MINIREVIEW
Synchronization of Ca
2+
oscillations: involvement of ATP
release in astrocytes
Schuichi Koizumi
Department of Pharmacology, Faculty of Medicine, University of Yamanashi, Japan
Introduction
Glia, Greek for ‘glue’, was discovered by Rudolph
Virchow, a German anatomist, in the mid-nineteenth
century. The name reflects the original view that glia
played merely a structural or supportive role for neu-
rons. Glial cells, especially astrocytes, are much more
than ‘glue’ or merely quiescent, and display their own
set of activities. They can receive inputs, assimilate
information, and send instructive chemical signals
both to neurons and to other neighboring cells.
Astrocytic activities may be assessed by an observed
increase in the intracellular Ca
2+
concentration
([Ca
2+
]
i
), using fluorescent Ca
2+
imaging techniques.
Astrocytes show transient increases in [Ca
2+
]

important partners to neighboring cells, including neurons, vascular cells,
and other glial cells. Although glial cells are not excitable in terms of
electrophysiology, they have been shown to generate synchronized Ca
2+
transients (Ca
2+
oscillations) through mechanisms of chemical coupling.
Until recently, Ca
2+
transients in astrocytes were thought to be totally
dependent on neuronal activities, because astrocytes express a large vari-
ety of receptors for neurotransmitters and surround almost all synapses
at which neurotransmitters are spilled over to stimulate astrocytes. In
addition, however, astrocytes have been shown to release diffusible sub-
stances, so-called ‘gliotransmitters’, and Ca
2+
transients in astrocytes are
therefore also triggered by astrocytic activities, leading to propagation of
Ca
2+
transients or Ca
2+
waves. In these processes, the gliotransmitter
ATP and activation of P2Y receptors play central roles. Interestingly,
astrocytes evoke Ca
2+
transients when neurons are not present, suggest-
ing that astrocytes themselves can initiate and control Ca
2+
transients.

include glial cells [1,2]. Evidence suggests that such
Ca
2+
-mediated extracellular signaling between astro-
cytes and neurons could be involved in the regulation
of synaptic transmission both in physiological and in
pathophysiological conditions.
In this minireview, the mechanisms underlying syn-
chronous Ca
2+
transients in astrocytes are summarized
from the viewpoint of chemical coupling by ATP.
Using this coupling, astrocytes regulate neurons and
vice versa. Neuron–glia communication appears to be
accentuated in pathophysiological conditions such as
epilepsy. Thus, the involvement of astrocytic Ca
2+
transients in epileptiform discharge in neurons is also
discussed.
Astrocytic Ca
2+
transients in vitro
The development of video imaging techniques has
allowed us to observe dynamic spatiotemporal changes
in [Ca
2+
]
i
in neurons and glial cells simultaneously.
Unlike neurons, astrocytes do not produce action

even neurons (Fig. 1Ac) [5]. For some years, astrocytic
Ca
2+
waves have been thought to propagate via gap
junctions [6], with the internal messenger inositol 1,4,5-
trisphosphate being the diffusible substance that
induces Ca
2+
release in neighboring cells. However,
Ca
2+
waves are propagated between astrocytes even
when the cells do not have an absolute requirement for
functional contact with each other directly, and the
extent and direction of the Ca
2+
wave propagation are
significantly influenced by movement of the extracellu-
lar medium [7]. These more recent reports suggest that
substances released from astrocytes can activate recep-
tor systems on astrocytes, evoking the release of addi-
tional substances, and thus producing a synchronized
propagating Ca
2+
wave of activity. In 1999, Guthrie
et al. [7] demonstrated that astrocytes release ATP,
which is responsible for the spreading of Ca
2+
tran-
sients with a slight synchronization (Fig. 1Ab). The

transients. However, it should be noted that
astrocytes themselves evoke the propagating Ca
2+
transients. As shown in Fig 1Ba, which represents
changes in [Ca
2+
]
i
in the neuron–astrocyte cocultures,
astrocytes and neurons show Ca
2+
oscillations with
different frequency and temporal patterns; that is,
Ca
2+
oscillations in astrocytes are less frequent and
synchronous than those in neurons. Importantly, the
Ca
2+
transients in astrocytes do not disappear when
neuronal Ca
2+
oscillation is inhibited by tetrodotoxin
(TTX) (Fig. 1Ba). In addition, astrocytes show spon-
taneous Ca
2+
transients even when they have been
purified and cultured without neurons (Fig. 1Bb).
These findings suggest that astrocytes can initiate the
spontaneous Ca

vesicular structure expressing the vesicular glutamate
transporters, and release glutamate in a Ca
2+
-depen-
dent and SNARE-dependent manner [10]. These find-
ings strongly suggest that exocytotic machinery is
involved in glutamate release in astrocytes, although
nonvesicular mechanisms for glutamate release have
also been proposed. In contrast, the mechanisms
underlying the release of ATP from astrocytes are less
Astrocytes
ATP
ATP
dF/F0
=0.2
10 s
3
4
TTX
Apyrase
1
2
Apyrase
5
6
Purified astrocytes
Neuron–astrocyte coculture
Neurons
Astrocytes
a

transients in adjacent astrocytes to form intercellular Ca
2+
waves by a
mechanism of chemical coupling mediated by ATP. In both cases, an increase in [Ca
2+
]
i
is driven by neuronal activities. (c) Astrocytic Ca
2+
transients also affect neuronal activities through the gliotransmitter ATP. (B) Neuronal activity-independent Ca
2+
transients in astrocytes. (a)
Neuronal Ca
2+
oscillations seen in the hippocampal neurons (blue traces shown as 3 and 4) are highly synchronous and are inhibited by TTX.
Adjacent astrocytes (red traces shown as 1 and 2) also show slower and less synchronous Ca
2+
oscillations. However, the synchronous
Ca
2+
oscillations in astrocytes are unaffected even when neuronal activities are inhibited by TTX, suggesting that astrocytes have mecha-
nism(s) by which they form neuronal activity-independent Ca
2+
transients. (b) Astrocytes reveal synchronous Ca
2+
transients (red traces 5
and 6) when neurons are not present (purified astrocytes). Astrocytic Ca
2+
oscillations seen in the presence of TTX or in purified astrocytes
were abolished by the ATP-degrading enzyme apyrase. (c) Schematic cartoon of neuronal activity-independent astrocytic Ca

strongly suggest that astrocytes should release ATP
through a mechanism of exocytosis, which would be a
key event for neuron–astrocyte communication. How-
ever, we still did not know which molecules transport
ATP into astrocytic vesicles. Recently, this question
has been answered. Sawada et al. (2008) [19] demon-
strated that SLC17A9 or vesicular nucleotide trans-
porter, a novel member of an anion transporter family,
functions as a vesicular nucleotide transporter, and is
essential for the storage of nucleotides within vesicles.
Importantly, SLC17A9 is expressed in astrocytes.
These findings suggest that the mechanisms of ATP
release could also include exocytosis, although the nat-
ure of the signals released from astrocytes may vary
with varying physiological and pathological conditions
[17]. It is important to elucidate the mechanisms by
which astrocytes release ATP in response to distinct
stimuli, as this will further clarify the mechanisms
underlying the synchronized Ca
2+
transients.
Astrocytic Ca
2+
waves in vivo
With the recent development of multiphoton micros-
copy, we can observe astrocytic Ca
2+
transients in vivo.
Hirase et al. [20] were the first to analyze changes in
[Ca

ing mice, Ca
2+
responses in cerebellar Bergmann glia
(radial astrocytes) were more frequent and contained
ATP-mediated components. The less frequent chemical
coupling in vivo might result, in part, from the fact that
the activity of the ATP-degrading enzyme ectonucleo-
tidases is higher in vivo or in slice preparations than in
primary cultures in vitro [18,23]. Astrocytic Ca
2+
tran-
sients in vivo also include a component that is depen-
dent on neuronal activities. In the mouse or ferret,
whisker [24], limb [25] and visual stimulation [26] causes
more frequent Ca
2+
transients in astrocytes in the bar-
rel, the primary somatosensory cortex, and the visual
cortex, respectively. These Ca
2+
transients in astrocytes
were delayed by a few seconds as compared with the
Astrocyte
Exocytosis
Cl
–
channels
P2X7 receptors
P2Y1,P2Y2
Maxi-anion

stores. (B) Multiple pathways for the release of ATP. Hemichannels
of connexin or pannexin, maxi-anion channels, P2X7 receptors and
Cl
)
channels are pathways through which ATP can flow. In addi-
tion, the existence of exocytotic ATP is also suggested. ER, endo-
plasmic reticulum.
S. Koizumi Astrocytic Ca
2+
oscillations and neuronal activities
FEBS Journal 277 (2010) 286–292 ª 2009 The Author Journal compilation ª 2009 FEBS 289
neuronal responses [24,26], and nearly correlated with
the strength of the sensory stimulation. Thus, neuronal
activities also affect astrocytic Ca
2+
transients. More
importantly, the application of bicuculine, an antago-
nist of 4-aminobutyrate A receptor [20], or picrotoxin
[27] increases neuronal activity by triggering epileptic-
like discharges, which subsequently result in a great
increase in Ca
2+
transients that are often propagated
into nearby astrocytes [20]. Thus, neuronal activity-dri-
ven Ca
2+
oscillation occurs synchronously in multiple
astrocytes, and seems to be accentuated by pathological
activities of neurons such as epileptiform discharges.
Pathology and Ca

Neurons
SIC
SIC
Astrocytes
ATP
ATP/ado
ATP
ATP
Reactive
astrocytes
SIC
SIC
SIC
SIC
SIC
-
+
Neurons
glu
glu
glu
glu
glu
glu
glu
Epileptiform dischar
g
e
glu
Neurons

of several astrocyte-specific molecules are associated
with epilepsy-like firing in neurons [28]. A spatially
restricted seizure focus in the brain can be identified
for epilepsies acquired after head trauma, tumor, or
other severe focal insults to the brain, when astrocytes
often become reactive. Reactive astrocytes change in
their abilities to release, take up and metabolize glio-
transmitters, which would cause unusual excitation of
adjacent local neuronal networks [28]. Recently, it was
proposed that slow-inward currents (SICs) recorded in
the hippocampal neurons are caused by astrocytic glu-
tamate [29,30] (Fig. 3B). Although astrocyte-induced
SICs are not epileptiform bursts as such (Fig. 3B, left),
their synchronization is closely associated with the for-
mation of ictal bursts. Astrocytic Ca
2+
oscillation and
the subsequent glutamate release are key events in the
synchronization of SICs [31]. As shown in the right
panel of Fig. 3B, reactive astrocytes increase the
release of glutamate by facilitating Ca
2+
oscillations in
response to the gliotransmitter ATP (or glutamate),
and generate synchronous SICs in small groups of con-
tiguous neurons, followed by epilepsy-like firings [28].
Interestingly, several antiepileptic agents, including val-
proate, gabapentin, and phenytoin, reduce astrocytic
Ca
2+

zures. Interestingly, astrocytes also show spontaneous
Ca
2+
transients that are independent of neuronal
activities. Thus, astrocytes themselves have the ability
to produce their own Ca
2+
transients, and therefore
might control neuronal activities or synaptic transmis-
sions through a mechanism independent of neurons.
The physiological and pathophysiological significance
of the spontaneous Ca
2+
transients in astrocytes
remains unknown. Clarification of the detailed mecha-
nisms underlying the spontaneous Ca
2+
transients in
astrocytes, especially those occurring in vivo, would
shed light on this issue and further our understanding
of the neuron–glia interaction.
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