The role of astroglial connexin 30 in sleep homeostasis

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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 [q-bio.NC].

Université Pierre et Marie Curie - Paris VI, 2014. English. �NNT : 2014PA066232�. �tel-01085329�

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Universié Pierre et Marie Curie – Paris 6

The role of astroglial connexin 30 in sleep homeostasis

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Thèse présentée devant

l’Universié Pierre et Marie-Curie

pour obtenir le grade de Docteur en Neurosciences

par

Xinhe Liu

ED3C : Ecole Doctorale Cerveau, Cognition et Comportement

Jury constitué de:

Jean MARIANI Président du jury

Tommaso FELLIN Rapporteur Marc MESNIL Rapporteur Jean-Marie PETIT Examinatuer

Bruno WEBER Examinatuer

Christian GIAUME Directeur de Thèse

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Thèse 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 (Université de Poitiers), Jean-Marie Petit (Ecole Polytechnique Fédérale de Lausanne), Bruno Weber (Institute of Pharmacology and Toxicology) and Christian Giaume (Collège 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 Jérémie 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.

I’m 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 (École des Neurosciences Paris Île 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, I’m deep grateful to my family. My parents have always given

me the strongest support and trust in my choice of career and personal life. I’m

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.

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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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ROM A GLIAL SYNCYTIUM TO A MORE RESTRICTED AND SPECIFIC GLIAL NETWORKING

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STROGLIAL CONNEXINS AS ELEMENTS OF

SLEEP

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WAKE CYCLE REGULATION AND DYSFUNCTION

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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 Ca

²⁺

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 Ca

²⁺

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 Ca

²⁺

signaling, and the water channels named aquaporins (AQPs).

<= >+)+-&('%,

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 GABA

_B

receptor (GABA

_B

R) 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

⁺

/Ca

²⁺

exchangers (NCXs) that mediate the exchange of 2 Na

⁺

and 1 Ca

²⁺

, Na

⁺

/HCO

3-

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 Ca

²⁺

signaling systems, which allow them to respond to a variety of neurotransmitters and other signaling molecules in the form of intracellular Ca

²⁺

elevations (for reviews see Araque et al., 2001; Fiacco and McCarthy, 2006; Perea et al., 2009). Astroglial Ca

²⁺

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).

B28).A:,%(A')+%,.1,2%&'()*&+%,

Astrocyte intracellular Ca

²⁺

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 Ca

²⁺

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 Ca

²⁺

release from the ER is critical for the exocytosis of gliotransmitters (see below) from astrocytes (Malarkey and Parpura, 2009; Parpura et al., 2011).

Ca

²⁺

transients in astrocytes could also be caused by the entry of Ca

²⁺

from the extracellular space. The depletion of the ER Ca

²⁺

store triggers Ca

²⁺

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 Ca

²⁺

entry (Putney, 1986, 1990). In addition, extracellular Ca

²⁺

can enter the cell through Ca

²⁺

permeable ionotropic receptors and NCXs. The ionotropic receptors involved in astrocyte Ca

²⁺

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 Ca

²⁺

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

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Figure 3. Ca

²⁺

signaling in astrocytes. [Ca

²⁺

]

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 Ca

²⁺

-ATPase (SERCA). Another Ca

²⁺

source comes from the entry of extracellular Ca

²⁺

through ionotropic receptors, store-operated channels (SOC) or Na

⁺

/Ca

²⁺

exchangers (NCXs). Ca

²⁺

pumps/ATPases (PMCA) can extrude cytosolic Ca

²⁺

. [Ca

²⁺

]

i

is affected by cytosolic Ca

²⁺

-binding proteins (CBPs) and the mitochondria. Mitochondrial Ca

²⁺

uptake occurs through voltage-dependent anion channels (VDAC) in the outer membrane and the uniporter in the inner membrane, while Ca

²⁺

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 Ca

²⁺

entry (De Bock et al., 2014; Orellana et al., 2012).

C'(-+'&.+%,(4,)28).A:,%.0128.10,.1,2%&'()*&+%,

The Ca

²⁺

signals in astrocytes exhibit complex spatiotemporal properties. Slow (seconds to minutes) Ca

²⁺

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) Ca

²⁺

responses elicited by minimal synaptic activity are restricted to microdomains of perisynaptic processes (Di Castro et al., 2011; Panatier et al., 2011). Ca

²⁺

transients elicited in individual astrocytes can propagate across cell boundaries as intercellular Ca

²⁺

waves, which can travel long distances (<500 µm) at relatively low speed (approximately 14 µm/s) (Scemes and Giaume, 2006; Deitmer and Rose, 2010). In the neocortex Ca

²⁺

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 Ca

²⁺

waves (Haas et al., 2006; Schipke et al., 2002). Astrocytes Ca

²⁺

signaling has also been observed in vivo. Whisker stimulation increased astrocyte Ca

²⁺

transients in the mouse barrel cortex (Wang et al., 2006). Visual stimuli were shown to elicit astrocyte Ca

²⁺

signals in the ferret visual cortex (Schummers et al., 2008). During locomotor performance of mice, concerted Ca

²⁺

excitation occurred in networks of hundreds of cerebellar Bergmann glial cells extending across several hundred microns (Nimmerjahn et al., 2009).

<= D(3.A:,%.0128.10,

In addition to Ca

²⁺

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 10–25 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 Ca

²⁺

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; Na

x

, Na

⁺

channels activated by extracellular Na

⁺

; NCLX, mitochondrial Na

⁺

/Ca

²⁺

exchanger; NHE, Na

⁺

/H

⁺

exchanger; NKCC1, Na

⁺

/K

⁺

/Cl

^-

cotransporter; 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 Ca

²⁺

-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) 1–3, 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 Ca

²⁺

-dependent exocytosis have also been described, including reversal of neurotransmitter transporters and diffusion through Cx/Px hemichannels—the opening of Cx43 hemichannels is Ca

²⁺

-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

⁺

/Cl

^-

cotransporters. Local intracellar K

⁺

is dispersed from the site of activity into distant astrocytes

through gap junction channels (GJCs) and extruded through K

ir

4.1 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 Ca

²⁺

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

(2.7–3.5 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 K

ir

4.1 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. K

ir

4.1 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 K

_ir

4.1 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 (NH

4

+

) 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 Ca

²⁺

concentration ([Ca

²⁺

]

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 [Ca

²⁺

]

_i

induces release of glutamate, which acts on NR2B subunit-

!#%!

Figure 8. Functional effects of glutamate, ATP and D‑serine 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 GABA

B

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 [Ca

²⁺

]

_i

in astrocytes. The artificially induced [Ca

²⁺

]

_i

elevations,

however, may not capture the full characteristics of endogenous Ca

²⁺

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

²⁺

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

!#'!

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).

<= 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.