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Contents lists available at ScienceDirect

Neuroscience Research
journal homepage: www.elsevier.com/locate/neures

Review article

Sleep in vertebrate and invertebrate animals, and insights into the

function and evolution of sleep
Shinichi Miyazaki, Chih-Yao Liu, Yu Hayashi
International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Many mammalian species, including humans, spend a substantial fraction of their life sleeping. Sleep
Received 21 January 2017 deprivation in rats ultimately leads to death, indicating the essential role of sleep. Exactly why sleep is
Received in revised form 29 March 2017 so essential, however, remains largely unknown. From an evolutionary point of view, almost all animal
Accepted 29 March 2017
species that have been investigated exhibit sleep or sleep-like states, suggesting that sleep may benet
Available online xxx
survival. In certain mammalian and avian species, sleep can be further divided into at least two stages,
rapid eye movement (REM) sleep and non-REM sleep. In addition to a widely conserved role for sleep,
Keywords:
these individual sleep stages may have roles unique to these animals. The recent use of state-of-the-art
REM sleep
Slow wave activity
techniques, including optogenetics and chemogenetics, has greatly broadened our understanding of the
Mammals neural mechanisms of sleep regulation, allowing us to address the function of sleep. Studies focusing on
Reptiles non-mammalian animals species have also provided novel insights into the evolution of sleep. This review
Amniotes provides a comprehensive overview regarding the current knowledge of the function and evolution of
Zebrash (Danio rerio) sleep.
Roundworm (Caenorhabditis elegans) 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.
Fruit y (Drosophila melanogaster)

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. How to dene sleep . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. Sleep in certain vertebrate species can be further divided into REM sleep and NREM sleep . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4. Evolution of REM and NREM sleep explored by phylogenetic and genetic approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5. Sleep can happen locally in the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6. Genetic studies of sleep in the teleost zebrash (D. rerio) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
7. Sleep in invertebrate animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
7.1. General introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
7.2. Genetic studies of sleep in the fruit y (D. melanogaster) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
7.3. Genetic studies of sleep in the roundworm (C. elegans) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
8. Is there a conserved role for sleep? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
9. Concluding remarks and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

ing and drinking, however, the essential function of sleep remains

1. Introduction a mystery. In a radical experiment in which rats were not allowed
to sleep at all, the sleep-deprived rats exhibited drastic symp-
We spend nearly one-third of our life sleeping. Sleep, like eating toms such as excessive body weight loss and severe skin lesions,
or drinking, is essential for living. Almost everyone has experienced and invariably died within a couple of weeks (Rechtschaffen and
the feeling of falling to pieces after a night of poor sleep. Unlike eat- Bergmann, 2002; Rechtschaffen et al., 1989). The direct cause of
such symptoms resulting from a lack of sleep remains unknown.
During the night, sleep cycles between two distinct states, rapid
Corresponding author. eye movement (REM) sleep (when vivid dreams occur) and non-
E-mail address: hayashi.yu.fp@u.tsukuba.ac.jp (Y. Hayashi). REM (NREM) sleep. An abnormal balance between the two sleep

http://dx.doi.org/10.1016/j.neures.2017.04.017
0168-0102/ 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

Please cite this article in press as: Miyazaki, S., et al., Sleep in vertebrate and invertebrate animals, and insights into the function and
evolution of sleep. Neurosci. Res. (2017), http://dx.doi.org/10.1016/j.neures.2017.04.017
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Fig. 1. Representative human EEG patterns during each sleep stage. NREM sleep
is further divided into multiple stages. Numbers in parentheses indicate classical
NREM sleep stages. The EEG data were kindly provided by Dr. Makoto Satoh and Ms.
Kumiko Shimoyama (International Institute for Integrative Sleep Medicine (WPI-
IIIS), University of Tsukuba, Japan).
states is a common and early symptom of various psychiatric dis-
orders, suggesting that both sleep states have important roles.
Because these two sleep states are evident only in certain vertebrate
species, elucidating their functions might be key to understanding
our complex brain.
From the aspect of evolution, sleep or sleep-like states are
conserved across diverse animal species, suggesting that sleep
fullls a common purpose that benets animal survival. Until
now, however, the function of sleep in any animals has remained
largely unknown. Researchers have recently begun to study the
evolutionarily conserved components of sleep in simple genetic
animal models, including fruit ies (Drosophila melanogaster),
roundworms (Caenorhabditis elegans), and zebrash (Danio rerio)
(Hendricks et al., 2000; Raizen et al., 2008; Shaw et al., 2000; Singh
et al., 2014; Zhdanova et al., 2001).
New techniques, such as two-photon microscopy, optogenetics,
and chemogenetics, allow for the observation and manipulation
of neurons in vivo with unprecedented convenience and resolu-
tion. Partly due to these breakthrough techniques, recent studies
have provided evidence suggesting that sleep is involved in mem-
ory consolidation, clearance of brain metabolites, dendritic spine
remodeling, and brain development (Bushey et al., 2011; Donlea
et al., 2011; Xie et al., 2013; Yang et al., 2014). Further research is
needed, however, to elucidate the general function of sleep across
species.
Here, we rst introduce the general criteria for sleep that allow
for its denition in animals other than mammals. We then describe
REM sleep and NREM sleep, which are the two major stages of mam-
malian and avian sleep, and discuss studies and hypotheses related
to their evolution. Next, we briey describe the unique adaptive
changes in the sleep of aquatic mammals. We then discuss current
progress in studies using simple genetic animal models, namely
zebrash, fruit ies, and roundworms. Finally, the implicated func-
tions of sleep in mammals and invertebrate animals are compared
and discussed.
2. How to dene sleep
Sleep in mammals is a relatively evident state, as electroen-
cephalograms (EEG) and electromyograms (EMG) can be used to
easily distinguish sleep and wakefulness (Fig. 1). Such criteria based
on EEG and EMG patterns, however, cannot be applied well to other
animals. Therefore, more generally applicable criteria to dene
sleep in species across the animal kingdom have been proposed
(Campbell and Tobler, 1984; Rial et al., 2010). These criteria include
loss of locomotion, maintaining distinct postures, enhanced arousal
thresholds to environmental stimuli, rapid reversibility, preference
Please cite this article in press as: Miyazaki, S., et al., Sleep in vertebrate and invertebrate animals, and insights into the function and
evolution of sleep. Neurosci. Res. (2017), http://dx.doi.org/10.1016/j.neures.2017.04.017
Fig. 2. Typical human sleep architecture through the night. Sleep stage data were
kindly provided by Dr. Takashi Kanbayashi (Akita University, Japan).
for specic environmental locations, and homeostatic rebound to
sleep deprivation. Based on these denitions, sleep has been char-
acterized in fruit ies (D. melanogaster), roundworms (C. elegans),
zebrash (D. rerio), and various other animal species (Hendricks
et al., 2000; Raizen et al., 2008; Shaw et al., 2000; Singh et al.,
2014; Zhdanova et al., 2001). Within mammals, these criteria also
help to differentiate sleep from other quiescent states. For exam-
ple, hibernation is distinct from sleep in that reversibility to the
alert state is not rapid. Furthermore, the application of these deni-
tions to simple animal models led to intriguing ndings that several
genetic components related to sleep are conserved across different
species, including salt-induced kinase 3 (SIK3) (Funato et al., 2016),
epidermal growth factor signaling pathway (Foltenyi et al., 2007;
Kramer et al., 2003, 2001; Kushikata et al., 1998; Van Buskirk and
Sternberg, 2007), protein kinase G (Langmesser et al., 2009; Raizen
et al., 2008), cyclic adenosine monophosphate signaling pathway
(Graves, 2003; Hendricks et al., 2001; Raizen et al., 2008), dopamin-
ergic pathway (Singh et al., 2014; Wisor et al., 2001), histaminergic
pathway (Haas et al., 2008; Monnier et al., 1967; Nicholson et al.,
1985; Oh et al., 2013; Renier et al., 2007; Sundvik et al., 2011), and
N-methyl-d-aspartate (NMDA) receptors (Sunagawa et al., 2016;
Tomita et al., 2015). Cautious interpretation is necessary, however,
as many of these pathways, proteins, and neurotransmitters are
involved in a variety of cellular events, and not specically in sleep.
Genes involved in circadian rhythm also have roles independent
of the circadian rhythm in the regulation of sleep (Franken et al.,
2007; Monsalve et al., 2011; Naylor et al., 2000; Shaw et al., 2002;
Viola et al., 2007; Wisor et al., 2002). These ndings support the
feasibility of using the above-mentioned features to dissect sleep
in non-mammalian species.
3. Sleep in certain vertebrate species can be further divided
into REM sleep and NREM sleep
Human sleep can be roughly divided into two main stages, REM
sleep and NREM sleep. NREM sleep is further subdivided into mul-
tiple stages. During a nights sleep, a normal person cycles between
these stages (Figs. 1 and 2). Each sleep stage can be distinguished
by EEG, which detects the electrical activity derived from the brain
surface (Fig. 1) and EMG, which detects the electrical activity of
skeletal muscles. Each stage has a distinct pattern in EEG and EMG,
as well as other features. The EEG of an awake person shows mostly
alpha and beta activity (neural oscillations in the frequency range
of 7.512.5 Hz and 12.530 Hz, respectively) (Fig. 1). Beta activity
appears when the persons behavior requires attention. On the con-
trary, alpha activity appears during a relaxed and quiet state. After
sleep onset, a person rst enters NREM sleep, which can be further
divided into three stages, N1-N3 (classically four stages). In stage
N1, alpha activity gradually decreases as theta activity (48 Hz)
appears (Fig. 1 and 2). During stage N2, the EEG is featured by sleep
spindles (e.g., brief bursts of high frequency [714 Hz] activity) and
K-complexes (e.g., one cycle of slow oscillation) (Figs. 1 and 2).
When the person enters stage N3, delta activity (14 Hz) or so-
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called slow wave activity starts to dominate the EEG (Figs. 1 and 2).
Stage N3 is typically called slow wave sleep, and during this stage,
the person is in the deepest sleep and the threshold for awaken-
ing is highest. Classically, stage N3 is further divided into two stages
based on the intensity of the slow waves (Fig. 1). This series of NREM
sleep stages is usually followed by REM sleep, in which beta activity
again increases, along with theta activity (Figs. 1 and 2). In addition,
a person in REM sleep shows rapid eye movement, muscle atonia,
and partial loss of homeothermy. Therefore, REM sleep can be easily
distinguished from wakefulness by the low EMG signal, despite the
similar EEG patterns. At the end of REM sleep is when the person
can be most easily awakened with a small disturbance. Afterward,
the person again enters stage N1 of NREM sleep (Fig. 2). A typical
cycle of NREM sleep and REM sleep takes approximately 1.5 h on
average. A normal person sleeps approximately 8 h a day, during
which typically four or ve cycles of REM sleep and NREM sleep
occur.
REM sleep was rst reported in 1953 (Aserinsky and Kleitman,
1953). REM sleep is associated with the phenomenon of dreaming.
A similar state was discovered in cats (Dement, 1958; Jouvet et al.,
1959). REM sleep is also termed paradoxical sleep, as wake-like
EEG patterns and loss of muscle tone simultaneously occur during
this unique state. The discovery of REM sleep in cats led to intense
anatomic, pharmacologic, physiologic, and genetic studies by Jou-
vets group and other groups to elucidate the neural substrates of
REM and NREM sleep. These studies contributed to discoveries that
the brainstem pontine tegmental area and adjacent mesencephalic
and medullary regions contain neurons with key roles in the gener-
ation of REM and NREM sleep (Boissard et al., 2002; Crochet et al.,
2006; Hayashi et al., 2015; Lu et al., 2006; Sakai et al., 2001; Saper
et al., 2010; Vanni-Mercier et al., 1989; Weber et al., 2015).
4. Evolution of REM and NREM sleep explored by
phylogenetic and genetic approaches
REM and NREM sleep are observed in various mammalian
and avian species, but are not so evident in other vertebrates.
Understanding the evolutionary events that occurred during the
transitions from ancestral vertebrates to mammals and birds may
provide important insights into the function of each sleep state.
Reptiles may be key to elucidating these events, as they are
likely the closest ancestors to mammals and birds. Current reptiles
seem to share many features with their ancestors in metabolism
and brain architecture, including poikilothermy and simple telen-
cephalic structures. By contrast, mammals and birds are both
homeotherms and have advanced telencephalic structures, such as
the neocortex in mammals and the nidopallium (formerly neos-
triatum) in birds. Given that the reptilian EEG pattern is highly
dependent on body temperature and information processing is
largely different between reptiles and mammals/birds due to their
distinct brain architecture, some researchers hypothesize that the
transitions in sleep from the ancestral style to mammalian or avian
styles may be related to the evolution of homeothermy and telen-
cephalic structures (Nicol et al., 2000).
Despite a large number of studies seeking the origin of NREM
and REM sleep, solid and consistent conclusions cannot yet be
drawn. Until now, obvious signs of NREM and REM sleep could
only be detected in mammals and birds, and there is a possibility
that these sleep states emerged independently in each evolutionary
route of mammals and birds. Recent evidence, however, indicates
that reptiles also have multiple sleep stages that might correspond
to REM and NREM sleep. Here, to further explore this issue, we
introduce studies focusing on two kinds of animals, lizards and
monotremes.
Please cite this article in press as: Miyazaki, S., et al., Sleep in vertebrate and invertebrate animals, and insights into the function and
evolution of sleep. Neurosci. Res. (2017), http://dx.doi.org/10.1016/j.neures.2017.04.017
3
Monotremes are egg-laying mammals. Extant monotremes
are the echidna (Tachyglossus aculeatus) and the platypus
(Ornithorhynchus anatinus). During mammalian evolution, the
divergence of the monotreme lineage from other mammalian lin-
eages (the marsupial lineage and placental lineage) occurred ahead
of the divergence between marsupials and placental mammals.
Thus monotremes likely most closely resemble ancestral primitive
mammals. Early studies in which various measures of sleep/wake
were carefully recorded in the echidna suggested that this animal
undergoes NREM sleep with slow waves, but shows no sign of REM
sleep (Allison et al., 1972). One interpretation is that REM sleep
emerged in mammals after the divergence of the monotremes from
other mammals, and that NREM sleep closely resembles primitive
sleep. In studies by another group, however, simultaneous record-
ings of brainstem neural activity revealed a REM-like sleep pattern
although the cortical EEG detected slow wave activity resembling
NREM sleep (Siegel et al., 1996). This putatively primitive form of
sleep thus contains features of both REM and NREM sleep. In con-
trast to these studies, it was also reported that sleep in echidna
could be clearly differentiated as REM sleep or NREM sleep by cor-
tical EEG, but that REM sleep could only be detected at a certain
temperature range (Nicol et al., 2000). This report, however, is chal-
lenged by the view that quiet wakefulness and REM sleep were
not sufciently differentiated. Taken together, whether REM sleep
and NREM sleep are clearly differentiated in primitive mammals
remains to be solved.
Although reptiles do not exhibit obvious REM sleep and NREM
sleep stages, some characteristics of reptilian sleep patterns resem-
ble those of NREM sleep or REM sleep. In one such example, a lizard,
the Australian dragon (Pogona vitticeps), exhibits two distinct sleep
stages that share some features similar to NREM sleep and REM
sleep (Shein-Idelson et al., 2016). In more detail, during one sleep
stage, low-frequency activity (<4 Hz) resembling mammalian slow
wave activity is detected in a certain region of the brain, the dor-
sal ventricular ridge (DVR). In the other sleep stage, an activity
closer to wake-like activity is detected in the DVR. Moreover, this
latter sleep stage is accompanied by rapid eye movements. Thus,
the two sleep stages each exhibit some features that are similar to
mammalian NREM sleep and REM sleep, respectively. Throughout
the night, the Australian dragon cycles between these two sleep
stages. Interestingly, as with monotremes, the sleep architecture
in the Australian dragon is affected by ambient temperature. The
duration of each sleep stage episode is approximately 80 s at 27 C,
but when the room temperature is increased to 32 C, the average
episode duration decreases to 60 s. By contrast, when the temper-
ature is lowered to 25 C, the average episode duration increases
to over 100 s. Temperature-dependent changes in the duration of
NREM sleep and REM sleep episodes are also seen in mammals,
providing further support that the sleep stages in Australian drag-
ons might share a common origin with mammalian sleep stages
(Kumar et al., 2009). Because lizards, including Australian dragons,
belong to lepidosaurs, which is a subclass of reptiles most distant
from avians, REM and NREM sleep might have emerged early during
amniote evolution or even before that. It is important to note, how-
ever, that the DVR, in which sleep state-dependent neural activity is
observed, is a brain area anatomically distinct from the mammalian
neocortex or hippocampus, and thus whether the underlying mech-
anism of sleep stage-specic neural activities in lizards are similar
to those in mammals remains to be resolved.
Another long-debated question is which of the two sleep stages,
REM or NREM sleep, resembles the prototype of sleep. Several
arguments have been raised to support that REM sleep, rather
than NREM sleep, closely resembles a state in ancestral primi-
tive animals. During REM sleep, the thermoregulation in mammals
and birds is reduced; i.e., there is a partial loss of homeothermy.
Furthermore, REM sleep is generated in the brainstem, a struc-
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ture that is highly conserved among vertebrates. In fetuses and
infants of certain mammals, REM sleep is the major state of sleep,
perhaps because the rhombencephalon (hindbrain) matures in an
earlier developmental stage compared to other parts of the brain
(Finlay et al., 1998). In addition, the slow wave activity generated
in the cerebrum during NREM sleep is a brain activity unique to
animals with a well-developed telencephalon. Collectively, REM
sleep could be an ancient form of sleep. This hypothesis, however,
requires careful interpretation. Recent studies have clearly demon-
strated that the brainstem has crucial roles not only in generating
REM sleep but also in NREM sleep (Anaclet et al., 2014; Hayashi
et al., 2015). Sleep in neonates may qualitatively differ from that in
mature individuals, and some researchers have suggested that their
sleep should be classied as active sleep and quiet sleep, instead of
REM sleep and NREM sleep (Frank and Heller, 2003).
An effective approach to solving the evolutionary origin of REM
and NREM sleep might be to elucidate the molecular identity of the
neurons in the brainstem that regulate REM and NREM sleep. The
anatomic structures of the brainstem are highly conserved within
vertebrates, and thus such studies will allow for detailed compari-
son of sleep-regulating cells across animal species. For example, if
the neurons that generate REM or NREM sleep could be precisely
identied based on gene expression and cell lineage, it should be
possible to identify homologous cells in various animal species,
including monotremes and lizards. Functional manipulation of
these cells in these animals should provide substantial information
regarding whether the sleep states observed in these animals are
truly homologous to REM or NREM sleep. A recent study identied
and revealed the developmental origin of neurons in the brainstem
pons that negatively regulate REM sleep. These cells derive from
a group of proneural cells in the embryo hindbrain located in the
cerebellar rhombic lip and are positive for the transcription factor
gene Atoh1 (Hayashi et al., 2015). These Atoh1-positive proneural
cells also contribute to generating neurons in the brainstem that
strongly promote wakefulness. Thus, the Atoh1-positive cell lin-
eage is crucial for constructing anatomic structures that commit to
sleep-wake regulation. The Atoh1-positive proneural cells are dis-
tributed in the embryo hindbrain in a highly conserved manner
across vertebrates, even in zebrash. Perhaps, during evolution, a
subpopulation of Atoh1-positive proneural cells generated a func-
tionally novel group of neurons that could support the emergence
of REM and NREM sleep. Functional analyses of this cell lineage in
various animal species may help to clarify the evolutionary pro-
cesses of sleep.
Another key to solving the evolutionary origin of REM and NREM
sleep might be to identify genes involved in regulating either of the
two sleep states and addressing the roles of these genes in animals
that do not exhibit obvious REM or NREM sleep. Recently, a large-
scale forward genetics approach in mice revealed that a putative
gain-of-function mutation in the salt-induced kinase 3 (Sik3) gene
results in a large increase in the amount of NREM sleep, but not
REM sleep (Funato et al., 2016). Thus Sik3 strongly promotes NREM
sleep. Furthermore, as described in more detail below, homologues
of Sik3 in fruit ies and roundworms also promote sleep. This nd-
ing strongly supports that NREM sleep is regulated by a molecular
pathway that is likely involved in sleep-regulation across the ani-
mal phylum, even in invertebrates. Thus, at least some components
of NREM sleep seem to be controlled by molecular factors of an
extremely ancient origin.
5. Sleep can happen locally in the brain
Whales and dolphins, which spend their whole life under water,
may need to adopt a specialized form of sleep to avoid drowning or
predation while sleeping. Interestingly, dolphins circumvent such
Please cite this article in press as: Miyazaki, S., et al., Sleep in vertebrate and invertebrate animals, and insights into the function and
evolution of sleep. Neurosci. Res. (2017), http://dx.doi.org/10.1016/j.neures.2017.04.017
risks by sleeping in one hemisphere of the brain at a time, a phe-
nomenon known as unihemispheric sleep. During unihemispheric
sleep, one half of the brain generates slow waves, whereas the other
half retains a low level of alertness, allowing dolphins to keep swim-
ming. (Mukhametov et al., 1977). Another intriguing aspect of sleep
in dolphins is that the amount of REM sleep is very low, if any. REM
sleep in mammals is accompanied by loss of muscle tone, which
could be dangerous for whales and dolphins. Thus, they might have
evolved to minimize or even abandon REM sleep.
Fur seals also exhibit unihemispheric sleep (Mukhametov et al.,
1985). Unlike dolphins, fur seals live both on land and in water.
Interestingly, while sleeping on land, fur seals display bilateral
sleep, whereas when sleeping under water, they frequently dis-
play unihemispheric sleep. How the brain switches between these
two modes of sleep remains to be revealed.
Various bird species also show unihemispheric sleep
(Rattenborg et al., 2000). This might be benecial for reduc-
ing the risk of predation. Indeed, mallard ducks exhibit increased
unihemispheric sleep under a higher predation risk, which allows
for a rapid response to visual stimuli (Rattenborg et al., 1999). It
may also help migratory birds to retain consciousness during a
long period of ight (Rattenborg et al., 2016).
These studies in aquatic mammals and birds suggest that,
during evolution, unihemispheric sleep independently developed
multiple times in vertebrates. This suggests the innate nature of
vertebrate sleep to be locally regulated. Indeed, in humans, corti-
cal regions engaged in a learning task exhibit stronger slow wave
activity than other cortical regions during subsequent NREM sleep
(Huber et al., 2004). Moreover, in rats, prolonged wakefulness leads
to local emergence of slow waves even though, as a whole, the
individual appears to be awake (Vyazovskiy et al., 2011). Unihemi-
spheric sleep might be an extreme form of such local sleep.
6. Genetic studies of sleep in the teleost zebrash (D. rerio)
Along with fruit ies (D. Melanogaster) and roundworms (C. ele-
gans), the zebrash (D. rerio) is one of the most efcient animal
models for genetic analyses. The zebrash is a teleost sh that is
more closely related to mammals than invertebrate animal models.
For example, the architecture of the brain roughly resembles that of
mammals, especially in the brainstem, a key structure regulating
sleep. Moreover, many genes, including genes for neuropeptides
and their receptors, are conserved. Thus, to address the evolu-
tionary origin of wakefulness and REM/NREM sleep, the roles of
zebrash brain regions, genes, or neuromodulators homologous to
those involved in mammalian sleep are of particular interest.
Zebrash display a circadian quiescence that basically follows
the criteria for sleep (Yokogawa et al., 2007; Zhdanova et al., 2001).
The apparently conserved roles of histamine might provide clues to
the conservation of sleep/wake states across vertebrates. In mam-
mals, histamine is a well-studied wakefulness-inducing molecule.
Similarly, histamine seems to promote wakefulness in zebrash.
Mutants of histidine decarboxylase (hdc), a gene required for his-
tamine synthesis, exhibit increased sleep (Sundvik et al., 2011).
In addition, H1 histaminergic antagonists increase the amount of
sleep (Renier et al., 2007).
While the conserved roles of histamine support the conser-
vation between human sleep and zebrash sleep, the case is
not so simple for orexin (hypocretin), a neuropeptide important
for maintaining wakefulness in mammals (de Lecea et al., 1998;
Sakurai et al., 1998). In humans, a loss of neurons producing orexin
(hypocretin) leads to narcolepsy, a sleep disorder characterized
by excessive daytime sleepiness, cataplexy (sudden brief episodes
of muscle paralysis during wakefulness), and dream-like hallu-
cinations at sleep onset due to direct transition from wake to
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REM sleep (Peyron et al., 2000; Thannickal et al., 2000). Similar
phenotypes are observed in mice or dogs lacking either orexin
(hypocretin), orexin receptors, or the neurons producing orexin
(Chemelli et al., 1999; Hara et al., 2001; Lin et al., 1999; Willie et al.,
2003). On the other hand, administration of orexin (hypocretin)
to rats and mice increases wakefulness (Hagan et al., 1999; Mieda
et al., 2011; Piper et al., 2000). Orexinergic neurons re most dur-
ing wakefulness, and become silent upon entering sleep (Lee et al.,
2005; Mileykovskiy et al., 2005; Takahashi et al., 2008). Optoge-
netic activation of these neurons is sufcient to induce wakefulness
(Adamantidis et al., 2007). The orexin (hypocretin) receptors are
expressed in monoaminergic and cholinergic systems that promote
wakefulness. Thus, in mammals, orexin (hypocretin) seems to have
a role in maintaining wakefulness and suppressing REM sleep.
In contrast, zebrash mutants lacking the orexin (hypocretin)
receptor display no obvious phenotypes during the daytime, and
increased wakefulness and fragmented sleep during the nighttime
(Yokogawa et al., 2007). Even more surprising, the administration
of orexin (hypocretin) induces sleep in a receptor-dependent man-
ner (Yokogawa et al., 2007). Moreover, in contrast to mammals,
the orexin (hypocretin) receptor expression prole does not match
the monoaminergic and cholinergic arousal system, although there
seem to be some inconsistencies between studies (Mieda et al.,
2011; Prober et al., 2006; Yokogawa et al., 2007). Together, these
results suggest a somewhat different role for orexin (hypocretin)
in mammalian and teleost sh wakefulness. Cautious interpreta-
tion is required, however, as the function of orexin (hypocretin) in
mammals is itself complicated. Mice decient in orexin (hypocre-
tin) signaling, like the zebrash mutants, also display fragmented
sleep, but the total sleep amount seems to be unaffected (Mochizuki
et al., 2004). Future studies to assess the ring patterns of orexin-
ergic neurons in zebrash might be meaningful.
In mammals, the catecholamines dopamine and noradrenaline
(norepinephrine) also comprise a major component of the arousal
circuit. As described below, catecholamines also promote wake-
fulness in invertebrates. The roles of catecholamines in zebrash
sleep, however, are not well studied.
7. Sleep in invertebrate animals
7.1. General introduction
Silk moths are one of the most well-studied domesticated ani-
mals, and the life cycle of this insect is very well characterized. The
larvae (silkworms) undergo four molts before forming a cocoon.
Before each molt, they stop feeding and become immobile. In Japan,
this quiescent state is termed min, which means sleep. As in this
example, people have felt a certain commonness between sleep
and particular quiescent states in invertebrate animals. Moreover,
the recent identication of the NREM sleep-promoting gene Sik3 in
mice and the subsequent nding that the invertebrate homologues
of this gene also promote sleep-like states has provided evidence at
the molecular level that sleepiness is controlled by a highly con-
served molecular pathway (Funato et al., 2016). This breakthrough
nding is expected to lead to more evidence that sleep in mammals
and sleep-like states in invertebrates share similar mechanisms. If
this is the case, the nding will have at least two important impli-
cations. The rst is that sleep would be of an extremely ancient
origin, perhaps extending back to ancestral animals with a primi-
tive nervous system. The second is that we can utilize the fruit y
(D. melanogaster) and the roundworm (C. elegans), two of the most
efcient genetic animal models, to elucidate the molecular bases
underlying sleep. In fact, many researchers have begun intensive
studies of the molecular mechanisms and functions of sleep using
these two animal species.
Please cite this article in press as: Miyazaki, S., et al., Sleep in vertebrate and invertebrate animals, and insights into the function and
evolution of sleep. Neurosci. Res. (2017), http://dx.doi.org/10.1016/j.neures.2017.04.017
5
7.2. Genetic studies of sleep in the fruit y (D. melanogaster)
Adult fruit ies display a circadian rhythm of locomotor activ-
ity, with periods of rest at night. This model has been extensively
studied by circadian biologists, and such studies have made major
contributions to elucidating the molecular entity of our circadian
clock. Further investigations into the periods of rest displayed at
night will help to determine whether these periods of rest satisfy
the basic criteria for sleep. Rest in fruit ies is not only inuenced
by circadian rhythm, but also homeostatic regulation, as demon-
strated by a rebound after deprivation by mechanical stimulation
or social interaction (Hendricks et al., 2000; Shaw et al., 2000).
The arousal threshold is also increased. Thus, this behavior follows
the basic criteria for sleep, and cannot be explained simply as a
rest period regulated by the circadian clock. Moreover, like human
sleep, the rest period is decreased in old ies compared to young
ies, and caffeine, which promotes wakefulness in mammals, also
efciently promotes arousal in fruit ies (Hendricks et al., 2000; Wu
et al., 2009). In mammals, caffeine acts by antagonizing the recep-
tor for adenosine, one of the few known endogenous sleep-inducing
substances in mammals. In mice lacking the A2A adenosine recep-
tor, caffeine does not increase wakefulness (Huang et al., 2005).
While the apparently conserved effect of caffeine is intriguing, its
arousal effect in fruit ies is independent of the putative adeno-
sine receptor homologue, raising the possibility that the underlying
mechanism is largely different between mammals and fruit ies
(Wu et al., 2009).
Catecholamine neurotransmitters, namely dopamine and nora-
drenaline (norepinephrine) in the vertebrate central nervous
system, play important roles in mammalian sleep/wake regula-
tion. Dopamine promotes wakefulness in mammals. Mice lacking
the dopamine transporter gene, in which extracellular dopamine
is increased, exhibit increased wakefulness and reduced NREM
sleep (Wisor et al., 2001). Similarly, in fruit ies, a mutation in the
dopamine transporter gene results in increased activity and reduced
rest (Kume et al., 2005). Noradrenaline also enhances wakefulness
in mammals. Optogenetic activation of mouse noradrenaline-
releasing neurons in the brainstem locus coeruleus promotes
sleep-to-wake transitions, whereas optogenetic inhibition reduces
wakefulness (Carter et al., 2010). Although invertebrates do not
possess noradrenaline, the catecholamine octopamine is gener-
ally regarded as the invertebrate counterpart to noradrenaline, and
both are involved in conserved behaviors such as the ght-or-ight
response (Haller et al., 1997). Similar to the case of mouse nora-
drenaline, genetic excitation or silencing of octopamine-releasing
neurons in fruit ies increases or decreases wakefulness, respec-
tively (Crocker and Sehgal, 2008).
The roles of histamine in promoting wakefulness also seem con-
served. As in zebrash (D. rerio), mutants of hdc display increased
sleep (Oh et al., 2013). Like caffeine, however, the downstream
pathway is largely different. In mammals, the H1 receptor, which
is a G-protein coupled receptor, conveys the effect of histamine,
whereas in fruit ies, a histamine-gated chloride channel is crucial
(Oh et al., 2013).
In mammals, the inhibitory neurotransmitter gamma-amino
butyric acid (GABA) is involved in promoting sleep. GABAergic neu-
rons in the preoptic hypothalamic area or the brainstem promote
NREM sleep (Anaclet et al., 2014; Hayashi et al., 2015; Zhang et al.,
2015). In addition, the GABAA receptor is a major target for treating
insomnia in humans, although the responsible brain areas remain
to be claried (Equihua et al., 2013). GABA and the GABAA receptor
are also important for promoting sleep in fruit ies. Genetic hyper-
polarization of GABAergic neurons reduces sleep, and a mutation
in the GABAA receptor gene that increases the channel current leads
to increased sleep (Agosto et al., 2008).
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A disadvantage of using fruit ies (or roundworms) as a model
for sleep might be the low conservation of neuropeptides. While
orexin (hypocretin) has a crucial role in maintaining wakefulness in
mammals, invertebrates lack an obvious orexin (hypocretin) homo-
logue. Instead, in fruit ies, the neuropeptide pigment dispersing
factor, which is absent in vertebrates, plays a critical role in regulat-
ing circadian rhythms and maintaining wakefulness (Parisky et al.,
2008).
Perhaps the most direct evidence for the conservation of sleep-
regulating genes between mice and fruit ies comes from the recent
discovery of the involvement of Sik3 and its orthologue in sleep
regulation. As described above, a forward genetics approach was
carried out in mice to identify sleep-regulating genes in a non-
biased manner and revealed that a gain-of-function mutation in
Sik3 largely increases NREM sleep (Funato et al., 2016). Sik3 encodes
a protein kinase that belongs to the AMP-activated protein kinase
family. Intriguingly, in fruit ies, overexpression of the Sik3 ortho-
logue with a similar gain-of-function mutation drastically increases
the amount of sleep. Conversely, a hypomutation of the Sik3 ortho-
logue results in a dramatic decrease in the amount of sleep. These
ndings clearly demonstrate a conserved role for Sik3 in promoting
sleep.
Another interesting example is the conserved roles of the NMDA
receptor genes in promoting sleep. NMDA receptors comprise a
subclass of glutamate receptors and are well known for their con-
served roles in learning and memory across the animal phylum
(Roberts and Glanzman, 2003; Tsien, 2000; Xia et al., 2005). In fruit
ies, knockdown of the NMDA receptor gene Nmdar1 in the adult
nervous system reduces sleep during the night time (Tomita et al.,
2015). In mice, knockout of the NMDA receptor subunit gene Nr3a
reduces total sleep time (Sunagawa et al., 2016). NMDA receptors
are Ca2+ channels, thus intracellular Ca2+ signaling may have a role
in regulating sleep. Indeed, mutant fruit ies decient in the Ca2+ -
dependent phosphatase calcineurin exhibit a severe reduction in
sleep (Nakai et al., 2011; Tomita et al., 2011). Future studies to
address the roles of calcineurin in mammalian sleep may provide
interesting results.
7.3. Genetic studies of sleep in the roundworm (C. elegans)
Roundworms take less than a week to mature, and no obvious
circadian behavior has been reported. Recently, however, this small
animal has received much attention as a model for studying sleep.
Roundworms enter a quiescent state under certain conditions. The
most intensely studied is a state termed lethargus, which is actually
similar to the silkworm min state. Like silkworms, roundworm
larvae undergo four molts before becoming adults. Also like silk-
worms, before each molt, the larvae become quiescent, and in
roundworms this state is termed lethargus (Fig. 3). Although lethar-
gus is not a daily event but rather occurs at intervals of 712 h, this
state satises various sleep criteria, including an increased arousal
threshold and the display of a homeostatic rebound following its
disturbance by mechanical stimulation (Raizen et al., 2008).
For roundworm lethargus, the lack of a 24-h cycle and its restric-
tion to a particular immature stage largely separates it from sleep in
other animal models. The ndings from several studies, however,
support the notion that roundworm lethargus and fruit y sleep
are conserved at the molecular level. For example, the roundworm
homologue of pigment dispersing factor, PDF-1, negatively regu-
lates lethargus (Choi et al., 2013). A wide survey was conducted to
examine the extent of conservation between roundworm lethargus
and fruit y sleep (Singh et al., 2014). Of the 26 genes known to be
required for fruit y sleep, 20 orthologous roundworm genes were
examined for their involvement in lethargus. Surprisingly, all 20
genes were found to affect lethargus quantity and arousal thresh-
olds. Moreover, for 18 genes, including the dopamine transporter,
Please cite this article in press as: Miyazaki, S., et al., Sleep in vertebrate and invertebrate animals, and insights into the function and
evolution of sleep. Neurosci. Res. (2017), http://dx.doi.org/10.1016/j.neures.2017.04.017
Fig. 3. Schematic of the periodic emergence of lethargus, identied by the high frac-
tion of quiescence, during development of the roundworm (Caenorhabditis elegans)
larva. L1L4 indicate each larval stage. Each lethargus is followed by molting and
transition to the next larval stage.
GABAA receptor, and calcineurin genes, the direction of the effect
matched that of the fruit y genes. These studies highlight the deep
conservation between fruit y sleep and roundworm lethargus.
As in fruit ies, the orthologue of the mouse NREM sleep-
promoting gene Sik3, termed kin-29, also promotes quiescence
during lethargus. A putative null mutation in kin-29 results in
decreased quiescence during the lethargus period (Funato et al.,
2016). This nding suggests that, although lethargus is distinct from
sleep in other animals in that it lacks a 24-h cycle, the underly-
ing molecular mechanism that triggers the quiescent behavior is
preserved. How the same gene promotes sleep in both vertebrates
and invertebrates despite the lack of conservation of various neu-
ropeptides or anatomic structures is an interesting topic for future
studies.
8. Is there a conserved role for sleep?
The function of sleep is far less understood than its regulatory
mechanisms. Sleep deprivation eventually results in death in rats,
fruit ies (D. Melanogaster), and roundworms (C. elegans) (Driver
et al., 2013; Rechtschaffen and Bergmann, 2002; Shaw et al., 2002).
In all cases, the exact cause of the lethality is not clear. In mammals,
while the function of REM sleep remains largely unknown, NREM
sleep is involved in the secretion of growth hormone, synaptic plas-
ticity, memory consolidation, and clearance of brain metabolites
(Chauvette et al., 2012; Marshall et al., 2006; Rasch et al., 2007;
Takahashi et al., 1968; Xie et al., 2013; Yang et al., 2014). Although
slow waves that characterize NREM sleep are unique to mammals
and avian species, as discussed below, there seems to be some con-
servation between the roles of human sleep and sleep-like state in
invertebrates.
Novel imaging techniques using two-photon microscopy allow
for in vivo observation of the mouse brain at the synaptic level. Dur-
ing mammalian sleep, dendritic spines, which are post-synaptic
structures, undergo active remodeling (Maret et al., 2011; Yang
et al., 2014; Yang and Gan, 2012). Similarly, in fruit ies, sleep is
associated with structural changes in multiple brain areas, includ-
ing the mushroom body, which is important for learning and
memory (Bushey et al., 2011).
At the behavioral level, NREM sleep is related to memory con-
solidation. In humans, reactivation of a specic memory by means
of odor cues during NREM sleep, but not during wake or REM sleep,
improves memory consolidation (Rasch et al., 2007). Enhancing
slow wave activity during NREM sleep also improves memory con-
solidation (Marshall et al., 2006). Sleep also seems to be related to
learning and memory in fruit ies. In fruit ies, articial induction
of sleep by stimulating sleep-regulating neurons promotes the for-
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mation of long-term memories, and this effect is canceled by sleep
deprivation (Donlea et al., 2011).
As stated above, the function of REM sleep is much less under-
stood compared to NREM sleep. Various studies in humans and
animals have explored the function of REM sleep by forced awaken-
ing, i.e., immediately awakening the subject whenever the subject
enters REM sleep. Although this method can reduce the amount
of REM sleep, the repeated physical stimulation itself may cause
extreme stress, and eventually not only REM sleep but also the total
amount of sleep becomes largely reduced (Hayashi et al., 2015).
Therefore, it is difcult to interpret from these experiments the
exact roles of REM sleep. Many of the effects observed by these
experiments could not be differentiated from the effect of stress,
as the resulting symptoms overlap with various other stimuli that
increase stress. The application of optogenetics or chemogenetics
may help to overcome these problems. In one study, brainstem
neurons that negatively regulate REM sleep were either chemoge-
netically activated or inhibited to genetically suppress or increase
REM sleep, respectively, without any physical stimuli. The nd-
ings revealed that inhibiting or increasing REM sleep attenuates or
enhances slow wave activity in the subsequent NREM sleep, respec-
tively. This study suggested that REM sleep is involved in regulating
the cortical activity of the other sleep stage (Hayashi et al., 2015).
Slow wave activity promotes learning and memory consolidation
(Marshall et al., 2006; Miyamoto et al., 2016). At the cellular level,
slow waves enhance the potentiation of synapses (Chauvette et al.,
2012). Thus, REM sleep might indirectly regulate memory process-
ing in the cortex via slow wave enhancement. The hippocampus
plays a critical role in memory formation, and during REM sleep,
a strong theta oscillation is observed in the hippocampus. In mice,
when this theta oscillation during REM sleep was inhibited by opto-
genetic silencing of medial septum GABAergic neurons, the mice
could not properly consolidate what they learned prior to the sleep
(Boyce et al., 2016). Thus, the occurrence of hippocampal theta
waves during REM sleep is thought to be crucial for consolidat-
ing hippocampus-dependent memories. While these recent studies
suggest a critical role for REM sleep in learning and memory, other
physiologic functions of sleep remain obscure.
Chronic sleep deprivation in rats results in the inability to
regulate the body temperature, skin lesions, high metabolic rate,
and weight loss despite increased food intake. Within 23 weeks,
sleep-deprived rats die (Rechtschaffen and Bergmann, 2002;
Rechtschaffen et al., 1989). While this indicates the necessity of
sleep, the direct cause of lethality remains elusive. A study using
fruit ies was designed to address this issue (Shaw et al., 2002). The
study identied two strains that are extremely sensitive to sleep
deprivation. These strains carrying a mutation in either the circa-
dian clock gene cycle or the heat shock induced gene hsp83 showed
an exaggerated sleep rebound after 3 h of sleep deprivation. More-
over, individuals of these strains began to die after only 10 h of sleep
deprivation. These ndings might provide hints to the fundamen-
tal function of sleep at the molecular level. The hsp83 gene encodes
a chaperone protein, and thus sleep might be required for quality
management of certain proteins.
In roundworms, continuous disturbance of lethargus by
mechanical stimulation results in lethality (Driver et al., 2013). This
nding indicates that lethargus has an active role. In addition to
preparation for molting, synaptic remodeling and pruning events
in the nervous system coincide with lethargus timing (Hallam and
Jin, 1998; Hayashi et al., 2009; White et al., 1978), raising the pos-
sibility that, as with mammalian NREM sleep and fruit y sleep,
neural circuit remodeling is enhanced during lethargus.
The above-described sleep deprivation study in rats also sug-
gests that metabolism is tightly connected with sleep, as sleep
deprivation leads to a higher metabolic rate and enhanced appetite
(Rechtschaffen et al., 1989). Indeed, hypothalamic neurons such
Please cite this article in press as: Miyazaki, S., et al., Sleep in vertebrate and invertebrate animals, and insights into the function and
evolution of sleep. Neurosci. Res. (2017), http://dx.doi.org/10.1016/j.neures.2017.04.017
7
as the orexinergic neurons and melanin-concentrating hormone-
releasing neurons have dual roles in the regulation of sleep and
feeding, supporting their coordinated regulation (Chemelli et al.,
1999; Hassani et al., 2009; Jego et al., 2013; Konadhode et al.,
2013; Modirrousta et al., 2005; Qu et al., 1996; Sakurai et al., 1998;
Szentirmai and Krueger, 2006; Tsunematsu et al., 2014; Verret et al.,
2003). Starvation suppresses sleep in fruit ies, suggesting that
feeding and sleep also interact in invertebrates (Keene et al., 2010).
Sleep control might be an important strategy used to regulate the
energy distribution and conservation through the adjustment of
metabolism and feeding or reproductive behavior, although more
detailed examinations and accurate interpretations are needed.
9. Concluding remarks and future prospects
In this review, we describe recent ndings from studies of
various vertebrate or invertebrate animal species to clarify the mys-
teries of the evolution and function of sleep. Novel genetic tools
or simple animal models offer substantial advantages for identify-
ing neural circuits or genes that are engaged in sleep regulation.
Fundamental questions, however, are yet to be solved. Below, we
outline the important issues, together with a summary of the cur-
rent understanding and future directions to further address these
issues.
Is there a universal molecular pathway that encodes sleepi-
ness? The recent identication of some key sleep-regulating genes
may help to address this question about the molecular entity of
sleep. Our current understanding of the molecular basis of sleep,
however, is still weak compared to, for example, the circadian clock.
More intense high-throughput genetic and molecular approaches
might help to elucidate the overall picture of the core sleep-
regulatory pathway.
Why is sleep essential? Whether there is a general purpose
for sleep across animal species remains a question. The discovery
of unihemispheric sleep in aquatic mammals and birds, and local
sleep in rodents suggest that at least some aspects of sleep function
can be fullled without changing the state of the whole individ-
ual. Recent advances in genetic tools for precise manipulation of
neuronal activity, such as optogenetics and chemogenetics, have
led to the identication of neural circuits critical for sleep. In the
future, these tools are also expected to be effective for manipulat-
ing sleep/wake states and subsequent analyses of the phenotypic
outcome.
How did REM and NREM sleep stages evolve, and how are they
benecial? The identication and genetic manipulation of neural
circuits related to REM sleep or NREM sleep have revealed some
roles of each of these sleep stages in neural plasticity and mem-
ory consolidation. Future studies using the same approaches may
be effective for revealing other functions. In addition, identica-
tion and analyses of homologous neural circuits in animals that do
not exhibit obvious REM/NREM sleep states may provide clues to
the evolutionary origin of these sleep states. These approaches will
require the integration of multiple approaches, including genetics,
physiology, comparative neurology, behavioral studies, and devel-
opmental studies in various animal species.
Acknowledgements
This work was supported by the Japan Science and Technol-
ogy Agency CREST program (JPMJCR1655) and PRESTO program
(JPMJPR13AC); the Ministry of Education, Culture, Sports, Science,
and Technology of Japan (MEXT) World Premier International
Research Center Initiative (WPI); the MEXT KAKENHI for Scientic
Research on Innovative Areas Memory dynamism (16H01264);
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8 S. Miyazaki et al. / Neuroscience Research xxx (2017) xxxxxx
the Japan Society for the Promotion of Science KAKENHI grant
(16H06141) to Y.H.
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