Homeostatic function of astrocytes: Ca²⁺ and Na⁺ signalling

Abstract

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 the activation of respective fluxes through ionic channels or ion exchangers. Here, we provide a comprehensive review of astroglial Ca²⁺ and Na⁺ signalling.

1. Astroglia - the homeostatic cells of the brain

The nervous system in mammals represents complex network formed by several distinct cell types of neural crest and non-neural crest origin. In the central nervous system (CNS) the neural cells are neurones, astrocytes, NG2 glia and myelinating oligodendrocytes, whereas the non-neural cells are the microglia. In the peripheral nervous system the neural elements include sensory, sympathetic and parasympathetic as well as enteric neurones and highly diversified peripheral glia represented by satellite glial cells, enteric glia, and myelinating, non-myelinating and perisynaptic Schwann cells. All these diverse cells are unified through several levels of intercellular signalling accomplished by inter- and intracellular diffusion of various molecules that either bind and stimulate the plasmalemmal receptors or penetrate cellular membranes through specific ion channels, thus initiating electrical excitation (by virtue of charges carried by ions) or triggering local cytoplasmic responses by interacting with variety of intracellular targets.

Among many neural cells forming the CNS astroglial cells have a specific role of a main homeostatic element. Astrocytes (which means star-like cells, the name invented by Michael von Lenhossek [1,2]) represent a highly heterogeneous cell population, which include protoplasmic astrocytes of grey matter of the brain and the spinal cord, fibrous astrocytes localised in the white matter, classical radial glia that acts as a pluripotent neural precursor cell during development, 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 brain of higher primates also contains interlaminar, polarised and varicose projection astrocytes, with all these types being particularly developed in the human brain (see [3–14] for details and relevant references). In addition, astroglia cover several types of specialised cells such as ependymocytes, choroid plexus cells and retinal pigment epithelial cells.

Astrocytes appeared early in evolution, first as supporting cells and then gained their functional importance in the course of specialisation of the nervous system. Functions of astrocytes are many: i) they define the brain microanatomy and provide structural support for other cellular elements of the CNS; ii) they synthesise and store glycogen and produce lactate, the latter being preferred metabolic substrate for neurones; iii) they control homeostasis and turnover of several key neurotransmitters and neuromodulators (for example, glutamate and adenosine); and iv) they regulate synaptic connectivity through supporting synaptogenesis, controlling ion/transmitter concentrations in the synaptic cleft and regulating synaptic plasticity via secreting various neuroactive factors [14–16].

2. S pecific nature of astroglial excitability

Excitability of neurones and neuroglia are fundamentally different. Signalling in neuronal networks is mainly accomplished through propagating waves of transient opening of the plasmalemmal ion channels that provide fluxes of ions underlying electrical excitability. These propagating waves, manifested by action potentials, can rapidly (up to 100 m/s) convey excitation through neuronal axons. Electrical signals, when reaching neuronal terminals, activate local Ca²⁺ entry that initiates exocytotic release of neurotransmitters. The latter diffuse through the synaptic cleft and transfer excitation to the postsynaptic neuronal structures through the activation of specific receptors [17–22].

Glial cells, in contrast to neurones, cannot generate plasmalemmal action potentials, because of low densities of voltage-operated ion channels and high expression of K⁺ channels that prevent substantial depolarisations of glial membranes. Instead, glial cells use intracellular signalling routes where local gradients of ions interact with intracellular targets and trigger physiological reactions. In addition, glial cells, and astrocytes in particular, are physically connected into syncytia by means of intercellular contacts represented by gap junctions. The gap junctions are made by closely apposing plasmalemma of two neighbouring cells with a narrow (~ 2 – 2.5 nm) intercellular cleft. Here, specialised intercellular channels, which span both membranes and establish direct intercellular contacts, are concentrated. These junctional channels are composed of two aligned “hemichannels” or connexons each made up from 6 subunits or connexins [23,24]. Out of about 20 known connexins [25,26] astroglial cells express mainly connexins Cx43, and to a lesser extent Cx30 and Cx26; as minor constituents of their gap junctions, astrocytes can also express Cxs 45, 40 and 46 [27]. These gap junctional channels have a large pore (with a diameter of ~ 1.5 nm), which allows intercellular passage of ions and various active substances such as second messengers [e.g., inositol 1,4,5 trisphosphate (InsP₃)], nucleotides (ATP, ADP) or metabolic substrates (glucose). As a result, the gap junctions provide a specific route for intercellular and long-range signalling, which is manifested in propagating waves of ionic (Ca²⁺ or Na⁺) signals, or metabolic waves. The communication through gap junctions is of course much slower when compared with the spread of action potentials (on average, for example, Ca²⁺ waves propagate through astroglial syncytia with a speed of ~ 20 – 40 μm/s); however, it is more diversified and may provide a substrate for integrating intercellular signalling. It should be noted, however, that, although the above described gap junctional communication contributes to intercellular Ca²⁺ waves, it is ATP, which gets released from astrocytes, that serves as an extracellular signal to majorly support the intercellular Ca²⁺ waves [28–31].

Fast signalling in astroglial cells, which can be initiated by neuronal activity or many different neurotransmitters and neuromodulators, is mediated by intracellular ion gradients. In this essay, we shall concentrate on two ions, Ca²⁺ and Na⁺, which mediate signalling in astroglia and discuss in detail glial signalling mediated by them. It should be noted that the regulation of the Ca²⁺ dynamics (and possibly also of Na⁺) could differ in various subcellular locations of astrocytes, which could result in local signaling, rather than the above long-range intercellular waves. For instance, astrocyte perisynaptic processes are the sites where synapses can evoke local Ca²⁺ elevations that could result in a local feedback signalling via the gliotransmitter release [32–34].

3. Ca²⁺ signalling in astroglia

3.1. Molecular machinery of Ca²⁺ signalling

Evolutionary, Ca²⁺ has emerged 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, and rapid binding kinetics) and by its availability in the primordial ocean [35,36]. 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 plasmalemmal Ca²⁺-release activated Ca²⁺ channels (CRAC channels, that on a molecular level represent activity of Orai proteins) and cationic channels with various degrees of Ca²⁺ permeability (Figure 1). The latter cationic channels 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 (PMCA or plasmalemmal Ca²⁺ pumps), by SarcoEndoplasmic reticulum ATPases (SERCA or endoplasmic reticulum Ca²⁺ pumps) and by ion exchangers of which the Na⁺/Ca²⁺ exchanger (NCX) is by far the most important. Inside the cell, Ca²⁺ is buffered by Ca²⁺ binding proteins (CBPs), 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 K_D 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, CBPs allow almost free and long-distance Ca²⁺ diffusion that being instrumental for making ER Ca²⁺ tunnels [37,38]. 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 [39,40].

Figure 1.

Molecular cascades of Ca²⁺ and Na⁺ signalling in astroglia (see text for details). Abbreviations: AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; CBP, Ca²⁺ binding protein; EAAT, excitatory amino acid transporter; ER, endoplasmic reticulum; G, G-protein; GABA, γ-aminobutyric acid; GAT, GABA transporter; GPCR, G-protein coupled receptor; InsP3R, inositol 1,4,5 trisphosphate-gated Ca²⁺ channel/receptor; NCX, Na⁺/Ca²⁺ exchanger; NKA, Na⁺/K⁺ ATPase; NMDAR, N-methyl D-aspartate receptor; PLC, phospholipase C; PMCA, plasmalemmal Ca²⁺-ATPase; P2XR, purinergic 2X receptor, SERCA, sarco(endoplasmic) reticulum Ca²⁺ ATPase; SOCE, store-operate Ca²⁺ entry; TRP, transient receptor potential.

Effectors of Ca²⁺ signals are Ca²⁺ regulated enzymes (also known as “Ca²⁺ sensors”), binding 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 (Figure 1). Combinations of these 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 [41,42]. Another important feature of Ca²⁺ homeostatic/signalling system is its autoregulation by Ca²⁺ ions themselves, as transient changes in Ca²⁺ concentration establish multiple feedback mechanisms that modify the handling of itself. 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 SERCA 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 [43,44] for detailed discussion). Finally Ca²⁺ fluxes are modulated by mitochondria which, by providing ATP and dynamic Ca²⁺ buffering, regulate plasmalemmal Ca²⁺ entry and ER Ca²⁺ uptake [45,46].

3.2. 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 [47–52]. 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 [53–56]. 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 [13,16,52,57].

The ER is one of the largest intracellular organelles, which is involved in a variety of fundamental cellular processes such as protein synthesis, protein folding and trafficking haulage of secretory products etc. [58–61]. The ER is also a key organelle of Ca²⁺ 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 [62–66]. 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 [55,67]. Functional role of second type of ER Ca²⁺ release channel, the RyR, a Ca²⁺-gated Ca²⁺ channel, in astroglial Ca²⁺ dynamics remains controversial, although astrocytes express RyR both in vitro and in situ [68–70] and RyRs contribute to Ca²⁺ signals necessary for glutamate release via regulated exocytotic pathway [71]. The InsP₃Rs are simultaneously controlled by InsP₃ and Ca²⁺ ions and therefore local increase in [Ca²⁺]_i facilitates receptor opening and promotes Ca²⁺-induced Ca²⁺ release through InsP₃R. 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 and coordinated with the extracellular spread of ATP. These Ca²⁺ waves are important for astrocyte physiology; astroglial stimulation usually occurs at the level of distal processes, and Ca²⁺ waves convey this excitation to the soma. Furthermore, astroglial Ca²⁺ wave travels through astroglial syncytia, being therefore a substrate for astroglial long-range signalling [72,73]. In addition to InsP₃, astrocytes can also utilize other ER Ca²⁺-mobilizing second messengers in response to external stimuli, most notably cyclic adenosine diphosphoribose (cyclic ADP-ribose) [74,75] and nicotinic acid adenine dinucleotide phosphate, (NAADP) [76–78].

Ca²⁺ 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 [79,80] 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 [71]. 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 Ca²⁺ entry discussed next. The role for ER Ca²⁺ signalling cascade in controlling astroglial gliotransmission was subsequently corroborated in experiments in acute slices (e.g., [81–83]].

3.3. 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 (Figure 1). There is 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 [80] 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 a majority of electrically non-excitable cells. This Ca²⁺ influx pathway (initially described as a “capacitative” Ca²⁺ entry) [84,85] is controlled by the Ca²⁺ content in the ER lumen, where decrease in [Ca²⁺]_L results in the opening of plasmalemmal Ca²⁺-permeable channels [86]. 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 (I_CRAC) store-operated channels characterised by extremely high Ca²⁺ selectivity and very low single channel conductance. Activation of these channels reflects interaction of stromal interaction molecule (STIM) proteins (that detect ER Ca²⁺ concentration) with Orai (named after Greek gate-keeping goddesses [87]) proteins that form the plasmalemmal channel [88]. Alternatively store-operated Ca²⁺ influx may involve activation of TRPC channels [89].

The store-operated Ca²⁺ entry is functionally expressed in astroglia [90,91]. Initial experimental evidence indicates the role for TRPC channels. They are expressed in astrocytes at both mRNA and protein levels, and TRPC activity is involved in shaping astroglial Ca²⁺ signals [92–94]. Further analysis revealed that in astrocytes the TRPC channels are assembles of brain native heteromultimers [92,95] containing obligatory TRPC1 (channel forming subunit) and TRPC4 and/or TRPC5 (auxiliary subunits). Inhibition of TRPC1 channel expression by antisense 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 [92,95] and reduced plateau phase of ATP-activated [Ca²⁺]_i transients [95]. Likewise, immunological inhibition of TRPC1 protein substantially decreased mechanically-induced Ca²⁺ signalling in astrocytes and suppressed Ca²⁺-dependent glutamate release [95].

It has been recently demonstrated that astrocytes possess functional STIM1 and Orai1 molecules which play a role in thrombin-induced cytosolic Ca²⁺ dynamics [96]. This was demonstrated in cultured astrocytes, investigated using immunocytochemisty and Ca²⁺ imaging. Overexpression and silencing (using short interfering RNA) of STIM1/Orai1 led to enhanced or muted Ca²⁺ dynamics in astrocytes, respectively.

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 [97–99]. The most important astroglial ionotropic receptors are α-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 [100,101]. The NMDA receptors identified in cortical astrocytes [102–105] 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 (P_Ca/P_monovalent ~ 3 vs. ~10 in neurones [106]. Nonetheless, synaptic activation of astroglial NMDA receptors in cortical slices results in substantial Ca²⁺ signals [106].

Astrocytes express P2X_1/5 and P2X₇ purinoceptors, which may create Ca²⁺ fluxes [102,105,107]. The P2X_1/5 have moderate Ca²⁺ permeability (P_Ca/P_monovalent ~ 2), which is sufficient to produce physiologically relevant Ca²⁺ signals upon appropriate stimulation [106]. The P2X₇ receptors activation may result in massive Ca²⁺ influx, although this signalling is most likely present only in pathology [108].

Astrocytes also express TRPA1 channels, which generate frequent occurrence of local, punctate Ca²⁺ influx. This activity of TRPA1 contributes to the resting Ca²⁺ levels in astrocytes [34]. This ion channel is best known as a chemosensor for various environmental noxious stimuli causing pain [109–111], albeit it can also be activated by cold [112] or heat [113].

4. Na⁺ signalling in astroglia

4.1. Dynamic changes in cytoplasmic Na⁺ concentration in astrocytes

At 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 [114]; cultured astrocytes from visual cortex, 17 mM [115]; astrocytes in cortical slices, 17 – 20 mM [116]; see also [117] 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., [114,118–120]); 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 [121–123]. 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 resting level [124,125]; in hippocampus, glutamate induced [Na⁺]_i rise and astroglial Na⁺ waves [126]. Astroglial [Na⁺]_i in hippocampus was also reported to rise by ~ 7 mM following stimulation with γ-aminobutyric acid (GABA) [116]. Finally, astroglial [Na⁺]_i increases are induced by stimulation of synaptic inputs which has been detected in both cerebellum and hippocampus [125,127,128].

4.2. 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 (Figure 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 [124]. Na⁺ can also enter astrocytes through TRP channels, non-specific mechanosensitive cationic channels and possibly through Epithelial Na⁺ Channel (ENaC)/Degenerin family 21 channels or proton-activated Acid Sensing Ion Channels (ASICs); for review see [117]. Astrocytes in subfornical organ express specific type of Na⁺ channels sensitive to fluctuations in extracellular Na⁺ concentration. These channels (classified as Na_x channels) are involved in astroglial chemosensing and regulation of body Na⁺ homeostasis [129]. All in all, physiological stimulation of astrocytes trigger substantial Na⁺ influx, which is mainly mediated by ionotropic receptors and possibly by TRPC channels activated following depletion of ER Ca²⁺ stores.

The Na⁺- K⁺ pump or Na⁺/K⁺ ATPase (NKA) is the main energy-dependent astroglial Na⁺ transporter. Astrocytes throughout the CNS express the NKA a1/a2 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⁺ influx, which may represent a link between cytosolic Na⁺ fluctuations and K⁺ 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 [130,131]) that utilise the energy stored in pre-existing ion concentration gradients.

Arguably, the most physiologically important ion exchanger is the Na⁺/Ca²⁺ exchanger or NCX, which belongs to the SLC8 family [132]. 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 [133]. The NCX can mediate the transport of both 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 [117] 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 [115,124,134,135]. 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 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) act as the main sink for glutamate in the CNS accumulating ~80% of glutamate released in the course of synaptic transmission [136]. Glutamate accumulated into astrocytes is rapidly converted (by another astroglia-specific enzyme glutamine synthetase [137,138]) into glutamine; the latter is either transported 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 GABA-glutamine shuttles), or is utilised for astroglial energetics [137]. The stoichiometry of EAAT1/2 is 1 Glu⁻:3 Na⁺:1K⁺:1H⁺, of which Na⁺, proton and glutamate enter the cell in exchange to K⁺ efflux. As a result of this stoichiometry and transmembrane gradients of relevant ions, the reversal potential for glutamate transporters is more positive than 50 mV [117]. 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 can the glutamate transport change direction and provide additional glutamate, which may exacerbate excitotoxicity [139]. In physiological conditions, activation of glutamate transport in astrocytes triggers inward Na⁺ current which can elevate [Na⁺]_i by 10 – 20 mM [117,125]. Astrocytes also express GABA transporters of GAT1 and GAT3 types (SLC6 family), which are localised predominantly in astroglial processes surrounding inhibitory synapses. GABA transport via GAT3 can be affected by TRPA1 activity (decreased TRPA1 function leads to reduction in GABA uptake), which subsequently affects nearby GABA-ergic synaptic transmission [34]. GABA transporters provide for a transmembrane symport of 1 GABA molecule (uncharged in physiological conditions), 2 Na⁺ ions and 1 Cl⁻ anion. Activation of GABA transporters also result in Na⁺ influx that can elevate [Na⁺]_i by ~ 7 mM [116]. 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 reverse mode and hence facilitate GABA release from astrocytes; this release which can inhibit neuronal excitability was indeed detected in cortical slices [116].

Cellular Na⁺ homeostasis is also regulated by mitochondria which are able to accumulate Na⁺ through mitochondrial Na⁺/Ca²⁺ exchanger [40]. The NCLX, the solute carrier SLC24A6, is essential molecular component of this exchanger [140].

4.3. 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. 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 Na⁺-coupled neutral amino acid transporter SNAT3/SLC38A3 and is directly controlled by [Na⁺]_i [141].

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 by 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 [142]. 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 [124]. Such Ca²⁺ entry through NCX was even demonstrated to induce exocytotic release of neurotransmitters from astroglia [115,134,143].

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 Na⁺-proton exchanger and Na⁺-bicarbonate transporter, both being critical for pH homeostasis (see [117] for further discussion).

Finally, [Na⁺]_i controls one 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 [144–146]. 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.

5. Concluding remarks

Rapid astroglial signalling, that is fundamental for neuronal-glial communications, is mediated through fluctuations of cytoplasmic concentrations of two principal cations Ca²⁺ and Na⁺. Neuronal activity can trigger complex spatio-temporal changes of [Ca²⁺]_i and [Na⁺]_i in astrocytes, which in turn regulate multiple effector pathways that control homeostatic function of these glial cells.

Abbreviations

AMPA

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

[Ca²⁺]_i

cytoplasmic free Ca²⁺ concentration

[Ca²⁺]_L

intra-ER (or intraluminal) free Ca²⁺ concentration

CNS

central nervous system

CRAC

Ca²⁺-release activated Ca²⁺

ER

endoplasmic reticulum

GABA

γ-aminobutyric acid

InsP₃R

inositol 1,4,5 trisphosphate (InsP₃)-gated Ca²⁺ channel/receptor

[Na⁺]_i

cytoplasmic Na⁺ concentration

NCX

Na⁺/Ca²⁺ exchanger

NKA

Na⁺/K⁺ ATPase

NMDA

N-methyl D-aspartate

PLC

phospholipase C

PMCA

plasmalemmal Ca²⁺ ATPase

RyR

ryanodine receptor

SERCA

sarco(endoplasmic) reticulum Ca²⁺ ATPase

SLC

solute carrier

TRP

transient receptor potential