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Astrocyte Control of Synaptic Transmission
and Neurovascular Coupling
PHILIP G. HAYDON AND GIORGIO CARMIGNOTO
Silvio Conte Center for Integration at the Tripartite Synapse, Department of Neuroscience, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania; and Istituto di Neuroscienze, Centro Nazionale
Ricerche and Dipartimento di Scienze Biomediche Sperimentali, University of Padua, Padua, Italy
I. Introduction 1009
II. Astrocytic Calcium Signaling: The Biochemical Basis of Glial Excitability 1010
III. Neuron-to-Astrocyte Signaling in the Control of Cerebral Circulation 1011
IV. Neuronal Activity-Dependent Calcium Elevations in Astrocyte Endfeet 1013
V. Propagating Calcium Wave in Astrocytes May Contribute to Control Microcirculation 1014
VI. Activation of Astrocytes Can Also Trigger Arteriole Constriction 1014
VII. Discovery of Gliotransmission: Astrocytes Talk to Neurons 1016
VIII. Mechanisms of Glutamate Release From Astrocytes 1016
IX. Kiss-and-Run Release of Glutamate From Astrocytes 1017
X. The Tripartite Synapse: Astrocytes Modulate Neuronal Excitability and Synaptic Transmission 1018
XI. Release of Glutamate From Astrocytes 1018
XII. Astrocytes Activate Extrasynaptic NMDA Receptors 1019
XIII. Why Are Astrocyte-Evoked NMDA Currents So Large in Amplitude? 1020
XIV. Astrocytes Synchronously Activate Groups of Pyramidal Neurons 1021
XV. D-Serine: Selective Synthesis in and Release From Astrocytes 1021
XVI. Release of ATP From Astrocytes 1022
XVII. Glial-Derived ATP Modulates Neuronal Excitability 1023
XVIII. Purinergic Modulation of Synaptic Transmission 1023
XIX. Introduction of Molecular Genetics to Address the Roles of the Astrocyte in Neuronal Function 1024
XX. Gliotransmission Regulates Synaptic Cross-Talk 1025
XXI. Summary and the Future 1026
Haydon, Philip G., and Giorgio Carmignoto. Astrocyte Control of Synaptic Transmission and Neurovascular Coupling.
Physiol Rev 86: 1009–1031, 2006; doi:10.1152/physrev.00049.2005.—From a structural perspective, the predominant glial cell of
the central nervous system, the astrocyte, is positioned to regulate synaptic transmission and neurovascular coupling: the
processes of one astrocyte contact tens of thousands of synapses, while other processes of the same cell form endfeet on
capillaries and arterioles. The application of subcellular imaging of Ca
2⫹
signaling to astrocytes now provides functional data
to support this structural notion. Astrocytes express receptors for many neurotransmitters, and their activation leads to
oscillations in internal Ca
2⫹
. These oscillations induce the accumulation of arachidonic acid and the release of the chemical
transmitters glutamate, D-serine, and ATP. Ca
2⫹
oscillations in astrocytic endfeet can control cerebral microcirculation through
the arachidonic acid metabolites prostaglandin E
2
and epoxyeicosatrienoic acids that induce arteriole dilation, and 20-HETE
that induces arteriole constriction. In addition to actions on the vasculature, the release of chemical transmitters from
astrocytes regulates neuronal function. Astrocyte-derived glutamate, which preferentially acts on extrasynaptic receptors, can
promote neuronal synchrony, enhance neuronal excitability, and modulate synaptic transmission. Astrocyte-derived D-serine,
by acting on the glycine-binding site of the N-methyl-D-aspartate receptor, can modulate synaptic plasticity. Astrocyte-derived
ATP, which is hydrolyzed to adenosine in the extracellular space, has inhibitory actions and mediates synaptic cross-talk
underlying heterosynaptic depression. Now that we appreciate this range of actions of astrocytic signaling, some of the
immediate challenges are to determine how the astrocyte regulates neuronal integration and how both excitatory (glutamate)
and inhibitory signals (adenosine) provided by the same glial cell act in concert to regulate neuronal function.
I. INTRODUCTION
The nervous system consists of two classes of cell,
the neuron and glia. Although it is without doubt that
neurons are essential for nervous system function, studies
over the past decade are raising our awareness about the
diversity of roles played by glial cells in nervous system
function. In this review we focus on one of the subtypes
Physiol Rev 86: 1009–1031, 2006;
doi:10.1152/physrev.00049.2005.
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of glial cells, the astrocyte, and discuss our current un-
derstanding of how these cells operate hand in hand with
neurons to regulate integration in the central nervous
system. Necessarily, we restrict our focus to the roles of
astrocytes served by the release of three transmitters,
glutamate, D-serine, and ATP; how these gliotransmitters
regulate neuronal function; and how neuronal activity
can, through astrocytic signaling cascades, locally regu-
late vascular tone. We do not attempt to discuss the roles
of chemokines released from these glial cells, and instead
alert the reader to an excellent recent review on this topic
(219).
The discovery that chemical transmitters evoke Ca
2⫹
elevations in cultured astrocytes (37) sparked the imagi-
nation of a small group of neuroscientists who diverted
their attention to the investigation of this class of glial
cell. Although these cells play critical roles in supporting
neuronal function, astrocytic Ca
2⫹
excitability and the
consequent induced release of chemical transmitters,
which we now term gliotransmitters, has led to an emerg-
ing new understanding of the functional roles played by
these glial cells; we now appreciate that astrocytes listen
and talk to synapses and play roles in synaptic modulation
and in mediating synaptic cross-talk (9, 13, 30, 82, 148,
153, 205). In this review we have three goals: to provide a
view of nervous system activity from the perspective of
the astrocyte, to discuss how as an integrative hub the
astrocyte exerts control over cerebrovascular as well as
neuronal functions, and to discuss how the astrocyte and
gliotransmission play a fundamental role in shaping and
dynamically regulating the relative strengths of neighbor-
ing synaptic connections.
II. ASTROCYTIC CALCIUM SIGNALING:
THE BIOCHEMICAL BASIS OF
GLIAL EXCITABILITY
Anyone who has recorded, often by accident, from an
astrocyte in a brain slice preparation or in vivo knows that
these cells are electrically inexcitable, and offer little to
study with electrophysiological approaches. Generally,
astrocytes have a high resting K
⫹
conductance, respond
to depolarization with a linear current-voltage relation-
ship, and are coupled by gap junctions. Recent studies
have suggested that there may be many types of astro-
cytes. For example, Steinhauser’s group has identified
two classes of glial cell with distinct functional proper-
ties: one has the linear current-voltage relationship, high
resting K
⫹
conductance, and glutamate transporters ex-
pected of astrocytes, while the other expresses voltage-
gated conductances together with AMPA receptors (93,
132, 220). We await the results of future studies to deter-
mine whether this second class of cell type is a subtype of
astrocyte or a distinct glial cell type, and will therefore
focus the remainder of our discussion to the more tradi-
tional gap junction-coupled astrocyte with a linear cur-
rent-voltage relationship.
Because of the high resting K
⫹
conductance and gap
junction coupling, the first major function assigned to
these cells was in the clearance of extracellular K
⫹
fol-
lowing elevated periods of neuronal activity (158). Al-
though early studies showed that the application of neu-
rotransmitters can depolarize astrocytes (22, 100), per-
haps the most significant discovery that initiated renewed
vigor in studies of astrocytes was the discovery that the
application of the chemical transmitter glutamate induces
Ca
2⫹
oscillations and Ca
2⫹
waves between cultured hip-
pocampal astrocytes (32, 37, 61).
Glutamate-induced Ca
2⫹
oscillations in astrocytes re-
sult from the activation of class I metabotropic receptors
that induce the phospholipase-dependent accumulation
of inositol trisphosphate (IP
3
) that stimulates the release
of Ca
2⫹
from IP
3
-sensitive internal stores (99). Conse-
quently, by measuring Ca
2⫹
signaling, rather than mem-
brane potential, it was discovered that astrocytes are an
excitable system.
Since these initial observations it has been realized
that astrocytes express a plethora of metabotropic recep-
tors that can couple to second messenger systems (216,
217). For example, norepinephrine (48, 109), glutamate
(48, 165, 174, 175, 190), GABA (96), acetylcholine (11,
190), histamine (190), adenosine (173), and ATP (24, 171,
173) have all been shown to induce Ca
2⫹
elevations in
glial cells in brain slice preparations. In culture, the list of
metabotropic receptors is extensive. However, because
culturing astrocytes can lead to the misexpression of
proteins, it is not yet clear whether all of these receptors
are normally expressed in astrocytes in vivo.
The presence of Ca
2⫹
waves that propagate between
cultured astrocytes has intrigued several groups who
have attempted to identify the mechanism of signal prop-
agation. Although these waves occur in cell culture,
emerging evidence suggests that they do not occur under
physiological conditions in vivo (85). Nonetheless, under-
standing the mechanism of wave propagation has pro-
vided important insights into signals that can be released
from astrocytes. Two prominent hypotheses guided this
work: 1)IP
3
could diffuse through gap junctions to evoke
Ca
2⫹
signals in neighboring unstimulated astrocytes (87,
115, 183, 193, 214), and 2) a message, ATP, is released
from an astrocyte which, by activating P2Y receptors on
adjacent astrocytes, stimulates additional Ca
2⫹
signals
(38, 51, 72). Although it is likely that both pathways
contribute to wave propagation, that the wave can prop-
agate between physically disconnected cells (81) provides
compelling evidence for a role for the induced release of
ATP signaling to neighboring cells and mediating the
propagation of the wave.
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Although in cell culture waves of Ca
2⫹
elevation are
the norm, it is appropriate to ask whether long-range Ca
2⫹
waves could provide meaningful information if they were
to occur within a nervous system. Several studies have
now been performed using brain slice preparations, and
these studies indicate that Ca
2⫹
signals, under physiolog-
ical conditions, are less extensive. For example, photore-
lease of glutamate in hippocampal slices to stimulate
individual astrocytes demonstrated that activation of a
single cell was able to evoke Ca
2⫹
elevations in neighbor-
ing astrocytes (200). However, the range of this signal was
extremely small compared with those observed in cul-
tures. As will be discussed in section XVII, the extracellular
concentration of ATP is tightly regulated by ectonucleoti-
dases. It is likely that the difference in range over which
Ca
2⫹
signals propagate is in part regulated by the impact
of ectonucleotidases that more effectively hydrolyze ATP
to adenosine within the confined extracellular space of a
brain slice and in vivo (49).
Studies in brain slice preparations have led to the
proposal that astrocytes are functionally compartmental-
ized and Ca
2⫹
oscillations are predominantly restricted to
local microdomains. Imaging studies performed in hip-
pocampal slices showed that astrocytic Ca
2⫹
oscillations
occur in portions of a process of individual astrocytes
(165). Stimulation of the parallel fibers, which innervate
Purkinje cells and Bergmann glia, evoke Ca
2⫹
signals
restricted to microdomains of the Bergmann glial cell
processes (71). Three-dimensional reconstruction of the
processes of Bergmann glia shows that microdomains are
connected by extremely fine processes, providing a struc-
tural basis to support biochemical compartmentalization.
Because one hippocampal astrocyte has been calculated
to make contact with ⬃100,000 synapses (29), this local
Ca
2⫹
signaling provides the opportunity for astrocytes to
influence synaptic transmission in response to the glial
Ca
2⫹
signal while retaining synaptic specificity. The idea of
localized Ca
2⫹
signaling is supported by in vivo imaging of
astrocytes where synchronized Ca
2⫹
waves are not gen-
erally detected (85).
What is the stimulus for the astrocytic Ca
2⫹
signal?
Since chemical transmitters can induce Ca
2⫹
oscillations
in these glial cells, the ability of neuronal activity to
stimulate astrocytes was initially tested in studies in brain
slice preparations. Trains of activity in the Schaffer col-
lateral pathway of the hippocampus evoke Ca
2⫹
signals in
area CA1 astrocytes (165, 175). These signals result from
synaptically released glutamate acting on subtype 5 of
metabotropic glutamate receptors (mGluR5s), as well as a
contribution from ATP acting through P2Y receptors (24).
More recently, Newman (150) has shown that activation
of the retina by light, to stimulate photoreceptors and the
associated circuitry, does indeed stimulate Ca
2⫹
signals in
Mu¨ ller glial cells. In support of local signaling, Ca
2⫹
waves
were not detected, but instead Ca
2⫹
oscillations were
detected in endfeet of these glia. An intriguing observa-
tion by McCarthy’s group (149) suggests that astrocytes
do not rely solely on instructive cues from neurons, but
instead can intrinsically oscillate. With the use of a phar-
macological cocktail of antagonists and despite blocking
activity-dependent synaptic transmission, Ca
2⫹
oscilla-
tions persisted in hippocampal astrocytes. Thus, although
it is clear that neurons can activate astrocytic Ca
2⫹
sig-
nals, and that this can occur in vivo, it is also possible that
astrocytes have intrinsic capabilities of initiating Ca
2⫹
signals. However, at this point we are only at the begin-
ning of understanding the regulation of Ca
2⫹
signals
within astrocytes in vivo. Two-photon imaging in vivo
shows that long-range Ca
2⫹
signals are not the norm (85).
However, we have little idea about how neuronal activity
influences the spatial scale of glial Ca
2⫹
signals nor how
neuronal activity influences frequency encoding of glial
Ca
2⫹
signals. This is an extremely important area of in-
vestigation if we are to develop realistic models of the
role of the astrocyte in the control of synaptic transmis-
sion, neuronal excitability, as well as the control of the
cerebrovasculature.
These studies provide a different picture of Ca
2⫹
signaling in the astrocyte than was first described in cul-
tures. Oscillations are restricted to portions of the pro-
cesses of individual cells, the so-called microdomains.
They do not necessarily propagate over large distances,
even within one astrocyte. Since, as we will discuss later,
such Ca
2⫹
signals cause gliotransmitters to be released
that have feedback actions on neurons, localized re-
sponses to neuronal activity are likely to be of importance
in maintaining synaptic specificity, while permitting glio-
transmission to modulate neuronal function.
NEURON-TO-ASTROCYTE SIGNALING IN THE
CONTROL OF CEREBRAL CIRCULATION
From a purely structural perspective, the astrocyte is
situated much like a hub in which it receives inputs from
thousands of synapses and at the same time can make
contact with the local vasculature (Fig. 1). The combina-
tion of this structural relationship together with our new-
found appreciation of the presence of dynamic, activity-
dependent biochemical signaling between neurons and
astrocytes suggests that the astrocyte is an important
integrator of neuronal activity and consequently the local
control of cerebrovasculature.
The contact of astrocytic endfeet with arterioles and
capillaries, that was first described by Golgi at the end
1800s (66), has long been interpreted as an indication that
astrocytes can take up nutrients and metabolites from the
blood and then distribute them to other brain cells, in-
cluding neurons (7). Indirect support for such a view
derives from a number of more recent studies that de-
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scribe the structural association of astrocyte processes
with both synapses and cerebral vessels (172, 192, 215).
Beyond this physical relationship, subsequent studies sug-
gest that synaptic activity regulates astrocytic metabo-
lism, which consequently feeds these active neurons with
lactate. According to this view, the transport of glutamate
into the astrocyte following synaptic activity leads to the
activation of the Na
⫹
-K
⫹
-ATPase to restore the ion gradi-
ent that is necessary to drive glutamate transport. Astro-
cytic ATP is replenished from glucose derived from the
vasculature resulting in the accumulation of astrocytic
lactate. Monocarboxylate transporters are then believed
to shuttle lactate to synaptic terminals as a source of
neuronal ATP (170, 208). Though this process can occur,
its relative importance in relation to the direct use of
glucose as a neuronal energy source is the subject of
considerable debate (98, 127).
The increased energy demand of active neurons is
also met by local increases in blood flow in the area of
elevated neuronal activity. This phenomenon, which was
first described by A. Mosso in the late 1800s (142) and
later confirmed by Roy and Sherrington (180), is a funda-
mental event in brain function. Local increases in blood
flow result from the rapid dilation of arterioles and cap-
illaries of a restricted area in response to an episode of
high neuronal activity. As a consequence, blood flow in-
creases in that region within a few seconds, thereby en-
suring that most active neurons receive an adequate sup-
ply of oxygen and metabolic substrates for energy con-
sumption. Local accumulation of metabolic products has
been initially proposed to directly control blood flow.
Although under particular circumstances, such as brain
hypoxia or ischemia, this process may indeed affect blood
vessels, the time course of the neurovascular coupling
argues against this hypothesis (122). Results obtained
over the last few years provide conclusive support for the
view that blood flow is directly coupled to neuronal ac-
tivity rather than to local energy needs (16, 184). The
present knowledge on the multiple signaling pathways
that during activation lead to the production of vasoactive
factors suggests that the molecular mechanism at the
basis of functional hyperemia is highly complex and may
not necessarily be the same in all brain regions. Although
various aspects remain to be elucidated, most recent
studies highlight a central role of neuron-to-astrocyte sig-
naling in the local control of microcirculation (6, 60, 123,
145, 241, 242).
Because of their polarized anatomical structure and
of the vicinity of their endfeet with contractile elements of
blood vessels, such as smooth muscle cells in arterioles
and pericytes in capillaries, astrocytes have been long
proposed to contribute to the regulation of the blood flow
during neuronal activity. The ability of astrocytes to re-
move from the extracellular space around active synapses
FIG. 1. Neuronal synaptic activity can act
through the astrocyte network to regulate the
cerebrovasculature. The activity of glutamatergic
synapses can regulate astrocytic biochemical sig-
naling through the coactivation of metabotropic
glutamate and purinergic receptors to cause a
phospholipase C-dependent increase in astrocytic
Ca
2⫹
, which can propagate to the astrocytic end-
foot to exert local actions on the vasculature.
Through the activity of Ca
2⫹
-sensitive phospho-
lipase A
2
, accumulated arachidonic acid can
cause vasodilatory and vasoconstrictive actions
through at least two of its metabolic pathways.
Cyclooxygenase-2 (COX)-dependent accumula-
tion of PGE
2
leads to a vasodilation, while the
diffusion of arachidonic acid to the smooth mus-
cle, which contains high levels of CPY4A, leads to
the accumulation of 20-HETE that causes vaso-
constriction. While these two opposing actions
seem in conflict, since both have been seen to
occur in vivo, the challenge is to identify the
conditions that select for the respective actions.
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potassium ions increasingly concentrated there following
high neuronal activity and to redistribute them, through
the syncytium, to distal regions, was originally considered
a plausible mechanism to couple neuronal activity with
dilation of vessels (207). This hypothesis was substanti-
ated in the retina where a high potassium conductance
was found in astrocyte endfeet and Mu¨ ller cell processes
in contact with blood vessels (152, 168).
The demonstration that astrocytes produce a pleth-
ora of vasoactive substances, such as nitric oxide (NO)
(116, 146, 226), cyclooxygenase and epoxygenase activity-
derived products (2, 4, 157, 169), and ATP (15, 36, 176),
hints at the possibility that the control of microcirculation
by astrocytes could not be based simply on the “spatial
buffering” of K
⫹
hypothesis, but rather involves a more
complex mechanism and a number of different molecules.
Among these, are epoxyeicosatrienoic acids (EETs) that
cytochrome P-450 epoxygenase forms from arachidonic
acid (AA) (179). Support for a distinct role of EETs in
neurovascular coupling derives the observations that 1)
by acting on K
⫹
channels, EETs hyperpolarized smooth
muscle cells and trigger dilation of cerebral vessels (5, 65,
88); 2) pharmacological inhibition of P-450 epoxygenase
results in reduction of the basal blood flow in the cerebral
cortex as measured by laser Doppler flowmetry (2); and
3) stimulation of astrocytes in culture with glutamate
receptor agonists triggers formation of AA that is con-
verted to various AA metabolites including EETs (1, 19).
These observations led David Harper to propose a func-
tional role of astrocyte EETs in neuronal activity-depen-
dent regulation of blood flow (74, 75).
IV. NEURONAL ACTIVITY-DEPENDENT
CALCIUM ELEVATIONS IN
ASTROCYTE ENDFEET
Significant evidence in support of a distinct role of
astrocytes in neurovascular coupling was then obtained
in a series of experiments performed mainly in brain slice
preparations. In hippocampal and cortical slices it was
first observed that glutamate released at active synapses
triggered Ca
2⫹
oscillations in astrocytes that increased in
frequency according to increasing levels of neuronal ac-
tivity (165). These oscillations may represent a digital
signal in the control of cell activity as it was originally
proposed by Woods et al. (230) and Jacob et al. (94).
While this observation demonstrates that astrocytes are
sophisticated sensors of neuronal activity (243), it also
represents a clue to the possibility that astrocytes transfer
to blood vessels information on the level of neuronal
activity. Indeed, neuronal activity-dependent Ca
2⫹
eleva-
tions in astrocytes were observed to propagate to perivas-
cular endfeet (242). Such a signal provides a mechanistic
basis for the graded response of the blood flow to differ-
ent levels of neuronal activity, thereby strengthening the
idea of a distinct astrocytic role in neurovascular cou-
pling. Importantly, high-frequency stimulation of neuronal
afferents was found to trigger both Ca
2⫹
elevations in
astrocyte endfeet and dilation of cerebral arterioles. Fur-
thermore, Ca
2⫹
elevations triggered in astrocytes by ei-
ther t-ACPD, a mGluR agonist, or direct mechanical stim-
ulation of individual astrocytes by a patch pipette, also
evoked dilation of cortical arterioles, while inhibition by
mGluR antagonists of Ca
2⫹
oscillations evoked in astro-
cytes by synaptic glutamate, or the incubation with cyclo-
oxygenase (COX) inhibitors that block prostaglandin syn-
thesis, reduced neuronal activity-dependent dilation of
cerebral arterioles. Vasodilation appears to be mediated,
at least in part, by prostaglandin E
2
, since astrocytes in
culture were observed to release this powerful dilating
agent in a pulsatile manner according to the pattern of
t-ACPD-mediated Ca
2⫹
oscillations (244).
Activation of Ca
2⫹
elevations in astrocyte endfeet
has been also reported to suppress vasomotion (60), a
rhythmic fluctuation in the diameter of cerebral arterioles
that accompanies Ca
2⫹
oscillations in smooth muscle
cells (73, 224). Vasomotion, which is a natural property of
cerebral microcirculation in the intact brain, requires
some degree of tone that in arterioles from acute brain
slice preparations is seriously compromised. Vasomotion
could, however, recover upon treatment with agents that
induce arteriole constriction (123). Although its precise
functional significance and underlying mechanism remain
undefined, vasomotion is believed to contribute to micro-
vasculature hemodynamics by enhancing tissue oxygen-
ation especially when perfusion is compromised (209).
Interestingly, in arterioles from brain slice preparations,
vasomotion and the accompanied Ca
2⫹
oscillations in
smooth muscle cells are suppressed by stimulation of
neuronal afferents (27), suggesting that its inhibition or
reduction contributes to neuronal activity-dependent
blood flow changes. All together, these observations raise
the possibility that the suppression of vasomotion, which
accompanies Ca
2⫹
elevations in astrocyte endfeet, con-
tributes to the dilating action of astrocytes. In this action
of astrocytes, EET release may be involved since blocking
EET production with the epoxygenase inhibitor micon-
azole results in an increase in the frequency of vasomo-
tion (123).
Results from in vivo experiments that used the same
mGluR antagonists that in brain slices inhibited astrocyte-
mediated vasodilation corroborated the role of astrocytes
in functional hyperemia (242). By measuring the blood
flow in the somatosensory cortex by laser Doppler flow-
metry, the hyperemic response evoked by forepaw stim-
ulation was found to be markedly reduced after the sys-
temic application of mGluR antagonists. The action of the
mGluR antagonists was unrelated to unspecific effects on
the intensity of neuronal stimulation since the evoked
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somatosensory potential was unchanged. This is in agree-
ment with the unchanged amplitude of the Ca
2⫹
increase
triggered by neuronal afferent stimulation in neurons
from brain slices in the presence of the mGluR
antagonists.
According to these results, a model is proposed in
which astrocytes can encode different levels of neuronal
activity into defined Ca
2⫹
oscillation frequencies that, at
the level of perivascular endfeet, mediate the release of
dilating agents, such as EETs (2, 19, 123) and PGE
2
(242,
244) as well as constrictive agents such as 20-HETE (145;
see also below). Neuronal activity-dependent Ca
2⫹
oscil-
lations may ultimately represent the signaling system that
allows blood flow to vary in a manner proportional to the
intensity of neuronal activity.
V. PROPAGATING CALCIUM WAVE
IN ASTROCYTES MAY CONTRIBUTE
TO CONTROL MICROCIRCULATION
During functional hyperemia, the dilation of arte-
rioles in the area of activation will not increase blood flow
in that region effectively unless upstream vessels also
dilate. How vasodilator and vasoconstrictor responses are
conveyed from the initial site of activation to distant
locations is unclear. Coordinated vasoactive responses
may rely on coupling and communication between cells
within the vessel wall. Endothelial cells are indeed exten-
sively coupled (86, 118), and evidence has been also pro-
vided for an electronic coupling existing between endo-
thelial and smooth muscle cells (118, 185, 231). Hyperpo-
larization of an individual smooth muscle cell can thus
spread through the endothelial cells to other smooth mus-
cle cells via myoendothelial coupling and evoke a coor-
dinated dilating response along the length of an arte-
riole (70).
ACa
2⫹
wave propagating between perivascular as-
trocytes may also be involved. The Ca
2⫹
response in an
astrocyte in contact with a blood vessel, initially evoked
either by neuronal activity (242) or by direct electrical
stimulation (192), has been indeed observed to spread to
other perivascular astrocytes. Furthermore, connexin43
and purinergic receptors, i.e., the basic elements which
mediated the propagation of the Ca
2⫹
wave in cultured
astrocytes, are highly expressed at astrocyte endfeet
(192), and filling single astrocytes that are in the proxim-
ity of a blood vessel with Lucifer yellow results in the
diffusion of the dye to other astrocyte endfeet. Through
the release of vasoactive factors, activation of perivascu-
lar astrocytes by the Ca
2⫹
wave may affect the tone of
upstream and/or downstream blood vessels, thereby reg-
ulating the overall conductance of the vascular network
in a defined region.
VI. ACTIVATION OF ASTROCYTES CAN ALSO
TRIGGER ARTERIOLE CONSTRICTION
In hippocampal slices, Ca
2⫹
elevations in astrocyte
endfeet triggered by either photolysis of a Ca
2⫹
caged
compound or t-ACPD have been observed to evoke also
arteriole constriction (145). Studies in cultured cells show
that astrocytes can indeed produce, in addition to various
dilating agents, constrictive agents such as the COX prod-
ucts PGF
2
␣
(19, 195) and thromboxane A
2
(92, 169), en-
dothelins (126), and 20-hydroxyeicosatetraenoic acid (20-
HETE) (154). This latter compound, that derives from
-hydroxylation of AA by CYP4A, a cytochrome P-450
enzyme subtype (179), depolarizes smooth muscle cells
by inhibiting the opening of K
⫹
channels (112), and also
enhances Ca
2⫹
influx through voltage-dependent Ca
2⫹
channels (65). The formation of 20-HETE from AA in
smooth muscle cells is proposed to mediate the constric-
tive action of astrocytes in the hippocampus (145). This
action can also account for the constriction of cerebral
blood vessels associated with spreading depression and
ischemia (45), since Ca
2⫹
elevations and Ca
2⫹
waves are
known to occur in the astrocytes during these pathologi-
cal brain conditions (17, 110, 113, 114). The release of
20-HETE from astrocytes may, however, have a role also
under normal physiological conditions. For example, 20-
HETE has been proposed to play a crucial role in the
maintenance of myogenic tone in cerebral blood vessels
(64). The constrictive action of 20-HETE may also control,
together with that of dilating agents, the extent of neuro-
nal activity-dependent increases in blood flow (see also
below).
The results reported by Mulligan and MacVicar’s
study (145) are in conflict with those reported by Zonta et
al. (242) in which Ca
2⫹
elevations in astrocyte endfeet
were observed to trigger dilation of cerebral arterioles.
How can these conflicting results be reconciled in a uni-
fying hypothesis? In the latter study, most, although not
all, experiments were performed in cortical slices incu-
bated with N
G
-nitro-L-arginine methyl ester (L-NAME), a
NO synthase inhibitor that blocks the tonic action of NO
on arterioles and thus results in a long-lasting constriction
of arterioles. In contrast, this procedure was not applied
in most of the experiments described in Mulligan and
MacVicar’s study in hippocampal slices. Therefore, the
different initial state of contraction of cerebral arterioles
in the two studies may account, at least in part, for the
different results.
The central point here concerns the resting state of
pressurized arterioles in vivo. Under normal physiological
conditions in the intact brain, cerebral arteries are typi-
cally in a state of partial contraction (52). Although the
exact mechanisms at the basis of myogenic tone remain
uncertain, it is clear that this phenomenon is generated by
an interplay of pressure-mediated stretching of the
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smooth muscle cell membrane, intraluminal blood flow,
and various factors released by neurons, astrocytes, and
endothelial cells (43, 44, 84). A change in ion gating in
smooth muscle cell membrane, either directly or indi-
rectly via membrane depolarization, results in increased
levels of intracellular Ca
2⫹
concentration and activation
of the contractile process (84). The myogenic tone under-
lies cerebrovascular autoregulation, i.e., the ability of ves-
sels to respond to changes in transmural pressure with
either constriction when pressure increases or dilation
when pressure decreases (43, 188, 224). This property has
been also proposed to reflect a “regional blood reserve”
that various control mechanisms use to produce vasodi-
lation or vasoconstriction (62).
It is the importance of myogenic tone that likely
accounts for the discrepancy between the results of these
two studies. Accordingly, when the vascular tone is lost,
arterioles tend to be in a dilated state, and dilating agents
may be ineffective or less effective. Similarly, when
smooth muscles are excessively contracted, the inner
diameter of arterioles is reduced to such an extent that
constrictive agents can hardly induce a further constric-
tion. Therefore, given that in slice preparations the myo-
genic tone is lost, to detect a dilating effect of vasoactive
agents, a certain degree of constriction that mimics the
natural occurring myogenic tone of blood vessels in vivo
is pharmacologically induced (56, 57, 77, 78, 124, 182). In
agreement with this view, constriction of arterioles in-
duced by t-ACPD in hippocampal slices changed to dila-
tion when t-ACPD was applied after arterioles were pre-
constricted with L-NAME (145).
All together, these observations hint at the possibility
that astrocytes release both dilating and constrictive
agents. This ability of astrocytes seems, however, difficult
to reconcile with a distinct role of the astrocyte in neu-
rovascular coupling. Indeed, how can a Ca
2⫹
signal in
astrocyte endfeet lead to diametrically opposite changes
in arteriole diameter? The hypothesis can be advanced
that the ultimate effect of astrocyte activation may de-
pend on the balance between the action of dilating and
constrictive agents, on the one hand, and the resting state
of arterioles, on the other. The release of 20-HETE may
serve to generate a constrictive action that opposes the
powerful action of other dilating agents, released by neu-
rons and/or astrocytes themselves, ultimately modulating
the amplitude of the neuronal activity-dependent increase
in blood flow.
Certainly, to define the astrocyte’s role in the control
of cerebral blood flow, first of all, it will be important to
provide conclusive evidence for the release from astro-
cytes of both dilating and constrictive agents. In such a
case, are dilating and constrictive factors released simul-
taneously? If this corelease event does not occur, could it
be possible that a dilating, or a constrictive, agent might
be preferentially released according to a distinct pattern,
or amplitude or compartmentalization, of the Ca
2⫹
rise in
the astrocyte? An additional interesting issue would be
that of clarifying whether the various signaling pathways
that rely on diverse enzymes to metabolize AA, i.e., COXs,
lipoxygenases, epoxygenases, and
-hydroxylases, can be
differently regulated by modulatory factors. For example,
NO that is produced by neurons as well as glia has been
reported to downregulate the formation of the cyto-
chrome P-450 subtypes CYP2 that produce EETs (121,
211) as well as to inhibit 20-HETE formation (3).
A recent in vivo study provides further support for
the distinct role of astrocytes in neurovascular coupling
(204). After loading of astrocytes from the somatosensory
cortex of adult rats with both the Ca
2⫹
indicator rhod 2
and the Ca
2⫹
caged compound 1-(4,5-dimethoxy-2-nitro-
phenyl)-EDTA (DMNP-EDTA), and after labeling of blood
vessels with dextran-conjugated fluorescein, a Ca
2⫹
ele-
vation evoked in astrocyte endfeet by either photolysis of
DMNP-EDTA or stimulation of neuronal activity was fol-
lowed by a rapid (⬃1 s delay) and marked dilation of the
arteriole. Vasodilation and increase in blood flow, as mea-
sured by laser Doppler flowmetry, were sensitive to COX
inhibitors (204), thereby confirming the previous finding
that COX products are likely released from activated as-
trocyte endfeet to trigger arteriole dilation (242). When
the activation of astrocytes, which accompanies neuronal
activity, was blocked with the mGluR5 antagonist MPEP,
the vascular response induced by stimulation of neuronal
activity was significantly reduced, providing further evi-
dence for the central role of neuron-to-astrocyte signaling
pathway in the neurovascular coupling.
It should be noted in this in vivo study (204), as well
as in Mulligan and MacVicar’s brain slice investigation
(145), that a membrane-permeant EDTA-based caged
compound was used for the photolytic control of internal
Ca
2⫹
. As discussed by Ellis-Davies (50), this will result in
a compound that is 97% loaded with Mg
2⫹
rather than
Ca
2⫹
. Thus, at this time, it is not clear how photolysis
raised internal Ca
2⫹
. Nonetheless, Mulligan and MacVicar
(145) did perform an important control in which they
loaded DMNP-EDTA with Ca
2⫹
, then, after dialysis into a
single astrocyte, they showed that photolytic release of
Ca
2⫹
did replicate their results obtained using DMNP-
EDTA acetoxymethyl ester.
It is important to note that in these in vivo experi-
ments, Ca
2⫹
elevations in astrocyte endfeet occasionally
resulted in a significant arteriole constriction (204). Al-
though it was detected only in a few arterioles, this re-
sponse validates the hypothesis advanced above that ac-
tivation of astrocytes can release both dilating and con-
strictive agents.
The ability of astrocytes to trigger also arteriole con-
striction has been further confirmed in whole-mounted
retina preparations (135). Light stimulation as well as
photolysis of caged Ca
2⫹
that triggered a Ca
2⫹
rise in the
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stimulated glial cell, i.e., an astrocyte or a Mu¨ ller cell,
were indeed found to evoke both dilation and constriction
of retinal arterioles, mediated by different AA metabo-
lites, EETs, and 20-HETE, respectively. Additional evi-
dence for a role of glial cells as mediators of light-induced
response of arterioles was the observation that the inter-
ruption of neuron-to-glia signaling also blocked the re-
sponse of arterioles to light stimulation. Based on a series
of experimental observations, the authors suggest that
NO may play a modulatory role on arteriole responsive-
ness, favoring a vasodilating and vasoconstrictive re-
sponse to light when its level is low and high, respectively
(135).
While these observations confirm that the mecha-
nism that governs the blood flow response to neuronal
activity is complex and relies probably on different vaso-
active agents in different brain regions, they underline the
central role of astrocytes in functional hyperemia.
The important action of astrocytes in the control of
microvasculature raises also the possibility that an astro-
cyte dysfunction could be implicated in the dysregulation
of cerebral circulation in brain pathologies, for example,
in the defective neurovascular coupling that is associated
with Alzheimer’s disease (90, 155), as well as in the vas-
cular abnormal responses during stroke, trauma, and
spreading depression (89). The full characterization of the
molecular mechanisms that are at the basis of the astro-
cyte control on cerebral blood vessels in the normal and
pathological brain certainly represents one of the most
interesting challenges in neurobiological research in
years to come.
VII. DISCOVERY OF GLIOTRANSMISSION:
ASTROCYTES TALK TO NEURONS
In 1994 two studies (147, 160) provided the first
suggestion that the astrocytic Ca
2⫹
signals described by
Stephen Smith’s group do have functional consequences
on integration in the nervous system. In these studies it
was demonstrated that experimentally evoked Ca
2⫹
ele-
vations in astrocytes evoked elevations in the internal
Ca
2⫹
of adjacent neurons. These breakthrough discover-
ies, which were later reproduced in independent studies
performed by Andrew Charles’ (31) and Stan Kater’s lab-
oratories (80), provided a new insight into glial-neuron
interactions in the nervous system. Although one study
suggested an involvement of the gap junction-mediated
communication between the astrocyte and neuron (147),
the others offered a more compelling mechanistic insight
in which the Ca
2⫹
-dependent release of the excitatory
transmitter glutamate from the astrocyte evoked the de-
polarization of the neuron through the activation of iono-
tropic glutamate receptors (160). Indeed, ligands that el-
evated astrocytic Ca
2⫹
led to the release of glutamate
from pure cultures of astrocytes (95, 162), and optical
assays for glutamate demonstrated external waves of glu-
tamate elevation that followed the internal Ca
2⫹
waves in
culture (91).
After the demonstration of a glutamate-mediated as-
trocyte-neuron signaling pathway, it was several years
until it was feasible to document a similar pathway in a
more intact system to alleviate worries about potential
culture artifacts. In 1997, Pasti et al. (165) demonstrated
bidirectional signaling between astrocytes and neurons
and showed that the activation of astrocytic metabotropic
glutamate receptors to evoke glial Ca
2⫹
elevations caused
delayed neuronal Ca
2⫹
signals mediated by ionotropic
glutamate receptors. Given the previous culture studies,
this result was readily interpreted as being due to the
Ca
2⫹
-dependent release of glutamate from hippocampal
astrocytes, an observation that was later confirmed in
slice preparations by Bezzi et al. (19).
VIII. MECHANISMS OF GLUTAMATE RELEASE
FROM ASTROCYTES
Several mechanisms of glutamate release have been
proposed, and it is likely that more than one does operate
within an astrocyte. Evidence has been provided to sup-
port roles for the four pathways: exocytosis, hemi-chan-
nels, anion transporters, and P2X receptors. However,
under the condition of physiological Ca
2⫹
elevations,
there is a groundswell of support for an exocytotic mech-
anism. Although release through hemi-channels (234) and
P2X
7
receptors (46) has been proposed, effective release
requires the presence of low divalent saline to promote
opening of these channels (67, 101). Since under resting
conditions at the membrane potential of an astrocyte and
with normal divalent cation concentrations the open
probability of hemi-channels and of P2X
7
receptors is so
low, these pathways are unlikely to be utilized in physio-
logical conditions. However, it should be noted that dur-
ing neuronal activity, external Ca
2⫹
can fall substantially,
opening the possibility for these pathways of gliotrans-
mission to become activated under conditions of elevated
neuronal activity.
Further doubt has been cast on the potential role of
hemi-channel-mediated gliotransmission by the clear
demonstration that several antagonists that have been
used to block these channels are nonselective and also
block P2X
7
receptors. Additionally, using genetically mod-
ified astrocytoma cells, as well as astrocytes from con-
nexin43
⫺/⫺
and P2X
7
R
⫺/⫺
mice, the release of ATP under
low divalent cation conditions has been clearly demon-
strated to be mediated by P2X
7
R, not by hemi-channels
(199).
Pharmacological evidence has supported a P2X
7
mechanism of release (46); however, debate about
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whether this receptor is expressed in the nervous system
(108, 191), and because it is unlikely to be regulated by
elevations of internal Ca
2⫹
, limits enthusiasm for this
pathway. However, in hippocampal slices, sustained acti-
vation by BzATP of a receptor that has features similar to
the P2X
7
receptor has been recently reported to mediate
a sustained glutamate efflux from astrocytes (55). Under
pathological conditions, such as ischemia and brain
trauma, this P2X
7
-like receptor in astrocytes might be
activated by increasing concentrations of extracellular
ATP (47, 59, 125, 130, 134). The consequent glutamate
release may contribute to increasing the extracellular
concentration of glutamate to the abnormal levels that
cause excitotoxic cell death (35). Consistent with this
hypothesis are the observations that following an acute
spinal cord injury in rats, the functional recovery was
enhanced and the death of motoneurons decreased when
P2X
7
receptors were pharmacologically inhibited (222).
The role of anion transporters/channels is unclear at
this time. One of the problems with studying this pathway
is the poor selectivity of antagonists. For example, NPPB,
which inhibits transporters, can inhibit the filling of ves-
icles with transmitters (212). Nonetheless, under condi-
tions that promote swelling, it is likely that this pathway
could contribute to the release of glutamate from the
astrocyte (103, 104). The expression of dominant negative
vesicle proteins in astrocytes to inhibit Ca
2⫹
-regulated
exocytosis leaves a swelling-induced pathway of transmit-
ter release from astrocytes unaffected (236). Thus a volu-
metric release pathway likely exists in parallel to an exo-
cytotic pathway (203). A future challenge is to identify the
conditions that select for each of these two pathways.
There is now compelling evidence supporting an exo-
cytic mechanism of glutamate release from astrocytes.
These glial cells express a variety of vesicle proteins that
are essential for exocytosis (39, 83, 128, 161, 227, 236).
Clostridial toxins, when introduced into the astrocyte to
cleave target SNARE proteins, prevent glutamate release
(10). Ca
2⫹
elevations lead to an increase in membrane
surface area synchronous with the release of glutamate
(237). Bafilomycin A
1
, which inhibits the V-ATPase that
pumps protons into the vesicle (23) that are required to
drive the transport of glutamate, inhibits the uptake of
L-[
3
H]glutamate in astrocyte vesicles (39) and reduces the
release of glutamate (10, 166). Rose Bengal, an inhibitor
of vesicular glutamate transporters (VGLUT), similarly
blocks Ca
2⫹
-dependent glutamate release (140). Finally,
immunoelectron microscopy has revealed VGLUT ex-
pressing vesicles within the process of astrocytes in vivo
(20).
Volterra and colleagues (20) have used total internal
reflection fluorescence microscopy to reveal expressed
VGLUT-EGFP fusion proteins as well as acridine orange
(AO), a dye that accumulates in acidic organelles, and can
be used as a marker for exocytosis. Because of an absor-
bance shift in AO when located within vesicles compared
with physiological saline, fusion of an AO-filled vesicle
with the plasma membrane leads to a rapid change in AO
fluorescence emission, when excited at ⬃490 nm, as this
dye mixes with the extracellular saline. Stimuli that in-
duce Ca
2⫹
elevations were shown to cause a brief burst of
AO fluorescence events together with a reduction in the
numbers of VGLUT-EGFP fluorescent puncta, observa-
tions consistent with regulated exocytosis in astrocytes.
Furthermore, cocultured glutamate receptor expressing
reporter cells simultaneously detected the release of glu-
tamate providing strong evidence supporting exocytotic
release of this gliotransmitter from astrocytes. These re-
sults have recently been supported by an independent
study which has shown Ca
2⫹
-dependent fusion of vesicles
with the plasma membrane (39).
IX. KISS-AND-RUN RELEASE OF GLUTAMATE
FROM ASTROCYTES
Although this evidence is overwhelmingly in support
of a exocytic mechanism, there are still detractors who
argue that it is possible that the vesicle is inserted into the
membrane to provide a channel or transporter to mediate
the release of glutamate. Although we feel that the evi-
dence does not support their contention, there is one
troubling aspect to the vesicular hypothesis of glutamate
release from the astrocyte, since there are few vesicles
within astrocytic profiles in vivo. However, one recent
observation suggests that the mechanism of transmitter
release from the vesicle may be biased towards a kiss-
and-run fusion mechanism in which the vesicle does not
fully fuse with the plasma membrane but instead forms an
ephemeral pore that permits transmitter to be released
(33). This possibility is based on the observation that
synaptotagmin IV is essential for Ca
2⫹
-dependent gluta-
mate release from astrocytes (236). The synaptotagmin
gene family is known to play essential roles in the regu-
lation of vesicle fusion in different cell types (105). In
nerve terminals, synaptotagmin I plays a dominant role.
However, when synaptotagmin IV is expressed in place of
synaptotagmin I, fusion events are biased toward kiss-
and-run release rather than full fusion (221). An important
consequence is that the vesicle reacidifies, a critical step
for refilling of the vesicle with transmitter, up to 20 times
faster after kiss-and-run release compared with full fusion
events (63). Consequently, if synaptotagmin IV does in-
deed promote kiss-and-run release of glutamate from as-
trocytes, one can envision that one astrocytic vesicle is
equivalent to 20 nerve terminal vesicles.
Studies of exocytosis have been significantly ad-
vanced by the electrochemical detection of released
transmitters. This approach has now been turned to in-
vestigate the Ca
2⫹
-dependent release of transmitter from
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astrocytes (33). Because glutamate is not directly de-
tected by carbon fiber amperometry, astrocytes were pre-
loaded with dopamine, which, if released, is readily de-
tected electrochemically. Stimuli that elevate astrocytic
Ca
2⫹
were shown to cause rapid amperometric spikes.
Mechanical stimuli, which cause large prolonged Ca
2⫹
elevations in astrocytes, caused very large amperometric
signals. However, physiological stimuli only led to the
release of 1/10th of the vesicle content. During the brief
opening of a fusion pore (⬃2 ms), they showed quantal
transmitter release and suggest that relatively large vesi-
cles (⬃300 nm diameter) normally serve to mediate glu-
tamate release through a kiss-and-run mechanism that
only depletes a portion of the vesicular transmitter store.
Such a kiss-and-run mechanism could account for
transmission in vivo where there is a paucity of astrocytic
vesicles. Though they did demonstrate similar results
with acutely isolated astrocytes to answer concerns about
culture artifacts, and an independent study supports the
potential for large vesicular structures mediating trans-
mitter release from astrocytes in brain slices (97), it is not
yet clear where these large vesicles reside within an as-
trocyte in vivo. Indeed, immunoelectron microscopy has
shown the presence of 30-nm vesicles in an independent
study (20).
X. THE TRIPARTITE SYNAPSE: ASTROCYTES
MODULATE NEURONAL EXCITABILITY AND
SYNAPTIC TRANSMISSION
Following the discovery of the regulated release of
glutamate from astrocytes, many studies have gone on to
demonstrate that this gliotransmitter can modulate syn-
aptic transmission and neuronal excitability. In addition
to glutamate, glial-released D-serine and ATP have been
discovered to mediate powerful synaptic actions. To
maintain some coherence in our discussion, we present
information related to each gliotransmitter in indepen-
dent sections.
XI. RELEASE OF GLUTAMATE
FROM ASTROCYTES
Initially cell culture studies showed that astrocytes
can use glutamate to modulate neuronal excitability and
synaptic transmission. Whole cell recordings from a neu-
ron that was cocultured with astrocytes demonstrated
slow glutamate-mediated inward currents (SICs) when a
glial Ca
2⫹
elevation confronted the recorded neuron (12).
These results were later substantiated in thalamus and
hippocampus by showing pure N-methyl-D-aspartate
(NMDA) receptor-dependent neuronal currents following
astrocytic Ca
2⫹
elevations (54, 55, 163). In addition to
direct activation of neuronal currents, glial-released glu-
tamate was also shown to modulate synaptic transmis-
sion. Again in culture, astrocytic Ca
2⫹
elevations aug-
mented the frequency of miniature excitatory postsynap-
tic currents (EPSCs) and inhibitory postsynaptic currents
(IPSCs), an action that was judged to be due to the
gliotransmitter glutamate because effects were blocked
by the NMDA receptor antagonist D-AP5 (14). In brain
slice preparations, similar actions were observed because
GABAergic activation of Ca
2⫹
signals in astrocytes
caused an NMDA receptor-dependent increase in inhibi-
tory mIPSC frequency detected in pyramidal neurons and
a strengthening of certain inhibitory synapses (96). Sub-
sequently, other forms of synaptic modulation have been
identified in which metabotropic glutamate receptors and
kainate receptors mediate the actions of glial-released
glutamate. Single astrocyte photolysis of caged IP
3
in-
creases the frequency of excitatory mEPSCs, an action
that is blocked by the metabotropic receptor antagonists
LY367385 and 2-methyl-6-(phenylethynyl)-pyridine, sug-
gesting that glutamate release from astrocytes can act
through neuronal class I metabotropic glutamate recep-
tors to augment the release of transmitter from nerve
terminals (58). Flash photolysis of caged Ca
2⫹
also in-
creases spontaneous action potential-driven IPSCs, an
action that is mediated by kainate receptors containing
the GluR5 subunit (120).
These studies clearly show the potential for astro-
cytes to integrate neuronal activity and to provide feed-
back modulatory signals. The roles for these feedback
pathways are not yet known in the hippocampus. How-
ever, the first critical demonstration of the integrated
action of synaptic activity and synaptically associated
glial signals was provided by studies performed at the frog
neuromuscular junction. Associated with the neuromus-
cular junction are several perisynaptic Schwann cells that
are molecularly and functionally distinct cells from the
myelinating Schwann cell. Richard Robitaille (178) per-
formed elegant experiments in which he directly manip-
ulated GTP-binding protein signaling within these glia.
Direct microinjection of guanosine 5⬘-O-(3-thiotriphos-
phate) (GTP
␥
S) into the perisynaptic Schwann cell to
activate G protein signaling caused a reduction in the
strength of the neuromuscular junction. To ask whether
this glial-modulation pathway is recruited during physio-
logical conditions, he studied activity-dependent depres-
sion of this synapse. High-frequency stimulation of the
motoneuron axon causes a reversible depression of the
neuromuscular connection. However, after injection of
guanosine 5⬘-O-(2-thiodiphosphate) (GDP

S) into the
Schwann cell, to prevent G protein activation, stimulation
of the nerve trunk led to a diminished depression. Taken
together, these studies led to the proposal of the “tripar-
tite synapse” in which the astrocyte listens to synaptic
activity and provides feedback modulation of the strength
of the synaptic connection (13).
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XII. ASTROCYTES ACTIVATE EXTRASYNAPTIC
NMDA RECEPTORS
As discussed above, cell culture studies initially dem-
onstrated that astrocytes, by releasing glutamate, can ac-
tivate neuronal NMDA receptors. Substantiation of this
observation was recently provided in brain slice studies in
which glial glutamate was shown to selectively access a
specific class of NMDA receptor that contains the NR2B
subunit (54). In these studies a variety of stimuli, each of
which leads to astrocytic Ca
2⫹
oscillations, all caused
D-AP5-sensitive, NMDA receptor-mediated SICs in area
CA1 pyramidal neurons. Moreover, blockade of synaptic
transmission by brief incubation in tetanus toxin did not
prevent the detection of these SICs showing that they
were from a nonneuronal origin. (It should be noted that
while tetanus toxin can prevent glutamate release from
astrocytes when applied from the extracellular space, it
does so with such a slow time course, due to a paucity of
toxin receptors, that it is possible to selectively inactivate
nerve terminals with short-term treatment.) Finally, sin-
gle-cell stimuli such as flash photolysis of caged Ca
2⫹
,
specifically in the astrocyte, or single astrocyte depolar-
ization all evoked neuronally detected NMDA receptor
(NMDAR)-dependent SICs.
Where are the NMDARs located that mediate the
SIC? Using MK-801 to allow a use-dependent block of
synaptic NMDARs, initial culture studies demonstrated
that astrocytes predominantly talk to extrasynaptic
NMDARs (14). Measurement of the kinetics of SICs in
brain slice studies showed that they are very slow com-
pared with synaptic NMDA currents (8, 54). The rise time
of the astrocyte-evoked SIC is ⬃60 ms, although rise
times of 100–200 ms are not rare. The decay time is on the
order of 400–500 ms. Different subunit composition of the
NMDAR accounts for the different kinetics. Indeed, the
NMDAR is normally composed of two subunits: NR1 plus
an NR2 subunit and either NR2A, B, C, or D. Studies in
which the different NMDAR subunits are expressed in
heterologous systems have shown that certain subunit
combinations have distinguishing kinetic properties (41,
111, 133, 218). The same distinguishing kinetics are re-
ported from the native NMDARs (137, 141). Once syn-
apses have developed, synaptic NMDARs are primarily
comprised of NR1/NR2A subunits, while NR1/NR2B be-
come located at extrasynaptic locales (181, 206). Because
NR2B-containing NMDARs exhibit slowly decaying cur-
rents of similar kinetics to the SICs we observe, we asked
whether the NR2B selective antagonist ifenprodil would
attenuate the SICs. In agreement with the kinetic data,
ifenprodil selectively blocked the astrocyte-evoked SIC
with little or no effect on synaptic NMDA currents (54).
These data lend further support to the notion that the
glutamate released from astrocytes selectively acts on
extrasynaptic NMDARs that contain the NR2B subunit, an
observation which is supported by the results of double-
label immunoelectron microscopy (20) (Fig. 2).
Can this activation of extrasynaptic NR2B-NMDARs
hint at a distinct role of this astrocyte-to-neuron signal-
ing? There is increasing awareness that synaptic and ex-
trasynaptic NMDARs may subserve different, and to some
extent opposing, functional roles (42, 213). For example,
it has been reported that by shutting down activity of
cAMP response element binding protein (CREB), NR2B-
FIG. 2. Glutamate released from presynaptic terminals and from astrocytes acts on distinct NMDA receptors. The application of NMDA receptor
subunit-selective pharmacology to mature synapses has shown that synaptic glutamate preferentially acts on NR2A subunit-containing NMDA
receptors in addition to AMPA receptors, while astrocytic glutamate activates NR2B subunit-containing, extrasynaptic NMDA receptors. Here we
show the NR2B-containing receptors in an extrasynaptic locale of the spine. It should be noted that we provide this location for illustrative purposes
as they could equally well be located in the parent dendrite. Activation of NR2A- and NR2B-containing NMDA receptors leads to distinct cellular
responses: synaptic NR2A NMDA receptors lead to CREB activation, AMPA receptor recruitment, and LTP, while NR2B-containing receptors have
opposing actions potentially being involved in LTD, CREB shut-off, as well as promoting the synchronous activation of neurons.
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NMDAR activation promotes neuronal death, while by
inducing CREB activity, NR2A-NMDAR activation pro-
motes neuronal survival (76). Activation of the ERK sig-
naling pathway may also depend primarily on NR2B-con-
taining NMDARs (107), although this issue remains con-
troversial (102). A series of studies propose that NR2B-
containing NMDA receptors mediate synaptic cross-talk
(189), and the induction of long-term synaptic depression
(LTD), while NR2A-containing NMDARs seem more im-
portant in the induction of long-term potentiation (LTP)
(119, 131). As a plausible mechanism of NR2B-NMDAR-
mediated LTD, it has been observed that NR2A-NMDARs
promote, whereas NR2B-NMDARs inhibit, surface expres-
sion of AMPA receptors, essentially by regulating the
membrane insertion of GluR1 subunit (102). The conclu-
sions drawn in these studies rely mainly on the use of the
specific NR2B subunit antagonist ifenprodil, and the re-
cently developed antagonist of the NR2A subunit NPV-
AAM077. However, the observations that this latter com-
pound is relatively selective for the NR2A subunit and
decreases its selectivity with prolonged exposure cast
doubts about its use as a pharmacological tool to dissect
out the distinct role of the NR2A-NMDAR in LTP (18, 225).
While we need to improve our understanding of the
role of the different NMDAR subunits in the plastic
changes of synaptic strength, the available data allow us
to advance the hypothesis that NR2B-NMDA receptors,
which during early postnatal development are progres-
sively confined to extracellular locations, represent a
common, preferential target of either glutamate spilled
over from synapses and glutamate released from acti-
vated astrocytes (Fig. 2). It will be of extreme interest to
determine if and to what extent astrocytic glutamate,
while acting on NR2B-NMDARs, may contribute to funda-
mental events in neuronal transmission such as LTP and
LTD.
With the identification of the presence of the astro-
cyte-evoked NMDA current, it is important to determine
under which circumstances this current will be activated,
and whether these conditions could provide the synapse
with additional information. Specifically designed exper-
iments are needed to clarify these issues.
Given that along with NMDARs, AMPARs are also
expressed at extrasynaptic locations, the absence of
AMPA-mediated currents came initially with some sur-
prise. However, when cyclothiazide (CTZ) and D-AP5
were included in ACSF to prevent AMPA receptor desen-
sitization (167) and NMDA receptor activation, respec-
tively, AMPA receptor-mediated SICs were detected (54).
This result provides two important conclusions. First, it is
probably important that the astrocyte only talks to extra-
synaptic receptors; otherwise, synaptic access of glial
glutamate, by leading to a desensitization of synaptic
AMPA receptors, would reduce the fidelity of synaptic
transmission. Second, because there is no AMPA compo-
nent accompanying activation of NR2B-containing NMDA
receptors, the astrocyte alone is not able to cause neuro-
nal currents. Instead, a coincidence of an independent
depolarizing stimulus together with glutamate release
from the astrocyte will be required to admit current
through the extrasynaptic NMDA receptor. The exact con-
ditions that support such coincidence are yet to be de-
fined, although astrocyte-evoked SICs can be detected in
Mg
2⫹
-containing saline albeit at a lower frequency than in
Mg
2⫹
-free conditions.
Interestingly, the rise time of AMPA-mediated events
recorded in the presence of CTZ and D-AP5 is comparable
to that of NMDAR-mediated events. Apparently, the con-
centration of glia glutamate increases relatively slowly in
the extracellular space, and this determines the slow
activation of NMDARs and AMPARs in the neuronal mem-
brane. While a slow diffusion of glia glutamate in the large
extracellular space can reasonably account for the time
course of the increase in its extracellular concentration, it
cannot be excluded that the process of glutamate release
from the astrocyte could be somewhat slower than that
from neurons.
XIII. WHY ARE ASTROCYTE-EVOKED NMDA
CURRENTS SO LARGE IN AMPLITUDE?
Astrocyte-evoked NMDAR currents can be extremely
large in magnitude. The average current detected is ⬃100
pA (8, 53, 55). In contrast, the NMDA current due to the
fusion of a single vesicle in the synapse is of the order of
2–3 pA (156). If both are mediated by exocytosis, why are
SICs so large? The size of vesicles within astrocytes have
been reported to range from 30 to 300 nm (20, 33, 39). If
the vesicles that mediate the release of glutamate from
the astrocyte are indeed 300 nm, as suggested by Chen et
al. (33), rather than ⬃40 nm in diameter, then the astro-
cytic vesicle will contain 422 times as much transmitter as
a synaptic vesicle based purely on volumetric arguments.
Because extrasynaptic NMDARs are at most 1/10th the
density of synaptic receptors (156), the magnitude of an
astrocyte-evoked NMDA receptor current could be as
large as 85–126 pA (assuming a synaptic NMDA current of
2–3 pA), and an absence of receptor saturation. Thus an
average amplitude of the SIC of 100 pA is not out of the
question, although it likely involves glutamate diffusion
from the site of release to distant NMDARs that are not
already occupied by transmitter. Such a requirement for
diffusion would also account for the desensitization of
AMPA receptors.
These arguments hold for full-fusion of a 300-nm
vesicle. However, the studies of Chen et al. (33) show that
fusion-pore release of transmitter is the norm under phys-
iological conditions. If, as they identified for the false
transmitter dopamine, the vesicle normally only releases
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1/10th of its transmitter, then it is likely that astrocyte-
evoked NMDA currents are of a much smaller amplitude,
⬃8–12 pA in magnitude. Although it is possible to detect
currents of this magnitude when looking at stimulus
evoked events, this is a very difficult task when merely
scrolling through ongoing recordings and especially given
the slow kinetics of NMDAR-mediated SICs. Based on
these arguments, we conclude that the 100 pA SIC de-
tected in recordings from pyramidal neurons likely reflect
the full-fusion of a relatively large glutamate-filled vesicle
with the astrocytic plasma membrane, which activates
distant, extrasynaptic NMDARs. These SICs are rare, oc-
curring within a given pyramidal neuron at a frequency of
⬃1/min. Because physiological stimuli preferentially
cause fusion-pore release of transmitter, we anticipate
that these SICs represent large, supranormal events. In-
stead, higher frequency fusion-pore-related events are
likely to be ongoing that have been beneath the resolution
of our recording conditions.
XIV. ASTROCYTES SYNCHRONOUSLY ACTIVATE
GROUPS OF PYRAMIDAL NEURONS
The quantitative arguments just presented would
suggest that the release of a vesicle of glutamate from an
astrocyte would have the potential to act synchronously
on several adjacent dendrites, because to achieve the
magnitude of NMDA receptor current, the transmitter
must diffuse to activate unoccupied receptors. The notion
of synchronous activation of neurons has been tested
using confocal imaging as well as paired electrophysio-
logical recordings. Activation of class I mGluRs causes
Ca
2⫹
oscillations on astrocytes and to the delayed, syn-
chronous Ca
2⫹
accumulation in groups of pyramidal neu-
rons. When paired recordings are made from pyramidal
neurons, synchronous astrocyte-evoked SICs are detected
as long as the recorded cell bodies were within ⬃100
m
of one another (8, 54). We do not feel that this means that
glial-released glutamate is diffusing 100
m to access
these neurons. Rather, a dendrite of these paired neurons
likely occupies a similar volume within the neuropil, al-
lowing them to both detect the glial-released glutamate.
At this time the functional implications of this synchro-
nous NMDAR activation are not clear and await a further
understanding of whether the 100-pA SICs are the normal
amplitude event, or whether the majority are smaller in
amplitude with more spatially confined actions that might
not lead to synchronous activation of groups of neurons.
When one considers two distinct functions of the
astrocyte, the control of the cerebrovasculature and the
release of gliotransmitters, it becomes clear that an un-
derstanding of the spatiotemporal constraints on glial
Ca
2⫹
signaling is essential to develop an integrated view
of the function of these glial cells. On the one hand, we
have discussed the importance of the astrocyte in re-
sponding to synaptic activity and causing Ca
2⫹
elevations
in perivascular endfeet. Yet, on the other hand, we have
discussed the equal importance of local microdomain
Ca
2⫹
signals to maintain a degree of synaptic specificity
on glial glutamate signaling. How can these two conflict-
ing issues be resolved?
XV. D-SERINE: SELECTIVE SYNTHESIS IN
AND RELEASE FROM ASTROCYTES
It has been appreciated for a considerable time that
the NMDAR requires not only glutamate for its activation,
but also the coagonist glycine. More recent data suggest
that D-serine may actually be the endogenous ligand for
this modulatory glycine-binding site of the NMDAR (143).
Astrocytes express an enzyme, serine racemase, which
converts L-toD-serine (229). Since this enzyme is only
expressed in astrocytes (186, 187, 228), these glial cells
are the source of this endogeous ligand for the glycine-
binding site of the NMDAR.
Although little evidence is available concerning the
mechanism of release of this amino acid, one study pro-
vides strong evidence for a Ca
2⫹
-regulated exocytotic
release pathway. Similar to glutamate release, Ca
2⫹
ele-
vations are both necessary and sufficient for the release of
D-serine (144). Subcellular fractionation on sucrose gradi-
ents revealed D-serine in the same fractions as synapto-
brevin II and as glutamate raising the possibility of the
coloading of glutamate and D-serine in the same vesicles.
Further support for an exocytotic mechanism of release
was provided by a punctate immunolocalization and that
treatment of cultures with the clostridial toxin tetanus
toxin, which cleaved synaptobrevin II, caused a signifi-
cant attenuation of the Ca
2⫹
-dependent release of this
amino acid.
Because of this evidence supporting an exocytotic
release pathway for this transmitter, it is essential that in
studies of glial glutamate release, which have been shown
to act selectively on NMDARs, controls for actions medi-
ated by D-serine are included. Thus, in several of the
studies discussed concerning astrocyte-evoked NMDAR-
mediated SICs, glycine was added to slice preparations at
saturating concentrations to ensure that results could
be assigned to effects of glutamate rather than D-serine
(8, 54).
Because D-serine is selectively synthesized in astro-
cytes, it has been possible to ask whether this gliotrans-
mitter impacts synaptic transmission and plasticity (40).
In the retina, addition of exogenous D-serine augments
NMDA receptor currents, while addition of D-amino acid
oxygenase, which degrades D-serine, reduces the magni-
tude of these currents (196). In hippocampal cultures
devoid of astrocytes, addition of D-serine has been shown
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to be critical to enable the induction of synaptic plasticity,
while in brain slices and in mixed cultures of astrocytes
and neurons, degradation of D-serine blocks LTP induc-
tion (233). The crucial role of D-serine in LTP is strength-
ened by the finding that the impairment of LTP observed
in CA1 neurons from slices of 12-mo-old rats is overcome
by addition of D-serine (232).
In the hypothalamic supraoptic nucleus (SON), the
astrocytic coverage of glutamatergic synapses changes as
animals enter lactation, allowing the unique opportunity
to determine the role of astrocyte-derived D-serine in the
regulation of synaptic transmission and plasticity. As an-
imals begin lactation, the astrocytic coverage of these
synapses is drastically reduced. By comparing the NMDA
component of the synaptic current in virgin and lactating
rats, Oliet and colleagues (159) demonstrated a reduced
NMDA component of the synaptic connection that corre-
lates with the reduced glial coverage of the synapse.
Because exogenous addition of D-serine enhances the
NMDA component in lactating animals compared with
that seen in virgin rats, the authors conclude that glial
D-serine acts as the endogenous coagonist of the NMDAR
and locally regulates the degree to which synaptic gluta-
mate can activate these receptors. In addition to studying
synaptic transmission, they also ask whether glial-derived
D-serine regulates plasticity. Their results provide the ex-
citing observation that the astrocyte controls a form of
metaplasticity: whether a synapse exhibits LTP or LTD is
controlled by the glial coverage of the synapse and the
extent to which D-serine is provided to synaptic NMDARs.
When the astrocytic processes retract and the level of
synaptic D-serine is reduced, LTD is induced, whereas in
virgin rats that have a high degree of synaptic coverage,
the same stimulus induces LTP.
The degree to which D-serine is released in a tonic
manner, versus in an activity-dependent fashion, and thus
can dynamically regulate NMDAR function remains to be
demonstrated. However, that exogenous D-serine can aug-
ment NMDAR activity indicates that the glycine-binding
site is not saturated under resting conditions, and opens
the potential for dynamic regulation of this site by Ca
2⫹
signaling in astrocytes and the associated release of D-
serine. If glutamate and D-serine are copackaged in the
same vesicle, an interesting possibility is raised in which
the regulated release of agonist and coagonist will en-
sure maximal activation of the NR2B-containing NMDAR
(Fig. 3).
XVI. RELEASE OF ATP FROM ASTROCYTES
In addition to glutamate and D-serine, astrocytes also
release the chemical transmitter ATP. Though many
fewer studies have been performed in which this nucleo-
tide has been studied, significant advances have been
made in understanding its role in the regulation of synap-
tic transmission.
Because the studies are at such an early stage, the
mechanisms controlling the release of ATP are far from
FIG. 3. Astrocyte-derived signals act both
presynaptically and postsynaptically to regu-
late synaptic transmission. The release of glu-
tamate, D-serine, and ATP from astrocytes has
a diversity of synaptic actions. Presynaptically,
glutamate can access metabotropic glutamate
receptors (58) and kainate receptors (120) to
enhance synaptic transmission. Postsynapti-
cally, glutamate can act on extrasynaptic
NMDA receptors to depolarize the neuronal
membrane and promotes neuronal synchrony
(54), while D-serine acts on the glycine-binding
site of NMDA receptors and can regulate syn-
aptic plasticity (159). ATP may also act
postsynaptically on P2X receptors to depolar-
ize the neuronal membrane and regulate the
insertion of postsynaptic AMPA receptors (68).
After hydrolysis by ectonucleotidases to aden-
osine, ATP can have distant action on presyn-
aptic A1 receptors to cause heterosynaptic de-
pression of excitatory synaptic transmission
(164).
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resolved. Indeed, there is even debate about whether the
ATP is released through a Ca
2⫹
-dependent mechanism.
Until the proximate stimulus is understood, it will be
difficult to make headway in understanding the details of
this purinergic pathway.
The identification of ATP being a messenger of the
astrocyte was beautifully demonstrated in studies con-
cerning Ca
2⫹
waves that propagate between astrocytes in
culture. When waves confronted a cell-free region, they
could jump the gap due to the release of a diffusible
message (81). Later lucerifin/luciferase studies demon-
strated that ATP was released during Ca
2⫹
waves. How-
ever, chelation of Ca
2⫹
with BAPTA, a manipulation
which blocks glutamate release, did not affect the release
of ATP (223). Although it is clear that both ATP and
glutamate release require phospholipase C activity (223),
it is not certain whether the diacylglycerol or IP
3
arms of
this pathway are critically involved in regulating ATP
release. In support of a role for the diacylglycerol path-
way is the ability of phorbol esters to stimulate ATP and
to a lesser extent glutamate release from cultured astro-
cytes (36). The action of phorbol esters is, however, very
intriguing as it raises the potential for independent regu-
lation of the two primary transmitters that are released
from astrocytes. Whether the release of ATP is protein
kinase C (PKC) dependent is not clear because it is now
appreciated that phorbol esters stimulate a variety of
diacylglycerol effectors including PKC, RasGRP, protein
kinase D, and the vesicle-associated protein munc-13 (26,
177, 194). Of particular interest is munc-13. This vesicle-
associated protein is a homolog of the Caenorhabditis
elegans uncoordinated (unc)-13 protein that has been
shown to be essential for synaptic transmission. Munc-13
contains C1 diacylglycerol binding sites and acts by un-
folding the SNARE protein syntaxin so that it is able to
form the SNARE complex that is essential for vesicle
priming before exocytosis (26). Since the release of ATP
from astrocytes exhibits sensitivity to tetanus toxin and is
blocked by expression of a cytosolic SNARE domain
(164), an exocytic mechanism may underlie the release of
this nucleotide. Thus a candidate regulatory pathway for
ATP release is through diacylglycerol-dependent activa-
tion of munc-13.
XVII. GLIAL-DERIVED ATP MODULATES
NEURONAL EXCITABILITY
Although details of the regulatory pathway control-
ling ATP release are sketchy, what is clear is that follow-
ing its release it has powerful actions on adjacent neu-
rons. In the retina Eric Newman (151) has demonstrated
that activation of astrocytes and Mu¨ ller glial cells causes
ATP to be released. However, after ATP release, which
has been measured with luciferin/luciferase imaging in
the retina, neuronal actions are mediated by adenosine.
Once ATP is released into the extracellular space, it is
well known that there are a variety of ectonucleotidases
that are responsible for the hydrolysis of ATP to AMP, and
then a 5⬘-nucleotidase that is responsible for the final
hydrolysis of AMP to adenosine (240). This nucleoside
has potent actions mediated by A1, A2, and A3 receptors.
Of relevance to this discussion, however, are A1 receptors
that are activated by concentrations of adenosine of the
order of tens of nanomolar. Thus ATP can be released in
concentrations that might be subthreshold for the activa-
tion of neuronal P2 receptors (micromolar) yet suffi-
ciently high to allow an accumulation of adenosine that
will activate A1 receptors. In the retina, ATP that is re-
leased from glial cells is hydrolyzed to adenosine, where
it causes the activation of K
⫹
currents that hyperpolarize
retinal ganglion neurons (151). In hippocampal cultures,
the release of ATP from stimulated astrocytes was also
found to depress glutamatergic neuronal transmission
(106). The authors suggest that both ATP and the ATP
metabolite adenosine contribute by acting on presynaptic
P2 and A1 receptors, respectively. Activation of P2Y
1
receptors on CA1 interneurons and astrocytes from hip-
pocampal slices by ATP, probably released by both neu-
rons and astrocytes, was also found to cause an interneu-
ron excitation that leads to increased GABAergic synaptic
inhibition onto pyramidal neurons (24).
It is certainly worth mentioning that the control of
the relative levels of ATP, ADP, AMP, and adenosine is a
complex process in which several enzymes are involved
(28, 49). Of particular interest is the observation that the
5⬘-nucleotidase responsible for the hydrolysis of AMP to
adenosine is inhibited by nucleotides (239). Thus one
could envision that under low rates of release of ATP, this
nucleotide is effectively hydrolyzed to adenosine. How-
ever, with elevated levels of release it is possible that
nucleotides will accumulate since the 5⬘-nucleotidase will
be inhibited by their elevated levels.
XVIII. PURINERGIC MODULATION
OF SYNAPTIC TRANSMISSION
Since adenosine is known to cause a presynaptic
inhibition of transmitter release, it is possible that astro-
cytes by releasing ATP could exert a purine-dependent
regulation of synaptic transmission. Over a decade ago it
was realized that activity in a synaptic pathway acts lat-
erally to regulate the relative strength of neighboring
synapses (129). Stimulation of one pathway with a high-
frequency train (100 Hz for 1 s) caused a depression of the
neighboring unstimulated pathway. How did these two
pathways talk to one another? Through pharmacological
studies it was determined that the heterosynaptic sup-
pression was mediated by the accumulation of adenosine
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that acted through A1 receptors. However, the source was
unclear. In 2003, it was suggested that the adenosine was
derived from the astrocyte (235). These experiments were
based on the use of a metabolic poison, fluorocitrate,
which due to selective accumulation in astrocytes is al-
leged to selectively inhibit the tricarboxylic acid cycle of
the astrocyte (201, 202). Notwithstanding the concerns
about this poison, this study raised the intriguing possi-
bility that the astrocyte mediates heterosynaptic suppres-
sion of synaptic transmission (Fig. 3).
It has been unclear how adenosine accumulates in
the extracellular space. Several mechanisms have been
proposed including the direct release through equilibra-
tive transporters as well as through the hydrolysis of
released nucleotides (28, 49). These studies, as well as the
study of Newman (151), indicate the importance of the
hydrolysis of released ATP. In the presence of a P2 re-
ceptor antagonist to block direct actions of ATP, the
inhibition of ectonucleotidases reduced the adenosine-
dependent heterosynaptic suppression, as well as the
glial-evoked activation of neuronal K
⫹
currents (151).
In contrast to the suppressive effects of astrocytic
purines in the hippocampus, ATP has been shown to
potentiate the amplitude of glutamate-mediated mEPSCs
in the paraventricular nucleus (68). Although details still
remain to be determined, this study is consistent with the
idea that an extrinsic input to the astrocyte causes the
release of ATP from these glial cells which, by acting
through postsynaptic P2X receptors, causes the insertion
of postsynaptic AMPA receptors. Again, as discussed for
the studies by Zhang et al. (235), this study utilized fluo-
rocitrate as a glial metabolic poison. Following treatment
purinergic actions were lost. Importantly, however, an
independent analysis of stressed animals, in which dehy-
dration causes a retraction of the glial coverage of the
synapse, confirmed the importance of the glial cell in
mediating the purinergic signal (68). In this condition, and
in agreement with fluorocitrate treatment, actions of ATP
were lost. Although it is not clear how a persistent eleva-
tion of ATP is achieved in the face of the activity of
ectonucleotidases, these results point to a widespread
role for an astrocytic source of ATP in the regulation of
synaptic transmission.
XIX. INTRODUCTION OF MOLECULAR
GENETICS TO ADDRESS THE
ROLES OF THE ASTROCYTE IN
NEURONAL FUNCTION
A challenge in studying the role of gliotransmission in
the regulation of synaptic integration is that receptors
expressed on astrocytes are similarly expressed on neu-
rons, making cell-selective pharmacological manipula-
tions a challenge. Consequently, the use of a metabolic
poison, fluorocitrate, in brain slice studies, in combina-
tion with an analysis of purified cultures of astrocytes, has
been used to identify roles for gliotransmission in the
nervous system (235). The ideal way to overcome this
problem is to use cell type-specific molecular genetic
approaches to manipulate the astrocyte and then deter-
mine consequences for neuronal function. Several labora-
tories have been making great advances in this direction,
which has resulted in the first direct demonstration of the
roles of astrocyte-derived ATP in the control of synaptic
transmission, plasticity, and heterosynaptic cross-talk
(164). Before discussing the results of this study, the
general strategy that has been utilized is discussed.
To use molecular genetics, an astrocyte-specific pro-
moter is needed to direct gene expression. Michael Bren-
ner’s group has isolated the human glial fibrillary acidic
protein (GFAP) promoter and used it extensively in stud-
ies of the astrocyte (25, 198, 238). Initially, this promoter
was used to drive the expression of reporters, and they
were shown to be selective for the astrocyte. However,
further analyses of different transgenic animals have
shown that this cell type specificity is not absolute and
great caution must be used in characterizing each line of
animal that is produced (198).
Nonetheless, under stringent conditions astrocyte
specificity can be achieved. One powerful way to study
the astrocyte would be to employ the Cre recombinase
system in combination with the GFAP promoter to excise
specific genes selectively in astrocytes. Although initial
tests of this approach have been performed, there are
grave concerns about this strategy because neuronal pro-
genitors express GFAP. Consequently, the floxed allele is
excised in cells that become neurons as well as astrocytes
(238).
Perhaps the most powerful approach to be used is
the combination of cell type specificity together with
inducibility of transgenes. Gossen and Bujard (69) have
developed such an inducible transgenic system that can
be applied in a cell type-specific manner. This tetracy-
cline-regulated system requires the expression of two
transgenes. A cell type-specific promoter is used to ex-
press a tetracycline transactivator protein (tTA) which
binds to a tetracycline operator (tetO) that drives the
expression of the transgene of choice. In cells that do not
express tTA, tetO does not drive transgene expression.
Additionally, tTA binds doxicycline (dox), a tetracycline
analog that can be introduced into mice by addition to the
food or drinking water. In the presence of dox, tTA is
unable to bind to tetO preventing transgene expression.
Thus, in this system, one achieves both cell type specific-
ity and inducibility of transgene expression. Ken Mc-
Carthy and co-workers (117, 164) have now developed
this approach for the astrocyte and demonstrated astro-
cyte specificity of inducible transgene expression.
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Although the mechanism of release of ATP is not
definitively resolved, because its release is reduced by
tetanus toxin (36), the expression of a SNARE domain of
the SNARE protein synaptobrevin II, which we know is
expressed in astrocytes (237), should block the formation
of the endogenous SNARE complex and thus prevent
vesicle fusion with the plasma membrane and thus release
of ATP. Therefore, transgenic mice have been generated
in which tetO drives the expression of the dominant neg-
ative SNARE domain (dnSNARE) that has been crossed
with an astrocyte-specific mouse in which the human
GFAP promoter controlled the expression of tTA. Ani-
mals were reared on dox, to prevent transgene expression
until weaning whereupon transgene was expressed by
removal of dox from the drinking water. Extensive char-
acterization of these animals shows astrocyte-selective
transgene expression. Moreover, the GFAP-tTA animal
has been crossed with at least eight different tetO trans-
genic animals, and each transgene was only expressed in
astrocytes (164).
With the use of these mice, which are referred to as
dnSNARE mice, three fundamental observations were
made. First, expression of the transgene resulted in an
enhancement of the amplitude of basal synaptic transmis-
sion at the Schaffer collateral CA1 synapse. This effect
was determined to be due to a reduction in the extracel-
lular levels of adenosine because in slices from wild-type
animals A1 receptor antagonists mimicked the effect of
transgene, and in dnSNARE slices, antagonist had no
effect, while an A1 agonist fully reversed the effect of
transgene expression. Second, as a consequence of reduc-
ing extracellular adenosine levels, transgene expression
reduced the magnitude of theta-burst-induced LTP. This
reduction in LTP that was observed with dnSNARE ex-
pression resulted from a change in the range available for
plasticity. Under normal conditions, the persistent astro-
cyte-dependent suppression of synaptic transmission al-
lows a greater range for the synapse to be potentiated.
Third, in agreement with the results of Zhang et al. (235),
adenosine-dependent heterosynaptic suppression of un-
stimulated synaptic pathways was abolished (Fig. 3).
Through a series of pharmacological experiments it
was demonstrated that these actions were due to a block-
ade of ATP release from the astrocyte and that, under
normal circumstances, released ATP was hydrolyzed to
adenosine. This caused an A1 receptor-mediated presyn-
aptic inhibition of excitatory synaptic transmission. Ex-
pression of the dnSNARE by blocking ATP release re-
lieved this suppression of synaptic transmission.
These results were quite surprising as it is clear that
neurons can release adenosine through equilibrative
transporters. However, the astrocyte-selective expression
of dnSNARE shows that the astrocyte is the cell type with
the predominant control over extracellular adenosine,
and we conclude that this adenosine arises from the
hydrolysis of released ATP. As adenosine accumulates we
then propose it reenters the astrocyte through equilibra-
tive transporters where it is phosphorylated to replenish
intracellular ATP.
XX. GLIOTRANSMISSION REGULATES
SYNAPTIC CROSS-TALK
From the preceding discussion it should be clear that
although we are still at an early stage in our understand-
ing of the impact of gliotransmission in the nervous sys-
tem, some principles are emerging (Fig. 3). First, glio-
transmitters can have both excitatory and inhibitory ac-
tions. Excitation is provided by glial-derived glutamate
and D-serine, while inhibition is provided by the release of
ATP and the resulting accumulation of adenosine. There
is no evidence for GABA being released from astrocytes.
Second, gliotransmission mediates heterosynaptic cross-
talk. With glutamate this action is likely to be relatively
local because glutamate transporters will rapidly bind to
and take up this transmitter. With adenosine the distance
over which inhibition can be exerted is likely to be con-
siderably greater, since adenosine is cleared from the
extracellular space by equilibrate transporters. At this
point one transgenic system has been developed to iden-
tify the actions of gliotransmitters, and with surprising
results. As we develop a further understanding of the
pathways that differentially regulate the release of the
individual gliotransmitters, it should be possible to pin-
point how these transmitter systems contribute to brain
function.
In addition to their roles in human health, there is the
potential for the astrocyte and gliotransmission to have a
significant impact on human disease. In several neurolog-
ical conditions as well as psychiatric disorders, alter-
ations in astrocyte structure have been identified (136). In
depression, there are fewer astrocytes, whereas astro-
cytes are reactive in the brains of patients suffering from
epilepsy, Parkinson’s disease, and Alzheimer’s disease.
Are there corresponding consequences for neuronal func-
tion? The negative symptoms of schizophrenia have been
identified to result, at least in part, from hypofunction of
the NMDA receptor (138, 139). Consequently, D-serine is
receiving considerable attention in the development of a
therapeutic approach to treat these negative symptoms
(79, 197, 210). Adenosine and A1 receptors have been
shown to be critical for mediating the acute effects of
alcohol, and perturbation of this pathway leads to alcohol
preference (34). Do the astrocyte and the gliotransmitter
ATP, which we now recognize play a critical role in the
control of extracellular adenosine, serve any functions in
alcohol addiction? Adenosine is also known to have pow-
erful anticonvulsant actions (21). Because there is a sig-
nificant percentage of the epileptic population that do not
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respond well to current anticonvulsants, efforts are un-
derway to manipulate this nucleoside. However, because
of a diversity of peripheral actions of adenosine, systemic
administration is not a treatment of choice. Perhaps tar-
geting the astrocyte with therapeutics would offer an
alternative treatment strategy for these patients.
XXI. SUMMARY AND THE FUTURE
Since the discovery that a gliotransmitter glutamate
can be released from cultured astrocytes, we have over
the subsequent decade made many observations that
demonstrate that gliotransmission, either mediated by
glutamate, D-serine, or ATP/adenosine, is intimately
linked with neuronal function. However, a true under-
standing of how the astrocyte interacts with neurons is
still missing. Are these interactions critical for brain func-
tion? Although it is clear that the ratio of glia to neurons
increases through phylogeny (148), is this a natural pro-
cess required to accompany the growing brain or does it
provide the system with higher level functions that we
currently fail to appreciate? Now that a general strategy to
apply molecular genetics to the study of gliotransmission
has been elucidated, the next several years should pro-
vide the opportunity to begin to address these questions.
We now have the tools at hand to determine how glio-
transmission contributes to behavior, and whether glio-
transmission could contribute to neuropathological con-
ditions in which astrocytes become reactive.
ACKNOWLEDGMENTS
Address for reprint requests and other correspondence:
P. G. Haydon, Silvio Conte Center for Integration at the
Tripartite Synapse, Dept. of Neuroscience, Univ. of Pennsyl-
vania School of Medicine, Philadelphia, PA 19104 (e-mail:
pghaydon@mail.med.upenn.edu) or G. Carmignoto (e-mail:
gcarmi@bio.unipd.it).
GRANTS
P. G. Haydon is supported by grants from the National
Institute of Neurological Disorders and Stroke and National
Institute of Mental Health. G. Carmignoto is supported by grants
from the Italian University and Health Ministries.
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