The 6th Annual IIIS Symposium

Solving the mystery of sleep
December 14, 2017
Tokyo Conference Center Shinagawa
Tokyo, Japan

Organizer

Masashi Yanagisawa
International Institute for Integrative Sleep Medicine (WPI-IIIS)
University of Tsukuba

Supporting Organization
Ministry of Education, Culture, Sports, Science and Technology

Timetable
Opening Address
Welcome

Masashi Yanagisawa

WPI-IIIS, University of Tsukuba

Opening address 1

Akira Ukawa

WPI Program Director

Opening address 2

Tetsuya Kishimoto

Ministry of Education, Culture, Sports,
Science and Technology (MEXT)

9:40-10:10

Fast high-resolution miniature two-photon
microscopy for brain imaging in freelybehaving mice at the single-spine level

Liangyi Chen

Peking University

Page. 4

10:10-10:40

Sleep deprivation, circadian rhythms,
and alcohol

Joshua J. Gooley

Duke-NUS Medical
SchoolPage. 5

10:40-11:10

Hypothalamic circuits for predation and
Minmin Luo
evasionNational Institute of
Biological Sciences, Beijing

Page. 6

11:10-11:40

Cell assembly mechanisms underlying
memory association

Kaoru Inokuchi

University of Toyama

Page. 7

Francesca Siclari

Lausanne University
Hospital

Page. 8

Shigenobu Shibata

Waseda University

Page. 9

Molecular mechanism of sleep disorderunder neuropathic pain

Minoru Narita

Hoshi University

Page. 10

Critical time window for the improved
13:30-14:00 spatial memory by optogeneticallyinduced slow wave sleep

Shu-min Duan

Zhejiang University

Page. 11

9:30-9:40

Session 1 | Chair: Sakiko Honjoh

11:40-11:50 Break & Lunch preparation

Lunchon Session | Chair: Yu Hayashi
11:50-12:20 The neural correlates of dreaming
12:20-12:50

Chrono-nutrition and -exercise studies
from mice to human

12:50-13:00 BreakSession 2 | Chair: Robert Greene
13:00-13:30

Poster Session & Tea Break
14:00-15:10

Data Blitz and Poster Session

MSD Afternoon Seminar | Chair: Masashi Yanagisawa
15:10-15:40 Arc -haeology of long-term memory
15:40-16:10

Spatio-temporal dynamics of brain
activity during sleep

Neural circuits of orexin neurons:
16:10-16:40 interface of systems of emotion andarousal/vigilance
16:40-16:50 BreakPathophysiology of circadian rhythm
16:50-17:20
sleep-wake disorder

Haruhiko Bito

The University of Tokyo Page. 12

Vladyslav Vyazovskiy

University of Oxford

Page. 13

Takeshi Sakurai

WPI-IIIS, University ofTsukubaPage. 14

Akiko Hida

National Center of
Neurology and Psychiatry

Page. 15

Stanford University

Page. 16

Understanding of the impact of light on
17:20-17:50 circadian rhythms: Efficacious
Jamie M. Zeitzer
phototherapy of circadian sleep disorders
17:50

Closing

18:00-20:00 Reception

2

Information
Precautions
1. No outlets are available nearby audience seats
2. No smoking in symposium venues: please smoke at the designated area on each floor
3. Wireless LAN is available in the foyer.

LunchFree lunch will be provided for pre-registered participants. Some extra may be available fornon-pre-registered participants on a first-come-first-served basis (will be announced).

Reception
18:00 - 20:00 at Foyer on 5th floor
For MSD afternoon seminar attendees.

Floor Map (5th floor) | Tokyo Conference Center Shinagawa

Symposium / Poster sesion venue(Large Hall A + B)

Reception desk

Concierge

Foyer

Escalator

The 6th Annual IIIS Symposium3Session 1

9:40 - 10:10

Fast high-resolution miniature two-photon
microscopy for brain imaging in freely-behaving
mice at the single -spine level
ABSTRACT

Liangyi Chen
Peking University, China

The 6th Annual IIIS Symposium

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Recent developments in miniaturized microscopes havefurthered the quest to visualize brain activities and structuraldynamics in animals engaged in self-determined behaviors.
However, it remains an unmet challenge to resolve activity atsingle dendritic spines, the elemental units underlying
neuronal computation, in freely-behaving animals. Here, wereport the design, testing, and application of a fast, highresolution, miniaturized two-photon microscope (FHIRMTPM) that accomplishes this goal. With a headpiece
weighing 2.15 g and a new type of hollow-core photonic
crystal fiber to deliver 920-nm femtosecond laser pulses, theFHIRM-TPM is capable of imaging commonly usedbiosensors at high spatiotemporal resolution (0.64 µm
laterally and 3.35 µm axially, 40 Hz at 256 × 256 pixels). Its
micro-electromechanical systems scanner also enables
random-access capability and free-line scanning at up to
10,000 Hz. It compares favorably with benchtop two- photon
microscopy and miniature wide-field fluorescencemicroscopy in the structural and functional imaging of
Thy1-GFP- or GCaMP6f-labeled neurons. Further, wedemonstrate its unique application and robustness withhour-long recording of neuronal activities down to the levelof spines in mice experiencing vigorous body and head
movements or engaging in social interaction. Thus, our new
generation miniature microscope provides neuroscientists
the long-sought tool-of-choice for imaging the brain at thesynaptic level in freely-behaving animals.

Session 1

10:10 - 10:40

Sleep deprivation, circadian rhythms, and alcohol
ABSTRACT

Joshua J. Gooley
Duke-NUS Medical School,
Singapore

Most alcohol-related vehicular crashes occur at night, at atime when drivers are also likely to be sleep-deprived. It istherefore important to understand the interaction of alcoholand sleep loss on drowsiness and driving errors. My
laboratory is examining whether exposure to sleep
deprivation enhances the dose-dependent effects of alcoholon objective measures of sleepiness and driving simulator
performance. We have implemented the alcohol clamp
procedure, in which alcohol is administered intravenously toachieve a prescribed breath alcohol concentration (BrAC)
while subjects undergo sleep deprivation in the laboratory.
Our results indicate that combining a small dose of alcoholwith partial sleep deprivation can result in profound deficits
in driving performance. In other studies, we have examinedcircadian regulation of breath alcohol concentration byadministering small doses of alcohol at different phases of thecircadian cycle. Our results indicate that peak BrAC values
exhibit circadian variation with lower values in the evening
and higher values in the morning. Together, these studies
demonstrate that sleep deprivation and circadian rhythms
modulate alcohol responses. This work has potential
implications for road safety and alcohol toxicity.

The 6th Annual IIIS Symposium

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10:40 - 11:10

Hypothalamic circuits for predation and evasion
ABSTRACT

Minmin Luo
National Institute of Biological
Sciences, Beijing, China

The 6th Annual IIIS Symposium

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The interactions between predator and prey represent someof the most dramatic events in nature, and constitute a matterof life-and-death for both sides. The hypothalamus has been
implicated in driving predation and evasion; however, theexact underlying hypothalamic neural circuits remain poorly
defined. Here, we demonstrate that inhibitory and excitatory
projections from the mouse lateral hypothalamus (LH) to theperiaqueductal gray (PAG) in the midbrain drive, respectively,
predation and evasion. Optogenetically stimulating PAGprojecting LH GABA neurons drove strong predatory attack.
LH GABA neurons were activated during predation, andoptogenetically inhibiting these cells blocked predation. In
contrast, stimulating PAG-projecting LH glutamate neurons
drove evasion. LH glutamate neurons were activated during
evasion, and inhibiting them impeded the ability to makepredictive evasion. Therefore, the seemingly opposite
behaviors of predation and evasion are tightly regulated by
two dissociable modular command systems within a single
neural projection from the LH to the PAG.

Session 1

11:10 - 11:40

Cell assembly mechanisms underlying memory
association
ABSTRACT

Kaoru Inokuchi
University of Toyama, Japan

Memories are not stored in isolation from other memories but
are integrated into associative networks and are accumulated
based on prior experiences throughout life. A specificneuronal ensemble represents a given memory. Recent
studies suggest that synchronous activity of neuronalensembles encoding distinct memory traces mediates theassociation of memories, which is accompanied by thegeneration of a co-sharing neuronal subpopulation.
However, the functional role of this co-shared ensemble inmemory association remains elusive. Here, we show theexistence of a small population of co-shared neurons in thebasolateral amygdala (BLA) that mediates the link between
memories but is unnecessary for recalling individual
memories. Using two amygdala-dependent behaviouralparadigms, conditioned taste aversion (CTA) andauditory-cued fear conditioning (AFC), we find that
presenting the conditioned stimulus used for the CTA task
triggers the conditioned response of the AFC task, that is,freezing behaviour, after natural co-reactivation of theirmemory retrievals. Analyses using a cellular imaging
method, Arc/Homer1a catFISH, shows an increase in theratio of the co-shared neuronal subpopulation, an
overlapping ensemble. Optical silencing of the overlapping
ensemble suppresses CTA retrieval- induced freezing.
However, retrieval of the original CTA or AFC memory is notaffected. Thus, only the link between the CTA and AFC
memories is interrupted, without impairing either original
memory. Our findings provide new insight into a specificneuronal ensemble shared by two pre-stored memories,
generated through consecutive recall of the distinct
pre-stored memories. This finding may lead to thedevelopment of a way to dissociate unwanted connections
formed between traumatic memories and related daily events
without affecting the original memories.

The 6th Annual IIIS Symposium

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Lunchon Session

11:50 - 12:20

The neural correlates of dreaming
ABSTRACTFrancesca Siclari
Lausanne University Hospital,
Switzerland

The 6th Annual IIIS Symposium

8Dreaming is a form of consciousness that occurs during sleep,
while we are functionally disconnected from the environment.
Traditionally, dreaming has been linked to REM sleep, abehavioral state characterized by fast, desynchronizedelectroencephalographic activity similar to wakefulness. In
recent years however, it has become clear that dreaming canalso occur in NREM sleep, in which EEG activity is dominatedby slow waves and spindles. This has challenged theunderstanding of the neural correlates of conscious
experiences in sleep. In the present talk I will present a study
in which we investigated the neural correlates of dreaming
using a serial awakening paradigm and high-density EEG
recordings. We found that dreaming, irrespective of sleep
stage, is associated with a local reduction of low-frequency
activity in posterior cortical regions. By monitoring brain
activity in these regions in real time, we were able to predictthe presence or absence of dreaming in NREM sleep with ahigh accuracy. In addition, we found that specific dreamcontents were associated with localized high-frequency
increases within these posterior cortical areas. Taken together,
these local EEG correlates may account for the presence of
conscious experiences in behavioral states with radicallydifferent global EEG signatures.

Lunchon Session

12:20 - 12:50

Chrono-nutrition and -exercise studies frommice to human
ABSTRACT

Shigenobu Shibata
Waseda University, JapanThe rapid increase in adiposity is related to significant
changes in the human environment and lifestyle, including
less physical exercise and high-energy dietary choices. This
lifestyle is also related to locomotive syndrome, such as
sarcopenia, osteoporosis and joint problems. Thus, reducing
fat and/or sucrose and increasing protein and minerals in thediet and increasing physical exercise represent the primarymethods for prevention of and/or recovery from lifestylerelated diseases such as obesity and locomotive syndrome in
middle and old age persons. However the bet timing of
nutrient intake and exercise is still unknown. After discovery
of molecular mechanism of circadian rhythm, research fieldof chrono-biology has influenced on many other research
fields such as pharmacology, nutrition science and sports/exercise science.
Circadian clock systems play important roles inclock-related physiological functions including energy,
protein and mineral metabolisms. Breakfast skipping andhigh fat diet as for late supper cause obesity. Protein richbreakfast stimulates skeletal muscle volume in mice under 2meals per day schedule. In addition, inulin rich breakfast
reduced colon pH according with increase of short chain
fatty acids. Foods containing some nutrients and functional
components are reported to provide phase-resetting effects
on peripheral clocks. In addition, circadian rhythm system
controls physical exercise. The optimal timing of exercise forprotection of obesity may be late afternoon. Morning
exercise and protein rich breakfast prevented the soleus
muscle atrophy by tail suspension- induced muscle atrophy
mouse. The present results strongly suggest that the timing of
feeding and exercise is critical for protection of lifestyle
related diseases such as obesity and locomotive syndromes.
We are interested in elucidating how new approach such as
chrono-nutrition and chrono-exercise help to promote healthscience using animal and observation/intervention humanexperiments.

The 6th Annual IIIS Symposium

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13:00 - 13:30

Molecular mechanism of sleep disorder underneuropathic pain
ABSTRACT

Minoru Narita
Hoshi University School of
Pharmacy and Pharmaceutical Sciences, JapanThe 6th Annual IIIS Symposium

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It has been reported that sleep problems and daytime
sleepiness are commonly found in opioid-treated primarycare patients with chronic pain and cancer pain, and seem tobe related mainly to depression and the severity of pain.
Cumulative evidence demonstrates that the forebrain,including neurons in the anterior cingulate cortex (ACC)
plays an important role in pain-related perception andchronic pain. Sleep dysregulation was observed after sciatic
nerve ligation in mice. On the basis of the results of
EEG/EMG, the hypnotic effects of midazolam and zolpidem,
were dramatically suppressed in mice with sciatic nerve
ligation. We also found that sciatic nerve ligation caused amorphological change in GFAP-labelled astrocytes, and also
GFAP-positive immunoreactivity was significantly increasedin the ACC of mice. Under these conditions, membranebound GABA transporters (GATs) on activated GFAP-positive
astrocytes were significantly increased in the ACC, andextracellular GABA levels in this area after depolarization
were rapidly decreased. We next used functional magnetic
resonance imaging to visualize the increased blood
oxygenation level-dependent signal intensity in the ACC ofmice with sciatic nerve ligation under mild noxious stimuli.
Such stimuli significantly increased the release of glutamatein the ACC. In addition, sciatic nerve ligation and mild
noxious stimuli changed the morphology of astrocytes in the
ACC. Furthermore, glutamate induced the translocation of
GAT-3 to astrocyte cell membranes using primary cultured
glial cells from the mouse cortex. Moreover, the GABA levelat the synaptic cleft in the ACC of nerve-ligated mice wassignificantly decreased exposure to mild noxious stimuli. We
also demonstrated that selective optical stimulation of theseastrocytes in vivo triggered sleep disturbance. Taken together,
the present findings provide novel evidence that astrocytic
overactivation in the ACC can mimic sleep disturbance inmice.

Session 2

13:30 - 14:00

Critical time window for the improved spatial memoryby optogenetically-induced slow wave sleep
ABSTRACT

Shu-min Duan
Zhejiang University School of
Medicine, China

Sleep is thought to be involved in memory consolidation fora long time. However, most studies investigate the correlationof sleep and memory by means of sleep deprivation, which
may induce stress and thus secondarily interfere withmemory formation. Furthermore, the precise time window
for the correlation of sleep and memory consolidation
remains unclear. We found that optogenetic stimulation of
GABAergic neurons in parafacial zone (PZ) immediately
induced slow wave sleep (SWS) in (VGAT)-ChR2-EYFP mice.
Taking the advantage of this approach, we are able to control
SWS in these mice in precise timing. The 30 min SWS
induced by light stimulation of GABAergic neurons in PZ
immediately or 15 min after the sample phase of theobject-in-place task signiﬁcantly improved the memory of
mice. The improved memory was blocked when the lightstimulation was coupled with gentle touch to prevent SWS
induction, suggesting that SWS, but not light stimulation of
PZ itself, is crucial for the improved memory. Interestingly,
when SWS was induced 30 min or more after the learning
phase failed to improve the memory. The light-induced SWS
immediately after the learning phase also enhanced memoryin Y-maze spatial memory and contextual fear memory. The
light-induced SWS right before the test phase had no effecton memory performance, suggesting SWS does not affectmemory retrieval. Our results indicate that SWS play a
crucial role in the consolidation of hippocampus-dependent
spatial memory.

The 6th Annual IIIS Symposium

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MSD Afternoon Seminar

MSD Afternoon Seminar

15:10- 15:40

Arc-haeology of long-term memory
ABSTRACT

Haruhiko Bito
The University of Tokyo
Graduate School of Medicine,Japan

Deciphering the intricate and interactive relationship between theinformation encoded in the genome and the ongoing synaptic
activity is critical for understanding the molecular and cellularsignaling underlying long-term memory formation andmaintenance of long-lasting changes within the brain. To
systematically dissect this question, we investigated the molecularbasis of the signaling from synapses to the nucleus and from thenucleus to the synapses, which crucially determines thepersistence of synaptic plasticity. We thus uncovered an
activity-dependent protein kinase cascade CaMKK-CaMKIV thatcritically controls the amplitude and time course ofphosphorylation of a nuclear transcription factor CREB
downstream of synaptic activity, thereby activating a plethora ofadaptive transcriptional responses within an active neuronal
circuit. We also identified a novel “inverse” synaptic tagging
mechanism in which one of CREB’s target gene, Arc, acts as abrake that helps weaken the non-potentiated synapses during themaintenance phase of synaptic plasticity. In genomic parlance,
Arc’s rapid induction following strong physiological stimuli is
dictated by a potent synaptic activity-responsive element (SARE)
present in its enhancer/promoter region, which strikingly harbors aunique cluster of binding sites for CREB, MEF2 and SRF/TCF. Basedon this discovery, we created a synthetic promoter E-SARE which
now allows to map, label, record and manipulate active neuronalensembles in various areas of the brain in vivo. Recently, we
designed a new set of genetically encoded Ca2+ indicators (GECIs),such as R-CaMP2, which are molecular spies of neuronal activity
with most desirable properties such as fast speed and signal
linearity. This was largely achieved by exchanging the M13
sequence of classical GECIs with a sensitive CaM-binding
sequence engineered based on neuronal CaMKKs. These efforts
collectively start to illuminate key molecular and cellular events
that are essential in neuronal coding and information processing in
active neuronal circuits and systems in vivo.

The 6th Annual IIIS Symposium

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MSD Afternoon Seminar

15:40 - 16:10

Spatio-temporal dynamics of brain activity
during sleep
ABSTRACT

Vladyslav Vyazovskiy
University of Oxford, UK

The 6th Annual IIIS Symposium

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Sleep has been found in all animal species carefully studied
to date; yet, the biological function of sleep remains unclear.
Sleep can be defined on at least two distinct levels: thebehaviour of the whole organism and the spatiotemporal
patterns of neuronal activity in the brain. Upon falling asleep,
cortical networks alternate between periods of generalized
population firing and periods of relative silence. This patternof neuronal activity gives rise to electroencephalogram (EEG)
oscillations at a frequency of approximately 1-4 Hz, which
are termed slow waves. Although a prevailing view is thatbrain states are regulated in a global fashion, research overthe last decades has revealed that spontaneous brain activity
during sleep can be locally modulated. For example, slow
wave activity (SWA) is more intense in frontal compared tomore posterior areas, especially in early sleep or after sleep
deprivation, and regional differences are apparent at the levelof individual sleep slow waves. Although the alternation of
periods of increased neuronal activity and silence is usuallycorrelated across cortical regions and individual neurons,
up-states can sometimes be seen in one region of the cortex
while another region is in a down-state, with these statesoften spreading as travelling waves. Importantly, topographic
differences in brain activity during sleep are functional, as
peripheral stimulation or the spontaneous use of
circumscribed cortical areas leads to more intense local EEG
SWA. Such data demonstrate that not only waking duration
per se, but also strong activity in specific regions affects the‘intensity’ of subsequent sleep. Moreover, sleep deprivation isassociated with increased low-frequency EEG activity during
waking in both animals and humans, and recordings in ratssuggested that this EEG pattern reflects local neuronal OFF
periods. This notion suggests that sleep need accumulates atthe level of local cortical networks or even single neurons.
Thus, understanding the function of sleep requires one tobridge the gap between local and global levels - fromindividual cells to large-scale brain networks and global
behaviour.

MSD Afternoon Seminar

16:10- 16:40

Neural circuits of orexin neurons: interface ofsystems of emotion and arousal/vigilance
ABSTRACT

Takeshi Sakurai
WPI-IIIS, University of Tsukuba,
Japan

Animals shift their sleep-wakefulness state according to theirinternal state and the external environment by utilizing three major
influential elements, i.e., homeostatic, circadian and allostatic
factors. Among them, allostatic factors include the nutritional stateand external environment, which trigger emotion. For example,
stressful and emotionally-salient situations such as encountering
predators, adapting to novel situations or expecting a reward
require animals to shift their behavior to a vigilant state, along withalteration of their physiological condition through modulation ofautonomic and endocrine functions. Studies of efferent andafferent systems of orexin-producing neurons have shown that theorexin neuronal system has close interactions with systems that are
involved in the regulations of emotion, energy homeostasis,reward, and arousal. Many studies have suggested that orexin
neurons are activated during the behavioural expression of fear orin response to cues associated with danger or reward. Theseobservations suggest that orexin neurons are involved in control ofvigilance states in response to outer environment. On the other
hand, orexin system affects expression of behavioral response
against outer environment via control of monoaminergic neurons.
I will discuss functional interplay between the limbic system andarousal system, and involvement of orexin and orexin receptors inthese interactions.

The 6th Annual IIIS Symposium

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MSD Afternoon Seminar

16:50- 17:20

Pathophysiology of circadian rhythm
sleep-wake disorder
ABSTRACT

Akiko Hida
National Center of Neurology
and Psychiatry, JapanThe 6th Annual IIIS Symposium

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Circadian rhythm sleep-wake disorders (CRSWDs) are defined bypersistent or recurrent disturbed sleep–wake patterns and consistof several subtypes including delayed sleep–wake phase disorder(DSWPD) and non-24-hour sleep–wake rhythm disorder(N24SWD). CRSWDs are thought to result from impairment of thecircadian clock system and/or a misalignment between theendogenous circadian rhythm and exogenous entrainment factors.
In mammals, the central oscillator in the suprachiasmatic nucleusof the hypothalamus incorporates environmental cues, such as
light exposure, and coordinates the phase of oscillators inperipheral tissues. The molecular mechanisms underlying thecircadian clock system involve the transcription-translation
negative feedback loops of multiple clock genes. Evaluating thecircadian phenotype is crucial for establishing a precise clinicaldiagnosis and for understanding the pathophysiology of CRSWDs.
we examined the circadian phenotypes of patients with DSWPD
and N24SWD by evaluating Bmal1-luciferase (Bmal1-luc) rhythms
in skin fibroblast cells from individual patients. The period lengthof the Bmal1-luc rhythm (in vitro period) varied among individuals.
The N24SWD group showed a significantly longer in vitro period
than did the control or DSWPD group. Furthermore, in vitro period
was associated with response to chronotherapy in the N24SWD
group. Longer in vitro periods were observed in the
non-responders compared to the responders in the N24SWD
group. Our findings suggest that prolonged circadian periods
contribute to the onset and poor treatment outcome of N24SWD.
In vitro rhythm assays could be useful for predicting circadianphenotypes and clinical prognosis in patients with CRSWDs.

MSD Afternoon Seminar

17:20 - 17:50

Understanding of the impact of light on circadianrhythms: Efficacious phototherapy of circadiansleep disorders
ABSTRACTJamie M. Zeitzer
Stanford University, USA

Light is the most potent influence on the timing of the humancircadian clock and has been used for decades to treat
circadian-based sleep disorders. Under ideal laboratoryenvironments, light can shift the human circadian clock up to
three hours in a day, though the true value is much less underreal-world settings. Furthermore, light treatments in the realworld often are scheduled to occur during times at whichpeople are meant to be sleeping, further limiting its
effectiveness. Based on recent advances in our understandingof the neurobiology that underlies the transfer of lightinformation from the retina to the circadian clock, we haveundertaken a series of experiments to examine if we could
optimize light treatment such that larger shifts could be
evoked and that such shifting could occur when people are
sleeping. Our examination of sequences of brief flashes oflight has satisfied both of these criteria. Flash sequences are
far more potent at shifting circadian timing than is continuous
light. In a first optimization step, we generated phase shifts in
response to light flashes that are at least three-fold larger thanthose observed after traditional light therapy and are,photon-per-photon, 12,000 times more effective. Flashsequences can be administered during sleep without
necessarily interfering with sleep. Our most recent work
demonstrates the clinical effectiveness of this therapy in teenswith delayed sleep. In conjunction with behavioral therapy,
light flashes administered during sleep in teens is able tomove sleep to an earlier time, increasing nightly sleep by ~45
minutes and improving subjective sleep quality. Future work
focuses on further optimization of light flashes with the goalof generating very large (>8 hr.) phase shifts.

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