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MINIREVIEW Synchronization of Ca 2+ oscillations: involvement of ATP release in astrocytes Schuichi Koizumi Department of Pharmacology, Faculty of Medicine, University of Yamanashi, Japan Introduction Glia, Greek for ‘glue’, was discovered by Rudolph Virchow, a German anatomist, in the mid-nineteenth century. The name reflects the original view that glia played merely a structural or supportive role for neu- rons. Glial cells, especially astrocytes, are much more than ‘glue’ or merely quiescent, and display their own set of activities. They can receive inputs, assimilate information, and send instructive chemical signals both to neurons and to other neighboring cells. Astrocytic activities may be assessed by an observed increase in the intracellular Ca 2+ concentration ([Ca 2+ ] i ), using fluorescent Ca 2+ imaging techniques. Astrocytes show transient increases in [Ca 2+ ] i (Ca 2+ transients) that spread into adjacent astrocytes and neurons to form synchronous Ca 2+ oscillations or Ca 2+ waves. Initially, such Ca 2+ events were believed Keywords astrocytes; ATP; Ca 2+ release; gliotransmitter; neuron–glia; neurons; P2 receptors Correspondence S. Koizumi, Department of Pharmacology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan Fax: +81 55 273 6739 Tel: +81 55 273 9503 E-mail: skoizumi@yamanashi.ac.jp (Received 25 May 2009, revised 25 August 2009, accepted 28 September 2009) doi:10.1111/j.1742-4658.2009.07438.x Glial cells, especially astrocytes, are not merely supportive cells, but are important partners to neighboring cells, including neurons, vascular cells, and other glial cells. Although glial cells are not excitable in terms of electrophysiology, they have been shown to generate synchronized Ca 2+ transients (Ca 2+ oscillations) through mechanisms of chemical coupling. Until recently, Ca 2+ transients in astrocytes were thought to be totally dependent on neuronal activities, because astrocytes express a large vari- ety of receptors for neurotransmitters and surround almost all synapses at which neurotransmitters are spilled over to stimulate astrocytes. In addition, however, astrocytes have been shown to release diffusible sub- stances, so-called ‘gliotransmitters’, and Ca 2+ transients in astrocytes are therefore also triggered by astrocytic activities, leading to propagation of Ca 2+ transients or Ca 2+ waves. In these processes, the gliotransmitter ATP and activation of P2Y receptors play central roles. Interestingly, astrocytes evoke Ca 2+ transients when neurons are not present, suggest- ing that astrocytes themselves can initiate and control Ca 2+ transients. Astrocytic Ca 2+ transients are observed even in vivo, through mechanisms of chemical coupling by gliotransmitters, but they are less frequent and synchronous than those in vitro. Although we have not yet clarified their significance in the central nervous system, astrocytic Ca 2+ transients are dramatically affected by pathological conditions, suggesting that, in addi- tion to physiological events, they might be closely involved in disorders in the central nervous system. Abbreviations [Ca 2+ ] i , intracellular Ca 2+ concentration; SIC, slow-inward current; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TTX, tetrodotoxin. 286 FEBS Journal 277 (2010) 286–292 ª 2009 The Author Journal compilation ª 2009 FEBS to be solely triggered by neurotransmitters that are spilled over at the synapses. However, astrocytes themselves integrate and form synchronized Ca 2+ transients by mechanisms of chemical coupling. In this, extracellular ATP and activation of P2 receptors play central roles. In addition, although rapid neuro- transmission was believed to be restricted solely to neuron–neuron communication, it has been found to include glial cells [1,2]. Evidence suggests that such Ca 2+ -mediated extracellular signaling between astro- cytes and neurons could be involved in the regulation of synaptic transmission both in physiological and in pathophysiological conditions. In this minireview, the mechanisms underlying syn- chronous Ca 2+ transients in astrocytes are summarized from the viewpoint of chemical coupling by ATP. Using this coupling, astrocytes regulate neurons and vice versa. Neuron–glia communication appears to be accentuated in pathophysiological conditions such as epilepsy. Thus, the involvement of astrocytic Ca 2+ transients in epileptiform discharge in neurons is also discussed. Astrocytic Ca 2+ transients in vitro The development of video imaging techniques has allowed us to observe dynamic spatiotemporal changes in [Ca 2+ ] i in neurons and glial cells simultaneously. Unlike neurons, astrocytes do not produce action potentials, and thus they were thought to be quiescent. However, they have since been found to be busy or noisy in terms of ‘Ca 2+ excitability’. About 20 years ago, elevations in [Ca 2+ ] i in individually cultured astrocytes in response to neurotransmitters were first reported [3]. After initial observations, it became apparent that many neurotransmitters stimulate [Ca 2+ ] elevations in astrocytes by activating specific receptors expressed on these cells. Astrocytes express a wide range of receptors for different neurotransmitters, sur- round almost all synapses, and therefore respond to neurotransmitters spilled over at synapses when neurons are activated [1]. Thus, Ca 2+ transients in astrocytes were initially thought to be totally depen- dent on neurons (Fig. 1A). Subsequently, it was demonstrated that these Ca 2+ transients could, in turn, stimulate the release of chem- ical transmitters from astrocytes, which mediates com- munication between astrocytes (Fig. 1Ab) [3,4] and even neurons (Fig. 1Ac) [5]. For some years, astrocytic Ca 2+ waves have been thought to propagate via gap junctions [6], with the internal messenger inositol 1,4,5- trisphosphate being the diffusible substance that induces Ca 2+ release in neighboring cells. However, Ca 2+ waves are propagated between astrocytes even when the cells do not have an absolute requirement for functional contact with each other directly, and the extent and direction of the Ca 2+ wave propagation are significantly influenced by movement of the extracellu- lar medium [7]. These more recent reports suggest that substances released from astrocytes can activate recep- tor systems on astrocytes, evoking the release of addi- tional substances, and thus producing a synchronized propagating Ca 2+ wave of activity. In 1999, Guthrie et al. [7] demonstrated that astrocytes release ATP, which is responsible for the spreading of Ca 2+ tran- sients with a slight synchronization (Fig. 1Ab). The spreading of Ca 2+ transients in astrocytes appears to be mediated by chemical coupling by ATP, for the fol- lowing reasons. First, ATP is released from astrocytes during Ca 2+ wave propagation [7,8]. Second, the prop- agation can be reduced or even abolished by a puriner- gic antagonist [5,7–9] or the ATP-degrading enzyme apyrase [5,7]. Third, visualization of the release of ATP demonstrates that the velocity of ATP release correlates well with that of the Ca 2+ wave in astro- cytes [5]. All of these findings suggest that the extra- cellular molecule ATP could be a primary signal for the Ca 2+ wave propagation, and highlight the impor- tance of ATP in cross-talk among astrocytes and even other cell types in the central nervous system (Fig. 1Ac). Such glial chemical couplings are termed ‘gliotransmissions’, and ATP serves a central role as a gliotransmitter. Now we understand that astrocytes receive and respond to neurotransmitters, and release gliotrans- mitter ATP to form synchronized and spreading Ca 2+ transients. However, it should be noted that astrocytes themselves evoke the propagating Ca 2+ transients. As shown in Fig 1Ba, which represents changes in [Ca 2+ ] i in the neuron–astrocyte cocultures, astrocytes and neurons show Ca 2+ oscillations with different frequency and temporal patterns; that is, Ca 2+ oscillations in astrocytes are less frequent and synchronous than those in neurons. Importantly, the Ca 2+ transients in astrocytes do not disappear when neuronal Ca 2+ oscillation is inhibited by tetrodotoxin (TTX) (Fig. 1Ba). In addition, astrocytes show spon- taneous Ca 2+ transients even when they have been purified and cultured without neurons (Fig. 1Bb). These findings suggest that astrocytes can initiate the spontaneous Ca 2+ transients by a neuronal activity- independent mechanism. Such Ca 2+ transients were almost abolished by apyrase (Fig. 1B) and P2 recep- tor antagonists, suggesting that extracellular ATP and activation of P2 receptors are responsible for the spontaneous Ca 2+ events. S. Koizumi Astrocytic Ca 2+ oscillations and neuronal activities FEBS Journal 277 (2010) 286–292 ª 2009 The Author Journal compilation ª 2009 FEBS 287 Mechanisms of ATP release from astrocytes The P2 receptors responsible for increases in [Ca 2+ ] i in astrocytes are well characterized, and G-protein-cou- pled P2Y1 and P2Y2 receptors have been shown to play central roles in the event (Fig. 2B). However, the mechanism underlying the release of ATP from astro- cytes is controversial and a still matter of debate (Fig. 2B). With regard to the release of glutamate, astrocytes express soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), have a vesicular structure expressing the vesicular glutamate transporters, and release glutamate in a Ca 2+ -depen- dent and SNARE-dependent manner [10]. These find- ings strongly suggest that exocytotic machinery is involved in glutamate release in astrocytes, although nonvesicular mechanisms for glutamate release have also been proposed. In contrast, the mechanisms underlying the release of ATP from astrocytes are less Astrocytes ATP ATP dF/F0 =0.2 10 s 3 4 TTX Apyrase 1 2 Apyrase 5 6 Purified astrocytes Neuron–astrocyte coculture Neurons Astrocytes a ATP ATP Neurons Astrocytes b ATP ATP Astrocytes Neurons c a b c A B Initiator Fig. 1. Synchronized Ca 2+ transients in astrocytes and neurons. (A) Cartoon of neuronal activity-dependent Ca 2+ transients in astrocytes. (a) When neurotransmitters such as glutamate are released from nerve terminals, some are spilled over and stimulate adjacent astrocytes (peri- synaptic astrocyte) to evoke Ca 2+ transients in the cell. (b) If neurons are excessively excited, perisynaptic astrocytes causes frequent Ca 2+ transients or Ca 2+ oscillations, followed by the spreading of Ca 2+ transients in adjacent astrocytes to form intercellular Ca 2+ waves by a mechanism of chemical coupling mediated by ATP. In both cases, an increase in [Ca 2+ ] i is driven by neuronal activities. (c) Astrocytic Ca 2+ transients also affect neuronal activities through the gliotransmitter ATP. (B) Neuronal activity-independent Ca 2+ transients in astrocytes. (a) Neuronal Ca 2+ oscillations seen in the hippocampal neurons (blue traces shown as 3 and 4) are highly synchronous and are inhibited by TTX. Adjacent astrocytes (red traces shown as 1 and 2) also show slower and less synchronous Ca 2+ oscillations. However, the synchronous Ca 2+ oscillations in astrocytes are unaffected even when neuronal activities are inhibited by TTX, suggesting that astrocytes have mecha- nism(s) by which they form neuronal activity-independent Ca 2+ transients. (b) Astrocytes reveal synchronous Ca 2+ transients (red traces 5 and 6) when neurons are not present (purified astrocytes). Astrocytic Ca 2+ oscillations seen in the presence of TTX or in purified astrocytes were abolished by the ATP-degrading enzyme apyrase. (c) Schematic cartoon of neuronal activity-independent astrocytic Ca 2+ oscillations. One or some initiator astrocyte(s) release ATP, and this is followed by ATP-dependent Ca 2+ transients that propagate into adjacent astrocytes. Astrocytic Ca 2+ oscillations and neuronal activities S. Koizumi 288 FEBS Journal 277 (2010) 286–292 ª 2009 The Author Journal compilation ª 2009 FEBS well understood. The release of ATP is reduced by inhibitors of several anion channels [11,12], ATP-bind- ing cassette proteins or cystic fibrosis transmembrane conductance regulator [13], gap junctions [14], and P2X7 receptors, suggesting the involvement of multiple pathways in the release. In addition to these, the release of ATP is partly dependent on Ca 2+ [15] and SNARE proteins [16], and astrocytes possess vesicles that contain ATP [16,17]. Inhibition of ATP release by vesicular ATPase inhibitors has also been reported [17]. Interestingly, Pascual et al. (2005) [18] generated inducible transgenic mice that express a dominant-neg- ative SNARE domain selectively in astrocytes to block the exocytotic events in astrocytes. Using the trans- genic mice, they demonstrated that astrocytes released ATP through a mechanism of exocytosis, and that the astrocytic ATP and its metabolite adenosine tonically suppressed synaptic transmissions. These results strongly suggest that astrocytes should release ATP through a mechanism of exocytosis, which would be a key event for neuron–astrocyte communication. How- ever, we still did not know which molecules transport ATP into astrocytic vesicles. Recently, this question has been answered. Sawada et al. (2008) [19] demon- strated that SLC17A9 or vesicular nucleotide trans- porter, a novel member of an anion transporter family, functions as a vesicular nucleotide transporter, and is essential for the storage of nucleotides within vesicles. Importantly, SLC17A9 is expressed in astrocytes. These findings suggest that the mechanisms of ATP release could also include exocytosis, although the nat- ure of the signals released from astrocytes may vary with varying physiological and pathological conditions [17]. It is important to elucidate the mechanisms by which astrocytes release ATP in response to distinct stimuli, as this will further clarify the mechanisms underlying the synchronized Ca 2+ transients. Astrocytic Ca 2+ waves in vivo With the recent development of multiphoton micros- copy, we can observe astrocytic Ca 2+ transients in vivo. Hirase et al. [20] were the first to analyze changes in [Ca 2+ ] i in cortical astrocytes from living, anesthetized rats using this technique. Over 60% of the imaged astrocytes showed spontaneous Ca 2+ transients with complex spatiotemporal patterns. The spontaneous Ca 2+ transients in vivo were very complex and occurred with a relatively low frequency under basal conditions. They showed a limited degree of correlation with nearby astrocytes when compared with those seen in vitro [20]. However, like Ca 2+ transients in vitro, astrocytic Ca 2+ transients in vivo occurred, in large part, intrinsically rather than being neuronal activity- driven, although ATP-mediated chemical coupling was not demonstrated [21]. However, a recent in vivo Ca 2+ imaging experiment by Nimmerjahn et al. (2009) [22] demonstrated that anesthetic agents greatly decreased Ca 2+ responses in glial cells and that, in awake behav- ing mice, Ca 2+ responses in cerebellar Bergmann glia (radial astrocytes) were more frequent and contained ATP-mediated components. The less frequent chemical coupling in vivo might result, in part, from the fact that the activity of the ATP-degrading enzyme ectonucleo- tidases is higher in vivo or in slice preparations than in primary cultures in vitro [18,23]. Astrocytic Ca 2+ tran- sients in vivo also include a component that is depen- dent on neuronal activities. In the mouse or ferret, whisker [24], limb [25] and visual stimulation [26] causes more frequent Ca 2+ transients in astrocytes in the bar- rel, the primary somatosensory cortex, and the visual cortex, respectively. These Ca 2+ transients in astrocytes were delayed by a few seconds as compared with the Astrocyte Exocytosis Cl – channels P2X7 receptors P2Y1,P2Y2 Maxi-anion channels Connexin/pannexin hemichannels N Gq PLC ATP, ADP, UTP ER Ca 2+ C Ins(1,4,5)P 3 Ins(1,4,5)P 3 -R A B Fig. 2. P2 receptors and secretory pathways of ATP in astrocytes. (A) The predominant P2 receptors that produce Ca 2+ transients in astrocytes are P2Y1 and P2Y2 receptors, both of which are coupled with Gq–phospholipase C (PLC), and activation of which results in the formation of Ins(1,4,5)P 3 , leading to Ca 2+ release from Ca 2+ stores. (B) Multiple pathways for the release of ATP. Hemichannels of connexin or pannexin, maxi-anion channels, P2X7 receptors and Cl ) channels are pathways through which ATP can flow. In addi- tion, the existence of exocytotic ATP is also suggested. ER, endo- plasmic reticulum. S. Koizumi Astrocytic Ca 2+ oscillations and neuronal activities FEBS Journal 277 (2010) 286–292 ª 2009 The Author Journal compilation ª 2009 FEBS 289 neuronal responses [24,26], and nearly correlated with the strength of the sensory stimulation. Thus, neuronal activities also affect astrocytic Ca 2+ transients. More importantly, the application of bicuculine, an antago- nist of 4-aminobutyrate A receptor [20], or picrotoxin [27] increases neuronal activity by triggering epileptic- like discharges, which subsequently result in a great increase in Ca 2+ transients that are often propagated into nearby astrocytes [20]. Thus, neuronal activity-dri- ven Ca 2+ oscillation occurs synchronously in multiple astrocytes, and seems to be accentuated by pathological activities of neurons such as epileptiform discharges. Pathology and Ca 2+ transients in astrocytes Ca 2+ waves in astrocytes propagate into neurons and affect the excitability of neurons via the release of neuroactive gliotransmitters such as ATP and gluta- mate, and affect synaptic transmission [5,23]. Both ATP and glutamate are gliotransmitters through which astrocytes can actively regulate synaptic transmission. ATP differs from glutamate in that it inhibits rather than potentiates synaptic transmission. Thus, we hypothesize that the opposing actions of astrocytic glu- tamate and ATP represent a means by which astro- cytes can dynamically modulate neuronal activity by releasing distinct transmitters, which can either excite or inhibit synaptic transmission (Fig. 3B, left). There- fore, one can easily imagine that dysfunctional astro- cytes in certain pathological conditions could result in an imbalance in neuronal excitability, leading to excess neuronal excitation, such as in epilepsy (Fig. 3A, right). Epileptic seizures are sudden uncontrolled attacks of a convulsive or a nonconvulsive nature asso- ciated with unusually intense neuronal firings. Although epilepsy is a neurocentric disorders, the involvement of astrocytes in its pathophysiology is the Neurons Astrocytes ATP Neurons SIC SIC Astrocytes ATP ATP/ado ATP ATP Reactive astrocytes SIC SIC SIC SIC SIC - + Neurons glu glu glu glu glu glu glu Epileptiform dischar g e glu Neurons TP/ado - + glu ATP ATP Epileptiform discharge A B A Fig. 3. Neuronal activities or epileptiform discharges induced by astrocytic Ca 2+ transients. (A) Ca 2+ transients in perisynaptic astrocytes result in the release of multiple gliotransmitters, i.e. ATP (or its metabolite adenosine, ado) and glutamate (glu). Astrocytic ATP inhibits neu- ronal excitation, whereas glutamate increases it. If one of these dual effects of astrocytes is impaired, adjacent neurons will cause excess excitability, owing to an imbalance of inhibitory and excitatory modulation, leading to epileptiform discharges in neurons. (B) Release of gluta- mate induced by astrocytic Ca 2+ transients induces SICs in neighboring neurons. Reactive astrocytes show increased Ca 2+ transients and release of glutamate, subsequently inducing synchronous SICs in adjacent neuronal networks. This synchronization of SICs is able to cause epileptiform discharges in neurons. Astrocytic Ca 2+ oscillations and neuronal activities S. Koizumi 290 FEBS Journal 277 (2010) 286–292 ª 2009 The Author Journal compilation ª 2009 FEBS subject of growing interest and, in fact, the functions of several astrocyte-specific molecules are associated with epilepsy-like firing in neurons [28]. A spatially restricted seizure focus in the brain can be identified for epilepsies acquired after head trauma, tumor, or other severe focal insults to the brain, when astrocytes often become reactive. Reactive astrocytes change in their abilities to release, take up and metabolize glio- transmitters, which would cause unusual excitation of adjacent local neuronal networks [28]. Recently, it was proposed that slow-inward currents (SICs) recorded in the hippocampal neurons are caused by astrocytic glu- tamate [29,30] (Fig. 3B). Although astrocyte-induced SICs are not epileptiform bursts as such (Fig. 3B, left), their synchronization is closely associated with the for- mation of ictal bursts. Astrocytic Ca 2+ oscillation and the subsequent glutamate release are key events in the synchronization of SICs [31]. As shown in the right panel of Fig. 3B, reactive astrocytes increase the release of glutamate by facilitating Ca 2+ oscillations in response to the gliotransmitter ATP (or glutamate), and generate synchronous SICs in small groups of con- tiguous neurons, followed by epilepsy-like firings [28]. Interestingly, several antiepileptic agents, including val- proate, gabapentin, and phenytoin, reduce astrocytic Ca 2+ oscillations, thereby leading to inhibition of the synchronization of SICs [30]. Thus, Ca 2+ transients in reactive astrocytes could be closely related to the observed pathophysiology of epilepsy and could be therapeutic targets for the treatment of epilepsy or, potentially, for other brain disorders. Conclusion and perspective Astrocytes show synchronous Ca 2+ transients by mechanisms of chemical coupling that are mainly mediated by ATP. Asastrocytic Ca 2+ transients are, in part, dependent on neuronal activities, excess exci- tation of neurons results in highly frequent and prop- agating Ca 2+ transients in adjacent multiple astrocytes, which in turn send feedback signals to neurons and control their excitation. These neuron– astrocyte communications mediated by astrocytic Ca 2+ transients are accentuated in pathological con- ditions, suggesting the involvement of astrocytic Ca 2+ transients in brain disorders, including epileptic sei- zures. Interestingly, astrocytes also show spontaneous Ca 2+ transients that are independent of neuronal activities. Thus, astrocytes themselves have the ability to produce their own Ca 2+ transients, and therefore might control neuronal activities or synaptic transmis- sions through a mechanism independent of neurons. The physiological and pathophysiological significance of the spontaneous Ca 2+ transients in astrocytes remains unknown. 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MINIREVIEW Synchronization of Ca 2+ oscillations: involvement of ATP release in astrocytes Schuichi Koizumi Department of Pharmacology, Faculty of Medicine,. suggesting the involvement of multiple pathways in the release. In addition to these, the release of ATP is partly dependent on Ca 2+ [15] and SNARE proteins

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