Importance of astrocytes for potassium ion (K+) homeostasis in brain and glial effects of K+ and its transporters on learning

Accepted Manuscript Title: Importance of Astrocytes for Potassium Ion (K+ ) homeostasis in Brain and glial Effects of K+ and its Transporters on Learning Author: Leif Hertz Ye Chen Dr. PII: DOI: Reference:

S0149-7634(16)30252-4 http://dx.doi.org/doi:10.1016/j.neubiorev.2016.09.018 NBR 2604

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26-4-2016 12-8-2016 23-9-2016

Please cite this article as: Hertz, Leif, Chen, Ye, Importance of Astrocytes for Potassium Ion (K+) homeostasis in Brain and glial Effects of K+ and its Transporters on Learning.Neuroscience and Biobehavioral Reviews http://dx.doi.org/10.1016/j.neubiorev.2016.09.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Importance of Astrocytes for Potassium Ion (K+) homeostasis in Brain and glial Effects of K+ and its Transporters on Learning

Leif Hertz1 and Ye Chen2*

1

Laboratory of Metabolic Brain Diseases, Institute of Metabolic Disease Research and Drug

Development, China Medical University, Shenyang, P. R. China, 2

Henry M. Jackson Foundation, Bethesda, MD 20817, USA

Running title: K+ homeostasis affects learning

*Please send correspondence to Dr. Ye Chen, Henry M. Jackson Foundation, Bethesda, MD 20817, USA. Email: [email protected]

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Highlights     

Increased extracellular K+ is initially cleared by astrocytic Na+,K+-ATPase activity Subsequent dispersion and Kir4.1 mediated release of K+ allow neuronal accumulation The astrocytic NKCC1 creates the post-stimulatory K+ undershoot enhancing excitation Transmitters regulate K+ channels and gap junctions with consequences for learning Abnormal extracellular K+ concentrations or astrocytic Na+,K+-ATPase impair memory

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Abstract Initial clearance of extracellular K+ ([K+]o) following neuronal excitation occurs by astrocytic uptake, because elevated [K+]o activates astrocytic but not neuronal Na+,K+-ATPases. Subsequently, astrocytic K+ is re-released via Kir4.1 channels after distribution in the astrocytic functional syncytium via gap junctions. The dispersal ensures widespread release, preventing renewed [K+]o increase and allowing neuronal Na+,K+-ATPase-mediated re-uptake. Na+,K+ATPase operation creates extracellular hypertonicity and cell shrinkage which is reversed by the astrocytic cotransporter NKCC1. Inhibition of Kir channels by activation of specific PKC isotypes may decrease syncytial distribution and enable physiologically occurring [K+]o increases to open L-channels for Ca2+, activating [K+]o-stimulated gliotransmitter release and regulating gap junctions. Learning is impaired when [K+]o is decreased to levels mainly affecting astrocytic membrane potential or Na+,K+-ATPase or by abnormalities in its 2 subunit. It is enhanced by NKCC1-mediated ion and water uptake during the undershoot, reversing neuronal inactivity, but impaired in migraine with aura in which [K+]o is highly increased. Vasopressin augments NKCC1 effects and facilitates learning. Enhanced myelination, facilitated by astrocyticoligodendrocytic gap junctions also promotes learning. Keywords: astrocytic syncytium; connexins; Kir4.1 inhibition by PKC; L-channels for Ca2+; learning; memory; migraine; Na+,K+-ATPase; Na+,K+-ATPase-mediated exit of 3 Na+ versus entry of 2 K+ creates extracellular hypertonicity and cell shrinkage; NKCC1, a cotransporter of Na+, K+, 2 Cl- and water; pannexin; post-stimulatory undershoot; potassium; regulatory volume increase; traumatic brain injury; vasopressin,

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1. Introduction Effects of K+ on cognitive processes such as prey catching in frogs were discussed in an excellent review in this journal (Laming et al., 2000), but it stopped short of discussing learning as such. However, during the last 16 years much progress has been made in the understanding of the role of astrocytes in cellular re-accumulation of K+ after its neuronal release during brain activation, making such a discussion a possibility to-day. To this end we will i) describe the magnitude of the increases in extracellular K + concentration ([K+]o) evoked by neuronal excitation and conclude that they normally will selectively activate the Na+,K+-ATPase; ii) describe the differences between the astrocytic and neuronal Na+,K+-ATPases that cause initial activation of the astrocytic but not the neuronal Na+,K+-ATPase by elevated [K+]o; iii) discuss stimulation of astrocytic and neuronal Na+,K+-ATPases by different noradrenergic subtypes and its dependence on [K+]o; iv) examine whether the ion and water transporter NKCC1 under special, but perhaps common, conditions also might be activated by normally occurring increases in [K+]o, which would have important functional consequences; v) debate the mechanism triggering NKCC1 stimulation during the undershoot, including stimulation of the Na+,K+-ATPase needed to create the ion gradients driving NKCC1; vi) document the high, mainly Kir4.1-mediated K+ conductance of astrocytic cell membranes, allowing depolarization by even very small increases in [K+]o, its role in return of astrocytically accumulated K+ to the extracellular space and its regulation by protein kinase C (PKC); vii) describe the role of connexins and pannexins in moving K+ through an astrocytic network and their regulation by high [K+]o; and viii) provide an integrated description of the processes involved in K+ homeostasis at the cellular level of the mammalian brain

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following neuronal K+ release, i.e., Na+-K+-ATPase, NKCC1, Kir4.1 and -adrenergic activities as well as K+ transport via connexins and pannexins. Following this, the paper will describe i) potential involvement of astrocytes in effects of stimulation-induced increase in K+ in the synapse; ii) adverse effects on learning by deviations of [K+]o or iii) by abnormalities in the astrocytic 2 subunit of the Na+,K+-ATPase; iv) relief of poststimulatory neuronal hyperpolarization and inactivity by correction of extracellular hyperosmolarity by -adrenergic stimulation of the Na+,K+-ATPase driving NKCC1; and v) potential NKCC1 stimulation by normal elevations of [K+]o, due to a decreased threshold for NKCC1 or vi) concomitant vasopressinergic activity and its functional consequences.

2. K+ homeostasis in mammalian brain 2.1. Brain activity increases [K+]o An increase in [K+]o is a hallmark of neuronal excitation. During action potential propagation the depolarizing entry of Na+ is followed by repolarizing K+ exit (Hodgkin and Huxley, 1952; Traub et al., 1994). Stimulation of ionotropic glutamate receptors is well known to cause intracellular increases of Na+ in neurons (Teichberg et al., 1981; Rose and Konnerth, 2001; Yu, 2006; Bennay et al., 2008). At the same time it raises [K+]o (Prince et al., 1973; Rice and Nicholson, 1990), and activation of postsynaptic NMDA receptors is the major source of synaptic K+ efflux (Shih et al., 2013). The increase at different subcellular levels is not known, but in their most recent ‘budget’ for energy utilization in gray matter (which to a large degree reflects active transport) the Attwell group estimates that glutamatergic stimulation causes twice as much energy use 5

as action potentials (Howarth et al., 2012). However, some of the released cellular K+ following glutamatergic stimulation enters the synaptic cleft (Shih et al. 2013), which may reduce the difference between the increase in [K+]o caused by glutamatergic stimulation and action potential propagation. Based on determination of volume and energy metabolism of different neural components it has been estimated that dendrites and astrocytes have the highest rates of energy metabolism (discussed and refereed in Hertz, 2011), suggesting that most K+ is released from dendrites (probably following both glutamatergic stimulation and action potential propagation). Dendritic spines are covered by astrocytic processes and synaptic glutamate increases the astrocytic coverage (Genoud et al., 2006). Pronounced and complex astrocytic coverage of Bergmann glia has also been firmly established (Grossche et al., 2002). Increases in [K+]o also occur after GABA-ergic stimulation of CA1 hippocampal neurons due to release of K+ by KCC2, a K+, Cl− cotransporter (Viitanen et al., 2010). Finally, K+ can also originate from astrocytes since uptake of glutamate, which almost exclusively takes place in astrocytes (Danbolt 2001; Zhou and Danbolt, 2013; Danbolt et al., 2016) occurs together with Na+ uptake (providing its driving force) and with the release of one K+ (Rauen et al., 1992; Levy et al., 1998). Quantitatively more important, K+ initially accumulated in astrocytes is returned to the extracellular space as will be discussed in 2.2 and 2.5. During normal brain activity [K+]o in mammalian brain increases relatively little from its resting level of ~3.0 mM. Thus, visual stimulation causes an increase of 0.5 mM in occipital cortex and the largest increase in response to physiological activity amounts to 3 mM after prolonged cutaneous stimulation (Lothman et al., 1975; Heinemann and

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Lux 1977; Somjen 2002). However, these values migh be considerably higher in the very narrow space between neurons and the covering glia cells. Below it will be discussed that low increases of [K+]o stimulate the astrocytic Na+,K+-ATPase, but not the neuronal Na+,K+-ATPase or NKCC1. Much larger increases up to a ceiling level of ~10-12 mM are evoked by seizures, and during ischemia and spreading depression [K+]o can reach levels of 80 mM (Lothman et al., 1975; Heinemann and Lux, 1977; Somjen 2002). Spreading depression is a peculiar phenomenon where short-lasting waves of electrophysiological hyperactivity followed by minute-long waves of inhibition traverse the cortical surface. It can be elicited by strong electrical stimulation or administration of large amounts of K+ or glutamate (Leao, 1944; Bures et al., 1974), but it also occurs spontaneously in certain diseases (see section 3.3). A smaller and relatively short-lasting increase in [K+]o is seen after traumatic brain injury (Filippidis et al., 2014).

2.2. Initial cellular reuptake of elevated [K+]o occurs mainly into astrocytes and in mammalian brain it is mostly mediated by Na+,K -ATPase activity A role of glial cells in redistribution of the increase in [K+]o following local neuronal activity was first shown in leech ganglia and necturus nerve ~50 years ago (Kuffler and Potter, 1964; Orkand et al., 1966; Kuffler et al., 1966). The K+ redistribution in these non-mammalian nervous tissues is current-carried and occurs through electrically coupled glial cells with high membrane permeability for K+. It re-distributes excess K+ to larger areas of the extracellular space in which [K+]o was initially not increased, and K+ can be re-accumulated into neurons from these locations which show little or no increase in [K+]o. 7

It was long believed that a similar mechanism redistributes neuronally released K+ in mammalian brain, although an active uptake of K+ in glia cells was suggested (Hertz, 1965) and later demonstrated in mouse astrocytes in primary cultures (Hertz, 1979). From 2000 and onwards it has been firmly established that cellular re-uptake of K+ in mammalian brain occurs predominantly by active transport (Xiong and Stringer, 2000; D’Ambrosio et al., 2002; Larsen et al., 2014), at the moderately elevated [K+]o described above perhaps normally exclusively mediated by the Na+,K+-ATPase. The importance of active K+ uptake in astrocytes in mammalian brain tissue and optic nerve has also been firmly established (Ransom et al., 2000; Somjen et al., 2008; Dufour et al., 2011; Bay and Butt, 2012; Macaulay and Zeuthen, 2012; Larsen et al., 2014, 2016a; Larsen and MacAulay, 2014). The cellular increase in K+ creates a problem since astrocytically accumulated K+ must be returned to neurons. The solution to this problem is that the astrocytic increase in K+ content is transient (Walz, 2000; Dufour et al. 2011). This is because astrocytically accumulated K+ is returned to the extracellular space via Kir.4.1 channels (Bay and Butt, 2012), which are specifically inhibited by the low concentrations of Ba2+ (Walz et al., 1984a) used in the study by Bay and Butt. The release is probably preceded by K+ redistribution and dilution through astrocytic connexins and/or pannexins. It will therefore not cause any significant increase in [K+]o, which will further neuronal re-uptake, as will be discussed later. Together these processes accomplish the same as the re-distribution through glial cells in non-mammalian brain, but the removal from the extracellular space is mainly by active transport. Operation of the Na+,K+-ATPase requires simultaneous stimulation of an extracellular K+-stimulated site and an intracellular Na+-stimulated site (Skou 1957,

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2004). Its operation mediates efflux of 3 Na+ and influx of 2 K+ (Thomas, 1972; Clarke et al., 1989). The Na+,K+-ATPase is expressed both in neurons and astrocytes, but it is stimulated by elevated [K+]o only in astrocytes (Henn et al. 1972; Grisar et al., 1980; Hajek et al., 1996). This is because the subunit composition is different in neurons and astrocytes. Among the catalytic  subunits both cell types express 1, astrocytes express 2 and neurons 3 (McGrail et al., 1991; Cameron et al., 1994; Li et al., 2013) as illustrated in Fig. 1. The non-catalytic  subunits, which control  subunit function and membrane expression (Geering, 2001), are also differentially distributed (Fig. 1), and the combination of  and  subunits regulates the affinities of the extracellular K+-stimulated site and the intracellular Na+-stimulated site (Crambert et al., 2000; Larsen et al., 2014, 2016a). The 2/2 subunit expression, which is specific for astrocytes, has the lowest K+ affinity, enabling stimulation of specifically the astrocytic Na+,K+-ATPase by elevated [K+]o. This has been shown both for mechanically isolated astrocytes (Henn et al, 1972; Grisar et al., 1980) and mouse astrocyte cultures in which the Km value for K+ is 1.9 mM in astrocytes versus 0.43 mM in neurons, and Vmax in astrocytes is two times higher than in neurons (Hajek et al., 1996). Also in skeletal muscle the 2 subunit of the Na+,K+ATPase is stimulated by elevated [K+]o (DiFranco et al., 2015). In addition to subunit composition FXYD proteins regulate ion affinity. FXYD7 is selectively expressed in brain (Beguin et al., 2002), where it is present in both neurons and astrocytes (Beguin et al., 2002: Li et al., 2013). It decreases the affinity of the 1/1 isozyme by one half and also that of the astrocytic 2/1, but not that of the neuronal 3/1 isozyme (Beguin et al., 2002).

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The stimulation of the astrocytic but not the neuronal Na+,K+-ATPase by elevated [K+]o after neuronal excitation is the main reason for the K+ uptake in astrocytes in intact brain tissue. The astrocytic uptake is transient (Dufour et al., 2011), and the recovery of [K+]o in optic nerve has two phases, a brief, initial phase during which [K+]o falls rapidly, and a second, more prolonged phase during which [K+]o falls more slowly (Ransom et al., 2000). These differences can be explained by the lower affinity but higher Vmax value of the astrocytic than of the neuronal Na+,K+-ATPase described above (Hajek et al., 1996). In cultured astrocytes a small increase in [K+]o also increases K+ uptake (Hertz, 1979) and transiently also K+ content (Xu et al., 2013; Hertz et al., 2015a,c, 2016a) (Fig. 2A). Since astrocytes are non-excitable cells the intracellular Na+ concentration does not increase during neuronal stimulation as such, and intracellular Na+ in astrocytes is not increased at elevated [K+]o (White et al. 1992). As previously mentioned stimulation of the intracellular Na+-sensitive site is necessary for the ability of elevated [K+]o to increase Na+,K+-ATPase activity. An increase in intracellular Na+ concentration by the drug monensin, which facilitates exchange between intracellular H+ and extracellular Na+ (Mollenhauer et al., 1990), stimulates deoxyglucose phosphorylation in astrocytes, reflecting increased glucose utilization and energy production (Yarowsky et al.,1986; Peng et al., 1994; Wang et al., 2012). This is mainly due to ouabain-inhibited stimulation of the Na+- sensitive site of the Na+,K+-ATPase, triggering Na+ extrusion, although the simultaneous intracellular alkalinisation also has some effect (Erecinska et al. 1995). An increase in extracellular Na+ is also able to counteract the abolishment of K+-stimulated K+ uptake in astrocytes during inhibition of glycogenolysis described below (Xu et al., 2013; DiNuzzo et al, 2013). The increased Na+ concentration probably does so by

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opening a Na+ channel which is normally opened by glycogenolysis-dependent signaling by nanomolar concentrations of endogenous ouabains and allows sufficient entry of Na+ for simulataneous stimulation of the Na+,K+-ATPase’s intracellular site when the extracellular site is stimulated by increased [K+]o. Besides activating the Na+,K+-ATPase’s catalytic activity K+ stimulation of the Na+,K+-ATPase

also

activates

a

signaling

pathway mediated

by nanomolar

concentrations of endogenous ouabain(s) (Xu et al., 2013). Operation of this pathway via Src and a metalloproteinase stimulates the epidermal growth factor receptor (EGFR), which in turn opens two different signaling pathways (Xu et al., 2013). One of these leads via stimulation of the inositoltrisphoshate (IP3) receptor to an increase in [Ca2+]i and glycogenolysis, while the other via the Ras-Raf-Mek cascade phosphorylates and activates extracellular regulated kinases 1 and 2 (ERK1/2). The increase in [Ca2+]i and in glycogenolysis leads to opening of a Na+ channel (Xu et al., 2013; DiNuzzo et al., 2013; Hertz et al., 2015a, 2016a), which provides intracellular Na+ needed for stimulation of the Na+,K+-ATPase. Accordingly the astrocytic K+ uptake is abolished by inhibition of glycogenolysis (Fig. 2B). An attempt to confirm this mechanism in brain slices in collaboration with the Christine Rose group in Düsseldorf did unfortunately not confirm the cell culture observations. However, the experiments were carried out 30 min after the preparation of the slices, although glycogen is depleted from astrocytes in freshly prepared slices, only begins to reappear by 1 hour, and is not fully recovered until after 3 hours (Fiala et al. 2003), for which reason we in other experiments have used an incubation period of 5 hrs (Xu et al., 2014). Moreover, stimulation was carried out by superfusion with 0.5 mM glutamate and co-transport of Na+ during astrocytic

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accumulation of glutamate (Drejer et al., 1982; Cholet et al., 2002; Matos et al., 2013) might have supplied sufficient intra-astrocytic Na+ using this approach. This is consistent with the finding by Larsen et al. (2016b) that the contribution of the Na+,K+-ATPase to clearance of stimulus-induced [K+]o transients in hippocampal slices is reduced by inhibition of the mainly astrocytic glutamate transporters. Testing different isozyme constellations in oocytes these authors also established that the intracellular Na+-sensitive site of the Na+,K+-ATPase was not fully saturated at the basal intracellular Na+ concentration at any constellation of isozymes. These observations strongly support the necessity of either glutamate- or glycogen-mediated astrocytic Na+ uptake into astrocytes in order for increased [K+]o to stimulate their K+ uptake. What remains unresolved is whether glycogenolysis may be necessary only for astrocytic uptake of action potentialmediated increase in [K+]o or also could contribute to re-uptake of K+ released by glutamatergic stimulation at sites further away from the glutamate uptake sites. Since neurons are excitable cells accumulating Na+ during the excitation they have probably no need for similar mechanisms. There is good evidence for the importance of glycogenolysis for astrocytic K+ uptake in the brain in vivo during spreading depression (SD), which as previously mentioned is associated with a huge increase in [K+]o. During SD the wave of cellular depolarization shows an increased propagation rate in mouse hippocampal slices during selective inhibition of the breakdown of glycogen (Seidel and Shuttleworth, 2011), which is compatible with a decreased cellular K+ accumulation. Moreover, Feuerstein et al. (2015) determined glucose and glycogen utilization and extracellular levels of glucose and lactate during spreading depression in rat brain. They could only develop a successful

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model of energy metabolism using a two cell model consisting of glycogen-metabolizing astrocytes and neurons metabolizing no glycogen and using lactate only after its extracellular concentration became highly elevated. This model showed an excellent fit with their measured values and indicated that each wave of spreading depression increased neuronal glucose utilization 4 times and astrocytic utilization of glucose plus glycogen 8 times. The level of glycogen decreased rapidly, consistent with an almost 60year-old finding by Krivanek (1958), and it accounted for more than two thirds of astrocytic metabolism. Finally, an increase in mRNA expression of the 2 subunit of the Na+,K+-ATPase decreases susceptibility for spreading depression and increases K+ clearance (Seidel et al., 2015), which during spreading depression occurs into astrocytes (Seidel et al., 2016). On the other hand SD waves propagate faster, spread more widely and last longer during treatment with fluoroacetate (Largo et al., 1997a), a specific inhibitor of astrocytic energy metabolism. Together, these observations strongly support a role of glycogenolysis for astrocytic K+ uptake in intact brain.

2.3. The Na+, K+-ATPase is also stimulated by noradrenaline The Na+,K+-ATPases in both astrocytic and neuronal cultures are also stimulated by noradrenaline (Fig. 3), but only when [K+]o is not simultaneously elevated or decreased. The astrocytic enzyme is stimulated by isoproterenol, a β-adrenergic agonist acting via the adenylate cyclase, and -adrenergic stimulation of the K+ analogue

86

Rb

has also been demonstrated in pig hearts. The astrocytic Na+,K+-ATPase is also stimulated by serotonin and dopamine (I. Hajek and L. Hertz, unpublished experiments) probably by stimulation of D1 and 5-HT-7 receptors which like isoproterenol activates

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the adenylate cyclase (Neve et al., 2004; Hoyer et al., 2002). In contrast the neuronal stimulation observed by Hajek et al. (1996) was not -adrenergic but probably adrenergic. This is consistent with findings by Baskey et al. (2009) in freshly obtained neurons which have shown that stimulation of the neuronal enzyme is -adrenergic and inhibited by prazosin. These authors found similar activities in neurons and astrocytes, whereas Hajek et al. (1996) found higher Vmax in astrocytes. The involvement of different adrenergic subtypes in astrocytes and neurons is consistent with both - and -adrenergic Na+,K+-ATPase stimulation in brain tissue (Phillis, 1992; Vizi and Oberfrank, 1992). Baskey et al. also observed that the neuronal Na+,K+-ATPase activity increased after REM sleep deprivation, while the glial enzyme activity decreased and that these changes were mediated by an effect of noradrenaline on the 1-adrenoceptor. These observations suggest that although the recent findings of altered [K+]o during sleep by Ding et al. (2016) also show that changes in ion homeostasis alter behavior, the changes observed by these authors are exerted on neurons and not on glia. Correlations between sleep stages and locus coeruleus activity have been known for more than 40 years (Chu and Bloom, 1973), and Aston-Jones and Bloom (1981) concluded that the noradrenalinelocus coeruleus system may globally influence target neurons and thereby influence overall behavioral orientation. This was long before the importance of glial cells in behavior was generally acknowledged, but effects of noradrenaline on both sleep and learning is consistent with this conclusion. In the kidney and in neurons interactions between K+, Na+, and Ca2+ concentrations, noradrenaline and Na+,K+-ATPase activity can be explained by changes in [Ca2+]i which activate calcineurin and enhance Na+,K+-ATPase activity by

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dephosphorylation, whereas activation of protein kinase C (PKC) causes phosphorylation and decrease in Na+,K+-ATPase activity. This topic is beyond the scope of the present paper but it is discussed with references in Hertz et al., 2016a.

2.4. NKCC1 stimulation by higher [K+]o might be physiologically important Besides Na+,K+-ATPase also NKCC1, a cotransporter of Na+, K+, 2Cl- and water, is expressed in brain (Epstein and Silva, 1985; Dawson, 1987; Hamann et al., 2010; Macaulay and Zeuthen, 2012). The cellular localization of NKCC1 is disputed, but immunological determination of NKCC1 can be misleading (Blaesse et al., 2009), and immunochemical and in situ hybridization determination of many genes in brain seems to be especially uncertain in astrocytes (Peng et al., 2013). Moreover cellular expression of NKCC1 changes during postnatal development (Jantzie et al., 2015). There is no doubt that cultured astrocytes (Jayakumar et al., 2008), glioma cell (Zhu et al.2014), cultured oligodendrocytes (Wang et al., 2003; Chen et al., 2007; Fu et al., 2015) and both mature and immature GABAergic neurons (Khirug et al., 2008; Dzhala et al., 2010; Rangroo Thrane et al., 2013) express Slc2a, the gene for NKCC1, but in the present context the important question is its astrocytic expression in the mature brain. Expression of mRNA for NKCC1 in glia cells was shown by Kanaka et al. (2001) in both Bergmann glia (an astrocytic cell) and in corpus callosum. MacVicar et al. (2002) clearly showed NKCC1 expression in optic nerve and its colocalization with GFAP immunopositive astrocytes. Both mRNA and protein expression of NKCC1 has been determined in astrocytes of the dorsal root and trigeminal ganglia of the rat (Price et al., 2006).

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NKCC1 is normally activated by either an elevation of [K+]o of at least 10 mM (Walz and Hertz, 1984; Walz and Hinks, 1985; Xu et al., 2013) or extracellular hypertonicity leading to cellular shrinkage (Akar et al., 1999; Qusous et al., 2011). The requirement for a high [K+]o for its activation is supported by K+ effects on swelling in brain slices (Lund-Andersen and Hertz, 1970). However in rat cultures NKCC is stimulated by non-elevated [K+]o (Walz and Kimelberg, 1985; Tas et al., 1987; Larsen et al., 2014), possibly a result of the extremely low cell permeability for K+ found in these cultures (Hertz, 2015b). Its function is dependent upon ion gradients created by the Na+,K+-ATPase, making it a secondary active transporter (Pedersen et al., 2006). It is therefore also activated by 1-adrenergic stimulation of the Na+,K+-ATPase (Song et al., 2015).

The increases in [K+]o during normal neuronal stimulation in brain slices may not high enough to activate NKCC1 (Larsen et al., 2014), although it should be kept in mind that the increase in [K+]o in the very narrow space between neurons and astrocytes might be considerably higher than those described in section 2.1. Also, Ransom et al. (1985) observed an activity-dependent decrease in extracellular space in rat optic nerve, which paralleled the increase in [K+]o, and ontogenetically developed pari passu with the appearance of glia cells. A comparison between the ontogenetic development of the extracellular shrinkage and the development of astrocytes and oligodendrocytes suggested that swelling of both of these two glia cell types may be responsible for the reduced extracellular space. Exposure to highly elevated [K+]o and resulting depolarisation of the astrocytic cell membrane leads to nimodipine-inhibited opening of L-channels for Ca2+ (Hertz et al.,

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1989) (Fig. 4A,B) and activation of a signaling pathway that eventually leads to phosphorylation and activation of NKCC1 and astrocytic swelling (Cai et al, 2011) (Fig. 4C). Although not shown in the Figure this pathway also depends on glycogenolysis, and its dependence on Na+,K+-ATPase activity makes it dependent on signaling

by

nanomolar concentrations of endogenous ouabains. During brain ischemia/re-perfusion, combination with analogous events in brain endothelial cells (O’Donnell et al., 2004a,b; Khanna et al., 2014; Hertz., 2015b) leads to brain edema, which often is fatal. During acute liver failure and in ammonia-treated astrocyte cultures a considerably smaller increase in the ammonium ion, NH4+, combined with inflammatory damage of NKCC1 leads to a similar but more gradually developing edema (Jayakumar et al., 2008, 2011; Dai et al., 2013; Song et al., 2014a; Hertz et al., 2015b). This is because NH4+ can potently replace K+ in stimulation of the Na+,K+-ATPase, active ion transport, and thus also of the Na+,K+-ATPase dependent NKCC1 (Post et al., 1960; Moser, 1987; Jayakumar and Norenberg, 2010) as well as the associated increase in oxygen consumption (Hertz and Schou, 1962; Kurtz and Balaban, 1986). The higher potency is possibly related to the ability of ammonia to inhibit K+ channels (Bleich et al., 1995), a correlation which will be discussed in some detail later. Ammonia-induced cell swelling can be prevented by canrenone, an antagonist of the cytosolic aldosterone receptor and of ouabain (Dai et al. 2013; Song and Du. 2014). Since inhibition of ERK1/2 partly blocks oxidative/nitrosactive stress involved in astrocytic swelling (Rao et al., 2010), the branch of the pathway by endogenous ouabains that leads to ERK1/2 phosphorylation may be responsible for the inflammatory damage of NKCC1, which facilitates ammonia-induced swelling. However, there is also evidence

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that aldosterone in a cell line can regulate NKCC1 protein expression by reducing its ubiquitination (Ding et al., 2014). Since NKCC1 operates at lower [K+]o in cultured rat astrocytes than in cultured mouse cells, and this co-incides with a reduced membrane conductance for K+ (Walz and Kimelberg, 1985) NKCC1 might possibly contribute more to clearance of [K+]o in astrocytes in the brain if their K+ permeability was decreased. Reduced expression of Kir4.1 channels, which are the main reason for the high membrane conductance of normal astrocytes is seen in glutamate-overexposed astrocytes in vitro and in the brain of rats with acute liver failure (Obara-Michlewska, 2015). Diminished Kir4.1 opening may also occur under physiological conditions, since glutamate acting on AMPA receptors can inhibit Kir4.1 channels (Schröder et al., 2002). In section 2.6 it will be shown that activation of protein kinase C (PKC) can inhibit channel mediated K+ uptake in astrocytes. Simultaneous exposure to normally occurring elevations of [K+]o and glutamate or transmitters selectively increasing PKC might therefore enable opening of L-channels for Ca2+ and subsequent NKCC1 stimulation in brain by normally occurring increases in [K+]o. A paper by MacVicar and Tse (1988) showing that inhibition of Lchannel-like currents were maximized by inhibition with Ba2+ is unfortunately not relevant because these authors used Ba2+ concentration of 2.5-10 mM. This was in spite of the demonstration by Walz et al. (1984a) that Ba2+ concentrations of this magnitude almost abolish ouabain-sensitive K+ uptake and Na+,K+-ATPase activity in cultured astrocytes. However, the essential effects of [Ca2+ ]i for astrocytic function (Zorec et al., 2012) makes it of major importance for brain function to establish if reduced astrocytic K+ conductance by itself may promote L-channel activation. The following sections will

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show that this is likely if the decreased membrane permeability for K+ also reduces gapjunction-mediated K+ transport, which it may well do. It is in further support of a physiological role of depolarization-mediated opening of L-channels for Ca2+ that mRNA for the L-channels Cav1.2 and Cav1.3 are expressed both in cultured and freshly isolated astrocytes (Yan et al., 2013; Du et al., 2014). Moreover, 2 weeks of fluoxetine treatment increases Cav1.2 expression not only in cultured astrocytes but also in astrocytes in brain (Du et al., 2014), which probably would not have been the case if the channel was not operating. Cav1.2 mRNA is also upregulated in hyperammonemic mice (Wang et al., 2015), consistent with the ability of NH4+ to potently stimulate NKCC1 via opening of L-channels. Nevertheless it is often believed that astrocytes have no functional L-channels, a concept partly due to the failure of Carmignoto et al. (1998) to demonstrate voltage-dependent Ca2+ channels in astrocytes in brain slices. However these authors used 2 mM lactate as the only metabolic substrate, and L-channel activity is controlled by metabolism (Kostyuk, 1984), in cultured astrocytes specifically degradation of glycogen (Xu et al., 2013), which is only slowly formed from lactate. Moreover, other authors have demonstrated Ca2+ channel activity in retinal glial cells (Newman, 1985), and Duffy and MacVicar (1994) described an increase in [Ca2+]i in hippocampal astrocytes in situ which was inhibited by the L-channel blocker verapamil.

2.5. Importance of NKCC1 in regulatory volume increase NKCC1 is also important in regulatory volume increase in the brain, because extracellular hypertonicity leads to cellular shrinkage which is reversed by regulatory

19

volume increase (Pedersen et al., 2006; Hoffmann et al., 2007; Sid et al., 2010). Extracellular hypertonicity is known to develop after neuronal excitation (Dietzel et al., 1982, 1989), perhaps due to the asymmetric ion transport by the Na+,K+-ATPase, which catalyzes entry of 2 K+ and exit of 3 Na+ (Thomas, 1972; Clarke et al., 1989). However Dietzel et al. explain the hypertonicity as a result of spatial buffer currents with sinks and sources at different cortical layers. Hypernatremia secondary to brain damage or less frequently to diabetes insipidus leads to severe, life-theatening extracellular hypertonicity (Spatenkova et al., 2011; Payen et al., 2014). The extracellular hypertonicity is accompanied by a large decrease in astrocyte cell volume (Risher et al., 2009) and by associated neuronal gene expression changes which are blocked by the astrocyte-specific toxin fluoroacetate (Yuan et al., 2010). Extracellular hyperosmolarity depresses neuronal population spikes and extracellular synaptic potentials (Huang and Somjen, 1995; Somjen, 2002). In cultured astrocytes NKCC1-mediated regulatory volume increases, which are enhanced by β-adrenergic Na+,K+-ATPase stimulation, reverses cell shrinkage during extracellular hypertonicity (Song et al., 2015). The undershoot in [K+]o developing after neuronal excitation, especially after intense or long-lasting stimulation (Ransom et al., 2000), depends on NKCC1 activity as indicated by its inhibition by furosemide (Xiong and Stringer, 2000) (Fig. 5A), an inhibitor of NKCC1, although not as specific as bumetanide, since it inhibits both NKCC1 and KCC2, a different cotransporter (Blaesse et al., 2009 ; Ko et al., 2014). Subsequently cellularly accumulated K+ is released via Kir4.1 channels (Fig. 5B), as shown by an increased undershoot when these channels are inhibited (D’Ambrosio et al., 2002; Bay and Butt, 2012) or in mice with a null mutation in the Kir.4.1 gene (Neusch et

20

al., 2006). The involvement of both NKCC1, and of Kir4.1 channels indicates that the undershoot is secondary to K+ uptake into astrocytes, not neurons. Similarly to the NKCC1-mediated regulatory volume increase after osmotically induced cell shrinkage in astrocyte cultures (Song et al., 2015) the accumulation of ions and water in astrocytes during the undershoot must contribute to

normalization of

extracelllar hypertonicity (Hertz et al., 2013) and astrocytic cell volume. It is therefore likely also to normalize the reduced neuronal activity resulting from extracellular hyperosmolarity (Huang and Somjen, 1995) and thus play a role in reversal of neuronal slow afterhyperpolarization, sAHP (Hertz et al., 2013, 2015a, 2016a). These effects are consistent with the conclusion by Forrest (2014) that the Na+,K+-ATPase processes information by integrating spike

numbers and causing hyperpolarisation. However,

Forrest (2014) does not consider the possibility that non-neuronal Na+,K+-ATPases could be involved. Na+,K+-ATPase requirement has also been demonstrated for afterhyperpolarization of cortical layer 5 neurons and hippocampal CA pyramidal neurons (Gulledge et al., 2013), and the amplitude of the neuronal hyperpolarization in Drosophilia larvae depends on the number of spikes in the burst (Glanzman, 2010; Pulver and Griffith, 2010). As previously mentioned regulatory volume increase by NKCC1 is greatly stimulated by -adrenergic stimulation of the Na+,K+-ATPase, which creates the ionic gradients allowing operation of NKCC1 and thus astrocytic uptake of K+ together with Na+ and Cl-. Since noradrenergic Na+,K+-ATPase stimulation selectively occurs at normal [K+]o and only the astrocytic enzyme responds to -adrenergic stimulation (Hajek et al., 1996), it is interesting that β-adrenergic termination of sAHP in isolated thalamic tissue

21

also only takes place at non-elevated [K+]o (McCormick and Prince, 1988). This observation strongly supports that β-adrenergic acceleration of regulatory volume increase in astrocytes leads to not only the post-excitatory undershoot in [K+]o but also to termination of slow neuronal hyperpolarization. Since NKCC1 transports two molecules of Cl- the extracellular Cl- concentration and thus the intracellular/extracellular Cl- ratio is of major importance for its activation. In addition to expressing NKCC1 astrocytes possess several other types of Cl- transport systems, such as voltage-sensitive and ligand-gated channels and a HCO3-/Cl- exchanger. They are responsible for astrocytic volume regulation, and in the case of the exchanger also partly for intracellular pH regulation (Baba et al., 1992). Although most papers dealing with effect of Cl- concentrations on brain function mainly deal with GABAergic neurons Bekar and Walz (2002) showed the importance of the intracellular Clconcentrations in astrocytes for activation of one of the astrocytic K+ channels.

2.6.

Membrane conductance and Kir 4.1 channel downregulation

by PKC

activation The K+ channels operating during impulse propagation are repolarizing (Hodgkin and Huxley, 1952). A different type of K+ channel with a reduced current flow at depolarized membrane potential was described by Katz (1949) and the currents eventually characterized as inwardly rectifying K+ currents (Kir). The most common is the Kir 4.1 channel, which is highly expressed in these cells (Butt and Kalsi, 2006; Olsen and Sontheimer, 2008). However, many additional K+ channels and currents are also found in astrocytes (Bekar and Walz, 2002; Zhou et al., 2009; Olsen et al., 2015). The 22

astrocytic K+ channels provide a very high membrane permeability (conductance) for K+, and in spite of its name Kir 4.1 can function in both directions, depending on membrane potential and the difference between intracellular and extracellular K+ concentrations.. In most studies it is evaluated as K+ currents (Olsen, 2012). However, we have instead measured uptake of

42

K, which in cultured mouse astrocytes at 5 mM [K+]o amounts to

1200- 2000 nmol.min per mg protein, to a large extent due to homoexchange with intracellular non-labeled K+ (Hertz, 1978). Active, Na+,K+-ATPase-mediated K+ uptake, measured in cultures previously depleted for intracellular K+, is 5-10 times slower (Hertz, 1979). Due to the high K+ conductance the slope of the membrane potential of the cells when [K+]o is changed between 1.5 and 100 mM could have been expected to be 61 mV/10-fold change at 37oC, which would indicate coherence with the Nernst equation. However, as seen in Fig 6A, the simultaneous increase of active transport with rising [K+]o reduced this value to 51 mV/10-fold change (Walz et al., 1984b). The large differences in membrane potential seen by very minor alteration in [K+]o around physiological levels contrast membrane potential response in neurons, which in spite of a relatively high K+ conductance is only slightly altered at physiological levels of [K+]o (Fig. 6B). This is due to the fact that Na+ conductance in neurons also is relatively high, and much higher [K+]o is required for a depolarization (Somjen, 2002). Nevertheless minor increases in [K+]o do have some effects also on neurons (Walz, 2000). Kir4.1 channels are concentrated around small blood vessels and capillaries (Neusch et al., 2006) and are also positioned close to synapses (Higashi et al., 2001; Kurachi and Hibino, 2003). They can under certain conditions contribute to K+ uptake also in mammalian brain (Somjen et al., 2008; Bay and Butt 2012; Larsen et al., 2014;

23

Larsen and MacAulay, 2014), but their most important role is to re-release astrocytically accumulated K+ to the extracellular space, which allows a secondary, neuronal K+ reuptake (Bay and Butt, 2012). They are also the reason for the high negative membrane potential in astrocytes and thereby the ability of astrocytes to accumulate neuronally released glutamate (Anderson and Swanson, 2000). The driving force for this uptake is glutamate’s co-transport with 2 Na+, and as a consequence glutamate accumulation is most effective at high negative resting potentials and reduced after astrocyte specific knockout of Kir4.1 (Olsen and Sontheimer, 2008). One hour after addition of phorbol 12-myristate-13 acetate (PMA) the rate of channel-mediated K+ uptake in cultured astrocytes is potently and dose-dependently lowered (Fig. 7) without any effect on active uptake until very high concentrations are reached (Hertz, 1989). This observation is analogous to the finding by MacVicar et al. (1987) that another phorbolester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), induces membrane potential oscillations in astrocytes in hippocampal slices associated with large decreases in membrane conductance during the depolarizing phase. Some other phorbolesters have little or no effect on K+ uptake (A. Bender and L. Hertz, unpublished experiments), reflecting that different phorbolesters interact with different PKC isozymes (MacEwan et al., 1999). That PKA seems to have the opposite effect on Kir channels is described below, and that PKC and PKA exert similar effects on connexin-mediated transport is described in section 2.7. Administration of serotonin or noradrenaline had at most marginal effects on K+ uptake, consistent with a lack of effect of serotonin on membrane potential or input resistance in hippocampal slice astrocytes found by Walz and MacVicar (1988). However, there is electrophysiological evidence for Kir channel

24

inhibition by serotonin in leech glia (Britz et al., 2005), perhaps indicating different expression of subtype 5-HT receptors. The reason for the lack of effects by serotonin and noradrenaline in our mammalian astrocytes may be their simultaneous activation of various receptor subtypes with different signaling pathways. That this could be important is suggested by the observation that Kir2.1 channels cloned from mouse skeletal muscles and expressed in oocytes are inhibited by protein kinase C (PKC), whereas protein kinase A (PKA) activity supports channel function (Fakler et al., 1994). Noradrenaline and cAMP also enhance a nifedipine-sensitive calcium current in cultured rat astrocytes (MacVicar and Tse, 1988). Reactive astrocytes in the brain in vivo and in culture express Kir2.3 channels (Perillán et al., 2000), which in cardiomyocytes are inhibited by 1A receptor activation, an inhibition that is attenuated by inhibition of PKC (Zitron et al., 2008). PKC-mediated downregulation of Kir4.1 channel current has been demonstrated in cortical collecting ducts in the mouse kidney cortex following stimulation of dopaminergic D2 receptors (Zaika et al., 2013). The cytokine tumor necrosis factor  (TNF ) similarly induces a PKC-dependent reduction in astrocytic K+ conductance (Köller et al. 1998). On the other hand increasing cAMP in astrocytes in the intact rat or mouse optic nerve with dBcAMP or forskolin hyperpolarizes their membrane potential by ~15 mV, whereas inhibition of PKA causes depolarization (Bolton et al., 2006). Inhibition of Kir4.1 opening by 100 M BaCl2 blocked dBcAMP’s hyperpolarizing effect showing that cAMP and PKA act on these channels and supporting that Kir4.1 opening in astrocytes can be regulated by the PKC/PKA ratio.

25

2.7.

Gap-junction-mediated K+ redistribution, connexins and pannexins Astrocytes are extensively coupled through gap junctions into a functional syncytium, which provides electrical and ionic coupling. Gap junctions are aqueous channels between the cytoplasm of two neighboring cells formed by two hemichannels or connexons, which mediate electrical and metabolic coupling by allowing cytoplasmic exchange of ions (K+, Ca2+, Na+ ), second messengers (cAMP, IP3) and metabolic substrates and products (Pannasch and Rouach, 2013). Ma et al. (2016) showed that the membrane potential of an individual astrocyte in a hippocampal syncytium is wellmaintained at quasi-physiological levels even when recorded with K+-free pipette solutions that alter the K+ equilibrium potential to non-physiological voltages, whereas this is not the case for an individual isolated astrocyte. Thus, the astrocyte's electrical and ionic coupling within the syncytium makes its membrane potential similar to that of its neighbors and minimizes polarization changes during local alterations of [K+]o. Since gap junction coupling is membrane potential-dependent (see below) this may explain why decreased membrane conductance makes NKCC1 activation possible at much lower [K+]o as described above, and it would make PKC-induced inhibition of membrane permeability for K+ a potentially very important, but presently under-recognized mechanism, capable of regulating Kir-mediated K+ transport. Unfortunately this system is not easily studied in brain slices where no spontaneous activity can be expected of transmitters activating PKC. Different connexins are expressed in astrocytes, oligodendrocytes, microglia and endothelial cells, whereas they all express pannexin 1, which does not form gap junctions but like connexin hemichannels functions as one of several routes for release of 26

gliotransmitters (Decrock et al., 2015; Dahl, 2015). These authors also describe the many important roles of connexin and pannexin signaling pathways. Exposure of cultured astrocytes to high [K+]o (>10-20 mM) induces a dosedependent increase in dye coupling whose magnitude depends upon the extracellular Ca2+ concentration and outlasts the application of K+ by at least 90 min (Scemes and Spray, 2012). The mechanism differs for connexins and pannexins. For connexins the long-term increase in coupling is attenuated by reducing the extracellular Ca2+ concentration, prevented by nifedipine, and potentiated by the L-channel agonist Bay-K-8644 (Silverman et al., 2009), i.e., induced via the pathway opened by NKCC1-mediated K+ uptake and mediated by influx of Ca2+ through L-type Ca2+ channels (Fig. 4C). The long duration of the response is probably due to participation of the CaM kinase which can maintain an autonomous activity for a prolonged period of time dependent on the amplitude, frequency, and duration of the activating increase in [Ca2+]i (De Pina-Benabou et al., 2001). For pannexin1 in astrocytes (and neurons) the effect of elevated [K+]o is due to release of ATP which subsequently acts on purinergic P2 receptors in other astrocytes or in neurons (Scemes and Spray 2012). Degradation of ATP to adenosine can further mediate activation of P1 adenosine receptors. ATP is an important gliotransmitter (Butt, 2011; Heinrich et al., 2012) which is released from vesicles (Gucek et al., 2012) and hemichannels (Chever et al., 2014). It integrates neuronal and glial networks (Butt, 2011), and in white matter tracts axonal electrical activity triggers astrocytic Ca2+ signals and propagation of released ATP to amplify the initial Ca2+ signal through the glial network (Hamilton et al., 2008; Butt et al., 2014).

27

The demonstration by Enomoto and Yamasaki (1985) that a phorbolester can reduce Cx43-mediated coupling has repeatedly been confirmed (Bao et al., 2004; Laird et al., 2005; Solan and Lampe, 2009). The membrane-permeable PMA which inhibited Kir4.1 channels in mouse astrocytes was found by Alstrøm et al. (2015a) to inhibit mouse Cx30 but not rat Cx43. However, the previously mentioned interaction of different phorbolesters with different PKC isozymes and their activation by different phorbolesters should be kept in mind. Several PKC isoforms have been implied in the regulation of Cx43 and not always with identical results. However, experiments have been performed under many different conditions and the effects may not be identical on Cx channels and hemichannels, which might explain the inconsistencies in the literature (Alstrøm et al., 2015b). Moreover EGF can disrupt gap junctional communication and Cx43 phosphorylation independently of the phorbolester-sensitive PKC but with involvement of mitogen-activated protein (MAP) kinases (Kanemitsu and Lau, 1993). One possible explanation for the activity of EGF may be its promotion of astrocytic glutamate- or ATP-induced Ca2+ oscillations, which are reduced by pharmacological activation of PKC (Morita et al., 2003, 2015). This reduction might be related to the effect of PKC stimulation on K+ conductance and thus on membrane potential in astrocytes. An effect via EGF is highly relevant in astrocytes because many transmitters, e.g. the serotonergic 5-HT2B receptor (Li et al, 2008) act by transactivation of the EGF receptor after metalloproteinase-mediated release of a growth factor, and this signaling pathway is interrupted when PKC activity is inhibited. Both Cx30 and Cx43 opening measured by uptake of the dye ethidium is also potently inhibited by extracellular Ca2+ (Hansen et al., 2014). This is not in disagreement with the observations by Scemes and Spray (2012) that

28

opening of L-channels for Ca2+ increases dye coupling, since PKC-mediated Ca2+ release depends upon store-operated Ca2+ entry (Li et al., 2011). As a consequence the transmitter-induced release probably occurs in intracellular domains different from those accessible to Ca2+ entering the cells via depolarization–mediated L-channels. Moreover gap junction permeability is regulated by transjunctional voltage (Vj), i.e., the voltage difference between connected hemichannels, but in some connexins also by the membrane potential as such (Vm) (Nielsen et al., 2012; Fasciani et al., 2013). This would contribute to make the effect of PMA shown in Fig. 7 of importance for decreased Cx43 opening. However the only way to establish whether PKC inhibition of membrane permeability for K+ is associated wth reduced gap junction opening would be to measure both in the same preparation. Gap junctions are also important for spreading depression. Thus, the gap junction blockers heptanol or hexanol completely and reversibly prevent the propagation of spreading depression (Largo et al., 1997b). However, Tamura et al. (2011) reached an opposite result, i.e., that another gap junction blocker carbenoxolone enhanced spreading depression, whereas Peters et al. (2003) found it to decrease its rate of progression in brain slices. The observation by Largo et al. (1997b) is supported by an increase in spreading depression by deletion of Cx43 (in one study specifically located in astrocytes) in the mouse brain (Theis et al., 2003; Söhl et al., 2004). Astrocytes are connected with oligodendrocytes by gap junctions (Massa and Mugnaini, 1982; Nualart-Marti et al., 2013), which is important for myelination (Li et al., 2014; Lundgaard ert al. 2014). Transplantation of glial cells into neonatal myelindeficient rat spinal cords leads to myelination and a 3-fold increase in conduction

29

velocity (Utzschneider et al., 1994). Since a correlation between Kir channels and gap junction permeability was suggested it is of interest that oligodendrocytes express Kir4.1 and other K+ channels (Poopalasundaram et al., 2000; Neusch et al., 2001; Kalsi et al., 2004; Butt and Kalsi, 2006) and accurately sense local [K+]o in mature gray matter (Maldonado et al., 2013). The importance of depolarization of oligodendrocytes by elevated [K+]o for increased myelination and conduction velocity and therefore for facilitation of learning was first suggested by Roitbak (1983, 1984; Sotnikov and Roitbak 1980). Later research showed that small changes in myelin thickness profoundly affect neural network function and modify the rate of spike arrival, oscillation frequency and propagation of brain waves (Pajevic et al., 2014; Fields et al., 2014), whereas ablation of Kir4.1 channels inhibits myelination (Butt and Kalsi, 2006). It also confirmed that depolarization increases impulse conduction (Yamazaki et al., 2014). This was done by evoking light-induced depolarization of specifically oligodendrocytes in hippocampal slices from adult transgenic mice where exposure of alveus to blue light caused depolarization which triggered additional compound action potentials (CAPs). The effect was independent of protein synthesis but there was a significant correlation between area of depolarization and increase in CAPs. After 500 msec stimulation the response lasted 3 hrs, although with a gradual decrease in magnitude. Ba2+ (100 μM) greatly inhibited the response. Another K+ channel inhibitor, AP-4, which inhibits aminopyridine-sensitive K+ channels, prevented only the CAP increase during the first few min, but it also reduced conduction latency.

2.8.

Synopsis: K+ homeostasis in brain 30

Neuronal excitation increases [K+]o in vivo and in brain slices. The subsequent cellular K+ reuptake occurs mainly by active transport, at moderately elevated [K+]o generally supposed to be mediated exclusively by the Na+,K+-ATPase although this is not necessarily the case. The Na+,K+-ATPase is expressed both in neurons and astrocytes, but it is stimulated by elevated [K+]o only in astrocytes. Its stimulation leads to K+ uptake in astrocytes in vivo. A small increase in [K+]o also increases K+ uptake and content in cultured astrocytes. An increase in intracellular Na+ is necessary for simultaneous stimulation of the intracellular Na+-sensitive site of the Na+,K+-ATPase. It can be generated either by Na+ co-transport during astrocytic accumulation of glutamate or by a glycogenolysis-dependent opening of a Na+ channel. That K+ uptake into cultured astrocytes is increased after chronic treatment of the cells with a medium containing a reduced concentration of K+ suggests that the primary role of astrocytic K+ uptake is not to enhance removal of excess [K+]o after neuronal activity but to ensure K+ uptake in astrocytes and its further transport in a glial-neuronal-glial network. This transport is initiated by connexin- and pannexin-mediated redistribution to other astrocytes and followed by Kir4.1-mediated release and neuronal reuptake, mediated by the neuronal Na+,K+-ATPase (Fig. 8). The scenario illustrated in this Fig. is consistent with the recent conclusion by Larsen et al. (2016a) that astrocytes respond to the immediate release of K+ from neurons, whereas the neurons themselves are the primary K+ absorbers as activity ends. Astrocytes and oligodendrocytes are connected by gap junctions and depolarization of oligodendrocytes increases CAPs. Both the astrocytic and the neuronal Na+,K+-ATPase are in addition stimulated by noradrenaline (Fig. 3), but not when [K+]o is elevated; only the astrocytic enzyme is

31

stimulated by isoproterenol, a β-adrenergic agonist. This effect plays a role during the post-stimulatory undershoot, which is inhibited by furosemide, an inhibitor of NKCC1, a cotransporter of Na+, K+, 2Cl- and water. The undershoot may be triggered by extracellular hypertonicity and cellular shrinkage developed as a consequence of the 3/2 ratio between K+ uptake and Na+ extrusion by the Na+,K+-ATPase. The cellular uptake of ions and water leads to regulatory volume increase and it contributes to normalization of extracellular tonicity. The astrocytic membrane expresses a high density of Kir4.1 channels, making it highly permeable for K+. Even very small changes in [K+]o after neuronal excitation therefore have large effects on their membrane potential but much less effect on neuronal membrane potential (Fig. 6). In turn this affects several related astrocytic parameters, like intracellular K+ concentration. Depending on the magnitude of [K+]o, Kir4.1 channels can under certain conditions contribute to cellular K+ uptake, although their main function is to mediate the subsequent efflux of astrocytically accumulated K+ after clearance of the increase in [K+]o. Evidence is accumulating that channel activity and gap junction opening can be down-regulated by PKC and/or EGF activation, which might have the important consequence that NKCC1 activity could be triggered at physiologically occurring increases in [K+]o via K+-mediated depolarization and opening of L-channnels for Ca2+. This would be important for such phenomena as K+-mediated Ca2+ uptake, gliotransmitter release and K+-increase in connexin and pannexin function. On the other hand channel activity is up-regulated by cAMP and PKA. However, the importance of effects of G-protein-coupled receptors on Kir channels (see also Butt and Kalsi, 2006) needs further investigation.

32

The correlation between Kir channels and spreading depression may also not have been finally resolved as shown by divergent results, although most suggested that it required astrocytic Cx43 opening. Inhibition of propagation of spreading depression by gap junction closure suggests that K+ accumulated into astrocytes is transported onward via gap junctions. The dependence of spreading depression on astrocytic function and metabolism, including glycogenolysis and the astrocyte-specific2 subunit of the Na+,K+-ATPase is well-established. However, in section 3.2, evidence will be presented that an initial neuronal excitation and glutamatergic activity is also essential. Spreading depression accordingly shows many similarities with neuronal-astrocytic K+ homeostasis and might be a response to overloading the homeostatic mechanism by intense electrical or chemical stimulation. The pathway for clearance of elevated [K+]o (astrocytic uptake, astrocytic redistribution, astrocytic release, neuronal uptake) is reminiscent of that for de novo formation and degradation of transmitter glutamate (astrocytic production of metabolic precursor, transfer to neurons, neuronal transmitter release, astrocytic uptake, astrocytic degradation). It even reminds of that for neuronal re-use of released glutamate (neuronal release, astrocytic uptake, metabolic conversion to glutamine (which is not a transmitter), transfer of glutamine to neurons and re-generation of glutamate, neuronal transmitter release) (Hertz, 2013; Hertz and Rothman, 2016). These sequences are obviously energetically much more expensive than formation, release and re-uptake or degradation in the neurons themselves would have been a tell-tale that they are likely to be important for brain function. K+ homeostasis and glutamate turnover may also be directly connected since glutamate uptake in the mammalian brain is mainly mediated by the astrocytic

33

transporters GLT-1 and GLAST (EAAT2 and EAAT1) (Danbolt 2001, Zhou and Danbolt 2013). Robinson and Jackson (2016) have very recently discussed evidence that these transporters co-localize and co-precipitate with the Na+/K+-ATPase, the Na+/Ca2+ exchanger (essential for ouabain signaling [Song et al., 2013]), glycogen metabolizing enzymes, glycolytic enzymes and mitochondrial proteins.

3. Effects of K+ and its Transporters on Learning 3.1. Effects of glutamate-mediated K+ release in the synaptic cleft Retrograde synaptic signaling mediated by K+ efflux from postsynaptic NMDA receptors into the synaptic cleft (Shih et al, 2013) provides means for altering synaptic function and thus possibly learning by K+ effects not only on presynaptic function but also on astrocytes within the tripartite synapse. As stated by the authors, furher details of this interestng finding are needed to understand its importance, but besides affecting the release of gliotransmitters astrocytic uptake of glutamate might also be affected by astrocytic depolarization. These effects may, in turn, influence learning.

3.2. Inhibition of learning by change of [K+]o. The important role of astrocytes in clearance of [K+]o after neuronal excitation and probably also in creation of the post-stimulatory undershoot together with the effects of minor changes in [K+]o on astrocytic functions suggest that K+ effects on astrocytes may play an important role during learning. A direct adverse effect on learning due to an abnormal [K+]o has been shown in one-trial aversive learning experiments in day-old

34

chickens (Gibbs et al., 1978), a precocious animal with functioning astrocytes at this age (O’Dowd et al., 1995). A decrease of the normal [K+]o evoked by intracranial injection 5 min before training of 20 μl isotonic NaCl solution with low amounts of KCl inhibits learning, but injection of the NaCl solution alone does not have such an effect. This was shown at the memory test 3 hrs later by refusal of the chickens to peck at a shiny chromed lure which during the 10 sec training period had been tainted with a drug of aversive taste but was clean during the test (Fig. 9A). The learning impairment reaches its peak with 2 mM KCl, and addition of 7 mM KCl has no inhibitory effect. Whether the saline contains 140 or 154 mM Na+ makes little difference (Gibbs et al., 1978). The learning inhibition after injection of 2.0 to 4.0 mM KCl only becomes apparent from 15 min after training and onwards, i.e., it abolished the phase in this learning paradigm named intermediate learning, which lasts from 15 min till 1 hr after training (Gibbs et al., 1978). The authors pointed out that this delayed effect resembles that of ouabain (Gibbs and Barnett, 1976; Gibbs and Ng, 1976), suggesting an inhibition of Na+,K+-ATPase activity. Since the astrocytic Na+,K+-ATPase has a Km of 1.9 mM for K (Hajek et al., 1996) it will be only weakly stimulated at the low [K+]o, whereas the +

neuronal enzyme with a Km of 0.45 mM will be virtually fully stimulated. Accordingly, Na+,K+-ATPase activity specifically in astrocytes is likely to be the primary cause of the inhibition of learning. One may wonder why injection of NaCl alone did not abolish memory, but perhaps the larger hyperpolarization enabled channel-mediated K+ uptake to assist deficient Na+,K+-ATPase activity, The immediate inhibition of memory when 0.2-2.0 mM KCl was added to the NaCl solution must be caused by a different mechanism than that described above. Gibbs

35

et al. (1978) pointed to hyperpolarization of glial cells at reduced [K+]o, and focused on possible effects on K+ uptake. An effect on K+ uptake is, however, unlikely since inhibition of learning was immediate, whereas inhibition of the Na+,K+-ATPase as already mentioned only prevents learning from 15 min post-training and onwards. However, glutamate is released to the extracellular fluid soon after training and inhibition of its uptake abolishes learning from the beginning (Gibbs et al., 2004). Since glutamate uptake is metabolically driven by co-accumulation with Na+, and Na+ uptake is promoted by the hyperpolarization occurring after injection of NaCl alone or with only 0.1 mM KCl (which caused no significant inhibition of learning) deficient glutamate removal may have caused the learning impairment. This explanation is consistent with the previously mentioned inhibition of glutamate uptake in vivo after astrocyte-specific knockout of Kir4.1 (Olsen and Sontheimer, 2008). Moreover, early administration of 4 mM glutamate similarly impairs memory (Crowe et al., 1989). The astrocytic and oligodendrocytic depolarisation by K+ released from the axon during impulse propagation and the resulting increase in myelination also promotes learning (Fields, 2008, 2015; Bergles and Richardson, 2015; Chevalier et al., 2015; Yang et al., 2016) as originally proposed by Roitbak. Since this chapter primarily deals with astrocytes we will not further discuss this important and long virtually overlooked mechanism.

3.3. Inhibition of learning by interference with the Na+,K+-ATPase Inhibition of learning in the day-old chicken by the Na+,K+-ATPase inhibitor ouabain (at concentrations greatly exceeding those of endogenous ouabains) can be 36

prevented by subcutaneous injection of noradrenaline, the monoamine oxidase inhibitor pargyline or the 1-adrenergic drug metaraminol (Gibbs and Ng, 1979). This effect may partly be consistent with β-adrenergic stimulation of Na+,K+-ATPase from day-old chickens (Jeffrey, 1976), and the similar effect by noradrenaline and isoproterenol in mouse astrocytes (Hajek et al., 1996). However, 1-adrenergic stimulation of the neuronal Na+,K+-ATPase must also play a role in the rescue of the memory. Other authors have demonstrated learning inhibition by ouabain in different species and paradigms (e.g., Mizumori et al., 1987; Gornicka-Pawlak et al., 2015) without testing effects on glial cells. As previously mentioned the α2 isoform of the Na+,K+-ATPase is present in astrocytes but absent in mature neurons (Fig. 1), although it is expressed in immature neurons. In contrast α3 is only expressed in neurons (McGrail et al., 1991; Peng et al., 1997; Moseley et al., 2003; Li et al., 2013). For this reason changes in the α2-isoform suggest astrocytic effects, although this isotype is also expressed in myelinating oligodendrocytes (Fink et al., 1996; Knapp et al., 2000) and in microvessels, which in addition express 1 and 3 (Zlokovic et al., 1993). The observation by Tadi et al. (2015) that avoidance learning causes an upregulation of mRNA expression of the α2 subunit in the adult mouse hippocampus 24 and 72 hrs after training (Fig 9 B) therefore suggests participation of the astrocytic Na+,K+-ATPase in the learning process. This is also consistent with learning abnormalities found in animals haploinsufficient in this isoform, i.e., lacking a fully functional gene for the α2 isoform, which suggests astrocytic (and/or oligodendrocytic or vascular) defects described by Moseley et al. (2007). These authors tested learning in α2-

37

and α3-deficient mice in the Morris water maze, where the length of the path to reach a hidden platform is an indication of spatial learning and is reliably indicated by the latency to get to the platform. Both α2+/- and α3+/- mice needed longer time to reach the platform than wild-type mice (Fig. 9 C), and this was not primarily due to altered swimming speed, but an indication of learning impairment. Since the astrocytic Na+,K+-ATPase mediates initial cellular re-uptake of neuronally released K and also is necessary for +

NKCC1 activity driving the undershoot it cannot be concluded which of these functions – or both – showed an impairment. The KO was not conditional and interference with the neuronal Na+,K+-ATPase during development cannot be excluded. Mice in which the normally ouabain-sensitive α2 isoform is made resistant to ouabain but retains its basal Na+,K+-ATPase enzymatic function become deficient in learning of egocentric navigation (Schaefer et al., 2011), which in humans encodes routes and integrated paths and eventually becomes implicit or procedural memory. However, these mice were not impaired in spatial learning in the Morris water maze, which uses external cues and depends upon other brain structures than egocentric navigation (Vorhees and Williams, 2014). The reason why the ouabain-resistant mice are deficient in this type of spatial learning might be the dependence of astrocytic K+ uptake on the normally occurring signaling via endogenous ouabains during K+ stimulation of the Na+,K+-ATPase and the resulting stimulation of glycogenolysis (section 2.2). The effect of inhibition of ouabain sensitivity – in the absence of added ouabain – in addition supports the notion that the dependence of astrocytic K+ uptake on endogenous ouabain signaling demonstrated in cultured astrocytes also applies to the brain in vivo.

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Several diseases are associated with α2 abnormalities in the brain, e.g., some relatively rare genetic forms of migraine, including familial hemiplegic migraine type 2 (FHM2) (De Fusco et al., 2003; Segall et al., 2005; Lingrel et al., 2007). Although far from all migraine patients experience any aura (Sarrouilhe et al., 2014), migraine attacks are in

many patients preceded by an aura which is often visual (Farmer et al., 2001) with scintillating scotoma and blurry vision (Petrusic et al., 2014). This includes FHM2, which often is associated with cognitive impairment, language disorder, mental retardation and psychiatric manifestations (Bøttger et al., 2012; Isaksen and LykkeHartman, 2016). Mouse models carrying the human disease mutations display altered cortical spreading depression. Animals with heterozygous KO of the 2 gene are viable, but display altered spatial learning, and homozygous KO mice die shortly after birth by respiratory malfunction due to abnormal Cl- homeostasis in brainstem neurons (Isaksen and Lykke-Hartman, 2016). This may be associated with [K+]o reaching ~15 mM (del Negro et al., .2001). Also in other forms of migraine cognitive function and working memory are impaired during the attacks (Farmer et al., 2001). Moreover, migraine is associated with a greater risk of transient global amnesia (Lin et al., 2014), characterized by the sudden onset of anterograde amnesia with a good prognosis (Arena and Rabinstein, 2015). In patients with aura the characteristic spread of each symptom and the sequence of different symptoms suggest that spreading depression is closely associated with migraine (Russell and Olesen, 1996). This concept is supported by neuroimaging studies recording changes in cerebral blood flow or energy metabolism in migraine patients (Lauritzen, 1994, 2011; Hadjikhani et al, 2001) and with the ability of vagus nerve stimulation to

39

reduce migraine symptoms and inhibit spreading depression (Chen et al., 2016). However the expression of 2 in blood vessels and oligodendrocytes may also be important, since migraine is associated with vascular phenomena (Lauritzen 2011) and long-term chronic migraine patients show abnormalities in white matter tracts involved in pain, emotion and cognition (Gomez-Beldarrain et al., 2015). The spreading depression by itself and the associated cellular depolarization also affects memory. Buresova and Bures (1985) showed that both unilateral and bilateral spreading elicited by electrical stimulation interferes with learning by rats in a radial maze. The effect of spreading depression was small when it was elicited before the test but large when the stimulation occurred during the test. Analogously, although animals that are functionally decorticated due to spreading depression swim normally they are unable to find a visible island in a pool and to escape onto it; however, previous exposure in the pool improves the performance in animals with unilateral spreading depression (Panakhova et al., 1985). This differential effect in

naive and trained animals is

consistent with the view by Sutherland et al. (1983) that input to the dentate gyrus is not necessary for previously learnt spatial performance. Nevertheless, even after prolonged pre-training escape latencies were increased by unilateral spreading depression (Panakhova et al., 1985). In section 2 astrocytic involvement in spreading depression was emphasized. However, it should also be remembered that the propagation of spreading depression in vivo is synchronous with neuronal oscillations in a strip of tissue ahead of the depolarizing wave, which begins before the rise of [K+]o and is followed by a minutelong loss of neuronal activity before complete recovery (Herreras et al., 1994; Lauritzen,

40

2011). Blockade of ionotropic glutamate receptors decreases the frequency of prodromal oscillations, retards spreading depression and shortens the duration of depolarization, whereas inhibition of glutamate uptake increases the oscillatory frequency (Larossa et al., 2006). Selective metabolic poisoning of astrocytes first increases and later decreases oscillatory frequency and amplitude at a time when neuronal electrical properties are still normal (Larossa et al., 2006), and it lowers a largely increased release of glutamate (Szerb, 1991). These data suggest that glutamate released from neurons plays a role in the generation of oscillations and they are consistent with the original observation by van Harreveld and Fifkova (1970) that glutamate is released from retina during spreading depression. That poisoning of astrocytes initially increases the oscillations is probably due to deficient K+ and glutamate uptake by the poisoned astrocytes, whereas the later decrease is caused by lack of glutamate supply to neurons from the astrocytes.

3.4 . Importance of NKCC1 stimulation by extracellular hyperosmolarity As already mentioned the cotransporter NKCC1 is activated by extracellular hypertonicity (Pedersen et al., 2006; Hoffmann et al., 2007; Sid et al., 2010), which depresses neuronal population spikes and extracellular synaptic potentials (Huang and Somjen, 1995). Behavioral effects, including those exerted on learning by reduction of cAMP- and PKA- dependent after-hyperpolarization (Haug and Storm, 2000; Oh et al., 2009) may therefore depend upon neuronal-astrocytic interactions during restoration of normal ion distribution after action potential-mediated increases in [K+]o. This applies not only to -adrenergic signaling but also to dopamine which like isoproterenol stimulates the astrocytic Na+,K+-ATPase (section 2.3) and increases neuronal excitability by 41

suppressing neuronal hyperpolarization via cAMP (Pedarzani and Storm, 1995). Corticotropin releasing hormone (CRH), vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), histamine and serotonin also suppress slow neuronal hyperpolarization by activating cAMP and PKA (Haug and Storm, 2000). It was mentioned that dopamine probably acts on the D1 receptor and serotonin on the 5-HT-7 receptor (section 2,3). The D1 receptor activates astrocytic metabolism (Requardt et al., 2010) and the 5-HT-7 receptor is expressed and functionally important in astrocytes (Altabella et al., 2014). It is important for learning and memory (Romano et al., 2014; Nativio et al., 2015). If neuronal excitability regulates the strength of learning, interventions reducing the postburst afterhyperpolarization should enhance synaptic plasticity and learning (Disterhoft and Oh, 2006; Sehgal et al. 2013), whereas procedures preventing the reduction should counteract learning. This seems to be the case since administration of the specific NKCC1 inhibitor bumetanide (Blaesse et al., 2009; Ko et al., 2014), which prevents regulatory volume increase, blocks inhibitory avoidance learning in intact animals and attenuates LTP (Ko et al., 2014). The less specific inhibitors of NKCC1, ethacrynic acid and furosemide inhibit learning in day-old chickens (Gibbs and Ng, 1977), slightly older chickens (Rogers et al., 1977) and rats (Frieder and Allweis, 1982). Although synaptic activity is a prerequisite for learning it is the experiencedependent plasticity of the synaptic transmission which enables learning, and it includes structural and functional changes in dendrites and their spines (Sehgal et al., 2013). The morphological changes in dendritic spines are closely associated with astrocytic processes interacting with the spines by responding to neurotransmitters and by releasing

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gliotransmitters (Perez-Alvarez et al., 2014). The motility of these processes is rapidly increased by stimuli inducing hippocampal LTP, it reaches a maximum in 20-30 min, and it depends on presynaptic activity and on astrocytic increase in [Ca2+]i.

3.5. Roles of NKCC1-stimulated, L-channel-mediated Ca2+ uptake in astrocytes for memory and memory impairment It is important to establish whether opening of the L-channel for Ca2+ by stimulation of the astrocytic NKCC1 with sufficiently high [K+]o or by an NKCC1 stimulated by lower than normal [K+]o may play a role for K+ homeostasis under physiological conditions. This is because so many apparently important phenomena are only evoked by levels of [K+]o high enough to stimulate NKCC1. High [K+]o increases [Ca2+]i in cultured astrocytes and this effect can be inhibited by a blocker of L-channels for Ca2+ (Fig. 4). Zhao et al. (1996) showed that 10 mM K+ has no effect on [Ca2+]i and that a significant effect required 20 mM [K+]o. Exposure to [K+]o of a magnitude stimulating NKCC1 causes release of gliotransmitter ATP, glutamate and adenosine (Heinrich et al., 2012; Song et al., 2014b; Hertz et al., 2014b; Liu et al., 2015). Glycogenolysis is also stimulated to a much larger degree by a [K+]o of this magnitude than at levels stimulating only the Na+,K+-ATPase in brain (Hof et al., 1988), freshly isolated Müller cells (Reichenbach et al., 1993) and cultured astrocytes (Xu et al., 2014: Hertz et al., 2015d). As previously mentioned (section 2.7) [K+]o high enough to activate NKCC1 are also required for the Ca2+-dependent long lasting increase in gap junctionmediated dye transport (Scemes and Spray, 2012).There is evidence that gap junction

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blockers have prophylactic effect against migraine in patients with an aura, but not in those without any aura (Sarrouilhe et al., 2014). Other prophylactic agents include specific and non-specific inhibitors of the serotonergic 5-HT2B receptor (Schmuck et al., 1996), whose function is PKC-dependent (section 2.7). This receptor is expressed at two times higher density in astrocytes than in neurons (Li et al., 2012), and methysergide, which is one of the 5-HT2B antagonists used prophylactically in migraine, displaces serotonin from its astrocytic binding site (Hertz et al., 1979). It is therefore possible that the 5-HT2B antagonists may inhibit the aura by blocking PKC-induced inhibition of astrocytic Kir channels (section 2.6) and gap junction opening (section 2.7). However, they might also be effective in migraine without any aura by inhibiting 5-HT2B receptors in endothelial cells of the meninges (Schmuck et al., 1996). A gain-of-function missense variant in SLC12A2, the gene encoding NKCC1 has been identified in patients suffering from schizophrenia (Bernstein et al., 2015), a disease which besides its specific symptomatology causes learning impairment (Sharma and Antonova, 2003; Moustafa et al., 2016). Although the authors only discussed NKCC1 in GABAergic neurons, obsevations from other laboratories show a considerable involvement of astrocytes in this disease (Ma et al., 2013; Meng et al., 2016; Pinacho et al., 2016). Moreover, Reuss and Unsicker (2001) showed long ago that atypical neuroleptic drugs downregulate dopamine sensitivity in rat cortical and striatal astrocytes.

3.6. Role of vasopressin Vasopressin (AVP) also potently increases [Ca2+]i in astrocytes (Fig. 10A,B) but not in cultured neurons (Chen et al., 2000). It causes a large increase in water content in 44

cultured astrocytes exposed to a high [K+]o (Fig. 10C) but has no effect at non-elevated [K+]o (Chen et al., 1992). These findings are consistent with observations that vasopressinergic fibers reach not only the pituitary (from where AVP is released as an antidiuretic hormone) but also many other areas of the brain (De Vries et al., 1983; Doczi et al., 1990; Buijs, 1990) and that cultured astrocytes express AVP receptors (Hösli et al., 1991). The ability of AVP to increase astrocytic swelling has been confirmed by many authors and the astrocytic AVP receptor identified as a V1a receptor, which stimulates the phosphatidylinositide signaling system, increases [Ca2+]i and transactivates the EGF receptor (Du et al., 2008; Hertz et al., 2014a). The signaling pathway shows many similarities with that stimulated by [K+]o high enough to activate NKCC1 shown in Fig. 4 (Peng et al., 2012; Hertz et al, 2014a), and this could be the reason why vasopressin increases swelling induced by high [K+]o. AVP also causes an increase in water content in intact brain (Doczi et al., 1982, 1990), and an inhibition of the edema by a V1 antagonist first shown by Rosenberg et al. (1990) has repeatedly been confirmed (discussed and refereed in Hertz, 2014a). It also increases hypotonic swelling of astrocytes although its mechanism differs from that increasing water content during K+induced swelling and requires aquaporin 4 (Cai et al., 2011; Peng et al., 2012). AVP also has effects on the blood-brain barrier and choroid plexus (Chen and Spatz, 2004). AVP is of special interest because it has been found to improve learning (van Wimersma Greidanus et al., 1975; de Wied, et al., 1993). This has led to therapeutic use of vasopressin and its analogue desmopressin in cognitive disorders. However several authors (e.g., Jenkins et al., 1982) reported no therapeutic effect of desmopressin, and a review by Strupp and Levitsky (1985) concluded that both the initial enthusiasm for

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vasopressin’s use against cognitive impairment and the subsequent skepticism were premature. Although almost all negative reports involved cognitively-impaired individuals, vasopressin therapy may be beneficial for some patients, and a few controlled studies in unimpaired human subjects strongly suggest that vasopressin does affect cognition. Animal data do not disprove a putative mnemonic role by AVP (Strupp and Levitsky, 1985), and two studies from the Gibbs/Ng group support this conclusion. Crowe et al. (1990) showed that AVP administered intracranially at the time of training produced long-term memory in chicks trained on a diluted aversant, which without intervention leads to weakly reinforced memory which fades after 30 min. Gibbs et al. (1986) showed that animals pretreated with 2 mM KCl (Fig. 9A) which normally abolishes memory from the beginning were able to remember until 20 min after training when pretreated with AVP. This is the same length of time as in animals treated with slightly higher concentrations of KCl who retain the memory trace for 15 min (right part of Fig 9A). It therefore may suggest that the AVP-evoked increase in [K+]o (Fig. 10A,B) as such has a memory-enhancing effect. Moreover, since the uptake of water by NKCC1 is somewhat less that corresponding to an isotonic solution (Zeuthen and MacAulay, 2012), the extracellular fluid may become hypotonic, which enhances synaptic transmission and neuronal excitability (Somjen, 2002). The effects described above and the high expression of genes for the L-channel for Ca2+ in astrocytes (Yan et al., 2013; Du et al., 2014) support the concept that NKCC1 and Ca2+ channel activity in astrocytes are relevant for normal brain function. It is therefore possible that the inhibitory effect of PKC activation on Kir4.1 opening (Fig. 7) or of EGF can coincide with increases in [K+]o due to neuronal excitation. This would

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enable astrocytic NKCC1 stimulation at lower [K+]o, similar to the observation by Walz and Kimelberg (1985) in cultured rat astrocytes. Most studies of [K+]o responses to neuronal excitation have been performed in brain slices and to our knowledge such a possibility has never been tested in this preparation which cannot spontaneously activate transmitters acting on PKC. Already Butt and Kalsi (2006) suggested that Kir channels may be regulated by neurotransmitters and couple glial K+ regulation with neuronal activity. As discussed in section 2.7 activation of the V1a receptor by vasopressin which stimulates the phosphatidylinositol signaling system could also decrease gap junction opening by a PKC-mediated effect, facilitating K+-induced depolarization of individual astrocytes by reducing participation of the syncytium. Reduction of Kir4.1 activity may also have adverse effects. Deficiencies in K+ homeostasis may contribute to learning difficulties after traumatic brain injury (TBI) by reduction of K+ conductance in hippocampal glia and resulting failure of K+ homeostasis (D’Ambrosio et al. 1999; Sajja et al., (2016). However, Santhakumar et al. (2003) found no alteration in regulation of [K+]o after TBI, and the character of the trauma may be important. Inflammation secondary to opening of pannexin channels by highly elevated [K+]o leads to caspase-1 activation in primary cultures of both neurons and astrocytes (Silverman et al., 2009), which in intact brain may be causally involved in reduction of K+ conductance. Similarly TBI is accompanied by increased NKCC1 activity which is prevented by inhibition of inflammation-induced NF-B (Jayakumar et al., 2014). However, reactive astrocytes in hippocampal slices also acquire enhanced Clconductance allowing them to accumulate KCl both by Na+,K+-ATPase activation and

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passively when exposed to elevated [K+]o (Walz and Wuttke, 1999). Thus, they become able to take up K+ even under anoxic condition.

4. Concluding remarks Kuffler et al. (1966) suggested that ‘the spatial buffer’ might possibly also serve as a physiological signal, and Hertz (1965) suggested that the proposed active uptake of K+ in astrocytes enhanced learning. Research during the intervening 50 years has confirmed these suggestions. Our own work has mainly, but not exclusively, been performed using primary cultures of mouse astrocytes and we have recently documented that these cells due to the culturing technique used are very similar to their in situ counterparts in both gene expression and signaling pathways (Hertz et al., 2016b). Research by many other investigators was performed in vivo. A major reason why collaboration between astrocytes and neurons is essential for the establishment of memory is the different manner of communication between individual cells. Each neuron is an autonomous cell communicating with other neurons through synaptic activity. This ‘neuron doctrine’ is generally attributed to Ramon y Cajal (1888) and a one year earlier demonstration by the Norwegian zoologist, polar explorer and humanitarian Fridtjof Nansen (1887) is only now recognized (Edwards and Huntford, 1998; Compston, 2010). An occasionally cited assertion that Nansen also suggested involvement of glia in learning is less well founded. Inter-astrocytic communication is in contrast reticular, because individual astrocytes are connected in a syncytium by gap junctions made up of connexins. This astroglial network enables rapid intercellular exchange of ions, metabolites, and neuroactive substances in a selective and plastic 48

manner, and can regulate synapses, neuronal circuits, and behavior (Pannasch and Rouach, 2013). Whether brain function depends upon interactions between individual cells or is reticular was hotly debated between Ramon y Cajal and Camillo Golgi ~100 years ago. Now the answer can be given: it depends on both, neuronal interactions between individual cells and astrocytic syncytial activity which creates different functional connections that may be modified by previous activity and occasionally may occur faster than multisynaptic neuronal conduction (Hertz, 1965; Laming, 2000). Spreading depression shares many features with K+ homeostasis but in an exaggerated form. It is involved in migraine with aura, which in turn can cause severe learning impairment.

Acknowledgment The authors gratefully acknowledge the help of Susan Barr, Norway’s Rigsantikvar’s Direktorat for kulturminneforvaltning

and President of the International Arctic Science Committee in

establishing the role of Fridtjof Nansen in Neuroscience.

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receptors. J Mol Cell Cardiol 44, 84-94. Zlokovic, B.V., Mackic, J.B., Wang, L., McComb, J.G., McDonough, A., 1993. Differential expression of Na,K-ATPase alpha and beta subunit isoforms at the blood-brain barrier and the choroid plexus. J. Biol. Chem. 268, 8019-8025. Zorec, R., Araque, A., Carmignoto, G., Haydon, P.G., Verkhratsky, A., Parpura, V., 2012. Astroglial excitability and gliotransmission: an appraisal of Ca2+ as a signalling route. ASN Neuro 4.

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Figure legends Fig. 1. Subunit composition of Na+,K+-ATPase from freshly isolated astrocytes and neurons obtained from mice where either the astrocyte-specific marker GFAP (GFP) or a neuronal marker, YFPH, had been linked to a fluorescent compound, allowing isolation of an astrocytic, respective neuronal, cell fraction by fluorescence-activated cell sorting (FACS) as described by Lovatt et al. (2007). In each cell fraction mRNA expression was quantitated by reverse transcription polymerase chain reaction (RT-PCR) using the same amounts of each RNA. Products of PCR for α and β subunits and for the house-keeping gene TATA-binding protein (TBP) are shown from three astrocytic and three neuronal samples. Note that the 1 subunit shows a higher expression in astrocytes than in neurons, 2 subunit is virtually confined to astrocytes and the 3 subunit is only expressed in neurons. The 1 subunit is equally expressed in the two cells types and the 2 subunit is strongly expressed in astrocytes, but absent from neurons. The housekeeping gene TBP is equally expressed in both cell types as a further indication that equal amounts of astrocytic and neuronal RNA were analyzed. From Li et al., 2013.

Fig. 2. Mean K+ uptake ± SEM into astrocytes measured as increase in intracellular K+ concentration, determined in arbitrary units based on enhanced fluorescence of the K+ indicator benzofuran isophthalate (PBFI) in the presence of pluronic acid. A. After incubation of PBFI-AM-loaded cells in physiological saline solution for 2 min and subsequent wash, the cells were from zero time incubated either in a similar solution or in a solution to which an additional 5 mM KCl had been added isosmotically. Note a large, statistically significant (* P<0.05) initial increase followed by a smaller increase (where

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statistical significance was not tested) when [K+]o is increased by 5 mM. B. Similar experiments with addition of 10 mM 1,4-dideoxy-1,4-imino-d-arabinitol (DAB), an inhibitor of glycogenolysis, with or without 5 mM KCl. Note complete inhibition of the response to KCl by DAB, which alone has no effect. Results for each condition were based on measurements of fluorescence in 40–77 individual cells from two to five separate cultures. From Xu et al., (2013).

Fig. 3. Na+,K+-ATPase stimulation (indicated in the upward direction) and inhibition (indicated in the downward direction) by 10 M noradrenaline at 1, 3, 6, or 12 mM extracellular K+ concentration in homogenates of primary cultures of mouse cerebral cortical astrocytes (open bars) or neurons (solid bars). The activity in the same homogenates in the absence of any transmitter equals 0%. Results are means ± SEM for four to seven different homogenates. Note that the stimulatory effect is virtually restricted to the extracellular K+ concentration used during the culturing (5.4 mM) and the noradrenaline at other [K+]o values had an inhibitory effect, especially in neurons. From Hajek et al., 1996.

Fig. 4. A. Uptake of 45Ca into astrocytes in primary cultures as a function of time at 37oC at the normal extracellular K+ concentration of 5.4 mM (open squares) or at 60 mM extracellular K+ (filled diamonds) shown as means ± SEM. 45Ca (0.5 Ci) had been added to 1 ml serum-free medium 60 sec before the increase in K+ concentration. B. Effect of addition 1 hr before the measurements of nimodipine, a blocker of L-channels for Ca2+, on unstimulated

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Ca uptake (5.4 mM [K+]o, filled diamonds) and K+-stimulated

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Ca uptake

(60 mM K+, open quares) into cultured astrocytes shown as means ± SEM . Note that only

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the latter is strongly and potently inhibited. C. Diagram showing signaling pathways towards ERK1/2 phosphorylation activated by elevation of the extracellular K+ concentration and inhibition of this pathway by specific inhibitors (ovals). Elevation of the extracellular K+ concentration above 10–15 mM depolarizes the cell membrane sufficiently to lead to depolarization-mediated Ca2+ entry through voltage-dependent L-channels. This effect increases with the magnitude of the increase in [K+]o for which reason 60 mM [K+]o was used in this study. The increase in [Ca2+]i is necessary for ERK1/2 phosphorylation, which is inhibited by BAPTA-AM, and it leads to a Src-dependent (and PP1-inhibited), release of HB-EGF (shown by ELISA) from its membrane-bound precursor by the metalloproteinase ADAM17 (inhibited by GM6001) and by siRNA against ADAM17. The released HB-EGF activates (phosphorylates) the EGF receptor (inhibited by AG1478), leading to activation of the MAP kinase cascade, Ras (inhibited by bumetanide), Raf and MEK (inhibited by U0126), with activation of MEK causing ERK1/2 phosphorylation. ERK1/2 phosphorylation activates (phosphorylates) the cotransporter NKCC1 through pathways that were not studied and are only partly known. This leads to influx of Na+ and K+ together with 2 Cl- and water. Accordingly K+-induced swelling is contingent upon ERK1/2 phosphorylation. From Cai et al. 2011 (Color figure on Internet).

Fig. 5. A. Effect of furosemide, an inhibitor of NKCC1, in brain slices exposed to an extracellular K+ concentration high enough to elicit epileptic discharges in the additional absence of Ca2+ (8 mM K+; 0 mM Ca2+). The effects of the pathologically large elevation of the extracellular K+ concentration and furosemide-mediated inhibition of K+ clearance at these K+ concentration by the Na+,K+, 2Cl− and water cotransporter NKCC1 are of less

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relevance in the present context, although the increase in peak extracellular K+ concentration is consistent with NKCC1 activation. However, NKCC1 is also activated by hypertonicity at normal extracellular K+, which occurs after Na+,K+-ATPase activity due to the asymmetry between efflux of 3 Na+ and influx of 2 K+. The observation that furosemide decreases the undershoot shows NKCC1 activity at non-increased extracellular K+ concentration is important for the generation of the undershoot. This observation in intact tissue is consistent with experiments in astrocytic cultures showing involvement of NKCC1 in regulatory volume increase after cell shrinkage due to extracellular hypertonicity. B. On the other hand the amplitude of the composite action potential (CAP) and of the subsequent undershoot in rat optic nerve during 20 Hz stimulation for 120 sec in a physiological saline solution with or without 100 M BaCl2, an inhibitor of Kir4.1 channels, were both increased by Ba2+, an indication that Kir4.1 channels participate in return of astrocytically accumulated K+ to the extracellular fluid at both elevated and non-elevated [K+]o. A. from Xiong and Stringer, 2000, B. from Bay and Butt 2012.

Fig. 6. A. Membrane potential (mV) in mouse astrocytes (total number 21) in primary cultures as a function of the extracellular K+ concentration and measured at 37oC with a Clark glass microelectrode inserted intracellularly. The cells had been grown at 5.4 mM [K+]o and membrane potential was initially measured at this value, whereupon [K+]o was changed. The values are means with a SEM not exceeding 0.7 mV. The continuous line is the best fit through all experimental values (51 mV/10-fold change in extracellular K+ concentration), and the stippled line passing through the mean resting potential represents the theoretical slope expected from the Nernst potential (61 mV/10-fold change in

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extracellular K+ concentration). The ‘Supernernstian’ behaviour of the cells is due to concentration-dependent active uptake of K+, known to occur in these cells. B. Membrane potential rat dorsal root ganglia neurons in vitro at different extracellular K+ concentrations. Each point shows the mean±SD of measurements in 4-9 cells. Na+ was deleted as K+ was added to the bath solution. The straight line represents the slope of the Nernst potential at 35oC, assuming constant intracellular K+ concentration. From Somjen et al. 1981, p 168.

Fig. 7. Effect of the phorbolester phorbol-12myristate-13acetate (PMA) on uptake of 42K in primary cultures of mouse astrocytes. The uptake was measured in untreated cultures during a 10-sec. period when it represents an intense homo-exchange with non-labeled intracellular K+ via K+ channels, mainly Kir4.1 (open squares) and during a 30-sec period in similar cultures which during a 30-min period had been depleted for K+ by incubation in ice-cold K+-free medium so that only active uptake remained (closed squares). Note a potent inhibition of channel-mediated uptake and a non-potent and possibly non-specific inhibition of active uptake. From Hertz, 1989.

Fig. 8. Cartoon illustrating mechanisms presumably involved in interactions between astrocytes and neurons in clearance of extracellular K+ after its release from an excited neuron. For graphical reasons neurons and astrocytes are indicated at different sides of a capillary. After its release K+ is almost exclusively taken up by astrocytes, because only the astrocytic Na+,K+-ATPase is stimulated by elevated [K+]o, whereas the neuronal K+sensitive site is saturated already at resting levels of [K+]o. After its astrocytic uptake K+ is transported via connexins and pannexins to adjoining astrocytes (also away from the vessel),

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with only gradual release via Kir4.1 channels to the extracellular phase, preventing another significant increase in extracellular [K+]o. This allows the neuronal Na+,K+-ATPase with its higher K+ affinity to efficiently take up K+, a necessity in order not to deplete neurons for K+. The astrocytic Na+,K+-ATPase is probably prevented from competition at the normal [K+]o due to its dependence on uptake of Na+ into the non-excitable astrocytes, a process requiring either glycogenolysis, which only occurs at elevated [K+]o, or intense glutamate uptake. The requirement for glycogenolysis has been shown in well-differentiated cultures of astrocytes resembling their in situ counterparts in gene expression and signaling (Hertz et al., 2015c, 2016b), all other events both in cultures and in intact tissue. Although not illustrated in the figure, Na+,K+-ATPase activity leads to extracellular hypertonicity (due to uptake of 2 K+ and exit of 3 Na+) combined with astrocytic shrinkage, which may trigger the undershoot in [K+]o after neuronal activity by 1-adrenergic stimulation of K+ uptake into astrocytes during regulatory volume increase. Evidence that all the astrocytic events illustrated as well as the undershoot are of importance for learning is presented in section 3.

Fig. 9. A. Dose-dependent effect of extracellular K+ concentrations on memory, shown by the ability of day-old chickens to avoid pecking at a lure which had previously been tainted with an aversively tasting drug but was clean at the memory test. Twenty l of varying concentrations of KCl from 0.1 to 10.0 mM (in 140 or 154 mM NaCl) were administered intracranially 5 min before learning and retention tested 180 min after learning. SAL refers to NaCl only. Note that the Fig. shows the K+ concentrations injected not measured extracellular K+ concentrations. B. Gene upregulation of the alpha2 subunit of the Na+,K+ATPase 24 and 72 hrs after one trial aversive learning in the mouse. The differences are

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significant at the 0.05* and 0.01** levels. C. Haploinsufficient α2+/- and α3+/- mice were tested for spatial learning in the Morris water maze, measuring the latency (a measure of the distance they needed to swim) to reach a platform that had previously been above the water surface but was now hidden. Group comparisons with α1+/- and wild type mice showed longer latencies (see inset) to reach the platform in both α2+/- and α3+/- mice as an indication of impaired spatial learning based on external cues. A. From Gibbs et al., 1978. B. From Tadi et al., 2015. C. From Schaefer et al., 2011.

Fig. 10. A. Tracing showing the effect of 1 x 10-6 M vasopressin (AVP) on [Ca2+]i in primary cultures of mouse astrocytes during control conditions and in the absence of extracellular Ca2+. Such tracings were used to construct the concentration dependence for AVP-induced rise in [Ca2+]i by subtracting the value for [Ca2+]i in the absence of AVP from its peak value during exposure to AVP. B. Concentration dependence of AVP-induced increase in [Ca2+]i above its control value (89 ± 6.2 nM) in primary cultures of mouse astrocytes. Results are means ± SEM of 5–7 individual experiments. The increase in [Ca2+]i is statistically significant at all AVP concentrations except 10-9 M, with P<0.05 at 10-10 M, P<0.0005 at 10-8 M and P<0.0001 at 10-7 and 10-6 M. C. Changes of water content (determined as [14C] urea space) in astrocytes (a) and neurons (b) in primary cultures by an elevated extracellular K+ concentration and/or addition of AVP. The following incubation conditions were used during a 30-min period: control (5 mM potassium) ( ); AVP 10-12 M added to control ( ); 60 mM potassium ( ); 60 mM potassium plus 10-12 M AVP ( ). SEM values are indicated by vertical bars. *Significantly (p<0.05) different from the control; **Significantly different from value obtained at the same K+ concentration (60

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mM) in the presence of AVP. Note the AVP only increases water content in astrocytes and only when the extracellular K+ concentration is concomitantly increased. A and B from Chen et al., 2000. C from Chen et al., 1992.

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