REVIEW ARTICLE

Astrocyte Sodium Signaling and the Regulation of Neurotransmission Sergei Kirischuk,1 L aszl o H eja,2 Julianna Kardos,2 and Brian Billups3 The transmembrane Na1 concentration gradient is an important source of energy required not only to enable the generation of action potentials in excitable cells, but also for various transmembrane transporters both in excitable and non-excitable cells, like astrocytes. One of the vital functions of astrocytes in the central nervous system (CNS) is to regulate neurotransmitter concentrations in the extracellular space. Most neurotransmitters in the CNS are removed from the extracellular space by Na1-dependent neurotransmitter transporters (NeuTs) expressed both in neurons and astrocytes. Neuronal NeuTs control mainly phasic synaptic transmission, i.e., synaptically induced transient postsynaptic potentials, while astrocytic NeuTs contribute to the termination of phasic neurotransmission and modulate the tonic tone, i.e., the long-lasting activation of extrasynaptic receptors by neurotransmitter that has diffused out of the synaptic cleft. Consequently, local intracellular Na1 ([Na1]i) transients occurring in astrocytes, for example via the activation of ionotropic neurotransmitter receptors, can affect the driving force for neurotransmitter uptake, in turn modulating the spatio-temporal profiles of neurotransmitter levels in the extracellular space. As some NeuTs are close to thermodynamic equilibrium under resting conditions, an increase in astrocytic [Na1]i can stimulate the direct release of neurotransmitter via NeuT reversal. In this review we discuss the role of astrocytic [Na1]i changes in the regulation of uptake/release of neurotransmitters. It is emphasized that an activation of one neurotransmitter system, including either its ionotropic receptor or Na1-coupled co-transporter, can strongly influence, or even reverse, other Na1-dependent NeuTs, with potentially significant consequences for neuronal communication. GLIA 2015;00:000–000

Key words: glutamate transporters, GABA, glycine, glutamine, gliotransmitters

Introduction he Na1 gradient maintained by the plasma membrane Na1/K1-ATPase is of pivotal importance for neuronal and glial cell physiology. In neurons, the generation of action potentials, a “bit” of information in the central nervous system (CNS), requires an unremitting driving force for Na1 ions. Glial cells in contrast are non-excitable cells and although they maintain a high transmembrane Na1 gradient, they do not generate action potentials. Nevertheless, sodium ions can cross astrocytic membranes through voltage- and ligand-gated channels and transporters (for review see Kirischuk et al., 2012; Rose and Karus, 2013). As the membrane resistance of astrocytes is relatively low, transmembrane currents can produce relatively small changes of the membrane potential (Deitmer and Rose, 2010; Verkhratsky and Steinhauser, 2000). In contrast, activation of glial channels,

T

receptors or transporters can result in large [Na1]i transients, which influence a variety of cellular functions. Stimulation of astrocytes with agonists of ionotropic glutamate receptors elevates astrocytic [Na1]i by 10–25 mM (Kirischuk et al., 1997, 2007; Rose and Ransom, 1997), in a similar way to neuronal activity which also elicits large [Na1]i responses in astrocytes. Even short stimulation of neuronal fibers increases astrocytic [Na1]i by 10–15 mM (Bennay et al., 2008; Kirischuk et al., 2007; Langer and Rose, 2009) and, moreover, the spatiotemporal profile of synaptically induced [Na1]i transients appears to be input dependent (Bennay et al., 2008). Another important feature of astrocytic [Na1]i transients is their long duration. In contrast to [Ca21]i responses which last only hundreds of milliseconds, astrocytic [Na1]i transients persist for tens of seconds (Bennay et al., 2008; Kirischuk et al., 2007; Langer and Rose, 2009). As the resting [Na1]i in

View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22943 Published online Month 00, 2015 in Wiley Online Library (wileyonlinelibrary.com). Received Sep 14, 2015, Accepted for publication Oct 28, 2015. Address correspondence to Brian Billups, Eccles Institute of Neuroscience, John Curtin School of Medical Research, The Australian National University, 131 Garran Rd, Acton ACT 2601, Australia. E-mail: [email protected] or Sergei Kirischuk, University Medical Center of the Johannes Gutenberg University Mainz, Institute of Physiology, Duesbergweg 6, 55128 Mainz, Germany. E-mail: [email protected] From the 1University Medical Center of the Johannes Gutenberg University Mainz, Institute of Physiology, Mainz, Germany; 2Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary; 3Eccles Institute of Neuroscience, John Curtin School of Medical Research, The Australian National University, Acton, ACT, Australia.

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FIGURE 1: Stoichiometry of glutamate (EAATs; A), glutamine (SNAT3; B), GABA (GATs; C) and glycine (GlyT1 and GlyT2; D) transporters.

astrocytes amounts to 15–20 mM (Rose and Ransom, 1996; Unichenko et al., 2012), such large and long-lasting elevations of [Na1]i can strongly affect all Na1-dependent processes in astrocytes. The extracellular concentration of neurotransmitters in the CNS is mainly regulated by specific transporters which can rapidly bind a neurotransmitter molecule and relocate it inside cells. Most of neurotransmitter transporters (NeuTs) utilize the transmembrane Na1 gradient as a driving force for the uptake. Interestingly, stimulation of neuronal fibers can elevate astrocytic [Na1]i by 10–20 mM even in the presence of ionotropic glutamate receptor antagonists (Bennay et al., 2008; Kirischuk et al., 2007; Langer and Rose, 2009), indicating that the activity of NeuTs can significantly change [Na1]i. Thus, NeuT-mediated [Na1]i transients will decrease the driving force for further neurotransmitter clearance via NeuTs. Moreover, many NeuTs are electrogenic and their reversal potentials are determined by both ion and neurotransmitter transmembrane gradients. As a consequence astrocytic [Na1]i transients may theoretically even change the directionality of NeuT-mediated transport, i.e., leading to a NeuT-mediated release of neuroactive substances that can act as “gliotransmitters.” Considering the recent dispute over the existence of vesicular release from astrocytes (Barbour et al., 1991; Fujita et al., 2014; Hamilton and Attwell, 2010; Sloan and Barres, 2014), it is essential to better understand the NeuT-mediated astrocytic release mechanisms. In this review, we will discuss the role of astrocytic Na1 in the regulation of neurotransmitter uptake and gliotransmitter release. Glutamate Transporters Glutamate released from excitatory synapses must be rapidly removed from the extracellular space to allow continuous high-frequency neurotransmission and to avoid excitotoxicity. This vital task is accomplished by members of the excitatory amino acid transporter (EAAT) family, of which 5 (EAAT15) have been cloned (Danbolt, 2001). Rather than being transported directly back into presynaptic terminals, most of 2

the released glutamate is sequestered into neighboring astrocytes (Rothstein et al., 1996). This is accomplished by the EAAT1 (the product of the gene Slc1a3) and EAAT2 (Slc1a2) isoforms (Conti et al., 1998; Minelli et al., 2001), which are also known as GLAST and GLT-1, respectively. The relative proportions of these two transporters vary depending on brain region, for example EAAT2 expression is fourfold higher in rat hippocampal stratum radiatum and EAAT1 expression is sixfold higher in the cerebellar molecular layer (Chaudhry et al., 1995; Lehre and Danbolt, 1998). However, the properties of the two transporters are broadly similar and they both share the same stoichiometry: glutamate influx is driven by the cotransport of 3 Na1 and 1 H1 ions, and the countertransport of 1 K1 ion (Fig. 1A; Levy et al., 1998; Owe et al., 2006; Zerangue and Kavanaugh, 1996). The 3:1 ratio of Na1 to glutamate molecules transported causes a significant Na1 influx into glial cells when glutamate uptake is stimulated. This phenomenon has been directly observed in cultured astrocytes, where activation of glutamate uptake increases [Na1]i by 10–20 mM (Chatton et al., 2000, 2001; Kimelberg et al., 1989; Rose and Ransom, 1996). In situ fluorescent measurements of glial [Na1]i have been performed in a number of brain areas. In cerebellar Bergmann glia application of EAAT substrates increases the [Na1]i by up to 20 mM (Bennay et al., 2008; Kirischuk et al., 2007). Similar results are observed in neocortical astrocytes, where EAAT activation causes a rise in [Na1]i of 28 mM (Unichenko et al., 2012); in hippocampal astrocytes where brief EAAT activation results in a [Na1]i rise of 4.8 mM (Langer and Rose, 2009); and in perisynaptic astrocytes in brainstem slices where a somatic rise of 3.4 mM is observed (Uwechue et al., 2012). In a physiological context, EAATs mediate a 2–10 mM elevation of astrocytic [Na1]i in response to activity in adjacent synapses. This is evident for Bergmann glial responses upon parallel or climbing fiber stimulation (Bennay et al., 2008; Kirischuk et al., 2007); for hippocampal astrocytic responses following short bursts of Volume 00, No. 00

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Schaffer collateral stimulation (Langer and Rose, 2009); and for layer 2/3 neocortical astrocytes after stimulation in layer 4 (Unichenko et al., 2012). The spatio-temporal profile of the [Na1]i transient is dependent on the type, location and duration of the synaptic stimulation, with weak stimulation causing local rises that are confined to the astrocytic process adjacent to the active synapses and stronger stimulation causing a global [Na1]i rise (Bennay et al., 2008; Langer and Rose, 2009). The sodium signals can then spread in a wavelike manner throughout the astrocytes and via gap junctions into neighboring astrocytes over a distance of 100 mm (Langer et al., 2012). Similarly, regenerative Na1-waves have also been observed in cultured astrocytes, in response to mechanical or electrical stimulation (Bernardinelli et al., 2004). Although synaptic stimulation produces smaller rises in [Na1]i than global EAAT activation, it is likely that [Na1]i transients in the local near-membrane microdomains are actually much larger, but decay too rapidly to be reliably recorded by fluorescent sodium imaging experiments. While diffusion away from its entry site may reduce the local magnitude of the [Na1]i transient, ultimately the majority of the [Na1]i is removed from the cell by the action of the Na1/ K1-ATPase (Cholet et al., 2002). However, as discussed later, other Na1-dependent transporters may reverse and additionally remove a proportion of the accrued Na1. The [Na1]i rise mediated by EAATs can have a number of important physiological effects on astrocytes, via its influence on other transporter systems. One such key interaction is the close physical and functional association of EAATs with the Na1/K1-ATPase (Cholet et al., 2002; Rose et al., 2009; Sheean et al., 2013). Astrocytes express both the a1 and a2 containing forms of the Na1/K1-ATPase, of which the a2 form predominates. a2 containing isoforms have a higher sodium affinity than a1 (9–12 mM vs. 16 mM for a1) and a faster turnover rate (Blanco, 2005). It is known that EAATs compartmentalize with the a2 containing Na1/K1-ATPase in the astrocytic membrane, where both EAAT1 and EAAT2 have been shown to form macromolecular complexes with this and other proteins involved in energy metabolism (Bauer et al., 2012; Genda et al., 2011). EAAT activation induces a [Na1]i rise, which in turn stimulates ATP hydrolysis as the Na1/K1-ATPase restores the transmembrane Na1 gradient (Chatton et al., 2000; Magistretti and Chatton, 2005). This increases astrocytic glucose utilization and also stimulates lactate production (Pellerin and Magistretti, 1994), which can potentially support the metabolism of neighboring neurons (Pellerin et al., 2007). In addition to metabolic signaling via the Na1/K1-ATPase, the EAAT-induced [Na1]i rise influences Ca21 signaling in astrocytes via its action on the Na1Ca21 exchanger (NCX). NCX isoforms are present in astrocytic processes adjacent to synapses, where they are likely to Month 2015

colocalize with EAATs and be exposed to the microdomains of [Na1]i signaling (Minelli et al., 2007). The [Na1]i rise reverses NCX, causing a robust intracellular Ca21 signal, which is amplified by Ca21-induced Ca21 release from ryanodine sensitive stores (Rojas et al., 2007). These mechanisms highlight the central role of EAATs and [Na1]i in linking activity in neighboring synapses to the metabolic and intracellular signaling functions of astrocytes. The coupling of glutamate transport to 3 Na1 ions gives EAATs a very high accumulating power: at equilibrium they can theoretically reduce the extracellular glutamate concentration to 0.3 nM (Allen et al., 2004), which is similar to the observed value of 25 nM in hippocampal slices (Herman and Jahr, 2007). However, one disadvantage of their high dependence on the electrochemical Na1 gradient is that it makes EAATs significantly sensitive to alterations in the astrocytic [Na1]i, with increases in [Na1]i reducing the driving force for glutamate uptake. Using equations for the flux associated with EAAT transport (Allen et al., 2004; Zerangue and Kavanaugh, 1996) an increase of [Na1]i from 15 to 35 mM would reduce the driving force for glutamate transport by 12.7 fold. For glial EAATs this has been directly demonstrated in retinal M€ uller cells (Barbour et al., 1991), in neocortical astrocytes (Unichenko et al., 2012) and for EAAT2 in cell culture (Bergles et al., 2002). In addition to the direct effect of Na1 on the driving force of EAATs, an increase of [Na1]i down-regulates glutamate transporter function by promoting the internalization of astrocytic EAATs from the surface membrane (Nakagawa et al., 2008). Furthermore, increased [Na1]i makes the transporter more sensitive to inhibition by internal glutamate (Barbour et al., 1991), probably as a consequence of Na1 unbinding after glutamate from the internal surface of the transporter (Bergles et al., 2002; Koch et al., 2007; Larsson et al., 2004; Otis and Jahr, 1998). Because inhibition of EAATs has been shown to play a vital role in controlling levels of activation of NMDA and nonNMDA receptors at active synapses (Tzingounis and Wadiche, 2007), the negative effects of [Na1]i on EAAT function would be expected to have wide-reaching consequences for synaptic transmission. This has been demonstrated at cortical synapses where activation of astrocytic GABA transporters increases [Na1]i, inhibits EAATs and subsequently influences the activity of adjacent neuronal circuits (Unichenko et al., 2012). Accumulated glial [Na1]i may also have important clinical consequences, for example during hyperammonemia (Kelly et al., 2009), as would be observed for in hepatic encephalopathy following liver failure. The subsequent inhibition of EAATs under these conditions may thus directly contribute to the elevated extracellular glutamate levels observed in this condition (Rose, 2002). 3

The substrate inhibition of EAATs by accumulated [Na ]i may be relieved by other astrocytic Na1-coupled transporters, as they can rapidly remove a proportion of the accrued [Na1]i and therefore increase the EAAT transport rate and capacity. For example, the Na1/K1-ATPase extrudes a significant amount of Na1 following EAAT activation, and if this process is blocked the [Na1]i increase is greatly enhanced (Chatton et al., 2000), inhibiting EAATs. The same protective mechanism can potentially occur for other Na1coupled transporters, if they are able to reverse under physiological conditions. This has been proposed for the ability of GABA transport (Unichenko et al., 2012) and sodiumbicarbonate exchange (Deitmer and Schneider, 2000) to reduce [Na1]i and help maintain the driving force for glial glutamate transport. Similarly, astrocytic SNAT3 glutamine transporters operate to extrude Na1 under physiological conditions, as discussed below, and will also reduce substrate inhibition of EAATs. It is thus clear that the dynamic interplay between a range of different transporter systems controls the [Na1]i signals in astrocytes following EAAT activation. 1

Glutamate Recycling and Glutamine Release Glutamate released from synapses must be recycled or otherwise replenished to maintain adequate levels of excitatory neurotransmission (Marx et al., 2015). As most of the released glutamate is transported into astrocytes (Danbolt, 2001; Rothstein et al., 1996), astrocytic metabolism plays a key role in this process (Kreft et al., 2012). Sequestered glutamate is mainly amidated to form glutamine by the enzyme glutamine synthetase, although during periods of high neuronal activity up to 50% of it may alternatively be deaminated to form a-ketoglutarate and enter the TCA cycle (McKenna, 2007). Astrocytic glutamine is subsequently transported out of astrocytes and into neurons, where it is potentially used as a precursor for glutamate, forming a glutamate-glutamine cycle (see reviews by Bak et al., 2006; Hertz et al., 1999; Marx et al., 2015). The astrocytic glutamine release mechanism is therefore a central process in the synapses’ ability to maintain a sustained level of neurotransmission. Astrocytes express a variety of amino acid transporters that can transport small uncharged amino acids such as glutamine. The most prevalent ones include the system L transporters LAT1 and LAT2; system y1L transporter y1LAT2; system A transporters SNAT1 and SNAT2; system ASC transporter ASCT2; and system N transporters SNAT3 and SNAT5 (Deitmer et al., 2003; Hamdani et al., 2012; Heckel et al., 2003; Nagaraja and Brookes, 1996; Su et al., 1997). Of these transporters, under physiological conditions SNAT1, SNAT2, ASCT2 and y1LAT2 catalyze the uptake of glutamine, whereas LAT2 and SNAT3 are also capable of mediating glutamine release (Deitmer et al., 2003). LAT2 is a 4

Na1-independent obligate exchanger, which can release glutamine in exchange for another neutral amino acid (Meier et al., 2002). In contrast, glutamine transport by SNAT3 (Slc38a3) is sodium-dependent, being coupled to the co-transport of one Na1 ion and the counter-transport of one H1 (Fig. 1B; Broer et al., 2002; Chaudhry et al., 1999, 2001). The H1 countertransport balances the charge of the cotransported Na1, rendering transport electroneutral and preventing the membrane potential from contributing to the overall driving force of the transporter. As a consequence of this, the equilibrium of glutamine across the astrocytic membrane is governed mainly by the transmembrane Na1 gradient, and a rise in [Na1]i can stimulate glutamine efflux. From a resting [Na1]i of 15– 20 mM (Rose and Ransom, 1996; Unichenko et al., 2012), an increase of a further 20 mM will increase the driving force for the efflux of glutamine by up to 2.3 fold. At equilibrium this would increase the external glutamine concentration from 0.5 mM (Gjessing et al., 1972) to 1.2 mM (using the equation from Broer et al., 2002). The activity of astrocytic glutamine synthetase helps maintain a cytoplasmic glutamine pool of 5–8 mM (Patel and Hunt, 1985; Schousboe et al., 1979; Storm-Mathisen et al., 1992). It is evident that the [Na1]i rise that occurs as a consequence of astrocytic glutamate influx has the potential to directly stimulate the release of glutamine from this pool via SNAT3 transport (Fig. 2). In support of this hypothesis, EAAT activation in perisynaptic astrocytes in brain slices causes the release of glutamine within a few milliseconds (Uwechue et al., 2012), as is also observed over a longer time-scale in cultured Bergmann glial cells (Martinez-Lozada et al., 2013) and cortical astrocytes (Broer et al., 2004). Daspartate, which is a substrate for EAATs but not glutamine synthetase, causes a [Na1]i rise in astrocytes but would not be expected to increase the astrocytic glutamine supply (Bennay et al., 2008; Langer and Rose, 2009; Unichenko et al., 2012; Uwechue et al., 2012). The ability of D-aspartate to also stimulate the astrocytic release of glutamine further suggests that it is the [Na1]i rise that causes the immediate glutamine release rather than a glutamate metabolite (MartinezLozada et al., 2013; Uwechue et al., 2012). The close physical association observed between EAAT1 and SNAT3 in Bergmann glia (Martinez-Lozada et al., 2013) suggest that upon EAAT stimulation, a rapid local rise of [Na1]i close to the plasma membrane would have an immediate influence on SNAT3 and stimulate glutamine release. Because glutamate influx by EAATs is coupled to the influx of three Na1 ions (Zerangue and Kavanaugh, 1996) whereas glutamine efflux via SNAT3 is coupled to the efflux of only one Na1 (Broer et al., 2002; Chaudhry et al., 1999), there is potential for 3:1 amplification in the coupling of glutamate uptake to glutamine release. Volume 00, No. 00

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FIGURE 2: Localization of astrocytic glutamate (EAATs), glutamine (SNAT3) and GABA (GAT) transporters and regulation of their function by intracellular [Na1] changes.

Glutamine released from astrocytes can subsequently be transported directly into presynaptic terminals to support further glutamatergic neurotransmission (Billups et al., 2013). As astrocytic sodium plays a key role in controlling this glutamine release, dynamic Na1 signals in astrocytes could be central in controlling levels of excitatory synaptic transmission via this mechanism. GABA Transporters In contrast to glutamate which is predominantly taken up by astrocytes, GABA, the major inhibitory neurotransmitter is removed from the synaptic cleft mainly by the neuronal GABA transporter (GAT) subtype GAT1. Because of the high capacity and abundance of synaptic GAT1, GABA rarely escapes the synapse and consequently the role of nonneuronal GABA transport has long been overlooked (Zhou and Danbolt, 2013). GATs belong to the Slc6 family of Na1- and Cl2dependent neurotransmitter transporter proteins that also includes glycine transporters (Kristensen et al., 2011). To date, four distinct GAT subtypes have been cloned: GAT1 (Slc6a1), BGT-1 (Slc6a12), GAT-2 (Slc6a13), and GAT-3 (Slc6a11). Because the betaine-GABA transporter BGT-1 is found in leptomeninges and cerebral blood vessels (Borden, 1996; Conti et al., 2004; Zhou et al., 2012) and the astrocytic GAT-2 shows a very low expression level (Borden, 1996), it is the GAT-3 subtype that is responsible for the majority of astroglial GABA transport. Month 2015

The coupling of both EAATs (Zerangue and Kavanaugh, 1996) and GATs (Iversen and Neal, 1968) to Na1 transport (Fig. 1) has an important consequence: the two proteins can be functionally connected through the intracellular Na1 concentration if they shared a common pool in the intracellular space in which [Na1]i changes can affect the function of both proteins. Ultrastructural anatomical studies supported this possibility by demonstrating that GAT-3 is expressed on astrocytic processes ensheathing glutamatergic synapses (Minelli et al., 1996). These astrocytic processes have also been shown to express high level of EAAT1 and EAAT2 (Proper et al., 2002). The co-localization of GATs and EAATs (Heja et al., 2012) on the same astrocytes, therefore, makes the Na1-mediated functional linkage between glutamate and GABA transporters a viable possibility. In principle, the linkage between astrocytic EAATs and GATs through [Na1]i might manifest in a wide range of functional interactions and potential outcome on the neurotransmission in the surrounding cells. If the activity of EAATs or GATs only moderately affected the [Na1]i in the local microdomain of the other transporter (due to either long distance between the two transporter types or low influx rate of the activated transporter) the activity of one transporter may only reduce the transport rate of the other (Kirischuk et al., 1997). If more subtle [Na1]i changes occur in the vicinity of an EAAT or GAT, its transport direction may be reversed and the intracellular glutamate or GABA along with the coupled Na1 can be released to the extracellular space. This activityinduced transporter reversal helps restoring the transmembrane Na1 gradient without activating the Na1/K1-ATPase and using ATP, thereby saving energy and reducing the metabolic stress on astrocytes. Moreover, extensive neurotransmitter influx through one of the transporters might lead to a high level of transporter mediated neurotransmitter release through the other transporter, which significantly contributes to the activation of neurotransmitter receptors, and thus modulates neurotransmission. To explore whether these scenarios take place in practice under physiological (or pathophysiological) conditions, the driving forces of the transporters need to be investigated in detail. Theoretical calculations can provide a good estimate of the potential of transporter reversal (Richerson and Wu, 2003). GABA transport is electrogenic due to the cotransported Na1 and Cl2 ions (Fig. 1C), therefore the reversal potential of the transporter (i.e., where they are at equilibrium and no net transport occurs) can be calculated according to the Nernst Eq. (1). Vr;GAT 52

!  2 RT ½GABAi ½Na1 i ½Cl 2 i ln   : ð2ZNa1 1ZCl 2 ÞF ½Cl 2 o ½GABAo ½Na1 o

(1)

5

where Vr is the reversal potential, R is the universal gas constant, T is the temperature, ZX is the valence of X, F is Faraday’s constant, [X]i and [X]o are the intracellular and extracellular concentrations of X. Note that the main difference between the equations for GATs and EAATs is that the ratio of intracellular and extracellular Na1 concentrations is raised to 2nd (GAT) or 3rd (EAAT) power according to the number of co-transported Na1 ions. This difference ultimately leads to much higher reversal potential for EAATs and, importantly, makes [Na1]i the most influential factor in determining the driving force for transport. Assessing the intracellular and extracellular concentrations of GABA to be 2 mM (Lee et al., 2011) and 200 nM (Hagberg et al., 1985), respectively and calculating with astrocytic [Na1]i of 15 mM (Rose and Ransom, 1996; Unichenko et al., 2012) and [Cl-]i of 15 mM (Kettenmann et al., 1987), the reversal potential of GATs is estimated to be around 264 mV, very close to the resting membrane potential. Contrarily, EAATs reverse at positive membrane potentials at a wide range of intracellular and extracellular glutamate concentrations. Further elevation of [Na1]i by 10–20 mM (Bennay et al., 2008; Kirischuk et al., 2007; Langer and Rose, 2009) will easily reverse GATs while still maintaining the inward uptake direction of EAATs. Noteworthy, the reversal potential of the transporters is very sensitive to the value of the parameters involved in the Nernst equation which are very difficult or impossible to measure experimentally, especially considering that local concentrations in the vicinity of the proteins can be dynamically changing. This consideration is highlighted by a recent study (Savtchenko et al., 2015) showing that neuronal GAT1 does not reverse due to synaptic GABA release despite GAT1 on cultured neurons have been shown to be reversible (Wu et al., 2007). This observation also emphasizes that astrocytic GATs have a unique potential to be fine-tuned by intracellular Na1 dynamics, since they are exposed to more stable extracellular GABA level. Because the glial GAT-3 transporters are facing the extrasynaptic space instead of the synapse (Kinney and Spain, 2002) and the neuronal GAT-1 is synaptically localized, GABA released through the astrocytic GATs can spread through the extracellular space and activate different extrasynaptic GABA receptors without being taken up by adjacent neurons. The subsequent activation of extrasynaptically localized presynaptic GABAB receptors is expected to attenuate GABAergic signaling (Unichenko et al., 2015). Another important consequence of the GAT-mediated glial GABA release is the activation of extrasynaptic GABAA receptors that directly generate tonic inhibition on neurons (Kilb et al., 2013; Song et al., 2013; Unichenko et al., 2015). This pathway appears to be especially important, since the amount of 6

astrocytic GABA strictly correlates with the degree of tonic inhibition in various brain regions (Yoon et al., 2011). In the recent years, various lines of evidence have emerged confirming that the Na1-mediated linkage between astrocytic EAATs and GATs is capable to generate a critical efflux of GABA that can activate extrasynaptic GABAA receptors and exert significant control over neuronal excitability. It has been demonstrated (Heja et al., 2009, 2012) that during periods of intense excitation, astrocytic uptake of synaptically released glutamate triggers the reversal of GAT-3, constituting the glutamate/GABA exchange mechanism (Fig. 2). The released glial GABA generates tonic inhibition on the neighboring overexcited neurons (Heja et al., 2012). Moreover, the negative feedback provided by astrocytes is proportional to the network activity. In the low-[Mg21] epilepsy model, the glutamate/GABA exchange mechanism was demonstrated to significantly reduce the duration of seizure-like events (Heja et al., 2012). Additionally, it was shown that GAT-3 upregulation in sclerotic tissue is limited to areas with mostly preserved neuronal mass (Lee et al., 2006), further supporting the neuroprotective role of the glutamate/GABA exchange. In addition, expression of voltage-dependent Na1 channels and inward Na1 currents are also increased in astrocytes from temporal lobe epilepsy patients (Bordey and Spencer, 2004), fueling the compensatory GABA release and adding a new layer to the role of astrocytic Na1 homeostasis in CNS diseases. Importantly, astrocytes do not synthesize GABA from glutamate, therefore the released GABA is formed by an alternative pathway from putrescine (Heja et al., 2012), the level of which is markedly increased in epilepsy (Laschet et al., 1992; Najm et al., 1992). In addition, putrescine production can also be induced by glutamine (Baudry et al., 1988) and astrocytic GABA synthesis can be directly triggered by glutamate uptake (Jow et al., 2004), showing that several astrocytic mechanisms are available that maintain the intracellular GABA level in an activity-dependent manner. It was also demonstrated that epilepsy is not the only CNS disease in which the Na1-mediated astrocytic glutamate/GABA exchange may contribute to the pathophysiological mechanism. Two different mouse models of Huntington’s disease have been observed to exhibit strongly reduced GAT3-mediated GABAergic tonic inhibition which could be restored by activating the astrocytic EAATs with their substrate D-aspartate (Wojtowicz et al., 2013). However, increased tonic inhibitory drive through the reverse action of GAT-3 has been suggested to be a major barrier against the functional recovery after stroke (Clarkson et al., 2010), highlighting the fact that increased tonic inhibition is not always beneficial. Besides pathophysiological conditions, the glutamate/GABA exchange mechanism has also been demonstrated to play important physiological roles. The source of the Volume 00, No. 00

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paracrine GABA in the developing neocortex that constitutes the STOP signal during neuron migration has been suggested to be the GAT-3 mediated GABA release (Unichenko et al., 2013). Importantly, the astrocytic exchange of glutamate and GABA is strictly directed in one way and not the other. Because of the lower expression level of astrocytic GATs, the [Na1]i increase corresponding to GAT influx activity is much more limited than following EAAT activation (Chatton et al., 2003). Also, the coupling of EAATs to transport of 3 Na1 ions instead of the 1:2 stoichiometry of GABA: Na1 (Fig. 1C) results in significantly higher reversal potential of EAATs which is far from the physiologically available astrocytic membrane potential. Therefore, despite the GAT activitytriggered modulation of EAAT function (Unichenko et al., 2012), the shared astrocytic Na1 pool of EAATs and GATs allows only generation of inhibitory tonic current during overexcitation and does not allow conversion of inhibitory mechanisms to potentially harmful excitatory actions. Glycine Transporters Glycine is one of the most important inhibitory neurotransmitters in the posterior regions of the CNS, including the spinal cord and brain stem. Synaptically released glycine activates postsynaptic glycine receptors (GlyRs), inducing phasic postsynaptic potentials. As GlyRs are ligand-gated anion channels, their activation results in the generation of inhibitory postsynaptic potentials. In addition to its inhibitory action via ionotropic GlyRs, glycine can modulate glutamatergic neurotransmission (Johnson and Ascher, 1987). Here glycine serves as an essential co-agonist of glutamate at NMDA receptors. These receptors are usually formed from two GluN1 subunits and two GluN2 subunits, with glycine binding to GluN1 subunits and glutamate binding to the corresponding site on GluN2 subunits (Nakanishi, 1992). Glycine binding to the glycine binding site of the NMDA receptors: (1) facilitates glutamate-activated currents via NMDA receptors and (2) primes NMDA receptors to be exocytosed (Nong et al., 2003). Moreover, glycine itself can activate a purely excitatory conductance, namely an NMDA receptor consisting of only GluN1 and GluN3 subunits (Chatterton et al., 2002). This short overview of multiple glycine functions demonstrates that a precise control of glycine concentration both in the synaptic cleft and in the extrasynaptic space in general is of pivotal important for the CNS. Extracellular glycine concentration is regulated by glycine transporters. Two genes encoding glycine transporters, GlyT1 (Slc6a9) and GlyT2 (Slc6a5), have been cloned. Both GlyTs belong to the large family of Na1- and Cl2-dependent neurotransmitter transporter proteins that also includes GABA transporters (for review Kristensen et al., 2011). Month 2015

Interestingly, GlyT1 has a stoichiometry of 2 Na1:1 Cl2:1 glycine molecule, whereas GlyT2 co-transports one glycine molecule with three Na1 and one Cl2 (Fig. 1D; Roux and Supplisson, 2000). This stoichiometry allows GlyT2 to maintain a relatively high (20-40 mM) concentration of glycine in the cytosol, pointing out that GlyT2 may be responsible for glycine reuptake into the presynaptic terminal. Indeed, GlyT2 was originally shown to be exclusively localized in the presynaptic terminals of glycinergic neurons (Zafra et al., 1995), and electrophysiological data confirm the crucial role of this transporter in maintaining the phasic glycinergic transmission. Genetic ablation of GlyT2 reduces the amplitudes of miniature glycinergic postsynaptic currents without inducing any anatomical or biochemical abnormality, indicating that the neurotransmitter filling of presynaptic vesicles is compromised in the GlyT2-deficient mice. As a consequence GlyT2deficient mice reproduce the symptoms of the hereditary human motor diseases (Gomeza et al., 2003b). Later GlyT2 expression in astrocytes was also reported (Aroeira et al., 2014). GlyT1 was originally shown to be expressed in glial cells, in particular astrocytes, around both glycinergic and non-glycinergic synapses (Zafra et al., 1995). Later GlyT1 expression was also reported in glutamatergic neurons, including presynaptic terminals and dendrites. In addition, GlyT1 was reported to be physically associated with NMDA receptors, supporting the idea that GlyT1 can regulate NMDA receptor-mediated synaptic transmission (Cubelos et al., 2005). Electrophysiological data seem to confirm this hypothesis, because pharmacological blockade of GlyT1 potentiates NMDA receptor-mediated responses (Chen et al., 2003). In contrast, genetic elimination of GlyT1 selectively in neurons do not result in any detectable motor impairment, indicating that at least in caudal regions of the CNS neuronal GlyT1 makes a minor contribution to the regulation of glycinergic neurotransmission (Eulenburg et al., 2010). Newborn GlyT1-deficient mice demonstrate severe motor and respiratory deficits and dye during the first postnatal day (Gomeza et al., 2003a). Electrophysiological data show that GlyT1 ablation leads to prolongation of spontaneous glycinergic postsynaptic potential, indicating that GlyT1 contributes to the clearance of synaptically released glycine from the synaptic cleft. Moreover, lack of GlyT1-mediated glycine uptake leads to an increased accumulation of glycine in the extracellular space and persistent activation of extrasynaptic GlyRs, inducing tonic GlyR-mediated conductance and in turn sustained inhibition (Gomeza et al., 2003a). Mice, in which GlyT1 was selectively ablated only in glial cells, reproduce the symptoms of conventional GlyT1knockout mice, suggesting that glial GlyT1 strongly contributes to both termination of phasic glycinergic 7

neurotransmission and regulation of extrasynaptic glycine concentration (Eulenburg et al., 2010). As mentioned above GlyT1 has a stoichiometry of 2 1 Na :1 Cl-:1 glycine molecule, i.e. GlyT1 is an electrogenic transporter. Considering an astrocytic intracellular glycine concentration of 2 mM (Berger et al., 1977) and an extracellular one of 0.1 mM, the reversal potential of GlyT1 in astrocytes appears to be relatively close to the resting membrane potential (Supplisson and Roux, 2002).These modeling results reveal that GlyT1 may operate not only in uptake mode, but also in release mode. A cell depolarization and/or an elevation of astrocytic [Na1]i may reverse transport direction, resulting in a non-vesicular, GlyT1-mediated release of glycine from the cytosol into the extracellular space. Indeed, K1 depolarization- or veratridine-induced release of glycine from brain synaptic plasma membrane vesicles was reported to be Ca21independent (Aragon et al., 1988; for review see Adam-Vizi, 1992). An elevation of intracellular [Na1]i with veratridine, which directly opens voltage-gated Na1 channels, appears to stimulate transporter-mediated release much stronger than the high K1 depolarization, indicating the potency of [Na1]i levels not only for transporter-mediated uptake but for release as well (for review see Bernath, 1992). The reversal potential of GlyT1 depends on extracellular glycine concentration (Attwell et al., 1993). Therefore, at glycinergic synapses high glycine concentration during phasic transmission makes the GlyT1 reversal potential more positive, favoring thus glycine clearance. GlyT1-mediated glycine uptake not only reduces extracellular glycine concentration but also elevates astrocytic [Na1]i, making the reversal potential of GlyT1 more negative and favoring thus GlyT1mediated release. Thus, shortly after glycine exocytosis GlyT1 helps to decrease glycine concentration in the synaptic cleft to terminate the activation of low affinity GlyRs. But later GlyT1 might release glycine, providing it for the GlyT2mediated presynaptic uptake and refilling presynaptic glycine pool (Fig. 3A). At glutamatergic synapses GlyT1 obviously operates in uptake mode under resting conditions. Neuronal glutamate release activates the Na1-permeable glutamate receptors not only on the postsynaptic neuron but also on perisynaptic astrocytes, making the reversal potential of GlyT1 more negative, favoring glycine release and activation of neuronal NMDA receptors (Fig. 3B). Although the physiological role of astrocytic GlyT2 is still elusive, a recent study demonstrated that both GlyT1 and GlyT2 in astrocytes are modulated through BDNF/TrkB pathway (Aroeira et al., 2015). D-Serine

Release and Uptake another important gliotransmitter, also can bind to the glycine-binding site of the NMDA receptor, modulating

D-serine,

8

FIGURE 3: Role of GlyTs at glycinergic and glutamatergic synapses. (A) GlyTs at a glycinergic synapse. When glycine concentration in the synaptic cleft ([Gly]e) is high (left), both GlyT1 and GlyT2 take up glycine. When [Gly]e is low and astrocytic [Na1]i is elevated (right), GlyT1 releases glycine. (B) GlyTs at a glutamatergic synapse. During neurotransmission astrocytic [Na1]i is low, GlyT1 removes glycine (left). Activation of astrocytic glutamate receptors/transporters elevates astrocytic [Na1]i. GlyT1 reverses and releases glycine, a coagonist of NMDA receptors (right).

the glutamatergic neurotransmission. Initially serine racemase, the enzyme which converts L-serine to D-serine, was reported to be expressed only in glial cells. Stimulation of cultured astrocytes with glutamate results in a Ca21- and SNARE complex-dependent D-serine release (Henneberger et al., 2010; Mothet et al., 2005). More recently, however, serine racemase was also indentified in neurons (Kartvelishvily et al., 2006; Yoshikawa et al., 2007). Interestingly, neuronal release of D-serine seems to be Ca21 independent and occurs via the Na1-independent alanine-serine-cysteine transporter (Asc1; Slc7a10; Rosenberg et al., 2010, 2013). A Ca21-independent release of D-serine from astrocytes has been reported (Rosenberg et al., 2010). It was suggested that this release is mediated by volume-regulated anion channels. However, astrocytes also express a system ASC-like Na1dependent neutral amino acid transporter (ASCT2; Slc1a5). Although ASCT2 is structurally related to EAATs, the ASCT2-mediated transport is not electrogenic. The most probable stoichiometry is one neutral amino acid is cotransported with one Na1 and counter-transported with one K1 (Utsunomiya-Tate et al., 1996). Although astrocytic ASCT2mediated D-serine release has been shown in cultures (Ribeiro et al., 2002), the above stoichiometry suggests that ASCT2 operates in uptake mode and does not reverse under Volume 00, No. 00

Kirischuk et al.: Neurotransmission Regulation by Astrocytic [Na1]

physiological conditions. Nevertheless, an elevation of astrocytic [Na1]i will reduce the driving force of ASCT2-mediated D-serine transport, slowing down its uptake and facilitate Dserine-mediated modulation of NMDA receptors. One can also suggest that the neuronal Na1-independent Asc1mediated D-serine release and astrocytic Na1-dependent ASCT2-mediated D-serine uptake regulate steady-state perisynaptic D-serine concentration, while the astrocytic vesicular Dserine release converts neuronal/astrocytic activity into the extracellular D-serine dynamics. Adenosine Release ATP, a well-established gliotransmitter, and its metabolites, including adenosine, are important signaling molecules in the CNS. Although it is generally believed that astrocytes first release adenine nucleotides, which are extracellularly metabolized to adenosine (Parkinson et al., 2002), direct astrocytic adenosine release has been reported (Martin et al., 2007). The release mechanism is not yet characterized. It was shown, however, that astrocytes express both equilibrative and concentrative nucleotide transporters (ENTs and CNTs, respectively). ENTs (Slc29 family) transport adenosine via facilitated diffusion, whereas CNTs are Na1-dependent symporters (King et al., 2006). CNT2 (Slc28a2) and CNT3 (Slc28a3) expression in astrocytes has been reported (Li et al., 2013). Although direct evidence is not available, one could suggest that astrocytic [Na1]i may affect CNT-mediated adenosine uptake and influence the extracellular adenosine level/ dynamics.

Conclusions Astrocytes possess a variety of molecular mechanisms which allow fast and local changes of [Na1]i, including neurotransmitter receptors and NeuTs. Most NeuTs utilize transmembrane Na1 gradient for regulation of neurotransmitter levels in the extracellular space. We conclude that local [Na1]i transients occurring especially in tiny perisynaptic astrocytic processes can strongly influence the clearance of synaptically released neurotransmitters and/or initiate a release of gliotransmitters. Via this mechanism, these astrocytic Na1 signals will have a profound impact on neuronal communication throughout the central nervous system. In addition, under pathological conditions changes in [Na1]i will strongly affect the activity of Na1-dependent transmitter uptake in astrocytes, although the final impact of astrocytic [Na1]i rise is difficult to predict. Because some NeuTs (GATs and GlyT1) operate close to their reversal potentials under resting conditions, even slight hyperactivity in the CNS can lead to release of GABA and glycine, in turn decreasing neuronal activity. In this regard, astroglial EAAToperated Na1 symport, subsequent GABA release and Month 2015

increased tonic inhibition have recently been placed in the perspective of antiepileptic drug (AED) design and other AED-responsive CNS diseases, such as anxiety, bipolar depression, neuropathic pain, migraine prophylaxis, tremor, tardive dyskinesia and obesity (Kardos et al., 2015). In contract, reversal potentials of some other Na1-dependent mechanisms (e.g., the Na1/Ca21-exchanger or Na1-HCO2 3 exchanger) are also close to the resting potential of astrocytes, indicating that astrocytic [Na1]i increase will affect intracellular milieu via [Ca21]i and pH changes. In the extreme case, strong astrocytic [Na1]i elevation, for example as a result of epileptic activity, can even reverse EAATs, leading to EAATmediated glutamate release. Thus, fine tuning of astrocytic [Na1]i has to be taken into account when devising an effective medication strategy.

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