt-inducible kinase 3 (Sik3) andhistone deacetylase (HDAC) 4, a SIK3 substrate, are
implicated in the sleep need signaling in glutamatergic
neurons. Here, we demonstrate that the SIK3-HDAC4
pathway also regulates circadian behaviors, such as thestrong arousal phase-locked to the beginning of the darkphase in laboratory mice, acting in the suprachiasmatic
nucleus (SCN). SIK3 deficiency in the gammaaminobutyric acid (GABA)-ergic neurons orneuromedin S (NMS)-producing neurons delayed thearousal peak phase and lengthened the behavioral
circadian cycle under both 12-h light: 12-h dark
condition (LD) and constant dark condition (DD)without changing daily sleep amounts. In contrast, theinduction of a gain-of-function mutant allele of Sik3 in
GABAergic neurons exhibited advanced activity onset
and a shorter circadian period. Loss of SIK3 in arginine
vasopressin (AVP)-producing neurons lengthened thecircadian cycle, but the arousal peak phase was similar
to that in control mice. Heterozygous deficiency of
HDAC4 shortened the circadian cycle, whereas micewith HDAC4 S245A mutation, which is resistant tophosphorylation by SIK3, delayed the arousal peakphase. Phase-delayed core clock gene expressions weredetected in the liver of mice lacking SIK3 in
GABAergic neurons. These results suggest that the
SIK3-HDAC4 pathway regulates the circadian period
length and the timing of arousal through NMS-positive
neurons in the SCN, independent of the regulation of
sleep amounts and depth.
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regulation of sleep remain to be elucidated. We
previously found that a gain-of-function Sleepy (Slp)mutation in the salt-inducible kinase 3 (Sik3) gene,which produces the mutant SIK3(SLP) protein,increases non-REM sleep (NREMS) and NREMS EEG
delta density, a marker for the level of sleep need. We
expect that SIK3 takes part in cellular signaling
conveying seep need. However, it remains to beelucidated which cells and brain regions are responsible
for the increased NREMS in Sleepy mutant mice. In thisstudy, we aim to determine the cell type and neuronalgroups that enhance quality and quantity of NREMS
through SIK3(SLP) expression. First, we showed that
Sik3(Slp) in mature neurons are sufficient to increase
NREMS amount and NREMS EEG delta density withsynapsin1CreERT2;Sik3Slp-flox/+ mice which express
Sik3(Slp) in neurons upon tamoxifen injections. Next,
we explored neuronal groups through which SIK3(SLP)
expression increases NREMS, by AAV-mediatedexpression of SIK3(SLP) in the hypothalamus. We
found that SIK3(SLP) expression in the medial part of
the hypothalamus increased NREMS. This result
implies that SIK3(SLP) expression in the specific brain
regions directly or indirectly modulates the activity ofneurons executing the switch of sleep/wake states,
which results in increased NREMS amounts. This will
be an important step to connecting the intracellularsignal representing sleep need with the circuit-levelmechanism for sleep/wake switch.
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SIK3 regulates sleep amount in hypothalamic
nuclei
Kanako Iwasaki1, Tran Phan Ngoc Thao1, Tomoyuki
Fujiyama1, Shinya Nakata1, Minjeong Park1, Chika
Miyoshi1, Noriko Hotta-Hirashima1, Aya Ikkyu1, Miyo
Kakizaki1, Yukiko Ishikawa1, Fumihiro Sugiyama1,
Seiya Mizuno1, Manabu Abe2, Kenji Sakimura2, Satoru
Takahashi1, Hiromasa Funato1,3, Masashi Yanagisawa1,4
1 University of Tsukuba
2 Niigata University
3 Toho University
4 University of Texas Southwestern Medical Center-----------------------------------------------------------------Sleep amount is regulated in a homeostatic manner.
Although a number of sleep/wake switching neuralcircuits have been identified, the mechanisms by whichthese neural circuits contribute to homeostatic
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