astrocyte calcium waves: what they are and what they do

Astrocyte Calcium Waves: What They Areand What They DoELIANA SCEMES1* AND CHRISTIAN GIAUME2*1Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York2College de France, Paris, France

KEY WORDSglial cells; gliotransmitters; ATP; gap junctions; connexins

ABSTRACTSeveral lines of evidence indicate that the elaborated cal-cium signals and the occurrence of calcium waves in astro-cytes provide these cells with a specific form of excitability.The identification of the cellular and molecular steps in-volved in the triggering and transmission of Ca21 wavesbetween astrocytes resulted in the identification of two path-ways mediating this form of intercellular communication.One of them involves the direct communication between thecytosols of two adjoining cells through gap junction channels,while the other depends upon the release of ‘‘gliotrans-mitters’’ that activates membrane receptors on neighboringcells. In this review we summarize evidence in favor of thesetwo mechanisms of Ca21 wave transmission and we discussthat they may not be mutually exclusive, but are likely towork in conjunction to coordinate the activity of a group ofcells. To address a key question regarding the functionalconsequences following the passage of a Ca21 wave, we list,in this review, some of the potential intracellular targets ofthese Ca21 transients in astrocytes, and discuss the func-tional consequences of the activation of these targets for theinteractions that astrocytes maintain with themselves andwith other cellular partners, including those at the glial/vas-culature interface and at perisynaptic sites where astrocyticprocesses tightly interact with neurons. VVC 2006 Wiley-Liss, Inc.

CALCIUM SIGNALS AS A SPECIFIC MODE OFEXCITABILITYAND TRANSMISSION IN

ASTROCYTES: HISTORICAL PERSPECTIVE

The findings that astrocytes express a variety of ionchannels and membrane receptors, which enable them torespond on a millisecond time scale to neuronal activitywith changes in membrane potential and/or increases inintracellular Ca21 levels (Barres et al., 1990; MacVicarand Tse, 1988; Marrero et al., 1989; McCarthy and Salm,1991; Salm and MacCarthy, 1990; Usowic et al., 1989) wasthe first step in the glia field leading to the hypothesisthat these cells could play a role in CNS information pro-cessing. It was based on these early reports that Cornell-Bell et al. (1990) and Charles et al. (1991) first reportedthat astrocytes were not only able to respond to externalstimulation with increases in intracellular calcium eleva-tions but, most importantly, they were be able to transmitthese calcium signals to adjacent non-stimulated astro-cytes, as intercellular Ca21 waves (ICWs). The presence of

such phenomenon of propagating waves of calcium lead tothe proposition that ‘‘networks of astrocytes constitute anextraneuronal pathway for rapid long-distance signaltransmission within the CNS.’’ Moreover, these authors(Cornell-Bell et al., 1990) proposed that ‘‘if Ca21 activityin the network of astrocytes constituted another form ofintercellular communication, such signaling should havea physiological relevance influencing neuronal activity,and thus being bi-directional.’’ Work by several independ-ent groups showed that indeed there is a reciprocal com-munication between neurons and astrocytes. Hippocam-pal neuronal activity was shown to trigger calcium wavesin astrocyte networks (Dani et al., 1992) and astrocyte cal-cium waves were shown to modulate neuronal activity(Dani et al., 1992; Kang et al., 1998; Nedergaard, 1994;Parpura et al., 1994; Parri et al., 2001). The mode bywhich astrocyte calcium signals affect synaptic transmis-sion was then shown to be dependent on regulated exocy-tosis of stored glutamate, ATP, and D-serine (Bezzi et al.,2004; Coco et al., 2003; Mothet et al., 2005; Parpura et al.,1994; Pascual et al., 2005). Consequently, the pre- andpost-synaptic components of neuronal transmission gaineda new partner, the perisynaptic glia, forming togetherwhat was initially termed the ‘‘tripartite-synapse’’ (Araqueet al., 1998a,b). Because these studies indicated that theelectrically silent astrocytes were active participants ofCNS information processing, it became plausible to con-sider that astrocytes are nevertheless ‘‘excitable’’ cells,with Ca21 fluctuations being the signal by which they re-spond, integrate, and convey information.

Although with the limitation of the studies performedin culture, pioneering experiments (Cornell-Bell et al.,1990) generated insightful ideas that, followed by experi-mental evidence, brought a new perspective to the role ofglial cells in CNS function. Of note is their report on thespatial and temporal pattern changes that occur after100 lM glutamate application. Following the initial Ca21

elevation induced by receptor activation, oscillatory Ca21

Grant sponsor: National Institutes of Health; Grant number: RO1-NS41023 toES; Grant sponsor: INSERM.

*Correspondence to: Eliana Scemes, Department of Neuroscience, Kennedy Center,Room No. 203, Albert Einstein College of Medicine, 1410 Pelham Parkway, Bronx, NY10461, USA. E-mail: [email protected]; Christian Giaume, INSERM-U587, Col-lege de France, 11 Place Marcelin Berthelot, Paris 75005, France.E-mail: christian.giaume@college-de-france

Received 21 March 2006; Accepted 23 May 2006

DOI 10.1002/glia.20374

Published online 26 September 2006 in Wiley InterScience (www.interscience.wiley.com).

GLIA 54:716–725 (2006)

VVC 2006 Wiley-Liss, Inc.

fluctuations preceded the intercellular spread of Ca21.This oscillatory behavior persisted for long periods oftime (5–30 min) with variable frequencies (10–110 mHz).A direct correlation between glutamate concentration andfrequency of oscillations was observed: at low concentra-tions (below 1 lM) intracellular Ca21 transients wereasynchronous and localized, whereas at higher concentra-tions (10–100 lM) intercellular Ca21 waves propagatedover long distances. This indicated that small fluctuationsin intracellular Ca21 could be integrated to generate aglobal intracellular response, which could be transmittedthroughout the astrocytic network. Recent development ofin vivo imaging, using two-photon microscopy, has pro-vided evidence for coordinated astrocyte Ca21 activity inthe neocortex of rats (Hirase et al., 2004).

Although many of the questions that were raised whenCa21 waves were first described have been answered,many are still debated and others have been generated.This review is not intended to revisit the concept and evi-dence that culminated in this new line of investigation, forwhich several recent reviews are available (Charles andGiaume, 2002; Nedergaard et al., 2003; Scemes, 2000).Instead, this review will focus on the characteristicsand consequences of intercellular Ca21 signaling inastrocytes and their relevance to CNS function. Because

‘‘gliotransmitter’’ released from astrocytes (the mechan-isms by which this release is accomplished are discussedin the other reviews), besides affecting synaptic transmis-sion and brain microcirculation, is also likely to serve asan autocrine signal, we will consider in this review someimportant aspects of this feedback mechanism for thetransmission of Ca21 waves between astrocytes.

CA21 WAVES IN VITRO AND IN VIVO: WHENAND WHERE DO THEY OCCUR?

A calcium wave is defined as a localized increase in cy-tosolic Ca21 that is followed by a succession of similarevents in a wave-like fashion. These Ca21 waves can berestricted to one cell (intracellular) or transmitted to neigh-boring cells (intercellular) (Fig. 1A).

The basic steps that lead to intracellular Ca21 waves inastrocytes usually involve the activation of G-protein-coupled receptors, activation of phospholipase C, and theproduction of IP3, which following IP3R activation leads toCa21 release from the endoplasmic reticulum (ER) (Golo-vina and Blaustein, 2000; Scemes, 2000; Sheppard et al.,1997). These intracellular Ca21 signals are spatially andtemporally complex events involving the recruitment of

Fig. 1. Intercellular Ca21 waves and their intracellular targets. (A)The transmission of intercellular Ca21 signals between astrocytes is illu-strated in the sequential images obtained from Fluo-3-AM loaded spinalcord astrocytes. Mechanical stimulation (arrow) of a single astrocyte inculture induces intracellular Ca21 elevation (displayed as an increase influorescence intensity) in the stimulated cells, which is then followed byCa21 increases in neighboring astrocytes. Images were acquired with anOrca-ER CCD camara attached to Nikon TE2000 inverted microscopeequipped with a 310 objective, using Metafluor software. Bar: 50 lm.(B) The diagram illustrates the major intracellular targets of cytosolicCa21 fluctuations in astrocytes. Elevation of intracellular Ca21 levels is

shown to affect (arrows) several plasma membrane proteins (symbolsrefer from left to right to metabotropic receptors, K1 (Ca21) channels,Na1/Ca21 exchanger, and Ca21-ATPase), as well as intracellular ones.The inositol-trisphosphate receptors (IP3R) are located at the endoplas-mic reticulum (ER) where Ca21 exert a cooperative action. Rises in[Ca21]i also target several cytoskeleton elements (cytosk), enzymes (E),and vesicles involved on the release of ‘‘gliotransmitters’’. Finally, Ca21

and the Ca21 liberating second messenger IP3 permeate gap junctionchannels and then act on similar intracellular targets in neighboringcoupled cells.

717INTERCELLULAR CALCIUM WAVES

GLIA DOI 10.1002/glia

elementary Ca21 release sites (Ca21 puffs: Parker andYao, 1994), which then propagate throughout the cell byan amplification mechanism. This amplification involvesfour components, two of which depend on positive andtwo on negative feedback mechanisms provided byreleased Ca21. These feedback mechanisms are: (a) theactivation of nearby IP3Rs due to the co-agonistic action ofCa21 on these receptors (Bezprovanny and Ehrlich, 1995;Finch et al., 1991; Yao et al., 1995), (b) the additional gen-eration of IP3 through the Ca21-dependent activation ofPLC (Berridge, 1993; Venance et al., 1997), (c) the buffer-ing power of mitochondria, attenuating the excess Ca21

levels at IP3R microdomains that otherwise would reducethe sensitivity of these receptors to IP3 (Boitier et al.,1999; Simpson et al., 1998), and (d) the presence of endog-enous low affinity Ca21 buffers (calcium binding proteins)that limit the diffusion of Ca21 ions within single astro-cytes (Wang et al., 1997).

Once triggered, intracellular Ca21 waves can be trans-mitted to neighboring cells as ICWs. Regardless of themechanism by which these waves may travel (see detailsthat follow), the mechanism that triggers Ca21 transientsin adjacent astrocytes relies on IP3 production and subse-quent release of Ca21 from the ER, as summarized abovefor intracellular Ca21 waves. Therefore, the extent towhich these intercellular Ca21 waves can travel are gov-erned by the effective diffusion properties of the Ca21

mobilizing signaling molecules within and between cells.ICW spread between astrocytes derived from cell cul-

ture, brain slice, and whole retina preparations has beenobserved following pharmacological, electrical, and me-chanical stimulation (for review see Boitier et al., 1999;Charles, 1998; Charles and Giaume, 2002; Giaume andVenance, 1998; Newman, 2004; Scemes, 2000; Simpsonet al., 1998). Although there are some differences regard-ing the distance and shape of ICWs generated by eachtype of stimulation, once they are initiated, the velocitiesby which they travel between astrocytes are fairly simi-lar, independent of the mode of stimulation and type ofpreparation. For instance, pharmacological, mechanical,and electrical stimulation of rat retina induced ICWs thattraveled at a mean speed of 23 lm/sec (Newman andZahs, 1997); pharmacologically and mechanically-inducedintercellular Ca21 waves between cultured astrocytestravel at a mean velocity of 18 lm/sec (for reviews seeGiaume and Venance, 1998; Scemes, 2000), and in theelectrically stimulated brain slice preparations, the meanCa21 wave velocity is 15 lm/sec (Dani et al., 1992; Hasset al., 2005; Schipke et al., 2002). In contrast to the veloc-ity of Ca21 wave spread, the extent to which ICW travelsis highly variable, even when comparing a single type ofstimulation among different types of preparations. Forexample, differences in the number of cells participatingin ICW transmission following mechanical stimulationwere observed among cultures of astrocytes prepared fromdistinct brain regions; ICWs spread to twice as many cor-tical and hippocampal astrocytes as between astrocytesfrom the hypothalamus and brain stem (Blomstrandet al., 1999). Similarly, mechanically induced Ca21 wavesbetween cultured telencephalic astrocytes spread radially

to an area of 450 lm2 comprising about 400 cells whilewaves traveling between cultured diencephalic astrocytesspread unevenly to an area of 130 lm2 recruiting about100 cells (Peters et al., 2005).

Regardless of the notorious differences between cultureand in situ conditions (e.g., morphology of astrocytes, sizeof the extracellular space), some generalizations can bemade with respect to the properties of ICWs. Based on thestudies reported above, it is likely that some of the charac-teristics of Ca21 waves are governed by the intrinsic prop-erties of the astrocytic Ca21 signaling toolkit (Berridgeet al., 2000a,b)—G-coupled membrane receptors, Ca21

mobilizing second messengers, ER, Ca21 buffering mole-cules and intracellular organelles—that once fully activatedto produce an intracellular Ca21 wave can be transmittedto an adjacent astrocyte, with a velocity that is independ-ent of the type of preparation used, i.e., independent ofthe morphological differences. Moreover, even consideringthat the extent of ICW spread is vastly larger in cultureconditions than in brain slices, most likely due to the re-stricted extracellular space in the latter, the studies per-formed in cultured astrocytes from different brain regionsdescribed above suggest that the distance of transmissionof ICW is likely related to heterogeneous populations ofastrocytes. Differences in type and number of membranereceptors and gap junction channels, main components ofastrocyte intercellular Ca21 signaling (see following), arelikely responsible for defining boundaries of communicat-ing networks.

Although these in vitro studies indicate that astrocytescan to some variable extent and degree transmitICWs, the magnitude of the stimuli necessary to triggerthis form of Ca21signaling is usually considerably higherthan would be expected to occur under physiologicalsituation. Thus, the existence and relevance of this formof astrocytic communication for CNS function may bequestioned at least in normal, physiological situations. Inwhole-mount retinas, flickering light stimulation indu-ces Ca21 transients, but not intercellular Ca21 waves inMueller cells of the inner plexiform layer; however, in thepresence of adenosine, light flashes enhance Mueller cellresponses and induce ICW spread between these glialcells (Newman, 2005). This study suggested that ICWpropagation between Mueller cells may occur under non-physiological conditions, such as under hypoxic condi-tions where adenosine levels are significantly increased(Ribelayga and Mangel, 2005). However, it should benoted that adenosine levels in retinas also fluctuate in acircadian and light-dependent manner (Ribelayga andMangel, 2005) and therefore, ICW spread between Muel-ler cells may occur under physiological conditions. Simi-larly, using two-photon laser scanning microscopy tomonitor in vivo Ca21 dynamics in astrocytes from superfi-cial cortical layer of rat brains, Hirase et al. (2004)recorded low frequency Ca21 transients in single astro-cytes that could be spread, although within a very limitedrange, between few neighboring cells with a low but non-zero level of coordination; the degree of such coordinatedCa21 activity between neighboring astrocytes was foundto be positively correlated with neuronal discharge, as

718 SCEMES AND GIAUME


observed following blockade of neuronal GABAA recep-tors with bicuculline. Thus, this study provided evidencethat enhanced but not seizure-related neuronal activityinfluences intercellular glia communication in the intactbrain.

In view of what is known so far about ICWs in astro-cytes, it is likely that this form of Ca21 signal transmis-sion play important role under pathological but not underphysiological conditions. With the exception of early CNSdevelopmental stages, when spontaneous Ca21 waves arefrequently observed and implicated in the generation, dif-ferentiation, and migration of neural cells (Feller et al.,1996; Gu and Spitzer, 1995; Kumada and Komuro, 2004;Scemes et al., 2003; Weissman et al., 2004; Wong et al.,1995), spontaneous ICWs are rarely seen in the matureCNS, even following physiological stimulation.

PATHWAYS FOR INTERCELLULARTRANSMISSION OF CA2+ SIGNALS

IN ASTROCYTES

There are two possible pathways by which Ca21 signalscan be transmitted between cells; one involves the trans-fer of Ca21 mobilizing second messengers directly fromthe cytosol of one cell to that of an adjacent one throughgap junction intercellular channels and the other involvesthe ‘‘de novo’’ generation of such messengers in neighbor-ing cells through the activation of membrane receptorsdue to extracellular diffusion of agonists. These two path-ways are not mutually exclusive but are likely to work inconjunction to provide coordinated activity within groupsof cells.

Gap junction mediated transmission of ICWs was thefirst pathway identified in astrocytes (Finkbeiner, 1992).In this study it was shown that neither the direction northe velocity of glutamate-induced intercellular Ca21

waves were affected by rapid superfusion and that twogap junction channel blockers impaired Ca21 wave spreadbetween astrocytes without affecting this spread withincells. This finding together with several others performedin different systems (Blomstrand et al., 1999; Charleset al., 1991, 1992; Enkvist and McCarthy, 1992; Guanet al., 1997; Leybaert et al., 1998; Nedergaard, 1994; Saezet al., 1989; Scemes et al., 1998; Venance et al., 1995) pro-vided a strong basis supporting the view that gap junctionchannels played a crucial role in the transmission of Ca21

signals between astrocytes. It should be mentioned, how-ever, that some of these early experiments performedusing compounds that block gap junction channels mayhave not allowed the selective discrimination between gapjunction-dependent and -independent pathways for ICWtransmission. For instance, several gap junction channel/hemichannel blockers (heptanol, octanol, carbenoxolone,flufenamic acid, and mefloquine) have been recentlyreported to prevent ATP-dependent amplification of ICWsunder low divalent cation solutions by acting on P2X7

receptors (Suadicani et al., 2006).Evidence for the participation of an extracellular path-

way for the spread of intercellular Ca21 waves in astro-

cytes, first described in the non-coupled mast cells (Osip-chuk and Cahalan, 1992), was provided by Enkvist andMcCarthy (1992), showing that Ca21 waves could crosscell bare areas in confluent cultures of cerebral astrocytes.Later, Hassinger et al. (1996) showed that electrically-induced Ca21 waves in cultured astrocytes were able tocross cell-free areas up to 120 lm and travel with similarvelocity between confluent cells and through the acellularlanes; moreover, the extent and direction of waves travel-ing in confluent cultures were shown to be affected bysuperfusion (Hassinger et al., 1996). It was then demon-strated that ATP was the extracellular molecule releasedby stimulated astrocytes and that this messenger alsomediated the acellular transmission of Ca21 waves (Guthrieet al., 1999). Such findings provided an explanation forthe results showing that Ca21 waves were not totally abol-ished in conditions where coupling was reduced, as in thecase of astrocytes derived from Cx43-null mice (Nauset al., 1997; Scemes et al., 1998), or astrocytes treatedwith oleomide and anandamide, a condition that totallyprevented dye- and electrical-coupling (Guan et al., 1997),and why blocking purinergic receptors with the P2R an-tagonist suramin totally prevented this spread (Guanet al., 1997; Zanotti and Charles, 1997).

As mentioned above, calcium signals can be transmittedthrough the diffusion of an extracellular molecule actingon membrane receptors. Except for striatal astrocytes, whichdisplay a prominent Ca21 response to glutamate (Cornell-Bell et al., 1990; Finkbeiner, 1992), astrocytes from otherbrain regions, including the cortex (Guthrie et al., 1999),retina (Newman and Zahs, 1997) and spinal cord (Scemeset al., 2000; Scemes, unpublished observations), are moreresponsive to ATP than to glutamate. Astrocytes in situand in vitro express, at different levels, several ionotropicand metabotropic P2 purinergic receptors, some of whichhave been implicated in the transmission of calcium sig-nals (Fumagalli et al., 2003). Among the metabotropicP2Y receptors, the P2Y1R and P2Y2R subtypes are likelythose predominantly expressed in astrocytes (Ho et al.,1995; Idestrup and Salter, 1998; Zhu and Kimelberg,2001, 2004). Although both of these G-coupled P2Rs gen-erate PLC and IP3 upon stimulation and thus contributeto generating Ca21 elevations, they differ with regard totheir sensitivity and selectivity for nucleotides; with theexception of ATP that is an agonist (in the micromolarrange) at both receptors, the purine diphosphate nucleo-tide ADP is a potent agonist (in the nanomolar range)at the P2Y1R while the pyrimidine triphosphate nucleo-tide UTP stimulates (in the micromolar range) P2Y2Rbut not P2Y1R (Burnstock and Knight, 2004). Given thepresence of ectonucleotidases at the cell surface thatreadily hydrolyze ATP to form ADP before the full hydro-lysis into adenosine (Wink et al., 2006), signaling throughP2Y1R activation is likely to predominate.

Using a P2R-null astrocytoma (1321N1) cell line toselectively express P2YR, Gallagher and Salter (2003)and Suadicani et al. (2004) reported that both P2Y1R andP2Y2R were equally efficient to sustain Ca21 wave spreadand that, however, the properties (velocity, extent, andshape) of these waves differed. Although differences in



sensitivity of these two P2 receptor subtypes to ATP canin part explain why ICWs traveling between P2Y1R andP2Y2R differ (Gallagher et al., 2003), it is also likely thatgap junction channels contribute to modulating the veloc-ity, distance, and the shape of these extracellular gener-ated waves. For instance, by overexpressing the Cx43 inthis poorly coupled astrocytoma cell line stably expres-sing P2Y1R and P2Y2R subtypes, a 40% decrease in therate of ICW transmission was observed (Suadicani et al.,2004). Moreover, overexpression of Cx43 in P2Y1R andP2Y4R-cells caused dramatic changes in the shape anddistance of ICW spread; the non-wave shape, saltatorybehavior of Ca21 waves traveling amongst poorly coupledP2Y1R expressing cells assumed a more homogeneous,wave-shaped form in better coupled P2Y1R astrocytomacells, while the limited spread of Ca21 signals providedby P2Y4R, was practically abolished by increasing gapjunctional communication (Suadicani et al., 2004).

These studies illustrate that the properties of Ca21 sig-nal transmission not only depend on the (sub)-type ofmembrane receptors but also on the degree of gap junc-tion mediated intercellular coupling. If, for example, anagonist (at its maximal effective concentration at a parti-cular receptor) is not sufficient to generate appropriatequantities of Ca21 mobilizing second messengers (e.g.,IP3) to sustain long distance Ca21 signal transmission,the increase in the effective volume of the intracellularcompartment provided by the gap junction channels willcertainly dissipate the gradient to levels below thresholdand the wave will be terminated (Giaume and Venance,1998; Suadicani et al., 2004). Alternatively, gap junctionchannels could recruit non-responsive cells into a net-work of cells expressing receptors that more efficientlygenerate second messengers (Venance et al., 1998). Thus,as initially proposed (Hassinger et al., 1996), both gapjunction dependent and independent pathways partici-pate in the transmission of Ca21 signals between astro-cytes; however, the relative contribution of each of thesepathways is likely to depend upon developmental, re-gional, and physiological states. Accordingly, it has beenrecently shown in acute brain slices that depending onthe brain regions (cortex versus hippocampus and corpuscallosum), the pathway mediating the transmission ofCa21 signals in brain slices is different (Haas et al.,2006). Another example illustrating that Ca21 waves canutilize different routes when traveling between glial cellswas provided in whole mounts of mouse retina, whereastrocyte-to-astrocyte Ca21 waves are mainly mediatedby the diffusion of second messengers through gap junc-tion channels, whereas astrocyte-to-Mueller cells are ba-sically dependent on the diffusion of ATP through theextracellular space (Newman, 2001, 2003, 2004).

REGENERATIVE VS NON-REGENERATIVEINTERCELLULAR CA21 WAVE TRANSMISSION

For both gap junction-dependent and -independentpathways discussed above, the basic law governing Ca21

signal transmission is provided by diffusion equations,

and therefore, diffusional parameters are expected to playa significant role limiting the extent to which these signalspropagate. However, regenerative models for Ca21 trans-mission have been proposed. These models are derivedfrom evidence obtained from several independent groupsindicating that, upon stimulation, astrocytes were able torelease ATP and glutamate (Ballerini et al., 1996; Kimel-berg et al., 1990, 1995; Parpura et al., 1994, 1995; Queirozet al., 1997, 1999). The release of these Ca21-mobilizing‘‘gliotransmitters’’ can potentially feedback on the astrocy-tic population, in an autocrine fashion, thus amplifyingthe extent to which these Ca21 signals are transmitted(Stout et al., 2002; Suadicani et al., 2006). In the studiesperformed by Hassinger et al. (1996) and Guthrie et al.(1999) showing that astrocyte Ca21 waves depended uponthe release of the extracellular messenger ATP, theauthors also proposed a mechanistic model by which ATPreleased from the stimulated cells would activate P2Rreceptors, which would then lead to mobilization of intra-cellular Ca21 in the neighboring cell that in turn would befollowed by the release of ATP from this neighboring cell.This succession of events would then occur sequentiallyalong the ICW path. Although recent evidence supportsthe hypothesis that ATP induces ATP release from astro-cytes (Anderson et al., 2004), this regenerative ATPrelease model, however, does not explain why Ca21 wavestravel within defined limits. By taking advantage of thedominant role of gap junctions in the propagation of cal-cium waves in rat striatal astrocytes, Giaume andVenance (1998) proposed that limiting factors linked to in-tracellular calcium signaling (PLC activity, calcium buf-fering, filling and re-filling of internal stores) contribute tosetting a threshold in cells at the edge of the ICW thatresulted in stopping the propagation. However, this pro-cess of ICW spread through gap junctions is not totallypassive but requires a regenerative process dependentupon calcium activation of PLCg to produce new IP3 inthe receiving cells. This regenerative but limited intercel-lular signaling was then tested by a mathematicalapproach that reproduced the propagation properties ofICWs (Hofer et al., 2002).

As a counter-proposal for such regenerative model ofCa21 wave transmission, Nedergaard’s group (Arcuinoet al., 2002; Cotrina et al., 1998, 2000) suggested a non-re-generative model based on a point source of ATP release,such that ATP released from a single cell would diffuseand stimulate a limited number of nearby cells. Evidencein favor of such a non-regenerative model of Ca21 wavetransmission is their observation that only cells located atthe epicenter of a spontaneously generated Ca21 wavewere permeable to molecules (propidium iodide; 562 Da)large enough to allow the release of ATP (Arcuino et al.,2002) and that the distance traveled by waves crossingcell-free areas was the same as those traveling betweencells (Arcuino et al., 2004). This point source release mech-anism of initiation of ICWs from the stimulated cell, to-gether with P2R activation and gap junction-mediated dif-fusion of IP3, have been incorporated into a recent mathe-matical model of ICW transmission in astrocytes (Iacobaset al., 2006).



Although all evidence so far generated indicates thatCa21 waves are spatially restricted, it is technically diffi-cult to distinguish between the regenerative and non-re-generative models. For instance, it is possible that due tothe presence of ectonucleotidases at the membrane sur-face of cells, and/or the hydrolysis of ATP on the extracel-lular solution, the concentration of extracellular ATPdecreases with distance of stimulation and therefore theconcentration of the agonist will not attain the levelsnecessary to trigger ATP release in cells located at thewave front. In favor of this hypothesis are the observa-tions that the concentration of ATP in the extracellularsolution declines 10-fold (from 78 lM to 6.8 lM) over adistance of 100 lm (Newman, 2001) and that the EC50

value necessary for ATP to induce ATP release fromastrocytes is 144 lM (Anderson et al., 2004).

Also important for the evaluation of whether or notand the extent to which ICW transmission involves a re-generative process is certainly dependent upon studiesaimed to identify the pathways of gliotransmitter releaseas well as the stimulation parameters necessary to acti-vate a particular pathway. In this regard, several sites ofgliotransmitter release have been identified, includingexocytotic vesicles, anion channels, pore-forming P2X7

receptors, and connexin hemichannels. The properties,relevance, and evidence in favor or against each of thepathways are reviewed in this special issue.

POTENTIAL TARGETS FOR CALCIUMWAVES IN ASTROCYTES

If one considers that calcium signaling is a major wayby which astrocytes encode and transmit information, it islikely that during the passage of a wave several calcium-dependent targets would be activated leading to changesin astrocytes engaged in the wave. Moreover, in certaincases the passage of a wave could lead to the priming ofthe astrocytes, thus leaving a ‘‘print’’ that persists andmodifies forthcoming astrocytic responses, setting the cel-lular basis for plasticity in glial cells. The understandingof the consequences of the engagement of these calcium-dependent events should provide a more comprehensivepicture of what is the role of these ICWs. Of course toreach such a goal, the spatial distribution, frequency, am-plitude, and pattern of propagating events would have tobe considered. In addition, the possibility of interactionbetween several waves triggered from different siteswould also be of importance. Actually, we are far from thatand what can be realistically achieved first is the identifi-cation of potential targets that could be activated follow-ing the occurrence of a calcium wave. In fact, besides thegeneration of the calcium-dependent release of ‘‘gliotrans-mitters’’ that will be reviewed in the following sections,several categories of cellular and molecular events can betargets that are affected during or after the propagation ofcalcium waves (Fig. 1B). Such targets include membraneeffectors whose activity is calcium-dependent. First, thereis now abundant evidence indicating that astrocytesexpress various classes of calcium-dependent ion channels

(see Olsen, 2005), mainly potassium channels. Indeed,three K1 channel types (BK, IK, and SK) can be distin-guished by their biophysical properties, calcium-sensitiv-ity, and their differential pharmacology with regard to tox-ins. So far, BK and SK, which are sensitive to [Ca21]i inthe micromolar and nanomolar ranges, respectively, havebeen described in astrocytes (Armstrong et al., 2005;Byschkov et al., 2001; Gebremedhim et al., 2003; Nowaket al., 1987; Price et al., 2002; Quandt and MacVicar,1986). They constitute potential targets of propagatingcalcium waves and both channel types have been shownto be activated by endothelin-1 (Bychkov et al., 2001), avasoactive peptide that was also reported to trigger cal-cium waves in cultured astrocytes (Venance et al., 1997).Interestingly, this peptide induces a biphasic response inastrocytes, which results in a transient depolarization fol-lowed by a sustained hyperpolarization (Bychkov et al.,2001). Second, calcium waves may also be important toamplify calcium signals by activating calcium release frominternal stores mainly in the endoplasmic reticulum;indeed such rise in [Ca21]i can operate either through acalcium-induced calcium release process involving ryano-dine receptor type 3 (Matyash et al., 2001) or by a coopera-tive activation of IP3 receptors (Marchant and Taylor,1997; Meyer and Stryer, 1988). However, since in astro-cytes the main source of Ca21 release from internal storesis mediated through the activation of IP3 receptors(Charles et al., 1993, Venance et al., 1997), amplificationof a rise in [Ca21]i may operate through this pathway.Third, the activity of the Na1/Ca21 exchanger can beincreased as a secondary step of the rise in [Ca21]i (Cara-foli and Chiesi, 1992). In astrocytes, this exchanger hasbeen identified at the plasma and the endoplasmic reticu-lum membranes and may be an important mechanism bywhich glia regulate the ionic content within the cytoplasmand of the extracellular space (see Olsen, 2005).

Biochemical cascades could also be targeted followingICWs since Ca21 binds to various molecular targets thattrigger or contribute to intracellular signal transductionpathways. These include the Ca21-dependent phospholi-pases such as sub-classes of PLC and PLA2. These phos-pholipases also might provide a certain degree of facilita-tion, given that during the waves, their activation causesan increase in the efficacy of metabotropic receptors towhich they are coupled (Glowinski et al., 1994; Oertnerand Matus, 2005). Interestingly, this could be the case ofP2Y metabotropic receptors coupled to PLC that are in-volved in the control of the extent of ICW spread (seeabove). Moreover, downstream calcium-dependent elementssuch as the phosphatases and protein kinases, includingthe two most widely studied calcium-sensitive protein ki-nases PKC and CaM kinase, are also potential targets ofcalcium waves. These facilitatory mechanisms can lead tothe production of second or transmembrane messengersthat may act distally and affect the properties of neighbor-ing astrocytes and also those of surrounding neurons andsmooth muscles, and endothelial cells. Finally, thesetransduction pathways can also interact with cytoplasmicenzymes linked to calcium-dependent effectors and fur-ther transfer signals to the nucleus and thus control gene



expression. In addition, cytoskeletal elements can be in-direct targets of the propagating rises in [Ca21]i. Forinstance, in neurons many calcium-dependent proteinsthat regulate the turn-over of filamentous actin have beenproposed to transduce intracellular Ca21 levels into spineshape changes (Oertner and Matus, 2005). Interestingly,rapid spontaneous motility was recently reported to occurin astrocytes at processes close to active synaptic term-inals, a location expected to induce calcium responses inastrocytes (Hirrlinger et al., 2004). Thus, changes in[Ca21]i during the propagation of waves could be asso-ciated with rapid motility and morphological changes ofastrocytes.

Finally, the radius of action (<5 lm) and the life-time(<1 ms) of calcium ions are rather limited in the cytoplasmmaking Ca21 itself a restricted rather than a global mes-senger (Allbritton et al., 1992). However, when [Ca21]i isincreased in astrocytes it may diffuse to neighboring astro-cytes through gap junction channels, which are known tobe permeable to Ca21 (Saez et al., 1989), only if the site of[Ca21]i rise occurs in the vicinity of these intercellularchannels. Indeed, although the main permeating messen-ger is thought to be IP3 (Venance et al., 1997), Ca21 canalso contribute to the propagation process (Giaume andVenance, 1998). In this case, it is then expected that Ca21

entering within a coupled cells through this pathway cansecondarily activate the above listed elements.

FUNCTIONAL CONSEQUENCES OFINTERCELLULAR CALCIUM WAVES

As the result of the activation of several of these molec-ular targets, the propagation of calcium waves may havefunctional consequences in astrocytes themselves and inthe interactions that they achieve with other cellularpartners. A first obvious interaction is that the rise in[Ca21]i might directly spread from an astrocyte to an ad-jacent neuron through gap junction channels and thussupport a direct mode of neuroglial interaction. Such arelationship was initially reported in co-cultures of neu-rons and astrocytes in which calcium waves initiated inglia were shown to induce calcium responses in someneurons that were blocked by gap junction inhibitors(Nedergaard, 1994). Although this interpretation waschallenged by studies demonstrating that glutamate re-lease from astrocytes is also involved (Parpura et al.,1994), several subsequent studies have confirmed thatfunctional gap junctions between these two cell types canbe formed in co-cultures and brain slices under certainconditions (Alvarez-Maubecin et al., 2000; Bittman et al.,2002; Froes et al., 1999). However, up to now there is noevidence for functional coupling between mature neuronsand astrocytes; thus this property could be rather limitedto early stages of CNS development.

Another consequence of calcium waves that has beenrecently well documented is the control of synaptic activity.This pioneering observation was performed in co-culturesby recording neuronal spontaneous and evoked activityduring the passage of an ICW triggered in the underlying

carpet of astrocytes (Araque et al., 1998a). This was thenconfirmed and complemented by brain slice studies, lead-ing to a full picture of an interaction loop involving activeneurons, astrocytic responses, and release mechanisms ofgliotransmitter that finally results in changes in neuronalactivity (Fellin and Carmignoto, 2004; Haydon, 2001; Vol-terra and Meldolesi, 2005). Such findings have contributedto the emerging concept of an active glial role in the controlof synaptic transmission in which intra- and intercellularcalcium signaling in astrocytes are key elements.

A new role of calcium waves was also recently demon-strated in the generation of Na1-mediated metabolicwaves in astrocytes (Bernardinelli et al., 2004). Indeed,glutamate released in the synaptic cleft is rapidly takenup by surrounding astrocytes and one consequence of thisuptake is the triggering of a molecular cascade that pro-vides metabolic substrates to neurons (Magistretti et al.,1999). This glutamate uptake results in an increase in in-tracellular Na1 and the activation of Na1/K1-ATPase.Single cell stimulation in cultured astrocytes generatesNa1 waves in parallel with Ca21 waves although the spa-tial and temporal properties of the waves carried by thetwo pathways are different. Moreover, Na1 waves giverise to spatially correlated increases in glucose up-take,indicating the occurrence of metabolic waves (Bernardi-nelli et al., 2004). While maneuvers that inhibit calciumwaves also inhibit Na1 waves, the inhibition of the Na1/K1 co-transporter or the enzymatic degradation of extra-cellular glutamate do not affect the propagation of calciumwaves. All together, these observations suggest that astro-cytes, through their properties by which they propagateintercellular calcium signals, also mediate Na1 and meta-bolic waves that should affect feeding of neurons and sec-ondarily their activity.

Interestingly, a single astrocyte can enwrap a largenumber of synapses and also be in contact with cerebralvessels (Peters, 1991; Simard et al., 2003; Ventura andHarris, 1999). Accordingly, another important consequenceof ICW propagation concerns the interface between astro-cytes and the vasculature. Indeed, the intimate relation-ship between astrocytic processes, termed ‘‘end-feet,’’ andblood vessels is well-known, which together contribute acellular architecture of gliovascular units (Nedergaardet al., 2003). Recently, in vivo imaging has provided con-vincing demonstrations of the functional role of such inter-face units. Indeed, [Ca21]i increases in astrocyte end-feethave been reported to produce either a dilation or a con-striction of blood vessels (Mulligan and MacVicar, 2004;Zonta et al., 2003). Although these observations seem con-flicting, this could be due to difference in pharmacologicaltreatments of the brain slices used (Peppiatt and Attwell,2004). Moreover, a recent report indicates that in acutelyisolated retina, light flashes and glial stimulation can bothevoke dilatation or constriction of arterioles (Metea andNewman, 2006). Finally, astrocytes in the somatosensorycortex in vivo have been shown to possess a powerfulmechanism for rapid vasodilation, indicating that one oftheir physiopathological roles is to mediate the control ofmicrocirculation in response to neuronal activity and itsdysfunction (Takano et al., 2006). Altogether, these obser-



vations are important because they could provide the basisto explain the contribution of astrocytes to deregulation ofcerebral circulation in brain pathologies.

CONCLUSIONS

There are still many aspects of astrocyte Ca21 signalingthat need to be further explored to appreciate in moredetail the importance of astrocyte ICW for brain functionunder physiological and pathological conditions (for re-views see Butt et al., 2004; Charles and Giaume, 2002;Ostrow and Sachs, 2005). An essential question to be ad-dressed is whether these waves occur in vivo and in whichsituation those global intracellular Ca21 transients aretransmitted to neighboring astrocytes. This still waits forestimations of the size of astrocyte networks involved in anICW, by determining the distribution and properties ofCa21-mobilizing membrane receptors and ion channels aswell as the distribution and properties of gap junctionalcommunication. For instance, to better resolve whetherand how astrocytes can integrate Ca21 signals, one has todetermine the components and cellular locations of theCa21 microdomains and to resolve the conditions in whichthe elementary Ca21 events (puffs) are transformed into aglobal Ca21 response. For that, the readers are certainlygoing to benefit from the reviews in the first half of this spe-cial issue describing the dynamics of Ca21 signals in glia.

Understanding the role of astrocyte Ca21 signals in CNSfunction also involves the identification of the intracellulartargets activated by these Ca21 transients, and the func-tional consequences of these activated pathways for theinteractions that astrocytes maintain with themselves andwith other cellular types. Moreover, to provide a more com-plete description of astrocyte Ca21 waves, a better knowl-edge of mechanisms by which transmitters are releasedfrom these cells is required. ‘‘Gliotransmitters’’ are not onlyexpected to affect neuronal activity and brain microcircula-tion, but are also expected to feedback on astrocytes andthus modulate their ‘‘Ca21 excitability’’. Because severalmechanisms of gliotransmitter release have been proposedin the last few years, it is fundamental to define the proper-ties and conditions in which each of these release sites arebrought into action. Presently, this field still faces contro-versial data and interpretations that may or may not becontradictory, but certainly needs to be discussed and ana-lyzed from the different viewpoints of workers in theseareas. We certainly consider that such a critical evaluationis timely, and we hope that by bringing together in this spe-cial issue of Glia expertise from different laboratories wewill provide the readers with state-of-the-art discussions onthe ‘‘mechanisms of transmitter release from astrocytes.’’

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astrocyte calcium waves: what they are and what they do

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