Molecular and Cellular Endocrinology 353 (2012) 45–56

Contents lists available at SciVerse ScienceDirect

Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

Review

Calcium signalling in astroglia

Alexei Verkhratsky a,b,c,⇑, José J. Rodríguez b,c, Vladimir Parpura b,c,d,e,⇑
a
Faculty of Life Sciences, The University of Manchester, Manchester, UK
b
IKERBASQUE, Basque Foundation for Science, 48011 Bilbao, Spain
c
Department of Neurosciences, University of the Basque Country UPV/EHU, 48940 Leioa, Spain
d
Department of Neurobiology, Center for Glial Biology in Medicine, Civitan International Research Center, Atomic Force Microscopy & Nanotechnology Laboratories, and
Evelyn F. McKnight Brain Institute, University of Alabama, Birmingham, USA
e
School of Medicine, University of Split, 21000 Split, Croatia

a r t i c l e i n f o a b s t r a c t

Article history: Astroglia possess excitability based on movements of Ca2+ ions between intracellular compartments and
Available online 10 September 2011 plasmalemmal Ca2+ fluxes. This ‘‘Ca2+ excitability’’ is controlled by several families of proteins located in
the plasma membrane, within the cytosol and in the intracellular organelles, most notably in the endo-
Keywords: plasmic reticulum (ER) and mitochondria. Accumulation of cytosolic Ca2+ can be caused by the entry of
Astroglia Ca2+ from the extracellular space through ionotropic receptors and store-operated channels expressed in
Calcium signalling astrocytes. Plasmalemmal Ca2+ ATP-ase and sodium–calcium exchanger extrude cytosolic Ca2+ to the
Endoplasmic reticulum
extracellular space; the exchanger can also operate in reverse, depending of the intercellular Na+ concen-
Mitochondria
Ca2+ channels
tration, to deliver Ca2+ to the cytosol. The ER internal store possesses inositol 1,4,5-trisphosphate recep-
Ionotropic receptors tors which can be activated upon stimulation of astrocytes through a multiple plasma membrane
metabotropic G-protein coupled receptors. This leads to release of Ca2+ from the ER and its elevation
in the cytosol, the level of which can be modulated by mitochondria. The mitochondrial uniporter takes
up Ca2+ into the matrix, while free Ca2+ exits the matrix through the mitochondrial Na+/Ca2+ exchanger as
well as via transient openings of the mitochondrial permeability transition pore. One of the prominent
consequences of astroglial Ca2+ excitability is gliotransmission, a release of transmitters from astroglia
which can lead to signalling to adjacent neurones.
Ó 2011 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Astroglia: definition, evolution and function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

2. Principles of calcium signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3. Calcium signalling in astrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.1. Endoplasmic reticulum provides for glial Ca2+ excitability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.2. The store-operated Ca2+ entry: a role for TRPC channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3.3. Ionotropic Ca2+ permeable receptors in astrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3.4. The sodium/calcium exchanger in astroglia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.5. Mitochondria in astroglial Ca2+ signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.6. Voltage-gated Ca2+ channels (VGCCs) in astroglia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

1. Astroglia: definition, evolution and function

⇑ Corresponding authors. Addresses: The University of Manchester, Oxford Road,
Manchester M13 9PT, UK. Tel.: +44 (0)161 2757324 (A. Verkhratsky); Department The term ‘‘astroglia’’ (which was named after the word ‘‘astro-
of Neurobiology, 1719 6th Avenue South, CIRC 429, University of Alabama,
cyte’’ invented by Michael von Lenhossek in 1891 (Lenhossek,
Birmingham, AL 35294, USA. Tel.: +1 205 996 7369; fax: +1 205 975 6320 (V.
Parpura). 1891, 1893), for historic account see also (Kettenmann and
E-mail addresses: alex.verkhratsky@manchester.ac.uk (A. Verkhratsky), Verkhratsky, 2008; Verkhratsky, 2006b)) is used to define all
vlad@uab.edu (V. Parp. non-myelinating macroglial cells in the central nervous system.

0303-7207/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mce.2011.08.039
46 A. Verkhratsky et al. / Molecular and Cellular Endocrinology 353 (2012) 45–56
These cells are represented by many subtypes which differ sub-
stantially in their morphology, location, general physiology, and
function (Matyash and Kettenmann, 2010; Verkhratsky and Butt,
2007). In fact only a minority of astrocytes have star-like morphol-
ogy, which was used a typifying criterion for their definition
(astron kytos – star-like cells). These cells were the neuroglia that
Ramón y Cajal visualized in 1913 using a gold sublimate stain,
which, as we know today targeted intermediate filaments that
consist mainly of glial fibrillary acidic protein (GFAP), a protein
contemporary used as an astroglial marker. However not all astro-
cytes are GFAP positive. Astroglia as a class of neural cells embrace
the radial glia, classical protoplasmic and fibrous astrocytes, radial
Müller retinal glial cells, pseudo-radial cerebellar Bergmann glial
cells, laminar and polarised astrocytes of the primate brain, velate
astrocytes of cerebellum, tanycytes that connect ventricular walls
with parts of hypothalamus and spinal cord, pituicytes in the
neuro-hypophysis, and perivascular and marginal astrocytes
(Kimelberg, 2010; Kimelberg and Nedergaard, 2010; Verkhratsky
and Butt, 2007) As if this is not sufficient, astroglia also include sev-
eral types of cells that line the ventricles or the subretinal space
represented by ependymocytes, choroid plexus cells and retinal
pigment epithelial cells.
There is however one basic and fundamental function which
unites all these cells – their primary responsibility is the control
of homeostasis of the nervous system (Heneka et al., 2010; Kimel-
berg, 2010; Verkhratsky and Butt, 2007; Verkhratsky et al., 2011).
This homeostatic function is executed at many levels, and includes
organ homeostasis (astroglia control the emergence and mainte-
nance of brain–blood barrier), cellular homeostasis (astroglia is
intimately involved in neurogenesis as both stem element and reg-
ulator) morphological homeostasis (astroglia defines the migratory
pathways for neural cells during development and shapes the mi-
cro-architecture of grey matter and controls synaptogenesis/syn-
aptic pruning), molecular homeostasis (which is represented by
regulation of ion, neurotransmitter and neurohormone concentra-
tion in the CNS extracellular space), metabolic homeostasis
(astroglia stores energy substrates in a form of glycogen and sup-
plies neurones with lactate) and defensive homeostasis (astroglio-
sis is the fundamental adaptive/defensive reaction of neural
tissue). Moreover astrocytes are possibly the main chemosensitive
elements of the brain that perceive systemic fluctuations in CO2,
pH and Na+ and thus regulate behavioural and systemic homeo-
static physiological responses (Gourine and Kasparov, 2011;
Gourine et al., 2010; Huckstepp et al., 2010; Shimizu et al., 2007).
Astroglial cells share the common ancestry with neurones and
oligodendrocytes all of them being scions of neuroepithelial cell,
and as such astrocytes are capable of expressing majority of mole-
cules that for a long time were believed to be exclusively present in
neurones (the most trilling examples being neurotransmitter
receptors and components of exocytotic release of neurotransmit-
ter-containing vesicles (Lalo et al., 2011b; Montana et al., 2006;
Parpura et al., 1994, 2011; Verkhratsky and Kettenmann, 1996)).
At the same time astroglial cells (as well as all other types of neu-
roglia) are electrically non-excitable being thus fundamentally dif-
ferent from neurones. The electrical excitability and fast chemical
synaptic transmission are the defining function of neurones; the
absolute bulk (90–95%) of energy consumed by the brain is spent
for maintaining electrical excitability and synaptic transmission
(Magistretti, 2009). The neurones are thus perfect elements of fast
information transfer, this perfection, however, was earned at the
expense of shifting all the house-holding, homeostatic and
defensive responsibilities to neuroglia.
In evolution, astrocytes appeared simultaneously with the
formation of the first centralised nervous system when neural cells
begun to assemble into the conglomerates known as neuronal gan-
glia. The most ancient proto-astrocytes known to us are the glial
cells of the Caenorhabditis elegans. In this worm 302 neurones are
assisted by 50 proto-astrocytes that cover dendrites of sensory
neurones and envelop the nerve ring (Oikonomou and Shaham,
2011). The proto-astrocytes of C. elegans are still very close to
neurones from which they evolved; for example these glial cells
express voltage-gated Ca2+ channels, produce depolarisation-
induced Ca2+ signals (Oikonomou and Shaham, 2011; Stout and
Parpura, 2011). At the same type proto-astrocytes of the C. elegans
specialise on new homeostatic functions regulating development
and general well being of neurones. Complete ablation of glial cells
from C. elegans nervous system seriously affects its development
and functional abilities, and yet neurones in the worm are still able
to survive in the absence of astroglia as they presumably have no
much need for glial trophic support, and the glia-less animals,
although disabled, remain alive (Bacaj et al., 2008; Oikonomou
and Shaham, 2011; Procko and Shaham, 2010; Reichenbach and
Pannicke, 2008).
Further evolution of the nervous system required more special-
isation form neurones and neuronal centres and coincided with a
critical increase in the importance of neuroglia (Deitmer et al.,
1999; Edwards and Meinertzhagen, 2010; Laming et al., 2000). In
the Annelids and Arthropods astrocytes become diversified, which
resulted in appearance of several major classes of glia (for example,
in the insects they include surface, cortex, neuropile and tract glia;
with further subdivision within the classes (Edwards and Mein-
ertzhagen, 2010)). Thus evolved and more complex glia becomes
critical for organising the nervous system of Arthropods by isolat-
ing the CNS from the body through formation of blood–brain bar-
rier and by isolating functionally distinct nervous centres. The
insect astroglia is fully homeostatically competent being involved
in trophic support, regulation of ion and neurotransmitter concen-
tration, brain development, circadian rhythms, etc. Most impor-
tantly at this evolutionary stage astroglia becomes the main
element of brain defence as the first programmes of reactive
astrogliosis become operative. All these changes make neuroglia
indispensable for the nervous system survival and the deletion of
the glial cells renders the nervous system unviable. Further evolu-
tion of the nervous system deepened the specialisation: neurones
became ultimately excitable elements that formed numerous func-
tionally separated nervous centres, whereas astroglia increased
their numerical presence, diversification, morphological complex-
ity and functional competence (Kimelberg, 2010; Laming et al.,
2000; Oberheim et al., 2006, 2009).
The wealth of homeostatic functions of astroglia necessitates
highly sophisticated signalling system(s) that are able to monitor
extracellular space, couple extracellular cues with cellular pro-
cesses and coordinate astroglial networks. The neuronal and glial
signalling systems evolved in parallel, with neurones perfecting
the electrical excitability (in close association with oligodendrog-
lia, which made possible the saltatory propagation of action poten-
tials) and refining the synaptic mechanisms (with the help of
astrocytes controlling the levels of neurotransmitters in the synap-
tic cleft). The glial cells, however, did not acquire plasmalemmal
electrical excitability, relying instead on excitable endomembranes
and intercellular gap junctions. The intracellular calcium signalling
system expressed in astrocytes seemingly represents the key ele-
ment in astroglial ‘‘excitability’’ and physiological operation.
2. Principles of calcium signalling
Calcium ions are arguably the most widespread and ubiquitous
signalling molecules operating in all types of living cells (Berridge
et al., 2000; Carafoli, 2002; Heilbrunn, 1943; Heilbrunn and
Wiercinsky, 1947; Petersen et al., 2005). Calcium was appointed
to this role very early in the evolution (Case et al., 2007), possibly
 A. Verkhratsky et al. / Molecular and Cellular Endocrinology 353 (2012) 45–56 47

when ATP was chosen as a main energy substrate: most of
reactions needed for ATP metabolism can proceed only at very
low Ca2+ concentrations (Burnstock and Verkhratsky, 2009). Fur-
ther, Ca2+ ions eagerly interact with various biological molecules
because of special properties of Ca2+ that include flexible coordina-
tion chemistry, high affinity for carboxylate oxygen, which is the
most frequent motif in amino acids, rapid binding kinetics, and ef-
fects on fluidity and fusion of cellular membranes (Jaiswal, 2001).
High Ca2+ concentrations, at the same time, are harmful for biolog-
ical material: excess of Ca2+ causes aggregation of proteins and nu-
cleic acids, affects the integrity of lipid membranes and initiates
the precipitation of phosphates. Therefore from the very dawn of
evolution the tight control over Ca2+ concentration in the cellular
compartments executed through containment of diffusional Ca2+
fluxes paired with active transport systems that pumped Ca2+ out
of the intracellular space (Case et al., 2007; Shemarova and Neste-
rov, 2005a,b). This tight control over Ca2+ is present throughout all
the life forms, and in all the living cells cytosolic Ca2+ is maintained
at comfortably low concentrations – somewhere around 100 nM,
this being 10,000–20,000 times lower than that in the extracellu-
lar milieu.
The huge Ca2+ concentration gradient aimed at the cytoplasm
forms the backbone for Ca2+ signalling by allowing diffusion of
Ca2+ ions between external environment and the cell interior.
The second fundamental principle of Ca2+ signalling is represented
by the existence of several intracellular compartments, which con-
trol Ca2+ movements in a very different way, therefore creating
steep Ca2+ concentration gradients within the cell (see e.g. Berridge
et al., 2003; Bregestovski and Spitzer, 2005; Knot et al., 2005; Kos-
tyuk and Verkhratsky, 1995; Petersen, 2005; Petersen et al., 2005;
Verkhratsky et al., 1998 and Fig. 1). Movements of Ca2+ between
these compartments and between the cell and extracellular space
produce very rapid, and quite often much localised, fluctuations
of free Ca2+ in different regions of the cell and within different
intracellular organelles; these fluctuations in turn exert signalling
action (Petersen, 2002; Petersen et al., 2005; Rizzuto and Pozzan,
2006).
The living cells contain a relatively limited number of Ca2+ han-
dling systems. Those include membrane Ca2+ channels (both
plasmalemmal and intracellular), cytoplasmic Ca2+ buffers and sev-
eral sets of membrane Ca2+ transporters represented by Ca2+
pumps and Ca2+ exchangers (Berridge et al., 2003; Kostyuk and
Verkhratsky, 1994, 1995; Petersen et al., 1994). These systems
have ancient evolutionary roots and were conserved in phylogen-
esis (Case et al., 2007; Shemarova and Nesterov, 2005a,b). By com-
bining different molecular components the versatility and
adaptability of Ca2+ signalling system is achieved because cells
can create the context-dependent ‘‘Ca2+ signalling toolkits’’ (Ber-
ridge et al., 2003), that tailor Ca2+ signalling events to match envi-
ronmental challenges. Indeed, Ca2+ signals resulting from cell
activation appear in multiple shapes, from local microdomains,
which, for example regulate neurotransmitter release, postsynap-
tic plasticity, or cell process guidance (Bolsover, 2005; Cavazzini
et al., 2005; Hartmann and Konnerth, 2005; Holcman et al.,
2005; Lohmann and Wong, 2005; Oertner and Matus, 2005; Red-
mond and Ghosh, 2005; Spitzer et al., 2005), through global Ca2+
signals, which control excitation–contraction or excitation–secre-
tion coupling (Kiselyov et al., 2003; Petersen, 2005; Wray et al.,
2005), regulate gene expression (Fields et al., 2005; Webb et al.,
2005) and tissue development (Puceat and Jaconi, 2005), to a prop-
agating Ca2+ waves accomplishing integration in complex multicel-
lular structures, such as, for example, in astroglial syncytia in the

Fig. 1. Principles of calcium signalling in astroglia. [Ca2+]i accumulation could be caused by the entry of Ca2+ from the extracellular space through ionotropic receptors or
store-operated channels (SOC). Plasmalemmal Ca2+ pumps/ATP-ases (PMCA) can extrude cytosolic Ca2+, while the plasmalemmal sodium–calcium exchanger (NCX) can
operate in both directions depending of intercellular Na+ concentration. An additional source of Ca2+ is available from the ER internal store that possesses InsP3 receptors,
which can be activated by the activity of metabotropic G-protein coupled receptors (GPCRs) and PLC. The ER store is (re)filled by the activity of the store-specific Ca2+-ATPase
(SERCA). Cytosolic Ca2+ levels can be affected by a variety of cytosolic Ca2+-binding proteins (CBPs) and by the action of mitochondria. A negative membrane potential exists
across the inner mitochondrial membrane. Mitochondrial Ca2+ uptake occurs through voltage-dependent anion channels (VDAC) present in the outer membrane and by the
uniporter in the inner membrane as the electrochemical gradient drives Ca2+ into the matrix, while free Ca2+ exits the mitochondrial matrix through the mitochondrial Na+/
Ca2+ exchanger and transient opening of the mitochondrial permeability transition pore (MPTP).
48 A. Verkhratsky et al. / Molecular and Cellular Endocrinology 353 (2012) 45–56
brain (Verkhratsky, 2006a; Verkhratsky and Toescu, 2006). At the
same time, the Ca2+ signalling machinery is responsible for the
control of cell survival, and numerous Ca2+-dependent pathways
trigger various modes of cell death, which are fundamental for nor-
mal tissue development and homeostasis (Nicotera et al., 2007;
Verkhratsky, 2007). Importantly, molecular components of Ca2+
homeostatic/signalling system are also controlled by Ca2+ ions
themselves, so that that changes in Ca2+ concentrations in different
cellular compartments affect the function and/or availability of
both Ca2+ channels and Ca2+ transporters (for example, plasmalem-
mal Ca2+ channels are generally subject for Ca2+-dependent inacti-
vation (Imredy and Yue, 1994; Morad et al., 1988; Rycroft and
Gibb, 2004a,b)); Ca2+ concentration gradient over the ER mem-
brane controls both availability of Ca2+ release channels for activa-
tion and efficacy of Ca2+ pumping through the endomembrane
(Burdakov et al., 2005; Guerrero-Hernandez et al., 2010; Koizumi
et al., 1999; Oldershaw and Taylor, 1993; Shmigol et al., 1996).
At the receiving end of Ca2+ signalling system ‘‘Ca2+ sensors’’
control cellular reactions. These sensors are represented by a mul-
titude of enzymes, which all are endowed with Ca2+ binding sites,
albeit with different affinities; Ca2+ binding to these binding sites
alters activity of the enzymes and therefore triggers or discontin-
ues diverse biochemical processes (Burgoyne et al., 2004; Carafoli,
2004; Carafoli et al., 2001). The differences in Ca2+ affinity of differ-
ent enzymatic systems allow for amplitude coding of Ca2+ signals.
The Ca2+-sensitive enzymes are often specifically concentrated in
cellular subcompartments, thus providing for precise spatial cod-
ing of the Ca2+ signals. Finally, the duration and kinetics of intracel-
lular Ca2+ fluctuations control the timing of Ca2+-binding/
unbinding to the ‘‘Ca2+ sensors’’ hence forming the basis for tempo-
ral coding of Ca2+ signalling events (Toescu and Verkhratsky, 1998).
Three main intracellular compartments in which Ca2+ signals
emerge and fall are (Fig. 1): the cytoplasm, the endoplasmic retic-
ulum and the mitochondria (although some other intracellular
organelles, such as e.g. Golgi complex, lysosomes and secretory
granules may participate in intracellular Ca2+ homeostasis and sig-
nalling – see e.g. Gerasimenko et al., 1996; Michelangeli et al.,
2005; Zhu et al., 2010). These compartments are formed by the
membranes represented by the plasmalemma that makes the out-
er cell border, by the endomembrane, which creates the endoplas-
mic reticulum and by the double mitochondrial membrane. Each of
these membranes contain distinct Ca2+ channels (that allow down-
hill Ca2+ diffusion) and transporters (which generally are responsi-
ble for Ca2+ transport against Ca2+ gradient); the properties of these
channels and transporters determine the impact on the shape of
Ca2+ signals (Carafoli et al., 2001; Toescu and Verkhratsky, 1998).
The second important determinant is the Ca2+ concentration and
electrochemical gradient. The free Ca2+ concentration in the cytosol
([Ca2+]i) is set in the range of 30–100 nM; the Ca2+ concentration in
the extracellular space normally ranges between 1 and 2 mM,
whereas Ca2+ concentration in the lumen of endoplasmic reticulum
([Ca2+]L) can be as high as 0.2–1 mM (Alonso et al., 1999; Mogami
et al., 1998; Solovyova and Verkhratsky, 2002; Solovyova et al.,
2002). The pre-existing concentration/electrochemical gradients
favour Ca2+ influx into the cytosol. Cell activation opens plasma-
lemmal or intracellular Ca2+ channels, which results in spatially
and temporally organised [Ca2+]i fluctuations. The intracellular
route for Ca2+ signalling generation is predominant in the electri-
cally non-excitable cells, as well as in electrically excitable cells
such as for instance in cardiac and skeletal muscle cells; conversely
in neurones the plasmalemmal Ca2+ pathway assumes higher
importance (Toescu and Verkhratsky, 1998). The rich complement
of voltage- and ligand-operated Ca2+ channels expressed in neuro-
nal membrane allows rapid transformation of membrane events
into cytosolic Ca2+ signals, which is critical for fast signalling idio-
syncratic for neuronal networks (Bregestovski and Spitzer, 2005;
Kostyuk and Verkhratsky, 1995). The neurones are also in posses-
sion of Ca2+-induced Ca2+ release which rapidly amplifies Ca2+ sig-
nals (Verkhratsky, 2005; Verkhratsky and Petersen, 2002;
Verkhratsky and Shmigol, 1996).
The spatial organisation of Ca2+ signals is determined by the cel-
lular distribution of Ca2+ channels and by intracellular Ca2+ biding
proteins generally known as Ca2+ buffers. In the cytosol Ca2+ buf-
fers are characterised by high (in a low nanomolar range) affinity
to Ca2+ ions (Ikura et al., 2002; Lewit-Bentley and Rety, 2000).
The cytosolic high affinity Ca2+ buffers tend to localise cytosolic
Ca2+ events and assist in forming a focal micro- or even nano-do-
mains of high (1–100 lM) cytosolic Ca2+, which in turn control
highly localised and rapid cellular reactions, such as for example
neurotransmitter release (Neher and Sakaba, 2008; Rizzuto and
Pozzan, 2006). The Ca2+ buffers expressed in the lumen or the ER
have low affinity (KD  0.5–1 mM), which permits relatively free
Ca2+ diffusion through the lumen of the endoplasmic reticulum
thus allowing long-range intracellular Ca2+ signalling through
‘‘Ca2+ tunnels’’ (Petersen and Verkhratsky, 2007; Solovyova and
Verkhratsky, 2003).
The termination of cytosolic Ca2+ signals is achieved by energy-
dependent Ca2+ transport against concentration gradients. In both
electrically excitable and non-excitable cells Ca2+ can be expelled
to the extracellular space by plasmalemmal Ca2+ pumps/ATP-ases
(PMCA), which utilities the ATP energy directly, or by sodium–cal-
cium exchanger (NCX), which uses the same ATP energy indirectly
in the form of preset transmembrane Na+ gradient (Guerini et al.,
2005). Alternatively Ca2+ can be taken up back to the ER lumen,
via sarco-(endo)plasmic reticulum Ca2+ ATP-ases (SERCA) (Van-
gheluwe et al., 2005).
Another cellular organelle critical for Ca2+ signalling and Ca2+
homeostasis is represented by mitochondria (Nicholls, 2005; Toe-
scu, 2000). The double membrane of mitochondria is endowed
with two main types of Ca2+-permeable channels. The outer mem-
brane contains the voltage-dependent anion channels that have
considerable Ca2+ permeability, whereas the inner membrane pos-
sesses the highly selective Ca2+channel usually referred to as Ca2+
uniporter (Rimessi et al., 2008). The mitochondria are hugely elec-
tronegative with regard to the cytosol (the mitochondrial mem-
brane potential, the Dw, created by the electron transport is
about 180 to 200 mV). This in turn generates the electric force
for Ca2+ aimed towards the mitochondrial matrix, when cytosolic
Ca2+ exceeds 300–400 nM (Miyata et al., 1991; Simpson and Rus-
sell, 1998). Therefore elevation of [Ca2+]i triggers substantial Ca2+
influx into the mitochondria; this Ca2+ influx depolarises the mito-
chondrial membrane hence stimulating ATP production (Rimessi
et al., 2008). In addition Ca2+ accumulation by the mitochondria
provides an additional Ca2+ buffer, which prevents large cytosolic
Ca2+ loads. The overload of mitochondria with free Ca2+ is danger-
ous and may initiate cell damage and various cell death programs
(Duchen et al., 2008; Nicotera and Orrenius, 1998; Toescu and Ver-
khratsky, 2004).
3. Calcium signalling in astrocytes
3.1. Endoplasmic reticulum provides for glial Ca2+ excitability
It is generally acknowledged that all types of glia operate a spe-
cial form of Ca2+ excitability (Verkhratsky et al., 1998). Numerous
experiments on neuroglial cells in vitro, in situ and in vivo have
identified the expression of multiple metabotropic receptors that,
when activated by physiological stimulation, trigger production
of inositol 1,4,5-trisphosphate (InsP3) and subsequent InsP3-
induced Ca2+ release from the ER (Finkbeiner, 1993; Hamilton
et al., 2008; Kastritsis et al., 1992; Kirischuk et al., 1996; McCarthy
 A. Verkhratsky et al. / Molecular and Cellular Endocrinology 353 (2012) 45–56 49

and Salm, 1991; Porter and McCarthy, 1995). Generally, Ca2+ sig-
nals induced by activation of metabotropic receptors (most notably
by ATP and glutamate) share the similar set of properties. These
[Ca2+]i transients persist in Ca2+-free extracellular solutions and
are sensitive to inhibition of SERCA pumps (by thapsigargin or by
cyclopiazonic acid) and to blockade of InsP3 receptors by intracel-
lular administration of heparin (Fig. 2, Kirischuk et al., 1995). It was
also found that intercellular diffusion of InsP3 and InsP3-induced
Ca2+ release represent the main mechanism of glial intercellular
Ca2+ waves that arguably underlie long-range signalling in glial
syncytia in vitro and in brain slices (Cornell Bell et al., 1990; Dani
et al., 1992; Giaume and Venance, 1998). Recently, synchronous
Ca2+ waves that embrace hundreds of astrocytes were also re-
corded in vivo; these Ca2+ waves were sensitive to inhibition of
gap junctions and purinoceptors suggesting their InsP3/ER origin
(Kuga et al., 2011).
The glial Ca2+ excitability is provided by the endomembrane en-
dowed with Ca2+ pumps and Ca2+ release channels both types of
molecules being regulated by cytosolic Ca2+. Regenerative opening
of intracellular Ca2+ channels creates the propagating wave of
endomembrane excitation; subsequent activation of SERCA pumps
terminates Ca2+ signal. The InsP3 receptors are also regarded as pri-
mary molecules that initiate Ca2+ release following physiological
stimulation. It seems that the type 2 InsP3 receptor is predomi-
nantly expressed in astroglial cells (Holtzclaw et al., 2002; Petra-
vicz et al., 2008; Sheppard et al., 1997). The InsP3 receptors are
often concentrated in the distal processes of astroglial cells (Holtz-
claw et al., 2002), where Ca2+ signals in response to metabotropic
stimulation seems to be initiated (Fig. 2D, Kirischuk et al., 1995).
The role for Ca2+-gated Ca2+ release channels/ryanodine receptors
(RyRs) in Ca2+ signalling in astroglia remains debatable. Activation
of RyRs by caffeine was observed in astrocytes cells in thalamus
(Parri and Crunelli, 2003). In contrast, little evidence was found
for RyRs-mediated Ca2+ signalling in hippocampal astrocytes (Beck
et al., 2004), despite the fact that astrocytes in the hippocampus
express RyRs receptors at both the mRNA and protein levels (Mat-
yash et al., 2002; Verkhratsky et al., 2002).
There has been a general consensus that Ca2+ ions released from
the ER via InsP3 induced Ca2+ release are critically important for the
exocytosis of transmitters from astroglial cells (Malarkey and Par-
pura, 2009; Parpura et al., 2011). The role for the ER Ca2+ release in
regulated vesicular release of neurotransmitters from astroglia was
initially demonstrated in cell cultures. It was found that inhibition
of SERCA pumps by thapsigargin, which lowers ER Ca2+ concentra-
tion due to an opposed background leakage, almost completely
eliminated Ca2+-dependent release of glutamate from astrocytes
(Innocenti et al., 2000; Jeremic et al., 2001). In depth analysis of
the contribution of ER calcium store to Ca2+-dependent glutamate
release from astrocytes was performed on cultured astrocytes (Hua
et al., 2004). It was found that thapsigargin reduced mechanically
induced glutamate release. Similarly, glutamate exocytosis was
inhibited by membrane-permeable antagonist of InsP3 receptors
(and of the store-operated Ca2+ entry) diphenylboric acid 2-amino-
ethyl ester (2-APB). Exposure of cultured astrocytes to 10 lM ryan-
odine for 10 min also decreased mechanically-induced glutamate
release. The pre-incubation of astrocytes with 10 mM of caffeine
(which acts as both inhibitor of InsP3 receptors and activator of
RyRs (Ehrlich et al., 1994; Wakui et al., 1990)) for 10 min similarly

Fig. 2. ATP-induced Ca2+ signalling in Bergmann glial cells results exclusively from inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release from ER Ca2+ stores. (A) ATP-
induced [Ca2+]i transients were measured from ‘‘bulk-loaded’’ Bergmann glial cells stained by incubating cerebellar slices in fura-2 acetoxymethyl ester (AM)-containing
solutions. Addition of ATP triggered an increase in [Ca2+]i that persisted in Ca2+-free extracellular solution. (B) In a similar experiment, incubation of slice with 500 nM
thapsigargin completely and irreversibly blocked ATP-induced Ca2+ signalling. (C) Intracellular administration of heparin via intracellular dialysis with a patch pipette
inhibited [Ca2+]i increase induced by ATP. Control [Ca2+]i transient was recorded from fura 2-AM-loaded cells before commencing intracellular dialysis. (D) Illustration of
spatial distribution of [Ca2+]i at time of maximum ATP response. Note higher levels of [Ca2+]i in Bergmann glial cell processes as compared with cell body. Modified from
Kirischuk et al. (1995).
50 A. Verkhratsky et al. / Molecular and Cellular Endocrinology 353 (2012) 45–56
reduced glutamate release. When astrocytes were pre-treated with
mixtures of 2-APB with ryanodine or caffeine no additive effect on
mechanically-induced glutamate release was found. Finally incu-
bation of astrocytes with 100 lM of Cd2+ (Cd2+ ions are known as
a broad spectrum antagonist of Ca2+ permeable plasma membrane
channels), reduced mechanically-induced glutamate release to 55%
of control. All these pharmacological agents not only inhibited glu-
tamate release but also reduced the mechanically induced [Ca2+]i
transients. These data confirm that Ca2+ release from the ER repre-
sents the main source of Ca2+ for controlling release of glutamate,
although plasmalemmal Ca2+ influx also has a role. The release of
glutamate and/or D-serine activated by the ER Ca2+ release was also
implicated in regulation of synaptic activity in Schaffer collateral-
CA1 neuronal synapses (Angulo et al., 2004; Fellin et al., 2004;
Henneberger et al., 2010; Perea and Araque, 2005; Rusakov et al.,
2011; Shigetomi et al., 2008).
Calcium signals originated in the ER are instrumental for astrog-
lial regulation of local blood flow. Glutamate-induced astroglial
[Ca2+]i transients induce release of vasoactive substances such as
derivatives of arachidonic acid or carbon monoxide that in turn af-
fect the tone of cerebral arterioles (Mulligan and MacVicar, 2004;
Xi et al., 2011; Zonta et al., 2003). The ER Ca2+ release can also con-
trol astroglial apoptosis through transactivation of proapoptotic
factor Bax (Morales et al., 2011).
Recently, however, the role of metabotropic receptors as well as
the InsP3-induced Ca2+ release in astroglia-dependent regulation of
neuronal networks was questioned. In experiments on mice with
genetically modified Ca2+ signalling pathways in astrocytes (which
either overexpressed Mas-related gene A1, MrgA1, metabotropic
receptor normally present only in sensory neurones, or did not ex-
press type 2 InsP3 receptors (Agulhon et al., 2010; Fiacco et al.,
2007; Petravicz et al., 2008)) has demonstrated that neither ampli-
fication nor occlusion of astroglial metabotropic Ca2+ signalling af-
fects synaptic transmission/plasticity in hippocampus. This
however remains debatable and recent experiments described spe-
cific conditions, under which astroglial InsP3/Ca2+ signalling initi-
ates modulation of synaptic plasticity (available in preliminary
form Haydon and Lee, 2011).
It is important to consider also that the ER is a multifunctional
organelle, which is involved not only in Ca2+ signalling but also
controls various aspects of cell biochemistry and the intracellular
transport of molecules (Berridge, 2002; Bootman et al., 2002).
Importantly, fluctuations on the ER Ca2+ that are affected by Ca2+
release also act as a signalling event inside the ER by changing
the activity of intra-ER enzymes such as chaperones (Michalak
et al., 2002; Verkhratsky and Petersen, 2002) thus providing the
link between cell activity and protein synthesis, post-translational
modification and trafficking..
The pre-eminence of intracellular Ca2+ as the source in glial sig-
nalling, however, does not exclude a role for plasmalemmal path-
ways for Ca2+ entry in shaping Ca2+ signals.
3.2. The store-operated Ca2+ entry: a role for TRPC channels
In the majority of electrically non-excitable cells the depletion
of the ER Ca2+ store triggers secondary Ca2+ influx that involves
opening of plasmalemmal channels. This mechanism is generally
referred to as store-operated or capacitative Ca2+ entry (Putney,
1986, 1990). Physiologically, the activation of the store-operated
Ca2+ entry is important for both replenishing the ER store (capaci-
tative function) and for producing the plateau phase of the Ca2+
transient which may outlast the period of metabotropic activation.
At the molecular level this mechanism is operated through specific
store-operated plasmalemmal channels (which may involve for-
mation of Orai/Stim1 protein complexes (Putney, 2007)) or some
types of TRP channels (Smyth et al., 2006).
The store-operated Ca2+ entry (SOCE) is present in all types of
neuroglial cells (Pivneva et al., 2008; Toescu et al., 1998; Tuschick
et al., 1997). The molecular identity of SOCE-related channels in
astrocytes are yet to be fully clarified. Expression of Orai/STIM1
proteins was detected only in astroglial cell line U373 MG (Barajas
et al., 2008). The Ca2+-release activated Ca2+ channel currents
(ICRAC) associated with SOCE in various immune cells also were
not detected in astrocytes; there are only some indications of sim-
ilarities in the pharmacological profiles of SOCE in astroglia and in
the rat basophilic leukemia cell line (Pizzo et al., 2001).
The expression of transient receptor potential (TRP) channels
has been identified in astrocytes (Golovina, 2005; Grimaldi et al.,
2003; Pizzo et al., 2001), and these channels were found to partic-
ipate in shaping of Ca2+ signals in astroglia (Golovina, 2005;
Grimaldi et al., 2003; Pizzo et al., 2001). It appears that the brain
native and functional canonical type TRP (TRPC) channels are het-
eromultimeric and formed by association of an obligatory TRPC1
with ancillary TRPC4 and/or TRPC5 proteins (Hofmann et al.,
2002; Strubing et al., 2001, 2003). Thus, affecting the activity/
expression of TRPC1 protein alone should unveil the role of this
pathway in intracellular Ca2+ dynamics in astrocytes should they
express these TRPC isoforms. Indeed, freshly isolated and cultured
astrocytes express all the three above TRPC isoforms (Golovina,
2005; Malarkey et al., 2008). Looking at the function of these pro-
teins, antisense knock down of the TRPC1 gene or immunological
approach by using a blocking antibody directed at an epitope in
the pore forming region of the TRPC1 protein reduced SOCE in cul-
tured astrocytes (Golovina, 2005; Malarkey et al., 2008). Addition-
ally, immunological block of TRPC1-mediated SOCE resulted in
reduction of purinergic receptor activation induced cytoplasmic
Ca2+ elevations in astrocytes (Fig. 3A) (Malarkey et al., 2008).
Hence, stimulation of astrocytes with ATP caused a biphasic
response, where an initial transient increase in cytoplasmic Ca2+
level was followed by a sustained (plateau) Ca2+ elevation
(Fig. 3A), the latter known to represent Ca2+entry from the extra-
cellular space (Tuschick et al., 1997) via stimulated SOCE channels
(Kresse et al., 2005). Interestingly, preincubation of astrocytes with
TRPC1 antibody resulted in a 34% reduction in the ATP-induced
transient Ca2+ elevation, along with an abolishment of the Ca2+ pla-
teau. These data identify TRPC1 protein as a likely component of
the SOCE channel activity upon purinergic receptor stimulation
in astrocytes (Malarkey et al., 2008). Similarly, mechanically-
induced Ca2+ response in astrocytes was greatly reduced by an
acute immunological inhibition of TRPC1 protein resulting in
reduced Ca2+-dependent glutamate release from astrocytes
(Malarkey et al., 2008) (Fig. 3B and C).
3.3. Ionotropic Ca2+ permeable receptors in astrocytes
The next important pathway involved in astroglial Ca2+ signal-
ling is associated with ionotropic Ca2+ permeable receptors. The
Ca2+ permeable ligand-gated ion channels were identified in
freshly isolated astroglial cells, in astrocytes in cultures and in
brain slices (Lalo et al., 2011b; Verkhratsky and Steinhauser,
2000). The main ionotropic receptors involved in astroglial Ca2+
signalling are represented by the a-amino-3-hydroxy-5-methyl-
4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate
(NMDA) glutamate receptors and P2X purinoceptors (Lalo et al.,
2006, 2008, 2011b,c; Verkhratsky and Kirchhoff, 2007). In many
types of astrocytes the AMPA receptors do not express GluR-B
(GluR2) subunit (Glaum et al., 1990; Muller et al., 1992), which
makes these receptors Ca2+ permeable (PCa/Pmonovalent  1,
(Burnashev et al., 1992). Rapid desensitization of AMPA receptors
in physiological conditions, however severely limits Ca2+ influx.
The second class of glutamate receptors expressed in astrocytes
is the NMDA receptors. These NMDA receptors were found in
 A. Verkhratsky et al. / Molecular and Cellular Endocrinology 353 (2012) 45–56
Fig. 3. TRPC1 plays a role in intracellular Ca2+ elevations in astrocytes. (A)
Application of ATP (100 lM) to astrocytes results in a biphasic response: the initial
transient Ca2+ elevation and sustained (plateau) Ca2+ elevation. Vertical dashed line
indicates the initial point of a sustained plateau Ca2+ response. The time of ATP
application is indicated by the horizontal bar. Both phases are reduced when TRPC1
containing channels are blocked with an antibody (Ab) against TRPC1. (B and C)
Mechanical stimulation of astrocytes evokes cytoplasmic Ca2+ elevations (B) and
consequential glutamate release (C). Arrows indicate the time when the mechanical
stimulus occurred. Both measurements are reduced by incubating astrocytes with
TRPC1 antibody. Traces in (A) and (B), reporting on intracellular Ca2+ levels,
represent mean time courses of fluo-3 and X-rhod-1 fluorescence expressed as DF/
F0 ± SEMs (percentage). Traces in (C) report on extracellular glutamate levels and
are expressed as mean DF/F0 ± SEMs of NADH fluorescence. Modified from
Malarkey et al. (2008).
acutely isolated cortical astrocytes as well as in astrocytes in corti-
cal slices (Lalo et al., 2006, 2011a; Palygin et al., 2011). Astroglial
NMDA receptors have specific gating and pharmacology. First they
demonstrate rather weak Mg2+ block which start to develop at
membrane potentials negative to 80 mV (Palygin et al., 2011).
Second, glial NMDA receptors were significantly more sensitive
to memantine and GluNR2C/D subunit-selective antagonist
UBP141 (Palygin et al., 2011). Third, NMDA receptors in astroglia
had a substantially lower Ca2+ permeability (PCa/Pmonovalent 3 vs.
10 in neurones (Palygin et al., 2010)); although this was sufficient
to produce cytoplasmic Ca2+ transients in response to both exoge-
nous agonists and synaptic stimulation (Fig. 4, Palygin et al., 2010).
The complex of biophysical and pharmacological properties indi-
cates that astroglial NMDA receptors are assembled from NR1,
NR2C/D and NR3 subunits.
The ATP-gated P2X1/5 purinoceptors are also present in cortical
astrocytes (Lalo et al., 2008). These receptors are exceptionally sen-
sitive to ATP (EC50  50 nM), and have very weak desensitisation in
the presence of agonist. The P2X1/5 receptors are Ca2+ permeable
(PCa/Pmonovalent  2) and their activation by ATP or by synaptic
51
stimulation trigger cytosolic Ca2+ signals (Lalo et al., 2008). There
is also some evidence for functional expression of a7 Ca2+ perme-
able nicotinic cholinoreceptors in cultured astrocytes (Oikawa
et al., 2005; Sharma and Vijayaraghavan, 2001); although the pres-
ence of these receptors in astroglial cells in situ has not been hith-
erto confirmed.
The ionotropic receptors expressed in astroglia are cationic
channels, and their activation results not only in Ca2+ influx but
also in very substantial Na+ entry. Changes in cytoplasmic Na+ con-
centration modulate another important component of Ca2+ homeo-
static machinery linked to the sodium/calcium exchanger (NCX)
that is also relevant for shaping astroglial Ca2+ signals.
3.4. The sodium/calcium exchanger in astroglia
All three types of mammalian Na+/Ca2+ exchangers, the NCX1,
NCX2 and NCX3 are expressed in astroglia and are localized in peri-
synaptic processes, especially in those covering excitatory syn-
apses (Minelli et al., 2007). The NCX have complex effect on
[Ca2+]i being involved in both Ca2+ extrusion and Ca2+ entry; the
mode of the NCX operation (forward vs. reverse) is determined
by transmembrane Na+ gradient and membrane potential (increase
in [Na+]i and depolarisation favour the reverse mode). The NCX-
mediated Ca2+ fluxes in both forward and reverse modes were
found in both cultured astrocytes and astroglial cells in situ (Gold-
man et al., 1994; Kirischuk et al., 1997; Takuma et al., 1994). Acti-
vation of astroglial ionotropic receptors induce substantial [Na+]i
elevation, which turns the exchanger into the reverse mode (Kiris-
chuk et al., 1997). Similar effects could be induced by activation of
glutamate transporter that transports three Na ions with one glu-
tamate and may trigger [Na+]i increase (Kirischuk et al., 2007; Ro-
jas et al., 2007). The reverse mode of the NCX can also be triggered
by mild depolarisation of cultured astrocytes (Paluzzi et al., 2007).
Astroglial Ca2+ signals produced by the reverse NCX were shown to
initiate exocytotic glutamate release (Paluzzi et al., 2007).
3.5. Mitochondria in astroglial Ca2+ signalling
Mitochondria in astrocytes were shown to sequester or release
Ca2+ to affect cytosolic Ca2+ changes and consequently modulate
exocytotic release of glutamate (Reyes and Parpura, 2008). When
mitochondrial Ca2+ sequestration via the uniporter was inhibited
by Ruthenium 360, mechanically-induced [Ca2+]i increase and glu-
tamate release were augmented in rat astrocytes. The converse
treatment of blocking the mitochondrial Ca2+ release reduced exo-
cytotic release of glutamate from astrocytes. Hence, the inhibition
of the mitochondrial Na+/Ca2+ exchanger with 7-chloro-5-(2-chlo-
rophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157)
reduced [Ca2+]i elevation and Ca2+-regulated glutamate release.
Additional pathway for Ca2+ release from mitochondria is via the
mitochondrial permeability transition pore (MPTP), a high conduc-
tance channel, whose transient openings may serve a physiological
role in the rapid Ca2+ removal from mitochondria (Altschuld et al.,
1992). Cyclophilin D (CypD), a matrix mitochondrial protein is in-
volved in modulation of the MPTP affinity for Ca2+. The agent cyclo-
sporin A (CsA) inhibits opening of the MPTP after binding to CypD
(Basso et al., 2005). The application of CsA to rat astrocytes caused
a reduction in [Ca2+]i elevation and Ca2+-depenent glutamate re-
lease (Reyes and Parpura, 2008). Interestingly, mouse CypD
knock-out astrocytes had attenuated mechanically-induced Ca2+
responses, but their exocytotic release of glutamate was unexpect-
edly augmented (Reyes et al., 2011). This enhancement of the
glutamatergic output had been attributed to extramitochondrial
effects via a downstream of Ca2+ modulation, likely at the level of
secretory machinery.
52 A. Verkhratsky et al. / Molecular and Cellular Endocrinology 353 (2012) 45–56

Fig. 4. Synaptically induced ionotropic Ca2+ signals in protoplasmic cortical astrocytes in situ. (A and B) Cortical layer II astrocytes were loaded with Fura-2 in situ via patch
pipette. Fluorescent images were recorded simultaneously with glial currents evoked by neuronal afferent stimulation in presence of CNQX (control) and after application of
30 lM D-AP5 (A) and 10 nM NF-449, selective antagonist of P2X receptors (B). Representative images (pseudo-colour, pipette image subtracted; warmer colours correspond
to higher [Ca2+]i levels) and glial synaptic currents (right column) were recorded from two different cells before (images on the top) and at the peak of the response. [Ca2+]i-
transients (middle columns) are expressed as F340/F380 ratio. Note significant enhancement of glia currents and Ca2+-transients upon high-frequency (HFS) stimulation.
Modified from Palygin et al. (2010).

3.6. Voltage-gated Ca2+ channels (VGCCs) in astroglia
We have recently reviewed the expression of VGCCs in astrocytes
(Parpura et al., 2011). Briefly, astrocytes in cell culture express five
main types of VGCCs: L, N, P/Q, R and T types (Barres et al., 1988,
1990; D’Ascenzo et al., 2004; Latour et al., 2003; MacVicar, 1984;
MacVicar et al., 1991; MacVicar and Tse, 1988). The level of expres-
sion or functionality of VGCCs was often associated with various
treatments of astrocytes, such as addition of dibutyryl-cAMP, co-
culturing them with neurones, or inducing acute oxidative stress
(Barres et al., 1990; Bond and Greenfield, 2007; Corvalan et al.,
1990; MacVicar and Tse, 1988). Indeed, activity of VGCCs in cultured
or freshly isolated astrocytes can lead to Ca2+ signalling (Duffy and
MacVicar, 1994; Eriksson et al., 1993; Jalonen et al., 1997).
Functional expression of VGCCs in astrocytes in situ has been
studied in developing/young animals with variable outcomes. In
acute slices from the visual cortex and the CA1 hippocampal region
of post-natal 5–18 day rats, patch-clamp whole-cell recordings
from astrocytes fail to detect any VGCC currents (Carmignoto
et al., 1998). Contrary, voltage-clamp experiments on glutamine
synthetase-positive cells, likely immature astroglia, in hippocam-
pus of 9- to 12-old-day mice demonstrated VGCC currents (Akopi-
an et al., 1996). Similarly, calcium dynamics mediated by VGCCs
were detected in astrocytes from neurogenic subventricular zone
(Young et al., 2010) and the ventrobasal thalamus (Parri and Cru-
nelli, 2003; Parri et al., 2001).
Thus far, the potential functional role of VGCCs expression in
astrocytes in situ remains limited to the example from the
ventrobasal thalamus (Parri and Crunelli, 2003; Parri et al.,
2001). In this brain region, it has been demonstrated that astro-
cytes in acute slices prepared from 5- to 17-day-old rats show
spontaneous [Ca2+]i oscillations, which dually draw Ca2+ from: (i)
the ER intracellular store (SERCA blockers blocked them) and (ii)
the extracellular space by the Ca2+ entry via VGCCs (nifedipine
blocked them, while BayK8644 enhanced them). Since astrocytic
oscillations lead to glutamate release and consequential NMDA
receptor-mediated neuronal excitability, it appears that, at least
in part, astrocytic VGCCs can play a role in modulation of neuronal
activity. NMDA receptors are essential for neuronal differentiation,
migration and synaptogenesis in the developing brain (Komuro
and Rakic, 1993, 1998), which raises a notion that astrocytic VGCCs
might be important in mediating these developmental events.
What might be the role of astrocytic VGCCs in mature brain, it re-
mains elusive at the moment. However, it is tempting to speculate
that VGCCs could be recruited in service at times when regenera-
tion and re-establishment of synaptic contacts are necessary. In
support of such possibility, reactive hippocampal astrocytes of
juvenile mice (12–14 weeks, based on reported weight) submit-
ted to pilocarpine-induced status epilepticus showed the up-regu-
lation of expression of L- and P/Q-type channels at 1 week and 2
months following such an insult (Xu et al., 2007).
4. Conclusions
The intent of this review was to summarize the Ca2+ signalling
in astroglia. These cells are heterogeneous in morphology and
 A. Verkhratsky et al. / Molecular and Cellular Endocrinology 353 (2012) 45–56
function and Ca2+ signalling is no exception. We discussed general
features of Ca2+ signalling mainly in astrocytes. Many of ap-
proached used by astrocytes are common to those utilised by other
electrically non-excitable cells. Yet, there are some specifics related
to the context of the astrocytic residence in the brain parenchyma.
Hence, we briefly discussed astrocytic exocytosis which critically
depends on intracellular Ca2+ variations. This pathway leads to
gliotransmission and signalling to adjacent neurones. There are,
however, many issues that remain to be resolved. For example,
the activation of ionotropic receptors expressed by astrocytes can
lead to increases in cyotosolic Ca2+, yet whether this excitation is
coupled to the exocytotic gliotransmission from astrocytes has
not been determined. Furthermore, it will be necessary to deter-
mine the role of VGCCs as the conduit for Ca2+ entry from the
extracellular space. For instance, does this pathway operate under
physiological conditions or it primarily operates under pathologi-
cal conditions, such as acute oxidative stress. It appears that
addressing these questions will require combining cell culture,
slice and in vivo approaches that will be labour intensive efforts
until we get more detailed picture.
Acknowledgements
Authors research was supported by Alzheimer’s Research Trust
(UK) Programme Grant (ART/PG2004A/1) to A.V. and J.J.R.; by Na-
tional Science Foundation (CBET 0943343) grant to V.P., by the
Grant Agency of the Czech Republic (GACR 309/09/1696) to J.J.R.
and (GACR 305/08/1384) to A.V.
References
Agulhon, C., Fiacco, T.A., McCarthy, K.D., 2010. Hippocampal short- and long-term
plasticity are not modulated by astrocyte Ca2+ signaling. Science 327, 1250–
1254.
Akopian, G., Kressin, K., Derouiche, A., Steinhauser, C., 1996. Identified glial cells in
the early postnatal mouse hippocampus display different types of Ca2+ currents.
Glia 17, 181–194.
Alonso, M.T., Barrero, M.J., Michelena, P., Carnicero, E., Cuchillo, I., Garcia, A.G.,
Garcia-Sancho, J., Montero, M., Alvarez, J., 1999. Ca2+-induced Ca2+ release in
chromaffin cells seen from inside the ER with targeted aequorin. J. Cell Biol. 144,
241–254.
Altschuld, R.A., Hohl, C.M., Castillo, L.C., Garleb, A.A., Starling, R.C., Brierley, G.P.,
1992. Cyclosporin inhibits mitochondrial calcium efflux in isolated adult rat
ventricular cardiomyocytes. Am. J. Physiol. 262, H1699–H1704.
Angulo, M.C., Kozlov, A.S., Charpak, S., Audinat, E., 2004. Glutamate released from
glial cells synchronizes neuronal activity in the hippocampus. J. Neurosci. 24,
6920–6927.
Bacaj, T., Tevlin, M., Lu, Y., Shaham, S., 2008. Glia are essential for sensory organ
function in C. elegans. Science 322, 744–747.
Barajas, M., Andrade, A., Hernandez-Hernandez, O., Felix, R., Arias-Montano, J.A.,
2008. Histamine-induced Ca2+ entry in human astrocytoma U373 MG cells:
evidence for involvement of store-operated channels. J. Neurosci. Res. 86, 3456–
3468.
Barres, B.A., Chun, L.L., Corey, D.P., 1988. Ion channel expression by white matter
glia: I. Type 2 astrocytes and oligodendrocytes. Glia 1, 10–30.
Barres, B.A., Koroshetz, W.J., Chun, L.L., Corey, D.P., 1990. Ion channel expression by
white matter glia: the type-1 astrocyte. Neuron 5, 527–544.
Basso, E., Fante, L., Fowlkes, J., Petronilli, V., Forte, M.A., Bernardi, P., 2005. Properties
of the permeability transition pore in mitochondria devoid of Cyclophilin D. J.
Biol. Chem. 280, 18558–18561.
Beck, A., Nieden, R.Z., Schneider, H.P., Deitmer, J.W., 2004. Calcium release from
intracellular stores in rodent astrocytes and neurons in situ. Cell Calcium 35,
47–58.
Berridge, M.J., 2002. The endoplasmic reticulum: a multifunctional signaling
organelle. Cell Calcium 32, 235–249.
Berridge, M.J., Bootman, M.D., Roderick, H.L., 2003. Calcium signalling: dynamics,
homeostasis and remodelling. Nat. Rev. Mol. Cell Biol. 4, 517–529.
Berridge, M.J., Lipp, P., Bootman, M.D., 2000. The versatility and universality of
calcium signalling. Nat. Rev. Mol. Cell Biol. 1, 11–21.
Bolsover, S.R., 2005. Calcium signalling in growth cone migration. Cell Calcium 37,
395–402.
Bond, C.E., Greenfield, S.A., 2007. Multiple cascade effects of oxidative stress on
astroglia. Glia 55, 1348–1361.
Bootman, M.D., Petersen, O.H., Verkhratsky, A., 2002. The endoplasmic reticulum is
a focal point for co-ordination of cellular activity. Cell Calcium 32, 231–234.
53
Bregestovski, P., Spitzer, N., 2005. Calcium in the function of the nervous system:
new implications. Cell Calcium 37, 371–374.
Burdakov, D., Petersen, O.H., Verkhratsky, A., 2005. Intraluminal calcium as a
primary regulator of endoplasmic reticulum function. Cell Calcium 38, 303–
310.
Burgoyne, R.D., O’Callaghan, D.W., Hasdemir, B., Haynes, L.P., Tepikin, A.V., 2004.
Neuronal Ca2+-sensor proteins: multitalented regulators of neuronal function.
Trends Neurosci. 27, 203–209.
Burnashev, N., Khodorova, A., Jonas, P., Helm, P.J., Wisden, W., Monyer, H., Seeburg,
P.H., Sakmann, B., 1992. Calcium-permeable AMPA-kainate receptors in
fusiform cerebellar glial cells. Science 256, 1566–1570.
Burnstock, G., Verkhratsky, A., 2009. Evolutionary origins of the purinergic
signalling system. Acta Physiol. (Oxf.) 195, 415–447.
Carafoli, E., 2002. Calcium signaling: a tale for all seasons. Proc. Natl. Acad. Sci. USA
99, 1115–1122.
Carafoli, E., 2004. The ambivalent nature of the calcium signal. J. Endocrinol. Invest.
27, 134–136.
Carafoli, E., Santella, L., Branca, D., Brini, M., 2001. Generation, control, and
processing of cellular calcium signals. Crit. Rev. Biochem. Mol. Biol. 36, 107–
260.
Carmignoto, G., Pasti, L., Pozzan, T., 1998. On the role of voltage-dependent calcium
channels in calcium signaling of astrocytes in situ. J. Neurosci. 18, 4637–4645.
Case, R.M., Eisner, D., Gurney, A., Jones, O., Muallem, S., Verkhratsky, A., 2007.
Evolution of calcium homeostasis: from birth of the first cell to an omnipresent
signalling system. Cell Calcium 42, 345–350.
Cavazzini, M., Bliss, T., Emptage, N., 2005. Ca2+ and synaptic plasticity. Cell Calcium
38, 355–367.
Cornell Bell, A.H., Finkbeiner, S.M., Cooper, M.S., Smith, S.J., 1990. Glutamate induces
calcium waves in cultured astrocytes: long-range glial signaling. Science 247,
470–473.
Corvalan, V., Cole, R., de Vellis, J., Hagiwara, S., 1990. Neuronal modulation of
calcium channel activity in cultured rat astrocytes. Proc. Natl. Acad. Sci. USA 87,
4345–4348.
D’Ascenzo, M., Vairano, M., Andreassi, C., Navarra, P., Azzena, G.B., Grassi, C., 2004.
Electrophysiological and molecular evidence of L-(Cav1), N-(Cav2.2), and R-
(Cav2.3) type Ca2+ channels in rat cortical astrocytes. Glia 45, 354–363.
Dani, J.W., Chernjavsky, A., Smith, S.J., 1992. Neuronal activity triggers calcium
waves in hippocampal astrocyte networks. Neuron 8, 429–440.
Deitmer, J.W., Rose, C.R., Munsch, T., Schmidt, J., Nett, W., Schneider, H.P., Lohr, C.,
1999. Leech giant glial cell: functional role in a simple nervous system. Glia 28,
175–182.
Duchen, M.R., Verkhratsky, A., Muallem, S., 2008. Mitochondria and calcium in
health and disease. Cell Calcium 44, 1–5.
Duffy, S., MacVicar, B.A., 1994. Potassium-dependent calcium influx in acutely
isolated hippocampal astrocytes. Neuroscience 61, 51–61.
Edwards, T.N., Meinertzhagen, I.A., 2010. The functional organisation of glia in the
adult brain of Drosophila and other insects. Prog. Neurobiol. 90, 471–497.
Ehrlich, B.E., Kaftan, E., Bezprozvannaya, S., Bezprozvanny, I., 1994. The
pharmacology of intracellular Ca2+-release channels. Trends Pharmacol. Sci.
15, 145–149.
Eriksson, P.S., Nilsson, M., Wagberg, M., Hansson, E., Ronnback, L., 1993. Kappa-
opioid receptors on astrocytes stimulate L-type Ca2+ channels. Neuroscience 54,
401–407.
Fellin, T., Pascual, O., Gobbo, S., Pozzan, T., Haydon, P.G., Carmignoto, G., 2004.
Neuronal synchrony mediated by astrocytic glutamate through activation of
extrasynaptic NMDA receptors. Neuron 43, 729–743.
Fiacco, T.A., Agulhon, C., Taves, S.R., Petravicz, J., Casper, K.B., Dong, X., Chen, J.,
McCarthy, K.D., 2007. Selective stimulation of astrocyte calcium in situ does not
affect neuronal excitatory synaptic activity. Neuron 54, 611–626.
Fields, R.D., Lee, P.R., Cohen, J.E., 2005. Temporal integration of intracellular Ca2+
signaling networks in regulating gene expression by action potentials. Cell
Calcium 37, 433–442.
Finkbeiner, S.M., 1993. Glial calcium. Glia 9, 83–104.
Gerasimenko, O.V., Gerasimenko, J.V., Belan, P.V., Petersen, O.H., 1996. Inositol
trisphosphate and cyclic ADP-ribose-mediated release of Ca2+ from single
isolated pancreatic zymogen granules. Cell 84, 473–480.
Giaume, C., Venance, L., 1998. Intercellular calcium signaling and gap junctional
communication in astrocytes. Glia 24, 50–64.
Glaum, S.R., Holzwarth, J.A., Miller, R.J., 1990. Glutamate receptors activate Ca2+
mobilization and Ca2+ influx into astrocytes. Proc. Natl. Acad. Sci. USA 87, 3454–
3458.
Goldman, W.F., Yarowsky, P.J., Juhaszova, M., Krueger, B.K., Blaustein, M.P., 1994.
Sodium/calcium exchange in rat cortical astrocytes. J. Neurosci. 14, 5834–5843.
Golovina, V.A., 2005. Visualization of localized store-operated calcium entry in
mouse astrocytes. Close proximity to the endoplasmic reticulum. J. Physiol. 564,
737–749.
Gourine, A.V., Kasparov, S., 2011. Astrocytes as brain interoceptors. Exp. Physiol. 96,
411–416.
Gourine, A.V., Kasymov, V., Marina, N., Tang, F., Figueiredo, M.F., Lane, S.,
Teschemacher, A.G., Spyer, K.M., Deisseroth, K., Kasparov, S., 2010. Astrocytes
control breathing through pH-dependent release of ATP. Science 329, 571–575.
Grimaldi, M., Maratos, M., Verma, A., 2003. Transient receptor potential channel
activation causes a novel form of [Ca2+]i oscillations and is not involved in
capacitative Ca2+ entry in glial cells. J. Neurosci. 23, 4737–4745.
Guerini, D., Coletto, L., Carafoli, E., 2005. Exporting calcium from cells. Cell Calcium
38, 281–289.
54 A. Verkhratsky et al. / Molecular and Cellular Endocrinology 353 (2012) 45–56
Guerrero-Hernandez, A., Dagnino-Acosta, A., Verkhratsky, A., 2010. An intelligent
sarco-endoplasmic reticulum Ca2+ store: release and leak channels have
differential access to a concealed Ca2+ pool. Cell Calcium 48, 143–149.
Hamilton, N., Vayro, S., Kirchhoff, F., Verkhratsky, A., Robbins, J., Gorecki, D.C., Butt,
A.M., 2008. Mechanisms of ATP- and glutamate-mediated calcium signaling in
white matter astrocytes. Glia 56, 734–749.
Hartmann, J., Konnerth, A., 2005. Determinants of postsynaptic Ca2+ signaling in
Purkinje neurons. Cell Calcium 37, 459–466.
Haydon, P.G., Lee, S., 2011. The alleged glial controversy is no more: conditions
under which astrocytic IP3/Ca2+ signals do modulate synaptic plasticity. Trans.
Am. Soc. Neurochem. 15.
Heilbrunn, L.V., 1943. An Outline of General Physiology. Saunders, Philadelphia.
Heilbrunn, L.V., Wiercinsky, F.J., 1947. Action of various cations on muscle
protoplasm. J. Cell. Comp. Physiol. 19, 15–32.
Heneka, M.T., Rodriguez, J.J., Verkhratsky, A., 2010. Neuroglia in neurodegeneration.
Brain Res. Rev. 63, 189–211.
Henneberger, C., Papouin, T., Oliet, S.H., Rusakov, D.A., 2010. Long-term potentiation
depends on release of D-serine from astrocytes. Nature 463, 232–236.
Hofmann, T., Schaefer, M., Schultz, G., Gudermann, T., 2002. Subunit composition of
mammalian transient receptor potential channels in living cells. Proc. Natl.
Acad. Sci. USA 99, 7461–7466.
Holcman, D., Korkotian, E., Segal, M., 2005. Calcium dynamics in dendritic spines,
modeling and experiments. Cell Calcium 37, 467–475.
Holtzclaw, L.A., Pandhit, S., Bare, D.J., Mignery, G.A., Russell, J.T., 2002. Astrocytes in
adult rat brain express type 2 inositol 1,4,5-trisphosphate receptors. Glia 39,
69–84.
Hua, X., Malarkey, E.B., Sunjara, V., Rosenwald, S.E., Li, W.H., Parpura, V., 2004. Ca2+-
dependent glutamate release involves two classes of endoplasmic reticulum
Ca2+ stores in astrocytes. J. Neurosci. Res. 76, 86–97.
Huckstepp, R.T., id Bihi, R., Eason, R., Spyer, K.M., Dicke, N., Willecke, K., Marina, N.,
Gourine, A.V., Dale, N., 2010. Connexin hemichannel-mediated CO2-dependent
release of ATP in the medulla oblongata contributes to central respiratory
chemosensitivity. J. Physiol. 588, 3901–3920.
Ikura, M., Osawa, M., Ames, J.B., 2002. The role of calcium-binding proteins in the
control of transcription: structure to function. Bioessays 24, 625–636.
Imredy, J.P., Yue, D.T., 1994. Mechanism of Ca2+-sensitive inactivation of L-type Ca2+
channels. Neuron 12, 1301–1318.
Innocenti, B., Parpura, V., Haydon, P.G., 2000. Imaging extracellular waves of
glutamate during calcium signaling in cultured astrocytes. J. Neurosci. 20,
1800–1808.
Jaiswal, J.K., 2001. Calcium – how and why? J. Biosci. 26, 357–363.
Jalonen, T.O., Margraf, R.R., Wielt, D.B., Charniga, C.J., Linne, M.L., Kimelberg, H.K.,
1997. Serotonin induces inward potassium and calcium currents in rat cortical
astrocytes. Brain Res. 758, 69–82.
Jeremic, A., Jeftinija, K., Stevanovic, J., Glavaski, A., Jeftinija, S., 2001. ATP stimulates
calcium-dependent glutamate release from cultured astrocytes. J. Neurochem.
77, 664–675.
Kastritsis, C.H., Salm, A.K., McCarthy, K., 1992. Stimulation of the P2Y purinergic
receptor on type 1 astroglia results in inositol phosphate formation and calcium
mobilization. J. Neurochem. 58, 1277–1284.
Kettenmann, H., Verkhratsky, A., 2008. Neuroglia: the 150 years after. Trends
Neurosci. 31, 653–659.
Kimelberg, H.K., 2010. Functions of mature mammalian astrocytes: a current view.
Neuroscientist 16, 79–106.
Kimelberg, H.K., Nedergaard, M., 2010. Functions of astrocytes and their potential as
therapeutic targets. Neurotherapeutics 7, 338–353.
Kirischuk, S., Kettenmann, H., Verkhratsky, A., 1997. Na+/Ca2+ exchanger modulates
kainate-triggered Ca2+ signaling in Bergmann glial cells in situ. FASEB J. 11, 566–
572.
Kirischuk, S., Kettenmann, H., Verkhratsky, A., 2007. Membrane currents and
cytoplasmic sodium transients generated by glutamate transport in Bergmann
glial cells. Pflugers Arch. 454, 245–252.
Kirischuk, S., Moller, T., Voitenko, N., Kettenmann, H., Verkhratsky, A., 1995. ATP-
induced cytoplasmic calcium mobilization in Bergmann glial cells. J. Neurosci.
15, 7861–7871.
Kirischuk, S., Tuschick, S., Verkhratsky, A., Kettenmann, H., 1996. Calcium signalling
in mouse Bergmann glial cells mediated by a1-adrenoreceptors and H1
histamine receptors. Eur. J. Neurosci. 8, 1198–1208.
Kiselyov, K., Shin, D.M., Muallem, S., 2003. Signalling specificity in GPCR-dependent
Ca2+ signalling. Cell. Signal. 15, 243–253.
Knot, H.J., Laher, I., Sobie, E.A., Guatimosim, S., Gomez-Viquez, L., Hartmann, H.,
Song, L.S., Lederer, W.J., Graier, W.F., Malli, R., Frieden, M., Petersen, O.H., 2005.
Twenty years of calcium imaging: cell physiology to dye for. Mol. Interv. 5, 112–
127.
Koizumi, S., Lipp, P., Berridge, M.J., Bootman, M.D., 1999. Regulation of ryanodine
receptor opening by lumenal Ca2+ underlies quantal Ca2+ release in PC12 cells. J.
Biol. Chem. 274, 33327–33333.
Komuro, H., Rakic, P., 1993. Modulation of neuronal migration by NMDA receptors.
Science 260, 95–97.
Komuro, H., Rakic, P., 1998. Orchestration of neuronal migration by activity of ion
channels, neurotransmitter receptors, and intracellular Ca2+ fluctuations. J.
Neurobiol. 37, 110–130.
Kostyuk, P., Verkhratsky, A., 1994. Calcium stores in neurons and glia. Neuroscience
63, 381–404.
Kostyuk, P., Verkhratsky, A., 1995. Calcium Signalling in the Nervous System. Wiley
& Sons, Chichester.
Kresse, W., Sekler, I., Hoffmann, A., Peters, O., Nolte, C., Moran, A., Kettenmann, H.,
2005. Zinc ions are endogenous modulators of neurotransmitter-stimulated
capacitative Ca2+ entry in both cultured and in situ mouse astrocytes. Eur. J.
Neurosci. 21, 1626–1634.
Kuga, N., Sasaki, T., Takahara, Y., Matsuki, N., Ikegaya, Y., 2011. Large-scale calcium
waves traveling through astrocytic networks in vivo. J. Neurosci. 31, 2607–2614.
Lalo, U., Palygin, O., North, R.A., Verkhratsky, A., Pankratov, Y., 2011a. Age-
dependent remodelling of ionotropic signalling in cortical astroglia. Aging Cell
10, 392–402.
Lalo, U., Pankratov, Y., Kirchhoff, F., North, R.A., Verkhratsky, A., 2006. NMDA
receptors mediate neuron-to-glia signaling in mouse cortical astrocytes. J.
Neurosci. 26, 2673–2683.
Lalo, U., Pankratov, Y., Parpura, V., Verkhratsky, A., 2011b. Ionotropic receptors in
neuronal–astroglial signalling: what is the role of ‘‘excitable’’ molecules in non-
excitable cells. Biochim. Biophys. Acta 1813, 992–1002.
Lalo, U., Pankratov, Y., Wichert, S.P., Rossner, M.J., North, R.A., Kirchhoff, F.,
Verkhratsky, A., 2008. P2X1 and P2X5 subunits form the functional P2X
receptor in mouse cortical astrocytes. J. Neurosci. 28, 5473–5480.
Lalo, U., Verkhratsky, A., Pankratov, Y., 2011c. Ionotropic ATP receptors in neuronal–
glial communication. Semin. Cell Dev. Biol. 22, 220–228.
Laming, P.R., Kimelberg, H., Robinson, S., Salm, A., Hawrylak, N., Muller, C., Roots, B.,
Ng, K., 2000. Neuronal–glial interactions and behaviour. Neurosci. Biobehav.
Rev. 24, 295–340.
Latour, I., Hamid, J., Beedle, A.M., Zamponi, G.W., Macvicar, B.A., 2003. Expression of
voltage-gated Ca2+ channel subtypes in cultured astrocytes. Glia 41, 347–353.
Lenhossek, M.v., 1891. Zur Kenntnis der Neuroglia des menschlichen
Ruckenmarkes. Verh. Anat. Ges. 5, 193–221.
Lenhossek, M.v., 1893. Der feinere Bau des Nervensystems im Lichte neuester
Forschung. Fischer’s Medicinische Buchhandlung H. Kornfield, Berlin.
Lewit-Bentley, A., Rety, S., 2000. EF-hand calcium-binding proteins. Curr. Opin.
Struct. Biol. 10, 637–643.
Lohmann, C., Wong, R.O., 2005. Regulation of dendritic growth and plasticity by
local and global calcium dynamics. Cell Calcium 37, 403–409.
MacVicar, B.A., 1984. Voltage-dependent calcium channels in glial cells. Science
226, 1345–1347.
MacVicar, B.A., Hochman, D., Delay, M.J., Weiss, S., 1991. Modulation of intracellular
Ca++ in cultured astrocytes by influx through voltage-activated Ca++ channels.
Glia 4, 448–455.
MacVicar, B.A., Tse, F.W., 1988. Norepinephrine and cyclic adenosine 30 :50 -cyclic
monophosphate enhance a nifedipine-sensitive calcium current in cultured rat
astrocytes. Glia 1, 359–365.
Magistretti, P.J., 2009. Neuroscience. Low-cost travel in neurons. Science 325, 1349–
1351.
Malarkey, E.B., Ni, Y., Parpura, V., 2008. Ca2+ entry through TRPC1 channels
contributes to intracellular Ca2+ dynamics and consequent glutamate release
from rat astrocytes. Glia 56, 821–835.
Malarkey, E.B., Parpura, V., 2009. Mechanisms of transmitter release from
astrocytes. In: Parpura, V., Haydon, P.G. (Eds.), Astrocytes in
(Patho)physiology of the Nervous System. Springer, New York, pp. 301–350.
Matyash, M., Matyash, V., Nolte, C., Sorrentino, V., Kettenmann, H., 2002.
Requirement of functional ryanodine receptor type 3 for astrocyte migration.
FASEB J. 16, 84–86.
Matyash, V., Kettenmann, H., 2010. Heterogeneity in astrocyte morphology and
physiology. Brain Res. Rev. 63, 2–10.
McCarthy, K.D., Salm, A.K., 1991. Pharmacologically-distinct subsets of astroglia can
be identified by their calcium response to neuroligands. Neuroscience 41, 325–
333.
Michalak, M., Robert Parker, J.M., Opas, M., 2002. Ca2+ signaling and calcium binding
chaperones of the endoplasmic reticulum. Cell Calcium 32, 269–278.
Michelangeli, F., Ogunbayo, O.A., Wootton, L.L., 2005. A plethora of interacting
organellar Ca2+ stores. Curr. Opin. Cell Biol. 17, 135–140.
Minelli, A., Castaldo, P., Gobbi, P., Salucci, S., Magi, S., Amoroso, S., 2007. Cellular and
subcellular localization of Na+–Ca2+ exchanger protein isoforms, NCX1, NCX2,
and NCX3 in cerebral cortex and hippocampus of adult rat. Cell Calcium 41,
221–234.
Miyata, H., Silverman, H.S., Sollott, S.J., Lakatta, E.G., Stern, M.D., Hansford, R.G.,
1991. Measurement of mitochondrial free Ca2+ concentration in living single rat
cardiac myocytes. Am. J. Physiol. 261, H1123–H1134.
Mogami, H., Tepikin, A.V., Petersen, O.H., 1998. Termination of cytosolic Ca2+
signals: Ca2+ reuptake into intracellular stores is regulated by the free Ca2+
concentration in the store lumen. EMBO J. 17, 435–442.
Montana, V., Malarkey, E.B., Verderio, C., Matteoli, M., Parpura, V., 2006. Vesicular
transmitter release from astrocytes. Glia 54, 700–715.
Morad, M., Davies, N.W., Kaplan, J.H., Lux, H.D., 1988. Inactivation and block of
calcium channels by photo-released Ca2+ in dorsal root ganglion neurons.
Science 241, 842–844.
Morales, A.P., Carvalho, A.C., Monteforte, P.T., Hirata, H., Han, S.W., Hsu, Y.T., Smaili,
S.S., 2011. Endoplasmic reticulum calcium release engages Bax translocation in
cortical astrocytes. Neurochem. Res. 36, 829–838.
Muller, T., Moller, T., Berger, T., Schnitzer, J., Kettenmann, H., 1992. Calcium entry
through kainate receptors and resulting potassium-channel blockade in
Bergmann glial cells. Science 256, 1563–1566.
Mulligan, S.J., MacVicar, B.A., 2004. Calcium transients in astrocyte endfeet cause
cerebrovascular constrictions. Nature 431, 195–199.
Neher, E., Sakaba, T., 2008. Multiple roles of calcium ions in the regulation of
neurotransmitter release. Neuron 59, 861–872.
 A. Verkhratsky et al. / Molecular and Cellular Endocrinology 353 (2012) 45–56
Nicholls, D.G., 2005. Mitochondria and calcium signaling. Cell Calcium 38, 311–317.
Nicotera, P., Orrenius, S., 1998. The role of calcium in apoptosis. Cell Calcium 23,
173–180.
Nicotera, P., Petersen, O.H., Melino, G., Verkhratsky, A., 2007. Janus a god with two
faces: death and survival utilise same mechanisms conserved by evolution. Cell
Death Differ. 14, 1235–1236.
Oberheim, N.A., Takano, T., Han, X., He, W., Lin, J.H., Wang, F., Xu, Q., Wyatt, J.D.,
Pilcher, W., Ojemann, J.G., Ransom, B.R., Goldman, S.A., Nedergaard, M., 2009.
Uniquely hominid features of adult human astrocytes. J. Neurosci. 29, 3276–
3287.
Oberheim, N.A., Wang, X., Goldman, S., Nedergaard, M., 2006. Astrocytic complexity
distinguishes the human brain. Trends Neurosci. 29, 547–553.
Oertner, T.G., Matus, A., 2005. Calcium regulation of actin dynamics in dendritic
spines. Cell Calcium 37, 477–482.
Oikawa, H., Nakamichi, N., Kambe, Y., Ogura, M., Yoneda, Y., 2005. An increase in
intracellular free calcium ions by nicotinic acetylcholine receptors in a single
cultured rat cortical astrocyte. J. Neurosci. Res. 79, 535–544.
Oikonomou, G., Shaham, S., 2011. The glia of Caenorhabditis elegans. Glia 59, 1253–
1263.
Oldershaw, K.A., Taylor, C.W., 1993. Luminal Ca2+ increases the affinity of inositol
1,4,5-trisphosphate for its receptor. Biochem. J. 292 (Pt. 3), 631–633.
Paluzzi, S., Alloisio, S., Zappettini, S., Milanese, M., Raiteri, L., Nobile, M., Bonanno, G.,
2007. Adult astroglia is competent for Na+/Ca2+ exchanger-operated exocytotic
glutamate release triggered by mild depolarization. J. Neurochem. 103, 1196–
1207.
Palygin, O., Lalo, U., Pankratov, Y., 2011. Distinct pharmacological and functional
properties of NMDA receptors in mouse cortical astrocytes. Br. J. Pharmacol.
163, 1755–1766.
Palygin, O., Lalo, U., Verkhratsky, A., Pankratov, Y., 2010. Ionotropic NMDA and P2X1/
2+
5 receptors mediate synaptically induced Ca signalling in cortical astrocytes.
Cell Calcium 48, 225–231.
Parpura, V., Basarsky, T.A., Liu, F., Jeftinija, K., Jeftinija, S., Haydon, P.G., 1994.
Glutamate-mediated astrocyte–neuron signalling. Nature 369, 744–747.
Parpura, V., Grubisic, V., Verkhratsky, A., 2011. Ca2+ sources for the exocytotic
release of glutamate from astrocytes. Biochim. Biophys. Acta 1813, 984–991.
Parri, H.R., Crunelli, V., 2003. The role of Ca2+ in the generation of spontaneous
astrocytic Ca2+ oscillations. Neuroscience 120, 979–992.
Parri, H.R., Gould, T.M., Crunelli, V., 2001. Spontaneous astrocytic Ca2+ oscillations
in situ drive NMDAR-mediated neuronal excitation. Nat. Neurosci. 4, 803–812.
Perea, G., Araque, A., 2005. Properties of synaptically evoked astrocyte calcium
signal reveal synaptic information processing by astrocytes. J. Neurosci. 25,
2192–2203.
Petersen, O.H., 2002. Calcium signal compartmentalization. Biol. Res. 35, 177–182.
Petersen, O.H., 2005. Ca2+ signalling and Ca2+-activated ion channels in exocrine
acinar cells. Cell Calcium 38, 171–200.
Petersen, O.H., Michalak, M., Verkhratsky, A., 2005. Calcium signalling: past, present
and future. Cell Calcium 38, 161–169.
Petersen, O.H., Petersen, C.C., Kasai, H., 1994. Calcium and hormone action. Annu.
Rev. Physiol. 56, 297–319.
Petersen, O.H., Verkhratsky, A., 2007. Endoplasmic reticulum calcium tunnels
integrate signalling in polarised cells. Cell Calcium 42, 373–378.
Petravicz, J., Fiacco, T.A., McCarthy, K.D., 2008. Loss of IP3 receptor-dependent Ca2+
increases in hippocampal astrocytes does not affect baseline CA1 pyramidal
neuron synaptic activity. J. Neurosci. 28, 4967–4973.
Pivneva, T., Haas, B., Reyes-Haro, D., Laube, G., Veh, R.W., Nolte, C., Skibo, G.,
Kettenmann, H., 2008. Store-operated Ca2+ entry in astrocytes: different spatial
arrangement of endoplasmic reticulum explains functional diversity in vitro
and in situ. Cell Calcium 43, 591–601.
Pizzo, P., Burgo, A., Pozzan, T., Fasolato, C., 2001. Role of capacitative calcium entry
on glutamate-induced calcium influx in type-I rat cortical astrocytes. J.
Neurochem. 79, 98–109.
Porter, J.T., McCarthy, K.D., 1995. Adenosine receptors modulate [Ca2+]i in
hippocampal astrocytes in situ. J. Neurochem. 65, 1515–1523.
Procko, C., Shaham, S., 2010. Assisted morphogenesis: glial control of dendrite
shapes. Curr. Opin. Cell Biol. 22, 560–565.
Puceat, M., Jaconi, M., 2005. Ca2+ signalling in cardiogenesis. Cell Calcium 38, 383–
389.
Putney Jr., J.W., 1986. A model for receptor-regulated calcium entry. Cell Calcium 7,
1–12.
Putney Jr., J.W., 1990. Capacitative calcium entry revisited. Cell Calcium 11, 611–
624.
Putney Jr., J.W., 2007. Recent breakthroughs in the molecular mechanism of
capacitative calcium entry (with thoughts on how we got here). Cell Calcium 42,
103–110.
Redmond, L., Ghosh, A., 2005. Regulation of dendritic development by calcium
signaling. Cell Calcium 37, 411–416.
Reichenbach, A., Pannicke, T., 2008. Neuroscience. A new glance at glia. Science 322,
693–694.
Reyes, R.C., Parpura, V., 2008. Mitochondria modulate Ca2+-dependent glutamate
release from rat cortical astrocytes. J. Neurosci. 28, 9682–9691.
Reyes, R.C., Perry, G., Lesort, M., Parpura, V., 2011. Immunophilin deficiency
augments Ca2+-dependent glutamate release from mouse cortical astrocytes.
Cell Calcium 49, 23–34.
Rimessi, A., Giorgi, C., Pinton, P., Rizzuto, R., 2008. The versatility of mitochondrial
calcium signals: from stimulation of cell metabolism to induction of cell death.
Biochim. Biophys. Acta 1777, 808–816.
55
Rizzuto, R., Pozzan, T., 2006. Microdomains of intracellular Ca2+: molecular
determinants and functional consequences. Physiol. Rev. 86, 369–408.
Rojas, H., Colina, C., Ramos, M., Benaim, G., Jaffe, E.H., Caputo, C., DiPolo, R., 2007.
Na+ entry via glutamate transporter activates the reverse Na+/Ca2+ exchange
and triggers Cai2+-induced Ca2+ release in rat cerebellar type-1 astrocytes. J.
Neurochem. 100, 1188–1202.
Rusakov, D.A., Zheng, K., Henneberger, C., 2011. Astrocytes as regulators of synaptic
function: a quest for the Ca2+ master key. Neuroscientist, in press.
Rycroft, B.K., Gibb, A.J., 2004a. Inhibitory interactions of calcineurin (phosphatase
2B) and calmodulin on rat hippocampal NMDA receptors. Neuropharmacology
47, 505–514.
Rycroft, B.K., Gibb, A.J., 2004b. Regulation of single NMDA receptor channel activity
by alpha-actinin and calmodulin in rat hippocampal granule cells. J. Physiol.
(Lond.) 557, 795–808.
Sharma, G., Vijayaraghavan, S., 2001. Nicotinic cholinergic signaling in hippocampal
astrocytes involves calcium-induced calcium release from intracellular stores.
Proc. Natl. Acad. Sci. USA 98, 4148–4153.
Shemarova, I.V., Nesterov, V.P., 2005a. Evolution of Ca2+ signaling mechanisms. Role
of calcium ions in signal transduction in lower eukaryotes. Zh. Evol. Biokhim.
Fiziol. 41, 303–313.
Shemarova, I.V., Nesterov, V.P., 2005b. Evolution of mechanisms of calcium
signaling: the role of calcium ions in signal transduction in prokaryotes. Zh.
Evol. Biokhim. Fiziol. 41, 12–17.
Sheppard, C.A., Simpson, P.B., Sharp, A.H., Nucifora, F.C., Ross, C.A., Lange, G.D.,
Russell, J.T., 1997. Comparison of type 2 inositol 1,4,5-trisphosphate receptor
distribution and subcellular Ca2+ release sites that support Ca2+ waves in
cultured astrocytes. J. Neurochem. 68, 2317–2327.
Shigetomi, E., Bowser, D.N., Sofroniew, M.V., Khakh, B.S., 2008. Two forms of
astrocyte calcium excitability have distinct effects on NMDA receptor-mediated
slow inward currents in pyramidal neurons. J. Neurosci. 28, 6659–6663.
Shimizu, H., Watanabe, E., Hiyama, T.Y., Nagakura, A., Fujikawa, A., Okado, H.,
Yanagawa, Y., Obata, K., Noda, M., 2007. Glial Nax channels control lactate
signaling to neurons for brain [Na+] sensing. Neuron 54, 59–72.
Shmigol, A., Svichar, N., Kostyuk, P., Verkhratsky, A., 1996. Gradual caffeine-induced
Ca2+ release in mouse dorsal root ganglion neurons is controlled by cytoplasmic
and luminal Ca2+. Neuroscience 73, 1061–1067.
Simpson, P.B., Russell, J.T., 1998. Role of mitochondrial Ca2+ regulation in neuronal
and glial cell signalling. Brain Res. Brain Res. Rev. 26, 72–81.
Smyth, J.T., Dehaven, W.I., Jones, B.F., Mercer, J.C., Trebak, M., Vazquez, G., Putney Jr.,
J.W., 2006. Emerging perspectives in store-operated Ca2+ entry: roles of Orai,
Stim and TRP. Biochim. Biophys. Acta 1763, 1147–1160.
Solovyova, N., Verkhratsky, A., 2002. Monitoring of free calcium in the neuronal
endoplasmic reticulum: an overview of modern approaches. J. Neurosci.
Methods 122, 1–12.
Solovyova, N., Verkhratsky, A., 2003. Neuronal endoplasmic reticulum acts as a
single functional Ca2+ store shared by ryanodine and inositol-1,4,5-
trisphosphate receptors as revealed by intra-ER [Ca2+] recordings in single rat
sensory neurones. Pflugers Arch. 446, 447–454.
Solovyova, N., Veselovsky, N., Toescu, E.C., Verkhratsky, A., 2002. Ca2+ dynamics in
the lumen of the endoplasmic reticulum in sensory neurons: direct
visualization of Ca2+-induced Ca2+ release triggered by physiological Ca2+
entry. EMBO J. 21, 622–630.
Spitzer, N.C., Borodinsky, L.N., Root, C.M., 2005. Homeostatic activity-dependent
paradigm for neurotransmitter specification. Cell Calcium 37, 417–423.
Stout Jr., R.F., Parpura, V., 2011. Voltage-gated calcium channel types in cultured C.
elegans CEPsh glial cells. Cell Calcium 50, 98–108.
Strubing, C., Krapivinsky, G., Krapivinsky, L., Clapham, D.E., 2001. TRPC1 and TRPC5
form a novel cation channel in mammalian brain. Neuron 29, 645–655.
Strubing, C., Krapivinsky, G., Krapivinsky, L., Clapham, D.E., 2003. Formation of novel
TRPC channels by complex subunit interactions in embryonic brain. J. Biol.
Chem. 278, 39014–39019.
Takuma, K., Matsuda, T., Hashimoto, H., Asano, S., Baba, A., 1994. Cultured rat
astrocytes possess Na+–Ca2+ exchanger. Glia 12, 336–342.
Toescu, E.C., 2000. Mitochondria and Ca2+ signaling. J. Cell. Mol. Med. 4, 164–175.
Toescu, E.C., Moller, T., Kettenmann, H., Verkhratsky, A., 1998. Long-term activation
of capacitative Ca2+ entry in mouse microglial cells. Neuroscience 86, 925–935.
Toescu, E.C., Verkhratsky, A., 1998. Principles of calcium signalling. In: Verkhratsky,
A., Toescu, E.C. (Eds.), Integrative Aspects of Calcium Signalling. Plenum Press,
New York, pp. 2–22.
Toescu, E.C., Verkhratsky, A., 2004. Ca2+ and mitochondria as substrates for deficits
in synaptic plasticity in normal brain ageing. J. Cell. Mol. Med. 8, 181–190.
Tuschick, S., Kirischuk, S., Kirchhoff, F., Liefeldt, L., Paul, M., Verkhratsky, A.,
Kettenmann, H., 1997. Bergmann glial cells in situ express endothelin B
receptors linked to cytoplasmic calcium signals. Cell Calcium 21, 409–419.
Vangheluwe, P., Raeymaekers, L., Dode, L., Wuytack, F., 2005. Modulating
sarco(endo)plasmic reticulum Ca2+ ATPase 2 (SERCA2) activity: cell biological
implications. Cell Calcium 38, 291–302.
Verkhratsky, A., 2005. Physiology and pathophysiology of the calcium store in the
endoplasmic reticulum of neurons. Physiol. Rev. 85, 201–279.
Verkhratsky, A., 2006a. Calcium ions and integration in neural circuits. Acta Physiol.
(Oxf.) 187, 357–369.
Verkhratsky, A., 2006b. Patching the glia reveals the functional organisation of the
brain. Pflugers Arch. 453, 411–420.
Verkhratsky, A., 2007. Calcium and cell death. Subcell. Biochem. 45, 465–480.
Verkhratsky, A., Butt, A., 2007. Glial Neurobiology. A Textbook. John Wiley & Sons,
Chichester.
56 A. Verkhratsky et al. / Molecular and Cellular Endocrinology 353 (2012) 45–56
Verkhratsky, A., Kettenmann, H., 1996. Calcium signalling in glial cells. Trends
Neurosci. 19, 346–352.
Verkhratsky, A., Kirchhoff, F., 2007. NMDA receptors in glia. Neuroscientist 13, 28–
37.
Verkhratsky, A., Orkand, R.K., Kettenmann, H., 1998. Glial calcium: homeostasis and
signaling function. Physiol. Rev. 78, 99–141.
Verkhratsky, A., Parpura, V., Rodriguez, J.J., 2011. Where the thoughts dwell: the
physiology of neuronal–glial ‘‘diffuse neural net’’. Brain Res. Rev. 66, 133–151.
Verkhratsky, A., Petersen, O.H., 2002. The endoplasmic reticulum as an integrating
signalling organelle: from neuronal signalling to neuronal death. Eur. J.
Pharmacol. 447, 141–154.
Verkhratsky, A., Shmigol, A., 1996. Calcium-induced calcium release in neurones.
Cell Calcium 19, 1–14.
Verkhratsky, A., Solovyova, N., Toescu, E.C., 2002. Calcium excitability of glial cells.
In: Volterra, A., Haydon, P., Magistretti, P. (Eds.), Glia in Synaptic Transmission.
OUP, Oxford, pp. 99–109.
Verkhratsky, A., Steinhauser, C., 2000. Ion channels in glial cells. Brain Res. Brain
Res. Rev. 32, 380–412.
Verkhratsky, A., Toescu, E.C., 2006. Neuronal–glial networks as substrate for CNS
integration. J. Cell. Mol. Med. 10, 826–836.
Wakui, M., Osipchuk, Y.V., Petersen, O.H., 1990. Receptor-activated cytoplasmic Ca2+
spiking mediated by inositol trisphosphate is due to Ca2+-induced Ca2+ release.
Cell 63, 1025–1032.
Webb, S.E., Moreau, M., Leclerc, C., Miller, A.L., 2005. Calcium transients and neural
induction in vertebrates. Cell Calcium 37, 375–385.
Wray, S., Burdyga, T., Noble, K., 2005. Calcium signalling in smooth muscle. Cell
Calcium 38, 397–407.
Xi, Q., Tcheranova, D., Basuroy, S., Parfenova, H., Jaggar, J.H., Leffler, C.W., 2011.
Glutamate-induced calcium signals stimulate CO production in piglet
astrocytes. Am. J. Physiol. Heart Circ. Physiol. 301, H428–433.
Xu, J.H., Long, L., Tang, Y.C., Hu, H.T., Tang, F.R., 2007. Cav1.2, Cav1.3, and Cav2.1 in
the mouse hippocampus during and after pilocarpine-induced status
epilepticus. Hippocampus 17, 235–251.
Young, S.Z., Platel, J.C., Nielsen, J.V., Jensen, N.A., Bordey, A., 2010. GABA(A) increases
calcium in subventricular zone astrocyte-like cells through L- and T-type
voltage-gated calcium channels. Front. Cell. Neurosci. 4, 8.
Zhu, M.X., Ma, J., Parrington, J., Galione, A., Evans, A.M., 2010. TPCs: endolysosomal
channels for Ca2+ mobilization from acidic organelles triggered by NAADP. FEBS
Lett. 584, 1966–1974.
Zonta, M., Angulo, M.C., Gobbo, S., Rosengarten, B., Hossmann, K.A., Pozzan, T.,
Carmignoto, G., 2003. Neuron-to-astrocyte signaling is central to the dynamic
control of brain microcirculation. Nat. Neurosci. 6, 43–50.