Review
Astrocytes and the regulation of
cerebral blood flow
Raymond C. Koehler1, Richard J. Roman2 and David R. Harder2,3
1
Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University, Baltimore, MD 21287, USA
2
Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226, USA
3
Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA
Moment-to-moment changes in local neuronal activity
lead to dynamic changes in cerebral blood flow. Emer-
ging evidence implicates astrocytes as one of the key
players in coordinating this neurovascular coupling.
Astrocytes are poised to sense glutamatergic synaptic
activity over a large spatial domain via activation of
metabotropic glutamate receptors and subsequent
calcium signaling and via energy-dependent glutamate
transport. Astrocyte foot processes can signal vascular
smooth muscle by arachidonic acid pathways involving
astrocytic cytochrome P450 epoxygenase, astrocytic
cyclooxygenase-1 and smooth muscle cytochrome
P450 v-hydroxylase activities, and by astrocytic and
smooth muscle potassium channels. Non-glutamatergic
transmitters released from neurons, such as nitric oxide,
cyclooxygenase-2 metabolites and vasoactive intestinal
peptide, might modulate neurovascular signaling at the
level of the astrocyte or smooth muscle. Thus, astrocytes
have a pivotal role in dynamic signaling within the
neurovascular unit. Important questions remain on
how this signaling is integrated with other pathways
in health and disease.
Introduction
Steady-state regional cerebral blood flow (CBF) is matched
to regional energy metabolism. However, increases in local
neuronal activity lead to dynamic increases in CBF that
exceed the increases in oxidative metabolism. This relative
hyperemic response provides an increased oxygen gradient
between blood vessels and tissue for assuring adequate
oxygen diffusion to the most distant mitochondria when
oxygen demand fluctuates. Here, we focus on the role of
astrocytes in coordinating the vascular response to
neuronal activation.
Our understanding of the multifunctional role of astro-
cytes and their relationships with neurons, blood vessels
and other astrocytes has grown substantially in recent
years. Astrocytes are now known to have more of a spongi-
form appearance than stellate appearance because of the
many fine processes that extend beyond the large processes
that typically stain for intermediate filaments. These fine
processes have minimal spatial overlap with the processes
of other astrocytes and result in an individual astrocytic
domain that is thought to contain 300–600 neuronal
dendrites [1] and 105 synapses in the rodent cortex and
Corresponding author: Koehler, R.C. (rkoehler@jhmi.edu).
160 0166-2236/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tins.2008.11.005 Available online 21 January 2009
hippocampus [2–4]. In the human cortex, a single astrocyte
might sense the activity and regulate the function of more
than one million synapses within its domain [4]. Moreover,
each astrocyte has at least one process with endfeet sur-
rounding a blood vessel [5]. By contrast, processes from
neurons are rarely in direct contact with intraparenchymal
blood vessels. This anatomical relationship has led to the
hypothesis that astrocytes have a pivotal role in the
dynamic regulation of the cerebral circulation [6,7].
Blood delivered to the major cerebral arteries is dis-
tributed over the cortical surface through pial arteries that
give rise to penetrating arterioles. Pial arteries are in close
proximity to the underlying glia limitans, and intrapar-
enchymal vessels emerging from penetrating arterioles
beyond the space of Virchow are in direct contact with foot
processes of astrocytes. In capillaries and venules without
smooth muscle, integrins maintain adhesion of foot pro-
cesses with the basal lamina surrounding the endothelium
and pericytes. Signaling between astrocytes and endo-
thelium is important for angiogenesis, maintenance of
endothelial tight junctions and transport across the
blood–brain barrier. More recently, signaling between
astrocytes and vascular smooth muscle (VSM) in intrapar-
enchymal arterioles and between glia limitans and pial
arteries has assumed increasing importance in the regu-
lation of CBF. Changes in CBF are actively controlled by
changes in VSM tone, leading to dilation or constriction of
pial arteries and intraparenchymal arterioles. With wide-
spread neuronal activation, dilation of pial arteries is
coordinated with intraparenchymal dilation to help main-
tain a constant perfusion pressure in penetrating arter-
ioles and prevent a passive decrease in CBF in other brain
regions. This coordination is thought to depend on sig-
naling through astrocytes [8] and possibly occur by ascend-
ing dilation propagated through the endothelium [9].
Measurements with laser-Doppler flowmetry (LDF),
which measures red-blood-cell flux integrated in cortical
tissue typically over 1 mm depth, show increases in CBF
in the primary somatosensory cortex within 1–2 s of sen-
sory stimulation [10]. Similar response times are evident
with intrinsic monitoring of tissue hemoglobin concen-
tration and with functional magnetic resonance imaging
(fMRI), which uses the blood-oxygen-level-dependent sig-
nal as a surrogate for CBF coupled to neuronal activity [11–
13]. More recent work has employed Ca2+ imaging in brain
slices and two-photon imaging in the intact brain to reveal
involvement of astrocytes in the coupling of CBF with
 Review Trends in Neurosciences Vol.32 No.3

neuronal activation. One way astrocytes might monitor
excitatory presynaptic activity is by glutamate activation
of metabotropic glutamate (mGlu) receptors, which are
known to stimulate phospholipase C and increase astro-
cytic Ca2+. Here, we summarize some of these recent
findings that illustrate the complexity of dynamic com-
munication between cell types within the neurovascular
unit with an emphasis on signaling by arachidonic acid
metabolites and the integration with other signaling path-
ways.
Cytochrome P450 epoxygenase metabolites
By the mid-1990s, several groups suggested a role for nitric
oxide (NO) derived from neurons and for adenosine in
contributing to vasodilation during neural activation
[14–16]. However, these molecules did not account for
the entire vascular response [17]. In 1998, Harder et al.
[7] proposed that astrocyte signaling involving epoxygen-
ase metabolites of arachidonic acid could have an import-
ant role in neurovascular coupling. Cyclooxygenase (COX)
products of arachidonic acid metabolism had traditionally
been thought to be the predominant eicosanoids involved
in vascular signaling. However, in the 1980s, work in the
peripheral circulation began to reveal an important role of
arachidonic acid metabolites derived from specific cyto-
chrome P450 (CYP) proteins [18]. One class of CYP-derived
eicosanoids formed by epoxygenation of arachidonic acid
comprises the four regioisomers of epoxyeicosatrienoic
acids (EETs). EETs were shown to hyperpolarize VSM
by opening calcium-sensitive potassium (KCa) channels
[19]. The mechanism is thought to involve EETs activation
of vanilloid transient receptor potential 4 (TRPV4) chan-
nels, which produce ryanodine-receptor-dependent
increases in local Ca2+ sufficient to activate nearby KCa
channels [20], although ADP ribosylation and phosphoryl-
ation mechanisms might also be involved [21,22]. In the
peripheral circulation, EETs might serve as one of several
endothelial-derived hyperpolarizing factors that relax
VSM. In the brain, astrocytes might serve a similar role
in providing glial-derived hyperpolarizing factors. EETs
are synthesized in astrocytes and can be further metab-
olized to less bioactive dihydroxyeicosatrienoic acids by
soluble epoxide hydrolase [23] or stored in membrane
phospholipid pools [24]. The epoxygenase enzyme CYP
2C11 is expressed in rat astrocytes and produces EETs
[25], whereas soluble epoxide hydrolase is enriched in

Figure 1. Simplified schematic diagram of selected aspects of glutamatergic signaling in an astrocytic synaptic process that results in arachidonic acid (AA) metabolite
signaling in an astrocyte foot process with vascular smooth muscle (VSM). Presynaptic release of glutamate (Glu) acts on postsynaptic AMPA receptors (GluRs), NMDA
receptors (NMDARs) and metabotropic glutamate receptors (mGluRs). Glu is taken up in the synaptic astrocyte process by the glutamate transporter GLT-1 with cotransport
of Na+, which stimulates energy-dependent Na+,K+-ATPase and consequent glucose uptake. Glu also activates astrocytic mGluR coupled to phospholipase C (PLC) and
Ins(1,4,5)P3 (IP3)-induced Ca2+ increases, which can be propagated throughout the astrocyte by release of ATP through hemichannels. Extracellular ATP acts on astrocytic
purinergic-2 receptors (P2Rs) to promote further intracellular Ca2+ release and is also metabolized to adenosine, which can stimulate astrocytic A2B receptors (A2BRs) and
which might also promote Ca2+ release. Increased Ca2+ in the astrocyte foot process is thought to stimulate phospholipase A2 (PLA2) and mobilize AA. Mobilized AA can
serve as a substrate for (i) astrocytic cyclooxygenase-1 (COX-1) and generate prostaglandin E2 (PGE2), which can stimulate vasodilatory cAMP formation in VSM, (ii)
cytochrome P450 2C11 (CYP 2C11) and generate vasodilatory epoxyeicosatrienoic acids (EETs), which are known to activate hyperpolarizing Ca2+-sensitive K+ (KCa)
channels, and (iii) cytochrome P450 4A (CYP 4A) in VSM and generate 20-HETE, which closes KCa channels and results in depolarization and constriction. In a small fraction
of postsynaptic neurons, NMDARs are coupled to neuronal nitric oxide (NO) synthase (nNOS), which generates highly diffusible NO that stimulates VSM guanylyl cyclase
(GC) and inhibits CYP 4A, thereby enabling astrocytic EETs to activate KCa channels. Baseline VSM constriction is also countered by release of NO from endothelial NOS
(eNOS) and by prostaglandin I2 (PGI2) from endothelial COX-1, both of which are sensitive to the mechanical shear stress of moving blood on the luminal wall. The broken
arrow indicates an inhibitory effect.

161
Review
neurons [26]. Addition of glutamate to the media of cul-
tured astrocytes releases both arachidonic acid and EETs
into the media [27]. It was observed that, in isolated
cerebral VSM, EETs open KCa channels and result in
hyperpolarizion and vasorelaxation [7,19,25] and that, in
vivo, topical application of EETs on pial arterioles
increases their diameter [28–30]. These observations led
to the hypothesis that stimulation of glutamate receptors
on astrocytes leads to the formation and release of EETs,
which then act on cerebral VSM to cause vasodilation
(Figure 1).
In vivo support for this hypothesis is derived from
several studies on rats. N-methyl-D-aspartic acid (NMDA)
perfusion through microdialysis probes implanted in the
striatum elicited an increase in local CBF that was blocked
not only by an inhibitor of NO synthase (NOS) but also by
inhibitors of epoxygenase [31]. Physiologic activation of the
cerebral cortex by movement of the whiskers or by elec-
trical forepaw stimulation produced increases in cortical
CBF that were attenuated by inhibitors of EETs synthesis
[32,33]. Moreover, an EET antagonist also suppressed the
evoked increase in CBF [34]. Therefore, EETs participate
in the signaling that couples neuronal activation with
vasodilation.
In addition to acting as a paracrine signaling molecule,
EETs might also serve as an intracellular messenger. One
of the regioisomers, 5,6-EET, acts as a calcium influx factor
to restore Ca2+ stores after depletion by thapsigargin in
astrocytes [30]. Application of 11,12-EET to cortical slices
increased astrocytic Ca2+ [35], although the mechanism for
the increase remains to be elucidated. Other evidence
indicates that EETs facilitate the opening of KCa channels
within astrocytes exposed to glutamate [36] or hypoxia
[37]. Intermittent hypoxia increases the expression of CYP
2C11 and the formation of EETs in astrocytes [37,38]. This
increase might contribute to the increased tolerance to
oxygen-glucose deprivation and ischemia [38,39]. More-
over, increased formation of EETs in hypoxic astrocytes
is associated with the formation of superoxide anion [37],
which might then serve as an intracellular signaling mol-
ecule or, under severe conditions, produce oxidative stress.
Therefore, the role of EETs as an intracellular messenger
within astrocytes and as an intercellular messenger be-
tween astrocytes and other cells is complex and requires
further investigation.
Signaling initiated by mGlu receptor activation
More direct evidence of a role for astrocytes in neurovas-
cular coupling has relied on observations in brain slices. In
vitro, astrocytes are known to propagate Ca2+ waves
throughout their processes by the release of ATP through
connexin hemichannels and consequent activation of pur-
inergic receptors on their own processes and on adjacent
astrocytes [40–42]. Adjacent foot processes can also com-
municate through gap junctions and lead to the spread of
signals along the vascular wall [5]. By acting in a coordi-
nated manner, astrocytes might integrate neuronal
activity over large spatial domains and thereby coordinate
vasodilation within microcirculatory units. Neuronal
stimulation of brain slices was known to produce dilation
of non-perfused blood vessels [43,44]. In 2003, Zonta et al.
162
Trends in Neurosciences Vol.32 No.3
[45] reported that neuronal stimulation resulted in an
increase in Ca2+ in a fraction of the astrocytes surrounding
blood vessels and that the increase in Ca2+ was associated
with an increase in blood vessel diameter. Subtype 1 and 5
of group I mGlu receptor antagonists blocked the increase
in astrocyte Ca2+ and the vasodilation without attenuating
the increase in neuronal Ca2+. Because aspirin attenuated
the dilation [45] and because cultured astrocytes release
prostaglandin (PG)E2 in response to glutamate [46], COX-
derived PGE2 is thought to trigger the vasodilation. One
limitation of this study was that the time course of the
astrocyte Ca2+ response and vasodilation was much slower
than the in vivo response. Nevertheless, work in vivo
demonstrated that the mGlu receptor antagonists attenu-
ated the increase in CBF during whisker stimulation
[34,45]. The role for astrocytic ionotropic glutamate recep-
tors in neurovascular coupling has yet to be determined.
Mechanisms of Ca2+ signaling might differ in astrocyte
processes directed at blood vessels and in processes
directed at synapses or in the astrocyte soma. Straub
et al. [47] focused on Ca2+ responses within endfeet and
observed them to be spatially heterogeneous and to
precede vasodilation in mouse cortical slices. The
responses depended on inositol (1,4,5)-trisphosphate
[Ins(1,4,5)P3] rather than on ryanodine receptors [47].
Retrograde Ca2+ waves were not observed. The increase
in Ca2+ spread through the foot process longitudinally
along the vessel wall but seemed to recover last at the
point of origin, which is presumed to be a locus of high
Ins(1,4,5)P3 receptor density. Whether these loci colocalize
with the expression of phospholipase A2 and KCa channels
remains to be determined.
Potassium signaling
Astrocytes have long been known to have high per-
meability to K+. Indeed, maintenance of a constant level
of extracellular K+ during neuronal activity is an import-
ant function of astrocytes for maintaining neuronal excit-
ability. Early observations that astrocyte foot processes on
blood vessels had a high density of K+ channels led to the
hypothesis that neuronal release of K+ during action poten-
tials resulted in the uptake of K+ by astrocyte processes at
synapses that was counterbalanced by release of K+ at
astrocyte foot processes surrounding blood vessels [6,48].
Because of the tight interstitial space between astrocyte
foot processes and blood vessels [49], small amounts of K+
release could result in transient elevation of extracellular
K+ to levels sufficient to produce hyperpolarization of VSM
by activation of inward rectifier potassium (Kir) channels.
This process of K+ siphoning was based on work in Mueller
cells in the retina. However, the more recent work demon-
strating intact neurovascular coupling in retina after gene
deletion of the Kir 4.1 channel subtype expressed in Muel-
ler cells casts some doubt on the K+ siphoning mechanism
as an important player in neurovascular coupling in retina
[50].
Astrocyte foot processes are enriched with multiple
types of K+ channels, including Kir, voltage-dependent
K+ (Kv) and KCa channels [51,52]. A set of key studies
elucidating direct communication between astrocytes and
the vasculature via K+ was performed by Filosa et al.
Review Trends in Neurosciences Vol.32 No.3

[53,54]. By increasing vascular tone with the use of the
thromboxane agonist U46619 in cortical slices, spon-
taneous oscillations in Ca2+ were induced in VSM. With
neuronal stimulation or application of a mGlu receptor
agonist, increases in astrocyte Ca2+ were observed on a
timescale of a few seconds [53], which was more rapid than
that reported by Zonta et al. [45] who studied slices from
younger animals at lower temperature. Interestingly, the
increase in astrocyte Ca2+ was associated with an immedi-
ate suppression of Ca2+ oscillations in VSM and with
vasodilation. To further understand the mechanism of
astrocyte–VSM coupling, the authors performed a patch
clamp study of the astrocyte foot process [54]. As expected,
large K+ currents were obtained in the foot process. How-
ever, a significant portion of K+ current was attributed to
KCa channels. Others have also shown that inhibitors of
KCa channels reduce the CBF response in vivo to whisker
stimulation [55]. Release of K+ through KCa channels on
foot processes is postulated to increase extracellular K+ in
the small space surrounding VSM.
To evaluate the role of Kir channels in the vascular
response, Filosa et al. [54] showed that barium, an inhibi-
tor of Kir channels, reduces the evoked dilation in cortical
brain slices, whereas the COX inhibitor indomethacin has
no effect. Therefore, the working hypothesis derived from
this work is that activation of the astrocyte mGlu receptor
produces an increased Ca2+ that is transmitted to the
astrocyte foot process, where KCa channels are activated
and sufficient K+ is released to open Kir 2.1 channels
expressed on adjacent VSM, thereby leading to vasorelaxa-
tion (Figure 2).
20-Hydroxyeicosatetraenoic acid signaling
Another eicosanoid, known as 20-hydroxyeicosatetraenoic
acid (20-HETE), produces depolarization of VSM at sub-
nanomolar concentrations by closing KCa channels and
opening voltage-dependent Ca2+ channels [56,57]. CYP
enzymes that possess v-hydroxylase activity generate
20-HETE from arachidonic acid. Isoforms of the CYP 4A
family possess v-hydroxylase activity and are expressed in
VSM, including cerebral VSM, where they produce 20-
HETE [58,59]. Interestingly, work in hippocampal slices
by Mulligan and MacVicar [60] demonstrated that pho-
tolysis of astrocytic caged Ca2+ often produces constriction
of neighboring arterioles that were blocked by either a
phospholipase A2 or a 20-HETE-synthesis inhibitor. The
predominance of a constrictor rather than a dilator
response in this preparation was probably related to the

Figure 2. Expanded view of selected aspects of potassium signaling in the astrocyte foot process and potential neuronal modulation. Increased Ca2+ in the astrocyte foot
process mediated by Ins(1,4,5)P3 (IP3) can activate KCa channels on the endfoot and release K+ into the small extracellular space, where it is postulated to transiently
accumulate to levels sufficient to stimulate VSM inward-rectifier K+ (Kir) channels and lead to VSM hyperpolarization and relaxation. EETs are also thought to promote
opening of astrocytic KCa channels in addition to VSM KCa channels. Because stimulation of perivascular interneurons containing vasoactive intestinal peptide (VIP) are
known to cause vasodilation and because VIP can increase Ca2+ in astrocyte endfeet, activation of these neurons might produce vasodilation through astrocytic Ca2+-
dependent mechanisms, such as activation of KCa channels and the PLA2 cascade. Subpopulations of neurons also express COX-2, which is required for the overall vascular
response to sensory activation, but the particular prostaglandin metabolite, receptor subtype and location of action on the astrocyte process, endfoot, vascular smooth
muscle or other neurons remains to be determined (as indicated by ‘?’). The broken arrow indicates an inhibitory effect.

163
Review
low vascular tone of non-pressurized vessels because
increasing vascular tone with an NOS inhibitor converted
the constrictor response to a dilator response. Although the
low vascular tone in brain slices is not physiologic, these
results raise the possibility that an increase in astrocytic
Ca2+ can stimulate Ca2+-sensitive phospholipase A2 to
mobilize sufficient arachidonic acid for diffusion to VSM,
where it can act as a substrate for 20-HETE synthesis
(Figure 1). Further support for the involvement of CYP
metabolites of arachidonic acid has been obtained in
retinal slices, in which light or glial activation resulted
in 20-HETE-dependent constriction in a portion of arter-
ioles and EETs-dependent dilation in a different set of
arterioles [61]. More recently, others have demonstrated
that application of a mGlu receptor agonist in cortical slices
produces constriction in vessels with low baseline tone and
dilation in vessels preconstricted with a thromboxane
analog [35]. Here, the constrictor response was reduced
by inhibitors of phospholipase A2, 20-HETE synthesis and
COX-1, whereas the dilator responses were suppressed by
inhibitors of phospholipase A2, EETs synthesis and COX-1.
Thus, the type of response elicited by astrocyte activation
depends on the baseline activation state of the arteriole.
These findings in brain slices raise the question of the
relative importance of these mediators in vivo. Application
of a 20-HETE synthesis inhibitor in vivo had no effect on
the increase in CBF measured by LDF during whisker
stimulation in mice and had only a small effect on enhan-
cing the response in rats [10]. Thus, 20-HETE does not
substantially constrain the evoked dilatory response in
intact cortex. However, activation of discrete cortical col-
umns can result in decreased perfusion in inhibitory-sur-
round cortex [62]. Even within the activated region, the
changes in red-blood-cell flux among capillaries are hetero-
geneous and a small proportion of capillaries exhibit a
decreased flux [63]. Whether the decreased perfusion is
the result of decreased tonic release of vasodilators or
increased release of vasoconstrictors or both is unknown,
but a role for 20-HETE in the inhibitory-surround response
or in the heterogenous response in the activated region is
plausible.
The lack of a large effect of a 20-HETE inhibitor on the
overall CBF response in vivo could be the result of other
mechanisms suppressing 20-HETE synthesis or the effi-
cacy of 20-HETE on downstream signaling. After inhi-
bition of neuronal NOS (nNOS), the CBF response to
whisker stimulation is decreased, and 20-HETE-synthesis
inhibition has a larger effect in enhancing the response
than without nNOS inhibition [10]. Moreover, the 20-
HETE-synthesis inhibitor was more effective in restoring
the CBF response after nNOS inhibition than after gua-
nylyl cyclase inhibition. Because NO can inhibit CYP v-
hydroxylase activity [64,65] and can dilate cerebral
arteries by both increasing cGMP and by inhibiting 20-
HETE synthesis [66,67], one interpretation is that neu-
ronally derived NO during activation increases VSM NO
concentration above the basal level provided by endothelial
NOS and suppresses 20-HETE synthesis locally in neigh-
boring arterioles. Suppressed 20-HETE production per-
mits vasodilation evoked by other mediators. Consistent
with this interpretation, the inhibitory effect of an EETs
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Trends in Neurosciences Vol.32 No.3
antagonist on the CBF response to whisker stimulation is
lost when NOS is inhibited and 20-HETE synthesis is
postulated to be disinhibited, whereas efficacy of the EETs
antagonist is restored when both nNOS and 20-HETE
synthesis are simultaneously inhibited [10]. Thus, unop-
posed 20-HETE synthesis might keep EETs from opening
vasodilatory KCa channels. By suppressing 20-HETE syn-
thesis, NO might permit EETs-dependent vasodilation,
thereby indicating a complex interplay of signaling among
neurons, astrocytes and VSM (Figure 1). Spatial hetero-
geneity in local vasodilator and constrictor responses
might be related to spatial heterogeneity in the local NO
concentrations above and below that necessary to inhibit
CYP 4A.
Adenosine signaling
Adenosine is a potent vasodilator and has long been impli-
cated as a mediator of neurovascular coupling [15,17].
Indeed, adenosine A2A receptors have a role in dilation
of upstream pial arterioles during neuronal activation or
direct activation of glutamatergic receptors [68,69]. In
addition to acting on vascular adenosine receptors, adeno-
sine could act on astrocytes, which also express various
types of adenosine receptors [70]. Activation of A2B recep-
tors can increase intracellular Ca2+ in astrocytes and
might participate in the propogation of increased Ca2+
throughout the astrocyte processes [71]. Ordinarily, an
increase in Ca2+ is associated with release of ATP through
connexin hemichannels, and A2B receptors potentiate the
ATP-evoked Ca2+ response [72,73]. In addition to acting on
astrocyte purinergic receptors, ATP can be hydrolyzed
sequentially to adenosine by ecto-enzymes that are
enriched at hemichannel sites [70,74]. Localized increases
in extracellular adenosine might be sufficient to activate
the A2B receptors (Figure 1), which have lower affinity for
adenosine than do A1 and A2A receptors. Support for a role
of A2B receptors in neurovascular coupling is derived from
the use of different A2B antagonists that inhibit the CBF
response to whisker stimulation [34]. Moreover, the lack of
an additive effect of an EETs antagonist with an A2B
antagonist in reducing the CBF response is consistent with
the A2B signaling acting sequentially with EETs signaling
within astrocytes. Another possibility is that ATP is
released at astrocyte endfeet and hydrolyzed to adenosine
[5,75], which could then activate VSM A2A or A2B receptors.
However, a VSM A2A-dependent mechanism might be
more important in pial arterioles than in parenchymal
VSM because A2A antagonists limit pial arteriolar dilation
[76] but not CBF measured by LDF [34] during sensory
activation. Furthermore, they do not limit ATP-induced
dilation of penetrating arterioles [77].
Carbon monoxide signaling
Carbon monoxide (CO) derived from heme oxygenase
metabolism of heme has a prominent role in various
aspects of cerebrovascular regulation in the newborn pig
[78]. Application of glutamate to the cortical surface of
piglet brain in vivo generates CO and dilates pial arteries
in a glial-dependent manner [79]. The dilation depends on
activation of VSM KCa channels [80], where CO binds to a
heme ligand [81]. In cortical slices from piglet brain,
Review
production of CO, activation of VSM KCa channels and
intraparenchymal arteriolar dilation in response to gluta-
mate are dependent on heme oxygenase in astrocytes [82].
Thus, CO might have a role as an astrocyte-derived sig-
naling molecule in the cerebral circulation of the newborn.
The role of CO in neurovascular coupling in mature brain is
less certain, although some evidence indicates a role
during seizures [83] and activation of a-amino-3-
hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) recep-
tors [69].
Neuronal modulation of astrocyte responses
During neuronal stimulation, NMDA receptors coupled to
nNOS are activated and lead to NO production by a
mechanism dependent on tissue plasminogen activator
[84]. Neuronally derived NO is usually considered to
directly signal the VSM. However, NO might also modu-
late signaling within astrocytes, although this possibility
has not been well investigated. Other neurotransmitters
might also interact with astrocyte signaling. In cortical
slices, stimulation of vasoactive intestinal peptide (VIP)-
positive interneurons in the vicinity of microvessels often
produces vasodilation, whereas stimulation of somato-
statin- or neuropeptides-Y-positive interneurons some-
times produces vasoconstriction [85]. Neuronal processes
from these interneurons can make close contact with astro-
cyte endfeet [86]. Both VIP and somatostatin application
increase Ca2+ in astrocyte endfeet [47], although VIP
causes dilation and somatostatin causes constriction. In
areas enriched with dopaminergic neurons, dopamine is
thought to modulate CBF, and the mechanism could
involve interactions with perivascular astrocytes [87]. Nor-
epinephrine has been demonstrated to increase Ca2+ in
astrocyte endfeet [60], which raises the possibility that
vasoconstriction evoked by locus coeruleus stimulation is
modulated by astrocyte coupling. Likewise, diffuse cholin-
ergic vasodilation evoked by basal forebrain stimulation
might also be modulated by astrocyte coupling. Thus, an
interesting possibility is that different populations of
neurons modulate the vasodilator and vasoconstrictor
responses to local and diffuse activation differentially by
modulating signaling within astrocyte endfeet (Figure 2).
Segmental changes in vascular blood volume
The vascular volume within arterioles comprises a small
fraction of the total cerebral blood volume, yet substantial
changes in blood volume occur during neuronal activation.
Increases in blood volume within capillaries and small
venules have been reported to accompany the increase
in red-blood-cell flux [11]. Because the increase in capillary
and venular volume lag behind the brisk increase in arter-
iolar volume, the increased volume in the these down-
stream vessels without smooth muscle is likely to be the
result of an increase in intraluminal distending pressure
secondary to upstream arteriolar dilation. The change in
diameter of capillaries during neuronal activation has been
estimated to be 10% [63]. Although this dilation amounts
to only 0.5 mm in a typical 5-mm-diameter capillary, this
change could have a physiological effect on decreasing the
viscous drag required for passage of a 7-mm-diameter red
blood cell or a larger and less deformable leukocyte.
Trends in Neurosciences Vol.32 No.3
In addition to pressure-induced changes in capillary
volume due to upstream dilation of arterioles, pericytes
have also been implicated in controlling vascular dimen-
sions and flow in capillaries [88,89]. Pericytes are in great
abundance around capillaries and small venules, where
they often have a longitudinal orientation and might
migrate axially along a blood vessel. However, a subset
has a circumferential orientation, and these pericytes
selectively contain the smooth muscle isoform of actin
[90]. Often observed at branch points, this subset is specu-
lated to adjust the distribution of red-blood-cell flux among
capillaries. Because pericytes and astrocyte foot processes
are separated by only the basal lamina, communication
between these cell types is likely to be crucially important
but remains a relatively unexplored area of research.
Flow-induced changes in shear stress along the endo-
thelial wall are sensed by mechanoreceptors. In arterioles,
the endothelium can signal surrounding smooth muscle
and change vascular tone to help restore shear stress.
Whether the endothelium in capillaries sends similar sig-
nals to surrounding astrocytes, which conceivably could
then signal neurons, is an interesting hypothesis that has
recently been proposed [91]. Furthermore, dilation of the
various segments of the microcirculation leads to distortion
of surrounding cells, including astrocytes. Whether
mechanoreceptors are present in endfeet and sense the
distortion is another open question. Endfeet are enriched
in aquaporin-4 water channels, which seem to colocalize
with K+ channels [49,52]. Transient release of K+ during
activation might be accompanied by transient shifts in
water, which would be consistent with MRI data that
indicate that water is relocated out of tissue and into
the vascular compartment during functional activation
[92]. Thus, many unanswered questions remain about
the role of astrocytes in sensing or responding to segmental
changes in vascular volume.
Astrocyte responses in vivo
Two-photon imaging has enabled a closer examination of
the relationship of neuronal activation, astrocytic Ca2+ and
vasodilation in the cerebral cortex of intact anesthetized
animals. Photolysis of caged Ca2+ in mouse cortical astro-
cytes produced dilation of some of the neighboring arter-
ioles with a latency of 1–2 s and a peak response within 4 s
[93]. In contrast to some studies using brain slices, con-
striction was not observed in vivo. Perivascular astrocytes
expressed COX-1, and the dilation to Ca2+ uncaging was
inhibited by a COX-1 inhibitor. Thus, a COX-1 metabolite,
such as PGE2, is presumed to participate in the dilator
response to increased astrocytic Ca2+.
Whisker stimulation in mouse produced an increase in
astrocytic Ca2+ with a latency of 3 s to the initial increase,
6–12 s to the peak response in the cell body and a slightly
longer delay in the foot process [94]. This response to
physiologic activation in vivo was blocked by mGlu re-
ceptor antagonists. Others using mechanical limb stimu-
lation observed faster Ca2+ responses within 1 s from
stimulation in the cell body and a variable delay in the
endfeet [12]. In the visual cortex of ferrets, astrocytic Ca2+
increased with an onset latency of 3–4 s after the start of
the visual stimulus and reached a peak at 6–7 s [13].
165
Review
Interestingly, astrocytic Ca2+ responses displayed sharper
tuning to orientation and spatial frequency than did
neuronal Ca2+ responses, possibly because an astrocyte
might sample presynaptic glutamate release from a large
number of synapses or have a lower threshold for eliciting a
Ca2+ response throughout the cell. Another unexpected
finding was that the astrocytes responded to visual acti-
vation individually and not as a spatial syncytium. It
remains to be determined whether this lack of spread of
astrocytic Ca2+ waves is specific for the primary visual
cortex or a secondary effect of the isoflurane anesthetic. A
key finding of this study was that the hemodynamic
response tracked the astrocytic Ca2+ response better than
the neuronal Ca2+ response during visual stimulation
when the astrocyte response was selectively suppressed
by a glutamate transport inhibitor or increased depth of
anesthesia. Elegant work in glomeruli in the olfactory bulb
indicated that the hemodynamic response to odor stimu-
lation is specifically related to presynaptic glutamate
release rather than to electrical activity per se. [95]. The
hemodynamic response partly depended sequentially on
activation of astrocytic mGlu5 receptors, increased Ca2+, a
COX-1 metabolite and partly on activation of glutamate
transporters. However, signaling among specialized cell
types in sensory nuclei, such as those in the olfactory bulb,
might not necessarily be generalized to other brain regions
where postsynaptic effects on interneurons are more com-
plex [96]. Nevertheless, the results from the various in vivo
studies confirm the observations in brain slices and demon-
strate that cortical astrocytes can be activated with physio-
logic sensory stimulation and that dilation of arterioles is
linked to the astrocytic Ca2+ response.
Issues to be resolved
Although the preponderance of evidence summarized
before indicates a crucial role for astrocytes in the control
of CBF, many questions remain to be answered.
First, vasodilation, as assessed by LDF, blood-oxygen-
level-dependent signals or the intrinsic optical signal,
begins to occur in <2 s from the onset of sensory stimu-
lation, whereas the astrocytic Ca2+ response often has a
delay of >2 s and a peak response that occurs a few seconds
later [13,94]. Therefore, the initial vasodilation seems to
precede the astrocytic Ca2+ response and, hence, does not
seem to require an increase in astrocytic Ca2+. The astro-
cyte Ca2+ response might be more important for sustaining
the vasodilation during prolonged activation.
Second, the observation that a glutamate-transport
inhibitor blocked the astrocytic Ca2+ response to visual
stimulation [13] is somewhat unexpected because excessive
synaptic glutamate resulting from transport inhibition
would be expected to enhance astrocytic mGlu receptor
stimulation. This result indicates that the astrocytic Ca2+
response to mGlu receptor stimulation is coordinated with
glutamate uptake and possibly metabolism by glutamine
synthetase or glutamate export through connexin hemi-
channels and reuptake by glutamate transporters [97]. In
the olfactory bulb, the astrocyte Ca2+ response does not
depend completely on glutamate transporter activity, which
can regulate the hemodynamic response independently of
the astrocyte Ca2+ response [95].
166
Trends in Neurosciences Vol.32 No.3
Third, administration of mGlu receptor antagonists,
EETs synthesis inhibitors and antagonists, and A2B
antagonists, individually or in combination, inhibit the
steady-state CBF response to whisker stimulation by
50% without affecting the CBF response in the first
few seconds [34]. Thus, drugs that are likely to disrupt
astrocyte signaling do not interfere with the initial vaso-
dilation, which might account for approximately half of the
response. Because neuronal activation is often episodic,
understanding the mechanisms of initial vasodilation
remains an important subject of research. With sustained
activation, the percentage increase in CBF and glucose
consumption exceeds the percentage increase in oxygen
consumption. Some of the excess glucose consumption and
lactate production is purported to occur in astrocytes. The
stimulus for increased astrocytic glucose consumption is
unclear but might be related to electrolyte and water
shifts, glutamate uptake, glutamate metabolism and glu-
tamate release through hemichannels and the associated
increase in Na+ [97], which is likely to stimulate glycolytic-
dependent Na+,K+-ATPase activity. The observations that
astrocytic glutamate release is associated with increased
NADH fluorescence [98] and that blocking glutamate
uptake reduces the vasodilatory response [13,95] are con-
sistent with a metabolic signal initiating part of the vas-
cular response. In this regard, the NADH:NAD+ ratio has
been proposed as a metabolic sensor [99].
Fourth, whereas COX-1 localized in astrocytes has been
implicated in astrocyte–vascular coupling [93,95], inhibi-
tors of COX-2 (which is localized in a subset of neurons
[100]) are actually more effective in reducing the CBF
response to sensory activation than are inhibitors of
COX-1 [101–103]. Furthermore, COX-1-null mice have
an intact response, whereas COX-2-null mice have a
decreased response. High concentrations of a COX-1
inhibitor increase baseline vascular tone, which could
reduce the dynamic response to activation. For example,
the tonic release of a COX-1 metabolite from the endo-
thelium might be required to maintain the minimal level of
cAMP in VSM that might be necessary for full expression of
the dynamic response to neuronal activation. Moreover,
SC-560, which is selective for inhibiting COX-1 in isolated
systems, seems to lose its selectivity in cell-culture systems
and inhibits COX-2 at nanomolar concentrations [104].
Thus, the 500 mM concentration of SC-560 used in some
studies [93,95] might result in nonspecific effects. More-
over, non-astrocytic expression of COX-1 and non-neuronal
expression of COX-2 complicate interpretation of the role of
each isoform [105]. Hence, the precise role of each COX
isoform in each cell type and their associated metabolites
need to be better characterized, and the role of neuronally
derived COX-2 metabolites on astrocyte and VSM PG re-
ceptor subtypes needs to be better discerned (Figure 2).
Fifth, the precise role of EETs as an autocrine-signaling
molecule within astrocytes versus a paracrine-signaling
molecule with VSM needs to be better defined. In contrast
to COX metabolites, de novo synthesis of EETs might not
be required during each episode of activation because
preformed EETs are stored in the phospholipid membrane
[24]. Hence, further elucidation is necessary concerning the
relative role of newly synthesized EETs versus EETs
Review
released from the phospholipid membrane and their mech-
anism of release.
Sixth, most work on neurovascular coupling has focused
on the cerebral cortex. However, the signaling mechanisms
differ in different brain regions. For example, NO is
required as a mediator of neurovascular coupling in the
cerebellum [106], whereas NO acts as a modulator in the
cerebral cortex [10,107]. Non-glutamate neurotransmit-
ters in different brain regions might have selective roles
in modulating astrocyte responses. Moreover, the state of
attention, anticipation or stress might change the release
of neuronal and glial modulators and thereby modulate the
gain of neurovascular coupling.
Seventh, anesthesia affects the magnitude of the CBF
response [108] and could affect the processing of signals in
selective pathways. For example, isoflurane has been
reported to suppress the astrocytic Ca2+ response more
than the neuronal Ca2+ response [13]. In the cerebral
cortex, hemodynamic responses seem to depend on local
field potentials with high-frequency activity in the gamma
range [109]. Background neuronal activity is affected by
anesthetics, and dynamic changes in neuronal activity on
different background activities are likely to alter the gain
of the responses in a nonlinear manner.
Conclusions
Emerging evidence from work in brain slices and in intact
anesthetized animals indicates that astrocytes play a
crucial part in gathering information on presynaptic
activity integrated over a large and discrete spatial
domain and in using that information to dynamically
regulate local perfusion. The astrocyte signaling mechan-
isms are complex but seem to involve increases in astro-
cytic Ca2+ that are initiated by activation of the mGlu
receptor, phospholipase C and Ins(1,4,5)P3 and that are
transmitted by release of ATP, which acts on astrocyte
purinergic receptors, and possibly by catabolism of ATP to
adenosine, which then acts on A2B receptors. An increase
in Ins(1,4,5)P3 within astrocyte endfeet causes the spread
of Ca2+ along the endfoot process, activation of phospho-
lipase A2 and mobilization of arachidonic acid. The ara-
chidonic acid can be used to support synthesis of (i) EETs,
which can help to sustain increases in astrocyte Ca2+ and
open KCa channels within astrocytes or on VSM to produce
vasorelaxation, (ii) PGE2, which can act on VSM to
increase cAMP, and (iii) 20-HETE, which closes KCa chan-
nels on VSM and produces vasoconstriction, especially
when the concentration of NO is too low to inhibit 20-
HETE synthesis. The increase in Ca2+ in astrocyte endfeet
also promotes opening of astrocytic KCa channels and
release of K+, which then could act on vascular Kir chan-
nels to produce vasorelaxation.
However, these mechanisms that are dependent on
astrocytic Ca2+ signaling seem to account for only approxi-
mately half of the steady-state increase in CBF, and other
mechanisms might initiate vasorelaxation within the first
few seconds. Other astrocytic mechanisms might include
energy-metabolism-dependent signals, such as those aris-
ing from glutamate transport. Other mechanisms include
neuronal release of COX-2 metabolites, NO and polypep-
tide neurotransmitters such as VIP. Thus, neurovascular
Trends in Neurosciences Vol.32 No.3
coupling is a highly integrated process involving multiple
signaling pathways.
Understanding how these mechanisms might be dis-
turbed in various neurologic and vascular disease states is
an important area of current research. Because inflam-
mation induces an increase in COX-2, inducible NOS and
reactive oxygen species, diseases that are associated with
inflammation are likely to disrupt normal neurovascular
coupling responses. Increases in angiotensin-associated
arterial hypertension and in b-amyloid associated with
Alzheimer’s disease can stimulate NADPH oxidase gener-
ation of superoxide in animal models and interfere with
vascular dilation to neuronal activation [110–112]. More-
over, aged rodents without overt disease have an impaired
vascular response that is related to increased NADPH
oxidase activity [113]. Impaired vascular responses to
neuronal activation in various disease states could influ-
ence the progression of the disease process. Furthermore,
fMRI is being used extensively for cognitive mapping in
various disease states. Knowing whether the disease state
alters neuronal activation or neurovascular signaling will
be crucial for proper interpretation of fMRI results.
Acknowledgements
We are supported in this area of research by a grant from the National
Institutes of Health (HL59996; www.nih.gov).
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