The name astroglia unifies many non-excitable neural cells that act as primary homeostatic cells in the nervous system. Neuronal activity triggers multiple homeostatic responses of astroglia that include increase in metabolic activity and synthesis of neuronal preferred energy substrate lactate, clearance of neurotransmitters and buffering of extracellular K+ ions to name but a few. Many (if not all) of astroglial homeostatic responses are controlled by dynamic changes in the cytoplasmic concentration of two cations, Ca2+ and Na+. Intracellular concentration of these ions is tightly controlled by several transporters and can be rapidly affected by activation of respective fluxes through ionic channels or ion exchangers. Here we provide a comprehensive review of astroglial Ca2+ and Na+ signalling.
Mammalian nervous system consists of electrically excitable neurones that are capable of generation of action potentials and fast chemical (synaptic) transmission and electrically non-excitable neuroglia that control brain homeostasis and defence. These two cell types form complex neuronal-glial networks, witch, by working in concert provide the cellular basis for multiple functions of the nervous system. Neuroglia of the central nervous system (CNS) is generally classified into macroglia (astrocytes and oligodendrocytes) and microglia, which are scions of foetal macrophages invading the neural tissue very early in embryonic development and providing for innate immunity of the CNS.
Astrocytes are arguably the most heterogeneous neuroglial cells; their main role is to maintain homeostasis of the CNS and they indeed do so at all levels from organ to molecules. The term astrocyte (astron kytos, - the “star-like” cell in Greek), was coined by Michael von Lenhossek in 1895 (Lenhossek, 1895; Kettenmann & Verkhratsky, 2008) is somewhat misleading because most of these cells in the in vivo brain do not have star-like appearance, displaying a spongioform morphology associated with highly elaborated processes. Astrocytic processes can be subdivided into astroshafts, relatively thick processes containing organelles, and thin flat lamellae, void of organelles, that enwrap synapses (Patrushev et al., 2013). There are many different types of astroglia in the mammalian CNS; these for example include protoplasmic astrocytes of grey matter of the brain and the spinal cord, fibrous astrocytes localised in the white matter, radial Müller retinal glial cells, pseudo-radial cerebellar Bergmann glial cells, velate astrocytes of cerebellum, tanycytes that connect ventricular walls with parts of hypothalamus and spinal cord, pituicytes in the neuro-hypophysis, and perivascular and marginal astrocytes; the human brain and the brain of higher primates also contains interlaminar, polarised and varicose projection astrocytes (Oberheim et al., 2006; Verkhratsky & Toescu, 2006; Kimelberg, 2010; Kimelberg & Nedergaard, 2010; Verkhratsky, 2010; Verkhratsky et al., 2011; Oberheim et al., 2012; Verkhratsky & Butt, 2013).
Astrocytes do not generate action potentials, and yet they are very much capable of perceiving their neurochemical and ionic environment and of mounting specific physiological responses to neuronal activity. Similarly to neurones astrocytes express several classes of plasmalemmal ion channels, including voltage-gated channels, although the densities of Ca2+ and Na+ channels are exceedingly low in vivo rendering current –voltage relationship linear (Verkhratsky & Steinhauser, 2000). However, in cultured astrocytes voltage dependent channels are expressed in higher quantities and regulate intracellular Ca2+ dynamics (Yaguchi & Nishizaki, 2010; Letellier et al., 2016). This fact opens a possibility that upon certain physiological or pathological conditions the expression of ion channels in astrocytes may change. In addition astrocytes express full complement of ionotropic and metabotropic receptors to neurotransmitetrs and neurohormones; expression of which however is highly region-specific (Verkhratsky et al., 1998). Astroglial neurotransmitter receptors are activated in response to neuronal activity being an important pathway in the integration of neuronal-glial networks (Verkhratsky, 2010). In contrast to neurones, however, activation of glial receptors does not trigger plasmalemma-associaetd action potentials, but rather induce complex fluctuations in the cytosolic concentrations of major cations, K+, Na+ and Ca2+; these ions in turn control astroglial functions and hence represents the specific mechanism of astroglial excitability. Importantly, changes in cytosolic concentrations of these ions are not restricted to a single cell but rather propagate though glial syncytia. These astroglial syncytia are formed through gap junctions that physically connect adjacent cells.
The gap junctions occur at in between apposing astrocytic membranes of neighbouring cells separated by quite narrow (~ 2 - 2.5 nm) intercellular cleft. The gap junctional permeability is mediated by specialised intercellular channels that protrude membranes of each cell and establish direct contact between cell interiors. These intercellular channels are composed of two precisely aligned connexons each made up from 6 subunits defined as connexins (Dermietzel & Spray, 1993; Dermietzel, 1998). Astrocytes express several types of connexins of which connexins Cx43 dominates and connexins Cx30, Cx40 and Cx45 are expressed to a lesser extent. Connexon channel has a large pore (with a diameter of ~ 1.5 nm), which allows intercellular transfer of ions second messengers [(e.g. inositol 1,4,5 trisphosphate (InsP3)], nucleotides such as ATP and ADP) or metabolic substrates (glucose). As a result the gap junctions form a distinct route for intercellular and long-range signalling which underlie propagating waves of ionic (Ca2+, Na+ or K+) signals, or even metabolic waves. Astrocytes use this intracellular signalling route to convey signals over long distances.
In this paper we shall provide a concise overview of astroglial ion signals, molecular mechanisms of their generation and their functional significance.
K+ mediated neuron-glia interactions
Although K+ concentration in the brain extracellular space changes in activity-dependent manner is maintained at a low millimolar range (2.5-5 mM). Excessive increase in extracellular concentration of K+ ([K+]o) often reflects a pathological process associated with extreme brain activity or suppression of potassium clearance mechanisms. For example, ischemia leads to intrinsic modifications in Na+, K+-ATPase, the major molecule involved in potassium clearance (Jamme et al., 1997a; Jamme et al., 1997b). Elevated extracellular K+ evokes seizures, spreading depression, migraine and in extreme case cell death. Maintenance of K+ gradient across plasma membrane is essential for many physiological processes such as membrane potential, repolarization of action potential, neurotransmitters uptake etc. Although global elevations in [K+]o are pathological, under physiological conditions [K+]o can undergo local and transient changes that can have signalling functions (Shih et al., 2013). Such elevations can occur around axon in association with propagating action potential and in the synaptic cleft during synaptic transmission. Increase in the [K+]o within the synaptic cleft can depolarize presynaptic terminal thus changing the probability of glutamate release (Shih et al., 2013). Indeed, presynaptic depolarization is shown to widen arriving action potential that causes enhancement of [Ca2+]i transient and increases probability of neurotransmitter release (Geiger & Jonas, 2000; Hori & Takahashi, 2009; Sasaki et al., 2011). Elevation of [K+]o in the synaptic cleft can also regulate astrocytic glutamate uptake by depolarizing perisynaptic astrocytic processes (PAPs) and affecting voltage-dependent glutamate transporters(Grewer et al., 2008).
What are the sources of K+ in or around the synapse? General assumption is that is comes as result of K+ efflux during action potential repolarising phase. However, in glutamate synapses, K+ can efflux also through postsynaptic AMPA and NMDA receptors(Ge & Duan, 2007). The NMDA receptors are recruited in activity-dependent manner and serve as a major source of K+ in the synaptic cleft (Shih et al., 2013). Although AMPA receptors responsible for larger peak current than do NMDA receptors, AMPA receptors inactivate very quickly. In contrast, activation of NMDA receptors typically lasts for hundreds of milliseconds(Attwell & Gibb, 2005). Moreover, NMDA receptors have larger single-channel conductance than AMPA receptors (Spruston et al., 1995). Nevertheless, contribution of AMPA receptors increases after induction of long-term potentiation that is associated with insertion of additional postsynaptic AMPA receptors (Ge & Duan, 2007). Moreover, neuronal and astrocytic transporters, ion exchangers and K+ channels can be involved in regulation of perisynaptic K+ concentration. For example, K+/Cl- co-transporter 2 (KCC2) extrudes KCl from the neurons following prolonged neuronal activity when cell depolarization is associated with activation of Cl- permeable GABAA receptors (Rivera et al., 1999). KCC2 mediated extracellular K+ accumulation depolarises the cells and may serve as a mechanism for their synchronization (Viitanen et al., 2010). Notably, high densities of KCC2 have been found in dendritic spines suggesting its interaction with the other K+ regulating pathways at the synaptic level synapse (Gulyas et al., 2001).
Neurotransmitters uptake by astrocytes may also contribute to dynamic fluctuations in the [K+]o. For example, glutamate transporters, exchange glutamate and Na+ for K+ and thus can potentially cause local K+ accumulation (Grewer et al., 2008). Ca2+ elevation in astrocytic endfeet activates large-conductance Ca2+ dependent potassium channels (BK channels)(Girouard et al., 2010). Because the endfeet touches arterioles, K+ elevation triggers arteriolar dilation or constriction depending on the concentration. However, K+ removal is also a major function of astrocytes (Walz, 2000). There are several mechanisms employed by astroglia for extracellular K+ buffering. Fast K+ influx through K+ channels according to electrochemical gradient begins immediately after ambient [K+]o increases and shifts K+ reversal potential towards more depolarising values. This process quickly depolarises astrocytic process ceasing further influx. The rest (and the bulk) of K+ can be taken into astrocytes by slower energy dependent mechanism represented by Na+/K+ ATPase. Spatial buffering of K+ has also been suggested as the capacity of astrocytes to redistribute locally elevated extracellular K+ trough gap-junctions that connect these cells to each other (Kofuji & Newman, 2004). Thus, complex interplay between K+ release mechanisms by neurons and K+ removal by astrocytes shapes spatio-temporal profile of [K+]o dynamics.
Within this profile, extracellular K+ elevations form various intercellular communication pathways. In glutamate synapses postsynaptic K+ release serves as retrograde signal regulating presynaptic release probability and responsible for activity dependent facilitation. K+ that depolarizes perisynaptic astrocytic lamellae may reduce the efficiency of uptake, and, therefore, glutamate spillover. K+ efflux due to KCC2 activity synchronises neighbouring neurons, which is important for information processing on the network level. Additionally, K+ released by astrocytes has a role in cortical neuro-vascular coupling.
Calcium signalling in astroglia
Molecular machinery of Ca2+ signalling
Calcium has been chosen by the evolution as one of the most universal intracellular second messengers, due to its unique qualities (flexible coordination chemistry, high affinity for carboxylate oxygen, which is the most frequent motif in amino acids, rapid binding kinetics) and by its availability in the primordial ocean (Petersen et al., 2005; Case et al., 2007). At high concentrations Ca2+ ions cause numerous anti-life effects such as protein and nucleic acid aggregations or precipitation of phosphates; in addition ATP-based energetics require low levels of Ca2+ in the cytosol. These factors stipulated the general principle of Ca2+ signalling, which is based around steep concentration gradients for Ca2+ between the cytosol and the extracellular environment as well as various intracellular compartments. These concentration gradients create electro-driving force for Ca2+ aimed at the cytosol where resting Ca2+ concentration is kept at level between 50 and 100 nM. Ca2+ movements across cellular membranes occur either via diffusion through Ca2+ permeable channels or by transport with ATP-consuming pumps or ion-dependent exchangers; the former underlie downhill Ca2+ translocation (i.e. in the direction of electro-chemical gradient) whereas the latter provides for up-hill (i.e. against electro-chemical gradient) Ca2+ flux. Ca2+-permeable ion channels are represented by several families, which include highly Ca2+ selective voltage-gated Ca2+ channels, intracellular Ca2+ channels (InsP3 receptors or InsP3Rs and ryanodine receptors or RyRs) and Ca2+-release activated Ca2+ channels (or CRAC channels, that on a molecular level represent activity of Orai proteins) and cationic channels with various degrees of Ca2+ permeability. These cationic channels in turn are represented by ligand-gated channels (or ionotropic neurotransmitter receptors such as for example glutamate, ATP or nicotinic acetylcholine receptors), by extended family of transient receptor potential (TRP) channels and some other types of cationic channels. Ca2+ transport against concentration gradients is mainly accomplished by plasmalemmal Ca2+ ATPases (PMCAs or plasmalemmal Ca2+ pumps), by SarcoEndoplasmic reticulum ATPases (SERCAs or endoplasmic reticulum Ca2+ pumps) and by ion exchangers of which the Na+/Ca2+ exchanger or NCX is by far the most important. Inside the cell Ca2+ is buffered by Ca2+ biding proteins (CBP), affinity of which to Ca2+ differs in different cellular compartments. For example, Ca2+ affinity of cytosolic CBPs lies in a low nM range, whereas endoplasmic reticulum (ER) CBPs have KD for Ca2+ at ~ 0.5 mM. These different affinities determine the range of diffusion of Ca2+ ions. In the cytosol CBPs limit diffusion and favour development of local high-Ca2+ concentration microdomains, whereas in the ER CBP allow almost free and long-distance Ca2+ diffusion that being instrumental for making ER Ca2+ tunnels (Solovyova & Verkhratsky, 2003; Petersen & Verkhratsky, 2007). Cellular Ca2+ homeostasis is also regulated by mitochondria which are able to accumulate Ca2+ (via electrochemically driven diffusion through Ca2+ selective channel generally referred to as Ca2+ uniporter) and to release Ca2+ through mitochondrial Na+/Ca2+ exchanger as well as transient openings of mitochondrial permeability transition pore (Altschuld et al., 1992; Nicholls, 2005).
Effectors of Ca2+ signals are Ca2+ regulated enzymes (also known as “Ca2+ sensors”), biding of Ca2+ to which affects functional activity. These Ca2+ sensors are many; they have different affinities to Ca2+ and are heterogeneously distributed between cellular compartments. These specificities of Ca2+ sensors sensitivity to Ca2+ and their cellular distribution underlie amplitude and spatial coding of Ca2+ signals.
The shape and spatio-temporal organisation of Ca2+ signals are defined by the interplay between Ca2+ diffusional fluxes and Ca2+ transport (Fig. 1). Combinations of those are multiple and labile; as was conceptualised by Michael Berridge, cells can create and rapidly modify "Ca2+ signalling toolkits" that adapt Ca2+ signalling to the environmental requirements (Berridge et al., 2000; Berridge et al., 2003). Another important feature of Ca2+ homeostatic/signalling system is its autoregulation by Ca2+ ions themselves as transient changes in Ca2+ concentration establish multiple feedbacks modifying the performance of Ca2+ handling molecule. As a rule most of Ca2+ permeable channels are subject to Ca2+-dependent inactivation, which develops either through direct binding of Ca2+ ions to the channel or Ca2+-dependent channel phosphorylation. Similarly, Ca2+ pumping by SERCAs is regulated by Ca2+ concentration within the ER lumen; this intraluminal Ca2+ concentration also controls the availability of intracellular Ca2+ channels for activation. Conceptually, lowering Ca2+ concentration in the ER facilitates Ca2+ uptake and reduces channels activation, whereas increase in intra-ER Ca2+ concentration facilitates channels opening and reduces SERCA activity ( see (Burdakov et al., 2005; Guerrero-Hernandez et al., 2010) for detailed discussion). Finally Ca2+ fluxes are regulated by mitochondria, which by providing ATP and dynamic Ca2+ buffering act regulate plasmalemmal Ca2+ entry and ER Ca2+ uptake (Parekh, 2008; Kopach et al., 2011).
Endoplasmic reticulum as a main source of astroglial Ca2+ signalling
Astroglial cells respond with intracellular Ca2+ elevation to a broad variety of external stimuli from direct mechanical stimulation to a multitude of neurotransmitters, neuromodulators, hormones and other biologically active substances. The ability of astroglia to react with [Ca2+]i elevation to almost every neuroligands it encounters was firmly established in experiments in cell cultures (Cornell Bell et al., 1990; Charles et al., 1991; Charles et al., 1993; Finkbeiner, 1993; Verkhratsky & Kettenmann, 1996; Verkhratsky et al., 1998). These early experiments were fundamental for glial research because they demonstrated that astrocytes are potentially capable of expressing virtually every receptor modality and that most of these receptors are coupled to ER through the phospholipase C (PLC)/InsP3 signalling cascade. These experiments also highlighted remarkable plasticity of astroglial cells in vitro, as indeed, these cells were able to rapidly modify receptor expression pattern. First studies of astrocytes in situ, in brain slices confirmed the primary importance of InsP3-ER link in generation of astroglial Ca2+ signals (Kirischuk et al., 1995; Porter & McCarthy, 1995; Kirischuk et al., 1996; Kirischuk et al., 1999). At the same time these experiments also found that receptor expression in astroglia in neural tissue is restricted to match that of neurons, i.e. immediate neurotransmitter environment (Verkhratsky et al., 1998; Parpura & Verkhratsky, 2012; Verkhratsky et al., 2012; Zorec et al., 2012).
The ER is one of the largest intracellular organelles, which involved in a variety of fundamental cellular processes such as protein synthesis, protein folding and trafficking haulage of secretory products etc. (Baumann & Walz, 2001; Berridge, 2002; Michalak et al., 2002; Verkhratsky, 2005). The ER is also a key organelle of calcium signalling being arguably the largest dynamic Ca2+ store able to accumulate, store and release Ca2+ ions in response to (patho)physiological stimulation. Ca2+ accumulation into the ER lumen is accomplished by SERCA pumping; the Ca2+ concentration in the ER at rest (also known as intraluminal Ca2+ concentration, [Ca2+]L) is maintained at 0.2 - 1.0 mM range (Mogami et al., 1998; Alonso et al., 1999; Solovyova & Verkhratsky, 2002; Solovyova et al., 2002; Verkhratsky & Petersen, 2002). Release of Ca2+ from ER in astroglia is primarily mediated by InsP3 receptors, and their inhibition by pharmacological agents (e.g., heparin) or by genetic deletion often prevents development of Ca2+ signals in astrocytes (Kirischuk et al., 1999; Agulhon et al., 2008). Functional role of second type of ER Ca2+ release channel, the ryanodine receptor (a Ca2+-gated Ca2+ channel) in astroglial Ca2+ dynamics remains controversial, although astrocytes do express RyRs both in vitro and in situ (Matyash et al., 2002; Verkhratsky et al., 2002; Beck et al., 2004). The InsP3Rs are simultaneously controlled by InsP3 and Ca2+ ions and therefore local increase in [Ca2+]i facilitates receptor opening thus promoting Ca2+-induced Ca2+ release through InsP3Rs. This feature underlies the occurrence of propagating Ca2+ waves which in essence represent a wave of ER membrane excitation manifested by propagating recruitment of InsP3 receptors. These Ca2+ waves are important for astrocyte physiology; astroglial stimulation usually occurs at the level of distal processes, and Ca2+ waves convey this excitation down to the soma. Furthermore, astroglial Ca2+ wave travel through astroglial syncytia, being therefore a substrate for astroglial long-range signalling (Giaume & Venance, 1998; Scemes & Giaume, 2006).
Calcium signals produced by activation of ER Ca2+ release control many functions of astroglia. In particular ER-originated Ca2+ signals are critical for inducing exocytotic release of neurotransmitters (such as for example ATP, glutamate or D-serine) from astrocytes (see (Malarkey & Parpura, 2009; Parpura et al., 2011) for review and references). Inhibition of Ca2+ accumulation into the ER by specific blockade of SERCA pumps with thapsigargin that leads to exhaustion of the ER Ca2+ content due to an unopposed leak through the endomembrane effectively eliminated Ca2+-dependent release of glutamate from cultured astrocytes (Hua et al., 2004). The same effect was achieved after inhibition of InsP3 receptors by membrane-permeable antagonist diphenylboric acid 2-aminoethyl ester (2-APB), which can also affect the store-operated calcium entry discussed next. The role for ER Ca2+ signalling cascade in controlling astroglial release of neuroactive substances was subsequently corroborated in experiments in situ (Fellin et al., 2004; Shigetomi et al., 2008; Henneberger et al., 2010; Rusakov et al., 2011).
Plasmalemmal Ca2+ influx in astrocytes: role of TRP channels and ionotropic receptors
Despite the fact that ER Ca2+ store acts as a main source for astroglial Ca2+ signalling, astrocytes also possess several mechanisms for Ca2+ entry that produce physiologically relevant Ca2+ signals (Fig. 1). There are little evidence that astrocytes in situ can express functional voltage-gated Ca2+ channels, although these channels have been detected in several in vitro experiments (see (Parpura et al., 2011) for detailed review). Two major pathways controlling plasmalemmal Ca2+ entry in astroglial cells are represented by store-operated and ligand-operated ion channels.
The store-operated Ca2+ entry is generally present in the majority of electrically non-excitable cells. This Ca2+ influx pathway (initially described as a ”capacitative” Ca2+ entry - (Putney, 1986, 1990) is controlled by the Ca2+ content in the ER lumen, when decrease in [Ca2+]L results in the opening of plasmalemmal Ca2+-permeable channels (Parekh & Putney, 2005). Activation of the store-operated Ca2+ entry fulfils two functions: first, it provides Ca2+ for replenishment of the ER store (the capacitative function), and second, it is important for producing the sustained (“plateau”) phase of the Ca2+ signal that often outlasts the period of cell stimulation. There are several molecular determinants of the store-operated Ca2+ entry. Many types of cells express specific (ICRAC) store-operated channels characterised by extremely high Ca2+ selectivity and very low single channel conductance. Activation of these channels reflects interaction of STIM proteins (that detect ER Ca2+ concentration) with Orai proteins (Putney, 2007) that form the plasmalemmal channel (these latter proteins were named after Greek gate-keeping goddesses (Feske et al., 2006)). Alternatively store-operated Ca2+ influx may involve activation of TRP channels (Smyth et al., 2006).
The store-operated Ca2+ entry is functionally expressed in astroglia (Tuschick et al., 1997; Pivneva et al., 2008). At the same time neither ICRAC channels nor STIM/Orai complexes were hitherto detected in astrocytes. In contrast, experimental evidence indicates the role for TRP channels. They are expressed in astrocytes at both mRNA and protein levels, and TRP activity is involved in shaping astroglial Ca2+ signals (Pizzo et al., 2001; Grimaldi et al., 2003; Golovina, 2005). Further analysis revealed that in astrocytes the TRP channels are assembles of brain native heteromultimers (Golovina, 2005; Malarkey et al., 2008) containing obligatory TRPC1 (channel forming subunit) and TRPC4 and/or TRPC5 (auxiliary subunits). Inhibition of TRPC1 channel expression by antisence mRNA or its occlusion with blocking antibody directed at an epitope in the pore forming region of the TRPC1 protein significantly decreased store-operated Ca2+ influx in cultured astrocytes (Golovina, 2005; Malarkey et al., 2008) and reduced plateau phase of ATP-activated [Ca2+]i transients (Malarkey et al., 2008). Likewise, immunological inhibition of TRPC1 protein substantially decreased mechanically-induced Ca2+ signalling in astrocytes and suppressed Ca2+-dependent glutamate release (Malarkey et al., 2008).
The second pathway for plasmalemmal Ca2+ entry in astrocytes is associated with ionotropic receptors (ligand-gated Ca2+-permeable channels). Several types of ionotropic receptors are present in astrocytes in vitro, in situ and in vivo (see (Verkhratsky & Steinhauser, 2000; Verkhratsky et al., 2009; Lalo et al., 2011b). The most important astroglial ionotropic receptors are -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl D-aspartate (NMDA) glutamate receptors and P2X purinoceptors. Often astroglial AMPA receptors do not express GluR-B (GluR2) subunit, which makes these receptors moderately Ca2+ permeable (Muller et al., 1992; Seifert & Steinhauser, 2001). The NMDA receptors identified in cortical astrocytes (Lalo et al., 2006; Verkhratsky & Kirchhoff, 2007; Lalo et al., 2011a; Oliveira et al., 2011) differ in their biophysics and pharmacology from the neuronal ones. In particular, astroglial NMDA receptors are weakly (if at all) sensitive to Mg2+ block at physiological resting potential, and their Ca2+ permeability is ~ 2 times lower as compared to neurones (PCa/Pmonovalent ~ 3 vs. ~10 in neurones (Palygin et al., 2010). Nonetheless synaptic actication of astroglial NMDA receptors in cortical slices results in substantial Ca2+ signals (Palygin et al., 2010).
Astrocytes also express P2X1/5 and P2X7 purinoceptors, which may create Ca2+ fluxes (Lalo et al., 2008; Lalo et al., 2011a; Oliveira et al., 2011). The P2X1/5 have moderate Ca2+ permeability ((PCa/Pmonovalent ~ 2), which is sufficient to produce physiologically relevant, Ca2+ signals upon appropriate stimulation (Palygin et al., 2010). The P2X7 receptors activation may result in massive Ca2+ influx, although this signalling is most likely present only in pathology (Franke et al., 2012).
Sodium signalling in astroglia
Dynamic changes in cytoplasmic Na+ concentration in astrocytes
At the rest, astrocytes have relatively high cytosolic Na+ concentration ([Na+]i); in various astroglial preparations (i.e., in culture and in acute slices) it was determined at ~ 15 - 20 mM (cultured hippocampal astrocytes - 15 - 16 mM (Rose & Ransom, 1996); cultured astrocytes from visual cortex - 17 mM (Reyes et al., 2012); astrocytes in cortical slices - 17 - 20 mM (Unichenko et al., 2012), see also (Kirischuk et al., 2012) for comprehensive review). These levels of resting [Na+]i in astrocytes are almost twice higher when compared to neurones (~ 4 - 10 mM see e.g. (Kiedrowski et al., 1994; Rose & Ransom, 1996; Knopfel et al., 1998; Pisani et al., 1998)); high cytosolic Na+ in astrocytes also has functional consequences because it sets reversal potential for many Na+-dependent transporters/exchangers, which shall be discussed below.
Stimulation of astrocytes (either mechanical or chemical) induces transient and complex changes in [Na+]i. For example, application of glutamate to astrocytes in vitro evoked local [Na+]i transients and propagating Na+ waves spreading though astroglial syncytium (Kimelberg et al., 1989; Rose & Ransom, 1997; Bernardinelli et al., 2004). Similarly, both single-cell [Na+]i transients and astroglial Na+ waves were observed in astroglial preparations in situ. In cerebellar Bergmann glia glutamate induced [Na+]i increase by 10 - 25 mM above the restring level (Kirischuk et al., 1997, 2007); in hippocampus glutamate induced [Na+]i rise and astroglial Na+ waves (Langer et al., 2012). Astroglial [Na+]i in hippocampus was also reported to rise by ~ 7 mM following stimulation with g-aminobutyric acid (GABA) (Unichenko et al., 2012). Finally, astroglial [Na+]i increases are induced by stimulation of synaptic inputs as has been detected in both cerebellum and hippocampus (Kirischuk et al., 2007; Bennay et al., 2008; Langer & Rose, 2009).
Molecular mechanisms controlling [Na+]i in astroglia
The cytosolic Na+ concentration in astrocytes is regulated by Na+ diffusion through plasmalemmal channels, by Na+ transport through ATP-dependent pumps and by Na+ translocation by multiple ion exchangers (Fig. 1). Main route for plasmalemmal diffusion of Na+ across the plasmalemma is associated with ionotropic glutamate and purinoceptors, which produce substantial Na+ fluxes upon activation. In Bergmann glia, for example, stimulation of AMPA receptors with kainate increases [Na+]i by ~ 20 -25 mM (Kirischuk et al., 1997). Sodium can also enter astrocytes through TRP channels, non-specific mechanosensitive cationic channels and possibly through Epithelial Sodium Channel (ENaC)/Degenerin family 21 channels or proton-activated Acid Sensing Ion Channels (ASICs), for review see (Kirischuk et al., 2012). Astrocytes in subfornical organ express specific type of sodium channels sensitive to fluctuations in extracellular Na+ concentration. These channels (classified as Nax channels) are involved in astroglial chemosensing and regulation of body Na+ homeostasis (Shimizu et al., 2007). All in all, physiological stimulation of astrocytes trigger substantial Na+ influx, which is mainly mediated by ionotropic receptors and possibly by TRP channels activated following depletion of ER Ca2+ stores.
The sodium-potassium pump or Na+/K+ ATPase (NKA) is the main energy-dependent astroglial Na+ transporter. Astrocytes throughout the CNS express the NKA 1/2 subunits. The Na+/K+ ATPase is activated following an increase in [Na+]i and hence every transient [Na+]i elevation promotes Na+ efflux in exchange for K+, which may represent a link between cytosolic Na+ fluctuations and potassium buffering. Astrocytes are also in possession of multiple ion exchangers or solute carriers (SLC; of which more than 50 families embracing ~ 380 members are known (Hediger et al., 2004; Ren et al., 2007)) that utilise the energy stored in pre-existing ion concentration gradients.
Arguably the most physiologically important is the sodium-calcium exchanger or NCX, which belongs to the SLC8 family (Lytton, 2007). All 3 main isoforms, NCX1, NCX2 and NCX3 are expressed in astroglia. Importantly, the NCX proteins are often concentrated in astroglial perisynaptic processes where they co-localise with NKA and plasma membrane glutamate transporters (Minelli et al., 2007). The NCX can mediate both transport of Na+ and Ca2+ in both directions; generally NCX may operate either in the forward mode (Ca2+ extrusion associated with Na+ influx) or in the reverse mode (Ca2+ entry associated with Na+ extrusion). This is determined by (i) stoichiometry of the exchanger, which is 3Na+:1Ca2+, (ii) transmembrane concentration gradients for Na+ and Ca2+, and (iii) the level of membrane potential. High resting [Na+]i in astrocytes sets the reversal potential of the NCX ~ -80 mV (see (Kirischuk et al., 2012) for calculations and further details), which is very close to the resting membrane potential. Consequently, the NCX in astrocytes dynamically fluctuates between forward/reverse modes and mediates both Ca2+ entry and [Ca2+]i clearance as well as Na+ influx/efflux (Kirischuk et al., 1997; Paluzzi et al., 2007; Rojas et al., 2007; Reyes et al., 2012). The reverse mode of the NCX is triggered by mild depolarisation and by Na+ influx through either ionotropic receptors or neurotransmitter transporters discussed below.
The Na+-dependent neurotransmitter transporters in astrocytes are mainly represented by plasma membrane transporters for glutamate and GABA. The glutamate transporters, generally classified as the excitatory amino acid (mainly glutamate) transporters 1 to 5 (EAAT1 to EAAT5 belonging to SLC1 family) are fundamental for glutamate homeostasis. Astrocytes, which specifically express EAAT1 and EAAT2 (homologues of which in rodents are known as glutamate transporter 1, or GLT1, and glutamate-aspartate transporter or GLAST, respectively) act as the main sink for glutamate in the CNS accumulating ~80% of glutamate released in the course of synaptic transmission (Danbolt, 2001). Glutamate accumulated into astrocytes is rapidly converted (by another astroglia-specific enzyme glutamine synthetase (Hertz & Zielke, 2004; Olabarria et al., 2011)) into glutamine; the latter is either transported back to neurones where it acts as the major precursor for glutamate and GABA and thus is indispensable for sustained synaptic activity (the glutamate-glutamine or GANA-glutamine shuttles) or is utilised for astroglial energetic (Hertz & Zielke, 2004). The stoichiometry of EAAT1/2 is 1 Glu-:3 Na+:1K+:1H+, of which Na+, protons and glutamate enter the cell in exchange to K+ efflux. At the result of this stoichiometry and transmembrane gradients of relevant ions the reversal potential for glutamate transporters is more positive than 50 mV (Kirischuk et al., 2012). This makes the reversal of glutamate transport impossible in physiological conditions; only during strong pathological insults accompanied by massive [Na+]i overload and very high extracellular K+ accumulation the glutamate transport can change direction and provide additional glutamate, which may exacerbate excitotoxicity (Attwell et al., 1993). In physiological conditions activation of glutamate transport in astrocytes triggers inward Na+ current which can elevate [Na+]i by 10 - 20 mM (Kirischuk et al., 2007; Kirischuk et al., 2012). Astrocytes also express GABA transporters of GAT1 and GAT3 types (SLC6 family), which are localised predominantly in astroglial processes surrounding inhibitory synapses. GABA transporters provide for a transmembrane transport of 1 GABA molecule (uncharged in physiological conditions) in exchange for 2 Na+ ions and 1 Cl- anion. Activation of GABA transporters also result in Na+ influx that can elevate [Na+]i by ~ 7 mM (Unichenko et al., 2012). Importantly the reversal potential for GABA transporters lies very close to astrocytic resting membrane potential and therefore even small elevation in [Na+]i can switch the transporter into the reverse mode and hence facilitate GABA release from astrocytes; this release which can inhibit neuronal excitability was indeed detected in cortical slices (Unichenko et al., 2012).
Cellular Na+ homeostasis is also regulated by mitochondria which are able to accumulate Na+ through mitochondrial Na+/Ca2+ exchanger (Nicholls, 2005). The NCLX, the solute carrier SLC24A6, is essential molecular component of this exchanger (Palty et al., 2010).
Functional role of astroglial Na+ signalling
Dynamic fluctuations in cytoplasmic Na+ concentration can affect surprisingly wide array of molecular targets and cascades that are critical for the homeostatic function of astroglia (Fig. 2). First of all, [Na+]i modulates homeostasis of several neurotransmitters, that include principal excitatory transmitter glutamate and inhibitory transmitters GABA and glycine. Glutamate uptake is critical for termination of excitatory transmission and as the first step in glutamate-glutamine/GABA glutamine shuttle. Increase in [Na+]i decreases the efficacy of glutamate transport; as it were glutamatergic transmission activates Na+ influx into astrocytes via ionotropic receptors and EAATs. Thus increased [Na+]i which coincides with the peak of glutamatergic synaptic transmission event temporarily decreases glutamate uptake, thus transiently increasing the effective glutamate concentration in the synaptic cleft. Levels of [Na+]i also influence glutamine synthetase as well as export of glutamine from astrocytes to neurones. The latter is mediated by sodium-coupled neutral amino acid transporter SNAT3/SLC38A3 and is directly controlled by [Na+]i (Mackenzie & Erickson, 2004).
Astroglial [Na+]i also regulates GABA-ergic transmission through (i) controlling astroglial GABA uptake via GAT1/3 pathway and (ii) by maintaining GABA synthesis in neuronal terminals through supplying glutamine. Astroglial GABA transport system is easy to reverse, because (as mentioned before) its reversal potential is set close to the resting potential of astrocyte. Thus mild depolarisation and even small increases in [Na+]i may reverse the GAT-dependent transport making astrocytes a source of GABA. Additionally, GABA-ergic transmission turned out very sensitive to astroglial glutamine supply, and inhibition of glutamine synthetase substantially suppresses GABA-ergic inhibitory transmission (Ortinski et al., 2010). Similarly astroglial [Na+]i regulates the efficacy of glycine clearance from the relevant synapses.
Dynamic changes in astroglial [Na+]i modulate Ca2+ signalling by defining the mode of operation of NCX. Increase in [Na+]i were shown to induce additional Ca2+ influx that contributed to neurotransmitter-evoked [Ca2+]i transients (Kirischuk et al., 1997). Such calcium entry through NCX was even demonstrated to induce exocytotic release of neurotransmitters from astroglia (Benz et al., 2004; Paluzzi et al., 2007; Reyes et al., 2012).
Astroglial Na+ signals are coupled to several important homeostatic pathways. In particular [Na+]i levels directly control the activity of NKA and Na+/K+/Cl- co-transporter NKCC1 thus regulating K+ buffering. The [Na+]i controls the activity of sodium-proton exchanger and sodium-bicarbonate transporter, both being critical for pH homeostasis (see (Kirischuk et al., 2012) for further discussion).
Finally, [Na+]i controls on of the most fundamental astroglial functions - that is the metabolic support of neurones. The latter occurs in the form of astrocyte-neurone lactate shuttle, when astrocytes supply active neurones with their preferred energy substrate lactate (Belanger et al., 2011; Suzuki et al., 2011; Pellerin & Magistretti, 2012). Neuronal-activity induced elevation of astroglial [Na+]i triggers lactate synthesis mediated through NKA; and therefore astroglial Na+ signalling is fundamental for neuronal metabolic support.
Rapid astroglial signalling that is fundamental for neuronal-glial communications is mediated through fluctuations of cytoplasmic concentrations of two principal cations calcium and sodium. Neuronal activity triggers complex and highly organised in both temporal and spatial domains changes in [Ca2+]i and [Na+]i, which in turn regulate multiple effector pathways that control homeostatic function of astroglia.
The research was supported by the Russian Science Foundation (grant project 16-14-00201). It was also partially supported in the framework of performing a state assignment, project component 6.2619.2014/K of 17.07.2014.
Agulhon C, Petravicz J, McMullen AB, Sweger EJ, Minton SK, Taves SR, Casper KB, Fiacco TA & McCarthy KD. (2008). What is the role of astrocyte calcium in neurophysiology? Neuron 59, 932-946.
Alonso MT, Barrero MJ, Michelena P, Carnicero E, Cuchillo I, Garcia AG, Garcia-Sancho J, Montero M & Alvarez J. (1999). Ca2+-induced Ca2+ release in chromaffin cells seen from inside the ER with targeted aequorin. J Cell Biol 144, 241-254.
Altschuld RA, Hohl CM, Castillo LC, Garleb AA, Starling RC & Brierley GP. (1992). Cyclosporin inhibits mitochondrial calcium efflux in isolated adult rat ventricular cardiomyocytes. Am J Physiol 262, H1699-1704.
Attwell D, Barbour B & Szatkowski M. (1993). Nonvesicular release of neurotransmitter. Neuron 11, 401-407.
Attwell D & Gibb A. (2005). Neuroenergetics and the kinetic design of excitatory synapses. Nat Rev Neurosci 6, 841-849.
Baumann O & Walz B. (2001). Endoplasmic reticulum of animal cells and its organization into structural and functional domains. Int Rev Cytol 205, 149-214.
Beck A, Nieden RZ, Schneider HP & Deitmer JW. (2004). Calcium release from intracellular stores in rodent astrocytes and neurons in situ. Cell calcium 35, 47-58.
Belanger M, Allaman I & Magistretti PJ. (2011). Brain energy metabolism: focus on astrocyte-neuron metabolic cooperation. Cell Metab 14, 724-738.
Bennay M, Langer J, Meier SD, Kafitz KW & Rose CR. (2008). Sodium signals in cerebellar Purkinje neurons and Bergmann glial cells evoked by glutamatergic synaptic transmission. Glia 56, 1138-1149.
Benz B, Grima G & Do KQ. (2004). Glutamate-induced homocysteic acid release from astrocytes: possible implication in glia-neuron signaling. Neuroscience 124, 377-386.
Bernardinelli Y, Magistretti PJ & Chatton JY. (2004). Astrocytes generate Na+-mediated metabolic waves. Proc Natl Acad Sci U S A 101, 14937-14942.
Berridge MJ. (2002). The endoplasmic reticulum: a multifunctional signaling organelle. Cell calcium 32, 235-249.
Berridge MJ, Bootman MD & Roderick HL. (2003). Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4, 517-529.
Berridge MJ, Lipp P & Bootman MD. (2000). The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1, 11-21.
Burdakov D, Petersen OH & Verkhratsky A. (2005). Intraluminal calcium as a primary regulator of endoplasmic reticulum function. Cell calcium 38, 303-310.
Case RM, Eisner D, Gurney A, Jones O, Muallem S & Verkhratsky A. (2007). Evolution of calcium homeostasis: from birth of the first cell to an omnipresent signalling system. Cell calcium 42, 345-350.
Charles AC, Dirksen ER, Merrill JE & Sanderson MJ. (1993). Mechanisms of intercellular calcium signaling in glial cells studied with dantrolene and thapsigargin. Glia 7, 134-145.
Charles AC, Merrill JE, Dirksen ER & Sanderson MJ. (1991). Intercellular signaling in glial cells: calcium waves and oscillations in response to mechanical stimulation and glutamate. Neuron 6, 983-992.
Cornell Bell AH, Finkbeiner SM, Cooper MS & Smith SJ. (1990). Glutamate induces calcium waves in cultured astrocytes: long- range glial signaling. Science 247, 470-473.
Danbolt NC. (2001). Glutamate uptake. Progr Neurobiol 65, 1-105.
Dermietzel R. (1998). Gap junction wiring: a 'new' principle in cell-to-cell communication in the nervous system? Brain research Brain research reviews 26, 176-183.
Dermietzel R & Spray DC. (1993). Gap junctions in the brain: where, what type, how many and why? Trends in neurosciences 16, 186-192.
Fellin T, Pascual O, Gobbo S, Pozzan T, Haydon PG & Carmignoto G. (2004). Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors. Neuron 43, 729-743.
Feske S, Gwack Y, Prakriya M, Srikanth S, Puppel SH, Tanasa B, Hogan PG, Lewis RS, Daly M & Rao A. (2006). A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature 441, 179-185.
Finkbeiner SM. (1993). Glial calcium. Glia 9, 83-104.
Franke H, Verkhratsky A, Burnstock G & Illes P. (2012). Pathophysiology of astroglial purinergic signalling. Purinergic Signal 8, 629-657.
Ge WP & Duan S. (2007). Persistent enhancement of neuron-glia signaling mediated by increased extracellular K+ accompanying long-term synaptic potentiation. J Neurophysiol 97, 2564-2569.
Geiger JR & Jonas P. (2000). Dynamic control of presynaptic Ca(2+) inflow by fast-inactivating K(+) channels in hippocampal mossy fiber boutons. Neuron 28, 927-939.
Giaume C & Venance L. (1998). Intercellular calcium signaling and gap junctional communication in astrocytes. Glia 24, 50-64.
Girouard H, Bonev AD, Hannah RM, Meredith A, Aldrich RW & Nelson MT. (2010). Astrocytic endfoot Ca2+ and BK channels determine both arteriolar dilation and constriction. Proc Natl Acad Sci U S A 107, 3811-3816.
Golovina VA. (2005). Visualization of localized store-operated calcium entry in mouse astrocytes. Close proximity to the endoplasmic reticulum. J Physiol 564, 737-749.
Grewer C, Gameiro A, Zhang Z, Tao Z, Braams S & Rauen T. (2008). Glutamate forward and reverse transport: from molecular mechanism to transporter-mediated release after ischemia. IUBMB Life 60, 609-619.
Grimaldi M, Maratos M & 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. The Journal of neuroscience : the official journal of the Society for Neuroscience 23, 4737-4745.
Guerrero-Hernandez A, Dagnino-Acosta A & Verkhratsky A. (2010). An intelligent sarco-endoplasmic reticulum Ca2+ store: release and leak channels have differential access to a concealed Ca2+ pool. Cell calcium 48, 143-149.
Gulyas AI, Sik A, Payne JA, Kaila K & Freund TF. (2001). The KCl cotransporter, KCC2, is highly expressed in the vicinity of excitatory synapses in the rat hippocampus. Eur J Neurosci 13, 2205-2217.
Hediger MA, Romero MF, Peng JB, Rolfs A, Takanaga H & Bruford EA. (2004). The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteinsIntroduction. Pflugers Arch 447, 465-468.
Henneberger C, Papouin T, Oliet SH & Rusakov DA. (2010). Long-term potentiation depends on release of D-serine from astrocytes. Nature 463, 232-236.
Hertz L & Zielke HR. (2004). Astrocytic control of glutamatergic activity: astrocytes as stars of the show. Trends Neurosci 27, 735-743.
Hori T & Takahashi T. (2009). Mechanisms underlying short-term modulation of transmitter release by presynaptic depolarization. J Physiol 587, 2987-3000.
Hua X, Malarkey EB, Sunjara V, Rosenwald SE, Li WH & Parpura V. (2004). Ca2+-dependent glutamate release involves two classes of endoplasmic reticulum Ca2+ stores in astrocytes. J Neurosci Res 76, 86-97.
Jamme I, Petit E, Gerbi A, Maixent JM, MacKenzie ET & Nouvelot A. (1997a). Changes in ouabain affinity of Na+, K+-ATPase during focal cerebral ischaemia in the mouse. Brain Res 774, 123-130.
Jamme I, Trouve P, Maixent JM, Gerbi A, Charlemagne D & Nouvelot A. (1997b). Regulation of Na+,K(+)-ATPase alpha subunit isoforms in mouse cortex during focal ischemia. Ann N Y Acad Sci 834, 658-660.
Kettenmann H & Verkhratsky A. (2008). Neuroglia: the 150 years after. Trends in neurosciences 31, 653-659.
Kiedrowski L, Wroblewski JT & Costa E. (1994). Intracellular sodium concentration in cultured cerebellar granule cells challenged with glutamate. Mol Pharmacol 45, 1050-1054.
Kimelberg HK. (2010). Functions of mature mammalian astrocytes: a current view. Neuroscientist 16, 79-106.
Kimelberg HK & Nedergaard M. (2010). Functions of astrocytes and their potential as therapeutic targets. Neurotherapeutics 7, 338-353.
Kimelberg HK, Pang S & Treble DH. (1989). Excitatory amino acid-stimulated uptake of 22Na+ in primary astrocyte cultures. The Journal of neuroscience : the official journal of the Society for Neuroscience 9, 1141-1149.
Kirischuk S, Kettenmann H & 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 & Verkhratsky A. (2007). Membrane currents and cytoplasmic sodium transients generated by glutamate transport in Bergmann glial cells. Pflugers Arch 454, 245-252.
Kirischuk S, Kirchhoff F, Matyash V, Kettenmann H & Verkhratsky A. (1999). Glutamate-triggered calcium signalling in mouse bergmann glial cells in situ: role of inositol-1,4,5-trisphosphate-mediated intracellular calcium release. Neuroscience 92, 1051-1059.
Kirischuk S, Moller T, Voitenko N, Kettenmann H & Verkhratsky A. (1995). ATP-induced cytoplasmic calcium mobilization in Bergmann glial cells. The Journal of neuroscience : the official journal of the Society for Neuroscience 15, 7861-7871.
Kirischuk S, Parpura V & Verkhratsky A. (2012). Sodium dynamics: another key to astroglial excitability? Trends Neurosci 35, 497-506.
Kirischuk S, Tuschick S, Verkhratsky A & Kettenmann H. (1996). Calcium signalling in mouse Bergmann glial cells mediated by a1-adrenoreceptors and H1 histamine receptors. Eur J Neurosci 8, 1198-1208.
Knopfel T, Guatteo E, Bernardi G & Mercuri NB. (1998). Hyperpolarization induces a rise in intracellular sodium concentration in dopamine cells of the substantia nigra pars compacta. Eur J Neurosci 10, 1926-1929.
Kofuji P & Newman EA. (2004). Potassium buffering in the central nervous system. Neuroscience 129, 1045-1056.
Kopach O, Kruglikov I, Pivneva T, Voitenko N, Verkhratsky A & Fedirko N. (2011). Mitochondria adjust Ca(2+) signaling regime to a pattern of stimulation in salivary acinar cells. Biochim Biophys Acta 1813, 1740-1748.
Lalo U, Palygin O, North RA, Verkhratsky A & Pankratov Y. (2011a). Age-dependent remodelling of ionotropic signalling in cortical astroglia. Aging Cell 10, 392-402.
Lalo U, Pankratov Y, Kirchhoff F, North RA & Verkhratsky A. (2006). NMDA receptors mediate neuron-to-glia signaling in mouse cortical astrocytes. The Journal of neuroscience : the official journal of the Society for Neuroscience 26, 2673-2683.
Lalo U, Pankratov Y, Parpura V & Verkhratsky A. (2011b). 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, Pankratov Y, Wichert SP, Rossner MJ, North RA, Kirchhoff F & Verkhratsky A. (2008). P2X1 and P2X5 subunits form the functional P2X receptor in mouse cortical astrocytes. The Journal of neuroscience : the official journal of the Society for Neuroscience 28, 5473-5480.
Langer J & Rose CR. (2009). Synaptically induced sodium signals in hippocampal astrocytes in situ. J Physiol 587, 5859-5877.
Langer J, Stephan J, Theis M & Rose CR. (2012). Gap junctions mediate intercellular spread of sodium between hippocampal astrocytes in situ. Glia 60, 239-252.
Lenhossek Mv. (1895). Der feinere Bau des Nervensystems im Lichte neuester Forschung. Second edition. Fischer’s Medicinische Buchhandlung H. Kornfield, Berlin.
Letellier M, Park YK, Chater TE, Chipman PH, Gautam SG, Oshima-Takago T & Goda Y. (2016). Astrocytes regulate heterogeneity of presynaptic strengths in hippocampal networks. Proc Natl Acad Sci U S A 113, E2685-2694.
Lytton J. (2007). Na+/Ca2+ exchangers: three mammalian gene families control Ca2+ transport. Biochem J 406, 365-382.
Mackenzie B & Erickson JD. (2004). Sodium-coupled neutral amino acid (System N/A) transporters of the SLC38 gene family. Pflugers Arch 447, 784-795.
Malarkey EB, Ni Y & Parpura V. (2008). Ca2+ entry through TRPC1 channels contributes to intracellular Ca2+ dynamics and consequent glutamate release from rat astrocytes. Glia 56, 821-835.
Malarkey EB & Parpura V. (2009). Mechanisms of transmitter release from astrocytes. In Astrocytes in (patho)physiology of the nervous system, ed. Parpura V & Haydon PG, pp. 301-350. Springer, New York.
Matyash M, Matyash V, Nolte C, Sorrentino V & Kettenmann H. (2002). Requirement of functional ryanodine receptor type 3 for astrocyte migration. Faseb J 16, 84-86.
Michalak M, Robert Parker JM & Opas M. (2002). Ca2+ signaling and calcium binding chaperones of the endoplasmic reticulum. Cell calcium 32, 269-278.
Minelli A, Castaldo P, Gobbi P, Salucci S, Magi S & Amoroso S. (2007). Cellular and subcellular localization of Na+-Ca2+ exchanger protein isoforms, NCX1, NCX2, and NCX3 in cerebral cortex and hippocampus of adult rat. Cell calcium 41, 221-234.
Mogami H, Tepikin AV & Petersen OH. (1998). Termination of cytosolic Ca2+ signals: Ca2+ reuptake into intracellular stores is regulated by the free Ca2+ concentration in the store lumen. Embo J 17, 435-442.
Muller T, Moller T, Berger T, Schnitzer J & Kettenmann H. (1992). Calcium entry through kainate receptors and resulting potassium- channel blockade in Bergmann glial cells. Science 256, 1563-1566.
Nicholls DG. (2005). Mitochondria and calcium signaling. Cell calcium 38, 311-317.
Oberheim NA, Goldman SA & Nedergaard M. (2012). Heterogeneity of astrocytic form and function. Methods Mol Biol 814, 23-45.
Oberheim NA, Wang X, Goldman S & Nedergaard M. (2006). Astrocytic complexity distinguishes the human brain. Trends in neurosciences 29, 547-553.
Olabarria M, Noristani HN, Verkhratsky A & Rodriguez JJ. (2011). Age-dependent decrease in glutamine synthetase expression in the hippocampal astroglia of the triple transgenic Alzheimer's disease mouse model: mechanism for deficient glutamatergic transmission? Mol Neurodegener 6, 55.
Oliveira JF, Riedel T, Leichsenring A, Heine C, Franke H, Krugel U, Norenberg W & Illes P. (2011). Rodent cortical astroglia express in situ functional P2X7 receptors sensing pathologically high ATP concentrations. Cereb Cortex 21, 806-820.
Ortinski PI, Dong J, Mungenast A, Yue C, Takano H, Watson DJ, Haydon PG & Coulter DA. (2010). Selective induction of astrocytic gliosis generates deficits in neuronal inhibition. Nat Neurosci 13, 584-591.
Palty R, Silverman WF, Hershfinkel M, Caporale T, Sensi SL, Parnis J, Nolte C, Fishman D, Shoshan-Barmatz V, Herrmann S, Khananshvili D & Sekler I. (2010). NCLX is an essential component of mitochondrial Na+/Ca2+ exchange. Proc Natl Acad Sci U S A 107, 436-441.
Paluzzi S, Alloisio S, Zappettini S, Milanese M, Raiteri L, Nobile M & Bonanno G. (2007). Adult astroglia is competent for Na+/Ca2+ exchanger-operated exocytotic glutamate release triggered by mild depolarization. J Neurochem 103, 1196-1207.
Palygin O, Lalo U, Verkhratsky A & Pankratov Y. (2010). Ionotropic NMDA and P2X1/5 receptors mediate synaptically induced Ca2+ signalling in cortical astrocytes. Cell calcium 48, 225-231.
Parekh AB. (2008). Mitochondrial regulation of store-operated CRAC channels. Cell calcium 44, 6-13.
Parekh AB & Putney JW, Jr. (2005). Store-operated calcium channels. Physiological reviews 85, 757-810.
Parpura V, Grubisic V & Verkhratsky A. (2011). Ca2+ sources for the exocytotic release of glutamate from astrocytes. Biochim Biophys Acta 1813, 984-991.
Parpura V & Verkhratsky A. (2012). The astrocyte excitability brief: From receptors to gliotransmission. Neurochem Int.
Patrushev I, Gavrilov N, Turlapov V & Semyanov A. (2013). Subcellular location of astrocytic calcium stores favors extrasynaptic neuron-astrocyte communication. Cell calcium 54, 343-349.
Pellerin L & Magistretti PJ. (2012). Sweet sixteen for ANLS. J Cereb Blood Flow Metab E-pub ahead of print, doi: 10.1038/jcbfm.2011.149.
Petersen OH, Michalak M & Verkhratsky A. (2005). Calcium signalling: past, present and future. Cell calcium 38, 161-169.
Petersen OH & Verkhratsky A. (2007). Endoplasmic reticulum calcium tunnels integrate signalling in polarised cells. Cell calcium 42, 373-378.
Pisani A, Calabresi P, Tozzi A, Bernardi G & Knopfel T. (1998). Early sodium elevations induced by combined oxygen and glucose deprivation in pyramidal cortical neurons. Eur J Neurosci 10, 3572-3574.
Pivneva T, Haas B, Reyes-Haro D, Laube G, Veh RW, Nolte C, Skibo G & Kettenmann H. (2008). Store-operated Ca2+ entry in astrocytes: different spatial arrangement of endoplasmic reticulum explains functional diversity in vitro and in situ. Cell calcium 43, 591-601.
Pizzo P, Burgo A, Pozzan T & Fasolato C. (2001). Role of capacitative calcium entry on glutamate-induced calcium influx in type-I rat cortical astrocytes. J Neurochem 79, 98-109.
Porter JT & McCarthy KD. (1995). Adenosine receptors modulate [Ca2+]i in hippocampal astrocytes in situ. JNeurochem 65, 1515-1523.
Putney JW, Jr. (1986). A model for receptor-regulated calcium entry. Cell calcium 7, 1-12.
Putney JW, Jr. (1990). Capacitative calcium entry revisited. Cell calcium 11, 611-624.
Putney JW, Jr. (2007). Recent breakthroughs in the molecular mechanism of capacitative calcium entry (with thoughts on how we got here). Cell calcium 42, 103-110.
Ren Q, Chen K & Paulsen IT. (2007). TransportDB: a comprehensive database resource for cytoplasmic membrane transport systems and outer membrane channels. Nucleic Acids Res 35, D274-279.
Reyes RC, Verkhratsky A & Parpura V. (2012). Plasmalemmal Na+/Ca2+ exchanger modulates Ca2+-dependent exocytotic release of glutamate from rat cortical astrocytes. ASN neuro 4, e00075.
Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa K, Pirvola U, Saarma M & Kaila K. (1999). The K+/Cl- co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature 397, 251-255.
Rojas H, Colina C, Ramos M, Benaim G, Jaffe EH, Caputo C & DiPolo R. (2007). Na+ entry via glutamate transporter activates the reverse Na+/Ca2+ exchange and triggers Cai2+-induced Ca2+ release in rat cerebellar Type-1 astrocytes. J Neurochem 100, 1188-1202.
Rose CR & Ransom BR. (1996). Intracellular sodium homeostasis in rat hippocampal astrocytes. J Physiol 491 ( Pt 2), 291-305.
Rose CR & Ransom BR. (1997). Gap junctions equalize intracellular Na+ concentration in astrocytes. Glia 20, 299-307.
Rusakov DA, Zheng K & Henneberger C. (2011). Astrocytes as Regulators of Synaptic Function: A Quest for the Ca2+ Master Key. Neuroscientist.
Sasaki T, Matsuki N & Ikegaya Y. (2011). Action-potential modulation during axonal conduction. Science 331, 599-601.
Scemes E & Giaume C. (2006). Astrocyte calcium waves: what they are and what they do. Glia 54, 716-725.
Seifert G & Steinhauser C. (2001). Ionotropic glutamate receptors in astrocytes. Prog Brain Res 132, 287-299.
Shigetomi E, Bowser DN, Sofroniew MV & Khakh BS. (2008). Two forms of astrocyte calcium excitability have distinct effects on NMDA receptor-mediated slow inward currents in pyramidal neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience 28, 6659-6663.
Shih PY, Savtchenko LP, Kamasawa N, Dembitskaya Y, McHugh TJ, Rusakov DA, Shigemoto R & Semyanov A. (2013). Retrograde Synaptic Signaling Mediated by K Efflux through Postsynaptic NMDA Receptors. Cell reports.
Shimizu H, Watanabe E, Hiyama TY, Nagakura A, Fujikawa A, Okado H, Yanagawa Y, Obata K & Noda M. (2007). Glial Nax channels control lactate signaling to neurons for brain [Na+] sensing. Neuron 54, 59-72.
Smyth JT, Dehaven WI, Jones BF, Mercer JC, Trebak M, Vazquez G & Putney JW, Jr. (2006). Emerging perspectives in store-operated Ca2+ entry: roles of Orai, Stim and TRP. Biochim Biophys Acta 1763, 1147-1160.
Solovyova N & Verkhratsky A. (2002). Monitoring of free calcium in the neuronal endoplasmic reticulum: an overview of modern approaches. J Neurosci Methods 122, 1-12.
Solovyova N & Verkhratsky A. (2003). Neuronal endoplasmic reticulum acts as a single functional Ca2+ store shared by ryanodine and inositol-1,4,5-trisphosphate receptors as revealed by intra-ER [Ca2+] recordings in single rat sensory neurones. Pflugers Arch 446, 447-454.
Solovyova N, Veselovsky N, Toescu EC & Verkhratsky A. (2002). Ca2+ dynamics in the lumen of the endoplasmic reticulum in sensory neurons: direct visualization of Ca2+-induced Ca2+ release triggered by physiological Ca2+ entry. Embo J 21, 622-630.
Spruston N, Schiller Y, Stuart G & Sakmann B. (1995). Activity-dependent action potential invasion and calcium influx into hippocampal CA1 dendrites. Science 268, 297-300.
Suzuki A, Stern SA, Bozdagi O, Huntley GW, Walker RH, Magistretti PJ & Alberini CM. (2011). Astrocyte-neuron lactate transport is required for long-term memory formation. Cell 144, 810-823.
Tuschick S, Kirischuk S, Kirchhoff F, Liefeldt L, Paul M, Verkhratsky A & Kettenmann H. (1997). Bergmann glial cells in situ express endothelinB receptors linked to cytoplasmic calcium signals. Cell calcium 21, 409-419.
Unichenko P, Myakhar O & Kirischuk S. (2012). Intracellular Na+ concentration influences short-term plasticity of glutamate transporter-mediated currents in neocortical astrocytes. Glia 60, 605-614.
Verkhratsky A. (2005). Physiology and pathophysiology of the calcium store in the endoplasmic reticulum of neurons. Physiological reviews 85, 201-279.
Verkhratsky A. (2010). Physiology of neuronal-glial networking. Neurochem Int 57, 332-343.
Verkhratsky A & Butt AM. (2013). Glial Physiology and Pathophysiology. Wiley-Blackwell, Chichester.
Verkhratsky A & Kettenmann H. (1996). Calcium signalling in glial cells. Trends Neurosci 19, 346-352.
Verkhratsky A & Kirchhoff F. (2007). NMDA receptors in glia. Neuroscientist 13, 28-37.
Verkhratsky A, Krishtal OA & Burnstock G. (2009). Purinoceptors on neuroglia. Mol Neurobiol 39, 190-208.
Verkhratsky A, Orkand RK & Kettenmann H. (1998). Glial calcium: homeostasis and signaling function. Physiological reviews 78, 99-141.
Verkhratsky A, Parpura V & Rodriguez JJ. (2011). Where the thoughts dwell: the physiology of neuronal-glial "diffuse neural net". Brain Res Rev 66, 133-151.
Verkhratsky A & Petersen OH. (2002). The endoplasmic reticulum as an integrating signalling organelle: from neuronal signalling to neuronal death. Eur J Pharmacol 447, 141-154.
Verkhratsky A, Rodriguez JJ & Parpura V. (2012). Calcium signalling in astroglia. Mol Cell Endocrinol 353, 45-56.
Verkhratsky A, Solovyova N & Toescu EC. (2002). Calcium excitability of glial cells. In Glia in synaptic transmission, ed. Volterra A, Haydon P & Magistretti P, pp. 99-109. OUP, Oxford.
Verkhratsky A & Steinhauser C. (2000). Ion channels in glial cells. Brain Res Brain Res Rev 32, 380-412.
Verkhratsky A & Toescu EC. (2006). Neuronal-glial networks as substrate for CNS integration. J Cell Mol Med 10, 826-836.
Viitanen T, Ruusuvuori E, Kaila K & Voipio J. (2010). The K+-Cl cotransporter KCC2 promotes GABAergic excitation in the mature rat hippocampus. J Physiol 588, 1527-1540.
Walz W. (2000). Role of astrocytes in the clearance of excess extracellular potassium. Neurochem Int 36, 291-300.
Yaguchi T & Nishizaki T. (2010). Extracellular high K+ stimulates vesicular glutamate release from astrocytes by activating voltage-dependent calcium channels. J Cell Physiol 225, 512-518.
Zorec R, Araque A, Carmignoto G, Haydon PG, Verkhratsky A & Parpura V. (2012). Astroglial excitability and gliotransmission: an appraisal of Ca2+ as a signalling route. ASN neuro 4.