The role of astroglial connexin 30 in sleep homeostasis

Xinhe Liu

To cite this version:

Xinhe Liu. The role of astroglial connexin 30 in sleep homeostasis. Neurons and Cognition.
Universite Pierre et Marie Curie - Paris VI, 2014. English. <NNT : 2014PA066232>. <tel01085329>

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Universi Pierre et Marie Curie Paris 6

The role of astroglial connexin 30

in sleep homeostasis
!
Thse prsente devant

lUniversi Pierre et Marie-Curie

pour obtenir le grade de

Docteur en Neurosciences
par

Xinhe Liu

ED3C : Ecole Doctorale Cerveau, Cognition et Comportement

Jury constitu de:

Jean MARIANI

Prsident du jury

Tommaso FELLIN

Rapporteur

Marc MESNIL

Rapporteur

Jean-Marie PETIT

Examinatuer

Bruno WEBER

Examinatuer

Christian GIAUME

Directeur de Thse

!
Thse soutenue le 23.09.2014

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ACKNOWLEDGEMENTS
Above all, I would like to express my gratitude to my thesis supervisor, Christian
Giaume, who has given me the greatest guidance and support throughout my thesis.
As his student, I deeply appreciate his genuine care for my progress at this critical
period of my scientific career. The rigorous training he has provided me will be
always be a precious assest for my career.
I would like to thank the members of the jury, Jean Mariani (UPMC), Tommaso
Fellin (Istutituto Italiano di Technologia), Marc Mesnil (Universit de Poitiers),
Jean-Marie Petit (Ecole Polytechnique Fdrale de Lausanne), Bruno Weber
(Institute of Pharmacology and Toxicology) and Christian Giaume (Collge de Fance)
for accepting to evaluate my thesis work. I specially thank the reporters, Tommaso
Fellin and Marc Mesnil, for their time and efforts in evaluating this manuscript.
I wish to express my appreciation to Jean-Marie Petit as the collaborator of my thesis
project. I greatly appreciate the guidance and discussions he offered me in an
unfamiliar research area and the huge amount of efforts he invested in this work. The
project would not have been possible without his assistance and contribution.
I would like to also thank all my colleagues for having created a pleasant and
stimulating lab life. Annette Koulakoff is always there to offer her patience and help
whenever I have problems with the daily work in the lab. Pascal Ezan has given me
important technical assistance in my experiments and broughter a great deal of
laughters to the lab. I thank Nathalie Rouach who has provided me help and advices,
especially with electrophysiology. I especially appreciate the huge efforts of
Anne-Cecile and Martine Cohen-Salmon to meet my constant demand for
experimental animals. I also give my thanks to Jrmie Sibille, Elena Dossi, Glenn
Dallerac and Gregory Ghezali for their discussions and assistance. I owe my thanks to
the staff of the animal facility, especially Jean-Charles Graziano: without their patient
assistance and diligent work, my behavioral experiments would not be possible. I
extend my special thanks to the former memers of the lab, Lisa Roux and Oana
Chever whose guidance and advices have greatly helped me in starting my project.
Im particularly grateful to the members of my collaborating laboratory at EPFL, for
the discussions and accompany they kindly offered me during my stay in Lausanne. I
sincerely thank Pierre Magistretti, whose scientific expertise and support has been
!

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indispensable for my project. Also, I deeply thank Valerie Eligert for her great help
with my daily work and life.
I would like to thank ENP (cole des Neurosciences Paris le de France), which has
offered me the marvelous PhD program in Paris. The faculty and staff of ENP are
extremely helpful with both the scientific and personal lives of its students. I will
never regret having chosen ENP for my PhD study.
I give many thanks to my friends, Yihui Cui, Chenju Yi, Lee Chun-Yao, who are also
my colleagues, for their accompany, help and encouragement in both my work and
daily life. It is a great fortune for me to have had them around.
Last but not the least, Im deep grateful to my family. My parents have always given
me the strongest support and trust in my choice of career and personal life. Im
particularly thankful to my mother, for her constant care, trust, encouragement and
help whenever I need it. And my grandmother, her mere presence gives me courage to
continue on my path.

!%!

Abstract

The role of astroglial connexin 30 in sleep homeostasis

Astrocytes play important roles in numerous brain functions and pathologies.
Recently, astrocytes have been demonstrated to regulate sleep homeostasis as a major
contributor of extracellular adenosine in the brain. A prominent feature of astrocytes
is that they are organized into networks via gap junction channels, which are mainly
constituted by connexin (Cx) 30 and Cx43. Based on the initial observation that the
mRNA expression of Cx30, but not Cx43, is enhanced after 6-hour sleep deprivations
in the mouse cortex and hippocampus, we hypothesize that Cx-based networking of
astrocytes might contribute to sleep homeostasis. Hence the goal of my thesis project
was to investigate whether and how astroglial Cxs, in particular Cx30, are involved in
sleep homeostatic regulation.
The first and second parts of my work employed in vivo electrophysiology and
pharmacological approaches to investigate the effects of sleep/wake-affecting
molecules (i.e., psychostimulants, sleep inducing drugs and anesthetics) on gap
junctional communication of astrocytes in acute slices of the mouse somatosensory
cortex. We found that superfusion of modafinil enhanced astroglial dye coupling,
which is a measure of gap junctional communication. In contrast, !-Hydroxybutyric
acid (GHB), a sleep-promoting agent, decreased astroglial coupling. The effect of
modafinil was abolished by TTX treatment, whereas that of GHB was not affected,
implicating involvement of neuronal activity in the effect of the former. In addition,
propofol and ketamine, two general anesthetics, also decreased astroglial coupling.
These results suggest that astroglial networks are bidirectionally regulated by
sleep/wake-affecting drugs.
The third part of my work addressed the role of Cx30 in the sleep-wake cycle by
utilizing Cx30 knockout (KO) mice. Compared to wild type (WT) mice, Cx30 KO
mice mainly exhibited a deficit in maintaining wakefulness during periods of high
sleep pressure, as manifested by the following observations: 1) they needed more
stimuli to be maintained awake during the 6-hour gentle sleep deprivation (SD); 2)
they exhibited an increase in slow wave sleep during 6-hour instrumental SD
monitored by electroencephalography and electromyography recordings. To probe the
possible causes these phenotypes of the Cx30 KO mice, we found that: 1) astroglial
dye coupling was enhanced in acute cortical slices from WT mice after SD, and such
enhancement depended on both neuronal activity and the presence of Cx30; 2) mRNA
!

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levels of several genes involved in brain energy metabolism were decreased in

multiple brain structures of the Cx30 KO mice.
In summary, these results suggest that astroglial Cx30 plays an important role in sleep
homeostasis, possibly by enhancing astroglial metabolic functions to fulfil the high
energy demand during periods of elevated sleep pressure.
!

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CONTENTS
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ADDITIONAL PUBLICATION 1: FROM A GLIAL SYNCYTIUM TO A MORE RESTRICTED
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ADDITIONAL PUBLICATION 2/ ASTROGLIAL CONNEXINS AS ELEMENTS OF

SLEEP-WAKE CYCLE REGULATION AND DYSFUNCTION!*****************************************************************!+75!

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Introduction
______________________

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Figure 1. Subtypes of astrocytes. Ia: pial tanycyte. Ib: vascular tanycyte. II: radial astrocyte
(Bergmann glia). III: marginal astrocyte. IV: protoplasmic astrocyte. V: velate astrocyte. VI:
fibrous astrocyte. VII: perivascular astrocyte. VIII: interlaminar astrocyte. IX: immature
astrocyte. X: ependymocyte. XI: choroid plexus cells. Reproduced from Reichenbach and
Wolburg, 2005.
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For a long time since their first discovery by Rudolf Virchow in 1858 (see
Kettenmann and Verkhratsky, 2008), glial cells were considered as mere passive,
structural cement of the brain. Their roles in brain physiology were largely
underestimated due to the fact that they are non-excitable and do not emit action
potentials. The turning point of the glia field was brought about by a series of
discoveries that astrocytes, a type of glial cells, actually express glutamate receptors
(Backus et al., 1989; Gallo et al., 1989) and ion channels that open in response to
glutamate (Usowicz et al., 1989), and that astrocytes respond to glutamate with
intracellular Ca2+ elevations (Cornell-Bell et al., 1990) and release glutamate that
stimulates nearby neurons (Parpura et al., 1994). These observations have led to an
explosion of research for the past two decades on glial cells, in particular, astrocytes
that are now recognized as indispensable, active partners of neurons involved in
numerous brain functions and diseases (see Kettenman and Ransom, 2013).
In the first part of Introduction, I summarize the current knowledge of the
physiological properties and functions of astrocytes with an emphasis on neuroglial
interactions. The aim of Introduction is to provide a background for succeeding
discussions of my thesis work and thus the topics covered here are chosen based on
their relevance to my thesis project.

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Astrocytes are a diverse and heterogeneous cell population that consists of many types
of glial cells (Figure 1), all of which differ to a more or less extent in their
morphologies, physiologies, gene expressions and functions (reviewed in Matyash
and Kettenmann, 2010; Zhang and Barres, 2010; Theis and Giaume, 2012).
Nevertheless, many astroglial cells conform to most of the following criteria: 1) being
electrically non-excitable; 2) having highly hyperpolarized membrane potentials (-75
to -90 mV); 3) expressing functional glutamate and g-aminobutyric acid (GABA)
transporters; 4) expressing glial fibrillary acidic protein (GFAP); 5) containing
glycogen granules; 6) possessing perivascular endfeet and/or perisynaptic processes; 6)
expressing abundantly gap junction proteins (Kimelberg, 2010).

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Table 1. Ion channels in astrocytes. Adapted from Verkhrasky and Butt 2013.

Table 2. Astroglial receptors of neurotransmitters and neuromodulators. Adapted from

Verkhrasky and Butt 2013.

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One major group of astrocytes is the protoplasmic astrocyte found in the gray matter
of the brain and the spinal cord. In rodents, protoplasmic astrocytes extend several
(5-10) primary processes more or less radially from the soma, and branch into
extensively ramified arborizations, which endow astrocytes with a spongiform shape
(Figure 2A). Astroglial process terminals are in close contact with the presynaptic
and postsynaptic components, forming structural basis of the tripartite synapse
(Araque et al., 1999a; Figure 2B). Apart from the perisynaptic processes, astrocytes
also extend processes that terminate on intraparenchymal vasculature (Figure 2C).
These specialized perivascular astroglial terminals are termed endfeet, which cover
almost completely the adluminal vascular surface (Kacem et al., 1998; Mathiisen et
al., 2010; McCaslin et al., 2011). The perivascular endfeet constitute a crucial
component of the neurovascular unit, where regulation of local cerebral blood flow
occurs (Attwell et al., 2010; Giaume et al., 2010; Petzold and Murthy, 2011).

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In the hippocampus and the cortex, it has been observed that astrocytes occupy
separate domains with minimal overlap (only 4-5% of cell volume) of the peripheral
processes between adjacent cells (Bushon et al., 2002; Ogata et al., 2002; Halassa et
al., 2007; Figure 2D). It is estimated in the rodent cortex, a single protoplasmic
astrocyte enwraps 4 to 8 neuron somata and ensheaths 30 to 600 dendrites (Halassa et
al., 2007), and an astrocyte covers 20,000 to 120,000 synapses within its domain
(Oberheim et al., 2009). It has also been shown that individual astrocytes interact
specifically with the synapses within their domains (Bushong et al., 2004). Despite
their distinct domain organization, protoplasmic astrocytes are not isolated from their
neighbors: they are highly interconnected by gap junction channels (GJCs), which
mediate direct intercellular exchanges of cytoplasmic substances. This network
organization adds another level to neuroglial interactions in addition to those that
occur at the single synapses and neurovascular units (see Giaume et al., 2010).

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Astrocytes express numerous types of ion channels, whose distributions and functions
are summarized in Table 1. Briefly, astrocytes are characterized by high passive K+
conductance and hyperpolarized resting membrane potentials, both of which are due
to K+ channel permeability. Several types of voltage-gated Na+ and Ca2+ channels are
expressed in astrocytes, although the density of these channels is too low to counteract
the large K+ conductance (Verkhratsky and Steinhauser, 2000). Astrocytes express
several isoforms of transient receptor potential (TRP) channels (Parpura et al., 2011),
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which are involved in Ca2+ signaling, and the water channels named aquaporins
(AQPs).

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Astrocytes express virtually all types of neurotransmitter receptors (Table 2;

Verkhratsky et al., 2010). Astrocytes from different brain regions express distinct sets
of receptors, which often match those present in neighboring neurons. For example,
the only astrocytes that express glycine receptors are those in the spinal cord, where
glycine is the main inhibitory neurotransmitter (Kirchhoff et al., 1996), and dopamine
receptors are expressed by astrocytes restricted to the basal ganglia, where
dopaminergic transmission is prominent (Miyazaki et al., 2004). This feature
represents one of the bases for astrocyte heterogeneity (Theis and Giaume, 2012).
The biophysical properties of astroglial receptors, however, have major differences
from their neuronal counterparts. Many astroglial receptors including the
metabotropic glutamate receptor (mGluR), muscarinic acetylcholine receptor
(mAChR), P2Y purinergic receptor (P2YR), and GABAB receptor (GABABR) are
high-affinity and slowly desensitizing, in comparison to the low-affinity and fast
desensitizing neuronal receptors (Panatier et al., 2011; Giniatullin et al., 2005; Bowser
and Khakh, 2004; Guthrie et al., 1999; Venance et al., 1997; Waldo and Harden,
2004). Such properties of astroglial receptors are thought to enable activation of
astrocytes by low concentrations of neurotransmitters that diffuse from the synaptic
cleft to the perisynaptic astrocyte processes (Araque et al., 2014).

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Astrocytes possess a wide variety of neurotransmitter transporters, which are involved

in the uptake, recycling and release of neurotransmitters at synaptic and extrasynaptic
sites. For example, astrocytes express two types of glutamate transporters, the
glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1)
(Danbolt, 2001). For GABA transporters (GAT), astrocytes predominately express
GAT3 (Conti et al, 2004). Astrocytes in the cortex, hippocampus, brain stem,
cerebellum and spinal cord express glycine transporter 1 (GlyT1) (Verkhartsky and
Butt, 2013). All these transporter mediate co-transport of Na+ with the
neurotransmitters, thus resulting in Na+ influx that can substantially increase
intracellular Na+ concentration ([Na+]i) (Kirichuk et al., 2012). Astrocytes also
express other ion transporters, including Na+/Ca2+ exchangers (NCXs) that mediate
the exchange of 2 Na+ and 1 Ca2+, Na+/HCO3- co-transporters (NBCs) that are
involved in pH homeostasis (Deitmer and Rose, 2010), and Na+/K+/Cl- co-transporters
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(NKCCs) that contribute to extracellular K+ homeostasis (Verkhartsky and Butt,

2013), etc.

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Astrocytes are unable to generate action potentials, but they possess highly
sophisticated Ca2+ signaling systems, which allow them to respond to a variety of
neurotransmitters and other signaling molecules in the form of intracellular Ca2+
elevations (for reviews see Araque et al., 2001; Fiacco and McCarthy, 2006; Perea et
al., 2009). Astroglial Ca2+ signaling plays a central role in modulation of synaptic
transmission (Agulhon et al., 2008; Perea et al., 2009) and is also involved in the
neurovascular coupling (Petzold and Murthy, 2011; Attwell et al., 2010).

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Astrocyte intracellular Ca2+ rise can either occur spontaneously in the absence of
neuronal activity (Aguado et al., 2002; Parri et al., 2001; Peters et al., 2003; Thrane et
al., 2012) or be triggered by neurotransmitters released during synaptic activity (Perea
and Araque, 2005a). Many astroglial receptors for neurotransmitters are the
metabotropic type (Table 2). Activation of these receptors triggers production of
inositol 1,4,5-trisphosphate (InsP3), which acts on InsP3 receptors to induce Ca2+
release from the endoplasmic reticulum (ER) (Hamilton et al., 2008; Kastritsis et al.,
1992; Kirischuk et al., 1996; McCarthy and Salm, 1991). Type 2 InsP3 receptors are
the predominant isoform expressed in astrocytes (Holtzclaw et al., 2002; Sheppard et
al., 1997). It is generally accepted that InsP3-induced Ca2+ release from the ER is
critical for the exocytosis of gliotransmitters (see below) from astrocytes (Malarkey
and Parpura, 2009; Parpura et al., 2011).
Ca2+ transients in astrocytes could also be caused by the entry of Ca2+ from the
extracellular space. The depletion of the ER Ca2+ store triggers Ca2+ influx through
specific plasmalemmal channels such as TRP channels (Golovina, 2005; Grimaldi et
al., 2003; Pizzo et al., 2001), a mechanism referred to as store-operated Ca2+ entry
(Putney, 1986, 1990). In addition, extracellular Ca2+ can enter the cell through Ca2+
permeable ionotropic receptors and NCXs. The ionotropic receptors involved in
astrocyte Ca2+ signaling are the "-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA), N-methyl-D-aspartate (NMDA) and P2X purinergic receptors (P2XRs)
(Lalo et al., 2006, 2008, 2011). The NCXs can mediate Ca2+ entry in the reverse mode
of operation, which is favored by increase in [Na+]i and depolarization (Figure 3).
Finally, connexin (Cx) and pannexin (Px) hemichannels (see Introduction II) are
!

"&!

Figure 3. Ca2+ signaling in astrocytes. [Ca2+]i increase can derive from the endoplasmic
reticulum (ER) that expresses InsP3 receptors, which are activated metabotropic G-protein
coupled receptors (GPCRs) and phospholipase C (PLC). The ER store is filled by the activity
of the store-specific Ca2+-ATPase (SERCA). Another Ca2+ source comes from the entry of
extracellular Ca2+ through ionotropic receptors, store-operated channels (SOC) or Na+/Ca2+
exchangers (NCXs). Ca2+ pumps/ATPases (PMCA) can extrude cytosolic Ca2+. [Ca2+]i is
affected by cytosolic Ca2+-binding proteins (CBPs) and the mitochondria. Mitochondrial Ca2+
uptake occurs through voltage-dependent anion channels (VDAC) in the outer membrane and
the uniporter in the inner membrane, while Ca2+ exits the mitochondria through the
mitochondrial NCX and the mitochondrial permeability transition pore (MPTP). Reproduced
from Verkhratsky et al., 2012a.
.

!"'!

permeable to calcium and could provide an alternative pathway for Ca2+ entry (De
Bock et al., 2014; Orellana et al., 2012).

C'(-+'&.+%,(4,)28).A:,%.0128.10,.1,2%&'()*&+%,
The Ca2+ signals in astrocytes exhibit complex spatiotemporal properties. Slow
(seconds to minutes) Ca2+ events in response to intense neuronal activity invade the
astrocyte somata (Kulik et al., 1999; Perea and Araque, 2005b; Fiacco and McCarthy,
2004; Rieger et al., 2007), whereas rapid (millisecond scale) Ca2+ responses elicited
by minimal synaptic activity are restricted to microdomains of perisynaptic processes
(Di Castro et al., 2011; Panatier et al., 2011). Ca2+ transients elicited in individual
astrocytes can propagate across cell boundaries as intercellular Ca2+ waves, which can
travel long distances (<500 m) at relatively low speed (approximately 14 m/s)
(Scemes and Giaume, 2006; Deitmer and Rose, 2010). In the neocortex Ca2+ wave
propagation depends on GJCs, whereas in hippocampus and corpus callosum release
of ATP and its activation of P2Y receptors are necessary for generation of Ca2+ waves
(Haas et al., 2006; Schipke et al., 2002). Astrocytes Ca2+ signaling has also been
observed in vivo. Whisker stimulation increased astrocyte Ca2+ transients in the
mouse barrel cortex (Wang et al., 2006). Visual stimuli were shown to elicit astrocyte
Ca2+ signals in the ferret visual cortex (Schummers et al., 2008). During locomotor
performance of mice, concerted Ca2+ excitation occurred in networks of hundreds of
cerebellar Bergmann glial cells extending across several hundred microns
(Nimmerjahn et al., 2009).

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D(3.A:,%.0128.10,

In addition to Ca2+ excitability of astrocytes, recent studies have demonstrated that

neuronal activity triggers Na+ transients in perisynaptic astrocytes. These Na+
transients are mediated by Na+-permeable channels and Na+-dependent transporters
(Figure 4). For example, ionotropic glutamate receptor agonists increases [Na+]i by
1025 mM (Rose and Ransom, 1996; Kirischuk et al., 1997; Kirischuk et al., 2007;
Langer and Rose, 2009). Electrical stimulation of neuronal afferents also elicits Na+
transients in Bergmann glia and hippocampal astrocytes (Kirischuk et al., 1997;
Kirischuk et al., 2007; Bennay et al., 2008). Focal initiation of astroglial Na+ rise was
shown to trigger propagating Na+ waves (Bernardinelli et al., 2004; Langer et al.,
2012), which depend on GJCs (Langer et al., 2012). Na+ dynamics influences diverse
astroglial functions such as metabolism of lactate, transmembrane transport of
neurotransmitters, K+ buffering and Ca2+ signaling (see review Kirischuk et al., 2012).
Based on these findings, Na+ signaling has been proposed to be another form of
astroglial excitability (Kirischuk et al., 2012).
!

"(!

Figure 4. Receptors, channels and transporters that contribute to astroglial [Na+]i changes and
the astroglial homeostatic functions regulated by [Na+]i changes. Abbreviations: ASIC,
acid-sensing ion channels; EAAT, excitatory amino acid transporters; ENac, epithelial
sodium channels; GAT, GABA transporters; GS, glutamine synthetase, iGluRs, ionotropic
glutamate receptors; mito, mitochondrion; Nax, Na+ channels activated by extracellular Na+;
NCLX, mitochondrial Na+/Ca2+ exchanger; NHE, Na+/H+ exchanger; NKCC1, Na+/K+/Clcotransporter; MCT1, monocarboxylase transporter 1; P2X, P2X purinergic receptor; TRP,
transient receptor potential channel. Reproduced from Kirischuk et al., 2012.

Figure 5. Pathways for gliotransmitter release from astrocytes. Abbreviations: Xc system,

cystine-glutamate exchanger; VRAC, volume-regulated anion channel. Reproduced from
Verkhratsky et al., 2012b.

!",!

!"#"E

$%&'(08.28,'+8+2%+,(4,08.(&'21%:.&&+'%,

Astrocytes are able to release a variety of neurotransmitters and neuromodulators

including glutamate, ATP, D-serine, GABA, tumor necrosis factor-" (TNF-"),
prostaglandins (PGs), and peptides that are referred to as gliotransmitters (Volterra
and Bezzi, 2002). However, the mechanisms of gliotransmission are still controversial
(for details, see Hamilton and Attwell, 2010; Nedergaard and Verkhratsky, 2012).
Numerous studies have reported that some gliotransmitters are released through
Ca2+-dependent exocytosis of vesicles (Bezzi et al., 2004; Jourdain et al., 2007;
Martineau et al., 2008) and lysosomes (Jaiswal et al., 2007; Li et al., 2008; Zhang et
al., 2007) (Figure 5). Most evidence indicates that astrocytes possess the exocytotic
molecular machinery including proteins of the soluble N-ethylmaleimide-sensitive
factor attachment protein receptor (SNARE) complex (Montana et al., 2006; Parpura
and Verkhratsky, 2012) and vesicular pumps and transporters such as the
vacuolar-type H+-ATPase (V-ATPase), vesicular glutamate transporters (VGLUTs)
13, and the vesicular nucleotide transporter (VNUT) (Parpura et al., 2010; Sawada et
al., 2008; Sreedharan et al., 2010). Furthermore, ultrastructural studies have shown
that astrocytes contain vesicles that range from 30 to 1,000 nm in diameter with clear
and dense cores (Parpura et al., 2010) and that 30-nm clear astroglial vesicles are
located closed to presynaptic terminals (Jourdain et al., 2007). There are, however,
some studies that reported contradictory findings, such as failing to detect VGLUT
expression in astrocytes by immunostaining (Graziano et al., 2008; Restani et al.,
2011; Li. D, et al., 2013) and by transcriptome studies (Cahoy et al., 2008), calling for
further investigations of the molecular of mechanisms of astroglial exocytosis.
Astroglial release modes other than Ca2+-dependent exocytosis have also been
described, including reversal of neurotransmitter transporters and diffusion through
Cx/Px hemichannelsthe opening of Cx43 hemichannels is Ca2+-dependent (Wang et
al., 2012), P2X7 receptors and volume-regulated anion channels (Figure 5;
Verkhratsky et al., 2012a). However, it remains unclear under what physiological or
pathological conditions different release mechanisms occur.

!"5 FA1)&.(1%,(4,2%&'()*&+%,
!"5"#

GH&'2)+88A82',.(1,213,I2&+',7(:+(%&2%.%,

The extracellular ion environment is extremely important for brain functions, because
any slight change in ion concentrations or in extracellular volumes affects membrane
potentials and excitability of neurons. Astrocytes play crucial roles in maintaining
extracellular ionic and water homeostasis.
!

"-!

Figure 6. Spatial K+ buffering: control of extracellular K+ homeostasis by astrocytes.

Generation of action potentials in neurons leads to rise in [K+]o. Locally, extracellular K+ is
taken up locally by individual astrocytes through Na+/K+ pumps and Na+/K+/Clcotransporters. Local intracellar K+ is dispersed from the site of activity into distant astrocytes
through gap junction channels (GJCs) and extruded through Kir4.1 channels back to the
extracellular space. Reproduced from Verkhartsky and Butt, 2013.

!#.!

2=

GH&'2)+88A82',-(&2%%.A:,7(:+(%&2%.%,

Neuronal activity is accompanied by the influx of Na+ and Ca2+ and efflux of K+,
resulting in variations of extracellular concentrations of these ions and in the volume
of the extracellular space (Sykova and Nicholson, 2008). In particular, the relative
changes for extracellular K+ concentrations ([K+]o) is especially high due to the low
resting [K+]o (2.73.5 mM) (Somjen, 2002). The increas in extracellular K+ is rapidly
removed by neighboring astrocytes through two major mechanisms: local K+ uptake
and spatial K+ buffering (Verkhartsky and Butt, 2013). Local K+ uptake is mainly
mediated by Na+/K+ pumps and NKCCs in individual cells. Spatial K+ buffering,
initially proposed by Orkand and Walz (Orkand, 1980; Walz, 1982), is based on
Kir4.1 channels that are highly K+ permeable and GJCs that connect individual
astrocytes into networks (Olsen and Sontheimer, 2008). Local entry of K+ creates an
electrical and chemical gradient in the astroglial network, along which K+ is dispersed
from the site of activity into distant cells. Kir4.1 channels are involved in extrusion of
K+ from the astroglial network back to the extracellular space (Figure 6).

<=

J2&+',213,+H&'2)+88A82',K(8A:+,7(:+(%&2%.%,

Astrocytes express water channels AQP4, which mediate water exchange between the
blood and the parenchyma. AQP4 and Kir4.1 channels are co-localized in the endfeet
(Wolburg et al, 2011) and are functionally coupled in movements of water and K+
(Amiry-Moghaddam and Ottersen, 2003). Extracellular volume changes are coupled
to synaptic activity, which has been shown to induce rapid shrinkage of the
extracellular space (Dietzel et al., 1980; Holthoff and Witte, 1996; Sykova et al.,
2003). Synaptic activity causes release of solutes such as glutamate and K+ that are
removed through co-transport with water by astrocytes (Haj-Yasein et al., 2012;
Nagelhus et al., 2004), resulting in swelling of astroglial processes and shrinkage the
extracellular space. Extracellular volume decrease has a significant impact on
synaptic transmission by increasing the local concentration of neurotransmitters.

!"5"5

L+A'(&'21%:.&&+',7(:+(%&2%.%,213,%*1&7+%.%,

Astrocytes are equipped with transporters and biosynthetic enzymes for key
neurotransmitters including glutamate and GABA, thus playing a paramount role in
homeostasis and synthesis of these transmitters. Astrocytes represent the main sink of
glutamate in the brain (Eulenburg and Gomeza, 2010). Due to its neurotoxicity when
it is present in excess, the majority of glutamate is removed from the synaptic cleft by
astroglial glutamate transporters. Glutamate in astrocytes is trafficked back to neurons

#"!

Figure 7. Glutamate-glutamine cycle and the GABA-glutamine cycle. (A) In glutamatergic

synapses, released glutamate is mostly taken up by astrocytes, where it is amidated to
glutamine by GS and trafficked back to the neurons. In neurons, glutamate is regenerated
from glutamine by PAG and ammonia (NH4+) is produced. The ammonia is transported back
to the astrocytes for detoxification. Glutamate might be also metabolized via the TCA cycle in
both neurons and astrocytes. (B) In GABAergic synapses, the released GABA is taken up by
both astrocytes and the pre-synaptic terminal. In astrocytes, GABA is metabolized to
succinate, which is further metabolized in the TCA cycle to a-ketoglutarate and then
glutamate. The following steps are similar to those in the glutamatergic synapse.
Abbreviations: GAD, glutamate decarboxylase; Glu, glutamate; Gln, glutamine; GS,
glutamine synthetase; "-KG, "-ketoglutarate; PAG, phosphate-activated glutaminase; Suc,
succinate; TCA, tricarboxylic acid. Adapted from Bak et al., 2006.

!##!

to replenish the presynaptic pools through the glutamate-glutamine shuttle (Figure 7).
Astrocytes are responsible for de novo synthesis of glutamate from glucose, using the
enzyme pyruvate carboxylase exclusively expressed in astrocytes (Hertz et al., 1999).
Also, astrocytes participate in the removal of GABA via GABA transporters GAT-1
and GAT-3, the latter being the major astroglial subtype (Madsen et al., 2007).
Similarly to glutamate, neuronal GABA is also derived from astrocytes through the
glutamate-glutamine shuttle: GABA is synthesized by glutamate decarboxylase (GAD)
in inhibitory neurons from glutamate, which in turn is transformed from glutamine
(Figure 7; Schousboe and Waagepetersen, 2007).

!"5"9

$%&'()*&+%,213,%*12-&.),&'21%:.%%.(1, ,

As discussed above, astrocytes are well equipped to sense, integrate, respond to and
regulate neuronal activities. Thus the perisynaptic astroglial processes are an
indispensable component of the synapse in addition to the presynaptic terminal and
the postsynaptic compartment, a concept referred to as the tripartite synapse (Araque
et al., 1999a). Astrocytes influence a large array of synaptic events in multiple ways
as discussed below.

2=

M8.(&'21%:.&&+','+8+2%+,213,%*12-&.),&'21%:.%%.(1, ,

For the past decade, how astroglial gliotransmittters affect synaptic transmission has
been a major focus of the glia field, which has revealed the remarkable diversity of
neuroglial interactions mediated by astroglial gliotransmittion (Table 3). Here are a
few examples of the signaling pathways involved in several types of astroglial
modulation of synaptic transmission.
Astroglial glutamate increases neuronal excitability (Figure 8A). Glutamate released
from neurons activates mGluRs in astrocytes to increase astrocyte intracellular Ca2+
concentration ([Ca2+]i) and trigger the release of glutamate. Astroglial glutamate
activates extrasynaptic NMDA receptors to evoke slow inward currents (SICs) in
nearby neurons, resulting in increased excitability of the neurons (Parri et al., 2001;
Fellin et al., 2004; Angulo et al., 2004; Perea and Araque, 2005a; D'Ascenzo et al.,
2007). As a single astrocyte contacts a large number of neurons (Halassa et al., 2007),
SICs can be generated in many neighboring neurons, thus promoting synchrony of
neuronal firing (Parri et al., 2001; Fellin et al., 2004; Angulo et al., 2004; Perea and
Araque, 2005).
Astroglial glutamate enhances presynaptic release probability (Figure 8B). Raising
astrocyte [Ca2+]i induces release of glutamate, which acts on NR2B subunit!

#$!

Figure 8. Functional effects of glutamate, ATP and Dserine released from astrocytes on
synaptic events (see text). Abbreviations: Pr, release probability; EctoATPase, extracellular
ATPase. Adapted from Hamilton and Attwell, 2010.

!#%!

containing NMDA receptors (Araque et al., 1998; Perea and Araque, 2007) or
mGluR1 on the presynaptic terminals (Perea and Araque, 2007; Fiacco and McCarthy,
2004) to increase the frequency of excitatory postsynaptic currents (EPSCs) recorded
in nearby neurons (Araque et al., 1998; Perea and Araque, 2005a; Fiacco and
McCarthy, 2004).
Astroglial ATP promotes heterosynaptic depression (Figure 8C). Repetitive activity
in hippocampal excitatory neurons activates interneurons and GABA release. GABA
acting on astroglial GABAB receptors triggers release of ATP (Serrano et al., 2006).
ATP is degraded extracellularly to adenosine, and adenosine acts on A1 receptors to
suppress transmitter release from nearby excitatory synapses (Serrano et al., 2006;
Pascual et al., 2005).
Astroglial D-serine co-activates NMDA receptors (Figure 8D). D-serine is
synthesized in astrocytes from L-serine by serine racemase (Wolosker et al., 1999)
and released in response to neuronal glutamate (Mothet et al., 2005). D-serine release
is crucial for the activation of NMDA receptors and the induction of long-term
potentiation (LTP) (Yang et al., 2003; Panatier et al., 2006).
The findings presented above indicate that release of glutamate, D-serine and ATP
from astrocytes plays important roles in regulation of many synaptic events from
basal transmission to synaptic plasticity. However, concerns have been raised over the
unspecificity of methods used to stimulate or suppress astroglial release of
gliotransmitters (Hamilton and Attwell, 2010; Nedergaard and Verkhratsky, 2012).
On the one hand, methods that aim to stimulate release of gliotransmitters might
simultaneously induce neurotransmitter release from nearby neuronal processes,
resulting in ambiguous origins of the transmitters. A method widely used is to
artificially raise [Ca2+]i in astrocytes. The artificially induced [Ca2+]i elevations,
however, may not capture the full characteristics of endogenous Ca2+ dynamics in
astrocytes. On the other hand, manipulations that aim to suppress gliotransmitter
release cannot avoid side effects on other signaling pathways, by which astrocytes can
also influence neuronal activity. The potential problems of research methods are well
illustrated by the studies utilizing the InsP3 receptor 2 knockout (KO) mice, which
lack InsP3-mediated Ca signaling in astrocytes: this deficiency did not impair
NMDA receptor-dependent LTP in the hippocampus (Agulhon et al., 2010), but it
blocked cholinergic transmission-induced LTP in both the hippocampus (Navarrete et
al., 2012) and cortex (Takata et al., 2011) as well as nucleus basalis-mediated
plasticity in the visual cortex (Chen, N. et al., 2012). These observations suggest that
2+

#&!

Table 3. Gliotransmitter-dependent regulations of synaptic transmission by astrocytes.

Abbreviations: EPSC, excitatory postsynaptic current; IPSC, inhibitory postsynaptic current;
LTD: long-term depression; LTP, long-term potentiation; NMDA, N-methyl-D-aspartate;
TNF, tumor necrosis factor. Adapted from Araque et al., 2014.
!#'!

InsP3 receptor 2 expression in astrocytes is essential for some but not all forms of
LTP, and, more importantly, they highlight the unresolved general issues in the field
of neuroglial interaction: how to reconcile the variety of gliotransmitters and signaling
pathways (summarized in Table 3) that are proposed to regulate synaptic transmission
and under what physiological conditions different types of regulations take place. To
answer these questions, research efforts are needed to better understand the temporal
and spatial specificity of astroglial receptor distribution and activation, and their
association with different mechanisms regulating the release of gliotransmitters
(Araque et al., 2014).

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N&7+',4(':%,(4,%*12-&.),'+0A82&.(1,

In addition to release of gliotransmitters, there are other modes in which astrocytes

can regulate synaptic transmission. Through active uptake of neurotransmitters such
as glutamate, GABA and monoamines via perisynaptic membrane transporters,
astrocytes control concentrations of these transmitters in the synaptic cleft and thus
modulate synaptic strength (Volterra, 2013). Also, the ability of astrocytes to lower
[K+]o, which is a key determinant of neuronal membrane potential, provides another
mechanism for astroglial regulation of neuronal excitability and synaptic transmission
(Nedergaard and Verkhratsky, 2012). These modes of regulation depend on the
astroglial coverage of the synapses, which undergo plastic changes controlled by the
physiological condition. For example, in the hypothalamus, astrocytes retract their
processes during lactation or dehydration, significantly reducing the astroglial
coverage of the magnocellular neurons (Theodosis et al., 2008). In the cortex, 24 h of
whisker stimulation elicited increases in the expression of glutamate transporters and
astroglial envelopment of excitatory synapses in the corresponding cortical column of
the barrel cortex (Genoud et al., 2006). A recent work found that Cx30 deletion
resulted in protrusion of hippocampal astroglial processes into the synaptic cleft,
facilitating glutamate clearance and thus decreasing synaptic strength (Pannasch et al.,
2014). Such structural changes of perisynaptic astroglial processes result in changes in
extracellular volume and the positions of astroglial transporters relative to the
synaptic cleft, which in turn affect the spillover or clearance of the neurotransmitters
and ultimately the synaptic transmission.

!"5";

L+A'(K2%)A82',)(A-8.10,

Increase in local neuronal activity triggers a rapid increase in local cerebral blood
flow, a phenomenon known as functional hyperemia or neurovascular coupling (Roy
and Sherrington, 1890). The functional hyperaemia takes place mostly at the level of
terminal arterioles and capillaries. The intraparenchymal vasculature is almost
completely (estimations vary between 80 to 100%) covered by astroglial endfeet
(Mathiisen et al., 2010; McCaslin et al., 2011). In the brain parenchyma, the walls of
penetrating arterioles and capillaries are also contacted by local interneurons and
intrinsic neurons (Golanov et al., 2001; Hamel, 2006; Rancillac et al., 2006; Yang et
al., 2000; Figure 9). Thus, local blood flow can be controlled by both neuronal and
!

#(!

Figure 9. Schematic representation of the neurovascular unit. Penetrating arterioles and

capillaries are covered by endfeet of astrocytes, which also extend perisynaptic processes that
cover synapses and detect synaptic activity. Vasoactive interneurons targeting the vasculature
synapse onto astrocyte endfeet rather than directly on the vessels. Inserts show the structures
of neurovascular units on the level of arterioles (top) and capillaries (down left) and of the
tripartite synapse (down right). Adapted from Petzold and Murthy, 2011.

!#,!

astroglial mechanisms (reviewed in Filosa, 2010; Petzold and Murthy, 2011; Attwell
et al., 2010). In neurons, synaptic glutamate acts on NMDA receptors to raise [Ca2+]i,
which induces production of nitric oxide (NO) by nitric oxide synthase (nNOS) and
arachidonic acid (AA) by phospholipase A2 (PLA2). NO activates guanylate cyclase
in the smooth muscle of the vessel and generates cyclic guanosine monophosphate
(cGMP) to dilate vessels. AA is converted to PGs that also dilate vessels. In astrocytes,
synaptic glutamate activating mGluRs raises [Ca2+]i , generating three types of
metabolite: PGs and epoxyeicosatrienoic acids (EETs) that dilate vessels, and
20-hydroxy-eicosatetraenoic acid (20-HETE) that constricts vessels. Also a rise of
[Ca2+]i in astrocyte endfeet may lead to release of K+, which dilates vessels (Figure
10).

!"5"@

O'2.1,+1+'0*,:+&2<(8.%:,

Sustenance of brain functions is energy expensive: 20% of the total energy produced
by the body is consumed by the brain which represents only 2% of the body mass
(Blanger et al., 2011). Most of the energy that the brain takes is spent on synaptic
transmission (Alle et al., 2009). It is estimated that glutamatergic transmission
consumes about 80% of the energy expended in the gray matter (Sibson et al., 1998;
Hyder et al., 2006; Attwell and Laughlin, 2001), highlighting the close relationship
between glutamatergic transmission and energy utilization. The brain derives energy
from oxidation of glucose and the increase in neural activity is coupled to the increase
in glucose utilization (Blanger et al., 2011). Astrocytes are ideally situated and fully
equipped to mediate this coupling: they possess perisynaptic processes that can
monitor synaptic activity while their endfeet express glucose transporters that deliver
glucose from blood circulation (Allaman et al., 2011). Besides, astrocytes are the only
brain cells that synthesize glycogen, which is the largest energy reserve of the brain
(Magistretti et al., 1993; Brown, 2004).
Although neurons can also directly uptake glucose from the extracellular space via
glucose transporters, experimental evidence indicates that astrocytes serves as the
major conduit that funnels energy substrates to neurons, especially during activation
of the brain (see Blanger et al., 2010). How astrocytes respond to neuronal activity
and supply energy to the neurons is theorized as the astrocyte-neuron lactate shuttle
hypothesis, which argues that neuronal activation triggers sustained production of
lactate by astrocytes and that lactate, instead of glucose, is the preferred energy
substrate of neurons (Pellerin and Magistretti 1994). This hypothesis has been
supported by numerous in vitro and in vivo studies (Barros et al., 2009; Cholet at al.,
2001; Voutsinos-Porche et al., 2003; Chuquet at al., 2010; Lin et al., 2010; Rouach et
al., 2008). The signaling pathway is as follows: glutamate released during synaptic
activity is cotransported with Na+ by astroglial glutamate transporters, resulting in an
increase in astroglial [Na+]i. The rise in [Na+]i stimulates Na+/K+ pump and increases
consumption of ATP. This in turn triggers in astrocytes glucose uptake from
circulation and glycolysis that produces lactate. Lactate is then released via
!

#-!

Figure 10. Major neuronal and astroglial pathways involved in the regulation of cerebral
blood flow. In astroyctes, synaptically released glutamate activates metabotropic glutamate
receptors (mGluRs) and raises [Ca2+]i, which activates phospholipase A2 (PLA2) to produce
arachidonic acid (AA) and its metabolites: prostaglandins (PGs) and epoxyeicosatrienoic
acids (EETs) in astrocytes, which dilate vessels, and 20-hydroxy-eicosatetraenoic acid
(20-HETE) in smooth muscle around the vessel, which constricts vessels. Elevated [Ca2+]i in
astrocyte endfeet also activate Ca2+-gated K+ channels (gK(Ca)), releasing K+, which dilates
vessels. In neurons, glutamate activating N-methyl-D-aspartate receptors (NMDARs) raises
[Ca2+]i, which stimulates neuronal nitric oxide synthase (nNOS) to produce nitric oxide (NO).
NO leads to production of cyclic guanosine monophosphate (cGMP) in smooth muscle to
dilate vessels. Raised [Ca2+]i, also results in generation of AA, which is converted to PGs that
dilate vessels. Adapted from Attwell et al., 2010.

!$.!

monocarboxylase transporters (MCTs) from astrocytes and taken up by neurons as the

energy substrate (Figure 11). Notably, this model does not apply to GABAergic
transmission, as experimental evidence (Chatton et al., 2003) and a modeling study
(Occhipinti et al., 2010) showed that GABA uptake by astrocytes is not coupled to
glucose utilization.

$"!

Figure 11. Schematic presentation of the astrocyte-neuron lactate shuttle. Glutamate (Glu)
released at the synapse is taken up by astrocytes via EAATs together with Na+, which is
extruded by Na+/K+ ATPase. Consumption of ATP triggers glucose uptake from the
circulation through the glucose transporter 1 (GLUT1) and production of pyruvate through
glycolysis in astrocytes. Pyruvate is converted to lactate by lactate dehydrogenase 5 (LDH5)
and shuttled to neurons through mainly MCT1 in astrocytes and MCT2 in neurons. In neurons,
lactate is converted to pyruvate by LDH1 and enters the Krebs cycle for oxidation. Neurons
can also take up glucose via GLUT3. Concomitantly, glutamate uptaken by astrocytes is
recycled back to the neurons through the glutamate-glutamine cycle. Abbreviations: GLS,
glutaminases. Reproduced from Blanger et al 2011.

!$#!

$$!

Figure 12. Gap junction channels (GJCs) and connexions (Cxs) (A) Schematic drawing of
GJCs, connexons and junctional plaques. Adapted from Kandel et al., 1995. (B) Topological
model of a Cx. The cylinders represent transmembrane domains (M1 M4). Each of the
extracellular loops (E1 and E2) has three conserved cysteine residues. Adapted from Kumar
and Gilula, 1996.

Table 4. The connexin gene family and its chromosomal distribution in mice and humans.
From Sohl and Willecke, 2004.

!$%!

""# $%&'(,-./-!)(00+1.0%!
Connexins (Cxs) are the molecular components of gap junctions (GJs) in vertebrates.
A hexamer of Cxs forms a hemichannel (HC) or a connexon. Two connexons on
adjacent plasma membrane docking together forms a GJ channel (GJC) (Figure 12A).
Aggregation of GJCs in closely apposed areas between neighboring cells forms
junctional plaques. Cxs are not found in invertebrates, but they express innexins,
which do not show sequence homology with Cxs but similarily form GJCs (Phelan
and Starich, 2001). Connexons can also function as HCs, allowing communication
between the cytoplasm and extracellular environment (Hofer and Dermietzel, 1998;
Contreras et al., 2002; Bennett et al., 2003). Under basal conditions, HCs are mostly
inactive (Bennett et al., 2003; Giaume et al., 2013). In astrocytes, several
experimental conditions including Ca2+-free medium, moderate increase in [Ca2+]i and
pro-inflammatory agents can lead to HC opening (see below). HCs can also be
composed of pannexins, a distinct family of membrane proteins that are homologous
to innexins (Scemes et al., 2007). Besides forming GJs and HCs, Cxs serve several
functions independent of channel activity (see below).

!!"# M+1+'28,2%-+)&%,(4,)(11+H.1%,
!!"#"# L(:+1)82&A'+,(4,)(11+H.1%,
Cxs are encoded by a gene family that includes 21 members in the human genome
and 20 in the mouse, and 11 of them have been identified in the brain (Nagy et al.,
2004; Sohl and Willecke, 2004). For one of the two nomenclatures in use, the Cxs are
named according to their molecular mass. For example, Cx43 means it is a Cx with a
molecular mass of 43 kDa (Beyer et al., 1990). The other nomenclature is the GJ
system, where Cxs are divided into subgroups (", # or !), abbreviated as Gj and
numbered according to the order of discovery (Eiberger et al., 2001). For example,
Cx43 was the first Cx of the a-group and hence named Gja1 (Table 4). As a common
practice, the former nomenclature is used to designate the Cx proteins while the latter
for the genes encoding Cxs.

!!"#"5 6(8+)A82',%&'A)&A'+,
Cxs are four-transmembrane proteins, consisting of one cytoplasmic and two
extracellular loops, four hydrophobic domains, and the cytoplasmatic N- and Ctermini (Figure 12B). The sequences of both extracellular loops are highly conserved,
including three cysteines that form disulfure bonds to stabilize the extracellular loops
during docking of two connexons (Kumar and Gilula, 1996). The N-terminus is
highly conserved whereas the C-terminus is variable for each Cx. The C-terminus of
Cxs harbors multiple phosphorylation sites and domains for protein interactions,
which are critical for the regulation of properties and functions of Cxs (Herv et al.,
2004; Laird, 2010).
!

$&!

Table 5. Relative immunolabelling densities of Cx30 and Cx43 in various regions of the rat
brain. From Nagy et al., 1999

!$'!

!!"#"9 GH-'+%%.(1,(4,2%&'(08.28,)(11+H.1%,
Cx43 and Cx30 are the main astroglial Cxs in the adult brain (Giaume et al., 1991;
Dermietzel et al., 1991; Nagy et al., 1999; Rash et al., 2001), although there is also a
minor expression of Cx26 (Mercier and Hatton, 2001; Nagy et al., 2001; Nagy et al.,
2011). Given the fact that GJ communication is completely abolished in knockout
(KO) mice of Cx43 and Cx30 (Wallraff et al., 2006; Rouach et al., 2008; Roux et al.,
2011), Cx43 and Cx30 are considered as the major components of astroglial GJCs
(Giaume et al., 2010).
These two atroglial Cxs have different developmental and tissue distribution patterns.
Expression of Cx43 begins around embryonic day 12 while Cx30 becomes detectable
during the third postnatal week in rodents (Kunzelmann et al., 1999). Both Cx
expression levels reach a maximum in adulthood and maintain relatively unchanged
through out the lifespan (Cotrina et al., 2001). The two astroglial Cxs exhibit distinct
distribution in the CNS. Expression of Cx43 is relatively homogeneous throughout
the brain, whereas Cx30 is far more abundant in diencephalic and hindbrain areas than
forebrain areas, and it is absent in the white matter (Nagy et al., 1999; Table 5).

!!"5 C'(-+'&.+%,(4,2%&'(08.28,)(11+H.1,)7211+8%, ,
!!"5"# /(I,&(,:+2%A'+,2)&.K.&*,(4,2%&'(08.28,)(11+H.1,)7211+8%,
2= G8+)&'(-7*%.(8(0.)28,:+2%A'+:+1&%,
Communications of GJs can be assessed by measuring junctional currents in pairs of
astrocytes with the double patch-clamp technique. It consists of imposing a voltage
step in one cell and monitor the currents induced in both cells. The ratio between the
currents in the two cells is termed coupling coefficient, which reflects the efficacy of
the GJ communication between the two cells (Dermietzel et al., 1991; Giaume et al.,
1991). The advantages of this electrophysiological measurement are its accuracy and
dynamic nature. For HCs, their opening can be monitoredd by a classical patch-clamp
approach (for details, see Giaume et al., 2012).

<=

P*+,)(A-8.10,213,A-&2Q+,

Dye coupling is an extensively used method to study GJCs. It is based on

GJC-dependent intracellular diffusion of low molecular weight dyes and tracers such
as Lucifer yellow, sulforhodamine B, and biocytin. It consists of injecting or dialysing
one astrocyte with a dye through the recording pipette during intracellular or
patch-clamp recordings and counting the number of coupled cells after a defined
period of time (in general less than 30 min). Dye coupling provides a qualitative
measure of the GJC status and information about the spatial organization of coupled
cells. This method, however, cannot measure the real time kinetics of GJ
communication (see Giaume et al., 2012).
!

$(!

HC activity can be measured by the dye uptake method. Low molecular weight
fluorescent dyes (Lucifer yellow, ethidium bromide, YoPro, etc) applied in the
extracellular medium are taken up by the cell via HCs. After a defined incubation
time (in general less than 30 min) the dye in the extracellular medium is washed out
and the level of fluorescence within the cells is measured as an indication of HC
activity (see Giaume et al., 2012).
The shortcoming of the dye coupling and uptake methods is that the dyes and tracers
mentioned above are biologically irrelevant and might not correctly reflect the
function of Cx channels in the diffusion of endogenous molecules. Therefore,
radiolabeled glucose and lactate (Tabernero et al., 1996) and fluorescent glucose
derivatives (Blomstrand and Giaume, 2006; Rouach et al., 2008) have been used to
follow the biological activity GJCs. Also, HC activity has been studied by monitoring
passage of endogenous molecules, such as ATP monitored by bioluminescent imaging
detection assay (Kang et al., 2008), fluorescent glucose derivatives (Retamal et al.,
2007) and glutamate by high-performance liquid chromatography (Ye et al., 2003).

!!"5"5 C+':+2<.8.&*,(4,2%&'(08.28,)(11+H.1,)7211+8%, ,
The central pore of GJCs and HCs is permeable to ions and small molecules, with a
cut-off molecular weight of 1-1.2 kDa. However, the permeability of GJCs depends
on not only the size of the molecule but also its shape, charge and interactions with
the Cxs in the channel (Giaume and Theis, 2010). It is known that most of GJCs are
permeable to second messengers, amino acids, nucleotides, Ca2+, siRNAs, and
glucose as well as its metabolites (Goldberg GS et al., 2004; Harris, 2007). Astrocyte
HCs have been shown to be permeable to glutamate, ATP (Cotrina et al., 2000; Ye et
al., 2003), glucose (Retamal et al., 2007) and glutathione (Rana and Dringen, 2007;
Stridh et al., 2008).

!!"5"9 >+0A82&.(1%,(4,2%&'(08.28,)(11+H.1,)7211+8,2)&.K.&*, ,
2= O*,.1&'2)+88A82',42)&('%,
The permeability of GJCs is tightly regulated by numerous factors including voltage,
Ca2+ concentration, pH and phosphorylation. Cx30 and Cx43 channels are gated by
and by both transjunctional voltage (Giaume et al., 1991) and the transmembrane
voltage (Gonzalez et al., 2007). GJC conductance increases upon membrane
depolarization and decreases upon membrane hyperpolarization (Enkvist and
McCarthy, 1994; De Pina-Benabou et al., 2001). Intracellular acidification (Duffy et
al., 2004) and high [Ca2+]i (Adermark and Lovinger, 2008) uncouples Cx43 GJCs
whereas Cx43 HCs opens in response to physiological rises in [Ca2+]i (Suadicani et al.,
2004; Wang et al., 2012). Finally, phosphorylation of Cx43 by different pathways can
either increase or decrease GJ communication (Nielsen et al., 2012). Astroglial HCs
are also regulated by these factors, although the direction of regulation can be
!$,!

different from GJCs. HCs are opened by membrane depolarization, [Ca2+]i elevation,
changes in their phosphorylation or redox status (Contreras et al., 2003 ).

<=

O*,)*&(Q.1+%,

Several pro-inflammatory cytokines have been demonstrated to reduce GJC activity

(reviewed in Orellana et al., 2013). Moreover, decreased expression of Cx43 in the
brain is observed in different models of pathologies associated with inflammation
(Brand-Schieber et al., 2005; Karpuk et al., 2011; Schalper et al., 2009; Takeuchi et
al., 2011). In contrast, pro-inflammatory agents (lipopolysaccharide, tumor necrosis
factor-", and interleukin-1#) have been shown to increase HC activity and ATP
HC-mediated release in astrocytes (Bennett et al., 2012; Orellana et al., 2011a, b).

)=

O*,1+A'(&'21%:.&&+'%,

Current evidence indicates that a number of neurotransmitters affect the activity of Cx

channels. Glutamate and several glutamate receptor agonists have been demonstrated
by several studies to increase GJC and HC activity in astrocytes (reviewed in Giaume
et al., 2010; Orellana et al., 2013). Endothelin 1 and 3 strongly inhibit dye coupling
among astrocytes (Giaume et al., 1992). ATP and 2-methylthio-ATP, a P2Y receptor
agonist decrease GJ coupling between cortical astrocytes (Meme et al., 2004). Finally,
noradrenaline exerts a dual regulation on dye coupling between cortical astrocytes
depending on the adrenergic receptor subtypes: activation of a1 receptor inhibits GJ
coupling while that of b receptor increases (Giaume et al., 1991).

3=

O*,1+A'(128,2)&.K.&*,

Considering the close interactions between neurons and astrocytes, it is not surprising
that GJC function in astrocytes is linked to neuronal activity. Gap junctional
communication between astrocytes is increased in epileptic human tissues (Lee et al.,
1995). Co-culture with neurons increases dye coupling in cerebellar and striatal
astrocytes (Fischer and Kettenmann, 1985; Rouach et al., 2000). In mouse
hippocampal slices, tetrodotoxin (TTX) decreases diffusion of 2-NBDG, a fluorescent
dexoyglucose analog, while evoked repetitive neuronal firing and epileptiform bursts
increase the diffusion of 2-NBDG, but not passive dyes, through astroglial GJs
(Rouach et al., 2008). In the mouse olfactory glomeruli, GJ coupling is reduced by
TTX and early sensory deprivation (Roux et al., 2011). Importantly, such regulation is
absent in Cx30 but not in Cx43 KO mice, indicating that Cx30 rather than Cx43
mediates this activity-dependent regulation (Roux et al., 2011).
The mechanisms underlying the activity-dependence of GJ coupling remain unclear.
In the hippocampus, activation of postsynaptic AMPA receptors is necessary for the
increased trafficking of 2-NBDG astroglial GJC (Rouach et al., 2008), while in the
olfactory glomerulus, extracellular K+ entry via astroglial Kir channels is a key player
in the activity dependence of dye coupling (Roux et al., 2011). The case of the
!

$-!

Figure 13. Schematic illustrations of the organizations of astroglial networks in different

brain regions. Red triangles, hippocampal pyramidal neurons. Red circles, neurons. Dark blue,
astrocytes. Light blue, astrocytes in the septa of the barrel cortex. Adapted from Giaume et al.,
2010.

!%.!

glomerulus corroborate observations that elevated [K+]o increase astroglial GJC

activity (Enkvist and McCarthy, 1994). In addition, it is shown that in the spinal cord
astrocytes [K+]o-dependent increase in GJ coupling involves phosphorylation of Cx43
by the calmodulin kinase II (De Pina-Benabou et al., 2001).

!!"5"; M2-,RA1)&.(1S:+3.2&+3,2%&'(08.28,1+&I('Q%,
Astroglial GJCs are the target of multiple constraints and regulations, thus conferring
GJ-mediated astroglial networks considerable spatial and functional specificity, as
revealed in dye coupling experiments (Giaume et al., 2010; Figure 13). For example,
in the hippocampus the pyramidal layer limits the number of astrocytes and dye
diffusion across this layer (Houades et al., 2006; Rouach et al., 2008; Figure 13A).
Astroglial processes within olfactory glomeruli are oriented toward the glomerulus
center, and few processes extend across the peri-glomerular neuronal soma (Roux et
al., 2011; Figure 13B). Moreover, Cx expressions are higher within glomeruli
compared to the extra-glomerular area, and there is a preferential coupling among
astrocytes within glomeruli and limited coupling with extra-glomerular astrocytes
(Roux et al., 2011). In the rodent barrel cortex, coupling within the barrels is
extensive and oriented towards the barrel center, whereas astrocytes located in the
septa between barrels are barely coupled (Houades et al., 2008; Figure 13C). This
feature may result from enriched Cx expression in the barrels and low expression in
the septa (Houades et al., 2008). !

!!"9 FA1)&.(1%,(4,2%&'(08.28,)(11+H.1%,
!!"9"# $--'(2)7+%,4(',%&A3*.10,&7+,4A1)&.(1%,(4,2%&'(08.28,)(11+H.1%,
Ideally, the methods to identify possible roles of astroglial Cxs in brain functions and
diseases should fulfill the following criteria: 1) having no side effects on neurons and
other brain cell types or on astroglial components other than Cxs; 2) specific to the Cx
in question; 3) capable of distinguishing among GJ, HC and non-channel functions of
Cxs; 4) capable of distinguishing between Cx and pannexin HC functions (see
Giaume and Theis, 2010). The currently existing experimental tools, however, have
failed to achieve at least some of these criteria (Table 6). Therefore, it is important to
use a combination of different tools to validate a conclusion and to bear in mind the
caveats and shortcomings of the methods in order to avoid over-interpretation the
data.

!!"9"5 $%&'(08.28,)(11+H.1%,'+0A82&+,1+A'(128,2)&.K.&*,
2= O*, :2.1&2.1.10, +H&'2)+88A82', .(1, 213, 1+A'(&'21%:.&&+',
7(:+(%&2%.%,
As discussed previously, astrocytes play critical roles in maintaining K+ spatial
buffering and glutamate clearance from the synapse, and the former requires diffusion
of K+ through GJCs (see Introduction I.2.1). The consequences of lacking the
!

%"!

Table 6. Loss-of-function approaches for studying astroglial Cxs. Abbreviations: Cre,

Cre-recombinase; Cx, Connexin; fl, floxed; GFAP, glial fibrillary acidic protein; GJ, gap
junction; T5M, substitution of a threonine by methionine at position 5 of the N-terminus.
Adapted from Pannasch and Rouach, 2013.

Figure 14. Proposed pathways of intercellular Ca2+ waves initiation and propagation. Local
stimulation leads to elevated IP3 in the initiator cell and generates an intracellular Ca2+ wave
by IP3 activated release of Ca2+ from the ER. Diffusion of IP3 through GJCs to the adjacent
cell elicits a second intracellular Ca2+ wave via IP3Rs. In addition, the initiator cell may
release ATP, via plasma membrane channels (hemichannels, maxi-anion channels) or
vesicular release, to activate P2Y receptors on adjacent cells, leading to IP3 production and
Ca2+ rise in the adjacent cell. Propagation of Ca2+ waves may involve the regenerative release
of ATP. Abbreviations: DAG, diacyl-glycerol; ER, endoplasmic reticulum; GPCR,
metabotropic G-protein coupled receptor; IP3, inositol 1,4,5-trisphosphate; PIP2,
phosphatidylinositol 4,5-bisphosphate; PLC, Phospholipase C; Adapted from Leybaert and
Sanderson, 2012.
!%#!

contribution of GJCs and possibly HCs to K+ and glutamate homeostasis are

highlighted in the study that investigated the synaptic transmission of the Cx30 and 43
double KO mice (Pannasch et al., 2011). In these mice, hippocampal excitatory
synaptic transmission was substantially increased due to insufficient K+ and glutamate
removal from the synapse. Moreover, the situation might be worsened by the
reduction in the extracellular space volume due to the swelling of disconnected
astrocytes. Intriguingly, a more recent study found that Cx30 KO mice exhibit a
phenotype distinct from that of the double KO (Pannasch et al., 2014). Cx30 KO mice
show decreased hippocampal excitatory synaptic transmission and impaired synaptic
plasticity due to enhanced glutamate clearance, which is a result of decreased distance
of astrocyte processes from the synaptic cleft. It was further demonstrated that Cx30
regulates astroglial morphology independent of GJC or HC functions (see II.3.5
below).

<=

O*,)(1&'.<A&.10,&(,2%&'(08.28,)28).A:,I2K+%T,

Astroglial Ca2+ waves have been proposed to reglulate synaptic transmission and, in
particular, coordination of neuronal assembly activity (Scemes and Giaume, 2006;
Leybaert and Sanderson, 2012), although there is only sporadic experimental evidence
(Fiacco and McCarthy, 2004). The astroglial Cxs can contribute to propagation of
Ca2+ waves through GJC and/or HC activities, depending on the brain region
(reviewed in Leybaert and Sanderson, 2012). Astroglial GJCs mediate intercellular
diffusion of Ca2+ and InP3 (see Giaume and Venance, 1998) while HCs participate in
regenerative release of ATP that mediates Ca2+ waves in astrocytes (Stout et al., 2002;
Figure 14).

!!"9"9 M2-,RA1)&.(1,)7211+8%,213,<'2.1,:+&2<(8.%:,
Astroglial GJCs have been demonstrated to play an important role in neurometabolic
coupling through supply of metabolites to neurons in response to synaptic glutamate.
In cultured astrocytes, GJCs are involved in glutamate-induced regenerative
propagation of Na+ waves, which gives rise to a spatially correlated increase in
glucose uptake (Bernardinelli et al., 2004). Direct evidence that GJ-mediated
astroglial networks contribute to the metabolic function of astrocytes is demonstrated
by the work of Rouach et al. (2008): in absence of extracellular glucose, delivery of
glucose or lactate into astroglial networks rescues glutamatergic synaptic transmission
and epileptiform activity in wild type mice but fails to do so in the double KO mice of
Cx43 and Cx30. In addition, astroglial Cxs may participate in neurovascular coupling.
There are high levels of Cx expression and GJ coupling in astroglial endfeet that
enwrap blood vessels (Nagy et al., 1999; Rouach et al., 2008). A few studies have
reported astroglial Ca 2+ waves occur in association with vasodilation or
vasoconstriction, but a causal link between the astroglial Ca2+ waves and the vascular
regulation is lacking. For example, in brain slices, electrical stimulation elicited
vasodilation and associated astroglial Ca2+ waves along blood vessels (Filosa et al.,
!

%$!

Table 7. Summary of the impact of astroglial Cx deficiency on animal behaviors. Adapted

from Pannasch and Rouach, 2013.

!%%!

2004), while in hippocampus spontaneous Ca2+ waves in vivo were associated with
decreased blood flow and this type of Ca2+ waves showed GJC dependence (Kuga et
al., 2011).

!!"9"; O+72K.(',
So far, there have been a few behavioral studies performed on KO mice of astroglial
Cxs, which are summarized in Table 7. Notably, the same Cx KO mice, e.g., the
Cx43 and Cx30 double KO (Dere et al., 2003; Lutz et al., 2009), have diverse
behavioral deficits, suggesting the Cxs have different functions depending on the
brain region. This calls for the development of conditional KO models to control the
temporal and spatial specificity of Cx inactivation. Morever, the Cx30 KO and the
Cx43 and Cx30 double KO mice exhibit differences in their phenotypes, which is not
surprising considering that Cx43 and Cx30 differ in their expression patterns,
permeability and regulatory pathways, etc. The distinct properties and potentially
distinct functionsthe latter have started to be revealedof Cx43 and Cx30 highlight
the necessity for further investigations on KO mice of single astroglial Cxs. Last but
not the least, few genetic tools exist to distinguish among GJC, HC and non-channel
functions of Cxs, resulting in difficulties for detailed investigations of mechanisms
underlying Cx functions in vivo.

!!"9"@ L(1S)7211+8,4A1)&.(1%,(4,2%&'(08.28,)(11+H.1%,
2= B+88,:.0'2&.(1,
An adhesive function of Cx43 has been demonstrated in vitro and in vivo: Cx43
expression was shown to increase aggregation of cells (Lin et al., 2002; Cotrina et al.,
2008) and during development Cx43 provides adhesive contacts necessary for
neuronal radial migration in the cortex (Elias et al., 2007). The adhesive function of
Cx43 is independent of its channel forming property (Lin et al., 2002; Cotrina et al.,
2008; Elias et al., 2007), but requires a direct interaction between extracellular loops
of Cx43 in the neural and radial glial cells (Elias et al., 2007). In addition, the
C-terminus of Cx43 is also involved the radial migration of cortical neurons (Cina et
al., 2009), possibly via interaction with cytoskeletal proteins (Kameritsch et al.,
2012).

<=

B+88,:('-7(8(0*,

Cx43 and Cx30 have been reported to associate with the cytoskeletal proteins: Cx30
directly interact with actin while Cx43 is thought to indirectly bind to actin; both
Cx43 and Cx30 also bind to tubulin (reviewed in Olk et al., 2009; Herv et al., 2012).
One of the functional consequences of such interactions may be modulation of cell
morphology. A few studies showed correlation between Cx43 expression and cell
morphological changes, although it was addressed whether such changes occurred
independent of its channel functions: expression of Cx43 in C6 glioma cells resulted
!

%&!

in reorganization of actin into stress fibers, associated with a more flattened cell
morphology (Naus et al., 1992; Cotrina et al., 2000). A recent study provided
evidence that Cx30 influences the morphology of astroglial processes in a
channel-independent manner. In Cx30 KO mice, the total volume of astroglial
processes the stratum radiatum region of the hippocampus increased due to enhanced
elongation and ramification (Pannasch et al., 2014). Moreover, such morphological
changes were inhibited by expression of full-length Cx30, but not C-terminally
truncated Cx30, indicating the Cx30 C-terminus was necessary for the role of Cx30 in
morphology (Pannasch et al., 2014).

!%'!

%(!

!
Figure 15. Illustrations of the typical electroencephalography (EEG) signals recorded during
different wake-sleep states in humans and rats. Adapted from Brown et al., 2012.

Figure 16. The typical structure of human sleep in a night. The hypnogram shows that sleep
proceeds in cycles of NREM and REM sleep, the order normally being NREM stage 1, stage
2, stage 3, stage 4, stage 2 and REM. There is more deep sleep (stage 3, 4) in early sleep,
while REM sleep increases before awakening. Adapted from Peplow, 2013.
!%,!

"""# 2-++3!
Sleep is of fundamental importance for animal and human survival. Indeed, even a
minor restriction in total sleep time can lead to significant cognitive impairments
(McCoy and Strecker, 2011; Killgore, 2010). Sleep disorders are highly prevailing in
modern society and sleep disturbances are associated with many neurodegenerative
diseases, psychiatric disorders, and other medical conditions (Parish, 2009). The
vitalness of sleep underscores the significance and necessity of sleep research. For the
past decades, intensive research efforts have largely deepened our understanding of
sleep-wake regulation and sleep functions, which will be the topics of this chapter.

!!!"# D8++-,4+2&A'+%,
!!!"#"# 62)'(%)(-.),213,<+72K.('28,4+2&A'+%,(4,%8++-,213,I2Q+, ,
Sleep is a reversible behavioral state characterized by reduced consciousness,
movement and responsiveness to sensory stimuli. Sleep is defined in laboratory
settings by the electroencephalography (EEG) and electromyography (EMG)
recordings. EEG records the electrical field activity of neurons (primarily cortical
neurons) and EMG records that of skeletal muscles. Based on EEG and EMG
activities, sleep is broadly divided into two stages: the non-rapid eye movement
(NREM) sleep or slow wave sleep (SWS), and the rapid eye movement (REM) sleep.
NREM sleep is characterized by low-frequency, high-amplitude EEG activity and
reduced muscle tone. In humans, NREM sleep is further subdivided into 4 stages. As
NREM sleep deepens and progresses from stage 1 to 4, EEG activity decreases in
frequency and increases in amplitude until it is dominated by slow wave activity
(SWA; 0.5-4 Hz). SWS refers to Stage 3 and 4 in humans. During REM sleep, the
EEG exhibits a wakefulness-like profile, with a high frequency and low amplitude,
but a complete loss of muscle tone (Figure 15). During REM sleep, humans report
active dreaming. Over the sleep period, an individual repetitively cycles through
different sleep stages, with gradually lighter NREM and longer REM sleep till a full
wakening (Figure 16).

!!!"#"5 O'2.1,'7*&7:%,2%%().2&+3,I.&7,3.44+'+1&,%8++-SI2Q+,%&2&+%,
2= J2Q+4A81+%%,
During wakefulness, the cortical EEG exhibits an 'activated' profile, dominated by fast,
locally generated rhythms of low amplitude beta (15-30 Hz) and gamma (30-120 Hz)
rhythms. Alpha rhythms (8-14 Hz) occur during relaxed wakefulness and are
suppressed by eye opening and visual stimuli (Palva and Palva, 2007). Theta (4-8 Hz)

<=

L>G6,%8++-,

%-!

Figure 17. Illustrations of several brain oscillatory patterns associated with sleep. The
dominant electrical field potentials during slow wave sleep (SWS) are the neocortical slow
oscillations (!0.8 Hz), thalamocortical spindles (1015 Hz), and the hippocampal sharp
wave-ripples (SWRs). In animals, REM sleep is characterized by ponto-geniculo-occipital
(PGO) waves, which originate from the pontine brain stem and propagate to the lateral
geniculate nucleus and visual cortex, and by hippocampal theta rhythms (48 Hz). In humans,
PGO waves and theta activity are less prominent. Adapted from Rasch and Born, 2013.

Figure 18. Example traces of a regular-spiking cortical neuron intracellularly recorded with
simultaneous EEG and electromyography (EMG) recordings during wake, SWS and REM
sleep in a freely moving cat. Periods marked by horizontal bars are expanded below (arrows).
Adapted from Steriade et al., 2001.rhythms occur during exploring behavior in rodents,

and during tasks requiring attention and memory in humans (Brown et al., 2012).
Lower frequency rhythms such as the theta rhythm occur over more widespread areas
and synchronize faster rhythms. These EEG rhythms are thought to temporally link
firing cortical neurons and synchronize cortical and subcortical areas for higher order
cognitive functions (Buzski and Draguhn, 2004).

!&.!

In human, NREM sleep stage 1 is a transition stage from wake and sleep. The EEG
exhibits a profile of relatively low-voltage rhythms and mixed frequencies, mainly
alpha and theta activity (Buzski, 2006). Stage 2 NREM sleep is characterized by the
appearance of sleep spindles (715 Hz) and K-complexes in the EEG (Figure 15).
Stages 3 and 4 of NREM sleep (deep sleep) are dominated by prominent,
high-amplitude delta waves (14 Hz). Fast rhythms, e.g., beta and gamma, also occur
during slow-wave sleep, and the slow oscillation (0.51 Hz), discovered by Steriade
and colleagues, groups fast and slow rhythms during NREM sleep (Steriade, 2006).

)=

>G6,%8++-, ,

During REM sleep, cortical EEG is activated and desynchronized. Theta rhythms are
typical of tonic REM sleep (periods in REM sleep without rapid eye movement)
(Buzski, 2002), while ponto-geniculo-occipital (PGO) waves appear simultaneously
with rapid eye movements during phasic REM sleep (periods in REM sleep with rapid
eye movements and muscle twitches) (Mignot, 2008). PGO waves are phasic field
potentials originating in the pontine tegmentum and propagating to the lateral
geniculate nucleus and occipital cortex (Figure 17).

!!!"#"9 B('&.)28,1+A'(128,2)&.K.&*,3A'.10,3.44+'+1&,%8++-SI2Q+,%&2&+%,
As mentioned above, EEG signals are mainly derived from electrical activity,
including synaptic currents and spiking, of cortical neurons (Buzski and Draguhn,
2004). Figure 18 shows simultaneous EEG, EMG and intracellular recordings of a
regular-spiking cortical pyramidal neuron during wake, SWS and REM sleep of a cat.
Neurons fire tonically during wakefulness indicated by activated EEG and increased
muscle tone. During SWS, they undergo cyclic hyperpolarized phases characterized
by lack of spontaneous firing ('down states') and depolarized phases with rich
spontaneous firing (the 'up states'). During REM sleep, indicated by muscular atonia
and EEG activation, neuron exhibit a tonic firing profile similar to that during
wakefulness. The extent of synchrony among cortical neurons also varies throughout
wake and sleep states. In NREM sleep, individual neurons begin and stop spiking
largely in synchronization with the population, while much less coordination among
neurons is detected in wakefulness and REM sleep (Figure 19).

!!!"5 L+A'(212&(:.)28,213,1+A'()7+:.)28,<2%.%,(4,%8++-,
213,I2Q+, ,
!!!"5"# J2Q+4A81+%%,
2= ?7+,2%)+13.10,'+&.)A82',2)&.K2&.10,%*%&+:,
The early work of Moruzzi and Magoun in the 1940s and 1950s (Moruzzi and
Magoun, 1949) established that the brainstem reticular formation (RF) is crucial for
maintaining cortical activation during wakefulness and REM sleep (Jones, 2005).
Groups of neurons in the midbrain, pons and mesencephalic RF, and their projections
!

&"!

Figure 19. Simultaneous EEG (upper panel) recordings and raster plots of spike activity of 6
cortical neurons (lower panel; each vertical line respresents a spike) in a rat during wake,
NREM and REM sleep. Note the high tonic spiking activity of the neurons in wake and REM
sleep. In NREM sleep, the rhythmic periods devoid of spiking are temporally associated with
negative phase of EEG slow waves. Adapted from Vyazovskiy et al., 2009.

Figure 20. The dorsal and ventral pathways of the ascending reticular activation system
(ARAS). The dorsal pathway (blue) originates in pontine and midbrain reticular formation,
most prominently the pedunculopontine and laterodorsal tegmental nuclei (LDT/PPT), which
project to the thalamic nuclei that innervate widespread areas of the cerebral cortex. The
ventral pathway (red) also originates in pontine/midbrain regions including the locus
coeruleus (LC) and the dorsal raphe nuclei (DRN). It is relayed by the lateral hypothalamic
(LH) and tuberomammillary (TMN) nuclei of the hypothalamus and the basal forebrain (BF),
all of which in turn project to the cortex. Adapted from Brown et al., 2012.

!&#!

via multiple relays to the forebrain where they stimulate cortical activation, constitute
the ascending reticular activating system (ARAS). The ARAS consists of the dorsal
and the ventral pathways (Figure 20). The dorsal pathway originates from glutamate
neurons in the midbrain, pontine, medullary RF and cholinergic neurons in the
pedunculopontine and laterodorsal tegmental nuclei (PPT/LDT) (Steriade et al., 1988;
Steriade and Glenn, 1982; Newman and Ginsberg, 1994; Cornwall and Phillipson,
1988; Hallanger et al., 1987), which project to the nonspecific intralaminar and
midline thalamic nuclei that diffusely innervate widespread neocortical areas. The
ventral pathway of the ARAS originates from multiple sites in the brain stem
including glutamatergic neurons of the parabrachial area, noradrenergic of the locus
coeruleus (LC), serotoninergic of the dorsal raphe nuclei (DRN), and dopaminergic of
the periaqueductal gray (Brown et al., 2012). These neurons project to the lateral and
posterior hypothalamus that contains the orexin/hypocretin neurons and histaminergic
neurons in the tuberomammillary nuclei (TMN). All these systems converge onto the
basal forebrain (BF), which in turn projects to the cortex (Detari et al., 1999;
Dringenberg and Vanderwolf, 1998; Semba, 2000).

<= L+A'()7+:.)28,<2%.%,(4,I2Q+4A81+%%,
$)+&*8)7(8.1+, ,
Two groups of cholinergic neurons located in the BF and LDT/PPT project to the
cortex directly and via the relay of thalamus, respectively. In vivo recording across
the sleep-wake cycle has shown that cholinergic neurons in the caudal BF fire during
wakefulness and REM sleep (Hassani et al., 2009; Lee et al., 2005a) and they increase
firing upon cortical activation: their firing rates correlate positively with the power of
fast gamma and theta activity typical of wakefulness and REM sleep and negatively
with slow delta activity (Duque et al., 2000; Manns et al., 2003; Lee et al., 2005).
Like in the BF, cholinergic neurons in the LDT/PPT discharge during waking,
decrease firing during SWS and increase firing during PS, with a firing rate correlated
to cortical activation (Boucetta and Jones, 2009; el Mansari et al., 1989; Kayama et al.,
1992; Steriade et al., 1990). Consistent with the firing patterns of cholinergic neurons,
acetylcholine level rises during wakefulness and REM sleep in the cortex and
thalamus where the cholinergic projections target (Celesia and Jasper, 1966; Jasper
and Tessier, 1971). The cholinergic system facilitates generation of fast rhythms
typical of wakefulness and REM sleep. Electrical stimulation of the LDT/PPT
enhances beta/gamma oscillations in thalamocortical system (Steriade et al., 1991).
Local application in the BF of agents that depolarize cholinergic neurons, such as
AMPA and norepinephrine, induces high-frequency cortical oscillations, while
lidocaine that inactivates cholinergic neurons abolishes fast rhythms (Jones, 2004).
Similarly, local injection of serotonin that hyperpolarizes BF cholinergic neurons
reduces gamma activity (Cape and Jones, 1998). Finally, lesion of PPT cholinergic
neurons by ibotenic acid leads of 30% reduction of waking time (Lu et al., 2006).

D+'(&(1.1,
!

&$!

Serotonergic neurons in the DRN fire during waking, decrease firing during NREM
sleep and cease firing during REM sleep (Jacobs and Fornal, 1991; McGinty and
Harper, 1976; Trulson and Jacobs, 1979), suggesting that serotonin is
wakefulness-promoting. Accordingly, systemic application of serotonergic receptor
agonists increases waking and reduces NREM and REM sleep (Monti and Jantos,
2008). However, unlike other wake-promoting neuromodulatory systems, the activity
of serotonergic neurons promotes quiet waking with reduced cortical activation.
Recordings in behaving animals reveal that serotonergic neurons exhibit highest
activity during feeding but decrease firing during active waking (Jacobs and Fornal,
1991). Furthermore, they reduce cortical activation through inhibition on the
cholinergic BF and brain stem neurons (Khateb et al., 1993. Thakkar et al., 1998).

L('+-.1+-7'.1+,
Norepinephrine neurons in the LC fire during waking, particularly during active
waking, decrease firing during NREM sleep and cease firing during REM sleep
(Aston-Jones and Bloom, 1981; Hobson, 1975). Norepinephrine neurons send diffuse
projections to the forebrain, brainstem and spinal cord to promote cortical activation
and behavioral arousal (Jones, 2005). LC neurons excite many other components of
the ARAS including thalamic relay neurons, serotonegic DRN neurons and
cholinergic BF neurons (Brown et al., 2012). Many drugs that are wakefulness
promoting, including amphetamine and modafinil, function partly by increasing
norepinephrinergic neurotransmission. Norepinephrine maintains the high muscle
tone during wakefulnessspinal motoneurons are depolarized and sensitized to
excitatory input by norepinephrine (White et al., 1991; White and Neuman, 1983).

/.%&2:.1+,
Histaminergic neurons in the TMN are active in wakefulness, slowly firing in NREM
sleep and silent in REM sleep (John et al., 2004; Takahashi et al., 2006;
Vanni-Mercier et al., 2003). Histamine excites most nuclei of the ARAS (Brown et al.,
2001; Haas et al., 2008) and injection of histamine into the ARAS promotes
wakefulness (Lin, 2000). Animals with deletion of histamine synthesizing enzyme,
histidine decarboxylase, have compromised arousal in face of a novel, potentially
dangerous environment (Parmentier et al., 2002; Anaclet et al., 2009).

N'+H.1,
Orexin neurons located in the lateral hypothalamus fire fastest during active waking,
decrease firing during quiet waking, and cease firing during sleep (Lee et al., 2005b;
Takahashi et al., 2008), and release of orexin in the hypothalamus is also higher
during waking than in sleep (Kiyashchenko et al., 2002). Orexin neurons project onto
and excite the cerebral cortex, other nuclei of the ARAS, and the spinal cord to
stimulate cortical activation, behavioral arousal and autonomic changes (Jones and
Muhlethaler, 2005). Intracerebroventricular injection of orexin increases wakefulness
!&%!

in rats in a dose-dependent manner (Piper et al., 2000). Photogenetic stimulation of

orexin neurons at frequencies above 5 Hz increases the probability of the animal
transiting from sleep to wakefulness (Adamantidis et al., 2007). In contrast,
administration of orexin receptor antagonists reduces wakefulness and increase both
NREM and REM sleep in animals and human subjects (Brisbare-Roch et al., 2007).
Orexin is necessary for the consolidation of wakefulness: transgenic mice and dogs
with spontaneous mutations that result in deficiency of orexin or orexin receptors
exhibit narcoleptic phenotypes and human patients of narcolepsy exhibit defected
orexin neurotransmission (Chemelli et al., 1999; Lin et al., 1999; Peyron et al., 2000).
Another function of the orexin system is to enhance wakefulness in response to
starvation: orexin neurons are activated by fasting in primates (Diano et al., 2003),
while orexin knockout mice do not respond to fasting with increased arousal
(Yamanaka et al., 2003).

P(-2:.1+,
Dopaminergic neurons in the ventral periaqueductal gray (vPAG) show Fos activity
during waking but not during sleep (Lu et al., 2006). These neurons project to other
components of the ARAS including the BF and thalamus (Lu et al., 2006).
Dopaminergic neurons of the ventral tegmental area (VTA) fire more bursts during
waking and REM sleep (Dahan et al., 2007) and increase bursting in the presence of
rewarding or aversive stimuli which require alertness in animals (Steinfels et al.,
1983). VTA neurons project to the striatum, BF and cortex, suggesting they may play
a role in stimulating arousal with emotional activation (Jones, 2005). The following
evidence supports that dopaminergic neurons play a role promoting wakefulness: 1)
the most potent wake-promoting substances, such as amphetamines and modafinil, are
considered to function mainly by increasing dopamine transmission due to the fact
that they lack effects in dopamine transporter knockout animals (Wisor et al., 2001); 2)
dopamine D2 receptor knockout mice have decreased waking and increased sleep (Qu
et al., 2010); 3) lesion of dopaminergic neurons in the vPAG results in a more than 20%
reduction in the amount of wakefulness per 24 hours (Lu et al., 2006).

M8A&2:2&+,
The nonspecific thalamic nuclei, which are an important relay of the dorsal ARAS,
send glutamatergic projections to the cortex to stimulate cortical activation (Hur and
Zaborszky, 2005). Some projections from the BF (Henny and Jones, 2008; Hur and
Zaborszky, 2005), VTA, LDT, and hypothalamus (Hur and Zaborszky, 2005) to the
cortex are also glutamatergic. In addition, the projections of the RF neurons to the
thalamus are mainly glutamatergic (Jones, 2005). Given the abundance and
importance of glutamate, it is not surprising that glutamate is involved in the control
of wakefulness. Glutamatergic neurotransmission is considered the backbone of the
wakefulness-promoting systems in the brain and it is regulated by other
neurotransmitters released by various arousal systems (Jones, 2005). In addition,
!

&&!

anesthetics such as ketamine inhibit glutamatergic neurotransmission (Rudolph and

Antkowiak, 2004). In rodents, lesion of the glutamatergic parabrachial neurons caused
a coma-like state, suggesting that these neurons are critical in maintaining
wakefulness (Fuller et al., 2011).

DA::2'*,
Multiple neurotransmitter systems of the ARAS contribute to the promotion of
wakefulness. They are highly interconnected and mutually excitatory. In light of the
fact that lesions of individual nuclei of the ARAS have generally minor effects on the
structure and amount of wakefulness (see Brown et al., 2012), there may be
considerable redundancy in the ARAS. The sheer number of the neurotransmissions
systems involved in promotion of wakefulness may guarantee wakefulness can remain
largely intact upon the loss of an individual system. In addition, different systems are
responsible for different aspects of wakefulness, e.g., acetylcholine for facilitation of
cortical activation, norepinephrine for maintenance of muscle tone, orexin for
consolidation of wakefulness, dopamine for arousal in the presence of rewards, etc.

!!!"5"5 L>G6,%8++-,
2= U+1&'(82&+'28,-'+(-&.),1A)8+A%,
An involvement of the basal forebrain and preoptic area (PO/BF) in the control of
sleep was first discovered by by Constantin von Economopatients suffering from
persistent insomnia had damage in this area caused by the influenza pandemic in the
early 20th century (Von Economo, 1930). Also, lesions of the PO/BF led to insomnia
in the cat (McGinty and Sterman, 1968; Sallanon et al., 1989). A population of
neurons in the ventrolateral preoptic nucleus (VLPO) that express Fos protein
specifically during sleep was later identified (Sherin et al., 1996). These neurons
contain the inhibitory neurotransmitters GABA and galanin and project heavily to the
nuclei of the ARAS (Sherin et al., 1996; Gaus et al., 2002; Sherin et al., 1998;
Steininger et al., 2001). Single-unit recordings confirmed that the VLPO contains
sleep-on and wake-off neurons (Szymusiak et al., 1998). Extensive lesions of the core
of the VLPO in the rat substantially decreased SWA and NREM sleep time (Lu et al.,
2000).
A prominent property of VLPO neurons is that they receive reciprocal projections
from many regions of the ARAS, including the TMN, DRN, vPAG, parabrachial
nucleus, and LC (Chou et al., 2002; Lu et al., 2006a). GABA and galanin were shown
to inhibit TMN, DRN, and LC neurons (Gervasoni et al., 1998; Gervasoni et al., 2000;
Pieribone et al., 1995; Schonrock et al., 1991), and the VLPO neurons are inhibited by
acetylcholine, norepinephrine, dopamine, and serotonin (Gallopin et al., 2000;
Gallopin et al., 2004). Histamine excites a subpopulation of inhibitory interneurons in
the VLPO, which in turn inhibit the projecting VLPO neurons (Lu et al., 2000). These
reciprocal inhibition between the VLPO and TMN/DRN/LC neurons led to the
!&'!

proposal of a flip-flop switch model of the sleep-wake alternation

mechanismactivation of the VLPO leads to silence of the nuclei of the ARAS and
promotes sleep, while activation of the ARAS nuclei leads to the shutdown of VLPO
neurons and promotes wakefulness (Figure 21; McGinty and Szymusiak, 2000; Saper
et al., 2001).

<=

6+3.21,-'+(-&.),2'+2,

Like VLPO neurons, neurons in median preoptic area (MnPO) send inhibitory
projections to the lateral hypothalamus, DRN, and LC (Suntsova et al., 2007;
Uschakov et al., 2007). Inhibition of the MnPO by GABAA receptor agonist led to
prolonged wakefulness, while electrical stimulation and application of glutamate and
the GABAA receptor antagonist enhanced NREM sleep in rats (Suntsova et al., 2007).
MnPO neurons often increase firing in advance of sleep, unlike VLPO neurons that
are activated at the same time as sleep onset (Szymusiak et al., 1998; Takahashi et al.,
2009). Fos expression pattern revealed that MnPO neurons are also active during
sleep deprivation, whereas VLPO neurons are mainly active during sleep (Gvilia et al.,
2006; Modirrousta et al., 2004; Peterfi et al., 2004). In addition, the MnPO projects
heavily to the VLPO (Chou et al., 2002; Uschakov et al., 2007), suggesting the MnPO
may drive VLPO activity. These observations indicate that MnPO neurons may play a
role in response to increased sleep pressure, whereas VLPO neurons may function to
maintain sleep (Gvilia et al., 2006).

>G6,D8++-,
The pons plays an essential role in REM sleeptransections of the pons and
extensive lesions of this region led to disruption of REM sleep (Jouvet, 1962; Webster
and Jones, 1988). Later Fos expression studies identified the sublaterodorsal nucleus
(SLD) in rodents, which are activated in REM sleep (Boissard et al., 2002; Lu et al.,
2006b). The majority of these REM-ON cells are glutamatergic (Luppi et al., 2011).
Disinhibition of the SLD neurons with GABA antagonists elicited REM sleep-like
behavior (Boissard et al., 2002). Lesions of the SLD in rats resulted in fragmentation
and reduction of REM sleep (Lu et al., 2006b). These observations indicate the SLD
is the core neural circuits in promotion of REM sleep.
The SLD receive major GABAergic inputs from the ventrolateral periaqueductal grey
matter (vlPAG) and lateral pontine tegmentum (LPT) (Boissard et al., 2003; Lu et al.,
2006b). Lesions (Lu et al., 2006b) and inhibition of the vlPAG/LPT with GABA
agonists (Crochet et al., 2006; Sapin et al., 2009; Sastre et al., 1996) increased REM
sleep. The retrograde labeling showed that vlPAG/LPT also receive GABAergic
projections from the SLD (Lu et al., 2006b), suggesting that the vlPAG/LPT and the
SLD are reciprocally inhibitory and their interaction may be the control of REM sleep
entry and exit.
!

&(!

Figure 21. Flip-flop switch model of wake-sleep control. (A) During wake, cholinergic (blue)
and monoaminergic neurons (green) of the ARAS are active and inhibit the VLPO/MnPO
neurons (magenta). (B) In NREM sleep, VLPO/MnPO neurons fire and inhibit the
components of the ARAS. Pathways under inhibition are illustrated as open circles and
dashed lines. Abbreviations: MnPO, median preoptic area; PB, parabrachial nucleus
(glutamate); PC, precoeruleus area; VLPO, ventrolateral preoptic nucleus; vPAG, ventral
periaqueductal gray. Adapted from Saper et al., 2010.

!&,!

Some REM-on neurons in the SLD send descending excitatory projections to

brainstem and spinal inhibitory nuclei that in turn inhibit motor neurons and cause
muscle atonia (Lu et al., 2006b; Luppi et al., 2004; Vetrivelan et al., 2009). Other
SLD neurons send ascending projections to the thalamic relay neurons, and innervate
the cortex together with cholinergic and glutamatergic neurons of the PPT/LDT and
BF to induce cortical activation during REM sleep (Luppi et al., 2011).
As discussed above, monoaminergic neurons, including those in the serotonergic
DRN, noradrenergic LC, and histaminergic TMN, cease firing specifically during
REM sleep, whereas the cholinergic neurons of the PPT/LDT also fire during REM
sleep. Application of cholinergic drugs to the mesopontine tegmentum can cause
REM sleep, whereas drugs that increase monoaminergic signaling inhibit REM sleep
(Luppi et al., 2006). Lesions of these nuclei, however, had very limited effects on
REM sleep (Blanco-Centurion et al., 2007; Lu et al., 2006b), suggesting the
cholinergic and monoaminergic inputs might not be essential for REM sleep
promotion but could modulate the alternation of NREM and REM sleep (Saper et al.,
2010).

!!!"9 /(:+(%&2%&.),)(1&'(8,(4,%8++-,
As proposed in the model of Borbely, two processes underlie the regulation of wake
and sleep (Borbely, 1982). Process C (circadian control) decides the timing of sleep
and wake. The circadian control of sleep is beyond the scope of this introduction and
will not be discussed here. Process S (homeostatic control) decides the depth and
duration of sleep as a function of sleep pressure accumulated in the preceding waking
period: the longer the waking period, the more sleep pressure is accumulated in brain
and the longer sleep is needed. In mammals, SWA in NREM sleep is a marker of
sleep pressureSWA increases with prolonged wakefulness (Dijk et al., 1987; Huber
et al., 2000; Lancel et al., 1991; Tobler and Borbely, 1986). Sleep pressure is reflected
by endogenous sleep factors that slowly build up during wakefulness to mediate the
homeostatic sleep response (Borbely and Tobler, 1989). The sleep factors should
induce sleep upon administration and their levels in the brain should increase with
increasing sleep pressure (Brown et al., 2012). In the following section, I will briefly
summarize current knowledge of several most studied sleep factorsadenosine, nitric
oxide, cytokines and prostaglandin D2, with adenosine being a common downstream
effector of the others.

III.3.1 $3+1(%.1+
The role of adensosine as a sleep factor is a well established. Administrations of
adenosine or adenosine analogs increase sleep in rodents and cats (Dunwiddie and
Worth, 1982; Portas et al., 1997; Radulovacki et al., 1984; Radulovacki et al., 1985;
Virus et al., 1983). Extracellular adenosine concentrations rise with time spent awake
in the BF (Blanco-Centurion et al., 2006; Kalinchuk et al., 2011; Porkka-Heiskanen et
!

&-!

al., 2000; Porkka-Heiskanen et al., 1997) and to a lesser extent in the cortex
(Kalinchuk et al., 2011; Porkka-Heiskanen et al., 2000) but not in other brain regions
(Porkka-Heiskanen et al., 2000). Extracellular adenosine is degraded by deaminase,
and inhibiting its activity increased sleep (Okada et al., 2003). In humans, a
polymorphism of the adenosine deaminase gene contributes to the individual
differences in SWS duration and intensity (Landolt, 2008). The extracellular
adenosine can be derived from multiple sources. Adenosine can be directly released
upon neuronal activation (Wall and Dale, 2008). ATP, co-released with other
neurotransmitters, is degraded to adenosine extracellularly (Krueger et al., 2008).
Adenosine can also originate from breakdown of ATP released by glia (Pascual et al.,
2005; Zhang et al., 2003).
The effects of adenosine are mainly mediated via two receptors in the brainA1
receptors that are largely inhibitory and A2a receptors that are excitatory (Brown et al,
2012). Conditional knockout mice of A1 receptor exhibited an attenuated SWA
rebound after sleep deprivation (Bjorness et al., 2009). In vitro studies have shown
that A1 receptors mediate inhibitory effects of adenosine on multiple wake-promoting
brain regions (Arrigoni et al., 2006; Liu and Gao, 2007; Arrigoni et al., 2001; Rainnie
et al., 1994). Infusion of A1 receptor agonists in these brain regions increased sleep
while infusion of antagonists decreased sleep (Alam et al., 2009; Portas et al., 1997;
Rai et al., 2010; Strecker et al., 2000; Thakkar et al., 2008). In addition, prolonged
sleep deprivation also upregulated A1 receptor expression in the BF and cortex
(Basheer et al., 2001, 2007; Elmenhorst et al., 2007, 2009). A2a receptors are also
implicated in mediating sleep-inducing effects of adenosine. The wake-promoting
effect of caffeine was blocked in A2a receptor knockout mice, but not in A1 knockout
mice (Huang et al., 2005), indicating caffeine acts on A2a receptors. In vitro,
adenosine was shown to excite a subpopulation of sleep-promoting VLPO neurons via
A2a receptors (Gallopin et al., 2005). In rats, selective A2A receptor agonists
administered close to the BF and VLPO induced NREM sleep (Methippara et al.,
2005; Satoh et al., 1998, 1999).

!!!"9"5 L.&'.),(H.3+,
Nitric oxide (NO) is a gaseous signaling molecule that has multiple roles the in central
nervous system (Calabrese et al., 2007) and here I will only discuss its role in sleep
homeostasis. In the brain, NO is synthesized under basal conditions by the neuronal
NO synthase (nNOS) and endothelial NO synthase (eNOS) (Calabrese et al., 2007).
Both NOS activity and concentration are higher during wake than during sleep in the
rat brain (Ayers et al., 1996), and NO concentration is highest during wake (Burlet
and Cespuglio, 1997). Systemic or local administrations of NOS inhibitors
consistently reduce sleep in the light period in rats (Cavas and Navarro, 2006; Kapas
et al., 1994; Monti et al., 1999; Monti and Jantos, 2004; Ribeiro and Kapas, 2005),
indicating NO is sleep promoting.
!'.!

During sleep deprivation, the increase of NO concentration in the BF is mediated by

the inducible NO-synthase (iNOS) in BF neurons (Kalinchuk et al., 2006b, 2010). The
iNOS is normally not present in the brain but is synthesized under pathological
conditions mainly by glia (Calabrese et al., 2007). Infusion of a selective iNOS
inhibitor in the rat BF during sleep deprivation prevented rebound in NREM sleep,
whereas a nNOS inhibitor decreased REM recovery but did not affect NREM
recovery (Kalinchuk et al., 2006b), suggesting iNOS and nNOS play differential roles
in sleep homeostasis.
One of the mechanisms by which NO regulates sleep homeostasis might be its
contribution to adenosine release. Indeed, in vitro studies have shown that NO donors
cause release of adenosine (Broome et al., 1994; Fallahi et al., 1996; Rosenberg et al.,
2000). In vivo, the increase of NO in the BF preceded that of adenosine during sleep
deprivation (Kalinchuk et al., 2011), and an NO donor infused in the BF induced an
increase in adenosine (Kalinchuk et al., 2006a). In addition, the BF is a crucial site
mediating the effects of NO and adenosinespecific lesion of the BF cholinergic
cells abolished the increases in adenosine and NO levels during sleep deprivation and
reduced recovery sleep (Kalinchuk et al., 2008), while infusion of an NO donor
mimicked the increase in NREM sleep after sleep deprivation (Kalinchuk et al.,
2006a).

!!!"9"9 B*&(Q.1+%,213,-'(%&208213.1,P5,
In addition to their well-known role in immune response, cytokines, especially
interleukin-1# (IL-1#) and tumor necrosis factor-" (TNF-"), are also involved in sleep
regulation in health and disease (Moldofsky et al., 1986). Many of the effects induced
by sleep loss, including sleepiness, fatigue, impaired cognition and increased
sensitivity to pain, can be mimicked by injection of IL-1# or TNF-" (Krueger, 2008).
The expressions of IL-1# and TNF-" increase with increasing sleep propensity in the
rat hypothalamus, hippocampus and cortex (Bredow et al., 1997; Floyd and Krueger,
1997; Taishi et al., 1998). In humans, plasma levels of IL-1# are highest at sleep onset
(Moldofsky et al., 1986). ATP released from neurons and glia acts on purine type 2
receptors to induce cytokine release from glia (Krueger, 2008). Cytokines activate
transcription factors such as nuclear factor kappa B, which in turn enhance expression
of the enzymes involved in the synthesis of other sleep factors including adenosine,
prostaglandins, and NO (Krueger, 2008).
Prostaglandin D2 (PGD2) also fulfills the criteria of sleep factors. As mentioned
above, PDG2 may act downstream of cytokines as the expression of cyclooxygenase,
the key enzyme in PGD2 synthesis, is induced by cytokines (Terao et al., 1998). The
sleep-promoting effects of PGD2 have been shown to be mediated by A2A receptors in
the VLPO. PGD2 acts on prostaglandin 1 receptors in the leptomeninges under the
basal forebrain and the hypothalamus to induce release of adenosine, which in turn
!

'"!

activates activates adenosine A2A receptor-expressing sleep-promoting VLPO neurons

(Urade and Hayaishi, 2011)

!!!"; FA1)&.(1%,(4,%8++-,
Sleep is a universal behavior conserved in distantly related animals, from some
invertebrates to humans. Considering that sleep is an inactive state that disenables
feeding, reproduction, and renders the animal vulnerable to predators, sleep should
have given animals significant adaptive advantages outweighing these apparent
detriments to be selected during evolution. Among the numerous putative functions of
sleep, I will discuss two that have found more experimental grounds: memory
consolidation and restoration.

!!!";"# D8++-,-'(:(&+%,:+:('*,)(1%(8.32&.(1,
A great number of behavioral studies have demonstrated that sleep supports primarily
the consolidation of all major types of memory, including declarative and
non-declarative memories. Indeed, compared with an equal length of waking interval,
post-learning sleep improves the performance in retests of the learning tasks
(Robertson et al., 2004; Smith, 2001; Marshall and Born, 2007). In humans, SWS
benefits the consolidation of hippocampus-dependent declarative memories (Plihal
and
Born,
1999;
Smith,
2001),
whereas
REM
sleep
benefits
hippocampus-independent, non-declarative memory, including procedural memory,
perceptual memory and emotional aspects of memory (Plihal and Born, 1999; Smith,
2001; Wagner et al., 2001). For detailed accounts of behavioral studies, please refer to
the following reviews of Diekelmann and Born (2010) and Rasch and Born (2013).
Here, I will focus on the mechanisms of memory consolidation by sleep.

2=

D8++-S2%%().2&+3,(%).882&.(1%,213,:+:('*,

SWS and REM sleep are characterized by specific neuronal oscillations, many of
which have been demonstrated to be the mechanisms conveying the beneficial effect
of sleep on memory consolidation at system and network levels, as discussed below.

D8(I, I2K+, 2)&.K.&*V, %-.138+%, 213, %72'-, I2K+S'.--8+%, (4, L>G6,

%8++-,
SWA, including slow oscillations, represents a central mechanism underlying the
effect of SWS on consolidation of the declarative memory. In animals, sensory
stimulation consistently increases SWA in the cortical areas involved in perception of
the stimulation during subsequent SWS (Kattler et al., 1994; Vyazovskiy et al., 2000;
Yasuda et al., 2005). In humans, the amplitude and slope of slow oscillations were
increased locally by learning experiences preceding SWS (Huber et al., 2004; Mlle
et al., 2004; Mlle et al., 2009) and decreased when learning was hindered by
immobilization of the arm used in a behavioral task (Huber et al., 2006). These
!'#!

studies indicate that SWA is homeostically regulated by learning and memory during
the pre-sleep wake period. Moreover, there are studies establishing a causal role of
SWA in memory consolidation. Suppression of SWA by acoustic stimuli without
waking up the subjects prevented the improvement by post-training sleep in
visuomotor learning (Landsness et al., 2009, 2011) as well as in visual texture
discrimination (Aeschbach et al., 2008). Transcranial direct current stimulation (tDCS)
that specifically enhances endogenous slow oscillations and spindle activity during
early SWS significantly improved the retention of declarative memories in humans
(Marshall et al., 2006). In contrast, tDCS at the theta frequency (5 Hz) that suppresses
endogenous slow oscillation and spindle activity impaired overnight retention of the
declarative memories (Marshall et al., 2011).
Spindle activity is a 10 to 15 Hz EEG oscillatory activity that is typical of human
NREM sleep stage 2 but also present during SWS (De Gennaro and Ferrara, 2003). It
originates from the thalamus and lasts for 0.5-3 s (De Gennaro and Ferrara, 2003).
Similar to SWA, many studies in both humans and animals have shown that spindle
activity levels are consistently correlated with the extent of improvement of
declarative memories by sleep (Berner et al., 2006; Clemens et al., 2005, 2006; Cox et
al., 2012; Genzel et al., 2009; Holz et al., 2012; Milner et al., 2006; Ruch et al., 2012;
Schabus et al., 2004; Schmidt et al., 2006). However, a causal role of spindles in
memory consolidation has not been demonstrated due to the lack of specific
manipulation method.
Hippocampal sharp waves are brief (50-100 ms), large deflections of the EEG signal
generated in CA3; they are superimposed by ripples, the high-frequency EEG
oscillations of 100-300 Hz originating in CA1, to form the sharp wave-ripple complex
(SWR) (Figure 17; Chrobak and Buzsaki, 1996; Buzsaki, 1986). SWRs occur mainly
during SWS but also during quiescent wakefulness. In rats, learning of association
tasks led to increased SWR activity during subsequent SWS (Eschenko et al., 2008;
Ramadan et al., 2009) that is correlated with the post-sleep improvement in the task
performance (Ramadan et al., 2009). In rats, two studies have shown that selective
disruption of ripple events by electrical stimulation during post-learning sleep
impaired consolidation of spatial memories, demonstrating a causal role of SWRs in
memory consolidation (Girardeau et al., 2009; Ego-Stengel and Wilson, 2010).
It has been shown that hippocampal SWRs, thalamic spindles, and cortical slow
oscillations are temporally aligned, which could favor hippocampal-neocortical
dialogue (ONeill et al., 2010). Generation of spindles and SWRs is suppressed during
the down state of slow oscillations, and they are concentrated during up states
(Clemens et al., 2007; Mlle et al., 2006; Sirota et al., 2003). In humans, spindle
activity enhanced by intense vocabulary learning concentrated on the up states of slow
oscillations and such enhancement could be an important mechanism by which
spindles contribute to consolidation of newly acquired memories during sleep (Mlle
et al., 2009, 2011). SWRs are also temporally coupled to spindles: ripples are tightly
!

'$!

phase-locked to the troughs of spindles (Clemens et al., 2011; Siapas and Wilson,
1998; Wierzynski et al., 2009). In addition, a strong increase in spindle activity was
associated with the occurrence of ripples (Mlle et al., 2006, 2009; Clemens et al.,
2007, 2011) and this increase was prolonged during SWS after learning (Mlle et al.,
2009). As these spindle-ripple events reach the neocortical networks during the up
states of slow oscillations, it may serve to transfer hippocampal memory traces
towards neocortical long-term stores, which is considered as a mechanism underlying
sleep-dependent memory consolidation in the active system consolidation model (See
below).

C(1&(S0+1.)A8(S()).-.&28,213,&7+&2,I2K+%,(4,>G6,D8++-,
PGO waves have been shown to increase robustly after training on an active
avoidance task, and changes in PGO waves were correlated with the improvement in
task performance in post-sleep retest (Datta, 2000; Datta et al., 2008; Ulloor and Datta,
2005). Moreover, PGO waves were necessary for training-induced increased
expression of plasticity-related genes and brain derived neurotrophic factor (BDNF)
in the hippocampus since these increases were abolished by selective suppression of
PGO waves (Datta et al., 2008; Ulloor and Datta, 2005).
So far, there is a lack of evidence for any essential function of theta activity during
REM sleep in memory consolidation (Rasch and Born, 2013). However, the
characteristics of REM sleep theta activity and faster EEG oscillations are distinct
from their counterparts during wakefulness or SWS: EEG activity during REM sleep
shows reduced coherence in theta and gamma frequencies between hippocampus and
neocortex, while there is a concurrent increase in theta and gamma coherence and
levels within local hippocampal and neocortical circuits during REM sleep
(Axmacher et al., 2008; Cantero et al., 2003). Thus, there might be diminished
information flow between the hippocampus and neocortex and enhanced local
information processing during REM sleep (Diekelmann and Born, 2010).

<=

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There are currently two major hypotheses for the mechanisms underlying the
consolidation of memory during sleep. The active system consolidation hypothesis
proposes that consolidation results from selective reactivation of memories during
sleep (Diekelmann and Born, 2010; Marshall and Born, 2007). The synaptic
homeostasis hypothesis holds that memory consolidation results from a global
synaptic downscaling during sleep (Tononi and Cirelli, 2006). In the following
sections, I will discuss these two hypotheses in detail in the attempt to provide an
overview of current status of the field and to identify what remains to be elucidated.

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The hypothesis that sleep supports the formation of long-term memory in an active
system consolidation process has been elaborated in several reviews (Born et al., 2006;
Diekelmann and Born 2010; Payne and Kensinger 2011; Lewis and Durrant 2011;
Rasch and Born, 2013). This hypothesis assumes that the coding of new memories
occurs in parallel in the neocortex and hippocampus during wake and that newly
encoded memories are consolidated during subsequent sleep through repeated
reactivation of the neural ensembles previously involved in encoding. Reactivation
occurs during SWS, accompanied by temporally coordinated slow oscillations,
spindles and SWRs, resulting in the redistribution of the temporarily stored memories
in hippocampus to long-term storage sites, i.e., the neocortex (system consolidation).
This process involves reorganization and thus plastic changes of neocortical networks,
which needs to be stabilized by consolidation at the synaptic level. Since the
neurophysiological and neurochemical milieu is not favorable for synaptic
potentiation during SWS, synaptic consolidation might occur during subsequent REM
sleep.

GK.3+1)+,4(',&7+,2)&.K+,%*%&+:,)(1%(8.32&.(1,7*-(&7+%.%,
The majority reactivation studies have focused on rat hippocampal place cells, which
code for the position of the animal in space (Burgess et al., 1998; O'Keefe et al.,
1979). Wilson and McNaughton first demonstrated that place cells firing together
while the rat was performing a spatial behavioral task exhibited an increased tendency
to fire together during subsequent SWS compared to pre-training sleep episodes
(Wilson and McNaughton, 1994). A subsequent study found that the reactivated cells
followed the same temporal sequence of spiking during the pre-sleep behavioral task
(Skaggs and McNaughton, 1996). Numerous studies have now demonstrated several
common features of reactivations of hippocampal place cells during SWS: 1) firing
patterns of reactivated cells are replayed at a much faster rate (10-20 times); 2) replay
activity decays after the first 20-40 min of sleep; 3) reactivations occur mostly during
SWRs (ONeil et al., 2010; Rasch and Born, 2013).
Reactivations have been observed to occur concomitantly with hippocampal neurons
after various learning tasks in non-hippocampal brain areas, including the parietal
cortex (Qin et al., 1997), medial frontal cortex (Peyrache et al., 2009), visual cortex
(Ji and Wilson, 2007) and striatum (Lansink et al., 2008, 2009; Pennartz et al., 2004).
In addition, reactivations in the neocortical areas (Ji and Wilson, 2007; Peyrache et al.,
2009) and the striatum (Lansink et al., 2008, 2009) were slightly preceded by
hippocampal reactivation. These findings indicate a leading role of hippocampal
memory replays for those occurring in neocortical and striatal areas. These
observations corroborate the view that newly acquired memories are redistributed
from the hippocampus, the supposedly temporary store, to neocortical and striatal
areas as the long-term stores, and the sequential reactivations might mediate the
information transfer from the hippocampus to the neocortex and the striatum.
!

'&!

Signs of memory reactivation during sleep in humans have been also observed by
imaging of brain activation with positron emission tomography or functional magnetic
resonance imaging in the area V1 in the visual cortex (Yotsumoto et al., 2009) and
hippocampal and parahippocampal areas (Peigneux et al., 2004), with the magnitude
of reactivations predicting performance at a later test.
So far, a few studies using the cueing procedures have provided the first set of direct
evidence that reactivations during sleep have functional significance (Rasch et al.,
2007; Rudoy et al., 2009; van Dongen et al., 2012). In these studies, contextual cues
(e.g., odors, sounds) presented in association with the learning materials were
redelivered during subsequent SWS. The cues did not disturb sleep of the subjects but
increased reactivation in brain areas involved in previous learning tasks. In one study,
subjects cued during SWS showed more improvement in performance than those cued
during waking or REM sleep (Rasch et al., 2007). In another study, different cues
were associated with different tasks and the subjects showed improvement only in the
task whose specific cue was redelivered during SWS (Rudoy et al., 2009). In
summary, the fact that experimentally induced reactivation by cueing enhances
retention of memories supports a causal role of reactivations in memory
consolidation.

D*12-&.),7(:+(%&2%.%,7*-(&7+%.%,
The synaptic homeostasis hypothesis proposed by Tononi and Cirelli (2003, 2006,
2013) assumes that learning during waking leads to widespread increase in synaptic
strength, which is renormalized by a global downscaling process during sleep.
Increasing synaptic strength would eventually reduce the selectivity of neuronal
responses, saturate the ability to learn and cause cellular stress. By renormalizing
synaptic strength, sleep is necessary to reduce the burden of increased synaptic
strength. Overall synaptic downscaling during sleep eliminates weakly potentiated
synapses, leading to enhancement of the signal-to-noise ratio, and as a result benefits
memory consolidation. Importantly, according to this hypothesis, slow oscillations
during SWS are the major contributor to synaptic downscaling.

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The synaptic homeostasis hypothesis has successfully integrated a large body of
experimental findings. Microarray studies have provided some indirect evidence
supporting the idea that wakefulness is associated with widespread synaptic strength
increase: they revealed transcripts of several genes pooled from large brain regions as
putative markers of synaptic potentiation, including several immediate early genes
and BDNF, were more expressed during wake than sleep (Cirelli et al., 2004;
Mackiewicz et al., 2007; Maret et al., 2007; Mongrain et al., 2010; Thompson et al.,
2010).

!''!

The insertion and removal of GluR1-containing AMPA receptors (AMPARs) in the

synaptic membrane are believed to lead to synaptic potentiation and depression,
respectively (Kessels and Malinow, 2009). Levels of GluR1-containing AMPARs
were about 40% higher after wakefulness than after sleep in the rat cerebral cortex
and hippocampus, and phosphorylation changes of GluR1 (Hinard et al., 2012;
Vyazovskiy et al., 2008), Ca2+/calmodulin-dependent protein kinase II and glycogen
synthase kinase 3 beta (Vyazovskiy et al., 2008) also indicated a net synaptic
potentiation during wake and depression during sleep. Moreover, similar sleep-wake
changes in AMPARs trafficking have been found in other studies showing the
insertion of GluA1-containing AMPAR in waking (Qin et al., 2005) and removal in
sleep (Lant et al., 2011).
Direct evidence supporting the synaptic homeostasis hypothesis has come from
electrophysiological experiments. Synaptic strength in vivo can be measured by the
slope of monosynaptic responses evoked by electrical stimulation, with the steeper
slope indicating the higher synaptic strength. In the rat frontal cortex (Vyazovskiy et
al., 2008) and the hippocampus (Lubenov and Siapas, 2008), the slope of the
monosynaptic response increased with time spent awake and decreased with time
spent asleep. Similar results have also been observed in humans: the response slope in
the frontal cortex increased progressively over 18 hours of continuous waking and
returned to baseline levels after sleep recovery (Huber et al., 2013). In acute slices of
the mouse prefrontal cerebral cortex (Liu et al., 2010), and at the excitatory synapses
on orexin hypothalamic neurons (Rao et al., 2007), the frequency and amplitude of
miniature excitatory postsynaptic currents were higher after sleep deprivation,
suggesting synaptic efficacy increases. In line with these findings, it was reported that
the firing rate of cortical neurons increased during prolonged waking and decreases
progressively during sleep (Vyazovskiy et al., 2009), also suggesting that synaptic
strength is enhanced by waking.
Spine turnover in adolescent animals has also been shown to be modulated by wake
and sleep. In one month-old and younger mice, both formation and elimination of
cortical spines occurred all day, while the summation is a net increase in spine density
during wake and a net decrease during sleep (Maret et al., 2011; Yang and Gan, 2012).
These studies have provided the structural evidence supporting the synaptic
homeostasis hypothesis.

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Although these two hypotheses are apparently supported by numerous experimental
observations, there are caveats for interpreting and drawing conclusions from the data.
For the active system consolidation hypothesis, concerns are raised over possible
overestimation of the importance of sleep-dependent neural reactivation for memory
consolidation (Tononi and Cirelli, 2013). On one hand, reactivation of spiking
patterns of place cells previously engaged in spatial tasks also occur in quiet
!

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wakefulness (Davidson et al., 2009; Diba and Buzsaki, 2007; Foster and Wilson, 2006;
Karlsson and Frank, 2009; Kudrimoti et al., 1999; Singer et al., 2013), suggesting that
wake-associated reactivations play a role in consolidation of the learned experiences.
Thus, it is necessary to identify the importance of reactivation in sleep distinct from
those during wake. On the other hand, reactivations during sleep are rare events,
making up often less than 10 percent of the total spontaneous activity of the
reactivated neurons during SWA and decline rapidly after the first hour of sleep (Ji
and Wilson, 2007; Kudrimoti et al., 1999; Nadasdy et al., 1999). What makes up the
rest of the spontaneous activity during SWS and why the reactivation events are rare
remain unresolved issues for the active consolidation hypothesis.
Despite the large body of evidence discussed above supports a net downscaling of
synaptic strength during sleep, as proposed by the synaptic homeostasis theory, these
findings are obtained in relation to total sleep, not specifically to SWS. Also, as
experimental evidence are lacking to state that synaptic downscaling solely explains
the memory consolidating benefits of SWS, it still remains unclear whether synaptic
potentiation occurs during SWS. Such a possibility has largely been neglected due to
the following experimental findings: electrical stimuli delivered in vivo can easily
induce long-term potentiation (LTP) in the hippocampus during wake and REM sleep
but not during SWS (Leonard et al., 1987; Bramham and Srebro, 1989); stimulation at
<1 Hz, the oscillation frequency range of the slow oscillations, is a standard protocol
to induce long-term depression (LTD) in vitro (Kemp and Bashir, 2001; Massey and
Bashir, 2007). Other studies using stimulation protocols that supposedly mimic
neuronal activity during SWS have produced mixed results. Burst firing, a
characteristic pyramidal neuron spiking pattern during up states in SWS, paired with
excitatory postsynaptic potentials was shown to lead to synaptic depression in vitro
(Czarnecki et al., 2007; Lante et al., 2011). Such stimulation, however, did not
consistently induced LTD or even induced LTP in vivo (Perrett et al., 2001; Hager
and Dringenberg, 2010). Stimulation near 10 Hz within the spindle range can result in
either LTP or LTD, depending on the intensity of the stimulation and the pattern of
cortical activity (Rosanova and Ulrich, 2005; Werk and Chapman, 2003; Werk et al.,
2006).
A recent work from Chauvette and colleagues provided the first direct evidence that
synaptic potentiation indeed occurs during SWS (Chauvette et al., 2012). In this study,
the authors showed that somatosensory evoked potentials by prethalamic electrical
stimulation in non-anesthetized cats were enhanced after the first SWS episode
compared to the previous wake episode. They further showed that in vitro SWS-like
stimulation patterns induced LTP in somatosensory pyramidal neurons only when the
stimulation pattern included an intracellular hyperpolarizing phase mimicking down
states of slow oscillations (Chauvette et al., 2012). This work implicates that the
discrepancies among the studies presented above could be largely a result of different
stimulation protocols. Therefore, it is important to use a protocol that mimics the
physiological reality of neuronal activities during SWS. In summary, conclusions
!',!

drawn from studies attempting to induce LTP or LTD should be viewed with more
scrutiny with respect to the experimental protocols, and more experimental evidence
is needed to improve our knowledge on how SWS affects synaptic strength.
Another big open issue concerns the role of REM sleep in synaptic scaling. As
proposed by the active system hypothesis, synaptic potentiation might take place
during REM sleep because SWS is largely viewed unfavorable to synaptic
potentiation (although this view is still under debate as just discussed above). Theta
oscillations characteristic of rodent REM sleep can modulate directionally
hippocampal plasticity: burst stimulation can induce LTP or LTD depending on
whether it occurs at the peak or the trough, respectively, of the theta waves (Holscher
et al., 1997; Pavlides et al., 1988). A recent study found increases in firing rates of
hippocampal CA1 neurons during SWS were outweighed by decreases during REM
sleep, leading to a net decrease in fire rates across sleep (Grosmark et al., 2012).
Compared to cortical neurons whose spiking rate decreased as a function of SWA
(Vyazovskiy et al., 2009), spiking rates of hippocampal neurons decreased as a
function of REM theta power (Grosmark et al., 2012). The work of Grosmark et al.
suggests that synaptic downscaling is brought about by theta activity during REM
sleep in the hippocampus. Considering the fact that hippocampal cells lack the slow
oscillation, it is intriguing to speculate that the hippocampus uses a different
mechanism to restore synaptic homeostasis.

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A long-held theory of sleep functions is that sleep restores the physiological and
neurological states that are compromised during wakefulness (Hartman, 1973; Oswald,
1970). There is now substantial evidence that sleep serves a restorative role.
Microarray studies have shown that prolonged wakefulness leads to increased
expression of genes encoding heat shock proteins and molecular chaperones in rodent
brains (Cirelli et al., 2004; Naidoo et al., 2005; Terao et al., 2003), indicating presence
of endoplasmic reticulum (ER) stress and activation of the unfolded protein response
(UPR). The UPR aims to restore normal ER function by reducing protein translation
and upregulating the expression of chaperones to assist in folding of protein and
degrading misfolded proteins (Schroder and Kaufman, 2005). The increase of stress
gene expression, however, is more limited during recovery sleep compared to sleep
deprivation (Terao et al., 2003), suggesting that extended wakefulness leads to ER
stress, which can be relieved by sleep.
In contrast with wakefulness, the expression of very different categories of genes are
upregulated during sleep (Cirelli et al., 2004; Terao et al., 2006; Mackiewicz et al.,
2007). These genes include those involved in: 1) biosynthetic pathways of heme,
cholesterol and lipids; 2) protein synthesis including translation regulation activity
and ribosome biogenesis; 3) intracellular transport of macromolecules; 4)
!

'-!

maintenance of synaptic vesicle pools; 5) functioning of antioxidant enzymes. These

observations suggest that a function of sleep is to replenish key components of
macromolecules synthesis, such as proteins and lipids used up and damaged by high
neural activity during wake. In addition, a recent study showed that there was a
striking increase in exchange of the cerebrospinal fluid with the interstitial fluid
during sleep, which in turn increased the rate of b-amyloid clearance from the brain
(Xie et al., 2013). This finding provided direct evidence that sleep serves a restorative
function partly as a result of enhanced removal of metabolic waste that accumulates in
the waking brain.

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Figure 22. Slow oscillations are reduced dnSNARE mice. (A) In vivo current-clamp
recordings from neurons showing slow oscillations in the cortex of anesthetized WT and
dnSNARE mice (B) Normalized power spectra from WT and dnSNARE animals. Adapted
from Fellin et al., 2009.

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"4# $%&'()*&+%!/05!%-++3!
As discussed in previous sections, astrocytes are indispensable participants in many
functions in healthy and diseased brains. Thus, it is not surprising that astrocytes are
found to be involved in sleep regulation. Early evidence came from the work of
Hyden and Lange, who found that the activity of succinooxidase (a key enzyme in the
Krebs cycle) in part of the reticular formation is high in neurons and low in glia
during sleep and the situation is reversed during wake (Hyden and Lange, 1965). In
recent years, important advances have been made in identifying a causal link between
astrocytes and sleep-wake cycle regulations. In this section, I will provide evidence
and discuss in detail how astrocytes and, in particular, astroglial connexins (Cxs)
could be involved in sleep regulation based on latest advances in sleep-wake
physiology research.

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7(:+(%&2%.%,
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As one of the most studied sleep factors, adenosine plays a key role in sleep-related
functions of astrocytes. Direct evidence demonstrating that ATP released from
astrocytes is a major source of extracellular adenosine in the brain comes from the
work of Haydon and colleagues. They generated an inducible transgenic mouse line
with astrocyte-specific expression of a dominant-negative domain of the soluble
N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), called
dnSNARE mice (Pascual et al., 2005). These mice have disrupted vesicular release of
gliotransmitters, and the extracellular ATP and adenosine concentrations in brain
slices of these mice are significantly reduced (Pascual et al., 2005; Schmitt et al.,
2012). In addition, astrocytes may regulate extracellular adenosine levels by an
adenosine cycle (Boison, 2008), which involves release of ATP, uptake of
extracellular adenosine, and conversion of intracellular adenosine to AMP by
adenosine kinase (AK) that are almost exclusively expressed in astrocytes in the adult
mouse brain (Studer et al., 2006).
As a result of impaired vesicular release, dnSNARE mice have reduced excellular
adenosine and thus a loss of tonic inhibition via the A1 receptor (Pascual et al., 2005;
Halassa et al., 2009). In addition, there is a hypofunction of postsynaptic NMDA
receptors due to decreased D-serine released from astrocytes and a reduction in
surface expression of the NR2 subunit of the NMDA receptor (Fellin et al., 2009).
Consequently, dnSNARE mice exhibit reduced cortical slow oscillations associated
with reduced duration of up states and prolonged down states in cortical neurons
(Fellin et al., 2009; Figure 22).

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Furthermore, EEG/EMG recordings revealed that the dnSNARE mice have reduced
slow wave activity (SWA) during basal NREM sleep and substantially attenuated
increase in recovery sleep duration and SWA rebound after sleep deprivation (SD)
(Halassa et al., 2009), indicating these mice have attenuated sleep pressure. The
observation that intracerebroventricular infusion of an A1 receptor antagonist
produced similar phenotype in wild type mice suggests that astrocyte-derived
adenosine acts on neuronal A1 receptors to modulate sleep pressure (Halassa et al.,
2009). In consistence with this view, both conditional knockout (KO) of A1 receptors
(Bjorness et al., 2009) and overexpression of AK that reduces extracellular adenosine
(Palchykova et al., 2010) cause similar sleep phenotypes to those of the dnSNARE
mice. In conclusion, astroglial release of ATP contributes to extracellular
accumulation of adenosine, which in turn modulate sleep pressure and sleep
homeostasis (Halassa and Haydon, 2010; Fellin et al., 2012).

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Besides ATP and D-serine, astrocytes release a variety of molecules including BNDF,
cytokines IL-1# and TNF-", PDG2 and NO (Volterra and Bezzi, 2002) that are known
to function as sleep factors (see Introduction III.3). However, whether astrocytes
actually release these molecules under physiological conditions to affect sleep in vivo
is not clear. Alternatively, astrocytes might influence sleep homeostasis indirectly via
affecting synaptic strength. For example, in cortical slices, astrocytes were shown to
release BDNF, which is involved in synaptic plasticity (Gomez-Palacio-Schjetnan and
Escobar, 2013), in an activity-dependent way (Bergami et al., 2008). Whisker
stimulation in awake rodents increased IL-1# immunoreactivity in astrocytes (Hallett
et al., 2010). Moreover, TNF-" derived from astrocytes in culture (Beattie et al., 2002;
Stellwagen and Malenka, 2006) and slices (Becker et al., 2013) is shown to be
involved in homeostatic synaptic plasticity. As the synaptic homeostasis hypothesis
proposes, a function of the slow wave sleep (SWS) is to downscale synaptic strength
increased during wakefulness and the homeostatic response of SWS reflects the extent
of synaptic strength up-scaling (Tononi and Cirelli, 2006; see Introduction III.4.1).
It is possible that these aforementioned molecules released from astrocytes might
indirectly modulate sleep homeostasis through modulating synaptic strength during
wakefulness.

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!U"5"# D8++-,213,:+&2<(8.%:,
Although largely remaining unclear, one of the proposed functions of sleep is to
restore energy spent during wakefulness (Frank, 2006). Indeed, overall metabolic rate
of the cerebral cortex is reduced during NREM sleep (Maquet, 1995), and the
extracellular concentration of glucose, which is the prime energy source of the brain,
increases during NREM sleep and decreases during wake and REM sleep
(Netchiporouk et al., 2001; Dash et al., 2013). Moreover, impairment of sleep is
!(%!

associated with higher risks of pathological conditions of metabolism, such as obesity,

diabetes, and hypertension (Porkka-Heiskanen et al., 2003; Spiegel et al., 2009).
Apart from peripheral conditions, functional imaging also revealed a variety of brain
areas that undergo abnormal metabolic states in patients of major sleep disorders
including insomnia and catalepsy (Desseilles, 2008).

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Considering the pivotal role of astrocytes in brain metabolism (see Introduction
I.2.5), it is tantalizing to hypothesize that astrocytes may be involved in sleep
physiology via their metabolic functions. Among the energy metabolites, there are
converging lines of evidence implying that lactate released by astrocytes might play a
role in sleep-wake regulation. Lactate is a suggested biomarker of sleep pressure:
cortical lactate concentration rises upon waking, maintains elevated during continuous
wakefulness, and declines persistently during NREM sleep (Naylor et al., 2012; Dash
et al., 2012). Also, cerebral SWA decreases the rate of glycolysis that produces lactate
(Wisor et al., 2013). Moreover, mRNA expression levels of several genes involved in
the astrocyte neuron lactate shuttle (Pellerin and Magistretti 1994) including Glut1,
"-2-Na+/K+ pump, Glt1, and Ldha are significantly increased following sleep
deprivation in astrocytes (Petit et al., 2013). Intriguingly, there is evidence that lactate
increases excitability of the wakefulness-promoting orexin neurons. In brain slices of
the hypothalamus, orexin neurons have been demonstrated to behave as essentially
lactate sensors: they specifically utilize lactate released by astrocytes; their
spontaneous firing rate is correlated with lactate concentration; lactate increases their
sensitivity to excitatory inputs (Parsons and Hirasawa, 2010). In addition to orexin
neurons, a recent study found that lactate released from astrocytes similarly excites
locus coeruleus (LC) neurons and triggers release of norepinephrine in a
dosage-dependent manner (Tang et al., 2014). Moreover, in vivo injections of
L-lactate in the LC evoke arousal, which is consistent with the role of norepinephrine
in promoting wakefulness (Tang et al., 2014). Intriguingly, the authors also showed
that these effects of lactate are independent of uptake into neurons and involve a
cAMP-mediated step. This implies that lactate might act like a gliotransmitter to
stimulate LC neurons rather than provide metabolic support to sustain LC neuronal
activity (Tang et al., 2014). These findings about orexin and LC neurons suggest that
astroglial lactate may play an important role in the regulation of wakefulness, which
can be tested by in vivo administration of lactate and simultaneous monitor of
sleep-wake states of the animal.

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Astrocytes through buffering ions in the extracellular space might participate in
shaping slow oscillations during sleep. Compelling evidence comes from studies with
simultaneous intracellular recording in neurons and astrocytes and the measurement
!

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Figure 23. Simultaneous recording of the field potential, a glia, and extracellular K
concentration ([K+]out) in the cortex of an anesthetized cat.!The slow oscillation in the field
potential is in-phase with variations in the [K+]out and intraglial potentials. Adapted from
Amzica et al., 2002.

!('!

of [K+]o and [Ca2+]o in the cat cortex (Amzica and Neckelmann, 1999; Amzica and
Massimini, 2002; Amzica, 2002). The authors showed that: 1) astroglial membrane
potential and capacitance oscillate in phase with up and down state oscillations of
neurons; 2) [K+]o and [Ca2+]o also oscillate at the same frequency of slow oscillations;
3) [K+]o oscillates in the way that suggests K+ is cleared from active spots and
released in the near vicinity, and oscillations in [Ca2+]o modulate synaptic efficacy
(Figure 23). In addition, using a KO mouse model of the astroglial Kir4.1 channel,
another study demonstrated in vivo that astroglial Kir4.1 plays a role in the clearance
of extracellular K+ after stimulations in the hippocampus (Chever et al., 2010). These
data suggest that astrocytes actively modulate the slow oscillations by buffering of
extracellular cations and the necessity of the Kir4.1 channel for an efficient uptake of
K+ by astrocytes.

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Given the important role of astrocytes in the regulation of sleep homeostasis as
discussed above, it is logical to speculate that astroglial connexins (Cxs) may play a
role. In the last part of the Introduction, I provide evidence and hypothesize about
how astroglial Cx may be regulated by changes in sleep homeostasis and reciprocally
influence sleep and wakefulness.

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244+)&.10,:(8+)A8+%, ,
There are several lines of evidence indicating that astroglial connexin channels are
regulated by sleep-wake affecting substances. General anesthetics (Mantz et al., 1993)
and oleamide, a sleep-inducing lipid (Guan et al., 1997), have been reported to inhibit
gap junctions in primary cultures of astrocytes. As discussed previously, several
cytokines including TNF-" and IL-1# that are known to regulate sleep homeostasis
(see Introduction III.3) have been demonstrated to reduce gap junction (GJ)
communications (reviewed in Orellana et al., 2013) and increase hemichannel (HC)
activity (Bennett et al., 2012; Orellana et al., 2011a, b). In addition, noradrenaline, a
neurotransmitter that promotes wakefulness (see Introduction III.2), exerts a dual
regulation on dye coupling between cortical astrocytes depending on the subtypes of
adrenergic receptors (Giaume et al., 1991b). These observations strongly suggest that
astroglial Cxs may undergo functional changes during different sleep wake states.
However, most of these studies were carried out in settings unrelated to sleep and
wake physiology and pharmacological experiments do not fully capture of the full
characteristics of natural sleep and wakefulness. Thus further investigations are
needed to directly address the question whether astroglial Cxs are regulated by
changes in sleep homeostasis.

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As discussed above, astrocytes can regulate sleep and wake through several pathways:
1) gliotransmission signaling; 2) metabolic support (or maybe lactate transmission),
which waits to be validated; 3) ionic buffering. As indicated in previous parts of this
Introduction, astroglial Cxs are likely to participate in all of these three functions of
astrocytes. For gliotransmission, HCs represent an alternative release pathway in
addition to vesicular release. HCs have been shown to mediate ATP release,
especially in pathological conditions (Kang et al., 2008; Garr et al., 2010; Iglesias et
al., 2009; Orellana et al., 2011; Giaume et al., 2013). Their permeability to glucose
and lactate and their crucial role in metabolic trafficking to sustain neuronal activity
in hypoglycemic conditions (Rouach et al., 2008) imply that astroglial GJCs might
contribute to potentially metabolic processes that affect the sleep-wake cycle, e.g.,
lactate-mediated effects on orexin or norepinephrine transmissions. Finally, GJCs are
indispensable players in the ion buffering functions of astrocytes (see Introduction
I.2.1) and thus may potentially contribute to modulation of slow oscillations during
sleep. In summary, the evidence discussed so far has provided the basis of my thesis
project, which is to investigate whether and how astroglial Cxs are involved in the
regulation of sleep-wake cycle.

!(,!

Results

________________

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Part I.

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The traditional view that astrocytes are mere passive supportive cells that glue the
neural tissue together has been totally transformed over the last two decades.
Mounting research evidence has demonstrated that astrocytes closely interact with
neurons to participate in a wide variety of brain functions, one of which is sleep
regulation (Halassa and Haydon, 2010; Fellin et al., 2012). The neuroglial interaction
at the cellular level that contributes to sleep regulation involves astroglial release of
gliotransmitters that act on neurons to modulate synaptic transmission (Fellin et al.,
2009; Halassa et al., 2009). Whether and how neuroglial interaction at the network
level contributes to sleep regulation, however, is largely unknown. The feature of
astrocytes that they form highly interconnected networks through gap junction
channels (GJCs) provides the structural basis for the occurrence of network-level
neuroglial interactions. Moreover, the functional plasticity of GJC-mediated astroglial
networks, especially its activity-dependence (Rouach et al., 2008; Roux et al., 2011),
opens the possibility that they can adapt to changes in activity of neuronal ensembles
during different behavioral states of the animal, such as those taking place over the
sleep-wake cycle.
Therefore, the aim of the first part of my thesis work was to determine whether
GJC-mediated astroglial networks are regulated in accordance with changes in
sleep-wake states. To induce such changes, we took advantages of compounds that are
known to affect sleep and wake in humans and animals. We also wanted to know
whether compounds that have opposite effects on sleep and wakewould induce
accordingly opposite effects on astroglial networks. Finally, given the
activity-dependence of astroglial networking (Rouach et al., 2008; Roux et al., 2011),
we also probed the role of neuronal activity in the effects sleep-wake affecting
molecules on astroglial network.

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Figure S1. Effects of several sleep-wake affecting molecules on astroglial gap junction
channels (GJCs). (A) The numbers of coupled astrocytes in slices treated with 100$M
oleamide (50.604.925; n = 5) or 4$M melatonin (43.335.783; n = 3) are not significantly
different from that in the control slices (50.001.125; n = 6). (B) 20$M Ritalin slightly
decrease the number of coupled astrocytes (36.202.458; n = 5) compared to the control
condition (43.802.437; n = 4).

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<.%)9%%.(0! !
In this study, we demonstrated that modafinil and GHB, two drugs that have opposite
effects on sleep and wakefulness, induced increase and decrease in astroglial gap
junctioanl communication, respectively. This finding indicates that GJC-mediated
astroglial networks are regulated by pharmacological agents that alter sleep-wake
states. However, these observations do not justify the generalization that
wakefulness-promoting drugs increase while sleep-promoting ones decrease astroglial
gap junctioanl communication. Indeed, we tested other compounds that influence
sleep and wakefulness, but did not find significant effects for oleamide and melatonin,
which are known sedative agents, and a slight decrease in dye coupling for Ritalin,
which is a treatment for narcolepsy and wakefulness-promoting (Figure S1). This is
probably due to the divergent operating mechanisms that underlie the actions of these
different molecules. Also, the pharmacologically induced sleep or wakefulness may
not fully mimic the properties of the natural ones. Thus the final test would be to
monitor the status of astroglial networking in vivo over spontaneous sleep-wake cycle.
In this work, we did not address the respective roles of Cx43 and 30. Nevertheless, the
enhancement of Cx30 expression suggests that Cx30 is more likely to be responsible
for the modafinil-induced increase in GJC coupling. In addition, Cx30 has been
shown to underlie the activity-dependence of astroglial networking in the olfactory
bulb (Roux et al., 2011). Thus the hypothesis that Cx30 is the molecular target of
modafinil is consistent with the finding that modafinil increased dye coupling in an
activity-dependent manner. Interestingly, the lack of effects by modafinil on cultured
astrocytes also implicates the involvement of Cx30 due to the fact that cultured
astrocytes only express Cx43 (Koulakoff et al., 2008).
How modafinil promotes wakefulness is still under debate. Multiple
neurotransmission systems including the dopaminergic, histaminergic and
glutamatergic systems are implicated in the action of modafinil (reviewed in Ballon
and Feifel, 2006). Since the acute brain slices we used do not contain the projections
of these systems to the cortex in the intact brain, modafinl-induced increase in
astroglial dye coupling is likely a local effect within the cortex. On the cortical level,
modafinil has been demonstrated to increase Cx36-containing GJC coupling among
GABAergic interneurons in cortical slices (Urbano et al., 2007). This may lead to a
decrease in the activity of GABAergic neurons and lateral inhibition, and in turn
result in the observed increase in excitatory neuronal activity under modafinil
treatment (Urbano et al., 2007). Thus in our scenario, the enhancement of astroglial
GJC coupling in cortical slices is likely a secondary result of increased GJC coupling
in cortical interneurons. Nevertheless, the mechanism that acts in vivo is probably
more complex due to the aforementioned effects of modafinil on multiple
neurotransmission systems.
It is known that exogenous GHB in the milimolar range activates GABAB receptors in
!

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the central nervous system (Lingenhoehl et al., 1999). As we have shown that GHB
decreases astroglial GJC coupling probably through a direct action on astrocytes, the
binding site of GHB is likely to be the astroglial GABAB receptors. The effects of
GABA and GABAB receptor agonists on astroglial gap junctioanl communications
have been investigated in cultured astrocytes, but no significant changes were found
(Rouach et al., 2000). The lack of effects might result from altered properties of
astrocytes in culture. Therefore, it awaits experiments in acute brain slices to
determine whether GABA decreases in astroglial GJC coupling.

!-.!

Part II.

=+0+'/-!/0+%&7+&.)%!7/>+!5.::+'+0&./-!.07.?.&('*!+::+)&%!(0!&7+!
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As a part of my thesis work, we first aimed to demonstrate changes in gap junction
(GJ)-mediated astroglial networks are correlated with changes in sleep-wake states. To induce
such changes, pharmacological treatments of experimental animals with sleep-wake affecting
molecules represent one important approach as we have employed in the first part of my
thesis work (see Results Part I). In the second part of my thesis work, we extended this
pharmacological approach to taking advantage of the general anesthetics that produce a brain
and behavioral state similar to that during the natural NREM sleep (Franks and Zecharia,
2011). Thus we instigated the effects of three selected general anesthetics on astroglial GJ
communication in an attempt to complement our preceding finding that sleep-wake affecting
drugs affect astroglial networking (see Results Part I).
Moreover, we also carried out a characterization study of the effects the three general
anesthetics on astroglial hemichannels, a question that has not been addressed yet according
to our knowledge. Thus this study bears another important goal unrelated to sleep-wake
regulation: to investigate the impact of general anesthetics on the astroglial connexin-based
channel functions in order to advance our knowledge on the non-neuronal effects of general
anesthetics. Given their wide application and paramount importance in both clinical and
research settings, our study also aims to contribute to a better understanding of the working
mechanisms of general anesthetics.

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Publication (submitted),

________________

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General anesthetics have differential inhibitory effects

on the two connexin channel functions in astroglia

Xinhe Liu1,2,3 , Ester Gangoso 1,2,3 , Stanislas Kandelman4,

Jean Mantz4 and Christian Giaume1,2,3

Collge de France, Center for Interdisciplinary Research in Biology (CIRB)/Centre National

de la Recherche Scientifique, Unit Mixte de Recherche 7241/ Institut National de la Sant et
de la Recherche Mdicale U1050, 75231 Paris Cedex 05, France

University Pierre et Marie Curie, ED, N158, 75005 Paris, France

MEMOLIFE Laboratory of Excellence and Paris Science Lettre Research University, 75005
Paris, France

Dpartement dAnesthsie et de Ranimation Bichat Beaujon Louis Mourier, Assistance

Publique Hpitaux de Paris, Universit Paris Diderot, INSERM UMR 1141, 75877 Paris
Cedex 18, France.

These two authors have contributed equally to the work.

-$!

Abstract
Astrocytes are a major non-neuronal cell population in the central nervous system and
actively involved in several brain functions and pathologies. They express a large
amount gap junction proteins allowing direct communication between adjacent cells and
forming astroglial networks. There are now several reports demonstrating that the
alteration of gap junctional communication impacts neuronal and synaptic activity. In
addition, these membrane proteins can also work as hemichannels, through which
gliotransmitters can be released and thus contribute to neuroglial interaction. Two
decades ago we reported that several anesthetics inhibited gap junctional
communication in primary cultures of astrocytes (Mantz et al., 1993). As there are more
and more attempts to study in vivo neuroglial interactions in anesthetized mice, we here
update our early study by employing acute cortical slices and incorporating the
investigation of their effects on hemichannel activity. For this purpose we selected two
anesthetics that are extensively used: propofol and ketamine previously investigated in
cultured astrocytes, and introduced the recently developed dexmedetomidine. We report
that these drugs have differential inhibitory effects on gap junctional communication as
well as hemichannel activity, and that dexmedetomine appears to be the least effective
compound on both channel functions when used in the clinically relevant range. Such
observations may contribute to optimize selection of the anesthetics for in vivo animal
studies.

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Introduction

During the last decade, one of the new fields in neuroscience has emerged from increasing
evidence indicating that glial cells, and in particular astrocytes, are engaged in bi-directional
dynamic interactions with neurons (see Agulhon et al., 2008; Halassa and Haydon, 2010;
Zorec et al., 2012; Araque et al., 2014). This statement stands on the demonstration that
astrocytes can sense neuronal signals, including their spiking activity (Roux et al., 2011), and
that in turn they can modulate neuronal activity and synaptic plasticity (Panatier et al., 2011;
Di Castro et al., 2011). Indeed, these glial cells express a large spectrum of membrane
receptors (see Verkhratsky and Butt, 2013) and they can also integrate and propagate
information generated by neuronal activity via sophisticated calcium signaling mechanisms
(Scemes and Giaume, 2006; De Bock et al., 2014). Finally, through calcium-dependent
processes astrocytes release gliotransmitters (Zorec et al., 2012) that impact synaptic
transmission (Araque et al., 2014). Interestingly, these neuroglial interactions occur not only
at the single cell level at the so-called tri-partite synapse (Araque et al., 1999) but also at the
more integrated level involving intercellular networks (Halassa and Haydon, 2010; Giaume,
2010). Taken as a whole, such neuroglial interactions are proposed to play a role in brain
functions and dysfunctions as well as in pathologies (Giaume et al., 2007; Verkhratsky et al.,
2013). Consequently, it is important to know whether and how the properties of astrocytes are
affected when therapeutic interventions are designed to target neurons. This issue may be
particularly relevant for anesthetic drugs that suppress consciousness by mechanisms that are
incompletely understood. Indeed, up-to-now whether non-neuronal targets contribute to
clinically perceptible anesthetic effects has received little attention.
A typical feature of astrocytes compared to neurons is their higher expression levels of
connexins, the membrane protein component of gap junction channels that allow direct
communication between adjacent cells (see Ransom and Giaume, 2013). Interestingly,
anesthetics for a long time have been used to inhibit gap junctional communication
(Siracusano et al., 2006; Rozental et al., 2001), including in astrocytes (Mantz et al., 1993).
More recently, it has been demonstrated that connexins also work as functional hemichannels
in normal as well as in pathological situations (see Giaume et al., 2013). However, so far
nothing is known about the sensitivity of astroglial hemichannels to anesthetics.
It is now established that the inhibition of connexin channels in astrocytes by pharmacological
or genetic approaches (see Giaume and Theis, 2010) affects neuronal activity (Pannasch et al.,
2011; 2014; Torres et al., 2012). Thus, it is important to determine how different classes of
general anesthetics act on connexin-based channel functions in astrocytes. More than 20 years
ago, we conducted a study with 10 general anesthetics to determine which of them were
effective on gap junctional communication in primary cultures of astrocytes (Mantz et al.,
1993). However, there is a critical need to determine whether general anesthetics affect
connexin-based channels, including hemichannels, in a more physiological model such as
acute brain slices that include neurons and preserve synaptic connections. In the present work,
we have investigated the effects of 3 anesthetics with distinct mechanisms of action (propofol,
an agonist of the gamma-aminobutyric acid A subtype receptors (GABAARs), ketamine, an
antagonist of the glutamate receptors, and dexmedetomidine, an agonist of the
alpha2-adrenergic receptors) on astroglial gap junction channels and hemichannels in acute
cortical slices of the mouse. We report that these drugs have differential efficiencies and
!

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effects on gap junction channels and hemichannels. Such observations may contribute to
optimal selection of anesthetics for in vivo animal studies and should improve the
understanding of the mechanisms by which a particular anesthetic acts on neuronal activity
Materials and Methods
All experiments involving animals have been carried out in accordance with the European
Community Council Directives of November 24th 1986 (86/609/EEC) and all efforts have
been made to minimize the number of animals used as well as their suffering.
Drugs
Propofol (Sigma-Aldrich, Buchs, Switzerland) and dissolved in DMSO, which was diluted to
a final concentration no more than 0.1%. The same dilution of DMSO in ACSF was used as
the control solution for experiments. Ketamine (Panpharma, Boulogne-Billancourt, France)
was diluted to experimental concentrations in ASCF. Finally, dexmedetomidine (Abcam,
Paris, France) powder was prepared in aliquots with water and diluted to final concentrations
in ASCF.

Electrophysiology and dye coupling experiments

Mice (P20-P27) were decapitated, and their brains were dissected and placed in ice-cold
artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 2.5 KCl, 25 glucose, 25
NaHCO3, 1.25 NaH2PO4, 2 CaCl2, and 1 MgCl2, bubbled with 95% O2/5% CO2 (pH 7.4).
Coronal brain slices (300 $m) containing the somatosensory cortex were cut using a
vibratome (Microm HM 650 V, Walldorf, Germany) filled with ice-cold ACSF. The slices
were incubated at 33 C for 30 min and transferred at room temperature (20-22 C) before use.
Thereafter, the slices were placed in a recording chamber and perfused continuously with
oxygenated ACSF (pH 7.4) at a rate of 2 ml/min. The somatosensory cortex was observed
using an up-right fixed stage microscope (Axioskop FS; Zeiss, Oberkochen, Germany)
equipped with Nomarski optics and an infrared video camera (Newvicon C2400; Hamamatsu,
Shizouka, Japan). Cells were identified as astrocytes based first on morphological criteria and
second on electrophysiological properties (see Results section). The pipette (3-8 M!)
solution contained (in mM): 105 K-Gluconate, 30 KCl, 10 Hepes, 10 Phospho-Creatine Tris,
4 ATP-Mg2+, 0.3 GTP-Tris, and 0.3 EGTA (adjusted to pH 7.4 with KOH). Whole-cell
membrane voltages and currents were amplified by a MultiClamp 700B amplifier, sampled by
a DIGIDATA 1322A Interface, and patch-clamp recordings (5 kHz sampling and 3 kHz
filtering) were performed with pClamp 9 software (Axon Instruments, Foster City, CA).
Series resistances were compensated at 80%. Input resistance (Rin) was measured in
voltage-clamp mode by applying hyperpolarizing voltage pulses (10 mV, 150 msec) from a
holding potential of -80 mV. To assess the level of gap junction coupling, sulforhodamine B
(1 mg/ml; Molecular Probes, Eugene OR, USA) was added to the pipette solution before each
experiment. Recorded cells were loaded passively with the dye for 10 min in current-clamp
mode. The duration of the whole-cell recording was kept constant to allow comparison among
experiments. Intercellular diffusion of sulforhodamine B was captured thereafter with the
CCD camera (Pixelfly QE, The Cook Corporation, Romulus, Michigan, USA) on different
focal planes and the number of surforhodamine B positive cells (i.e. cells dye coupled to the
recorded astrocyte) was determined using Image J software.
Dye Uptake experiments
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In slices derived from Adlh-L1-eGFP mice, cortical astrocytes were identified by GFP
fluorescence. Living slices were incubated with the HC permeable fluorescent tracer ethidium
bromide (Etd, 314 Da) for 10 min at RT and at 4 mM final concentration. General anesthetics
were added in the chamber 10 min before slices. Then, the slices were transferred in the
chamber and treated for 90 min with lipopolysaccharide (LPS, 100 ng/ml) in order to trigger
hemichannel activity in astrocytes (Retamal et al., 2007) and in the absence (control condition)
or in the presence of anesthetics. Finally, following 10 min incubation with Etd (4 mM),
slices were rinsed 15 min in ACSF to stop the uptake and reduce background labeling before
submerging them for 40 min in fixing solution (4% paraformaldehyde in 0.12 M buffer
phosphate). Fixed slices were then rinsed in PBS and mounted in Fluoromount-G mounting
medium until photomicrographs were taken. Images of cortical slices were taken with a 40x
objective at a confocal laser-scanning microscope (Leica TBCS SP2). Stacks of consecutive
confocal images for 20 mM at 1 mm intervals were acquired sequentially with two lasers
(argon 488 nm for eGFP and 561 nm for Etd). Images were processed with Image J. First,
cells that were eGFP positive were identified as astrocytes. Then Etd fluorescence intensity
was measured within the eGFP-positive cells. The total Etd fluorescence within each image
was calculated in arbitrary units (AU). Averages of at least three images of the cortical areas
for each slice were calculated as the final measurement of dye uptake in that slice.

Statistical analysis
For each data group, results are expressed as mean SEM and n refers to the number of
independent experiments. Unpaired two-tailed student test was used. Differences are
considered significant at *P < 0.05, **P < 0.01 and ***P < 0.001. GraphPad Prism 5 software
(GraphPad Software, La Jolla, CA, USA) was used for calculations.

Results
The level of gap junctional communication in the astroglial network was investigated by
performing whole-cell recording and dye coupling experiments in astrocytes located in layer I
of the somatosensory cortex from acute cortical slices. Dialyzed astrocytes were initially
selected based on their distinct morphology compared to neurons and then identified by their
electrophysiological properties, i.e., a hyperpolarized resting membrane potential (-81.70.5
mV, n = 66), a linear voltage/current relationship in response to current injections and a low
input resistance (25.70.7 M!, n = 66). Dye coupling was assessed by adding
sulforhodamine B to the pipette solution (see Giaume et al., 2012) and by counting the
number of stained cells after 10 min of whole-cell recording. In a previous work performed in
the somatosensory cortex we have demonstrated by double staining that dye coupling occurs
only between astrocytes when sulforhodamine dialysis was conducted in an astrocyte
(Houades et al., 2006; 2008). In control conditions, the number of coupled cells (45.81.8, n
= 10 for propofol; 44.91.6, n = 9 for ketamine and dexmedetomidine) stained by
sulforhodamine B and the shape of the coupling area (Fig. 1 A1) were identical to those
previously reported (Liu et al., 2013). To study the effect of general anesthetics on gap
junctional communication, slices were exposed to the compounds during a pre-incubation
period and whole-cell patch-clamp recording was achieved on single astrocytes in the
presence of the anesthetic. Propofol was found to be the most efficient gap junction channel
!

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inhibitor among the three tested general anesthetics. Indeed, its effect started to be
significantly effective with 24% inhibition of dye coupling at a concentration of 100 mM
(%15 min) while the inhibition reached 76% at 150 mM (Fig. 1 A2 and B), a concentration that
although superior to the EC50 for loss of consciousness, may be encountered during clinical
anesthesia in patients (Servin et al., 1990). When the slice was exposed to this later
concentration and then washed for 30 min, dye coupling recovered to 80% of the control,
demonstrating that the inhibition induced by propofol is reversible (Fig. 1 A3 and B). The
effect of ketamine was much less potent since its effect only reached 31% of inhibition even
when used at a high concentration of 300 mM (%30 min) (Fig. 2A). Finally, the least efficient
compound among the tested anesthetics was dexmedetomidine, with barely 17% inhibition
compared to control condition when used at 10 mM (%30 min), while lower concentrations
(0.5 and 1 mM) were virtually ineffective (Fig. 2B).

In normal situation, the basic feature for the functionality of connexin-based channels in
astrocytes is a high gap junctional communication and a weak hemichannel activity (Bennett
et al., 2003). However, in most pathological situations involving brain inflammation a
reactive gliosis is associated with elevated hemichannel activity in astrocytes (see Bennett et
al., 2012; Giaume et al., 2013). Thus, to mimic such pathological situation, microglial cells
were activated with the endotoxin lipopolysaccharide (LPS), which leads to the release
pro-inflammatory cytokines and triggers the opening of hemichannels in astrocytes (Retamal
et al., 2007). In these experiments hemichannel activity was investigated by using the
ethidium bromide (Etd) assay (see Giaume et al., 2012). In acute cortical slices, LPS
treatment (100 ng/ml, 90 min) increased by 567% (n=6) the uptake of Etd in eGFP-positive
astrocytes, indicating that hemichannels in astrocytes were activated. Interestingly, using this
protocol we found that the effect of the three selected general anesthetics was always
inhibitory when tested on the hemichannel function of connexins in astrocytes (Fig. 3 and 4,
A1-B3). However, their efficiency on hemichannels was distinct to that described above for
gap junctional communication. Indeed, ketamine, which had a weak effect on gap junctional
communication, was found to be the most efficient. Indeed, a 20% inhibitory action of this
compound on hemichannel activity started to be detected at a low concentration (10 mM) and
reached 67% at 50 mM (Fig. 3 A1-B3 and C). Propofol, which is a strong inhibitor of gap
junctional communication, started to be effective at 25 mM with a reduction of 25% and 77%
at 150 mM (Fig. 4). Finally, dexmedetomidine had no effect when used at 1 mM and was
found to a have a weak, but significant, inhibitory action (22%) at 10 mM (Fig. 3D). These
observations demonstrated for the first time that general anesthetics have an inhibitory effect
on hemichannel activity in astrocytes.

!-,!

Discussion
The present work has been undertaken to update the knowledge concerning the effect of
general anesthetics on two channel functions of connexins in astrocytes, i.e., gap junction
channels and hemichannels. For this purpose we have selected three anesthetic compounds,
propofol, ketamine and dexmedetomidine that are known to mediate their sedative action
through different pathways (see below). To determine whether these compounds affect
connexin channel functions in glia represents a critical issue because there is increasing
evidence that astrocytes interact actively with neurons and the vascular system (Carmignoto
and Haydon, 2006; Filosa and Iddings, 2013; Araque et al., 2014). Furthermore the two
channel functions provided by connexins have been shown to impact both types of cell
interactions. Indeed, impaired gap junctional communication (see Giaume et al., 2010) and
connexin hemichannel activity (see Giaume et al., 2013) have been demonstrated to affect
both neuroglial (Pannasch and Rouach, 2013) and gliovascular (Ezan et al., 2013; De Bock et
al., 2014) interactions. Such statement is based upon studies performed using either genetic or
pharmacological approaches (see Giaume and Theis, 2010) to suppress connexin-based
channel activities (Pannasch et al, 2011; Ezan et al., 2012; Orrelana et al., 2011).
Consequently, to determine whether general anesthetics impair connexin channels in
astrocytes is of primary importance since neuroglial and gliovascular interactions are now
frequently investigated using in vivo mouse models under anesthesia (see for instance Iliff et
al., 2013 and Kanemaru et al., 2014 for ketamine; Remsen et al., 1999 for propofol).
While it is known for a long time that anesthetics are inhibitors of gap junction channels
(Johnston et al., 1980; Rozental et al., 2001), so far there is no information available
concerning their effect on the other channel function of connexins, i.e., their hemichannel
activity. Two decades ago we have carried out a deep screening study related to the effect of
general anesthetics on gap junctional communication in primary astrocytes (Mantz et al.,
1993). In this work astrocytes were studied in cultures lacking their partner cells (i.e. in
particular neurons, micriglial and endothelial cells). Compared to this cell model, acute
cortical slices used in the present study have their anatomical structure and synaptic contacts
maintained intact. Consequently, this work provides important and more reliable information
that should orientate the choice of anesthetics for in vivo investigations of brain functions and
diseases involving astroglial connexins (see Giaume et al., 2010; Ransom and Giaume, 2013).
Moreover, recent studies have established that anesthetics, including ketamine, influence
calcium signaling in astrocytes (Nimmerjjahn et al., 2009; Thrane et al., 2012), a feature that
is considered as the modality of cell excitability for these glial cells devoid of action potential.
Considering that both connexin channels are involved in the propagation of intercellular
calcium waves (Scemes and Giaume, 2006; Leybeart and Sanderson, 2012) and that
hemichannels provide a pathway for calcium entry in astrocytes (Heidemann et al., 2005; De
Bock et al., 2012), our study may contribute to unraveling one of the mechanisms by which
anesthetics affect calcium signaling in astrocytes studied in anesthetized mice (see Thrane et
al., 2012).

Numerous studies have shown that anesthetics alter or block neurotransmission and
neuronal firing by acting as agonists or antagonists for various receptors on neurons
(Frank, 2008; Alkire et al., 2008; Brown et al., 2010). The three compounds tested in
this study have been selected considering their action on different signaling pathways.
!

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Propofol acts as GABA agonist in post-synaptic neuronal receptors, thus enhancing

the inhibitory GABAergic circuitry and decreasing the level of arousal (Jurd et al.,
2003). Ketamine acts as a NMDA glutamatergic receptor antagonist, thus blocking
the inhibitory effect of glutamatergic interneurons on GABAergic neurons in cortex,
hippocampus and limbic system (Franks, 2008). Finally, dexmedetomine, a more
recently developed anesthetic, is an "2-adrenergic agonist that acts via a neuronal
circuitry involving a2-adrenoceptors located on the locus coeruleus and enhancing the
activity of the pathways involved in non-rapid eye movement sleep (Nelson et al.,
2003). Thus the three anesthetics exert their sedative action on different neuronal and
possibly different astroglial targets that partly explain their differential effects on
connexin-based channel functions. However, the aim of the present work was not to
decipher the mode of action of general anesthetics but rather to determine their effects
on the two channel functions of connexins expressed in astrocytes and to establish a
hierarchy in their potency of action. Indeed, propofol was found to be the most
effective compounds that inhibit both gap junctional communication and hemichannel
activity by about 70% when used outside the usual clinical range but that have been
reported in surgical patients anesthetized with propofol (Servin et al., 1990). The
action of ketamine was characterized by a weak inhibition on gap junction channels
even at high concentrations (300 $M) but was very potent on hemichannels with a
strong inhibition (about 70%) at a much lower concentration (50 $M). Finally,
dexmedetomine was the least effective among the tested general anesthetics since its
effect on the two channel functions never exceeded 20% when used at the
concentration of 10 $M. These findings indicate that these three general anesthetics
have distinct efficiencies on connexin channels in astrocytes and that they have
differential effects on gap junctional communication compared to hemichannel
activity. Also, based on these observations dexmedetomine appears to be the
compound having minor effects on connexin channel functions at least when used in
the clinically relevant range. Taken together, these findings should help and guide
investigators in the design of experiments to investigate brain functions and
pathologies involving in vivo approaches with anesthetized animals.
Acknowledgments: This work was supported by the ANR grant NANR-12-BSV4-0013-01.

!"..!

References
Agulhon, C., Petravicz, J., McMullen, A.B., Sweger, E.J., Minton, S.K., Taves, S.R., Casper,
K.B., Fiacco, T.A., and McCarthy, K.D. (2008). What is the role of astrocyte calcium in
neurophysiology? Neuron 59, 932-946.
Alkire, M.T., Hudetz, A.G., and Tononi, G. (2008). Consciousness and anesthesia. Science
322, 876-880.
Araque, A., Parpura, V., Sanzgiri, R.P., and Haydon, P.G. (1999). Tripartite synapses: glia,
the unacknowledged partner. Trends Neurosci 22, 208-215.
Araque, A., Carmignoto, G., Haydon, P.G., Oliet, S.H., Robitaille, R., and Volterra, A. (2014).
Gliotransmitters travel in time and space. Neuron 81, 728-739.
Bennett, M.V., Contreras, J.E., Bukauskas, F.F., and Saez, J.C. (2003). New roles for
astrocytes: gap junction hemichannels have something to communicate. Trends Neurosci 26,
610-617.
Bennett, M.V., Garre, J.M., Orellana, J.A., Bukauskas, F.F., Nedergaard, M., and Saez, J.C.
(2012). Connexin and pannexin hemichannels in inflammatory responses of glia and neurons.
Brain Res 1487, 3-15.
Brown, E.N., Lydic, R., and Schiff, N.D. (2010). General anesthesia, sleep, and coma. N Engl
J Med 363, 2638-2650.
De Bock, M., Culot, M., Wang, N., da Costa, A., Decrock, E., Bol, M., Bultynck, G.,
Cecchelli, R., and Leybaert, L. (2012). Low extracellular Ca2+ conditions induce an increase
in brain endothelial permeability that involves intercellular Ca2+ waves. Brain Res 1487,
78-87.
De Bock, M., Decrock, E., Wang, N., Bol, M., Vinken, M., Bultynck, G., and Leybaert, L.
(2014). The dual face of connexin-based astroglial Ca communication: A key player in brain
physiology and a prime target in pathology. Biochim Biophys Acta 1843, 2211-2232.
Ezan, P., Andre, P., Cisternino, S., Saubamea, B., Boulay, A.C., Doutremer, S., Thomas,
M.A., Quenech'du, N., Giaume, C., and Cohen-Salmon, M. (2012). Deletion of astroglial
connexins weakens the blood-brain barrier. J Cereb Blood Flow Metab 32, 1457-1467.
Filosa, J.A., and Iddings, J.A. (2013). Astrocyte regulation of cerebral vascular tone. Am J
Physiol Heart Circ Physiol 305, H609-619.
Franks, N.P. (2008). General anaesthesia: from molecular targets to neuronal pathways of
sleep and arousal. Nat Rev Neurosci 9, 370-386.
Giaume, C., Kirchhoff, F., Matute, C., Reichenbach, A., and Verkhratsky, A. (2007). Glia: the
fulcrum of brain diseases. Cell Death Differ 14, 1324-1335.
Giaume, C., and Theis, M. (2010). Pharmacological and genetic approaches to study
connexin-mediated channels in glial cells of the central nervous system. Brain Res Rev 63,
160-176.
Giaume, C., Koulakoff, A., Roux, L., Holcman, D., and Rouach, N. (2010). Astroglial
networks: a step further in neuroglial and gliovascular interactions. Nat Rev Neurosci 11,
87-99.
!

"."!

Giaume, C., Leybaert, L., Naus, C.C., and Saez, J.C. (2013). Connexin and pannexin
hemichannels in brain glial cells: properties, pharmacology, and roles. Front Pharmacol 4, 88.
Halassa, M.M., and Haydon, P.G. (2010). Integrated brain circuits: astrocytic networks
modulate neuronal activity and behavior. Annu Rev Physiol 72, 335-355.
Haydon, P.G., and Carmignoto, G. (2006). Astrocyte control of synaptic transmission and
neurovascular coupling. Physiol Rev 86, 1009-1031.
Heidemann, A.C., Schipke, C.G., and Kettenmann, H. (2005). Extracellular application of
nicotinic acid adenine dinucleotide phosphate induces Ca2+ signaling in astrocytes in situ. J
Biol Chem 280, 35630-35640.
Houades, V., Rouach, N., Ezan, P., Kirchhoff, F., Koulakoff, A., and Giaume, C. (2006).
Shapes of astrocyte networks in the juvenile brain. Neuron Glia Biol 2, 3-14.
Houades, V., Koulakoff, A., Ezan, P., Seif, I., and Giaume, C. (2008). Gap junction-mediated
astrocytic networks in the mouse barrel cortex. J Neurosci 28, 5207-5217.
Iliff, J.J., Wang, M., Zeppenfeld, D.M., Venkataraman, A., Plog, B.A., Liao, Y., Deane, R.,
and Nedergaard, M. (2013). Cerebral arterial pulsation drives paravascular CSF-interstitial
fluid exchange in the murine brain. J Neurosci 33, 18190-18199.
Johnston, M.F., Simon, S.A., and Ramon, F. (1980). Interaction of anaesthetics with electrical
synapses. Nature 286, 498-500.
Jurd, R., Arras, M., Lambert, S., Drexler, B., Siegwart, R., Crestani, F., Zaugg, M., Vogt,
K.E., Ledermann, B., Antkowiak, B., et al. (2003). General anesthetic actions in vivo strongly
attenuated by a point mutation in the GABA(A) receptor beta3 subunit. FASEB J 17,
250-252.
Kanemaru, K., Sekiya, H., Xu, M., Satoh, K., Kitajima, N., Yoshida, K., Okubo, Y., Sasaki,
T., Moritoh, S., Hasuwa, H., et al. (2014). In Vivo visualization of subtle, transient, and local
activity of astrocytes using an ultrasensitive ca(2+) indicator. Cell Rep 8, 311-318.
Liu, X., Petit, J.M., Ezan, P., Gyger, J., Magistretti, P., and Giaume, C. (2013). The
psychostimulant modafinil enhances gap junctional communication in cortical astrocytes.
Neuropharmacology 75, 533-538.
Mantz, J., Cordier, J., and Giaume, C. (1993). Effects of general anesthetics on intercellular
communications mediated by gap junctions between astrocytes in primary culture.
Anesthesiology 78, 892-901.
Nelson, L.E., Lu, J., Guo, T., Saper, C.B., Franks, N.P., and Maze, M. (2003). The
alpha2-adrenoceptor agonist dexmedetomidine converges on an endogenous sleep-promoting
pathway to exert its sedative effects. Anesthesiology 98, 428-436.
Nimmerjahn, A., Mukamel, E.A., and Schnitzer, M.J. (2009). Motor behavior activates
Bergmann glial networks. Neuron 62, 400-412.
Orellana, J.A., Shoji, K.F., Abudara, V., Ezan, P., Amigou, E., Saez, P.J., Jiang, J.X., Naus,
C.C., Saez, J.C., and Giaume, C. (2011). Amyloid beta-induced death in neurons involves
glial and neuronal hemichannels. J Neurosci 31, 4962-4977.
!".#!

Panatier, A., Vallee, J., Haber, M., Murai, K.K., Lacaille, J.C., and Robitaille, R. (2011).
Astrocytes are endogenous regulators of basal transmission at central synapses. Cell 146,
785-798.
Pannasch, U., Vargova, L., Reingruber, J., Ezan, P., Holcman, D., Giaume, C., Sykova, E.,
and Rouach, N. (2011). Astroglial networks scale synaptic activity and plasticity. Proc Natl
Acad Sci U S A 108, 8467-8472.
Pannasch, U., and Rouach, N. (2013). Emerging role for astroglial networks in information
processing: from synapse to behavior. Trends Neurosci 36, 405-417.
Parpura, V., and Verkhratsky, A. (2013). Astrogliopathology: could nanotechnology restore
aberrant calcium signalling and pathological astroglial remodelling? Biochim Biophys Acta
1833, 1625-1631.
Ransom, B.R., and Giaume, C. (2013). Gap juunctions, hemichannels. In Neuroglia, third
edition, H. Kettenmann and B.R. Ransom, eds. (Oxford University Press), pp. 292-305.
Remsen, L.G., Pagel, M.A., McCormick, C.I., Fiamengo, S.A., Sexton, G., and Neuwelt, E.A.
(1999). The influence of anesthetic choice, PaCO2, and other factors on osmotic blood-brain
barrier disruption in rats with brain tumor xenografts. Anesth Analg 88, 559-567.
Retamal, M.A., Froger, N., Palacios-Prado, N., Ezan, P., Saez, P.J., Saez, J.C., and Giaume, C.
(2007). Cx43 hemichannels and gap junction channels in astrocytes are regulated oppositely
by proinflammatory cytokines released from activated microglia. J Neurosci 27,
13781-13792.
Roux, L., Benchenane, K., Rothstein, J.D., Bonvento, G., and Giaume, C. (2011). Plasticity of
astroglial networks in olfactory glomeruli. Proc Natl Acad Sci U S A 108, 18442-18446.
Rozental, R., Srinivas, M., Spray, D.C. (2001). How to close a gap junction channel. In
Connexin Methods and Protocoles, Methods in Molecular Biology, Vol. 154, R. Bruzzone,
and C. Giaume, eds. (Humana Press), pp. 447-476.
Scemes, E., and Giaume, C. (2006). Astrocyte calcium waves: what they are and what they do.
Glia 54, 716-725.
Servin, F., Cockshott, I.D., Farinotti, R., Haberer, J.P., Winckler, C., and Desmonts, J.M.
(1990). Pharmacokinetics of propofol infusions in patients with cirrhosis. Br J Anaesth 65,
177-183.
Siracusano, L., Girasole, V., Alvaro, S., and Chiavarino, N.D. (2006). Myocardial
preconditioning and cardioprotection by volatile anaesthetics. J Cardiovasc Med (Hagerstown)
7, 86-95.
Thrane, A.S., Rangroo Thrane, V., Zeppenfeld, D., Lou, N., Xu, Q., Nagelhus, E.A., and
Nedergaard, M. (2012). General anesthesia selectively disrupts astrocyte calcium signaling in
the awake mouse cortex. Proc Natl Acad Sci U S A 109, 18974-18979.
Torres, A., Wang, F., Xu, Q., Fujita, T., Dobrowolski, R., Willecke, K., Takano, T., and
Nedergaard, M. (2012). Extracellular Ca(2)(+) acts as a mediator of communication from
neurons to glia. Sci Signal 5, ra8.
Verkhratsky, A., and Butt, A. (2013) In Glial Physiology and Pathophysiology, A.
Verkhratsky and A. Butt, eds. (Wiley-Blackwell), pp. 527.
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Zorec, R., Araque, A., Carmignoto, G., Haydon, P.G., Verkhratsky, A., and Parpura, V.
(2012). Astroglial excitability and gliotransmission: an appraisal of Ca2+ as a signalling route.
ASN Neuro 4.

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Figure 1: Propofol strongly inhibits gap junctional communication in astroglial network from
acute cortical slices. A1-A3: Typical fluorescent images of dye-coupled astrocytes in acute
cortical slices under different conditions: control (A1), 15 min propofol 150 $M (A2) and 30
min washout after 15 min of propofol 150 $M (A3). Scale bar, 10 m. B: Summary diagram
indicating the numbers of dye coupled astrocytes under increasing concentrations of propofol.
Compared to the control (n = 10), no difference is found with 50 $M (n = 4), while there is a
significant decrease with 100$M (n = 5) and 150$M (n = 6).

".&!

Figure 2: Weak effects of ketamine and dexmedetomidine on gap junctional communication

in cortical astroglial networks. A: Dose-response curve for ketamine indicates that this drug is
ineffective at 50 $M (n = 5), while its inhibitory effect becomes but significant at 100 $M (n
= 6) and more pronounced at 300 $M (n = 4). B: Dose response curve for dexmedetomidine.
This anesthetic has no effect on astroglial coupling when used at a concentration of 500 nM
(n = 4) and has a weak, but statistically significant, inhibitory action at 1 $M and 10 $M (n =
4 and 5, respectively).

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Figure 3: Propofol inhibits hemichannel activity in astrocytes studied in acute hippocampal

slices treated with LPS. A1-A3 and B1-B3. Representative images of Etd uptake (red) in
astrocytes (green) in cortical slices from GFAP-eGFP transgenic mice, after LPS treatment
(100 ng/ml, 90 min) in the absence (A1 and A2) and in the presence (B1 and B2) of propofol
150 $M. Merge signals are illustrated in A3 and B3, respectively. Scale bars: 10 m. C.
Summary graph demonstrating that Etd uptake in astrocytes from LPS treated slices is
strongly inhibited by propofol used at concentration of 25, 50, 100 and 150 mM. Statistical
comparisons in B were done with the LPS stimulus condition without propofol (n= 5
independent experiments).

".(!

Figure 4: Inhibitory effects of ketamine and dexmedetomidine on hemichannel activity in

LPS treated astrocytes. A1-A3 and B1-B3. Representative images of Etd uptake (red) in
astrocytes (green) in cortical slices from GFAP-eGFP transgenic mice, after LPS treatment
(100 ng/ml, 90 min) in the absence (A1 and A2) and in the presence (B1 and B2) of ketamine
50 $M. Merge signals are shown in A3 and B3, respectively. Scale bars: 10 m. C: Summary
graph illustrating that the Etd uptake in LPS treated astrocytes is strongly inhibited by
ketamine used at concentration of 5, 10, 20 and 50 mM (n= 4 independent experiments). D:
Summary graph illustrating that the Etd uptake in LPS treated astrocytes is weakly, but
significantly, inhibited by dexmedetomidine used at concentration 10 mM while 1 mM is
ineffective (n= 4 independent experiments). Statistical comparisons in C and D were done
with the LPS stimulus condition without anesthetics.

!".,!

<.%)9%%.(0!
Like most general anesthetics, the three compounds we have tested in our study,
especially propofol and ketamine, have multiple targets in the central nervous system
(Brown et al., 2011). Therefore, the following discussion about the possible
mechanisms underlying their effects on astroglial connexin (Cx) channel functions
considers mainly their primary targets in the contest of our experiments carried out in
acute cortical brain slices.
The anesthetic action of propofol is mainly mediated by its activation of neuronal
GABAA receptor (Jurd et al., 2003). For astrocytes, activation of astroglial GABAA
receptors leads to depolarization in membrane potentials (Kettenmann et al., 1984),
which is consistent with our observation that propofol induces depolarization (5-10
mV) in astrocytes (data not shown), suggesting that propofol may directly activate
GABAA receptors in astrocytes. Therefore, the strong inhibition of gap junction
channels (GJCs) by proprofol may be a consequence of astroglial GABAA receptor
activation, although the effects of GABAA receptor activation on GJCs need to be
directly tested in acute brain slices. Similarly, despite that ketamine is known to act
primarily as a NMDA glutamatergic receptor antagonist (Franks, 2008), so far no
study has addressed the effects of NMDA receptor antagonism on astroglial GJCs. To
elucidate the mechanisms by which propofol and ketamine inhibit astroglial GJCs, the
action of glutamatergic and GABAergic transmission on astroglial GJCs needs to be
identified, particularly in the somatosensory cortex.
Dexmedetomidine is a "2-adrenergic receptor agonist (Nelson et al., 2003). Although
an early work in cultured astrocytes demonstrated that norepinephrine inhibits GJCs,
the inhibition is totally blocked by a "1-adrenergic receptors antagonist, indicating
"2-adrenergic receptor activation does not contribute to norepinephrine-induced
inhibition of GJCs (Giaume et al., 1991). This may explain the minute effects of
dexmedetomidine on astroglial dye coupling.
For the hemichannel experiments, we used LPS to induce opening of astroglial
hemichannels in acute cortical slices. The LPS-induced astroglial hemichannel
opening was shown to be triggered by inflammatory cytokines released from
LPS-activated microglia (Retamal et al., 2007). In agreement with our findings that
propofol and ketamine strongly reduced astroglial activation, several recent studies
showed that propofol and ketamine treatment suppressed release of the inflammatory
cytokines from cultured microglia and astrocytes (Peng et al., 2014; Tanaka et al.,
2013). In contrast, dexmedetomidine was also shown to reduce cytokine release from
cultured microglia but only at concentrations far beyond the clinically relevant range
(Peng et al., 2013), which may explain its minor inhibitory effect on hemichannels we
observed with clinically relevant dosages of dexmedetomidine.
Intriguingly, many general anesthetics have been reported to exhibit neuroprotective
properties (Wei and Inan, 2013) while astroglial hemichannel activation is associated
!

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various pathological conditions and may lead to amplification of neuronal death

(Bennett et al., 2012; Castellano and Eugenin, 2014). Accordingly, it is likely that the
strong inhibition of astroglial hemichannels by propofol and ketamine may contribute
to their neuroprotective effects and may hold therapeutic potentials. Indeed, it was
reported that the inhibition of cytokine-induced hemichannel activation is
neuroprotective against NMDA-induced excitotoxicity (Froger et al., 2010).

!"".!

Part III.

67+!'(-+!(:!)(00+1.0!AB!.0!%-++3!7(8+(%&/%.%!
!
Introduction
Over the last two decades, increasing research evidence has revealed that astrocytes
are indispensable participants in a wide variety of brain activities from brain
metabolism to synaptic transmission through their multifaceted dynamic interactions
with neurons (Perea et al., 2009; Petzold and Murthy, 2011; Belanger et al., 2011;
Araque et al., 2014). The consequences of such neuroglial interactions on the
molecular and cellular level have been addressed by numerous studies for the past two
decades, while in recent years the behavioral roles of astrocytes have begun to emerge.
Astrocytes have been implicated in memory consolidation (Suzuki et al., 2011; Lee et
al., 2014), motor coordination (Saab et al., 2012), regulation of respiration rhythm
generation (Gourine et al., 2010), and sleep homeostasisthe work from Haydon and
colleagues demonstrated that ATP released from astrocytes is a major source of brain
extracellular adenosine, which acts on neuronal A1 receptors to mediate the
homeostatic response of NREM sleep (Halassa et al., 2009).
Besides the mechanisms on the single cell level, I have been interested in the possible
contribution to sleep homeostasis by astroglial networks that are based on the high
level of intercellular connections through astroglial gap junction channels (GJCs)
(Giaume et al., 2010; Giaume and Liu, 2012). Previous works have shown that
GJC-mediated astroglial networks are spatially and functionally restricted (Houades et
al., 2008) and activity-dependent (Roux et al., 2011) and that they are involved in
energy metabolite trafficking to sustain neuronal activity (Giaume et al., 1997;
Blomstrand and Giaume, 2006; Rouach et al., 2008). Considering that sleep is a
function of neuronal ensembles and that NREM sleep is characterized by
synchronized oscillations involving large cortical areas and subcortical structures
(Steriade, 2006), we speculate that GJC-mediated network coordination of astrocytes
may contribute to the regulation of sleep. Indeed, there are lines of evidence showing
that GJCs are regulated by pharmacological agents that affect sleep and wake. On the
one hand, oleamide, a sleep-inducing lipid (Guan et al., 1997), and several general
anesthetics (Mantz et al., 1993) were reported to inhibit GJCs in primary cultures of
astrocytes. In addition, propofol, ketamine (see Results Part II) and
$-hydroxybutyric acid (GHB) (Liu et al., 2013; see also Results Part I) were shown
to inhibit astroglial GJCs in acute cortical slices. On the other hand, a potent
wakefulness-promoting drug, modafinil, was demonstrated to increase astroglial gap
junctioanl communication in an activity-dependent manner (Liu et al., 2013; see also
Results Part I). Importantly, this study also showed that the two connexins (Cxs) that
constitute GJCs in astrocytes, Cx30 and Cx43, are differentially regulated by
!

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modafinil: the mRNA and protein expression level of Cx30, but not Cx43, were
increased following modafinil administration. These results are in line with the
observation that Cx30 rather than Cx43 underlied the activity dependence of astroglial
networking in the olfactory glomeruli (Roux et al., 2011) and that gene expression
level of Cx30 was increased when mice were raised in an enriched environment
(Rampon et al., 2000).
Based on the evidence discussed above, the goal of the third part of my thesis work
was to investigate the role of Cx30 in the regulation sleep of homeostasis by using the
Cx30 knockout (KO) mouse (Boulay et al., 2013). So far, we have made the following
findings: 1) the mRNA expression of Cx30 but not Cx43 is increased by sleep
deprivation (SD); 2) gap junctioanl communication in cortical astrocytes is enhanced
by SD and Cx30 is necessary for such enhancement; 3) compared to wild type (WT)
mice, Cx30 KO mice exhibit higher sleep propensity, which is manifested as
compromised ability to maintain wakefulness during SD; 4) several genes related to
brain energy metabolism are down-regulated in multiple brain structures of the Cx30
KO mouse. Based on these results, we hypothesize that Cx30 is necessary to sustain
wakefulness during periods of high sleep pressure, likely through its active role in
neurometabolic coupling.

Materials and methods

Animals
Adult male mice were used in all experiments except for electrophysiology and dye
coupling experiments. Mice were housed in standard conditions (light-dark cycle:
12h/12h, temperature: 23 1C, humidity: 50% 10%) with food and water ad
libitum. We used a novel knockout mouse model of Cx30 developed by Boulay et al.
(2013). The authors generated a new mouse strain, in which the Cx30-coding exon
was replaced with a Cx30 floxed allele. The Cx30fl/fl mouse strain was crossed with
Pgk-Cre mice that ubiquitously express the Cre recombinase (Lallemand et al., 1998),
leading to global deletion of the Cx30 transcript sequence. This mouse model is
referred to as Cx30 knouckout (Cx30 KO) mice for the rest of this manuscript. The
Cx30 KO mice used in this study were on a mixed background of C57BL/6 and
129/Sv mouse strains. Since C57BL/6 consisted more than 90% of their genetic
background, C57BL/6 mice were used as the control for Cx30 KO mice.
All experiments were performed according to the European Community Council
Directives of November 24, 1986 (86/609/EEC) and all efforts were made to
minimize the number of animals used and their suffering.
Patch clamp and dye coupling of astrocytes
Experiments were carried out in acute brain slices prepared from animals aged
between P15 to P25. Coronal brain slices (300 $m thick) containing the
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somatosensory cortex were cut using a vibratome (Microm HM 650 V, Walldorf,

Germany) filled with ice-cold artificial cutting solution containing (in mM) 222
sucrose, 27 NaHCO3, 2.6 KCl, 1.25 NaH2PO4, 2 CaCl2, 7 MgSO4, and 0,1 ascorbic
acid. They were incubated at 32 C for 30 min in artificial cerebrospinal fluid (ACSF)
containing (in mM) 125 NaCl, 2.5 KCl, 25 glucose, 25 NaHCO3, 1.25 NaH2PO4, 2
CaCl2, and 1 MgCl2, continuously aerated with 95% O2/5% CO2 (pH 7.4). Then slices
were transferred at room temperature (2022 C) before use. The slices were placed in
a submerged recording chamber superfused with aerated ACSF at a rate of 2 mL/min
and observed using an up-right fixed stage microscope (Axioskop FS; Zeiss,
Oberkochen, Germany) equipped with Nomarski optics and an infrared video camera
(Newvicon C2400; Hamamatsu, Shizuoka, Japan). Whole-cell patch-clamp recording
of astrocytes was performed with pipettes (38 M&) filled with internal solution that
contains (in mM) 105 K-Gluconate, 30 KCl, 10 Hepes, 10 Phospho-Creatine Tris, 4
ATP-Mg2+, 0.3 GTP-Tris, and 0.3 EGTA (adjusted to pH 7.4 with KOH).
Experiments were conducted at 32 C. Membrane voltages and currents were
amplified by a MultiClamp 700B amplifier, sampled by a DIGIDATA 1322A
Interface, and patch-clamp recordings (5 kHz sampling and 3 kHz filtering) were
performed with Pclamp9 software (Axon Instruments, Foster City, CA). Series
resistances were compensated at 80%. Input resistance (Rin) was measured in
voltage-clamp mode by applying hyperpolarizing voltage pulses (10 mV, 150 ms)
from a holding potential of '80 mV. To assess the level of gap junctioanl
communication, dye coupling experiments were performed by adding sulforhodamine
B (1 mg/ml; Molecular Probes, Eugene OR, USA) to the pipette solution before
patch-clamp recording. Recorded cells were loaded with the dye for 10 min in the
whole-cell configuration and current-clamp mode to allow its diffusion among cells
connected by GJCs. Fluorescent images containing the dye coupled cells were
captured with the CCD camera (Pixelfly QE, The Cook Corporation, Romulus,
Michigan, USA) on different focal planes and the number of dye coupled cells was
determined using ImageJ software.
Gentle sleep deprivation (GSD) and SD with the CaResS Device
Both SD protocols began at light onset (ZT0 for zeitgeber time = 0) and lasted for 6
hours. For GSD, mice remained in their home cages and were constantly monitored
for behavioral signs of sleep onset, upon which they were aroused by gently moving
their nests and introducing new objects into the cage.The other type of SD was
performed using a device named CaResS (acronym for Cage for Restriction of Sleep)
that has been specially designed to diminish SD-induced stress (Baud et al., 2013).
The CaResS device consists of a cylindrical Plexiglas cage (30 cm diameter) with 3
inner radial walls and a mobile circular floor covered with sawdust. During SD, the
floor is rotated by a motor wheel at a speed that gradually increases from 1.5 to 2.5
rpm to compensate for increasing sleep pressure. The mouse is waken up when
carried by the rotating floor into contact with the inner walls (Figure 1). Although its
design leaves the possiblity that the mouse can fall asleep during the rotation time
before being touched by the inner walls, CaReSs device has been shown to keep the
!

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Figure 1. The schematic drawing of the CaResS device. Adapted from Petit et al., 2013.
Gene
Slc2a1
Slc2a3
Ldha
Ldhb
Ptg
Fos
MCT1
Mct2
MCT4
Slc1a3
Actb
Ppia

Primers
Forward: 5'-CCAGCTGGGAATCGTCGTT-3
Reverse: 5'-CTGCATTGCCCATGATGGA-3
Forward: 5'-GAGGAGAACCCTGCATATGATAGG-3
Reverse: 5'-CAAAGCTCATGGCTTCATAGTCA-3
Forward: 5'-TTGTCTCCAGCAAAGACTACTGTGT-3
Reverse: 5'-TTTCGCTGGACCAGGTTGAG-3
Forward: 5'-GCAGCACGGGAGCTTGTT-3
Reverse: 5'-CAATCTTAGAGTTGGCTGTCACAGA-3
Forward: 5(-TGCCTCTCGGTCCAATGAG-3
Reverse: 5-GGCAGTACGGAACTGTTCAA-3(
Forward: 5'-CGGAGGAGGGAGCTGACA-3
Reverse: 5'-CTGCAACGCAGACTTCTCATCT-3
Forward: 5(-AATGCTGCCCTGTCCTCCTA
Reverse: 5(-CCCAGTACGTGTATTTGTAGTCTCCAT
Forward: 5(-CAGCAACAGCGTGATAGAGCTT-3
Reverse: 5(-TGGTTGCAGGTTGAATGCTAAT-3
Forward: 5(-TCTGCAGAAGCATTATCCAGATCTA-3
Reverse: 5(-ATGATGAGGGAAGGCTGGAA-3
Forward: 5(-TGCCTTCGTTCTGCTCACGGT-3
Reverse: 5(-ACGGTCGGAGGGCAAATCCA-3
Forward: 5'-GCTTCTTTGCAGCTCCTTCGT-3
Reverse: 5'-ATATCGTCATCCATGGCGAAC-3
Forward: 5(-CAAATGCTGGACCAAACACAA-3
Reverse: 5(-GCCATCCAGCCATTCAGTCT-3

Table 1. Sequences of gene primers.

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mouse awake for 90% of the time during the 6h SD (Baud et al., 2013). Control
animals were similarly maintained in CaResS without rotation. The animals were
housed for at least 3 days for habituation to CaResS before SD.
Stress assessment for SD
The stress level of control and sleep-deprived animals was assessed by measuring
plasma glucocorticoid levels when animals were sacrificed at the end of SD. The
trunk blood was collected in heparinized tubes (microvette, Sarstedt, Nmbrecht,
Germany) and centrifuged to separate plasma. Glucocorticoid levels were determined
using a commercial kit (Corticosterone Enzyme Immunoassay Kit, Assay Designs,
LuBioScience, Lucerne, Switzerland). Data from mice that exhibited elevated
glucocorticoid levels were discarded.
Locomotor activity recordings
Passive infrared motion detectors were placed above animal cages and detected
rearing and lateral movements with a magnitude > 1-2 cm. Grooming and small
movements were not detected. Data were collected through a homemade interface and
recorded by bins of 1 min and pooled over every 15 min on a computer.
Surgery and electroencephalogram/electromyogram (EEG/EMG) recording
Anesthesia was induced with 4% and maintained with 1-1.5% isoflurane. Holes were
drilled in the skull over the frontal (bregma +1.5 mm and +1 mm laterally), parietal
(bregma -3 mm and +1 mm laterally) and contralateral parietal cortices to insert 2
gold-covered screws as EEG electrodes and another screw for anchorage, respectively.
Then the screws were fixed to the skull with a thin layer of dental resin (Relyx, 3M).
Two gold wires were placed onto the nuchal muscles as electrodes for the EMG
recording. The EEG and EMG electrodes were soldered to a connector and dental
cement (Paladur) was placed over the electrodes to fix the implant. After surgery, the
mice were placed in their home cages to recover for one week. For EEG/EMG
recording, the implant was plugged to a swivel-connected wire to allow free
movement of the animal. The EEG/EMG signals were recorded and digitized with an
Embla A10 amplifier (Medcare, USA), sampled at 100 Hz and filtered between 0.5
and 50 Hz.
Sleep stage scoring and spectral analysis
4-sec epochs of the EEG/EMG traces were visually scored in 3 stateswakefulness
(w), NREM sleep (n), and REM sleep (r)according to classical criteria (Tobler et al.,
1997). Spectral analysis of the EEG signal was performed for NREM sleep during
both the 4h recovery sleep (ZT6-ZT10) following SD and the baseline sleep
corresponding to the same time period to assess the rebound of slow wave activity
(SWA). Briefly, NREM sleep epochs were Fourier transformed (FFT) using
MATLAB (MathWorks Inc., Natick, USA) embedded FFT function and personalized
routines. For the SWA determination, the power spectrum of each 0.25-Hz bin of the
NREM-sleep EEG of the recovery period was summed from 0.5 Hz to 4 Hz and
!

""&!

expressed as the percentage of the total NREM spectrum. The rebound of SWA was
expressed as the percentage of increase compared to the baseline level.
Quantitative RT-PCR
For each mouse, different brain structures were dissected and homogeneized
(Ultraturax, IKA, Germany) in ice-cold TRIzol and stored at '80 C until RNA
extraction. Total RNA was extracted from tissue samples according to the TRIzol
manufacturer's protocol. The concentration of each sample was measured with
NanoDrop (NanoDrop Technologies, Wilmington, DE, USA), and the purity was
assessed using the ratios of optic density at 260/280 nm and 260/230 nm. Finally, the
RNA integrity was assessed using a 2100-Bioanalyzer (Agilent Technologies,
Waldbronn, Germany). The first strand of cDNA was synthesized from 200 ng of
total RNA using TaqMan RT-reagents (Applied Biosystem, Foster City, USA) after
incubation during 45 min at 48 C followed by 5 min at 95 C and finally stored at
4 C. Then, 2 L of RT-reagents were added to 0.5 L of forward and reverse primers
and to 23 L of Sybr-Green PCR MasterMix (Applied Biosystem, Foster City, USA).
Primer sequences were designed using Primer Express (3.0) software (Applied
Biosystem, Foster City, USA). Oligonucleotides primers were synthesized by
Microsynth (Balgach, Switzerland) and designed according to the published cDNA
sequences (NCBI database). The sequences of primers used in this study are listed in
Table 1. Forty cycles of amplification were then performed in an ABI Prism 7900
(Applied Biosystem, Foster City, USA) with 384-well plate, which allowed the
analysis of 6 genes in triplicate. Finally, data were obtained using the SDS sequence
detector software (Applied Biosystem, Foster City, USA). Expression level of target
genes was normalized against #-actin or cyclophilin A level according to geNorm
software (version 3.3; http://medgen.ugent.be/"jvdesomp/genorm/).
Statistical analysis
For all statical analysis, a nonparametric Mann-Whitney test was used. All data are
shown as mean SEM. A p value < 0.05 was considered statistically significant.
GraphPad Prism 5 software (La Jolla, CA, USA) was used for calculations.

Results
mRNA expression of Cx30 is increased by sleep deprivation
As we speculated that astroglial GJCs are involved sleep homeostasis regulation, we
first tested whether Cx30 and Cx43 are regulated by changes in sleep homeostasis. To
induce such changes, WT mice were deprived of sleep for 6 hours by GSD from the
beginning of the light period (ZT0) and then left undisturbed for 3 hours of recovery
sleep. Different groups of mice were sacrificed at the end of: 1) 6h spontaneous sleep
(ZT6), 2) 6h SD (SD6), 3) 9h of spontaneous sleep (ZT9), and 4) 6h SD and
subsequent 3h recovery sleep (SR3), respectively. The cortical and hippocampal
mRNA levels of the two Cxs were measured in each group. The mRNA levels of the

!""'!

SD6 and SR3 mice were normalized against and compared to those of the ZT6 and
ZT9 mice, respectively.
We found that Cx30 transcript levels in the SD6 group compared to the ZT6 group
were increased by 35% in the cortex (SD6 =135% !5%, n = 7 mice; ZT6 = 100%
3%, n =7 mice) and by 72% in the hippocampus (SD6 = 172% 12%, n = 7 mice;
ZT6 = 100% 7%, n =6 mice). Moreover, Cx30 level remained elevated by 36% in
the SR3 group compared to the ZT9 group in the cortex (SR3 = 136% 6%, n = 8
mice; ZT9 = 100% 2%, n =7 mice) and by 45% in the hippocampus (SR3 = 145%
10%, n = 5 mice; ZT9 = 100% 6%, n =6 mice). In contrast, Cx43 transcript levels
were unchanged in SD6 and SR3 groups compared to their respective controls, except
for a small increase of 15% in the SR3 group in the cortex (SR3 = 115% 4%, n = 8
mice; ZT9 = 100% 2%, n =7 mice) (Figure 2). These observations indicate that
Cx30 mRNA expression is strongly increased by high sleep pressure during SD,
whereas Cx43 is unaffected by this procedure.
Astroglial gap junctional communication is increased by sleep deprivation in a
neuronal activity- and Cx30-dependent manner
We then investigated whether increased Cx30 mRNA expression correlated with
simultaneous increase in gap junctional communication after SD. Acute cortical slices
were prepared from WT mice at the end the 6h SD protocol with the CaReSs device
(Petit et al., 2013; Figure 1) or 6h spontaneous sleep (the control). Dye coupling in
astroglial networks was then measured in layer I of the somatosensory cortex. We
found a 27% increase in the number of coupled cells in brain slices from mice after
SD (60 2, n = 7, Figure 3A2) compared to control mice (48 2, n = 8, Figure 3A1).
Moreover, 0.5 $M tetrodotoxin (TTX) application in the slices after SD brought down
the number of coupled cell (46 2, n = 4) to the same level as that in the control
(Figure 3C1), suggesting that SD may increase neuronal activity, which in turn
increases gap junctional communication in astrocytes. This feature is similar to that of
modafinal-induced increase in coupling (Liu et al., 2013; also see Results Part I). In
Cx30 KO mice, there was a 23% decrease in dye coupling (50 1, n = 6) compared to
WT mice (38 1, n = 5) under basal condition (Figure 3B), suggesting Cx43 is the
major mediator of gap junctional communication in cortical astrocytes. In contrast to
WT mice, however, dye coupling remained unchanged after SD (42 1, n = 5)
compared to controls (43 1, n = 6) in Cx30 KO mice (Figure 3C2). These results
altogether indicate that Cx30, not Cx43, is dynamically regulated by changes in sleep
homeostasis.
Cx30 KO mice exhibit less locomotor activity and minor sleep-wake cycle
changes in the baseline condition
To identify the possible roles of Cx30 in the regulation of sleep-homeostasis, we
characterized the sleep parameters of Cx30 KO mice. Under baseline conditions, the
Cx30 KO mice exhibited an overall decreasing tendency in locomotor activity during
!

""(!

Figure 2. Effects of sleep deprivation on mRNA expressions of astroglial connexins (Cxs).

For Cx30 in both the cortex (left) and hippocampus (right), its mRNA levels in the SD6 and
SR3 groups were substantially increased compared to the ZT6 and ZT9 groups, respectively.
For Cx43, a small increase was found only in the SR3 group in the cortex; no significant
differences were found for other groups (*p < 0.05, **p < 0.01, ***p < 0.001, ns: not
significant). Error bars = SEM, as in all other figures.

Figure 3. Dye coupling in cortical astrocytes under control condition and after sleep
deprivation (SD) in wild type (WT) and Cx30 knockout (Cx30 KO) mice. (A) Representative
fluorescent images of the dye coupled cells in acute brain slices from the control mice (A1)
and the mice after SD (A2). Note the faintly labeled cells in the periphery of the images. Scale
bar, 10 $m. (B) The number of dye coupled cells in Cx30 KO mice was moderately decreased
under control condition compared WT mice (**p < 0.01). (C) Dye coupling in WT and Cx30
KO mice after SD. In the WT mice, SD increased the number of coupled cells while TTX
treatment abolished the SD-induced increase (C1), whereas in Cx30 KO mice SD failed to
increase dye coupling in astrocytes (C2) (**p < 0.01).

!"",!

the dark (active) period and at the light-dark transition, with significant differences
compared to WT mice at 1h, 13h and 14h (Figure 4), indicating Cx30 KO mice are
less active than WT mice, especially during the dark period. We then carried out
EEG/EMG recordings to determine precisely the amount of sleep and wakefulness of
the Cx30 KO mice. We found that Cx30 KOs had a slight increase of 9% in NREM
and a 17% decrease in REM sleep in the light period (Figure 5). Thus, under baseline
conditions the Cx30 KO mice displayed minor changes in the duration of both types
of sleep.
Cx30 KO mice has compromised capability to maintain awake during SD
The minor changes under baseline conditions of Cx30 KOs point out the possibility
that these mice may show a more pronounced sleep response phenotype when
challenged by higher sleep pressure. To test this possibility, mice were subjected to 6h
SD in the CaReSs device with simultaneous EEG/EMG recordings. Despite the
continuous rotation of the CaReSs, the mice were able to fall asleep for very short
periods of time, mostly 10-20 sec long (see Materials and methods). The number
and episode duration of such short NREM episodes were measured. The Cx30 KO
mice showed an increasing tendency in the both parameters compared to the WT,
especially during the last 3 hours of the 6h SD (Figure 6A1, 6A2). Accordingly, the
total duration of NREM sleep for each hour of SD showed a significant increase in the
4th hour of SD (WT = 7.3 2.4 min, n = 6 mice; Cx30 KO = 20.4 3.4 min, n = 7
mice; Figure 6A3). Finally, the total NREM duration in Cx30 KOs drastically
increased by 1 fold compared to that in the WTs (58.1 12.6 min versus 28.1 6.1
min) during the last 3 hours of SD (Figure 6B). These findings indicate that Cx30 KO
mice may have difficulty to maintain the same arousal level as the WT mice,
especially during the second half of SD when sleep pressure is mounting so high that
they involuntarily enter more short NREM episodes than the WTs do. To confirm that
Cx30 KOs have compromised capability to maintain arousal in face of high sleep
pressure, we performed GSD, during which the animals are constantly maintained
awake, and thus the number of stimuli given during the SD period reflects the sleep
propensity of the animal. We found that the Cx30 KOs needed 50% more stimuli than
the WTs (WT = 8 1 stimuli, n = 6 mice; Cx30 KO = 12 1 stimuli, n = 6 mice)
during the 6 hours of GSD (Figure 7), indicating Cx30 KO mice indeed exhibited
higher sleep propensity and were less able to maintain arousal during high sleep
pressure.
Cx30 KO mice exhibit higher levels of slow wave activity during recovery sleep
As SWA (0-4 Hz) in NREM sleep reflects the amount of accumulated sleep pressure
during prior wakefulness and recovery sleep after sleep deprivation is associated with
a rebound of SWA (Dijk et al., 1987; Lancel et al., 1991; Tobler and Borbely, 1986),
we measured the percentage of SWA in the total NREM EEG spectrum (0.5-40 Hz)
of mice during the 4h recovery sleep (ZT6-ZT10) after the CaResS SD (Figure 8A).
!

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Figure 4. Hourly locomotor activity of WT and Cx30 KO mice over 24 hours under the
baseline condition. The shaded area represents the dark period from 19:00 to 7:00. The
activity level is expressed in arbitrary units (AU). Significant decreases were found at 13:00,
1:00 and 2:00 in Cx30 KO mice (n = 6) compared to WT mice (n = 6) (*p < 0.05, **p < 0.01).
Data for each mouse were collected and averaged over 6 consecutive days.

Figure 5. Cx30 KO mice displayed moderate changes in durations of baseline NREM and
REM sleep in the light period. The duration of SWS was significantly increased (A) while
that of PS was decreased (B) in the Cx30 KO mice (*p < 0.05).

!"#.!

The level of SWA was not different during baseline sleep between Cx30 KO and WT
mice (WT = 34.0% 0.3%, n = 6 mice; Cx30 = 37.1% 1.2%, n = 7 mice), while
both Cx30 KO and WT mice exhibited an enhancement in SWA during recovery
sleep (WT = 38.7% 0.8%; Cx30 = 44.7% 1.4%; Figure 8B). This rebound in the
SWA relatively to the baseline level was not different between WT and Cx30 KO
mice (13.3 2.4 versus 20.6 3.9%; Figure 8C, left panel). However, the percentage
of SWA in the total NREM spectrum was significantly bigger in the Cx30 KO mice
compared to that in WT mice, suggesting in the Cx30 KO mice experienced higher
sleep pressure in prior SD, which may partly explain their difficulty in maintaining
wakefulness during SD (Figure 8C, right panel).
Genes related to brain metabolism are downregulated in the Cx30 KO mice
As an attempt to identify the potential causes of the sleep-wake phenotypes of the
Cx30 KOs, we performed quantitative RT-PCR to examine the mRNA expression
levels of 14 selected genes related to brain energy metabolism with preferential
distributions in either astrocytes or neurons (Table 2). These genes mainly include
those involved in the astrocyte-neuron lactate shuttle (ANLS) (Pellerin and
Magistretti, 1994, 2012) and glycogen metabolism, which is exclusively carried out
by astrocytes (Brown, 2004). Two recent works have shown that many of these genes
are upregulated by SD (Petit et al., 2010, 2013), suggesting the metabolic pathways
involving these genes may be boosted to fulfil the metabolic demands during SD.
Given the substantially elevated sleep propensity of Cx30 KOs during SD, we asked
whether these genes have an altered expression in Cx30 KOs. We examined 6 brain
structures that are involved in the expression and/or regulation of sleep and
wakefulness: the frontal, sensory-motor and occipital cortices, hippocampus, thalamus,
and hypothalamus. We found that six of the genes listed in Table 2 showed changes
in mRNA expression: all of them were downregulated in Cx30 KOs compared WTs
under baseline conditions (Table 3). Among the six genes affected, four of them were
downregulated in multiple brain structures (Figure 9). Monocarboxylic acid
transporter 2 (Mct2) and protein targeting glycogen (PTG) are the most affected
genesthe former was downregulated in four structures and the latter in three. They
are followed by lactate dehydrogenase A (Ldha) and brain glycogen phosphorylase
(GPhos) that were affected in 2 structures. The extent of expression decreases in the
affected genes ranged from 9 to 20% (Table 3). Intriguingly, one of these genes,
Mct2, is mainly expressed by neurons. These results suggest that the ANLS and
astroglial glycogen metabolism pathways in Cx30 KOs might be hypofunctional,
which could be a potential cause of the sleep-wake behavioral deficits of the Cx30
KO mice.

Discussion
In this study, we showed that SD enhanced the mRNA expression of Cx30 and dye
coupling in cortical astrocytes, and the latter effect depended on neuronal activity and
the presence of Cx30. These observations corroborate our previous findings:
!

"#"!

Figure 6. Durations of NREM sleep during the 6-hour CaResS SD protocol. (A) Hourly
distributions of NREM sleep over the 6 hour of SD in Cx30 KO and WT mice. Compared to
WT mice, Cx30 mice exhibited an increasing tendency in both the number of NREM episodes
(A1) and the mean duration of NREM episodes (A2), and consequently a significant increase
in the duration of NREM sleep during the 4th hour of SD (A3) (*p < 0.05). (B) The total
NREM sleep duration during the last three hours of SD was significantly longer in the Cx30
KO compared to that in the WT mice (*p < 0.05).

Figure 7. Cx3O KO mice needed more stimuli than WT mice to be maintained awake during
gentle sleep deprivation (*p < 0.05).

!"##!

wakefulness-promoting modafinil enhanced Cx30 expression at mRNA and protein

levels and increased astroglial dye coupling in an activity dependent manner (Liu et
al., 2013; see Results Part I). Similarly, in the olfactory glomerulus astroglial dye
coupling is also regulated by neuronal activity and requires the presence of Cx30
(Roux et al., 2011). Therefore, increasing evidence indicates that Cx30 rather than
Cx43 mediates the response of the astroglial GJC as a function of neuronal activity
level, which is suggested to be elevated under conditions such as SD (Vyazovskiy et
al., 2009) and modafinil administration (Urbano et al., 2007).
We then studied the role of Cx30 in the regulation of sleep homeostasis by
characterizing the sleep phenotypes of Cx30 KOs. We found that compared to wild
type (WT) mice, Cx30 KO mice exhibited slightly higher amount of NREM but lower
amount of REM sleep during the light period under baseline conditions. More
strikingly, Cx30 KOs underwent substantially more NREM sleep than the WT mice
during instrumental SD in the CaReSs device, suggesting these mice are less capable
of maintaining arousal when challenged by high sleep pressure. This interpretation is
supported by the GSD experiment, showing the Cx30 KO mice needed more stimuli
to be maintained awake throughout the deprivation period. In addition, the differences
between WTs and Cx30 KOs were more pronounced during the last 3 hours of SD
(Figure 6). This may be due to the higher level of sleep pressure compared to the first
3 hours.
A few previous works have investigated other behavioral phenotypes of Cx30 KO
mice: these mice exhibit elevated anxiogenic behaviour in novel environments (Dere
et al., 2003) and impaired contextual fear memory (Pannasch et al., 2014). These
different behavioral deficits of Cx30 KO mice as well as the sleep-wake abnormality
we observed in our study point to the multi-functionality of Cx30, which is not
surprising given its expression throughout the brain (Nagy et al., 1999). Notably,
these studies used a Cx30 KO mouse model generated by Teubner et al. (2003), while
we used another recently developed model in our study (Boulay et al., 2013; see
Materials and methods). The former mouse model suffers from unspecific
elimination of Cx26 (Lynn et al., 2011), which is thought to be the cause of deafness
in these mice since half of Cx26 expression is preserved in the new model and these
mice have normal hearing (Boulay et al., 2013). Therefore, we took advantage of the
new mouse model to avoid potential unspecific deficits of the old model (Teubner et
al., 2003).
Finally, we probed the potential mechanisms by which Cx30 may affect sleep
homeostasis. We found that six genes related to brain energy metabolism had
decreased mRNA expression in multiple brain structures involved in sleep-wake
regulation of Cx30 KO mice. There are a few noteworthy points in this part of our
findings. First, the decreases in the gene expressions were relatively small, with the
highest reaching only 21% of reduction in PTG in the hippocampus. This is in
agreement with the lack of gross physiological deficits (by our observations; Dere et
!

"#$!

Figure 8. Slow wave activity (SWA) of Cx30 KO and WT mice during recovery sleep after SD. (A) The mean
NREM sleep spectrum (0.5-40 Hz) of 6 Cx30 KO and 7 WT mice during both baseline and recovery sleep from
ZT6 to ZT10. The data points represent the power spectrum of each 0.25-Hz bin expressed as the percentage of
the total EEG spectrum of NREM sleep. The area in light blue presents the frequency band of the SWA (0.5-4
Hz), and insert shows the EEG spectra in this frequency band in magnification. (B) The percentages of SWA,
which was summed from 0.5 Hz to 4 Hz, were increased in both Cx30 KO and WT mice after SD (*p < 0.05,
**p < 0.01). (C) During recovery sleep, the rebound in SWA relative to the baseline level was not different
between Cx30 KO and WT mice (left panel), whereas the percentage of SWA in the total NREM spectrum was
significant higher in the Cx30 KO mice compared the WT mice (right panel; *p < 0.05).

Table 2. The list of brain metabolism-related genes examined.

!"#%!

al., 2003; Pannasch et al., 2014) and minor baseline sleep-wake alterations in the
Cx30 KO mice: only when challenged by SD did these mice exhibit more pronounced
behavorial deficits. Second, one of the six genes affected, Mct2, is the isoform mainly
expressed by neurons. How does deletion of Cx30, a gene exclusively expressed by
astrocytes (Nagy et al., 1999; Kunzelmann et al., 1999), affect expression of these
neuronal genes? This might be a due to unknown functions of Cx30 in regulation of
gene expression, which has already been demonstrated for Cx43 (Iacobas et al., 2003,
2007). However, considering the absence of Cx30 in neurons, a more likely
explanation is that the downregulation in the neuronal genes is an indirect
consequence of impaired astroglial metabolic functions, or that neuronal genes are
downregulated as a result rather than a cause of sleep-wake phenotype of Cx30 KOs.
Third, genes expressed in the three cortical structures were less affected than those in
the three subcortical structures (Table 3). In particular, none of the examined genes
were affected in the frontal cortex, despite that it is the site which expresses the
highest sleep homeostatic response after SD (Finelli et al., 2000; Muzur et al., 2002).
A possible explanation may lie in the relative expression levels of Cx30 compared to
Cx43: the subcortical structures have generally higher relative expression of Cx30
compared the cortices, although with an exception of the occipital cortex (Figure 10).
Based on the results discussed above, we propose the following working hypothesis
of how Cx30 might contribute to regulation of sleep homeostasis: Cx30 mediates
neurometabolic coupling responses, either directly or indirectly, to sustain
wakefulness during periods of high sleep pressure. So far, our quantitative RT-PCR
experiments have identified that metabolism-related genes were affected in multiple
brain structures. The next step will be to determine which structure might be the
major contributor to the Cx30 KO phenotype by screening for neurotransmission
systemsin particular those known to promote wakefulnessthat could be altered in
Cx30 KO mice. Promising candidates include the orexinergic system in the
hypothalamus and the noradrenergic system in the locus coeruleus: the orexin neurons
(Parsons and Hirasawa, 2010) and the noradrenergic neurons (Tang et al., 2014) are
able to sense extracellular lactate and their firing rates are positively correlated with
lactate concentration. These observations suggest that lactate might play a role in
promoting wakefulness. Therefore, a direct test of our hypothesis would be to perform
in vivo injections of lactate immediately before SD to see whether lactate would
rescue the deficits of the Cx30 KOs during SD.

"#&!

Table 3. Levels of the affected genes in different brain structures of Cx30 KO mice expressed
as the percentages of those in the WT mice. The genes shown here were significantly
downregulated in one or more than one brain structures of the Cx30 KO mice (n = 7)
compared to the WT mice (n = 9) (*p < 0.05, **p < 0.01, ***p < 0.001). MCT2 that is mainly
expressed by neurons is marked in red. Note that none of the genes were affected in the
frontal cortex (marked in light green).

Figure 9. The expression levels of the four most frequently altered genes (MCT2, PTG, Ldha
and Gphos) in Cx30 KO mice in their respective affected brain regions. The mRNA level of
each gene was normalized against #-actin or cyclophilin A (*p < 0.05, **p < 0.01, ***p <
0.001).

!"#'!

Figure 10. Comparison of Cx30 and Cx43 mRNA expressions in the 6 brain structures
studied (***p < 0.001).

"#(!

.
.

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.
...................................

General discussion and

perspectives

________________

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!

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When considering the dynamic interactions between neurons and astrocytes, the fact
that these glial cells are organized into spatially and functionally restricted networks
through gap junction channels (GJCs) (Giaume et al., 2010) raises the possibility that
behaviors such as sleep involving large neuronal ensembles may need coordinated
action of astrocytes on the network scale. The behavioral consequences of possible
astroglial interactions at the network level have begun to be addressed in animal
knockout (KO) models of astroglial Cxs (e.g., in hippocampus-dependent learning
and memory, see Lutz et al., 2009). Therefore, the goal of my thesis work was to
investigate the reciprocal neuroglial interaction at the network level in sleep-wake
regulation. Based on this objective, I addressed the following questions: are the
GJC-mediated astroglial networks regulated by changes in sleep-wake states and do
they contribute to sleep-wake regulation? As presented in the Results part of my
thesis, I have provided the following answers to these two questions: 1)
GJC-mediated astroglial networks are bidirectionally regulated by pharmacological
agents affecting the sleep-wake cycle; 2) instrumentally-induced changes in sleep
homeostasis in mice also modulate GJC-mediated astroglial networks; 3) one of the
two astroglial Cxs, Cx30, play a key role in these regulations; 4) the inactivation of
the gene encoding for Cx30 affects sleep homeostasis of mice: Cx30 deficiency
impairs the capability of mice to maintain wakefulness when challenged by high sleep
pressure. How Cx30 may contribute to these observed phenomena is the focus of the
following discussion.

!"5 B(11+H.1,9X,2%,2,1+A'(128,2)&.K.&*,3+&+)&('T, ,
One significant feature of astroglial networking through GJCs is its dependence on
neuronal-activity: it suggests that the astroglial networking is able to respond to
changes in neuronal activity and to make adaptations to fulfill the roles of astrocytes
in important brain functions such as neurometabolic and neurovascular coupling. The
extent of the gap junctional communication differs according to the level of neuronal
activity has been demonstrated in the hippocampus for trafficking of energy
metabolites (Rouach et al., 2008) and in the olfactory glomerulus with dye coupling
(Roux et al., 2011). The findings of my thesis work that modafinil (Liu et al., 2013;
Results Part I) and sleep deprivation (SD) (Results Part III) increase astroglial dye
coupling in an activity-dependent manner further demonstrates that gap junctional
communication in astrocytes is capable of adapting to physiologically demanding
situations that involves elevated neuronal activity such as in the presence of high
sleep pressure. More importantly, a reoccurring observation of these and some other
studies is that the dynamic regulation of astroglial networking in response to neuronal
!"$.!

activity is mediated by Cx30, not Cx43: Cx30 underlies the activity-dependence of

astroglial dye coupling in the olfactory glomerulus (Roux et al., 2011); the sleep
deprivation-induced increase in astroglial dye coupling in the somatosensory cortex
depends on the presence of Cx30 (Results Part III); gene expression of Cx30 is
enhanced in the brains of mice raised in an enriched environment (Rampon et al.,
2000). Altogether, these observations led us to propose that Cx30 may act as a
neuronal activity sensor to dynamically regulate the extent GJC-mediated networking
in astrocytes according to the level of neuronal activity.
The functional consequences of this regulation may be multifaceted. First, considering
that astroglial networking via GJCs is engaged in energy metabolite trafficking
(Rouach et al., 2008), Cx30 may instruct the enhancement of intercellular trafficking
to fulfill the increased energy demand for sustaining elevated neuronal activity during
challenging situations such as SD. What remains unknown is whether Cx30 itself
constitutes part of the enhanced gap junctional communication or only serves as a
signal transducer. Second, Cx30 may as well mediate recruitment of intracellular
pathways other than gap junctional communication. This hypothesis is supported by
our finding that multiple genes related to brain energy metabolism are downregulated
at the transcription level in Cx30 KO mice (Results Part III). This finding also
suggests that Cx30 has an unidentified role in transcriptional regulation of gene
expression. So far the non-channel functions of Cxs have been better documented for
Cx43 (see Introduction II.3.5), which are thought to take place via interactions with
intracellular proteins such as cytoskeletal proteins and transcription factors (Herve et
al., 2012). There is evidence that Cx30 also interacts with cytoskeletal proteins
including actin and tubulin (Qu et al., 2009), which may mediate a recently identified
channel-independent role of Cx30 in astroglial morphology (Pannasch et al., 2014). In
addition, Cx30 has been demonstrated to bind to the PDZ domain-containing protein
zonula occludens-1 (Penes et al., 2005) that is involved in signal transduction and
transcriptional modulation (Bauer et al., 2010), suggesting that Cx30 may be part of
the molecular machinery that regulates gene expression. These converging lines of
evidence indicate that Cx30 may act on multiple levels including GJC-mediated
metabolite trafficking and intracellular metabolism pathways to boost energy supply
in response to elevated neuronal activity during challenging situations such as SD. In
addition to a global upregulation of brain metabolism that sustain neuronal function
during periods of high sleep pressure, Cx30 might exert a local but more powerful
influence on specific neural circuits where Cx30 expression is enriched and that are
known to control sleep and wake via enhanced metabolite production. The orexinergic
neurons in the hypothalamus and the noradrenergic neurons in the locus cereolus are
likely candidates: both types of neurons are known to promote wakefulness (see
Introduction III.2.1) and they both sense and are excited by astroglial lactate
(Parsons and Hirasawa, 2010; Tang et al., 2014) the preferred energy substrate of
neurons (Pellerin and Magistretti, 2012). Intriguingly, lactate excites noradrenergic
neurons like a gliotransmitter rather than as a metabolite (Tang et al., 2014). If it is
true that lactate is indeed a yet-unidentified gliotransmitter, it can be inferred that
!

"$"!

Cx30 may not be simply engaged in metabolic support but also mediate reciprocal
signaling with neurons via activity-dependent release of lactate.
My thesis work has focused on the regulation of sleep homeostasis. Considering the
ubiquitous expression of the Cx30 in the the grey matter (Nagy et al., 1999), it is not
surprising to find that Cx30 is implicated in other animal behaviors (reviewed in
Pannasch and Rouach, 2013; see also Introduction II.3.4). This is consistent with our
finding that multiple mouse brain structures show downregulation of
metabolism-related genes (Results Part III) in Cx30 KO mice. Apart from their roles
in sleep-wake regulation, all the brain structures we examined bear other important
functions: e.g., the hippocampus is involved spatial learning and memory; the
thalamus is the primary sensory relay to the cortex; the sensory-motor cortex mediate
sensory information processing and motor control. Thus the downregulated
expression of metabolism-related genes could be a universal mechanism underlying
the diverse behavioral deficits of Cx30 KO mice.
What remains to be identified is how Cx30 may sense the changes in neuronal
activity: what are the neuronal signals that Cx30 senses and what is the signaling
pathways in astrocytes that acts on Cx30? In addition, our finding that a neuronal
gene, MCT2, is downregulated in Cx30 KO mice (Results Part III) raises another
equally unclear issue: how does Cx30 located in astrocytes affect gene expression in
neurons?

!"9 P.44+'+1&.28,'(8+%,(4,BH9X,213,BH;9, ,
There is now accumulating evidence indicating that Cx30 and Cx43 play differential
roles in various brain functions. For example, Cx30 KO mice display a higher anxiety
level in novel environments (Dere et al., 2003), whereas Cx43 KO mice exhibit an
anxiolytic behavior (Frisch et al., 2003). Also, the Cx30 KO mice (Pannasch et al.,
2014) and double KO mice of both Cxs (Pannasch et al., 2011) show opposite
alterations in excitatory synaptic transmission, indicating Cx43 and Cx30 modulate
synaptic transmissions in the opposite directions. Our findings that the expression of
Cx30 but not 43 is upregulated by modafinil and SD as well as the Cx30-mediated
activity dependence of GJCs (Results Part I, III; Liu et al., 2013; Roux et al., 2011)
also point out the divergent roles of the two Cxs. These observations may partly
answer why two different Cxs are expressed in one cell type. Such functional
divergence of astroglial Cx30 and Cx43 probably originates from different functions
at the molecular level. For instance, Cx43 can form hemichannels HCs under both
physiological (Roux et al., in revision; Stehberg et al., 2012; Torres et al., 2012) and
pathological conditions (Kang et al., 2008; Garr et al., 2010; Orellana et al., 2011a,
b), whereas so far the HC function has not been reported for Cx30 in astrocytes,
although Cx30 can form functional hemichannels in transfected cells and in Xenopus
oocytes (Valiunas and Weingart, 2000; Hansen et al., 2014).
!"$#!

ATP, as an important gliotransmitter, has been shown to be released through astroglial

vesicles (Halassa et al., 2009) and modulate cortical slow oscillations in vivo (Fellin
et al., 2009) and cortical up states in vitro (Poskanzer and Yuste, 2011). Recently,
adenosine derived from Cx43 HC-mediated ATP release is observed to increase up
states of slow oscillations recorded in acute slices of the olfactory glomerulus (Roux
et al., in revision). This is consistent with a massive decrease of slow oscillations in
the olfactory bulb in the double KO mice of Cx43 and Cx30 (Roux et al., in revision).
In addition, slow oscillations are also decreased during NREM sleep in the prefrontal
cortex in the double KO mice (Roux et al., in revision). These findings suggest that
Cx43 HC-mediated ATP release might contribute to sleep-related slow oscillations
and NREM sleep is likely to be decreased in Cx43 KO mice, although it awaits
confirmation in the Cx43 KO mice and in vivo characterization of the sleep-wake
behavior of these mice. This is apparently contrary to what we found in the Cx30 KO
mice, which display more baseline NREM sleep and increased slow wave activity in
recovery sleep (Results Part III). As discussed above, the differential roles in the
regulation of slow oscillations/slow wave activity by Cx30 and Cx43 probably result
from their distinct functions in metabolic support and ATP release, respectively. The
unresolved issue is how to reconcile these apparently contrary functions of the two
astroglial Cxs. Do they act in competition? Does one of them take a dominant role
over the other under specific conditions? Such questions await more detailed
comparative analysis of single Cx KO mice for a particular brain function.
In addition to their molecular functions, Cx43 and Cx30 also show distinct expression
patterns. It is known that the expression of Cx43 begins around embryonic day 12
while Cx30 becomes detectable during the third postnatal week in rodents
(Kunzelmann et al., 1999). The early developmental emergence of Cx43 is consistent
with its crucial role in radial migration of cortical neurons (Elias et al., 2007; Cina et
al., 2009). Following the same line of reasoning, its late postnatal appearance may
imply a role of Cx30 in neurodevelopmental events that occur in the same time
window. Interestingly, while cultured astrocytes only express Cx43, they start to
express Cx30 when they are cocultured with neurons (Koulakoff et al., 2008)
suggesting that their presence and their differentiation is required. Taking the cortex
for example. At the late embryonic and early postnatal stages when cortical neurons
have finished migration into their target regions, neural stem cells migrate into the
cortex, proliferate locally and differentiate into astrocytes (Namihira and Nakashima,
2013). Then during the second and third postnatal week synaptogenesis and synaptic
pruning take place to wire the cortical neurons, a process in which astrocytes are
known to participate (Clarke and Barres, 2013; Chung et al., 2013). The facts that
Cx30 appears only after astrocyte differentiation (compared to the onset of Cx43 in
undifferentiated radial glia) and that its onset coincides with the period of
synaptogenesis imply a possible function of Cx30 in the wiring of neuronal circuits.
In addition, neuronal activity is necessary for guidance of wiring and fine-tuning of
neuronal connections (Yamamoto and Lopez-Bendito, 2012). Thus Cx30, being able
to mediate activity-dependent responses of astroglial GJCs, and perhaps other
!

"$$!

astroglial signaling pathways, might contribute to the coordination of astrocytes in

their regulation of neural circuit formation.
One prominent feature of astroglial networking is their preferential connections within
neuronal functional units compared to areas outside the units, which has been
demonstrated for the barrels of the somatosensory cortex (Houades et al., 2008) and
the olfactory glomerulus (Roux et al., 2011). Such compartmented spatial
organization of astroglial networking is based on the enrichment of the two astroglial
Cxs within these functional units. For the olfactory glomerulus, it is demonstrated that
Cx30 mRNA expression is higher than Cx43 (Nagy et al., 1999). Moreover, there is
even a sub-functional unit difference in the expression of the two Cxs: Cx43 is
preferentially expressed at the inner edge of the glomerular neuropile whereas Cx30 is
more enriched towards the central part of the glomerulus where neuronal activity
takes place (Roux et al., 2011). Considering the olfactory glomerulus as well as the
barrels of the somatosensory cortex is the site where synapses are enriched, the
activity-sensitive Cx30 may play a more important role than Cx43 in the spatial
organization of the astroglial networks. Combined with the above-discussed
implications of developmental onset of Cx30, it can be further speculated that such
overlapping between neural circuits and astroglial networks might be a result of
coordinated wiring of the two networks during development and that Cx30 might an
important contributor to such coordination.
Finally, Cx30 and Cx43 are differentially distributed in the brain. As shown by an
early study (Nagy et al., 1999) and confirmed by our observations (Results Part III),
Cx30 is generally more expressed in subcortical structures compared to Cx43. Their
differential distributions suggest that Cx30 and Cx43 may display different functional
importance depending on the brain region. Indeed, in contrast to neocortical
astrocytes in which Cx43 outweighs Cx30 in both the expression level and
contribution to GJC-mediated dye coupling (Results Part III), a recent study showed
that the thalamic astrocytes highly express Cx30 compared to Cx43, with the former
contributing to about 70% of astroglial gap junctional communication (Griemsmann
et al., 2014). Previously, the relative contribution of the two astroglial Cxs to gap
junctioanl communication have been rarely addressed in brain structures other than
the cortex and hippocampus (Cx30 KO mice preserve 50% dye coupling in the
hippocampus; see Rouach et al., 2008; Pannasch et al., 2014). Consequently, it was
generally thought that Cx30 constitutes a minor portion of gap junctioanl
communications in astrocytes. This recent work in thalamus point to the possibility
that Cx30 may play a primary role compared to Cx43 in certain brain functions
carried out by brain structures where Cx30 is enriched. This is likely the case for
sleep-wake homeostatic regulation. Most of the important nuclei that are involved in
sleep-wake control are located in subcortical structures such as the hypothalamus and
the brain stem (see Introduction III.2), where Cx30 is also abundantly expressed
(Nagy et al., 1999). Thus Cx30 may exert an influence on sleep and wake primarily
on the subcortical level, whereas Cx43 may act more on the cortical level likely
!"$%!

through hemichannel-dependent ATP release that contributes to adenosine

accumulation (for the role of adenosine in sleep homeostasis, see Introduction
III.3.1).

!"; D(:+,&+)71.)28,.%%A+%, ,
Although the initial goal of my thesis was to investigate the contribution of Cx30 as
part of astroglial GJCs in sleep regulations, our finding that Cx30 inactivation results
in downregulation of expression of some genes as discussed above strongly suggests a
non-channel function of Cx30. Therefore, to further elucidate whether Cx30 acts in
GJC-dependent or -independent manner, more specific genetic tools are needed, such
as the Cx30T5M/T5M mice that target only the channel function of Cx30 (Grifa et al.,
1999). As articulated above that the two astroglial Cxs very likely have different roles
depending on the brain region, and Cx43, in particular, has a crucial role in embryonic
development (Wiencken-Barger et al., 2007), one common problem faced by the
studies that investigate astroglial Cx functions in vivo is the use of constitutional Cx
KO models that suffer from developmental effects and lack of spatial and temporal
specificity. Therefore, conditional transgenetic models that have brain
region-specificity and are temporally controllable are necessary. Finally, a solution for
discriminating GJC and HC functions of Cx43 has begun to emerge: peptides such as
TAT-L2 and GAP19 that target the L2 region of the Cx43 C-terminus have been
shown to specially inhibit HCs without affecting GJCs (reviewed in Iyyathurai et al.,
2013; Abudara et al., in revision). These peptides are of great value for in vitro studies,
and they have been applied in vivo in several recent works (Stehberg et al., 2012;
Wang et al., 2013), although their stability and efficiency in vivo need to be further
tested.

""# C+'%3+)&.>+%! !
I have accomplished the aim of my thesis work to establish an association between
changes in sleep-wake states and GJC-mediated astroglial and to identify the role of
Cx30 in sleep homeostasis. The findings of my thesis work have also revealed a few
open questions that are worthy of future investigations.

!!"# P(, &7+, 2)&.(1%, (4, :(324.1.8, 213, 0+1+'28,

21+%&7+&.)%, (1, 2%&'(08.28, )(11+H.1, )7211+8%,
)(1%&.&A&+,-2'&,(4,&7+.',)8.1.)28,+44+)&%T,
As a recent work demonstrated that several general anesthetics specifically impair
astroglial Ca2+ signaling without affecting neuronal activity at low dosages (Thrane et
al., 2012), it is likely that astrocytes may independently contribute to the sedative
action of the anesthetics. Accordingly, one open issue of my thesis work is whether
the effects of modafilnil and the three general anesthetics on astroglial Cx channels
!

"$&!

contribute to their clinical effects. This possibility can be tested, for example, by
examining whether the clinical effects of modafinil and the anesthetics are
compromised or not in Cx KO mice. Such investigations would provide valuable
knowledge on the action mechanisms of these widely used drugs.

!!"5 $, '(8+, (4, )(11+H.1, 7+:.)7211+8%, .1, %8++-,

'+0A82&.(1T, ,
Although my thesis work has focused on Cx30, our finding that two general
anesthetics, propofol and ketamine, strongly inhibit astroglial HC activity raises the
possibility that astroglial HCs, which are composed by Cx43 and pannexins, might be
also involved in sleep regulations (Results Part II). Furthermore, as discussed above,
Cx43 HC-mediated ATP release modulates slow oscillations in the olfactory
glomerulus, and slow oscillations are decreased during NREM sleep in the prefrontal
cortex in the double KO mice (Roux et al., in revision). These findings suggest that
Cx43 HC-mediated ATP release might contribute to regulation of sleep homeostasis.
Therefore, it would be revealing to compare ATP release and adenosine concentration
in Cx43 KO mice before and during SD. Ideally, transgenetic models or mimetic
peptides that specifically targets hemichannel forming of Cx43 should be used.

!!"9 Y2)&2&+, 2%, 2, Q+*, :+3.2&(', (4, BH9X, 4A1)&.(1, .1,

1+A'(08.28,.1&+'2)&.(1%T, ,
The downregulated expression of the energy metabolism-related genes suggests a
hypofunction of brain metabolism and possibly decreased concentrations of energy
metabolites such as glucose and lactate in the Cx30 KO mice. We speculate that
lactate is likely the energy metabolite underlying the sleep phenotypes of the Cx30
KO mice, for several reasons: it plays a central role in the neurometabolic coupling
(see Introduction I.2.5); in Cx30 KO mice, the expressions of MCT2 and Ldha that
are directly associated with lactate metabolism and several genes in astroglial
glycogenolysis that can also lead to production of lactate are downregulated (see
Results Part III); it is closely associated with sleep homeostasis and it directly
influences the activity of the wakefulness-promoting orexin and noradrenergic
neurons (see Introduction IV.2.2).
Apart from sleep-wake regulation, alterations in lactate metabolism might also
contribute to other behavioral phenotypes of Cx30 KO mice, such as impairment in
hippocampus-dependent learning and memory (Pannasch et al., 2014). Production of
lactate from astroglial glycogenolysis and its trafficking to neurons have been
demonstrated to be necessary for long-term memory formation (Suzuki et al., 2011;
Newman et al., 2011). Moreover, we have found relatively big (15-20%) decreases in
the expressions of Ldha and another two genes related to glycogenolysis in the
hippocampus of Cx30 KO mice (see Results Part III), suggesting impaired
glycogenolysis and lactate production in the hippocampus of these mice.
!"$'!

Altogether, these observations suggest that lactate is very likely a downstream

effector of the action of Cx30 in mediating the coupling between astroglial energy
metabolism and neuronal activity. A direct test of this hypothesis would be to
compare in vivo concentrations of lactate in WT and Cx30 KO mouse brains and to
determine whether injection of lactate in vivo would rescue the behavioral deficits of
Cx30 KO mice.

!!"; $%&'(08.28, )(11+H.1%, .1, :(3A82&.(1, (4, <'2.1,

(%).882&.(1%T, ,
So far, I have presented and discussed evidence that both astroglial Cxs participate in
sleep regulations, and probably in learning and memory as well (as demonstrated by
studies of the double KO mice; see Introduction II.3.4). The remaining question is
how neurons behave in the absence of astroglial Cxs. One mechanism underlying the
behavioral phenotypes of Cx KO mice may be alterations of brain oscillations. In
agreement with this view, vesicular release of gliotransmitters in astrocytes has been
shown to impact on slow oscillations (Fellin et al., 2009; Halassa et al., 2009) and
gamma rhythms (Lee et al., 2014), consequently modulating sleep homeostasis
(Halassa et al., 2009) and recognition memory (Lee et al., 2014), respectively. For
astroglial Cxs, there are several possible mechanisms by which they may contribute to
astroglial modulations of brain oscillations. Both Cxs may function as GJCs to
regulate extracellular ion homeostasis to modulate slow oscillations (see
Introduction IV.3). As a study has shown that slow oscillations in brain slices
depend on neuronal metabolic states (Cunningham et al., 2006), GJCs may also
contribute via its role in energy metabolite trafficking (see Introduction II.3.3) and
Cx30 may act additionally through its transcriptional regulation of metabolism related
genes. Finally, Cx43 can contribute to gliotransmission, especially ATP release, as
hemichannels to regulate slow oscillations. What remains unaddressed is their role in
fast oscillations, such as theta, gamma, and beta, which are associated with learning
and memory (Colgin, 2013; Igarashi et al., 2014; Yamamoto et al., 2014).

"$(!

Bibliography

Abudara, V., Bechberger, J., Freitas-Andrade, M., De_Bock, M., Wang, N., Bultynck, G., Naus,
C., Leybaert, L and Giaume, C. (In revision). The connexin43 mimetic peptide Gap19
inhibits hemichannels without altering gap junctional communication in astrocytes.
Adamantidis, A.R., Zhang, F., Aravanis, A.M., Deisseroth, K., and de Lecea, L. (2007). Neural
substrates of awakening probed with optogenetic control of hypocretin neurons.
Nature 450, 420-424.
Adermark, L., and Lovinger, D.M. (2008). Electrophysiological properties and gap junction
coupling of striatal astrocytes. Neurochem Int 52, 1365-1372.
Aeschbach, D., Cutler, A.J., and Ronda, J.M. (2008). A role for non-rapid-eye-movement sleep
homeostasis in perceptual learning. J Neurosci 28, 2766-2772.
Aguado, F., Espinosa-Parrilla, J.F., Carmona, M.A., and Soriano, E. (2002). Neuronal activity
regulates correlated network properties of spontaneous calcium transients in
astrocytes in situ. J Neurosci 22, 9430-9444.
Agulhon, C., Petravicz, J., McMullen, A.B., Sweger, E.J., Minton, S.K., Taves, S.R., Casper,
K.B., Fiacco, T.A., and McCarthy, K.D. (2008). What is the role of astrocyte calcium
in neurophysiology? Neuron 59, 932-946.
Agulhon, C., Fiacco, T.A., and McCarthy, K.D. (2010). Hippocampal short- and long-term
plasticity are not modulated by astrocyte Ca2+ signaling. Science 327, 1250-1254.
Alam, M.N., Kumar, S., Rai, S., Methippara, M., Szymusiak, R., and McGinty, D. (2009). Role
of adenosine A(1) receptor in the perifornical-lateral hypothalamic area in
sleep-wake regulation in rats. Brain Res 1304, 96-104.
Allaman, I., Belanger, M., and Magistretti, P.J. (2011). Astrocyte-neuron metabolic
relationships: for better and for worse. Trends Neurosci 34, 76-87.
Alle, H., Roth, A., and Geiger, J.R. (2009). Energy-efficient action potentials in hippocampal
mossy fibers. Science 325, 1405-1408.
Amiry-Moghaddam, M., and Ottersen, O.P. (2003). The molecular basis of water transport in
the brain. Nat Rev Neurosci 4, 991-1001.
Amzica, F., and Neckelmann, D. (1999). Membrane capacitance of cortical neurons and glia
during sleep oscillations and spike-wave seizures. J Neurophysiol 82, 2731-2746.
Amzica, F., and Massimini, M. (2002). Glial and neuronal interactions during slow wave and
paroxysmal activities in the neocortex. Cereb Cortex 12, 1101-1113.
Amzica, F. (2002). In vivo electrophysiological evidences for cortical neuron-glia interactions
during slow (<1 Hz) and paroxysmal sleep oscillations. J Physiol Paris 96, 209-219.
Anaclet, C., Parmentier, R., Ouk, K., Guidon, G., Buda, C., Sastre, J.P., Akaoka, H., Sergeeva,
O.A., Yanagisawa, M., Ohtsu, H., et al. (2009). Orexin/hypocretin and histamine:
distinct roles in the control of wakefulness demonstrated using knock-out mouse
models. J Neurosci 29, 14423-14438.
Andersson, M., Blomstrand, F., and Hanse, E. (2007). Astrocytes play a critical role in
transient heterosynaptic depression in the rat hippocampal CA1 region. J Physiol
585, 843-852.
Angulo, M.C., Kozlov, A.S., Charpak, S., and Audinat, E. (2004). Glutamate released from glial
cells synchronizes neuronal activity in the hippocampus. J Neurosci 24, 6920-6927.
Araque, A., Sanzgiri, R.P., Parpura, V., and Haydon, P.G. (1998). Calcium elevation in
astrocytes causes an NMDA receptor-dependent increase in the frequency of
miniature synaptic currents in cultured hippocampal neurons. J Neurosci 18,
6822-6829.
Araque, A., Parpura, V., Sanzgiri, R.P., and Haydon, P.G. (1998). Glutamate-dependent
astrocyte modulation of synaptic transmission between cultured hippocampal
neurons. Eur J Neurosci 10, 2129-2142.
Araque, A., Parpura, V., Sanzgiri, R.P., and Haydon, P.G. (1999). Tripartite synapses: glia, the
unacknowledged partner. Trends Neurosci 22, 208-215.
!"$,!

Araque, A., Carmignoto, G., and Haydon, P.G. (2001). Dynamic signaling between astrocytes
and neurons. Annu Rev Physiol 63, 795-813.
Araque, A., Carmignoto, G., Haydon, P.G., Oliet, S.H., Robitaille, R., and Volterra, A. (2014).
Gliotransmitters travel in time and space. Neuron 81, 728-739.
Arrigoni, E., Rainnie, D.G., McCarley, R.W., and Greene, R.W. (2001). Adenosine-mediated
presynaptic modulation of glutamatergic transmission in the laterodorsal tegmentum.
J Neurosci 21, 1076-1085.
Arrigoni, E., Chamberlin, N.L., Saper, C.B., and McCarley, R.W. (2006). Adenosine inhibits
basal forebrain cholinergic and noncholinergic neurons in vitro. Neuroscience 140,
403-413.
Aston-Jones, G., and Bloom, F.E. (1981). Activity of norepinephrine-containing locus
coeruleus neurons in behaving rats anticipates fluctuations in the sleep-waking cycle.
J Neurosci 1, 876-886.
Attwell, D., and Laughlin, S.B. (2001). An energy budget for signaling in the grey matter of the
brain. J Cereb Blood Flow Metab 21, 1133-1145.
Attwell, D., Buchan, A.M., Charpak, S., Lauritzen, M., Macvicar, B.A., and Newman, E.A.
(2010). Glial and neuronal control of brain blood flow. Nature 468, 232-243.
Axmacher, N., Helmstaedter, C., Elger, C.E., and Fell, J. (2008). Enhancement of
neocortical-medial temporal EEG correlations during non-REM sleep. Neural Plast
2008, 563028.
Ayers, N.A., Kapas, L., and Krueger, J.M. (1996). Circadian variation of nitric oxide synthase
activity and cytosolic protein levels in rat brain. Brain Res 707, 127-130.
Backus, K.H., Kettenmann, H., and Schachner, M. (1989). Pharmacological characterization of
the glutamate receptor in cultured astrocytes. J Neurosci Res 22, 274-282.
Bak, L.K., Schousboe, A., and Waagepetersen, H.S. (2006). The glutamate/GABA-glutamine
cycle: aspects of transport, neurotransmitter homeostasis and ammonia transfer. J
Neurochem 98, 641-653.
Ballon, J.S., and Feifel, D. (2006). A systematic review of modafinil: Potential clinical uses and
mechanisms of action. J Clin Psychiatry 67, 554-566.
Bardoni, R., Ghirri, A., Zonta, M., Betelli, C., Vitale, G., Ruggieri, V., Sandrini, M., and
Carmignoto, G. (2010). Glutamate-mediated astrocyte-to-neuron signalling in the rat
dorsal horn. J Physiol 588, 831-846.
Barros, L.F., Courjaret, R., Jakoby, P., Loaiza, A., Lohr, C., and Deitmer, J.W. (2009).
Preferential transport and metabolism of glucose in Bergmann glia over Purkinje
cells: a multiphoton study of cerebellar slices. Glia 57, 962-970.
Basheer, R., Halldner, L., Alanko, L., McCarley, R.W., Fredholm, B.B., and Porkka-Heiskanen,
T. (2001). Opposite changes in adenosine A1 and A2A receptor mRNA in the rat
following sleep deprivation. Neuroreport 12, 1577-1580.
Basheer, R., Bauer, A., Elmenhorst, D., Ramesh, V., and McCarley, R.W. (2007). Sleep
deprivation upregulates A1 adenosine receptors in the rat basal forebrain.
Neuroreport 18, 1895-1899.
Baud, M.O., Magistretti, P.J., and Petit, J.M. (2013). Sustained sleep fragmentation affects
brain temperature, food intake and glucose tolerance in mice. J Sleep Res 22, 3-12.
Bauer, H., Zweimueller-Mayer, J., Steinbacher, P., Lametschwandtner, A., and Bauer, H.C.
(2010). The dual role of zonula occludens (ZO) proteins. J Biomed Biotechnol 2010,
402593.
Beattie, E.C., Stellwagen, D., Morishita, W., Bresnahan, J.C., Ha, B.K., Von Zastrow, M.,
Beattie, M.S., and Malenka, R.C. (2002). Control of synaptic strength by glial
TNFalpha. Science 295, 2282-2285.
Becker, D., Zahn, N., Deller, T., and Vlachos, A. (2013). Tumor necrosis factor alpha maintains
denervation-induced homeostatic synaptic plasticity of mouse dentate granule cells.
Front Cell Neurosci 7, 257.
Belanger, M., Allaman, I., and Magistretti, P.J. (2011). Brain energy metabolism: focus on
astrocyte-neuron metabolic cooperation. Cell Metab 14, 724-738.
Bennay, M., Langer, J., Meier, S.D., Kafitz, K.W., and Rose, C.R. (2008). Sodium signals in
cerebellar Purkinje neurons and Bergmann glial cells evoked by glutamatergic
synaptic transmission. Glia 56, 1138-1149.
!

"$-!

Bennett, M.V., Contreras, J.E., Bukauskas, F.F., and Saez, J.C. (2003). New roles for
astrocytes: gap junction hemichannels have something to communicate. Trends
Neurosci 26, 610-617.
Bennett, M.V., Garre, J.M., Orellana, J.A., Bukauskas, F.F., Nedergaard, M., and Saez, J.C.
(2012). Connexin and pannexin hemichannels in inflammatory responses of glia and
neurons. Brain Res 1487, 3-15.
Bergami, M., Santi, S., Formaggio, E., Cagnoli, C., Verderio, C., Blum, R., Berninger, B.,
Matteoli, M., and Canossa, M. (2008). Uptake and recycling of pro-BDNF for
transmitter-induced secretion by cortical astrocytes. J Cell Biol 183, 213-221.
Bernardinelli, Y., Magistretti, P.J., and Chatton, J.Y. (2004). Astrocytes generate
Na+-mediated metabolic waves. Proc Natl Acad Sci U S A 101, 14937-14942.
Berner, I., Schabus, M., Wienerroither, T., and Klimesch, W. (2006). The significance of sigma
neurofeedback training on sleep spindles and aspects of declarative memory. Appl
Psychophysiol Biofeedback 31, 97-114.
Beyer, E.C., Paul, D.L., and Goodenough, D.A. (1990). Connexin family of gap junction
proteins. J Membr Biol 116, 187-194.
Bezzi, P., Gundersen, V., Galbete, J.L., Seifert, G., Steinhauser, C., Pilati, E., and Volterra, A.
(2004). Astrocytes contain a vesicular compartment that is competent for regulated
exocytosis of glutamate. Nat Neurosci 7, 613-620.
Binder, D.K., and Steinhauser, C. (2006). Functional changes in astroglial cells in epilepsy.
Glia 54, 358-368.
Bjorness, T.E., Kelly, C.L., Gao, T., Poffenberger, V., and Greene, R.W. (2009). Control and
function of the homeostatic sleep response by adenosine A1 receptors. J Neurosci
29, 1267-1276.
Blanco-Centurion, C., Xu, M., Murillo-Rodriguez, E., Gerashchenko, D., Shiromani, A.M.,
Salin-Pascual, R.J., Hof, P.R., and Shiromani, P.J. (2006). Adenosine and sleep
homeostasis in the Basal forebrain. J Neurosci 26, 8092-8100.
Blanco-Centurion, C., Gerashchenko, D., and Shiromani, P.J. (2007). Effects of
saporin-induced lesions of three arousal populations on daily levels of sleep and
wake. J Neurosci 27, 14041-14048.
Blomstrand, F., and Giaume, C. (2006). Kinetics of endothelin-induced inhibition and glucose
permeability of astrocyte gap junctions. J Neurosci Res 83, 996-1003.
Boison, D. (2008). Adenosine as a neuromodulator in neurological diseases. Curr Opin
Pharmacol 8, 2-7.
Boissard, R., Gervasoni, D., Schmidt, M.H., Barbagli, B., Fort, P., and Luppi, P.H. (2002). The
rat ponto-medullary network responsible for paradoxical sleep onset and
maintenance: a combined microinjection and functional neuroanatomical study. Eur
J Neurosci 16, 1959-1973.
Boissard, R., Fort, P., Gervasoni, D., Barbagli, B., and Luppi, P.H. (2003). Localization of the
GABAergic and non-GABAergic neurons projecting to the sublaterodorsal nucleus
and potentially gating paradoxical sleep onset. Eur J Neurosci 18, 1627-1639.
Bonansco, C., Couve, A., Perea, G., Ferradas, C.A., Roncagliolo, M., and Fuenzalida, M.
(2011). Glutamate released spontaneously from astrocytes sets the threshold for
synaptic plasticity. Eur J Neurosci 33, 1483-1492.
Borbely, A.A. (1982). A two process model of sleep regulation. Hum Neurobiol 1, 195-204.
Borbely, A.A., and Tobler, I. (1989). Endogenous sleep-promoting substances and sleep
regulation. Physiol Rev 69, 605-670.
Born, J., Rasch, B., and Gais, S. (2006). Sleep to remember. Neuroscientist 12, 410-424.
Boucetta, S., and Jones, B.E. (2009). Activity profiles of cholinergic and intermingled
GABAergic and putative glutamatergic neurons in the pontomesencephalic
tegmentum of urethane-anesthetized rats. J Neurosci 29, 4664-4674.
Boulay, A.C., del Castillo, F.J., Giraudet, F., Hamard, G., Giaume, C., Petit, C., Avan, P., and
Cohen-Salmon, M. (2013). Hearing is normal without connexin30. J Neurosci 33,
430-434.
Bowser, D.N., and Khakh, B.S. (2004). ATP excites interneurons and astrocytes to increase
synaptic inhibition in neuronal networks. J Neurosci 24, 8606-8620.
Bramham, C.R., and Srebro, B. (1989). Synaptic plasticity in the hippocampus is modulated by
behavioral state. Brain Res 493, 74-86.
!"%.!

Brand-Schieber, E., Werner, P., Iacobas, D.A., Iacobas, S., Beelitz, M., Lowery, S.L., Spray,
D.C., and Scemes, E. (2005). Connexin43, the major gap junction protein of
astrocytes, is down-regulated in inflamed white matter in an animal model of multiple
sclerosis. J Neurosci Res 80, 798-808.
Bredow, S., Guha-Thakurta, N., Taishi, P., Obal, F., Jr., and Krueger, J.M. (1997). Diurnal
variations of tumor necrosis factor alpha mRNA and alpha-tubulin mRNA in rat brain.
Neuroimmunomodulation 4, 84-90.
Brisbare-Roch, C., Dingemanse, J., Koberstein, R., Hoever, P., Aissaoui, H., Flores, S.,
Mueller, C., Nayler, O., van Gerven, J., de Haas, S.L., et al. (2007). Promotion of
sleep by targeting the orexin system in rats, dogs and humans. Nat Med 13,
150-155.
Brockhaus, J., and Deitmer, J.W. (2002). Long-lasting modulation of synaptic input to Purkinje
neurons by Bergmann glia stimulation in rat brain slices. J Physiol 545, 581-593.
Broome, M.R., Collingridge, G.L., and Irving, A.J. (1994). Activation of the NO-cGMP signalling
pathway depresses hippocampal synaptic transmission through an adenosine
receptor-dependent mechanism. Neuropharmacology 33, 1511-1513.
Brown, R.E., Stevens, D.R., and Haas, H.L. (2001). The physiology of brain histamine. Prog
Neurobiol 63, 637-672.
Brown, A.M. (2004). Brain glycogen re-awakened. J Neurochem 89, 537-552.
Brown, E.N., Purdon, P.L., and Van Dort, C.J. (2011). General anesthesia and altered states
of arousal: a systems neuroscience analysis. Annu Rev Neurosci 34, 601-628.
Brown, R.E., Basheer, R., McKenna, J.T., Strecker, R.E., and McCarley, R.W. (2012). Control
of sleep and wakefulness. Physiol Rev 92, 1087-1187.
Burgess, N., Donnett, J.G., and O'Keefe, J. (1998). The representation of space and the
hippocampus in rats, robots and humans. Z Naturforsch C 53, 504-509.
Burlet, S., and Cespuglio, R. (1997). Voltammetric detection of nitric oxide (NO) in the rat brain:
its variations throughout the sleep-wake cycle. Neurosci Lett 226, 131-135.
Bushong, E.A., Martone, M.E., Jones, Y.Z., and Ellisman, M.H. (2002). Protoplasmic
astrocytes in CA1 stratum radiatum occupy separate anatomical domains. J
Neurosci 22, 183-192.
Bushong, E.A., Martone, M.E., and Ellisman, M.H. (2004). Maturation of astrocyte morphology
and the establishment of astrocyte domains during postnatal hippocampal
development. Int J Dev Neurosci 22, 73-86.
Buzsaki, G. (1986). Hippocampal sharp waves: their origin and significance. Brain Res 398,
242-252.
Buzsaki, G. (2002). Theta oscillations in the hippocampus. Neuron 33, 325-340.
Buzsaki, G., and Draguhn, A. (2004). Neuronal oscillations in cortical networks. Science 304,
1926-1929.
Cahoy, J.D., Emery, B., Kaushal, A., Foo, L.C., Zamanian, J.L., Christopherson, K.S., Xing, Y.,
Lubischer, J.L., Krieg, P.A., Krupenko, S.A., et al. (2008). A transcriptome database
for astrocytes, neurons, and oligodendrocytes: a new resource for understanding
brain development and function. J Neurosci 28, 264-278.
Calabrese, V., Mancuso, C., Calvani, M., Rizzarelli, E., Butterfield, D.A., and Stella, A.M.
(2007). Nitric oxide in the central nervous system: neuroprotection versus
neurotoxicity. Nat Rev Neurosci 8, 766-775.
Cantero, J.L., Atienza, M., Stickgold, R., Kahana, M.J., Madsen, J.R., and Kocsis, B. (2003).
Sleep-dependent theta oscillations in the human hippocampus and neocortex. J
Neurosci 23, 10897-10903.
Cape, E.G., and Jones, B.E. (1998). Differential modulation of high-frequency
gamma-electroencephalogram activity and sleep-wake state by noradrenaline and
serotonin microinjections into the region of cholinergic basalis neurons. J Neurosci
18, 2653-2666.
Castellano, P., and Eugenin, E.A. (2014). Regulation of gap junction channels by infectious
agents and inflammation in the CNS. Front Cell Neurosci 8, 122.
Cavas, M., and Navarro, J.F. (2006). Effects of selective neuronal nitric oxide synthase
inhibition on sleep and wakefulness in the rat. Prog Neuropsychopharmacol Biol
Psychiatry 30, 56-67.
!

"%"!

Cavelier, P., and Attwell, D. (2005). Tonic release of glutamate by a DIDS-sensitive

mechanism in rat hippocampal slices. J Physiol 564, 397-410.
Celesia, G.G., and Jasper, H.H. (1966). Acetylcholine released from cerebral cortex in relation
to state of activation. Neurology 16, 1053-1063.
Chatton, J.Y., Pellerin, L., and Magistretti, P.J. (2003). GABA uptake into astrocytes is not
associated with significant metabolic cost: implications for brain imaging of inhibitory
transmission. Proc Natl Acad Sci U S A 100, 12456-12461.
Chauvette, S., Seigneur, J., and Timofeev, I. (2012). Sleep oscillations in the thalamocortical
system induce long-term neuronal plasticity. Neuron 75, 1105-1113.
Chemelli, R.M., Willie, J.T., Sinton, C.M., Elmquist, J.K., Scammell, T., Lee, C., Richardson,
J.A., Williams, S.C., Xiong, Y., Kisanuki, Y., et al. (1999). Narcolepsy in orexin
knockout mice: molecular genetics of sleep regulation. Cell 98, 437-451.
Chen, N., Sugihara, H., Sharma, J., Perea, G., Petravicz, J., Le, C., and Sur, M. (2012).
Nucleus basalis-enabled stimulus-specific plasticity in the visual cortex is mediated
by astrocytes. Proc Natl Acad Sci U S A 109, E2832-2841.
Chen, M.J., Kress, B., Han, X., Moll, K., Peng, W., Ji, R.R., and Nedergaard, M. (2012).
Astrocytic CX43 hemichannels and gap junctions play a crucial role in development
of chronic neuropathic pain following spinal cord injury. Glia 60, 1660-1670.
Chever, O., Djukic, B., McCarthy, K.D., and Amzica, F. (2010). Implication of Kir4.1 channel in
excess potassium clearance: an in vivo study on anesthetized glial-conditional Kir4.1
knock-out mice. J Neurosci 30, 15769-15777.
Cholet, N., Pellerin, L., Welker, E., Lacombe, P., Seylaz, J., Magistretti, P., and Bonvento, G.
(2001). Local injection of antisense oligonucleotides targeted to the glial glutamate
transporter GLAST decreases the metabolic response to somatosensory activation.
J Cereb Blood Flow Metab 21, 404-412.
Chou, T.C., Bjorkum, A.A., Gaus, S.E., Lu, J., Scammell, T.E., and Saper, C.B. (2002).
Afferents to the ventrolateral preoptic nucleus. J Neurosci 22, 977-990.
Chrobak, J.J., and Buzsaki, G. (1996). High-frequency oscillations in the output networks of
the hippocampal-entorhinal axis of the freely behaving rat. J Neurosci 16,
3056-3066.
Chung, W.S., Clarke, L.E., Wang, G.X., Stafford, B.K., Sher, A., Chakraborty, C., Joung, J.,
Foo, L.C., Thompson, A., Chen, C., et al. (2013). Astrocytes mediate synapse
elimination through MEGF10 and MERTK pathways. Nature 504, 394-400.
Chuquet, J., Quilichini, P., Nimchinsky, E.A., and Buzsaki, G. (2010). Predominant
enhancement of glucose uptake in astrocytes versus neurons during activation of the
somatosensory cortex. J Neurosci 30, 15298-15303.
Cina, C., Maass, K., Theis, M., Willecke, K., Bechberger, J.F., and Naus, C.C. (2009).
Involvement of the cytoplasmic C-terminal domain of connexin43 in neuronal
migration. J Neurosci 29, 2009-2021.
Cirelli, C., Gutierrez, C.M., and Tononi, G. (2004). Extensive and divergent effects of sleep and
wakefulness on brain gene expression. Neuron 41, 35-43.
Clarke, L.E., and Barres, B.A. (2013). Emerging roles of astrocytes in neural circuit
development. Nat Rev Neurosci 14, 311-321.
Clemens, Z., Fabo, D., and Halasz, P. (2005). Overnight verbal memory retention correlates
with the number of sleep spindles. Neuroscience 132, 529-535.
Clemens, Z., Fabo, D., and Halasz, P. (2006). Twenty-four hours retention of visuospatial
memory correlates with the number of parietal sleep spindles. Neurosci Lett 403,
52-56.
Clemens, Z., Molle, M., Eross, L., Barsi, P., Halasz, P., and Born, J. (2007). Temporal coupling
of parahippocampal ripples, sleep spindles and slow oscillations in humans. Brain
130, 2868-2878.
Clemens, Z., Molle, M., Eross, L., Jakus, R., Rasonyi, G., Halasz, P., and Born, J. (2011).
Fine-tuned coupling between human parahippocampal ripples and sleep spindles.
Eur J Neurosci 33, 511-520.
Colgin, L.L. (2013). Mechanisms and functions of theta rhythms. Annu Rev Neurosci 36,
295-312.

!"%#!

Conti, F., Minelli, A., and Melone, M. (2004). GABA transporters in the mammalian cerebral
cortex: localization, development and pathological implications. Brain Res Brain Res
Rev 45, 196-212.
Contreras, J.E., Sanchez, H.A., Eugenin, E.A., Speidel, D., Theis, M., Willecke, K., Bukauskas,
F.F., Bennett, M.V., and Saez, J.C. (2002). Metabolic inhibition induces opening of
unapposed connexin 43 gap junction hemichannels and reduces gap junctional
communication in cortical astrocytes in culture. Proc Natl Acad Sci U S A 99,
495-500.
Contreras, J.E., Saez, J.C., Bukauskas, F.F., and Bennett, M.V. (2003). Gating and regulation
of connexin 43 (Cx43) hemichannels. Proc Natl Acad Sci U S A 100, 11388-11393.
Cornell-Bell, A.H., Finkbeiner, S.M., Cooper, M.S., and Smith, S.J. (1990). Glutamate induces
calcium waves in cultured astrocytes: long-range glial signaling. Science 247,
470-473.
Cornwall, J., and Phillipson, O.T. (1988). Afferent projections to the parafascicular thalamic
nucleus of the rat, as shown by the retrograde transport of wheat germ agglutinin.
Brain Res Bull 20, 139-150.
Cotrina, M.L., Lin, J.H., Lopez-Garcia, J.C., Naus, C.C., and Nedergaard, M. (2000).
ATP-mediated glia signaling. J Neurosci 20, 2835-2844.
Cotrina, M.L., Gao, Q., Lin, J.H., and Nedergaard, M. (2001). Expression and function of
astrocytic gap junctions in aging. Brain Res 901, 55-61.
Cotrina, M.L., Lin, J.H., and Nedergaard, M. (2008). Adhesive properties of connexin
hemichannels. Glia 56, 1791-1798.
Cox, R., Hofman, W.F., and Talamini, L.M. (2012). Involvement of spindles in memory
consolidation is slow wave sleep-specific. Learn Mem 19, 264-267.
Crochet, S., Onoe, H., and Sakai, K. (2006). A potent non-monoaminergic paradoxical sleep
inhibitory system: a reverse microdialysis and single-unit recording study. Eur J
Neurosci 24, 1404-1412.
Cummins, C.J., Lust, W.D., and Passonneau, J.V. (1983). Regulation of glycogen metabolism
in primary and transformed astrocytes in vitro. J Neurochem 40, 128-136.
Cunningham, M.O., Pervouchine, D.D., Racca, C., Kopell, N.J., Davies, C.H., Jones, R.S.,
Traub, R.D., and Whittington, M.A. (2006). Neuronal metabolism governs cortical
network response state. Proc Natl Acad Sci U S A 103, 5597-5601.
Czarnecki, A., Birtoli, B., and Ulrich, D. (2007). Cellular mechanisms of burst firing-mediated
long-term depression in rat neocortical pyramidal cells. J Physiol 578, 471-479.
D'Ascenzo, M., Fellin, T., Terunuma, M., Revilla-Sanchez, R., Meaney, D.F., Auberson, Y.P.,
Moss, S.J., and Haydon, P.G. (2007). mGluR5 stimulates gliotransmission in the
nucleus accumbens. Proc Natl Acad Sci U S A 104, 1995-2000.
Dahan, L., Astier, B., Vautrelle, N., Urbain, N., Kocsis, B., and Chouvet, G. (2007). Prominent
burst firing of dopaminergic neurons in the ventral tegmental area during paradoxical
sleep. Neuropsychopharmacology 32, 1232-1241.
Danbolt, N.C. (2001). Glutamate uptake. Prog Neurobiol 65, 1-105.
Dash, M.B., Tononi, G., and Cirelli, C. (2012). Extracellular levels of lactate, but not oxygen,
reflect sleep homeostasis in the rat cerebral cortex. Sleep 35, 909-919.
Dash, M.B., Bellesi, M., Tononi, G., and Cirelli, C. (2013). Sleep/wake dependent changes in
cortical glucose concentrations. J Neurochem 124, 79-89.
Datta, S. (2000). Avoidance task training potentiates phasic pontine-wave density in the rat: A
mechanism for sleep-dependent plasticity. J Neurosci 20, 8607-8613.
Datta, S., Li, G., and Auerbach, S. (2008). Activation of phasic pontine-wave generator in the
rat: a mechanism for expression of plasticity-related genes and proteins in the dorsal
hippocampus and amygdala. Eur J Neurosci 27, 1876-1892.
Davidson, T.J., Kloosterman, F., and Wilson, M.A. (2009). Hippocampal replay of extended
experience. Neuron 63, 497-507.
De Bock, M., Decrock, E., Wang, N., Bol, M., Vinken, M., Bultynck, G., and Leybaert, L. (2014).
The dual face of connexin-based astroglial Ca communication: A key player in brain
physiology and a prime target in pathology. Biochim Biophys Acta 1843, 2211-2232.
De Gennaro, L., and Ferrara, M. (2003). Sleep spindles: an overview. Sleep Med Rev 7,
423-440.
!

"%$!

De Pina-Benabou, M.H., Srinivas, M., Spray, D.C., and Scemes, E. (2001). Calmodulin kinase
pathway mediates the K+-induced increase in Gap junctional communication
between mouse spinal cord astrocytes. J Neurosci 21, 6635-6643.
Deitmer, J.W., and Rose, C.R. (2010). Ion changes and signalling in perisynaptic glia. Brain
Res Rev 63, 113-129.
Dere, E., De Souza-Silva, M.A., Frisch, C., Teubner, B., Sohl, G., Willecke, K., and Huston, J.P.
(2003). Connexin30-deficient mice show increased emotionality and decreased
rearing activity in the open-field along with neurochemical changes. Eur J Neurosci
18, 629-638.
Dermietzel, R., Hertberg, E.L., Kessler, J.A., and Spray, D.C. (1991). Gap junctions between
cultured astrocytes: immunocytochemical, molecular, and electrophysiological
analysis. J Neurosci 11, 1421-1432.
Desseilles, M., Dang-Vu, T., Schabus, M., Sterpenich, V., Maquet, P., and Schwartz, S. (2008).
Neuroimaging insights into the pathophysiology of sleep disorders. Sleep 31,
777-794.
Detari, L., Rasmusson, D.D., and Semba, K. (1999). The role of basal forebrain neurons in
tonic and phasic activation of the cerebral cortex. Prog Neurobiol 58, 249-277.
Di Castro, M.A., Chuquet, J., Liaudet, N., Bhaukaurally, K., Santello, M., Bouvier, D., Tiret, P.,
and Volterra, A. (2011). Local Ca2+ detection and modulation of synaptic release by
astrocytes. Nat Neurosci 14, 1276-1284.
Diano, S., Horvath, B., Urbanski, H.F., Sotonyi, P., and Horvath, T.L. (2003). Fasting activates
the nonhuman primate hypocretin (orexin) system and its postsynaptic targets.
Endocrinology 144, 3774-3778.
Diba, K., and Buzsaki, G. (2007). Forward and reverse hippocampal place-cell sequences
during ripples. Nat Neurosci 10, 1241-1242.
Diekelmann, S., and Born, J. (2010). The memory function of sleep. Nat Rev Neurosci 11,
114-126.
Dietzel, I., Heinemann, U., Hofmeier, G., and Lux, H.D. (1980). Transient changes in the size
of the extracellular space in the sensorimotor cortex of cats in relation to
stimulus-induced changes in potassium concentration. Exp Brain Res 40, 432-439.
Dijk, D.J., Beersma, D.G., and Daan, S. (1987). EEG power density during nap sleep:
reflection of an hourglass measuring the duration of prior wakefulness. J Biol
Rhythms 2, 207-219.
Ding, S., Fellin, T., Zhu, Y., Lee, S.Y., Auberson, Y.P., Meaney, D.F., Coulter, D.A.,
Carmignoto, G., and Haydon, P.G. (2007). Enhanced astrocytic Ca2+ signals
contribute to neuronal excitotoxicity after status epilepticus. J Neurosci 27,
10674-10684.
Dringenberg, H.C., and Vanderwolf, C.H. (1998). Involvement of direct and indirect pathways
in electrocorticographic activation. Neurosci Biobehav Rev 22, 243-257.
Duffy, H.S., Ashton, A.W., O'Donnell, P., Coombs, W., Taffet, S.M., Delmar, M., and Spray,
D.C. (2004). Regulation of connexin43 protein complexes by intracellular
acidification. Circ Res 94, 215-222.
Dunwiddie, T.V., and Worth, T. (1982). Sedative and anticonvulsant effects of adenosine
analogs in mouse and rat. J Pharmacol Exp Ther 220, 70-76.
Duque, A., Balatoni, B., Detari, L., and Zaborszky, L. (2000). EEG correlation of the discharge
properties of identified neurons in the basal forebrain. J Neurophysiol 84, 1627-1635.
Ego-Stengel, V., and Wilson, M.A. (2010). Disruption of ripple-associated hippocampal activity
during rest impairs spatial learning in the rat. Hippocampus 20, 1-10.
Eiberger, J., Degen, J., Romualdi, A., Deutsch, U., Willecke, K., and Sohl, G. (2001). Connexin
genes in the mouse and human genome. Cell Commun Adhes 8, 163-165.
el Mansari, M., Sakai, K., and Jouvet, M. (1989). Unitary characteristics of presumptive
cholinergic tegmental neurons during the sleep-waking cycle in freely moving cats.
Exp Brain Res 76, 519-529.
Elias, L.A., Wang, D.D., and Kriegstein, A.R. (2007). Gap junction adhesion is necessary for
radial migration in the neocortex. Nature 448, 901-907.
Elmenhorst, D., Meyer, P.T., Winz, O.H., Matusch, A., Ermert, J., Coenen, H.H., Basheer, R.,
Haas, H.L., Zilles, K., and Bauer, A. (2007). Sleep deprivation increases A1
!"%%!

adenosine receptor binding in the human brain: a positron emission tomography

study. J Neurosci 27, 2410-2415.
Elmenhorst, D., Basheer, R., McCarley, R.W., and Bauer, A. (2009). Sleep deprivation
increases A(1) adenosine receptor density in the rat brain. Brain Res 1258, 53-58.
Enkvist, M.O., and McCarthy, K.D. (1994). Astroglial gap junction communication is increased
by treatment with either glutamate or high K+ concentration. J Neurochem 62,
489-495.
Ernst, C., Nagy, C., Kim, S., Yang, J.P., Deng, X., Hellstrom, I.C., Choi, K.H., Gershenfeld, H.,
Meaney, M.J., and Turecki, G. (2011). Dysfunction of astrocyte connexins 30 and 43
in dorsal lateral prefrontal cortex of suicide completers. Biol Psychiatry 70, 312-319.
Eschenko, O., Ramadan, W., Molle, M., Born, J., and Sara, S.J. (2008). Sustained increase in
hippocampal sharp-wave ripple activity during slow-wave sleep after learning. Learn
Mem 15, 222-228.
Eulenburg, V., and Gomeza, J. (2010). Neurotransmitter transporters expressed in glial cells
as regulators of synapse function. Brain Res Rev 63, 103-112.
Fallahi, N., Broad, R.M., Jin, S., and Fredholm, B.B. (1996). Release of adenosine from rat
hippocampal slices by nitric oxide donors. J Neurochem 67, 186-193.
Faraguna, U., Vyazovskiy, V.V., Nelson, A.B., Tononi, G., and Cirelli, C. (2008). A causal role
for brain-derived neurotrophic factor in the homeostatic regulation of sleep. J
Neurosci 28, 4088-4095.
Fatemi, S.H., Folsom, T.D., Reutiman, T.J., Pandian, T., Braun, N.N., and Haug, K. (2008).
Chronic psychotropic drug treatment causes differential expression of connexin 43
and GFAP in frontal cortex of rats. Schizophr Res 104, 127-134.
Fellin, T., Pascual, O., Gobbo, S., Pozzan, T., Haydon, P.G., and Carmignoto, G. (2004).
Neuronal synchrony mediated by astrocytic glutamate through activation of
extrasynaptic NMDA receptors. Neuron 43, 729-743.
Fellin, T., Pozzan, T., and Carmignoto, G. (2006). Purinergic receptors mediate two distinct
glutamate release pathways in hippocampal astrocytes. J Biol Chem 281,
4274-4284.
Fellin, T., Halassa, M.M., Terunuma, M., Succol, F., Takano, H., Frank, M., Moss, S.J., and
Haydon, P.G. (2009). Endogenous nonneuronal modulators of synaptic transmission
control cortical slow oscillations in vivo. Proc Natl Acad Sci U S A 106, 15037-15042.
Fellin, T., Ellenbogen, J.M., De Pitta, M., Ben-Jacob, E., and Halassa, M.M. (2012). Astrocyte
regulation of sleep circuits: experimental and modeling perspectives. Front Comput
Neurosci 6, 65.
Fiacco, T.A., and McCarthy, K.D. (2004). Intracellular astrocyte calcium waves in situ increase
the frequency of spontaneous AMPA receptor currents in CA1 pyramidal neurons. J
Neurosci 24, 722-732.
Fiacco, T.A., and McCarthy, K.D. (2006). Astrocyte calcium elevations: properties, propagation,
and effects on brain signaling. Glia 54, 676-690.
Fiacco, T.A., Agulhon, C., Taves, S.R., Petravicz, J., Casper, K.B., Dong, X., Chen, J., and
McCarthy, K.D. (2007). Selective stimulation of astrocyte calcium in situ does not
affect neuronal excitatory synaptic activity. Neuron 54, 611-626.
Figiel, M., Allritz, C., Lehmann, C., and Engele, J. (2007). Gap junctional control of glial
glutamate transporter expression. Mol Cell Neurosci 35, 130-137.
Filosa, J.A., Bonev, A.D., and Nelson, M.T. (2004). Calcium dynamics in cortical astrocytes
and arterioles during neurovascular coupling. Circ Res 95, e73-81.
Filosa, J.A. (2010). Vascular tone and neurovascular coupling: considerations toward an
improved in vitro model. Front Neuroenergetics 2.
Finelli, L.A., Baumann, H., Borbely, A.A., and Achermann, P. (2000). Dual
electroencephalogram markers of human sleep homeostasis: correlation between
theta activity in waking and slow-wave activity in sleep. Neuroscience 101, 523-529.
Fischer, G., and Kettenmann, H. (1985). Cultured astrocytes form a syncytium after maturation.
Exp Cell Res 159, 273-279.
Floyd, R.A., and Krueger, J.M. (1997). Diurnal variation of TNF alpha in the rat brain.
Neuroreport 8, 915-918.

"%&!

Fossat, P., Turpin, F.R., Sacchi, S., Dulong, J., Shi, T., Rivet, J.M., Sweedler, J.V., Pollegioni,
L., Millan, M.J., Oliet, S.H., et al. (2012). Glial D-serine gates NMDA receptors at
excitatory synapses in prefrontal cortex. Cereb Cortex 22, 595-606.
Foster, D.J., and Wilson, M.A. (2006). Reverse replay of behavioural sequences in
hippocampal place cells during the awake state. Nature 440, 680-683.
Frank, M.G. (2006). The mystery of sleep function: current perspectives and future directions.
Rev Neurosci 17, 375-392.
Franks, N.P., and Zecharia, A.Y. (2011). Sleep and general anesthesia. Can J Anaesth 58,
139-148.
Fredholm, B.B., Battig, K., Holmen, J., Nehlig, A., and Zvartau, E.E. (1999). Actions of caffeine
in the brain with special reference to factors that contribute to its widespread use.
Pharmacol Rev 51, 83-133.
Frisch, C., Theis, M., De Souza Silva, M.A., Dere, E., Sohl, G., Teubner, B., Namestkova, K.,
Willecke, K., and Huston, J.P. (2003). Mice with astrocyte-directed inactivation of
connexin43 exhibit increased exploratory behaviour, impaired motor capacities, and
changes in brain acetylcholine levels. Eur J Neurosci 18, 2313-2318.
Froger, N., Orellana, J.A., Calvo, C.F., Amigou, E., Kozoriz, M.G., Naus, C.C., Saez, J.C., and
Giaume, C. (2010). Inhibition of cytokine-induced connexin43 hemichannel activity in
astrocytes is neuroprotective. Mol Cell Neurosci 45, 37-46.
Fuller, P.M., Sherman, D., Pedersen, N.P., Saper, C.B., and Lu, J. (2011). Reassessment of
the structural basis of the ascending arousal system. J Comp Neurol 519, 933-956.
Fuxe, K., Borroto-Escuela, D.O., Romero-Fernandez, W., Diaz-Cabiale, Z., Rivera, A., Ferraro,
L., Tanganelli, S., Tarakanov, A.O., Garriga, P., Narvaez, J.A., et al. (2012).
Extrasynaptic neurotransmission in the modulation of brain function. Focus on the
striatal neuronal-glial networks. Front Physiol 3, 136.
Gallo, V., Giovannini, C., Suergiu, R., and Levi, G. (1989). Expression of excitatory amino acid
receptors by cerebellar cells of the type-2 astrocyte cell lineage. J Neurochem 52,
1-9.
Gallopin, T., Fort, P., Eggermann, E., Cauli, B., Luppi, P.H., Rossier, J., Audinat, E.,
Muhlethaler, M., and Serafin, M. (2000). Identification of sleep-promoting neurons in
vitro. Nature 404, 992-995.
Gallopin, T., Luppi, P.H., Rambert, F.A., Frydman, A., and Fort, P. (2004). Effect of the
wake-promoting agent modafinil on sleep-promoting neurons from the ventrolateral
preoptic nucleus: an in vitro pharmacologic study. Sleep 27, 19-25.
Gallopin, T., Luppi, P.H., Cauli, B., Urade, Y., Rossier, J., Hayaishi, O., Lambolez, B., and Fort,
P. (2005). The endogenous somnogen adenosine excites a subset of
sleep-promoting neurons via A2A receptors in the ventrolateral preoptic nucleus.
Neuroscience 134, 1377-1390.
Garre, J.M., Retamal, M.A., Cassina, P., Barbeito, L., Bukauskas, F.F., Saez, J.C., Bennett,
M.V., and Abudara, V. (2010). FGF-1 induces ATP release from spinal astrocytes in
culture and opens pannexin and connexin hemichannels. Proc Natl Acad Sci U S A
107, 22659-22664.
Gaus, S.E., Strecker, R.E., Tate, B.A., Parker, R.A., and Saper, C.B. (2002). Ventrolateral
preoptic nucleus contains sleep-active, galaninergic neurons in multiple mammalian
species. Neuroscience 115, 285-294.
Genoud, C., Quairiaux, C., Steiner, P., Hirling, H., Welker, E., and Knott, G.W. (2006).
Plasticity of astrocytic coverage and glutamate transporter expression in adult mouse
cortex. PLoS Biol 4, e343.
Genzel, L., Dresler, M., Wehrle, R., Grozinger, M., and Steiger, A. (2009). Slow wave sleep
and REM sleep awakenings do not affect sleep dependent memory consolidation.
Sleep 32, 302-310.
Gervasoni, D., Darracq, L., Fort, P., Souliere, F., Chouvet, G., and Luppi, P.H. (1998).
Electrophysiological evidence that noradrenergic neurons of the rat locus coeruleus
are tonically inhibited by GABA during sleep. Eur J Neurosci 10, 964-970.
Gervasoni, D., Peyron, C., Rampon, C., Barbagli, B., Chouvet, G., Urbain, N., Fort, P., and
Luppi, P.H. (2000). Role and origin of the GABAergic innervation of dorsal raphe
serotonergic neurons. J Neurosci 20, 4217-4225.
!"%'!

Giaume, C., Fromaget, C., el Aoumari, A., Cordier, J., Glowinski, J., and Gros, D. (1991a).
Gap junctions in cultured astrocytes: single-channel currents and characterization of
channel-forming protein. Neuron 6, 133-143.
Giaume, C., Marin, P., Cordier, J., Glowinski, J., and Premont, J. (1991b). Adrenergic
regulation of intercellular communications between cultured striatal astrocytes from
the mouse. Proc Natl Acad Sci U S A 88, 5577-5581.
Giaume, C., Cordier, J., and Glowinski, J. (1992). Endothelins Inhibit Junctional Permeability in
Cultured Mouse Astrocytes. Eur J Neurosci 4, 877-881.
Giaume, C., Tabernero, A., and Medina, J.M. (1997). Metabolic trafficking through astrocytic
gap junctions. Glia 21, 114-123.
Giaume, C., and Venance, L. (1998). Intercellular calcium signaling and gap junctional
communication in astrocytes. Glia 24, 50-64.
Giaume, C., Kirchhoff, F., Matute, C., Reichenbach, A., and Verkhratsky, A. (2007). Glia: the
fulcrum of brain diseases. Cell Death Differ 14, 1324-1335.
Giaume, C., Koulakoff, A., Roux, L., Holcman, D., and Rouach, N. (2010). Astroglial networks:
a step further in neuroglial and gliovascular interactions. Nat Rev Neurosci 11, 87-99.
Giaume, C., and Theis, M. (2010). Pharmacological and genetic approaches to study
connexin-mediated channels in glial cells of the central nervous system. Brain Res
Rev 63, 160-176.
Giaume, C., Orellana, J.A., Abudara, V., and Saez, J.C. (2012). Connexin-based channels in
astrocytes: how to study their properties. Methods Mol Biol 814, 283-303.
Giaume, C., and Liu, X. (2012). From a glial syncytium to a more restricted and specific glial
networking. J Physiol Paris 106, 34-39.
Giaume, C., Leybaert, L., Naus, C.C., and Saez, J.C. (2013). Connexin and pannexin
hemichannels in brain glial cells: properties, pharmacology, and roles. Front
Pharmacol 4, 88.
Giniatullin, R., Nistri, A., and Yakel, J.L. (2005). Desensitization of nicotinic ACh receptors:
shaping cholinergic signaling. Trends Neurosci 28, 371-378.
Girardeau, G., Benchenane, K., Wiener, S.I., Buzsaki, G., and Zugaro, M.B. (2009). Selective
suppression of hippocampal ripples impairs spatial memory. Nat Neurosci 12,
1222-1223.
Golanov, E.V., Christensen, J.R., and Reis, D.J. (2001). Neurons of a limited subthalamic area
mediate elevations in cortical cerebral blood flow evoked by hypoxia and excitation of
neurons of the rostral ventrolateral medulla. J Neurosci 21, 4032-4041.
Goldberg, G.S., Valiunas, V., and Brink, P.R. (2004). Selective permeability of gap junction
channels. Biochim Biophys Acta 1662, 96-101.
Golovina, V.A. (2005). Visualization of localized store-operated calcium entry in mouse
astrocytes. Close proximity to the endoplasmic reticulum. J Physiol 564, 737-749.
Gomez-Gonzalo, M., Losi, G., Chiavegato, A., Zonta, M., Cammarota, M., Brondi, M., Vetri, F.,
Uva, L., Pozzan, T., de Curtis, M., et al. (2010). An excitatory loop with astrocytes
contributes to drive neurons to seizure threshold. PLoS Biol 8, e1000352.
Gomez-Palacio-Schjetnan, A., and Escobar, M.L. (2013). Neurotrophins and synaptic plasticity.
Curr Top Behav Neurosci 15, 117-136.
Gonzalez, D., Gomez-Hernandez, J.M., and Barrio, L.C. (2007). Molecular basis of voltage
dependence of connexin channels: an integrative appraisal. Prog Biophys Mol Biol
94, 66-106.
Gordon, G.R., Baimoukhametova, D.V., Hewitt, S.A., Rajapaksha, W.R., Fisher, T.E., and
Bains, J.S. (2005). Norepinephrine triggers release of glial ATP to increase
postsynaptic efficacy. Nat Neurosci 8, 1078-1086.
Gordon, G.R., Iremonger, K.J., Kantevari, S., Ellis-Davies, G.C., MacVicar, B.A., and Bains,
J.S. (2009). Astrocyte-mediated distributed plasticity at hypothalamic glutamate
synapses. Neuron 64, 391-403.
Gourine, A.V., Kasymov, V., Marina, N., Tang, F., Figueiredo, M.F., Lane, S., Teschemacher,
A.G., Spyer, K.M., Deisseroth, K., and Kasparov, S. (2010). Astrocytes control
breathing through pH-dependent release of ATP. Science 329, 571-575.
Graziano, A., Liu, X.B., Murray, K.D., and Jones, E.G. (2008). Vesicular glutamate
transporters define two sets of glutamatergic afferents to the somatosensory
!

"%(!

thalamus and two thalamocortical projections in the mouse. J Comp Neurol 507,
1258-1276.
Griemsmann, S., Hoft, S.P., Bedner, P., Zhang, J., von Staden, E., Beinhauer, A., Degen, J.,
Dublin, P., Cope, D.W., Richter, N., et al. (2014). Characterization of Panglial Gap
Junction Networks in the Thalamus, Neocortex, and Hippocampus Reveals a Unique
Population of Glial Cells. Cereb Cortex.
Grifa, A., Wagner, C.A., D'Ambrosio, L., Melchionda, S., Bernardi, F., Lopez-Bigas, N.,
Rabionet, R., Arbones, M., Monica, M.D., Estivill, X., et al. (1999). Mutations in GJB6
cause nonsyndromic autosomal dominant deafness at DFNA3 locus. Nat Genet 23,
16-18.
Grimaldi, M., Maratos, M., and Verma, A. (2003). Transient receptor potential channel
activation causes a novel form of [Ca 2+]I oscillations and is not involved in
capacitative Ca 2+ entry in glial cells. J Neurosci 23, 4737-4745.
Grosmark, A.D., Mizuseki, K., Pastalkova, E., Diba, K., and Buzsaki, G. (2012). REM sleep
reorganizes hippocampal excitability. Neuron 75, 1001-1007.
Guan, X., Cravatt, B.F., Ehring, G.R., Hall, J.E., Boger, D.L., Lerner, R.A., and Gilula, N.B.
(1997). The sleep-inducing lipid oleamide deconvolutes gap junction communication
and calcium wave transmission in glial cells. J Cell Biol 139, 1785-1792.
Guthrie, P.B., Knappenberger, J., Segal, M., Bennett, M.V., Charles, A.C., and Kater, S.B.
(1999). ATP released from astrocytes mediates glial calcium waves. J Neurosci 19,
520-528.
Gvilia, I., Xu, F., McGinty, D., and Szymusiak, R. (2006). Homeostatic regulation of sleep: a
role for preoptic area neurons. J Neurosci 26, 9426-9433.
Haas, B., Schipke, C.G., Peters, O., Sohl, G., Willecke, K., and Kettenmann, H. (2006).
Activity-dependent ATP-waves in the mouse neocortex are independent from
astrocytic calcium waves. Cereb Cortex 16, 237-246.
Haas, H.L., Sergeeva, O.A., and Selbach, O. (2008). Histamine in the nervous system. Physiol
Rev 88, 1183-1241.
Hager, A.M., and Dringenberg, H.C. (2010). Assessment of different induction protocols to
elicit long-term depression (LTD) in the rat visual cortex in vivo. Brain Res 1318,
33-41.
Haj-Yasein, N.N., Jensen, V., Ostby, I., Omholt, S.W., Voipio, J., Kaila, K., Ottersen, O.P.,
Hvalby, O., and Nagelhus, E.A. (2012). Aquaporin-4 regulates extracellular space
volume dynamics during high-frequency synaptic stimulation: a gene deletion study
in mouse hippocampus. Glia 60, 867-874.
Halassa, M.M., Fellin, T., Takano, H., Dong, J.H., and Haydon, P.G. (2007). Synaptic islands
defined by the territory of a single astrocyte. J Neurosci 27, 6473-6477.
Halassa, M.M., Florian, C., Fellin, T., Munoz, J.R., Lee, S.Y., Abel, T., Haydon, P.G., and
Frank, M.G. (2009). Astrocytic modulation of sleep homeostasis and cognitive
consequences of sleep loss. Neuron 61, 213-219.
Halassa, M.M., and Haydon, P.G. (2010). Integrated brain circuits: astrocytic networks
modulate neuronal activity and behavior. Annu Rev Physiol 72, 335-355.
Hallanger, A.E., Levey, A.I., Lee, H.J., Rye, D.B., and Wainer, B.H. (1987). The origins of
cholinergic and other subcortical afferents to the thalamus in the rat. J Comp Neurol
262, 105-124.
Hallett, H., Churchill, L., Taishi, P., De, A., and Krueger, J.M. (2010). Whisker stimulation
increases expression of nerve growth factor- and interleukin-1beta-immunoreactivity
in the rat somatosensory cortex. Brain Res 1333, 48-56.
Hamel, E. (2006). Perivascular nerves and the regulation of cerebrovascular tone. J Appl
Physiol (1985) 100, 1059-1064.
Hamilton, N., Vayro, S., Kirchhoff, F., Verkhratsky, A., Robbins, J., Gorecki, D.C., and Butt,
A.M. (2008). Mechanisms of ATP- and glutamate-mediated calcium signaling in
white matter astrocytes. Glia 56, 734-749.
Hamilton, N.B., and Attwell, D. (2010). Do astrocytes really exocytose neurotransmitters? Nat
Rev Neurosci 11, 227-238.
Han, J., Kesner, P., Metna-Laurent, M., Duan, T., Xu, L., Georges, F., Koehl, M., Abrous, D.N.,
Mendizabal-Zubiaga, J., Grandes, P., et al. (2012). Acute cannabinoids impair
!"%,!

working memory through astroglial CB1 receptor modulation of hippocampal LTD.

Cell 148, 1039-1050.
Han, Y., Yu, H.X., Sun, M.L., Wang, Y., Xi, W., and Yu, Y.Q. (2014). Astrocyte-restricted
disruption of connexin-43 impairs neuronal plasticity in mouse barrel cortex. Eur J
Neurosci 39, 35-45.
Hansen, D.B., Braunstein, T.H., Nielsen, M.S., and MacAulay, N. (2014). Distinct permeation
profiles of the connexin 30 and 43 hemichannels. FEBS Lett 588, 1446-1457.
Harris, A.L. (2007). Connexin channel permeability to cytoplasmic molecules. Prog Biophys
Mol Biol 94, 120-143.
Hartmann, E. (1973). Functions of Sleep. (Yale University Press).
Hassani, O.K., Lee, M.G., Henny, P., and Jones, B.E. (2009). Discharge profiles of identified
GABAergic in comparison to cholinergic and putative glutamatergic basal forebrain
neurons across the sleep-wake cycle. J Neurosci 29, 11828-11840.
Henneberger, C., Papouin, T., Oliet, S.H., and Rusakov, D.A. (2010). Long-term potentiation
depends on release of D-serine from astrocytes. Nature 463, 232-236.
Henny, P., and Jones, B.E. (2008). Projections from basal forebrain to prefrontal cortex
comprise cholinergic, GABAergic and glutamatergic inputs to pyramidal cells or
interneurons. Eur J Neurosci 27, 654-670.
Hertz, L., Dringen, R., Schousboe, A., and Robinson, S.R. (1999). Astrocytes: glutamate
producers for neurons. J Neurosci Res 57, 417-428.
Herve, J.C., Bourmeyster, N., and Sarrouilhe, D. (2004). Diversity in protein-protein
interactions of connexins: emerging roles. Biochim Biophys Acta 1662, 22-41.
Herve, J.C., Derangeon, M., Sarrouilhe, D., Giepmans, B.N., and Bourmeyster, N. (2012). Gap
junctional channels are parts of multiprotein complexes. Biochim Biophys Acta 1818,
1844-1865.
Hinard, V., Mikhail, C., Pradervand, S., Curie, T., Houtkooper, R.H., Auwerx, J., Franken, P.,
and Tafti, M. (2012). Key electrophysiological, molecular, and metabolic signatures
of sleep and wakefulness revealed in primary cortical cultures. J Neurosci 32,
12506-12517.
Hines, D.J., Schmitt, L.I., Hines, R.M., Moss, S.J., and Haydon, P.G. (2013). Antidepressant
effects of sleep deprivation require astrocyte-dependent adenosine mediated
signaling. Transl Psychiatry 3, e212.
Hobson, J.A., McCarley, R.W., and Wyzinski, P.W. (1975). Sleep cycle oscillation: reciprocal
discharge by two brainstem neuronal groups. Science 189, 55-58.
Hofer, A., and Dermietzel, R. (1998). Visualization and functional blocking of gap junction
hemichannels (connexons) with antibodies against external loop domains in
astrocytes. Glia 24, 141-154.
Holscher, C., Anwyl, R., and Rowan, M.J. (1997). Stimulation on the positive phase of
hippocampal theta rhythm induces long-term potentiation that can Be depotentiated
by stimulation on the negative phase in area CA1 in vivo. J Neurosci 17, 6470-6477.
Holthoff, K., and Witte, O.W. (1996). Intrinsic optical signals in rat neocortical slices measured
with near-infrared dark-field microscopy reveal changes in extracellular space. J
Neurosci 16, 2740-2749.
Holtzclaw, L.A., Pandhit, S., Bare, D.J., Mignery, G.A., and Russell, J.T. (2002). Astrocytes in
adult rat brain express type 2 inositol 1,4,5-trisphosphate receptors. Glia 39, 69-84.
Holz, J., Piosczyk, H., Feige, B., Spiegelhalder, K., Baglioni, C., Riemann, D., and Nissen, C.
(2012). EEG Sigma and slow-wave activity during NREM sleep correlate with
overnight declarative and procedural memory consolidation. J Sleep Res 21,
612-619.
Houades, V., Rouach, N., Ezan, P., Kirchhoff, F., Koulakoff, A., and Giaume, C. (2006).
Shapes of astrocyte networks in the juvenile brain. Neuron Glia Biol 2, 3-14.
Houades, V., Koulakoff, A., Ezan, P., Seif, I., and Giaume, C. (2008). Gap junction-mediated
astrocytic networks in the mouse barrel cortex. J Neurosci 28, 5207-5217.
Huang, Z.L., Qu, W.M., Eguchi, N., Chen, J.F., Schwarzschild, M.A., Fredholm, B.B., Urade, Y.,
and Hayaishi, O. (2005). Adenosine A2A, but not A1, receptors mediate the arousal
effect of caffeine. Nat Neurosci 8, 858-859.
Huber, R., Deboer, T., and Tobler, I. (2000). Effects of sleep deprivation on sleep and sleep
EEG in three mouse strains: empirical data and simulations. Brain Res 857, 8-19.
!

"%-!

Huber, R., Ghilardi, M.F., Massimini, M., and Tononi, G. (2004). Local sleep and learning.
Nature 430, 78-81.
Huber, R., Ghilardi, M.F., Massimini, M., Ferrarelli, F., Riedner, B.A., Peterson, M.J., and
Tononi, G. (2006). Arm immobilization causes cortical plastic changes and locally
decreases sleep slow wave activity. Nat Neurosci 9, 1169-1176.
Huber, R., Maki, H., Rosanova, M., Casarotto, S., Canali, P., Casali, A.G., Tononi, G., and
Massimini, M. (2013). Human cortical excitability increases with time awake. Cereb
Cortex 23, 332-338.
Huda, R., McCrimmon, D.R., and Martina, M. (2013). pH modulation of glial glutamate
transporters regulates synaptic transmission in the nucleus of the solitary tract. J
Neurophysiol 110, 368-377.
Hur, E.E., and Zaborszky, L. (2005). Vglut2 afferents to the medial prefrontal and primary
somatosensory cortices: a combined retrograde tracing in situ hybridization study
[corrected]. J Comp Neurol 483, 351-373.
Hyden, H., and Lange, P.W. (1965). Rhythmic Enzyme Changes in Neurons and Glia during
Sleep. Science 149, 654-656.
Hyder, F., Patel, A.B., Gjedde, A., Rothman, D.L., Behar, K.L., and Shulman, R.G. (2006).
Neuronal-glial glucose oxidation and glutamatergic-GABAergic function. J Cereb
Blood Flow Metab 26, 865-877.
Iacobas, D.A., Urban-Maldonado, M., Iacobas, S., Scemes, E., and Spray, D.C. (2003). Array
analysis of gene expression in connexin-43 null astrocytes. Physiol Genomics 15,
177-190.
Iacobas, D.A., Iacobas, S., and Spray, D.C. (2007). Connexin-dependent transcellular
transcriptomic networks in mouse brain. Prog Biophys Mol Biol 94, 169-185.
Igarashi, K.M., Lu, L., Colgin, L.L., Moser, M.B., and Moser, E.I. (2014). Coordination of
entorhinal-hippocampal ensemble activity during associative learning. Nature 510,
143-147.
Iglesias, R., Dahl, G., Qiu, F., Spray, D.C., and Scemes, E. (2009). Pannexin 1: the molecular
substrate of astrocyte "hemichannels". J Neurosci 29, 7092-7097.
Imeri, L., and Opp, M.R. (2009). How (and why) the immune system makes us sleep. Nat Rev
Neurosci 10, 199-210.
Iyyathurai, J., D'Hondt, C., Wang, N., De Bock, M., Himpens, B., Retamal, M.A., Stehberg, J.,
Leybaert, L., and Bultynck, G. (2013). Peptides and peptide-derived molecules
targeting the intracellular domains of Cx43: gap junctions versus hemichannels.
Neuropharmacology 75, 491-505.
Jacobs, B.L., and Fornal, C.A. (1991). Activity of brain serotonergic neurons in the behaving
animal. Pharmacol Rev 43, 563-578.
Jaiswal, J.K., Fix, M., Takano, T., Nedergaard, M., and Simon, S.M. (2007). Resolving vesicle
fusion from lysis to monitor calcium-triggered lysosomal exocytosis in astrocytes.
Proc Natl Acad Sci U S A 104, 14151-14156.
Jasper, H.H., and Tessier, J. (1971). Acetylcholine liberation from cerebral cortex during
paradoxical (REM) sleep. Science 172, 601-602.
Ji, D., and Wilson, M.A. (2007). Coordinated memory replay in the visual cortex and
hippocampus during sleep. Nat Neurosci 10, 100-107.
John, J., Wu, M.F., Boehmer, L.N., and Siegel, J.M. (2004). Cataplexy-active neurons in the
hypothalamus: implications for the role of histamine in sleep and waking behavior.
Neuron 42, 619-634.
Jones, B.E. (2004). Paradoxical REM sleep promoting and permitting neuronal networks. Arch
Ital Biol 142, 379-396.
Jones, B.E. (2005). From waking to sleeping: neuronal and chemical substrates. Trends
Pharmacol Sci 26, 578-586.
Jones, B.E., and Muhlethaler, M. (2005). Modulation of cortical activity and sleep-wake state
by hypocretin/orexin. In The Hypocretins: Integrators of Physiological Systems, L. de
Lecea and J.G. Sutcliffe, eds. (Springer), pp. 289301.
Jourdain, P., Bergersen, L.H., Bhaukaurally, K., Bezzi, P., Santello, M., Domercq, M., Matute,
C., Tonello, F., Gundersen, V., and Volterra, A. (2007). Glutamate exocytosis from
astrocytes controls synaptic strength. Nat Neurosci 10, 331-339.
!"&.!

Jouvet, M. (1962). [Research on the neural structures and responsible mechanisms in different
phases of physiological sleep]. Arch Ital Biol 100, 125-206.
Juge, N., Gray, J.A., Omote, H., Miyaji, T., Inoue, T., Hara, C., Uneyama, H., Edwards, R.H.,
Nicoll, R.A., and Moriyama, Y. (2010). Metabolic control of vesicular glutamate
transport and release. Neuron 68, 99-112.
Kacem, K., Lacombe, P., Seylaz, J., and Bonvento, G. (1998). Structural organization of the
perivascular astrocyte endfeet and their relationship with the endothelial glucose
transporter: a confocal microscopy study. Glia 23, 1-10.
Kalinchuk, A.V., Stenberg, D., Rosenberg, P.A., and Porkka-Heiskanen, T. (2006a). Inducible
and neuronal nitric oxide synthases (NOS) have complementary roles in recovery
sleep induction. Eur J Neurosci 24, 1443-1456.
Kalinchuk, A.V., Lu, Y., Stenberg, D., Rosenberg, P.A., and Porkka-Heiskanen, T. (2006b).
Nitric oxide production in the basal forebrain is required for recovery sleep. J
Neurochem 99, 483-498.
Kalinchuk, A.V., McCarley, R.W., Stenberg, D., Porkka-Heiskanen, T., and Basheer, R. (2008).
The role of cholinergic basal forebrain neurons in adenosine-mediated homeostatic
control of sleep: lessons from 192 IgG-saporin lesions. Neuroscience 157, 238-253.
Kalinchuk, A.V., McCarley, R.W., Porkka-Heiskanen, T., and Basheer, R. (2010). Sleep
deprivation triggers inducible nitric oxide-dependent nitric oxide production in
wake-active basal forebrain neurons. J Neurosci 30, 13254-13264.
Kalinchuk, A.V., McCarley, R.W., Porkka-Heiskanen, T., and Basheer, R. (2011). The time
course of adenosine, nitric oxide (NO) and inducible NO synthase changes in the
brain with sleep loss and their role in the non-rapid eye movement sleep homeostatic
cascade. J Neurochem 116, 260-272.
Kameritsch, P., Pogoda, K., and Pohl, U. (2012). Channel-independent influence of connexin
43 on cell migration. Biochim Biophys Acta 1818, 1993-2001.
Kandel, E.R., Schwartz, J.H., and Jessell T.M. (1995). Principles of Neural Science, third
edition. (Elsevier).
Kang, J., Jiang, L., Goldman, S.A., and Nedergaard, M. (1998). Astrocyte-mediated
potentiation of inhibitory synaptic transmission. Nat Neurosci 1, 683-692.
Kang, N., Xu, J., Xu, Q., Nedergaard, M., and Kang, J. (2005). Astrocytic glutamate
release-induced transient depolarization and epileptiform discharges in hippocampal
CA1 pyramidal neurons. J Neurophysiol 94, 4121-4130.
Kang, J., Kang, N., Lovatt, D., Torres, A., Zhao, Z., Lin, J., and Nedergaard, M. (2008).
Connexin 43 hemichannels are permeable to ATP. J Neurosci 28, 4702-4711.
Kapas, L., Fang, J., and Krueger, J.M. (1994). Inhibition of nitric oxide synthesis inhibits rat
sleep. Brain Res 664, 189-196.
Karlsson, M.P., and Frank, L.M. (2009). Awake replay of remote experiences in the
hippocampus. Nat Neurosci 12, 913-918.
Karpuk, N., Burkovetskaya, M., Fritz, T., Angle, A., and Kielian, T. (2011). Neuroinflammation
leads to region-dependent alterations in astrocyte gap junction communication and
hemichannel activity. J Neurosci 31, 414-425.
Kastritsis, C.H., Salm, A.K., and McCarthy, K. (1992). Stimulation of the P2Y purinergic
receptor on type 1 astroglia results in inositol phosphate formation and calcium
mobilization. J Neurochem 58, 1277-1284.
Kattler, H., Dijk, D.J., and Borbely, A.A. (1994). Effect of unilateral somatosensory stimulation
prior to sleep on the sleep EEG in humans. J Sleep Res 3, 159-164.
Kayama, Y., Ohta, M., and Jodo, E. (1992). Firing of 'possibly' cholinergic neurons in the rat
laterodorsal tegmental nucleus during sleep and wakefulness. Brain Res 569,
210-220.
Kemp, N., and Bashir, Z.I. (2001). Long-term depression: a cascade of induction and
expression mechanisms. Prog Neurobiol 65, 339-365.
Kessels, H.W., and Malinow, R. (2009). Synaptic AMPA receptor plasticity and behavior.
Neuron 61, 340-350.
Kettenmann, H., Backus, K.H., and Schachner, M. (1984). Aspartate, glutamate and
gamma-aminobutyric acid depolarize cultured astrocytes. Neurosci Lett 52, 25-29.
Kettenmann, H., and Verkhratsky, A. (2008). Neuroglia: the 150 years after. Trends Neurosci
31, 653-659.
!

"&"!

Kettenmann, H., and Ransom, B.R. (2013). Neuroglia, third edition. (Oxford University Press).
Khateb, A., Fort, P., Alonso, A., Jones, B.E., and Muhlethaler, M. (1993). Pharmacological and
immunohistochemical evidence for serotonergic modulation of cholinergic nucleus
basalis neurons. Eur J Neurosci 5, 541-547.
Killgore, W.D. (2010). Effects of sleep deprivation on cognition. Prog Brain Res 185, 105-129.
Kimelberg, H.K. (2010). Functions of mature mammalian astrocytes: a current view.
Neuroscientist 16, 79-106.
Kirchhoff, F., Mulhardt, C., Pastor, A., Becker, C.M., and Kettenmann, H. (1996). Expression of
glycine receptor subunits in glial cells of the rat spinal cord. J Neurochem 66,
1383-1390.
Kirischuk, S., Tuschick, S., Verkhratsky, A., and Kettenmann, H. (1996). Calcium signalling in
mouse Bergmann glial cells mediated by alpha1-adrenoreceptors and H1 histamine
receptors. Eur J Neurosci 8, 1198-1208.
Kirischuk, S., Kettenmann, H., and Verkhratsky, A. (1997). Na+/Ca2+ exchanger modulates
kainate-triggered Ca2+ signaling in Bergmann glial cells in situ. FASEB J 11,
566-572.
Kirischuk, S., Kettenmann, H., and Verkhratsky, A. (2007). Membrane currents and
cytoplasmic sodium transients generated by glutamate transport in Bergmann glial
cells. Pflugers Arch 454, 245-252.
Kirischuk, S., Parpura, V., and Verkhratsky, A. (2012). Sodium dynamics: another key to
astroglial excitability? Trends Neurosci 35, 497-506.
Kiyashchenko, L.I., Mileykovskiy, B.Y., Maidment, N., Lam, H.A., Wu, M.F., John, J., Peever,
J., and Siegel, J.M. (2002). Release of hypocretin (orexin) during waking and sleep
states. J Neurosci 22, 5282-5286.
Kondo, R.P., Apstein, C.S., Eberli, F.R., Tillotson, D.L., and Suter, T.M. (1998). Increased
calcium loading and inotropy without greater cell death in hypoxic rat cardiomyocytes.
Am J Physiol 275, H2272-2282.
Koulakoff, A., Ezan, P., and Giaume, C. (2008). Neurons control the expression of connexin 30
and connexin 43 in mouse cortical astrocytes. Glia 56, 1299-1311.
Kozlov, A.S., Angulo, M.C., Audinat, E., and Charpak, S. (2006). Target cell-specific
modulation of neuronal activity by astrocytes. Proc Natl Acad Sci U S A 103,
10058-10063.
Krueger, J.M., Rector, D.M., Roy, S., Van Dongen, H.P., Belenky, G., and Panksepp, J. (2008).
Sleep as a fundamental property of neuronal assemblies. Nat Rev Neurosci 9,
910-919.
Krueger, J.M. (2008). The role of cytokines in sleep regulation. Curr Pharm Des 14,
3408-3416.
Kudrimoti, H.S., Barnes, C.A., and McNaughton, B.L. (1999). Reactivation of hippocampal cell
assemblies: effects of behavioral state, experience, and EEG dynamics. J Neurosci
19, 4090-4101.
Kuga, N., Sasaki, T., Takahara, Y., Matsuki, N., and Ikegaya, Y. (2011). Large-scale calcium
waves traveling through astrocytic networks in vivo. J Neurosci 31, 2607-2614.
Kulik, A., Haentzsch, A., Luckermann, M., Reichelt, W., and Ballanyi, K. (1999). Neuron-glia
signaling via alpha(1) adrenoceptor-mediated Ca(2+) release in Bergmann glial cells
in situ. J Neurosci 19, 8401-8408.
Kumar, N.M., and Gilula, N.B. (1996). The gap junction communication channel. Cell 84,
381-388.
Kumar, S., Rai, S., Hsieh, K.C., McGinty, D., Alam, M.N., and Szymusiak, R. (2013).
Adenosine A(2A) receptors regulate the activity of sleep regulatory GABAergic
neurons in the preoptic hypothalamus. Am J Physiol Regul Integr Comp Physiol 305,
R31-41.
Kunze, A., Congreso, M.R., Hartmann, C., Wallraff-Beck, A., Huttmann, K., Bedner, P.,
Requardt, R., Seifert, G., Redecker, C., Willecke, K., et al. (2009). Connexin
expression by radial glia-like cells is required for neurogenesis in the adult dentate
gyrus. Proc Natl Acad Sci U S A 106, 11336-11341.
Kunzelmann, P., Schroder, W., Traub, O., Steinhauser, C., Dermietzel, R., and Willecke, K.
(1999). Late onset and increasing expression of the gap junction protein connexin30
in adult murine brain and long-term cultured astrocytes. Glia 25, 111-119.
!"&#!

Laird, D.W. (2010). The gap junction proteome and its relationship to disease. Trends Cell Biol
20, 92-101.
Lallemand, Y., Luria, V., Haffner-Krausz, R., and Lonai, P. (1998). Maternally expressed
PGK-Cre transgene as a tool for early and uniform activation of the Cre site-specific
recombinase. Transgenic Res 7, 105-112.
Lalo, U., Pankratov, Y., Kirchhoff, F., North, R.A., and Verkhratsky, A. (2006). NMDA receptors
mediate neuron-to-glia signaling in mouse cortical astrocytes. J Neurosci 26,
2673-2683.
Lalo, U., Pankratov, Y., Wichert, S.P., Rossner, M.J., North, R.A., Kirchhoff, F., and
Verkhratsky, A. (2008). P2X1 and P2X5 subunits form the functional P2X receptor in
mouse cortical astrocytes. J Neurosci 28, 5473-5480.
Lalo, U., Pankratov, Y., Parpura, V., and Verkhratsky, A. (2011). Ionotropic receptors in
neuronal-astroglial signalling: what is the role of "excitable" molecules in
non-excitable cells. Biochim Biophys Acta 1813, 992-1002.
Lalo, U., Palygin, O., Rasooli-Nejad, S., Andrew, J., Haydon, P.G., and Pankratov, Y. (2014).
Exocytosis of ATP from astrocytes modulates phasic and tonic inhibition in the
neocortex. PLoS Biol 12, e1001747.
Lancel, M., van Riezen, H., and Glatt, A. (1991). Effects of circadian phase and duration of
sleep deprivation on sleep and EEG power spectra in the cat. Brain Res 548,
206-214.
Landolt, H.P. (2008). Sleep homeostasis: a role for adenosine in humans? Biochem
Pharmacol 75, 2070-2079.
Landsness, E.C., Crupi, D., Hulse, B.K., Peterson, M.J., Huber, R., Ansari, H., Coen, M., Cirelli,
C., Benca, R.M., Ghilardi, M.F., et al. (2009). Sleep-dependent improvement in
visuomotor learning: a causal role for slow waves. Sleep 32, 1273-1284.
Landsness, E.C., Ferrarelli, F., Sarasso, S., Goldstein, M.R., Riedner, B.A., Cirelli, C., Perfetti,
B., Moisello, C., Ghilardi, M.F., and Tononi, G. (2011). Electrophysiological traces of
visuomotor learning and their renormalization after sleep. Clin Neurophysiol 122,
2418-2425.
Langer, J., and Rose, C.R. (2009). Synaptically induced sodium signals in hippocampal
astrocytes in situ. J Physiol 587, 5859-5877.
Langer, J., Stephan, J., Theis, M., and Rose, C.R. (2012). Gap junctions mediate intercellular
spread of sodium between hippocampal astrocytes in situ. Glia 60, 239-252.
Lansink, C.S., Goltstein, P.M., Lankelma, J.V., Joosten, R.N., McNaughton, B.L., and
Pennartz, C.M. (2008). Preferential reactivation of motivationally relevant information
in the ventral striatum. J Neurosci 28, 6372-6382.
Lansink, C.S., Goltstein, P.M., Lankelma, J.V., McNaughton, B.L., and Pennartz, C.M. (2009).
Hippocampus leads ventral striatum in replay of place-reward information. PLoS Biol
7, e1000173.
Lante, F., Toledo-Salas, J.C., Ondrejcak, T., Rowan, M.J., and Ulrich, D. (2011). Removal of
synaptic Ca(2)+-permeable AMPA receptors during sleep. J Neurosci 31,
3953-3961.
Le Meur, K., Mendizabal-Zubiaga, J., Grandes, P., and Audinat, E. (2012). GABA release by
hippocampal astrocytes. Front Comput Neurosci 6, 59.
Lee, S.H., Magge, S., Spencer, D.D., Sontheimer, H., and Cornell-Bell, A.H. (1995). Human
epileptic astrocytes exhibit increased gap junction coupling. Glia 15, 195-202.
Lee, M.G., Hassani, O.K., Alonso, A., and Jones, B.E. (2005a). Cholinergic basal forebrain
neurons burst with theta during waking and paradoxical sleep. J Neurosci 25,
4365-4369.
Lee, M.G., Hassani, O.K., and Jones, B.E. (2005b). Discharge of identified orexin/hypocretin
neurons across the sleep-waking cycle. J Neurosci 25, 6716-6720.
Lee, S., Yoon, B.E., Berglund, K., Oh, S.J., Park, H., Shin, H.S., Augustine, G.J., and Lee, C.J.
(2010). Channel-mediated tonic GABA release from glia. Science 330, 790-796.
Lee, H.S., Ghetti, A., Pinto-Duarte, A., Wang, X., Dziewczapolski, G., Galimi, F.,
Huitron-Resendiz, S., Pina-Crespo, J.C., Roberts, A.J., Verma, I.M., et al. (2014).
Astrocytes contribute to gamma oscillations and recognition memory. Proc Natl Acad
Sci U S A.
!

"&$!

Leonard, B.J., McNaughton, B.L., and Barnes, C.A. (1987). Suppression of hippocampal
synaptic plasticity during slow-wave sleep. Brain Res 425, 174-177.
Lewis, P.A., and Durrant, S.J. (2011). Overlapping memory replay during sleep builds
cognitive schemata. Trends Cogn Sci 15, 343-351.
Leybaert, L., and Sanderson, M.J. (2012). Intercellular Ca(2+) waves: mechanisms and
function. Physiol Rev 92, 1359-1392.
Li, D., Ropert, N., Koulakoff, A., Giaume, C., and Oheim, M. (2008). Lysosomes are the major
vesicular compartment undergoing Ca2+-regulated exocytosis from cortical
astrocytes. J Neurosci 28, 7648-7658.
Li, Y., Sacchi, S., Pollegioni, L., Basu, A.C., Coyle, J.T., and Bolshakov, V.Y. (2013). Identity of
endogenous NMDAR glycine site agonist in amygdala is determined by synaptic
activity level. Nat Commun 4, 1760.
Li, D., Herault, K., Silm, K., Evrard, A., Wojcik, S., Oheim, M., Herzog, E., and Ropert, N.
(2013). Lack of evidence for vesicular glutamate transporter expression in mouse
astrocytes. J Neurosci 33, 4434-4455.
Lin, L., Faraco, J., Li, R., Kadotani, H., Rogers, W., Lin, X., Qiu, X., de Jong, P.J., Nishino, S.,
and Mignot, E. (1999). The sleep disorder canine narcolepsy is caused by a mutation
in the hypocretin (orexin) receptor 2 gene. Cell 98, 365-376.
Lin, J.S. (2000). Brain structures and mechanisms involved in the control of cortical activation
and wakefulness, with emphasis on the posterior hypothalamus and histaminergic
neurons. Sleep Med Rev 4, 471-503.
Lin, J.H., Takano, T., Cotrina, M.L., Arcuino, G., Kang, J., Liu, S., Gao, Q., Jiang, L., Li, F.,
Lichtenberg-Frate, H., et al. (2002). Connexin 43 enhances the adhesivity and
mediates the invasion of malignant glioma cells. J Neurosci 22, 4302-4311.
Lin, A.L., Fox, P.T., Hardies, J., Duong, T.Q., and Gao, J.H. (2010). Nonlinear coupling
between cerebral blood flow, oxygen consumption, and ATP production in human
visual cortex. Proc Natl Acad Sci U S A 107, 8446-8451.
Lingenhoehl, K., Brom, R., Heid, J., Beck, P., Froestl, W., Kaupmann, K., Bettler, B., and
Mosbacher, J. (1999). Gamma-hydroxybutyrate is a weak agonist at recombinant
GABA(B) receptors. Neuropharmacology 38, 1667-1673.
Liu, Q.S., Xu, Q., Arcuino, G., Kang, J., and Nedergaard, M. (2004). Astrocyte-mediated
activation of neuronal kainate receptors. Proc Natl Acad Sci U S A 101, 3172-3177.
Liu, Z.W., Faraguna, U., Cirelli, C., Tononi, G., and Gao, X.B. (2010). Direct evidence for
wake-related increases and sleep-related decreases in synaptic strength in rodent
cortex. J Neurosci 30, 8671-8675.
Liu, X., Petit, J.M., Ezan, P., Gyger, J., Magistretti, P., and Giaume, C. (2013). The
psychostimulant modafinil enhances gap junctional communication in cortical
astrocytes. Neuropharmacology 75, 533-538.
Lu, J., Greco, M.A., Shiromani, P., and Saper, C.B. (2000). Effect of lesions of the ventrolateral
preoptic nucleus on NREM and REM sleep. J Neurosci 20, 3830-3842.
Lu, J., Sherman, D., Devor, M., and Saper, C.B. (2006). A putative flip-flop switch for control of
REM sleep. Nature 441, 589-594.
Lu, J., Jhou, T.C., and Saper, C.B. (2006). Identification of wake-active dopaminergic neurons
in the ventral periaqueductal gray matter. J Neurosci 26, 193-202.
Lubenov, E.V., and Siapas, A.G. (2008). Decoupling through synchrony in neuronal circuits
with propagation delays. Neuron 58, 118-131.
Luppi, P.H., Gervasoni, D., Boissard, R., Verret, L., Goutagny, R., Peyron, C., Salvert, D.,
Leger, L., Barbagli, B., and Fort, P. (2004). Brainstem structures responsible for
paradoxical sleep onset and maintenance. Arch Ital Biol 142, 397-411.
Luppi, P.H., Gervasoni, D., Verret, L., Goutagny, R., Peyron, C., Salvert, D., Leger, L., and
Fort, P. (2006). Paradoxical (REM) sleep genesis: the switch from an
aminergic-cholinergic to a GABAergic-glutamatergic hypothesis. J Physiol Paris 100,
271-283.
Luppi, P.H., Clement, O., Sapin, E., Gervasoni, D., Peyron, C., Leger, L., Salvert, D., and Fort,
P. (2011). The neuronal network responsible for paradoxical sleep and its
dysfunctions causing narcolepsy and rapid eye movement (REM) behavior disorder.
Sleep Med Rev 15, 153-163.
!"&%!

Lurtz, M.M., and Louis, C.F. (2007). Intracellular calcium regulation of connexin43. Am J
Physiol Cell Physiol 293, C1806-1813.
Lutz, S.E., Zhao, Y., Gulinello, M., Lee, S.C., Raine, C.S., and Brosnan, C.F. (2009). Deletion
of astrocyte connexins 43 and 30 leads to a dysmyelinating phenotype and
hippocampal CA1 vacuolation. J Neurosci 29, 7743-7752.
Lynn, B.D., Tress, O., May, D., Willecke, K., and Nagy, J.I. (2011). Ablation of connexin30 in
transgenic mice alters expression patterns of connexin26 and connexin32 in glial
cells and leptomeninges. Eur J Neurosci 34, 1783-1793.
Mackiewicz, M., Shockley, K.R., Romer, M.A., Galante, R.J., Zimmerman, J.E., Naidoo, N.,
Baldwin, D.A., Jensen, S.T., Churchill, G.A., and Pack, A.I. (2007). Macromolecule
biosynthesis: a key function of sleep. Physiol Genomics 31, 441-457.
Madsen, K., White, H.S., Clausen, R.P., Frlund, B., Larsson, O. M., Krogsgaard-Larsen, P.,
and Schousboe, A. (2007). Functional and pharmacological aspects of GABA
transporters. In Handbook of nerochemistry and molecular neurobiology: Neural
membranes and transport. A. Lajtha and M.E.A. Reith, eds. (Springer), pp. 285-304.
Magistretti, P.J., Sorg, O., Yu, N., Martin, J.L., and Pellerin, L. (1993). Neurotransmitters
regulate energy metabolism in astrocytes: implications for the metabolic trafficking
between neural cells. Dev Neurosci 15, 306-312.
Magistretti, P.J. (2008). The importance of a physiological approach to neuroscience. Nihon
Seirigaku Zasshi 70, 6 p preceding 267.
Malarkey, E.B., and Parpura, V. (2009). Mechanisms of transmitter release from astrocytes. In
Astrocytes in (Patho)physiology of the Nervous System, V. Parpura and P.G.
Haydon, eds. (Springer), pp. 301-350.
Mangia, S., Giove, F., and Dinuzzo, M. (2012). Metabolic pathways and activity-dependent
modulation of glutamate concentration in the human brain. Neurochem Res 37,
2554-2561.
Manns, I.D., Alonso, A., and Jones, B.E. (2003). Rhythmically discharging basal forebrain
units comprise cholinergic, GABAergic, and putative glutamatergic cells. J
Neurophysiol 89, 1057-1066.
Mantz, J., Cordier, J., and Giaume, C. (1993). Effects of general anesthetics on intercellular
communications mediated by gap junctions between astrocytes in primary culture.
Anesthesiology 78, 892-901.
Maquet, P. (1995). Sleep function(s) and cerebral metabolism. Behav Brain Res 69, 75-83.
Maret, S., Dorsaz, S., Gurcel, L., Pradervand, S., Petit, B., Pfister, C., Hagenbuchle, O.,
O'Hara, B.F., Franken, P., and Tafti, M. (2007). Homer1a is a core brain molecular
correlate of sleep loss. Proc Natl Acad Sci U S A 104, 20090-20095.
Maret, S., Faraguna, U., Nelson, A.B., Cirelli, C., and Tononi, G. (2011). Sleep and waking
modulate spine turnover in the adolescent mouse cortex. Nat Neurosci 14,
1418-1420.
Marshall, L., Helgadottir, H., Molle, M., and Born, J. (2006). Boosting slow oscillations during
sleep potentiates memory. Nature 444, 610-613.
Marshall, L., and Born, J. (2007). The contribution of sleep to hippocampus-dependent
memory consolidation. Trends Cogn Sci 11, 442-450.
Marshall, L., Kirov, R., Brade, J., Molle, M., and Born, J. (2011). Transcranial electrical
currents to probe EEG brain rhythms and memory consolidation during sleep in
humans. PLoS One 6, e16905.
Martin, E.D., Fernandez, M., Perea, G., Pascual, O., Haydon, P.G., Araque, A., and Cena, V.
(2007). Adenosine released by astrocytes contributes to hypoxia-induced modulation
of synaptic transmission. Glia 55, 36-45.
Martineau, M., Galli, T., Baux, G., and Mothet, J.P. (2008). Confocal imaging and tracking of
the exocytotic routes for D-serine-mediated gliotransmission. Glia 56, 1271-1284.
Mascetti, L., Muto, V., Matarazzo, L., Foret, A., Ziegler, E., Albouy, G., Sterpenich, V., Schmidt,
C., Degueldre, C., Leclercq, Y., et al. (2013). The impact of visual perceptual
learning on sleep and local slow-wave initiation. J Neurosci 33, 3323-3331.
Massey, P.V., and Bashir, Z.I. (2007). Long-term depression: multiple forms and implications
for brain function. Trends Neurosci 30, 176-184.

"&&!

Mathiisen, T.M., Lehre, K.P., Danbolt, N.C., and Ottersen, O.P. (2010). The perivascular
astroglial sheath provides a complete covering of the brain microvessels: an electron
microscopic 3D reconstruction. Glia 58, 1094-1103.
Matyash, V., and Kettenmann, H. (2010). Heterogeneity in astrocyte morphology and
physiology. Brain Res Rev 63, 2-10.
McCarthy, K.D., and Salm, A.K. (1991). Pharmacologically-distinct subsets of astroglia can be
identified by their calcium response to neuroligands. Neuroscience 41, 325-333.
McCaslin, A.F., Chen, B.R., Radosevich, A.J., Cauli, B., and Hillman, E.M. (2011). In vivo 3D
morphology of astrocyte-vasculature interactions in the somatosensory cortex:
implications for neurovascular coupling. J Cereb Blood Flow Metab 31, 795-806.
McCoy, J.G., and Strecker, R.E. (2011). The cognitive cost of sleep lost. Neurobiol Learn Mem
96, 564-582.
McGinty, D.J., and Sterman, M.B. (1968). Sleep suppression after basal forebrain lesions in
the cat. Science 160, 1253-1255.
McGinty, D.J., and Harper, R.M. (1976). Dorsal raphe neurons: depression of firing during
sleep in cats. Brain Res 101, 569-575.
McGinty, D., and Szymusiak, R. (2000). The sleep-wake switch: A neuronal alarm clock. Nat
Med 6, 510-511.
Meme, W., Ezan, P., Venance, L., Glowinski, J., and Giaume, C. (2004). ATP-induced
inhibition of gap junctional communication is enhanced by interleukin-1 beta
treatment in cultured astrocytes. Neuroscience 126, 95-104.
Mercier, F., and Hatton, G.I. (2001). Connexin 26 and basic fibroblast growth factor are
expressed primarily in the subpial and subependymal layers in adult brain
parenchyma: roles in stem cell proliferation and morphological plasticity? J Comp
Neurol 431, 88-104.
Methippara, M.M., Kumar, S., Alam, M.N., Szymusiak, R., and McGinty, D. (2005). Effects on
sleep of microdialysis of adenosine A1 and A2a receptor analogs into the lateral
preoptic area of rats. Am J Physiol Regul Integr Comp Physiol 289, R1715-1723.
Mignot, E. (2008). Why we sleep: the temporal organization of recovery. PLoS Biol 6, e106.
Milner, C.E., Fogel, S.M., and Cote, K.A. (2006). Habitual napping moderates motor
performance improvements following a short daytime nap. Biol Psychol 73, 141-156.
Min, R., and Nevian, T. (2012). Astrocyte signaling controls spike timing-dependent
depression at neocortical synapses. Nat Neurosci 15, 746-753.
Miyazaki, I., Asanuma, M., Diaz-Corrales, F.J., Miyoshi, K., and Ogawa, N. (2004). Direct
evidence for expression of dopamine receptors in astrocytes from basal ganglia.
Brain Res 1029, 120-123.
Mlinar, B., and Enyeart, J.J. (1993). Block of current through T-type calcium channels by
trivalent metal cations and nickel in neural rat and human cells. J Physiol 469,
639-652.
Modirrousta, M., Mainville, L., and Jones, B.E. (2004). Gabaergic neurons with
alpha2-adrenergic receptors in basal forebrain and preoptic area express c-Fos
during sleep. Neuroscience 129, 803-810.
Moldofsky, H., Lue, F.A., Eisen, J., Keystone, E., and Gorczynski, R.M. (1986). The
relationship of interleukin-1 and immune functions to sleep in humans. Psychosom
Med 48, 309-318.
Molle, M., Marshall, L., Gais, S., and Born, J. (2004). Learning increases human
electroencephalographic coherence during subsequent slow sleep oscillations. Proc
Natl Acad Sci U S A 101, 13963-13968.
Molle, M., Yeshenko, O., Marshall, L., Sara, S.J., and Born, J. (2006). Hippocampal sharp
wave-ripples linked to slow oscillations in rat slow-wave sleep. J Neurophysiol 96,
62-70.
Molle, M., Eschenko, O., Gais, S., Sara, S.J., and Born, J. (2009). The influence of learning on
sleep slow oscillations and associated spindles and ripples in humans and rats. Eur J
Neurosci 29, 1071-1081.
Molle, M., Bergmann, T.O., Marshall, L., and Born, J. (2011). Fast and slow spindles during the
sleep slow oscillation: disparate coalescence and engagement in memory
processing. Sleep 34, 1411-1421.
!"&'!

Mongrain, V., Hernandez, S.A., Pradervand, S., Dorsaz, S., Curie, T., Hagiwara, G., Gip, P.,
Heller, H.C., and Franken, P. (2010). Separating the contribution of glucocorticoids
and wakefulness to the molecular and electrophysiological correlates of sleep
homeostasis. Sleep 33, 1147-1157.
Montana, V., Ni, Y., Sunjara, V., Hua, X., and Parpura, V. (2004). Vesicular glutamate
transporter-dependent glutamate release from astrocytes. J Neurosci 24, 2633-2642.
Montana, V., Malarkey, E.B., Verderio, C., Matteoli, M., and Parpura, V. (2006). Vesicular
transmitter release from astrocytes. Glia 54, 700-715.
Monti, J.M., Hantos, H., Ponzoni, A., Monti, D., and Banchero, P. (1999). Role of nitric oxide in
sleep regulation: effects of L-NAME, an inhibitor of nitric oxide synthase, on sleep in
rats. Behav Brain Res 100, 197-205.
Monti, J.M., and Jantos, H. (2004). Microinjection of the nitric oxide synthase inhibitor L-NAME
into the lateral basal forebrain alters the sleep/wake cycle of the rat. Prog
Neuropsychopharmacol Biol Psychiatry 28, 239-247.
Monti, J.M., and Jantos, H. (2008). Activation of the serotonin 5-HT3 receptor in the dorsal
raphe nucleus suppresses REM sleep in the rat. Prog Neuropsychopharmacol Biol
Psychiatry 32, 940-947.
Moruzzi, G., and Magoun, H.W. (1949). Brain stem reticular formation and activation of the
EEG. Electroencephalogr Clin Neurophysiol 1, 455-473.
Mothet, J.P., Pollegioni, L., Ouanounou, G., Martineau, M., Fossier, P., and Baux, G. (2005).
Glutamate receptor activation triggers a calcium-dependent and SNARE
protein-dependent release of the gliotransmitter D-serine. Proc Natl Acad Sci U S A
102, 5606-5611.
Muzur, A., Pace-Schott, E.F., and Hobson, J.A. (2002). The prefrontal cortex in sleep. Trends
Cogn Sci 6, 475-481.
Nadasdy, Z., Hirase, H., Czurko, A., Csicsvari, J., and Buzsaki, G. (1999). Replay and time
compression of recurring spike sequences in the hippocampus. J Neurosci 19,
9497-9507.
Nagelhus, E.A., Mathiisen, T.M., and Ottersen, O.P. (2004). Aquaporin-4 in the central
nervous system: cellular and subcellular distribution and coexpression with KIR4.1.
Neuroscience 129, 905-913.
Nagy, J.I., Patel, D., Ochalski, P.A., and Stelmack, G.L. (1999). Connexin30 in rodent, cat and
human brain: selective expression in gray matter astrocytes, co-localization with
connexin43 at gap junctions and late developmental appearance. Neuroscience 88,
447-468.
Nagy, J.I., Li, X., Rempel, J., Stelmack, G., Patel, D., Staines, W.A., Yasumura, T., and Rash,
J.E. (2001). Connexin26 in adult rodent central nervous system: demonstration at
astrocytic gap junctions and colocalization with connexin30 and connexin43. J Comp
Neurol 441, 302-323.
Nagy, J.I., Dudek, F.E., and Rash, J.E. (2004). Update on connexins and gap junctions in
neurons and glia in the mammalian nervous system. Brain Res Brain Res Rev 47,
191-215.
Nagy, J.I., Lynn, B.D., Tress, O., Willecke, K., and Rash, J.E. (2011). Connexin26 expression
in brain parenchymal cells demonstrated by targeted connexin ablation in transgenic
mice. Eur J Neurosci 34, 263-271.
Naidoo, N., Giang, W., Galante, R.J., and Pack, A.I. (2005). Sleep deprivation induces the
unfolded protein response in mouse cerebral cortex. J Neurochem 92, 1150-1157.
Namihira, M., and Nakashima, K. (2013). Mechanisms of astrocytogenesis in the mammalian
brain. Curr Opin Neurobiol 23, 921-927.
Naus, C.C., Zhu, D., Todd, S.D., and Kidder, G.M. (1992). Characteristics of C6 glioma cells
overexpressing a gap junction protein. Cell Mol Neurobiol 12, 163-175.
Navarrete, M., and Araque, A. (2010). Endocannabinoids potentiate synaptic transmission
through stimulation of astrocytes. Neuron 68, 113-126.
Navarrete, M., Perea, G., Fernandez de Sevilla, D., Gomez-Gonzalo, M., Nunez, A., Martin,
E.D., and Araque, A. (2012). Astrocytes mediate in vivo cholinergic-induced synaptic
plasticity. PLoS Biol 10, e1001259.
Naylor, E., Aillon, D.V., Barrett, B.S., Wilson, G.S., Johnson, D.A., Harmon, H.P., Gabbert, S.,
and Petillo, P.A. (2012). Lactate as a biomarker for sleep. Sleep 35, 1209-1222.
!

"&(!

Nedergaard, M., Ransom, B., and Goldman, S.A. (2003). New roles for astrocytes: redefining
the functional architecture of the brain. Trends Neurosci 26, 523-530.
Nedergaard, M., and Verkhratsky, A. (2012). Artifact versus reality--how astrocytes contribute
to synaptic events. Glia 60, 1013-1023.
Nelini, C., Bobbo, D., and Mascetti, G.G. (2010). Local sleep: a spatial learning task enhances
sleep in the right hemisphere of domestic chicks (Gallus gallus). Exp Brain Res 205,
195-204.
Nestor, M.W., Mok, L.P., Tulapurkar, M.E., and Thompson, S.M. (2007). Plasticity of
neuron-glial interactions mediated by astrocytic EphARs. J Neurosci 27,
12817-12828.
Netchiporouk, L., Shram, N., Salvert, D., and Cespuglio, R. (2001). Brain extracellular glucose
assessed by voltammetry throughout the rat sleep-wake cycle. Eur J Neurosci 13,
1429-1434.
Newman, D.B., and Ginsberg, C.Y. (1994). Brainstem reticular nuclei that project to the
thalamus in rats: a retrograde tracer study. Brain Behav Evol 44, 1-39.
Newman, E.A. (2003). Glial cell inhibition of neurons by release of ATP. J Neurosci 23,
1659-1666.
Nie, H., Zhang, H., and Weng, H.R. (2010). Bidirectional neuron-glia interactions triggered by
deficiency of glutamate uptake at spinal sensory synapses. J Neurophysiol 104,
713-725.
Nielsen, M.S., Axelsen, L.N., Sorgen, P.L., Verma, V., Delmar, M., and Holstein-Rathlou, N.H.
(2012). Gap junctions. Compr Physiol 2, 1981-2035.
Nimmerjahn, A., Mukamel, E.A., and Schnitzer, M.J. (2009). Motor behavior activates
Bergmann glial networks. Neuron 62, 400-412.
O'Keefe, J., Nadel, L., and Willner, J. (1979). Tuning out irrelevancy? Comments on Solomon's
temporal mapping view of the hippocampus. Psychol Bull 86, 1280-1289.
O'Neill, J., Pleydell-Bouverie, B., Dupret, D., and Csicsvari, J. (2010). Play it again:
reactivation of waking experience and memory. Trends Neurosci 33, 220-229.
Oberheim, N.A., Takano, T., Han, X., He, W., Lin, J.H., Wang, F., Xu, Q., Wyatt, J.D., Pilcher,
W., Ojemann, J.G., et al. (2009). Uniquely hominid features of adult human
astrocytes. J Neurosci 29, 3276-3287.
Occhipinti, R., Somersalo, E., and Calvetti, D. (2010). Energetics of inhibition: insights with a
computational model of the human GABAergic neuron-astrocyte cellular complex. J
Cereb Blood Flow Metab 30, 1834-1846.
Ogata, K., and Kosaka, T. (2002). Structural and quantitative analysis of astrocytes in the
mouse hippocampus. Neuroscience 113, 221-233.
Okada, T., Mochizuki, T., Huang, Z.L., Eguchi, N., Sugita, Y., Urade, Y., and Hayaishi, O.
(2003). Dominant localization of adenosine deaminase in leptomeninges and
involvement of the enzyme in sleep. Biochem Biophys Res Commun 312, 29-34.
Olk, S., Zoidl, G., and Dermietzel, R. (2009). Connexins, cell motility, and the cytoskeleton.
Cell Motil Cytoskeleton 66, 1000-1016.
Olsen, M.L., and Sontheimer, H. (2008). Functional implications for Kir4.1 channels in glial
biology: from K+ buffering to cell differentiation. J Neurochem 107, 589-601.
Orellana, J.A., Shoji, K.F., Abudara, V., Ezan, P., Amigou, E., Saez, P.J., Jiang, J.X., Naus,
C.C., Saez, J.C., and Giaume, C. (2011a). Amyloid beta-induced death in neurons
involves glial and neuronal hemichannels. J Neurosci 31, 4962-4977.
Orellana, J.A., Froger, N., Ezan, P., Jiang, J.X., Bennett, M.V., Naus, C.C., Giaume, C., and
Saez, J.C. (2011b). ATP and glutamate released via astroglial connexin 43
hemichannels mediate neuronal death through activation of pannexin 1
hemichannels. J Neurochem 118, 826-840.
Orellana, J.A., von Bernhardi, R., Giaume, C., and Saez, J.C. (2012). Glial hemichannels and
their involvement in aging and neurodegenerative diseases. Rev Neurosci 23,
163-177.
Orellana, J.A., Martinez, A.D., and Retamal, M.A. (2013). Gap junction channels and
hemichannels in the CNS: regulation by signaling molecules. Neuropharmacology 75,
567-582.
Orkand, R.K. (1980). Extracellular potassium accumulation in the nervous system. Fed Proc
39, 1515-1518.
!"&,!

Oswald, I. (1970). Sleep. (Penguin Books).

Palchykova, S., Winsky-Sommerer, R., Shen, H.Y., Boison, D., Gerling, A., and Tobler, I.
(2010). Manipulation of adenosine kinase affects sleep regulation in mice. J Neurosci
30, 13157-13165.
Palva, S., and Palva, J.M. (2007). New vistas for alpha-frequency band oscillations. Trends
Neurosci 30, 150-158.
Panatier, A., Theodosis, D.T., Mothet, J.P., Touquet, B., Pollegioni, L., Poulain, D.A., and Oliet,
S.H. (2006). Glia-derived D-serine controls NMDA receptor activity and synaptic
memory. Cell 125, 775-784.
Panatier, A., Vallee, J., Haber, M., Murai, K.K., Lacaille, J.C., and Robitaille, R. (2011).
Astrocytes are endogenous regulators of basal transmission at central synapses.
Cell 146, 785-798.
Pannasch, U., Vargova, L., Reingruber, J., Ezan, P., Holcman, D., Giaume, C., Sykova, E.,
and Rouach, N. (2011). Astroglial networks scale synaptic activity and plasticity.
Proc Natl Acad Sci U S A 108, 8467-8472.
Pannasch, U., Freche, D., Dallerac, G., Ghezali, G., Escartin, C., Ezan, P., Cohen-Salmon, M.,
Benchenane, K., Abudara, V., Dufour, A., et al. (2014). Connexin 30 sets synaptic
strength by controlling astroglial synapse invasion. Nat Neurosci 17, 549-558.
Parish, J.M. (2009). Sleep-related problems in common medical conditions. Chest 135,
563-572.
Parmentier, R., Ohtsu, H., Djebbara-Hannas, Z., Valatx, J.L., Watanabe, T., and Lin, J.S.
(2002). Anatomical, physiological, and pharmacological characteristics of histidine
decarboxylase knock-out mice: evidence for the role of brain histamine in behavioral
and sleep-wake control. J Neurosci 22, 7695-7711.
Parpura, V., Basarsky, T.A., Liu, F., Jeftinija, K., Jeftinija, S., and Haydon, P.G. (1994).
Glutamate-mediated astrocyte-neuron signalling. Nature 369, 744-747.
Parpura, V., Baker, B.J., Jeras, M., and Zorec, R. (2010). Regulated exocytosis in astrocytic
signal integration. Neurochem Int 57, 451-459.
Parpura, V., Grubisic, V., and Verkhratsky, A. (2011). Ca(2+) sources for the exocytotic
release of glutamate from astrocytes. Biochim Biophys Acta 1813, 984-991.
Parpura, V., and Verkhratsky, A. (2012). The astrocyte excitability brief: from receptors to
gliotransmission. Neurochem Int 61, 610-621.
Parri, H.R., and Crunelli, V. (2001). Pacemaker calcium oscillations in thalamic astrocytes in
situ. Neuroreport 12, 3897-3900.
Parri, H.R., Gould, T.M., and Crunelli, V. (2001). Spontaneous astrocytic Ca2+ oscillations in
situ drive NMDAR-mediated neuronal excitation. Nat Neurosci 4, 803-812.
Parsons, M.P., and Hirasawa, M. (2010). ATP-sensitive potassium channel-mediated lactate
effect on orexin neurons: implications for brain energetics during arousal. J Neurosci
30, 8061-8070.
Pascual, O., Casper, K.B., Kubera, C., Zhang, J., Revilla-Sanchez, R., Sul, J.Y., Takano, H.,
Moss, S.J., McCarthy, K., and Haydon, P.G. (2005). Astrocytic purinergic signaling
coordinates synaptic networks. Science 310, 113-116.
Pascual, O., Ben Achour, S., Rostaing, P., Triller, A., and Bessis, A. (2012). Microglia
activation triggers astrocyte-mediated modulation of excitatory neurotransmission.
Proc Natl Acad Sci U S A 109, E197-205.
Pasti, L., Zonta, M., Pozzan, T., Vicini, S., and Carmignoto, G. (2001). Cytosolic calcium
oscillations in astrocytes may regulate exocytotic release of glutamate. J Neurosci 21,
477-484.
Patel, D., Zhang, X., and Veenstra, R.D. (2014). Connexin hemichannel and pannexin channel
electrophysiology: how do they differ? FEBS Lett 588, 1372-1378.
Pavlides, C., Greenstein, Y.J., Grudman, M., and Winson, J. (1988). Long-term potentiation in
the dentate gyrus is induced preferentially on the positive phase of theta-rhythm.
Brain Res 439, 383-387.
Payne, J.D., and Kensinger, E.A. (2011). Sleep leads to changes in the emotional memory
trace: evidence from FMRI. J Cogn Neurosci 23, 1285-1297.
Peigneux, P., Laureys, S., Fuchs, S., Collette, F., Perrin, F., Reggers, J., Phillips, C.,
Degueldre, C., Del Fiore, G., Aerts, J., et al. (2004). Are spatial memories
!

"&-!

strengthened in the human hippocampus during slow wave sleep? Neuron 44,
535-545.
Pellerin, L., and Magistretti, P.J. (1994). Glutamate uptake into astrocytes stimulates aerobic
glycolysis: a mechanism coupling neuronal activity to glucose utilization. Proc Natl
Acad Sci U S A 91, 10625-10629.
Pellerin, L., and Magistretti, P.J. (2012). Sweet sixteen for ANLS. J Cereb Blood Flow Metab
32, 1152-1166.
Penes, M.C., Li, X., and Nagy, J.I. (2005). Expression of zonula occludens-1 (ZO-1) and the
transcription factor ZO-1-associated nucleic acid-binding protein (ZONAB)-MsY3 in
glial cells and colocalization at oligodendrocyte and astrocyte gap junctions in mouse
brain. Eur J Neurosci 22, 404-418.
Peng, M., Wang, Y.L., Wang, C.Y., and Chen, C. (2013). Dexmedetomidine attenuates
lipopolysaccharide-induced proinflammatory response in primary microglia. J Surg
Res 179, e219-225.
Peng, M., Ye, J.S., Wang, Y.L., Chen, C., and Wang, C.Y. (2014). Posttreatment with propofol
attenuates lipopolysaccharide-induced up-regulation of inflammatory molecules in
primary microglia. Inflamm Res 63, 411-418.
Pennartz, C.M., Lee, E., Verheul, J., Lipa, P., Barnes, C.A., and McNaughton, B.L. (2004). The
ventral striatum in off-line processing: ensemble reactivation during sleep and
modulation by hippocampal ripples. J Neurosci 24, 6446-6456.
Peplow, M. (2013). Structure: the anatomy of sleep. Nature 497, S2-3.
Perea, G., and Araque, A. (2005). Properties of synaptically evoked astrocyte calcium signal
reveal synaptic information processing by astrocytes. J Neurosci 25, 2192-2203.
Perea, G., Navarrete, M., and Araque, A. (2009). Tripartite synapses: astrocytes process and
control synaptic information. Trends Neurosci 32, 421-431.
Perrett, S.P., Dudek, S.M., Eagleman, D., Montague, P.R., and Friedlander, M.J. (2001). LTD
induction in adult visual cortex: role of stimulus timing and inhibition. J Neurosci 21,
2308-2319.
Peterfi, Z., Churchill, L., Hajdu, I., Obal Jr, F., Krueger, J.M., and Parducz, A. (2004).
Fos-immunoreactivity in the hypothalamus: dependency on the diurnal rhythm, sleep,
gender, and estrogen. Neuroscience 124, 695-707.
Peters, O., Schipke, C.G., Hashimoto, Y., and Kettenmann, H. (2003). Different mechanisms
promote astrocyte Ca2+ waves and spreading depression in the mouse neocortex. J
Neurosci 23, 9888-9896.
Petit, J.M., Tobler, I., Kopp, C., Morgenthaler, F., Borbely, A.A., and Magistretti, P.J. (2010).
Metabolic response of the cerebral cortex following gentle sleep deprivation and
modafinil administration. Sleep 33, 901-908.
Petit, J.M., Gyger, J., Burlet-Godinot, S., Fiumelli, H., Martin, J.L., and Magistretti, P.J. (2013).
Genes involved in the astrocyte-neuron lactate shuttle (ANLS) are specifically
regulated in cortical astrocytes following sleep deprivation in mice. Sleep 36,
1445-1458.
Petzold, G.C., and Murthy, V.N. (2011). Role of astrocytes in neurovascular coupling. Neuron
71, 782-797.
Peyrache, A., Khamassi, M., Benchenane, K., Wiener, S.I., and Battaglia, F.P. (2009). Replay
of rule-learning related neural patterns in the prefrontal cortex during sleep. Nat
Neurosci 12, 919-926.
Peyron, C., Faraco, J., Rogers, W., Ripley, B., Overeem, S., Charnay, Y., Nevsimalova, S.,
Aldrich, M., Reynolds, D., Albin, R., et al. (2000). A mutation in a case of early onset
narcolepsy and a generalized absence of hypocretin peptides in human narcoleptic
brains. Nat Med 6, 991-997.
Phelan, P., and Starich, T.A. (2001). Innexins get into the gap. Bioessays 23, 388-396.
Pieribone, V.A., Xu, Z.Q., Zhang, X., Grillner, S., Bartfai, T., and Hokfelt, T. (1995). Galanin
induces a hyperpolarization of norepinephrine-containing locus coeruleus neurons in
the brainstem slice. Neuroscience 64, 861-874.
Piet, R., Poulain, D.A., and Oliet, S.H. (2004). Contribution of astrocytes to synaptic
transmission in the rat supraoptic nucleus. Neurochem Int 45, 251-257.
Piper, D.C., Upton, N., Smith, M.I., and Hunter, A.J. (2000). The novel brain neuropeptide,
orexin-A, modulates the sleep-wake cycle of rats. Eur J Neurosci 12, 726-730.
!"'.!

Pirttimaki, T.M., Hall, S.D., and Parri, H.R. (2011). Sustained neuronal activity generated by
glial plasticity. J Neurosci 31, 7637-7647.
Pizzo, P., Burgo, A., Pozzan, T., and Fasolato, C. (2001). Role of capacitative calcium entry on
glutamate-induced calcium influx in type-I rat cortical astrocytes. J Neurochem 79,
98-109.
Plihal, W., and Born, J. (1997). Effects of early and late nocturnal sleep on declarative and
procedural memory. J Cogn Neurosci 9, 534-547.
Plihal, W., and Born, J. (1999). Effects of early and late nocturnal sleep on priming and spatial
memory. Psychophysiology 36, 571-582.
Porkka-Heiskanen, T., Strecker, R.E., Thakkar, M., Bjorkum, A.A., Greene, R.W., and
McCarley, R.W. (1997). Adenosine: a mediator of the sleep-inducing effects of
prolonged wakefulness. Science 276, 1265-1268.
Porkka-Heiskanen, T., Strecker, R.E., and McCarley, R.W. (2000). Brain site-specificity of
extracellular adenosine concentration changes during sleep deprivation and
spontaneous sleep: an in vivo microdialysis study. Neuroscience 99, 507-517.
Porkka-Heiskanen, T., Kalinchuk, A., Alanko, L., Urrila, A., and Stenberg, D. (2003).
Adenosine, energy metabolism, and sleep. ScientificWorldJournal 3, 790-798.
Portas, C.M., Thakkar, M., Rainnie, D.G., Greene, R.W., and McCarley, R.W. (1997). Role of
adenosine in behavioral state modulation: a microdialysis study in the freely moving
cat. Neuroscience 79, 225-235.
Poskanzer, K.E., and Yuste, R. (2011). Astrocytic regulation of cortical UP states. Proc Natl
Acad Sci U S A 108, 18453-18458.
Putney, J.W., Jr. (1986). A model for receptor-regulated calcium entry. Cell Calcium 7, 1-12.
Putney, J.W., Jr. (1990). Capacitative calcium entry revisited. Cell Calcium 11, 611-624.
Qin, Y.L., McNaughton, B.L., Skaggs, W.E., and Barnes, C.A. (1997). Memory reprocessing in
corticocortical and hippocampocortical neuronal ensembles. Philos Trans R Soc
Lond B Biol Sci 352, 1525-1533.
Qin, Y., Zhu, Y., Baumgart, J.P., Stornetta, R.L., Seidenman, K., Mack, V., van Aelst, L., and
Zhu, J.J. (2005). State-dependent Ras signaling and AMPA receptor trafficking.
Genes Dev 19, 2000-2015.
Qu, C., Gardner, P., and Schrijver, I. (2009). The role of the cytoskeleton in the formation of
gap junctions by Connexin 30. Exp Cell Res 315, 1683-1692.
Qu, W.M., Xu, X.H., Yan, M.M., Wang, Y.Q., Urade, Y., and Huang, Z.L. (2010). Essential role
of dopamine D2 receptor in the maintenance of wakefulness, but not in homeostatic
regulation of sleep, in mice. J Neurosci 30, 4382-4389.
Radek, R.J., Decker, M.W., and Jarvis, M.F. (2004). The adenosine kinase inhibitor ABT-702
augments EEG slow waves in rats. Brain Res 1026, 74-83.
Radulovacki, M., Virus, R.M., Djuricic-Nedelson, M., and Green, R.D. (1984). Adenosine
analogs and sleep in rats. J Pharmacol Exp Ther 228, 268-274.
Radulovacki, M., Virus, R.M., Rapoza, D., and Crane, R.A. (1985). A comparison of the dose
response effects of pyrimidine ribonucleosides and adenosine on sleep in rats.
Psychopharmacology (Berl) 87, 136-140.
Rai, S., Kumar, S., Alam, M.A., Szymusiak, R., McGinty, D., and Alam, M.N. (2010). A1
receptor mediated adenosinergic regulation of perifornical-lateral hypothalamic area
neurons in freely behaving rats. Neuroscience 167, 40-48.
Rainnie, D.G., Grunze, H.C., McCarley, R.W., and Greene, R.W. (1994). Adenosine inhibition
of mesopontine cholinergic neurons: implications for EEG arousal. Science 263,
689-692.
Ramadan, W., Eschenko, O., and Sara, S.J. (2009). Hippocampal sharp wave/ripples during
sleep for consolidation of associative memory. PLoS One 4, e6697.
Rampon, C., Jiang, C.H., Dong, H., Tang, Y.P., Lockhart, D.J., Schultz, P.G., Tsien, J.Z., and
Hu, Y. (2000). Effects of environmental enrichment on gene expression in the brain.
Proc Natl Acad Sci U S A 97, 12880-12884.
Rana, S., and Dringen, R. (2007). Gap junction hemichannel-mediated release of glutathione
from cultured rat astrocytes. Neurosci Lett 415, 45-48.
Rancillac, A., Rossier, J., Guille, M., Tong, X.K., Geoffroy, H., Amatore, C., Arbault, S., Hamel,
E., and Cauli, B. (2006). Glutamatergic Control of Microvascular Tone by Distinct
GABA Neurons in the Cerebellum. J Neurosci 26, 6997-7006.
!

"'"!

Rao, Y., Liu, Z.W., Borok, E., Rabenstein, R.L., Shanabrough, M., Lu, M., Picciotto, M.R.,
Horvath, T.L., and Gao, X.B. (2007). Prolonged wakefulness induces
experience-dependent synaptic plasticity in mouse hypocretin/orexin neurons. J Clin
Invest 117, 4022-4033.
Rasch, B., Buchel, C., Gais, S., and Born, J. (2007). Odor cues during slow-wave sleep prompt
declarative memory consolidation. Science 315, 1426-1429.
Rasch, B., and Born, J. (2013). About sleep's role in memory. Physiol Rev 93, 681-766.
Rash, J.E., Yasumura, T., Dudek, F.E., and Nagy, J.I. (2001). Cell-specific expression of
connexins and evidence of restricted gap junctional coupling between glial cells and
between neurons. J Neurosci 21, 1983-2000.
Reichenbach, A., and Wolburg, H. (2005). Astrocytes and ependymal glia. In Neuroglia, H.
Kettenmann and B.R. Ransom, eds. (Oxford University Press), pp. 1935.
Restani, L., Antonucci, F., Gianfranceschi, L., Rossi, C., Rossetto, O., and Caleo, M. (2011).
Evidence for anterograde transport and transcytosis of botulinum neurotoxin A
(BoNT/A). J Neurosci 31, 15650-15659.
Retamal, M.A., Froger, N., Palacios-Prado, N., Ezan, P., Saez, P.J., Saez, J.C., and Giaume,
C. (2007). Cx43 hemichannels and gap junction channels in astrocytes are regulated
oppositely by proinflammatory cytokines released from activated microglia. J
Neurosci 27, 13781-13792.
Retey, J.V., Adam, M., Honegger, E., Khatami, R., Luhmann, U.F., Jung, H.H., Berger, W.,
and Landolt, H.P. (2005). A functional genetic variation of adenosine deaminase
affects the duration and intensity of deep sleep in humans. Proc Natl Acad Sci U S A
102, 15676-15681.
Retey, J.V., Adam, M., Khatami, R., Luhmann, U.F., Jung, H.H., Berger, W., and Landolt, H.P.
(2007). A genetic variation in the adenosine A2A receptor gene (ADORA2A)
contributes to individual sensitivity to caffeine effects on sleep. Clin Pharmacol Ther
81, 692-698.
Reyes, R.C., Verkhratsky, A., and Parpura, V. (2012). Plasmalemmal Na+/Ca2+ exchanger
modulates Ca2+-dependent exocytotic release of glutamate from rat cortical
astrocytes. ASN Neuro 4.
Reyes-Haro, D., Muller, J., Boresch, M., Pivneva, T., Benedetti, B., Scheller, A., Nolte, C., and
Kettenmann, H. (2010). Neuron-astrocyte interactions in the medial nucleus of the
trapezoid body. J Gen Physiol 135, 583-594.
Ribeiro, A.C., and Kapas, L. (2005). Day- and nighttime injection of a nitric oxide synthase
inhibitor elicits opposite sleep responses in rats. Am J Physiol Regul Integr Comp
Physiol 289, R521-R531.
Rieger, A., Deitmer, J.W., and Lohr, C. (2007). Axon-glia communication evokes calcium
signaling in olfactory ensheathing cells of the developing olfactory bulb. Glia 55,
352-359.
Robertson, E.M., Pascual-Leone, A., and Miall, R.C. (2004). Current concepts in procedural
consolidation. Nat Rev Neurosci 5, 576-582.
Rosanova, M., and Ulrich, D. (2005). Pattern-specific associative long-term potentiation
induced by a sleep spindle-related spike train. J Neurosci 25, 9398-9405.
Rose, C.R., and Ransom, B.R. (1996). Intracellular sodium homeostasis in rat hippocampal
astrocytes. J Physiol 491 ( Pt 2), 291-305.
Rosenberg, P.A., Li, Y., Le, M., and Zhang, Y. (2000). Nitric oxide-stimulated increase in
extracellular adenosine accumulation in rat forebrain neurons in culture is associated
with ATP hydrolysis and inhibition of adenosine kinase activity. J Neurosci 20,
6294-6301.
Rouach, N., Glowinski, J., and Giaume, C. (2000). Activity-dependent neuronal control of
gap-junctional communication in astrocytes. J Cell Biol 149, 1513-1526.
Rouach, N., Segal, M., Koulakoff, A., Giaume, C., and Avignone, E. (2003). Carbenoxolone
blockade of neuronal network activity in culture is not mediated by an action on gap
junctions. J Physiol 553, 729-745.
Rouach, N., Koulakoff, A., Abudara, V., Willecke, K., and Giaume, C. (2008). Astroglial
metabolic networks sustain hippocampal synaptic transmission. Science 322,
1551-1555.
!"'#!

Roux, L., Benchenane, K., Rothstein, J.D., Bonvento, G., and Giaume, C. (2011). Plasticity of
astroglial networks in olfactory glomeruli. Proc Natl Acad Sci U S A 108,
18442-18446.
Roux, L., Madar, A., Lacroix, M.M., Benchenane, K., and Giaume, C. (In revision). Astroglial
connexin 43 hemichannels modulate olfactory bulb slow oscillations.
Roy, C.S., and Sherrington, C.S. (1890). On the Regulation of the Blood-supply of the Brain. J
Physiol 11, 85-158 117.
Ruch, S., Markes, O., Duss, S.B., Oppliger, D., Reber, T.P., Koenig, T., Mathis, J., Roth, C.,
and Henke, K. (2012). Sleep stage II contributes to the consolidation of declarative
memories. Neuropsychologia 50, 2389-2396.
Rudolph, U., and Antkowiak, B. (2004). Molecular and neuronal substrates for general
anaesthetics. Nat Rev Neurosci 5, 709-720.
Rudoy, J.D., Voss, J.L., Westerberg, C.E., and Paller, K.A. (2009). Strengthening individual
memories by reactivating them during sleep. Science 326, 1079.
Saab, A.S., Neumeyer, A., Jahn, H.M., Cupido, A., Simek, A.A., Boele, H.J., Scheller, A., Le
Meur, K., Gotz, M., Monyer, H., et al. (2012). Bergmann glial AMPA receptors are
required for fine motor coordination. Science 337, 749-753.
Saez, J.C., and Leybaert, L. (2014). Hunting for connexin hemichannels. FEBS Lett 588,
1205-1211.
Sallanon, M., Denoyer, M., Kitahama, K., Aubert, C., Gay, N., and Jouvet, M. (1989).
Long-lasting insomnia induced by preoptic neuron lesions and its transient reversal
by muscimol injection into the posterior hypothalamus in the cat. Neuroscience 32,
669-683.
Santello, M., Bezzi, P., and Volterra, A. (2011). TNFalpha controls glutamatergic
gliotransmission in the hippocampal dentate gyrus. Neuron 69, 988-1001.
Sanzgiri, R.P., Araque, A., and Haydon, P.G. (1999). Prostaglandin E(2) stimulates glutamate
receptor-dependent astrocyte neuromodulation in cultured hippocampal cells. J
Neurobiol 41, 221-229.
Saper, C.B., Chou, T.C., and Scammell, T.E. (2001). The sleep switch: hypothalamic control of
sleep and wakefulness. Trends Neurosci 24, 726-731.
Saper, C.B., Fuller, P.M., Pedersen, N.P., Lu, J., and Scammell, T.E. (2010). Sleep state
switching. Neuron 68, 1023-1042.
Sapin, E., Lapray, D., Berod, A., Goutagny, R., Leger, L., Ravassard, P., Clement, O., Hanriot,
L., Fort, P., and Luppi, P.H. (2009). Localization of the brainstem GABAergic
neurons controlling paradoxical (REM) sleep. PLoS One 4, e4272.
Sasaki, T., Kuga, N., Namiki, S., Matsuki, N., and Ikegaya, Y. (2011). Locally synchronized
astrocytes. Cereb Cortex 21, 1889-1900.
Sastre, J.P., Buda, C., Kitahama, K., and Jouvet, M. (1996). Importance of the ventrolateral
region of the periaqueductal gray and adjacent tegmentum in the control of
paradoxical sleep as studied by muscimol microinjections in the cat. Neuroscience
74, 415-426.
Satoh, S., Matsumura, H., and Hayaishi, O. (1998). Involvement of adenosine A2A receptor in
sleep promotion. Eur J Pharmacol 351, 155-162.
Satoh, S., Matsumura, H., Koike, N., Tokunaga, Y., Maeda, T., and Hayaishi, O. (1999).
Region-dependent difference in the sleep-promoting potency of an adenosine A2A
receptor agonist. Eur J Neurosci 11, 1587-1597.
Savin, C., Triesch, J., and Meyer-Hermann, M. (2009). Epileptogenesis due to glia-mediated
synaptic scaling. J R Soc Interface 6, 655-668.
Sawada, K., Echigo, N., Juge, N., Miyaji, T., Otsuka, M., Omote, H., Yamamoto, A., and
Moriyama, Y. (2008). Identification of a vesicular nucleotide transporter. Proc Natl
Acad Sci U S A 105, 5683-5686.
Scammell, T.E., Gerashchenko, D.Y., Mochizuki, T., McCarthy, M.T., Estabrooke, I.V., Sears,
C.A., Saper, C.B., Urade, Y., and Hayaishi, O. (2001). An adenosine A2a agonist
increases sleep and induces Fos in ventrolateral preoptic neurons. Neuroscience
107, 653-663.
Scemes, E., and Giaume, C. (2006). Astrocyte calcium waves: what they are and what they do.
Glia 54, 716-725.
!

"'$!

Scemes, E., Suadicani, S.O., Dahl, G., and Spray, D.C. (2007). Connexin and pannexin
mediated cell-cell communication. Neuron Glia Biol 3, 199-208.
Schabus, M., Gruber, G., Parapatics, S., Sauter, C., Klosch, G., Anderer, P., Klimesch, W.,
Saletu, B., and Zeitlhofer, J. (2004). Sleep spindles and their significance for
declarative memory consolidation. Sleep 27, 1479-1485.
Schalper, K.A., Orellana, J.A., Berthoud, V.M., and Saez, J.C. (2009). Dysfunctions of the
diffusional membrane pathways mediated by hemichannels in inherited and acquired
human diseases. Curr Vasc Pharmacol 7, 486-505.
Schipke, C.G., Boucsein, C., Ohlemeyer, C., Kirchhoff, F., and Kettenmann, H. (2002).
Astrocyte Ca2+ waves trigger responses in microglial cells in brain slices. FASEB J
16, 255-257.
Schmidt, C., Peigneux, P., Muto, V., Schenkel, M., Knoblauch, V., Munch, M., de Quervain,
D.J., Wirz-Justice, A., and Cajochen, C. (2006). Encoding difficulty promotes
postlearning changes in sleep spindle activity during napping. J Neurosci 26,
8976-8982.
Schmitt, L.I., Sims, R.E., Dale, N., and Haydon, P.G. (2012). Wakefulness affects synaptic and
network activity by increasing extracellular astrocyte-derived adenosine. J Neurosci
32, 4417-4425.
Schonrock, B., Busselberg, D., and Haas, H.L. (1991). Properties of tuberomammillary
histamine neurones and their response to galanin. Agents Actions 33, 135-137.
Schousboe, A., and Waagepetersen, H.S. (2007). GABA: homeostatic and pharmacological
aspects. Prog Brain Res 160, 9-19.
Schroder, M., and Kaufman, R.J. (2005). The mammalian unfolded protein response. Annu
Rev Biochem 74, 739-789.
Schousboe, A., and Waagepetersen, H.S. (2007). In Handbook of Neurochemistry and
Molecular Neurobiology Neurotransmitter Systems, A. Lajtha, ed. (Springer), pp.
213-226.
Schummers, J., Yu, H., and Sur, M. (2008). Tuned responses of astrocytes and their influence
on hemodynamic signals in the visual cortex. Science 320, 1638-1643.
Schutz, M., Scimemi, P., Majumder, P., De Siati, R.D., Crispino, G., Rodriguez, L., Bortolozzi,
M., Santarelli, R., Seydel, A., Sonntag, S., et al. (2010). The human
deafness-associated connexin 30 T5M mutation causes mild hearing loss and
reduces biochemical coupling among cochlear non-sensory cells in knock-in mice.
Hum Mol Genet 19, 4759-4773.
Semba, K. (2000). Multiple output pathways of the basal forebrain: organization, chemical
heterogeneity, and roles in vigilance. Behav Brain Res 115, 117-141.
Serrano, A., Haddjeri, N., Lacaille, J.C., and Robitaille, R. (2006). GABAergic network
activation of glial cells underlies hippocampal heterosynaptic depression. J Neurosci
26, 5370-5382.
Serrano, A., Robitaille, R., and Lacaille, J.C. (2008). Differential NMDA-dependent activation
of glial cells in mouse hippocampus. Glia 56, 1648-1663.
Sheppard, C.A., Simpson, P.B., Sharp, A.H., Nucifora, F.C., Ross, C.A., Lange, G.D., and
Russell, J.T. (1997). Comparison of type 2 inositol 1,4,5-trisphosphate receptor
distribution and subcellular Ca2+ release sites that support Ca2+ waves in cultured
astrocytes. J Neurochem 68, 2317-2327.
Sherin, J.E., Shiromani, P.J., McCarley, R.W., and Saper, C.B. (1996). Activation of
ventrolateral preoptic neurons during sleep. Science 271, 216-219.
Sherin, J.E., Elmquist, J.K., Torrealba, F., and Saper, C.B. (1998). Innervation of histaminergic
tuberomammillary neurons by GABAergic and galaninergic neurons in the
ventrolateral preoptic nucleus of the rat. J Neurosci 18, 4705-4721.
Shigetomi, E., Bowser, D.N., Sofroniew, M.V., and Khakh, B.S. (2008). Two forms of astrocyte
calcium excitability have distinct effects on NMDA receptor-mediated slow inward
currents in pyramidal neurons. J Neurosci 28, 6659-6663.
Siapas, A.G., and Wilson, M.A. (1998). Coordinated interactions between hippocampal ripples
and cortical spindles during slow-wave sleep. Neuron 21, 1123-1128.
Sibson, N.R., Shen, J., Mason, G.F., Rothman, D.L., Behar, K.L., and Shulman, R.G. (1998).
Functional energy metabolism: in vivo 13C-NMR spectroscopy evidence for coupling
!"'%!

of cerebral glucose consumption and glutamatergic neuronalactivity. Dev Neurosci

20, 321-330.
Singer, A.C., Carr, M.F., Karlsson, M.P., and Frank, L.M. (2013). Hippocampal SWR activity
predicts correct decisions during the initial learning of an alternation task. Neuron 77,
1163-1173.
Sirota, A., Csicsvari, J., Buhl, D., and Buzsaki, G. (2003). Communication between neocortex
and hippocampus during sleep in rodents. Proc Natl Acad Sci U S A 100, 2065-2069.
Skaggs, W.E., and McNaughton, B.L. (1996). Replay of neuronal firing sequences in rat
hippocampus during sleep following spatial experience. Science 271, 1870-1873.
Smith, C. (2001). Sleep states and memory processes in humans: procedural versus
declarative memory systems. Sleep Med Rev 5, 491-506.
Sohl, G., and Willecke, K. (2004). Gap junctions and the connexin protein family. Cardiovasc
Res 62, 228-232.
Somjen, G.G. (2002). Ion regulation in the brain: implications for pathophysiology.
Neuroscientist 8, 254-267.
Spiegel, K., Tasali, E., Leproult, R., and Van Cauter, E. (2009). Effects of poor and short sleep
on glucose metabolism and obesity risk. Nat Rev Endocrinol 5, 253-261.
Sreedharan, S., Shaik, J.H., Olszewski, P.K., Levine, A.S., Schioth, H.B., and Fredriksson, R.
(2010). Glutamate, aspartate and nucleotide transporters in the SLC17 family form
four main phylogenetic clusters: evolution and tissue expression. BMC Genomics 11,
17.
Stehberg, J., Moraga-Amaro, R., Salazar, C., Becerra, A., Echeverria, C., Orellana, J.A.,
Bultynck, G., Ponsaerts, R., Leybaert, L., Simon, F., et al. (2012). Release of
gliotransmitters through astroglial connexin 43 hemichannels is necessary for fear
memory consolidation in the basolateral amygdala. FASEB J 26, 3649-3657.
Steinfels, G.F., Heym, J., Strecker, R.E., and Jacobs, B.L. (1983). Behavioral correlates of
dopaminergic unit activity in freely moving cats. Brain Res 258, 217-228.
Steininger, T.L., Gong, H., McGinty, D., and Szymusiak, R. (2001). Subregional organization
of preoptic area/anterior hypothalamic projections to arousal-related monoaminergic
cell groups. J Comp Neurol 429, 638-653.
Stellwagen, D., and Malenka, R.C. (2006). Synaptic scaling mediated by glial TNF-alpha.
Nature 440, 1054-1059.
Steriade, M., and Glenn, L.L. (1982). Neocortical and caudate projections of intralaminar
thalamic neurons and their synaptic excitation from midbrain reticular core. J
Neurophysiol 48, 352-371.
Steriade, M., Pare, D., Parent, A., and Smith, Y. (1988). Projections of cholinergic and
non-cholinergic neurons of the brainstem core to relay and associational thalamic
nuclei in the cat and macaque monkey. Neuroscience 25, 47-67.
Steriade, M., Datta, S., Pare, D., Oakson, G., and Curro Dossi, R.C. (1990). Neuronal activities
in brain-stem cholinergic nuclei related to tonic activation processes in
thalamocortical systems. J Neurosci 10, 2541-2559.
Steriade, M., Dossi, R.C., Pare, D., and Oakson, G. (1991). Fast oscillations (20-40 Hz) in
thalamocortical systems and their potentiation by mesopontine cholinergic nuclei in
the cat. Proc Natl Acad Sci U S A 88, 4396-4400.
Steriade, M., Timofeev, I., and Grenier, F. (2001). Natural waking and sleep states: a view
from inside neocortical neurons. J Neurophysiol 85, 1969-1985.
Steriade, M. (2006). Grouping of brain rhythms in corticothalamic systems. Neuroscience 137,
1087-1106.
Stevens, E.R., Esguerra, M., Kim, P.M., Newman, E.A., Snyder, S.H., Zahs, K.R., and Miller,
R.F. (2003). D-serine and serine racemase are present in the vertebrate retina and
contribute to the physiological activation of NMDA receptors. Proc Natl Acad Sci U S
A 100, 6789-6794.
Stout, C.E., Costantin, J.L., Naus, C.C., and Charles, A.C. (2002). Intercellular calcium
signaling in astrocytes via ATP release through connexin hemichannels. J Biol Chem
277, 10482-10488.
Strecker, R.E., Morairty, S., Thakkar, M.M., Porkka-Heiskanen, T., Basheer, R., Dauphin, L.J.,
Rainnie, D.G., Portas, C.M., Greene, R.W., and McCarley, R.W. (2000).
!

"'&!

Adenosinergic modulation of basal forebrain and preoptic/anterior hypothalamic

neuronal activity in the control of behavioral state. Behav Brain Res 115, 183-204.
Stridh, M.H., Tranberg, M., Weber, S.G., Blomstrand, F., and Sandberg, M. (2008). Stimulated
efflux of amino acids and glutathione from cultured hippocampal slices by omission
of extracellular calcium: likely involvement of connexin hemichannels. J Biol Chem
283, 10347-10356.
Studer, F.E., Fedele, D.E., Marowsky, A., Schwerdel, C., Wernli, K., Vogt, K., Fritschy, J.M.,
and Boison, D. (2006). Shift of adenosine kinase expression from neurons to
astrocytes during postnatal development suggests dual functionality of the enzyme.
Neuroscience 142, 125-137.
Suadicani, S.O., Flores, C.E., Urban-Maldonado, M., Beelitz, M., and Scemes, E. (2004). Gap
junction channels coordinate the propagation of intercellular Ca2+ signals generated
by P2Y receptor activation. Glia 48, 217-229.
Suntsova, N., Guzman-Marin, R., Kumar, S., Alam, M.N., Szymusiak, R., and McGinty, D.
(2007). The median preoptic nucleus reciprocally modulates activity of
arousal-related and sleep-related neurons in the perifornical lateral hypothalamus. J
Neurosci 27, 1616-1630.
Suzuki, A., Stern, S.A., Bozdagi, O., Huntley, G.W., Walker, R.H., Magistretti, P.J., and
Alberini, C.M. (2011). Astrocyte-neuron lactate transport is required for long-term
memory formation. Cell 144, 810-823.
Sykova, E., Vargova, L., Kubinova, S., Jendelova, P., and Chvatal, A. (2003). The relationship
between changes in intrinsic optical signals and cell swelling in rat spinal cord slices.
Neuroimage 18, 214-230.
Sykova, E., and Nicholson, C. (2008). Diffusion in brain extracellular space. Physiol Rev 88,
1277-1340.
Szymusiak, R., Alam, N., Steininger, T.L., and McGinty, D. (1998). Sleep-waking discharge
patterns of ventrolateral preoptic/anterior hypothalamic neurons in rats. Brain Res
803, 178-188.
Tabernero, A., Vicario, C., and Medina, J.M. (1996). Lactate spares glucose as a metabolic
fuel in neurons and astrocytes from primary culture. Neurosci Res 26, 369-376.
Taishi, P., Chen, Z., Obal, F., Jr., Hansen, M.K., Zhang, J., Fang, J., and Krueger, J.M. (1998).
Sleep-associated changes in interleukin-1beta mRNA in the brain. J Interferon
Cytokine Res 18, 793-798.
Takahashi, K., Lin, J.S., and Sakai, K. (2006). Neuronal activity of histaminergic
tuberomammillary neurons during wake-sleep states in the mouse. J Neurosci 26,
10292-10298.
Takahashi, J.S., Hong, H.K., Ko, C.H., and McDearmon, E.L. (2008). The genetics of
mammalian circadian order and disorder: implications for physiology and disease.
Nat Rev Genet 9, 764-775.
Takahashi, K., Lin, J.S., and Sakai, K. (2009). Characterization and mapping of sleep-waking
specific neurons in the basal forebrain and preoptic hypothalamus in mice.
Neuroscience 161, 269-292.
Takata, N., Mishima, T., Hisatsune, C., Nagai, T., Ebisui, E., Mikoshiba, K., and Hirase, H.
(2011). Astrocyte calcium signaling transforms cholinergic modulation to cortical
plasticity in vivo. J Neurosci 31, 18155-18165.
Takeuchi, A., Fukazawa, S., Chida, K., Taguchi, M., Shirataka, M., and Ikeda, N. (2012).
Semi-automatic counting of connexin 32s immunolocalized in cultured fetal rat
hepatocytes using image processing. Acta Histochem 114, 318-326.
Tanaka, M., Yamaguchi, K., Tatsukawa, T., Nishioka, C., Nishiyama, H., Theis, M., Willecke,
K., and Itohara, S. (2008). Lack of Connexin43-mediated bergmann glial gap
junctional coupling does not affect cerebellar long-term depression, motor
coordination, or eyeblink conditioning. Front Behav Neurosci 2, 1.
Tanaka, T., Kai, S., Matsuyama, T., Adachi, T., Fukuda, K., and Hirota, K. (2013). General
anesthetics inhibit LPS-induced IL-1beta expression in glial cells. PLoS One 8,
e82930.
Tang, F., Lane, S., Korsak, A., Paton, J.F., Gourine, A.V., Kasparov, S., and Teschemacher,
A.G. (2014). Lactate-mediated glia-neuronal signalling in the mammalian brain. Nat
Commun 5, 3284.
!"''!

Terao, A., Matsumura, H., and Saito, M. (1998). Interleukin-1 induces slow-wave sleep at the
prostaglandin D2-sensitive sleep-promoting zone in the rat brain. J Neurosci 18,
6599-6607.
Terao, A., Steininger, T.L., Hyder, K., Apte-Deshpande, A., Ding, J., Rishipathak, D., Davis,
R.W., Heller, H.C., and Kilduff, T.S. (2003). Differential increase in the expression of
heat shock protein family members during sleep deprivation and during sleep.
Neuroscience 116, 187-200.
Terao, A., Wisor, J.P., Peyron, C., Apte-Deshpande, A., Wurts, S.W., Edgar, D.M., and Kilduff,
T.S. (2006). Gene expression in the rat brain during sleep deprivation and recovery
sleep: an Affymetrix GeneChip study. Neuroscience 137, 593-605.
Teubner, B., Michel, V., Pesch, J., Lautermann, J., Cohen-Salmon, M., Sohl, G., Jahnke, K.,
Winterhager, E., Herberhold, C., Hardelin, J.P., et al. (2003). Connexin30
(Gjb6)-deficiency causes severe hearing impairment and lack of endocochlear
potential. Hum Mol Genet 12, 13-21.
Thakkar, M.M., Strecker, R.E., and McCarley, R.W. (1998). Behavioral state control through
differential serotonergic inhibition in the mesopontine cholinergic nuclei: a
simultaneous unit recording and microdialysis study. J Neurosci 18, 5490-5497.
Thakkar, M.M., Engemann, S.C., Walsh, K.M., and Sahota, P.K. (2008). Adenosine and the
homeostatic control of sleep: effects of A1 receptor blockade in the perifornical
lateral hypothalamus on sleep-wakefulness. Neuroscience 153, 875-880.
Theis, M., Jauch, R., Zhuo, L., Speidel, D., Wallraff, A., Doring, B., Frisch, C., Sohl, G.,
Teubner, B., Euwens, C., et al. (2003). Accelerated hippocampal spreading
depression and enhanced locomotory activity in mice with astrocyte-directed
inactivation of connexin43. J Neurosci 23, 766-776.
Theis, M., and Giaume, C. (2012). Connexin-based intercellular communication and astrocyte
heterogeneity. Brain Res 1487, 88-98.
Theodosis, D.T., Poulain, D.A., and Oliet, S.H. (2008). Activity-dependent structural and
functional plasticity of astrocyte-neuron interactions. Physiol Rev 88, 983-1008.
Thompson, C.L., Wisor, J.P., Lee, C.K., Pathak, S.D., Gerashchenko, D., Smith, K.A., Fischer,
S.R., Kuan, C.L., Sunkin, S.M., Ng, L.L., et al. (2010). Molecular and anatomical
signatures of sleep deprivation in the mouse brain. Front Neurosci 4, 165.
Thrane, A.S., Rangroo Thrane, V., Zeppenfeld, D., Lou, N., Xu, Q., Nagelhus, E.A., and
Nedergaard, M. (2012). General anesthesia selectively disrupts astrocyte calcium
signaling in the awake mouse cortex. Proc Natl Acad Sci U S A 109, 18974-18979.
Tian, G.F., Azmi, H., Takano, T., Xu, Q., Peng, W., Lin, J., Oberheim, N., Lou, N., Wang, X.,
Zielke, H.R., et al. (2005). An astrocytic basis of epilepsy. Nat Med 11, 973-981.
Tobler, I., and Borbely, A.A. (1986). Sleep EEG in the rat as a function of prior waking.
Electroencephalogr Clin Neurophysiol 64, 74-76.
Tobler, I., Deboer, T., and Fischer, M. (1997). Sleep and sleep regulation in normal and prion
protein-deficient mice. J Neurosci 17, 1869-1879.
Tononi, G., and Cirelli, C. (2003). Sleep and synaptic homeostasis: a hypothesis. Brain Res
Bull 62, 143-150.
Tononi, G., and Cirelli, C. (2006). Sleep function and synaptic homeostasis. Sleep Med Rev 10,
49-62.
Tononi, G., and Cirelli, C. (2013). Perchance to prune. During sleep, the brain weakens the
connections among nerve cells, apparently conserving energy and, paradoxically,
aiding memory. Sci Am 309, 34-39.
Torres, T., Velho, G., Sanches, M., and Selores, M. (2012). A case of erythrokeratodermia
variabilis with connexin 31 gene mutation (Cx31F137L). Int J Dermatol 51, 494-496.
Trulson, M.E., and Jacobs, B.L. (1979). Raphe unit activity in freely moving cats: correlation
with level of behavioral arousal. Brain Res 163, 135-150.
Ulloor, J., and Datta, S. (2005). Spatio-temporal activation of cyclic AMP response
element-binding protein, activity-regulated cytoskeletal-associated protein and
brain-derived nerve growth factor: a mechanism for pontine-wave generator
activation-dependent two-way active-avoidance memory processing in the rat. J
Neurochem 95, 418-428.

"'(!

Urade, Y., Eguchi, N., Qu, W.M., Sakata, M., Huang, Z.L., Chen, J.F., Schwarzschild, M.A.,
Fink, J.S., and Hayaishi, O. (2003). Sleep regulation in adenosine A2A
receptor-deficient mice. Neurology 61, S94-96.
Urade, Y., and Hayaishi, O. (2011). Prostaglandin D2 and sleep/wake regulation. Sleep Med
Rev 15, 411-418.
Urbano, F.J., Leznik, E., and Llinas, R.R. (2007). Modafinil enhances thalamocortical activity
by increasing neuronal electrotonic coupling. Proc Natl Acad Sci U S A 104,
12554-12559.
Uschakov, A., Gong, H., McGinty, D., and Szymusiak, R. (2007). Efferent projections from the
median preoptic nucleus to sleep- and arousal-regulatory nuclei in the rat brain.
Neuroscience 150, 104-120.
Usowicz, M.M., Gallo, V., and Cull-Candy, S.G. (1989). Multiple conductance channels in
type-2 cerebellar astrocytes activated by excitatory amino acids. Nature 339,
380-383.
Valiunas, V., and Weingart, R. (2000). Electrical properties of gap junction hemichannels
identified in transfected HeLa cells. Pflugers Arch 440, 366-379.
van Dongen, E.V., Takashima, A., Barth, M., Zapp, J., Schad, L.R., Paller, K.A., and
Fernandez, G. (2012). Memory stabilization with targeted reactivation during human
slow-wave sleep. Proc Natl Acad Sci U S A 109, 10575-10580.
Vanni-Mercier, G., Gigout, S., Debilly, G., and Lin, J.S. (2003). Waking selective neurons in
the posterior hypothalamus and their response to histamine H3-receptor ligands: an
electrophysiological study in freely moving cats. Behav Brain Res 144, 227-241.
Venance, L., Stella, N., Glowinski, J., and Giaume, C. (1997). Mechanism involved in initiation
and propagation of receptor-induced intercellular calcium signaling in cultured rat
astrocytes. J Neurosci 17, 1981-1992.
Verkhratsky, A., and Steinhauser, C. (2000). Ion channels in glial cells. Brain Res Brain Res
Rev 32, 380-412.
Verkhratsky, A., and Butt, A. (2013). Physiology and Pathophysiology of Neuroglia.
(Wiley-Blackwell's).
Verkhratsky, A. (2010). Physiology of neuronal-glial networking. Neurochem Int 57, 332-343.
Verkhratsky, A., Rodriguez, J.J., and Parpura, V. (2012a). Calcium signalling in astroglia. Mol
Cell Endocrinol 353, 45-56.
Verkhratsky, A., Rodriguez, J.J., and Parpura, V. (2012b). Neurotransmitters and integration in
neuronal-astroglial networks. Neurochem Res 37, 2326-2338.
Vetrivelan, R., Fuller, P.M., Tong, Q., and Lu, J. (2009). Medullary circuitry regulating rapid eye
movement sleep and motor atonia. J Neurosci 29, 9361-9369.
Virus, R.M., Djuricic-Nedelson, M., Radulovacki, M., and Green, R.D. (1983). The effects of
adenosine and 2'-deoxycoformycin on sleep and wakefulness in rats.
Neuropharmacology 22, 1401-1404.
Volterra, A., and Bezzi, P. (2002). Release of transmitters from glial cells. In The Tripartite
Synapse: Glia in Synaptic Transmission, A.Volterra, et al., eds. (Oxford University
Press), pp. 164-184.
Volterra, A. (2013). Astrocytes: modulation of synaptic function and network activity. In
Neuroglia, third edition, H. Kettenmann and B.R. Ransom, eds. (Oxford University
Press), pp. 481-493.
Von Economo, C. (1930). Sleep as a problem of localization. J Nerv Ment Dis 71, 249-259.
Voutsinos-Porche, B., Bonvento, G., Tanaka, K., Steiner, P., Welker, E., Chatton, J.Y.,
Magistretti, P.J., and Pellerin, L. (2003). Glial glutamate transporters mediate a
functional metabolic crosstalk between neurons and astrocytes in the mouse
developing cortex. Neuron 37, 275-286.
Vyazovskiy, V., Borbely, A.A., and Tobler, I. (2000). Unilateral vibrissae stimulation during
waking induces interhemispheric EEG asymmetry during subsequent sleep in the rat.
J Sleep Res 9, 367-371.
Vyazovskiy, V.V., Cirelli, C., Pfister-Genskow, M., Faraguna, U., and Tononi, G. (2008).
Molecular and electrophysiological evidence for net synaptic potentiation in wake
and depression in sleep. Nat Neurosci 11, 200-208.

!"',!

Vyazovskiy, V.V., Olcese, U., Lazimy, Y.M., Faraguna, U., Esser, S.K., Williams, J.C., Cirelli,
C., and Tononi, G. (2009). Cortical firing and sleep homeostasis. Neuron 63,
865-878.
Wagner, U., Gais, S., and Born, J. (2001). Emotional memory formation is enhanced across
sleep intervals with high amounts of rapid eye movement sleep. Learn Mem 8,
112-119.
Waldo, G.L., and Harden, T.K. (2004). Agonist binding and Gq-stimulating activities of the
purified human P2Y1 receptor. Mol Pharmacol 65, 426-436.
Wall, M., and Dale, N. (2008). Activity-dependent release of adenosine: a critical re-evaluation
of mechanism. Curr Neuropharmacol 6, 329-337.
Wallraff, A., Kohling, R., Heinemann, U., Theis, M., Willecke, K., and Steinhauser, C. (2006).
The impact of astrocytic gap junctional coupling on potassium buffering in the
hippocampus. J Neurosci 26, 5438-5447.
Walz, W. (1982). Do neuronal signals regulate potassium flow in glial cells? Evidence from an
invertebrate central nervous system. J Neurosci Res 7, 71-79.
Wang, X., Lou, N., Xu, Q., Tian, G.F., Peng, W.G., Han, X., Kang, J., Takano, T., and
Nedergaard, M. (2006). Astrocytic Ca2+ signaling evoked by sensory stimulation in
vivo. Nat Neurosci 9, 816-823.
Wang, N., De Bock, M., Antoons, G., Gadicherla, A.K., Bol, M., Decrock, E., Evans, W.H.,
Sipido, K.R., Bukauskas, F.F., and Leybaert, L. (2012). Connexin mimetic peptides
inhibit Cx43 hemichannel opening triggered by voltage and intracellular Ca2+
elevation. Basic Res Cardiol 107, 304.
Wang, N., De Vuyst, E., Ponsaerts, R., Boengler, K., Palacios-Prado, N., Wauman, J., Lai,
C.P., De Bock, M., Decrock, E., Bol, M., et al. (2013). Selective inhibition of Cx43
hemichannels by Gap19 and its impact on myocardial ischemia/reperfusion injury.
Basic Res Cardiol 108, 309.
Webster, H.H., and Jones, B.E. (1988). Neurotoxic lesions of the dorsolateral
pontomesencephalic tegmentum-cholinergic cell area in the cat. II. Effects upon
sleep-waking states. Brain Res 458, 285-302.
Wei, H., and Inan, S. (2013). Dual effects of neuroprotection and neurotoxicity by general
anesthetics: role of intracellular calcium homeostasis. Prog Neuropsychopharmacol
Biol Psychiatry 47, 156-161.
Werk, C.M., and Chapman, C.A. (2003). Long-term potentiation of polysynaptic responses in
layer V of the sensorimotor cortex induced by theta-patterned tetanization in the
awake rat. Cereb Cortex 13, 500-507.
Werk, C.M., Klein, H.S., Nesbitt, C.E., and Chapman, C.A. (2006). Long-term depression in
the sensorimotor cortex induced by repeated delivery of 10 Hz trains in vivo.
Neuroscience 140, 13-20.
White, S.R., and Neuman, R.S. (1983). Pharmacological antagonism of facilitatory but not
inhibitory effects of serotonin and norepinephrine on excitability of spinal
motoneurons. Neuropharmacology 22, 489-494.
White, S.R., Fung, S.J., and Barnes, C.D. (1991). Norepinephrine effects on spinal
motoneurons. Prog Brain Res 88, 343-350.
Wiencken-Barger, A.E., Djukic, B., Casper, K.B., and McCarthy, K.D. (2007). A role for
Connexin43 during neurodevelopment. Glia 55, 675-686.
Wierzynski, C.M., Lubenov, E.V., Gu, M., and Siapas, A.G. (2009). State-dependent
spike-timing relationships between hippocampal and prefrontal circuits during sleep.
Neuron 61, 587-596.
Wigren, H.K., Schepens, M., Matto, V., Stenberg, D., and Porkka-Heiskanen, T. (2007).
Glutamatergic stimulation of the basal forebrain elevates extracellular adenosine and
increases the subsequent sleep. Neuroscience 147, 811-823.
Wilhelmsson, U., Bushong, E.A., Price, D.L., Smarr, B.L., Phung, V., Terada, M., Ellisman,
M.H., and Pekny, M. (2006). Redefining the concept of reactive astrocytes as cells
that remain within their unique domains upon reaction to injury. Proc Natl Acad Sci U
S A 103, 17513-17518.
Willie, J.T., Chemelli, R.M., Sinton, C.M., Tokita, S., Williams, S.C., Kisanuki, Y.Y., Marcus,
J.N., Lee, C., Elmquist, J.K., Kohlmeier, K.A., et al. (2003). Distinct narcolepsy
!

"'-!

syndromes in Orexin receptor-2 and Orexin null mice: molecular genetic dissection
of Non-REM and REM sleep regulatory processes. Neuron 38, 715-730.
Wilson, M.A., and McNaughton, B.L. (1994). Reactivation of hippocampal ensemble memories
during sleep. Science 265, 676-679.
Wisor, J.P., Nishino, S., Sora, I., Uhl, G.H., Mignot, E., and Edgar, D.M. (2001). Dopaminergic
role in stimulant-induced wakefulness. J Neurosci 21, 1787-1794.
Wisor, J.P., Rempe, M.J., Schmidt, M.A., Moore, M.E., and Clegern, W.C. (2013). Sleep
slow-wave activity regulates cerebral glycolytic metabolism. Cereb Cortex 23,
1978-1987.
Witcher, M.R., Kirov, S.A., and Harris, K.M. (2007). Plasticity of perisynaptic astroglia during
synaptogenesis in the mature rat hippocampus. Glia 55, 13-23.
Wolburg, H., Wolburg-Buchholz, K., Fallier-Becker, P., Noell, S., and Mack, A.F. (2011).
Structure and functions of aquaporin-4-based orthogonal arrays of particles. Int Rev
Cell Mol Biol 287, 1-41.
Wolosker, H., Blackshaw, S., and Snyder, S.H. (1999). Serine racemase: a glial enzyme
synthesizing
D-serine
to
regulate
glutamate-N-methyl-D-aspartate
neurotransmission. Proc Natl Acad Sci U S A 96, 13409-13414.
Wu, Y., Li, W., Zhou, C., Lu, F., Gao, T., Liu, Y., Cao, J., Zhang, Y., Zhang, Y., and Zhou, C.
(2012). Ketamine inhibits lipopolysaccharide-induced astrocytes activation by
suppressing TLR4/NF-kB pathway. Cell Physiol Biochem 30, 609-617.
Xie, L., Kang, H., Xu, Q., Chen, M.J., Liao, Y., Thiyagarajan, M., O'Donnell, J., Christensen,
D.J., Nicholson, C., Iliff, J.J., et al. (2013). Sleep drives metabolite clearance from the
adult brain. Science 342, 373-377.
Yamamoto, N., and Lopez-Bendito, G. (2012). Shaping brain connections through
spontaneous neural activity. Eur J Neurosci 35, 1595-1604.
Yamamoto, J., Suh, J., Takeuchi, D., and Tonegawa, S. (2014). Successful execution of
working memory linked to synchronized high-frequency gamma oscillations. Cell 157,
845-857.
Yamanaka, A., Beuckmann, C.T., Willie, J.T., Hara, J., Tsujino, N., Mieda, M., Tominaga, M.,
Yagami, K., Sugiyama, F., Goto, K., et al. (2003). Hypothalamic orexin neurons
regulate arousal according to energy balance in mice. Neuron 38, 701-713.
Yang, G., Huard, J.M., Beitz, A.J., Ross, M.E., and Iadecola, C. (2000). Stellate neurons
mediate functional hyperemia in the cerebellar molecular layer. J Neurosci 20,
6968-6973.
Yang, Y., Ge, W., Chen, Y., Zhang, Z., Shen, W., Wu, C., Poo, M., and Duan, S. (2003).
Contribution of astrocytes to hippocampal long-term potentiation through release of
D-serine. Proc Natl Acad Sci U S A 100, 15194-15199.
Yang, G., and Gan, W.B. (2012). Sleep contributes to dendritic spine formation and elimination
in the developing mouse somatosensory cortex. Dev Neurobiol 72, 1391-1398.
Yasuda, T., Yasuda, K., Brown, R.A., and Krueger, J.M. (2005). State-dependent effects of
light-dark cycle on somatosensory and visual cortex EEG in rats. Am J Physiol Regul
Integr Comp Physiol 289, R1083-1089.
Ye, Z.C., Wyeth, M.S., Baltan-Tekkok, S., and Ransom, B.R. (2003). Functional hemichannels
in astrocytes: a novel mechanism of glutamate release. J Neurosci 23, 3588-3596.
Yotsumoto, Y., Sasaki, Y., Chan, P., Vasios, C.E., Bonmassar, G., Ito, N., Nanez, J.E., Sr.,
Shimojo, S., and Watanabe, T. (2009). Location-specific cortical activation changes
during sleep after training for perceptual learning. Curr Biol 19, 1278-1282.
Zhang, J.M., Wang, H.K., Ye, C.Q., Ge, W., Chen, Y., Jiang, Z.L., Wu, C.P., Poo, M.M., and
Duan, S. (2003). ATP released by astrocytes mediates glutamatergic
activity-dependent heterosynaptic suppression. Neuron 40, 971-982.
Zhang, Z., Chen, G., Zhou, W., Song, A., Xu, T., Luo, Q., Wang, W., Gu, X.S., and Duan, S.
(2007). Regulated ATP release from astrocytes through lysosome exocytosis. Nat
Cell Biol 9, 945-953.
Zhang, Z., Gong, N., Wang, W., Xu, L., and Xu, T.L. (2008). Bell-shaped D-serine actions on
hippocampal long-term depression and spatial memory retrieval. Cereb Cortex 18,
2391-2401.
Zhang, Y., and Barres, B.A. (2010). Astrocyte heterogeneity: an underappreciated topic in
neurobiology. Curr Opin Neurobiol 20, 588-594.
!"(.!

Zhang, X., Wang, J., Qian, W., Zhao, J., Sun, L., Qian, Y., and Xiao, H. (2014).
Dexmedetomidine inhibits tumor necrosis factor-alpha and interleukin 6 in
lipopolysaccharide-stimulated astrocytes by suppression of c-Jun N-terminal kinases.
Inflammation 37, 942-949.

"("!

Appendix

________________
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ADDITIONAL PUBLICATION 1:
From a glial syncytium to a more restricted and specific glial
networking

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ADDITIONAL PUBLICATION 2:
Astroglial connexins as elements of sleep-wake cycle
regulation and dysfunction
Liu, X., and Giaume, C. (2014). Astroglial connexins as elements of sleep-wake cycle
regulation and dysfunction. In Pathological Potential of Neuroglia, V. Papura and A.
Verkhratsky, eds. (Springer).

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