the temporal and spatial coupling between increased neuronal activity and cerebral blood flow ( cbf ) , known as functional hyperemia or neurovascular coupling , is a highly regulated phenomenon that ensures adequate supply of oxygen and glucose to the neurons at work during a given task . although intuitively appealing , a direct link between energy state and blood flow is not universally accepted , and the physiological basis of neurovascular coupling still remains uncertain . indeed , neither the lack of glucose or oxygen appears to fully justify the hemodynamic response ( powers et al . , 1996 ; wolf et al .
2010 ) , which may serve as a safety measure for substrate delivery during functional activation ( leithner et al . , 2010 ) . despite these limitations ,
the changes in hemodynamic signals ( bold response , cbf or cerebral blood volume , cbv ) are commonly used as surrogate markers to map changes in neural activity in brain imaging procedures , such as functional magnetic resonance ( fmri ) , positron emission tomography ( pet ) , or diffuse optical imaging ( doi ) under both physiological and pathological conditions .
accordingly , an adequate interpretation of imaging data imperatively requires understanding of the cellular basis of the activated neurocircuitry and its interaction with astrocytic and vascular targets .
in contrast to the innervation of large cerebral arteries by ganglia from the peripheral nervous system , which is mainly involved in autoregulation ( for a review , hamel , 2006 ) , the neuronal circuitry at play here refers to pathways of the central nervous system that interact with the brain microcirculation . as such , it is known that the changes in activity are triggered by the incoming afferents , but that it is their local processing by the targeted cells that drives the perfusion changes ( logothetis et al . , 2001 ;
these hemodynamic changes are mainly , if not exclusively , achieved by the control of the vasculature at the arteriolar level ( hillman et al . , 2007 ) , from the pial surface down to the precapillary level where vascular pericytes stand ( jones , 1970 ) , the latter contractile elements being likely involved in a localized control of capillary tone ( peppiatt et al . , 2006 ) .
additionally , it has been shown that the hemodynamic response correlates with synchronized synaptic activity , a highly energy consuming process ( arthurs et al . , 2000 ) , and that it is controlled by signaling molecules released during increased synaptic activity ( devor et al . , 2007 ) . recently , using the neurovascular coupling response to various sensory stimuli ( whisker , forepaw or hindpaw , visual ) , elegant experiments demonstrated that the spread of the hemodynamic activity accurately reflects the neural response ( berwick et al . , 2008 ) , that it is driven by synaptic activity generated by intracortical processing ( franceschini et al . , 2008 ) , and that the latter reflected the balance between excitatory and inhibitory signals ( devor et al . , 2005 , 2007 ; shmuel et al . , 2006 ) .
particularly , suppressed neuronal activity or functional neuronal inhibition has been associated with decrease in blood oxygenation and perfusion , which could explain the negative bold signals ( shmuel et al . , 2002 , 2006 ) as it occurred concurrently with arteriolar constriction ( devor et al . , 2007 ) .
as an attempt to identify the underlying neuronal circuitry , an interesting study by lu et al .
( 2004)showed that laminar bold and cbv responses to rat whisker stimulation spatially correlated with increased neuronal activity evaluated by c - fos upregulation . in an unrelated study in rats treated with the serotonin releasing drug m - chlorophenylpiperazine , positive bold signals and c - fos immunoreactivity correlated in areas of increased activity , but not in those that displayed diminished bold signals , presumably due to decreased neuronal signaling ( stark et al . , 2006 ) .
recently , it was shown that stimulation of corticocortical and thalamocortical inputs to the same area of the somatosensory cortex induced completely distinct frequency - dependent changes in cbf and oxygen consumption , and evoked activity in different populations of cortical excitatory pyramidal cells and inhibitory gaba interneurons ( enager et al . , 2009 ) .
stimulation intensity increases occurred with the silencing or recruitment of distinct inhibitory interneurons , indicating that neural network activation is both stimulus- and frequency - dependent .
particularly , the hemodynamic responses were attributed to activation of cyclooxygenase-2 ( cox-2 ) pyramidal cells and somatostatin ( som)/nitric oxide synthase ( nos ) inhibitory interneurons , consistent with pharmacological studies that implicated cox-2- and nos - derived vasodilator messengers in these neurovascular pathways ( niwa et al . , 2000 ;
moreover , increased activity in inhibitory interneurons has been associated with the initiation of the hemodynamic response triggered by synchronized cortical activity , as induced by activation of the basal forebrain ( niessing et al . , 2005 ) .
stimulation of this basalocortical afferent input further indicated selective activation of cholinoceptive layers ii to vi som and neuropeptide y ( npy ) interneurons , as well as layer i gaba interneurons , with widespread activation of pyramidal cells , including those that contain cox-2 ( kocharyan et al .
although it was demonstrated that the cbf response was triggered by cholinergic afferents , its full expression required gabaa - mediated transmission on neuronal , vascular and/or astrocytic targets ( kocharyan et al . , 2008 ) .
together these anatomical , neurochemical and functional studies demonstrate the importance of identifying the cellular ensemble that underlies hemodynamic signals , highlighting that specific subsets of neurons are activated by a given stimulus , depending on the afferent input they receive and integrate .
in addition to the difficulty in identifying the exact contribution of excitatory and inhibitory neurotransmissions in the evoked hemodynamic response , the effect of these neurotransmitter systems on perivascular astrocytes needs to be considered ( for a detailed review , carmignoto and gomez - gonzalo , 2009 ) .
the enwrapping of synapses and blood vessels ( kacem et al . , 1998 ) by glial processes and , particularly , their intervening endfeet in multiple neurovascular appositions identified at the ultrastructural level led to the concept of a neuronal - astrocytic - vascular tripartite functional unit ( vaucher and hamel , 1995 ; cohen et al . , 1996 ; paspalas and papadopoulos , 1996 ; vaucher et al . , 2000 ) .
the significance of these interactions in the regulation of cbf was first substantiated by the demonstration that astrocytes could synthesize vasodilatory messengers ( table 1 ) , particularly epoxyeicosatrienoic acids ( eets ) generated from p450 arachidonic acid epoxygenase activity ( alkayed et al . , 1996 )
that were involved in the cbf response to glutamate ( alkayed et al . , 1997 ; harder et al . ,
soon after came the first in vitro ( in cortical slices ) and in vivo demonstrations for a role of astrocytes , through metabotropic glutamate receptor ( mglurs)-induced ca transients , in microarteriolar dilation and the increase blood flow to forepaw stimulation , a response mediated by arachidonic acid products , possibly prostaglandin e2 ( pge2 ) ( zonta et al . ,
a role for astrocytes in neurovascular coupling has been reaffirmed in various paradigms ( carmignoto and gomez - gonzalo , 2009 ) .
neuronal and glial metabolism by - products ( e.g. , co2 , h , adenosine  ) are not included .
no , nitric oxide ; vip , vasoactive intestinal polypeptide ; pge2 , prostaglandin e2 ; eets , epoxyeicosatrienoic acids ; nos , nitric oxide synthase ; npy , neuropeptide y ; cox , cyclooxygenase , nmda - r , n - methyl d aspartate receptors , iglurs ; ionotropic glutamate receptors , mglur , metabotropic glutamate receptors ; vsmc , vascular smooth muscle cell , sgc , soluble guanylate cyclase , vpac1 , vip / pacap receptor type 1 ; ep , prostaglandin e2 receptors ; gpcr , g protein coupled receptor .
kca , ca+-activated k channels ; kir , inward rectifier k channels ; g , conductance .
another pathway activated in astrocytic endfeet following ca increases is the large - conductance , calcium - sensitive potassium ( bk ) channels that induce k release ( table 1 ) , activation of smooth muscle kir channels and relaxation ( filosa et al . , 2006 ) .
recently , it was shown that the extent of the ca increases in astrocytic endfeet determined the dilatory or contractile nature of the vascular response , both mediated by extracellular k ( girouard et al . , 2010 ) .
this novel mechanism would reunify previous  apparently  contradictory findings , in brain slices or in retina , of dilation and constriction being induced by increased ca signaling in astrocytes , and explained by the levels of no ( mulligan and macvicar , 2004 ; metea and newman , 2006 ) , oxygen ( gordon et al . , 2008 ) , or the pre - existing tone of the vessels ( blanco et al . , 2008 ) .
( 2010 ) showed that neuronal activation in vitro ( electrical field stimulation ) similarly acted through serial activation of astrocytic bk and smooth muscle kir channels . considering that gaba ( nilsson et al .
2008 ) and peptides such as som that colocalize with gaba ( somogyi et al . , 1984 ; cauli et al . , 2000
) in interneurons also increase ca signaling in astrocytes , ( straub et al . , 2006 ) , the latter could act as intermediaries to gaba in neurovascular coupling ( table 1 ) .
whether or not the astrocytic / smooth muscle bk and kir channel activation cascade is involved still remains to be determined .
similarly , it would be interesting to evaluate if the latter , or other contractile mechanisms of neuronal ( cauli et al .
2006 ) or vascular origins ( mulligan and macvicar , 2004 ) could explain the dilatory and constrictive phases seen in the central core of neuronal depolarization and surround region of hyperpolarization , respectively , after somatosensory stimulation ( devor et al .
, 2007 ) . to date the cellular and molecular mechanisms or even the functional significance of this response remains unknown
in addition to the astrocyte - derived vasodilatory messengers pge2 ( zonta et al . , 2003 ) , eets ( alkayed et al . , 1996 ) , or k ions ( filosa et al . , 2006 ) ,
iii pyramidal cells ( yamagata et al . , 1993 ; breder et al . ,
1995 ) , nitric oxide ( no ) ( gotoh et al . , 2001 ) whose synthetic enzyme is expressed by discrete subpopulations of cortical gaba interneurones ( kubota et al . , 1994 ) , vip ( yaksh et al . , 1987 ) , acetylcholine ( scremin et al . , 1973 ) and corticotropin - releasing factor ( de michele et al . , 2005 ) synthesized by bipolar / bitufted gaba interneurones ( morrison et al . , 1984 ; chdotal et al . , 1994a ; cauli et al . , 1997 ; gallopin et al . , 2006
for instance the highly ca permeable nmda receptors , expressed by cortical neurons ( monyer et al . , 1994 ; cauli et al . , 2000 ) , promote the release of pge2 ( pepicelli et al . ,
astrocytes , some gaba interneurons produce substances with vasocontractile properties , namely npy ( abounader et al . , 1995 ; cauli et al . , 2004
) and som ( long et al . , 1992 ; cauli et al . ,
cortical neurons producing these vasoactive peptides are intimately associated with blood vessels through neuronal - astrocytic - vascular appositions described above ( chdotal et al .
, 1994b ; abounader and hamel , 1997 ; estrada and defelipe , 1998 ; vaucher et al . ,
2000 ; wang et al . , 2005 ) , and their receptors are expressed by smooth muscle cells and astrocytes ( chalmers et al . , 1995 ; bao et al . , 1997 ; abounader et al . , 1999 ;
, 2000 ; cauli et al . , 2004 ; straub et al . , 2006 ; cahoy et al . ,
this raises the intriguing question of whether or not astrocytes are intermediaries for neuron - derived vasoactive messengers or , alternatively , if the latter exert direct effects on the microcirculation .
it is widely admitted that an increase in intracellular ca ( table 1 ) is a required early event for the production and/or release of vasoactive messengers from neurons ( lauritzen , 2005 ) and astrocytes ( straub and nelson , 2007 ) .
examination of ca dynamics in these cell types could provide a clue to decipher their relative and temporal contribution to functional hyperemia .
the general view is that rapid ca events reflect an entry following fast ( 1012  ms ) spiking response of neurons ( petersen et al . , 2003 ) and/or activation of ca permeable ionotropic receptors , whereas slower dynamics are mainly driven by activation of metabotropic receptors leading to the release of ca from intracellular stores ( perea and araque , 2005 ) .
hence , cortical neurons , which express more frequently and abundantly ionotropic glutamate receptors ( monyer et al . , 1994 ;
2008 ) , are likely to be responsible for the majority of fast ca responses .
in contrast , group i mglurs ubiquitously expressed by cortical neurons ( baude et al . , 1993 ; cauli et al . , 2000 ) and
astrocytes ( porter and mccarthy , 1996 ) would be responsible for slower ca dynamics in both cell types .
consistent with this , in somatosensory or visual cortex , evoked ca events in neurons are virtually locked with sensory stimulations and precede those in astrocytes by a few seconds ( stosiek et al .
wang et al . , 2006 ; schummers et al . , 2008 ; murayama et al . ,
2009 ) , although a small proportion ( 5% ) of astrocytes can exhibit ca responses as fast as neurons ( winship et al . , 2007 ) .
calcium uncaging in astrocytic endfeet in vivo showed that arterioles start to dilate 500  ms after the onset of ca increase ( takano et al . ,
2006 ) indicating that synthesis , release and effects of vasodilatory messengers must be achieved within this time window . since hemodynamic responses initiate 600  ms after the onset of sensory stimulations ( kleinfeld et al . , 1998 ;
2003 ) , it appears that only cell types exhibiting fast evoked ca events ( i.e. , less than 100  ms ) can account for the early phase of the hemodynamic response .
therefore , vasoactive messengers produced by neurons and , possibly , also by astrocytes with fast ca events , could explain this response ( figure 1 ) .
summary of the proposed regulation of cortical microvessels by pyramidal cells , gaba interneurons and astrocytes ( a ) , and how their respective effects can temporally regulate cbf changes ( b ) .
( a ) subcortical afferents from a variety of brain areas target distinct populations of neurons in the cerebral cortex .
these activated neuronal networks can either directly act on local microvessels , which are endowed with receptors ( geometric forms on the vessel wall ) for most neurotransmitters / neuromediators , or indirectly via astrocytes that act as intermediaries to both pyramidal cells and interneurons .
known direct vasoactive mediators released from pyramidal cells and interneurons correspond respectively to cox-2 derivatives like prostaglandin e2 ( pge2 ) and no and , possibly , gaba , whereas astrocytes act chiefly by releasing dilatory eets , an effect comparatively slow as opposed to that of no and pge2 ( or other neurally released vasoactive molecules or peptides ) .
the possibility that sub - cortical afferents directly contact and act upon cortical astrocytes or microvessels also has to be taken into consideration . modified from figure 3 in hamel ( 2006 ) .
( b ) schematic representation of the relative and temporal contributions of selected vasoactive mediators produced by pyramidal cells ( pge2 ) , interneurons ( no ) and astrocytes ( eets ) , to the cbf response evoked by sensory stimulation ( see table 1 for a more complete list ) .
brief stimulations ( 1  s ) are more likely to involve neurally - derived mediators whereas sustained stimulation ( 1  min ) are more susceptible to recruit astrocyte - derived messengers . no being transiently released its contribution to cbf response during sustained stimulation is minor and could account for its permissive role .
in the cerebral cortex , none of the vasoactive messengers implicated in neurovascular coupling ( girouard and iadecola , 2006 ) , whether of neuronal or astroglial origin , can individually account for the hemodynamic response , as demonstrated by genetic invalidation ( ma et al .
kitaura et al . , 2007 ) or synthesis inhibition ( lindauer et al . , 1999
; peng et al . , 2002 ; hoffmeyer et al . , 2007 ; leithner et al . , 2010 ) .
when individually summed the inhibition of these messengers largely exceeds the expected value of 100% ( iadecola , 2004 ) , which suggests that their kinetics of action , temporal and spatial recruitment must be carefully considered to elucidate their relative contributions .
alternatively , this may suggest that the activated pathways do not obligatorily operate independently from each other , and that , under certain circumstances , some may act like modulator rather than mediator of the perfusion responses , as documented for no in the somatosensory cortex ( lindauer et al . , 1999 ) .
no , one of the fastest diffusible ( wood and garthwaite , 1994 ) vasodilator produced by a subset npy - expressing interneurons ( dawson et al . ,
correspondingly , shibuki 's group showed that neuronal no can account for up to 50% of the cbf response evoked by a brief ( 1  s ) sensory stimulation ( kitaura et al . , 2007 ) .
in contrast , others only found a permissive role for no when long ( 1  min ) stimulations were used ( lindauer et al .
these differences likely reflect the fact that no release is transient ( buerk et al . , 2003 ) which can be explained by the no scavenging effect of hemoglobin and/or by the adaptation of no producing interneurons ( karagiannis et al . , 2009 ) .
in contrast , vasodilatory prostanoids produced by cox-2 , which is chiefly expressed by pyramidal cells ( yamagata et al . , 1993 ; breder et al . , 1995 ) , account for 50% of the cbf response evoked by both sustained ( niwa et al . , 2000 ) and brief stimulations ( kitaura et al . , 2007 ) .
consistent with an involvement of neuron - derived messengers in the early phase of neurovascular coupling , blockade of prostanoids and no synthesis almost completely abolished hemodynamic responses evoked by brief sensory stimulations ( kitaura et al . , 2007 ) .
during long lasting sensory stimulation , blockade of eets synthesis ( peng et al . , 2002 ) or their receptors ( liu et al . , 2008 )
blocked about 50% of the blood flow response , demonstrating that eets produced by astrocytes ( alkayed et al . , 1996 )
similarly , the local release of k from astrocytic endfeet ( filosa et al . , 2006 ) accounts for up to 50% of the cbf increase evoked by long lasting sensory stimulations ( girouard et al . , 2010 ; leithner et al . , 2010 ) surprisingly , combined blockade of nos , coxs , p450 epoxygenase , bk channels and adenosine receptors ( leithner et al . , 2010 ) did not reach a total inhibition of the late phase of the hemodynamic response as expected from individual blockades ( see above ) .
this suggests that either multiple inhibition was incomplete or that other long lasting vasodilatory messengers are involved ( cauli et al . , 2004 ) , possibly vip , which is contained within gaba interneurons targeted by thalamocortical afferents ( staiger et al . , 1996 ) and
similarly vasodilatory messengers derived from the endothelium ( rosenblum , 1986 ) such as no , prostacyclin ( faraci and heistad , 1998 ) but also eets ( campbell and fleming , 2010 ) , might be recruited under certain circumstances as it was reported for endothelial no after muscarinic m5 receptor activation ( elhusseiny and hamel , 2000 ; yamada et al . , 2001 ) .
current evidence suggests that neuronal and astroglial signals that transduce changes in neuronal activity into an integrated vascular response are highly dependent upon the neurotransmitter released by the incoming afferents , and strictly determined by the target neurons within the activated area .
particularly , depending on the nature of the afferent input ( i ) different neuronal or astroglial messengers , likely acting in sequence , mediate the hemodynamic changes , ( ii ) some recruited neurons release messengers that can directly alter blood vessel tone , ( iii ) others act by modulating neuronal and astroglial activity , and ( iv ) astrocytes may act as intermediaries for both excitatory and inhibitory neurotransmitters ( figure 1 ) . probably due to the large diversity of cortical neurons ( ascoli et al . , 2008 ) , to our knowledge
, no in vivo study has yet investigated ca dynamics in identified neurons producing vasoactive substances or the vascular effects of their stimulation .
the growing development of transgenic mice expressing genetically encoded fluorescent reporters and/or optogenetic tools ( cardin et al .
2009 ) in discrete subsets of cortical neurons ( heintz , 2001 ) together with the emergence of ultrafast multispectral imaging systems ( bouchard et al . , 2009 ) that allow simultaneous monitoring of ca events and hemodynamics should help evaluate the contribution of specific neuronal types in neurovascular coupling .
this should provide decisive conclusions on the temporal , spatial and extent of the neurally - driven hemodynamic alterations and how the latter can be interpreted in the context of brain imaging of normal or pathological physiology .
indeed , since astrocytes appear as intermediary effectors in conveying signals for sustained hemodynamic responses , their alteration  primarily expressed by a state of chronic activation  in several chronic diseases of the central nervous system such as alzheimer 's disease or epilepsy , has to be seriously considered .
altered perfusion signals detected by fmri , pet or doi may represent astroglial dysfunction and not necessary impaired neuronal activity . extending such thinking to the microcirculation itself , the functional endpoint in the intricate cascade of neuronal - astrocytic - vascular events evoked by increased brain activity , any diseases of the blood vessels themselves or alterations in their physical capacity to dilate or constrict ,
as seen in pathologies such as hypertension , diabetes , hypercholesterolemia and , even alzheimer 's disease ( iadecola , 2004 ; zlokovic , 2008 ) would hinder the correct vascular response to totally normal neuronal activities .
this further highlights that extreme caution should be applied to perfusion signals when making direct inference to altered neural activity ( schleim and roiser , 2009 ; ekstrom , 2010 ) .
hence , a careful understanding of the neuronal circuitry at work will need to be interpreted in the context of a healthy or sick brain taking neuroinflammation and vascular diseases as possible confound factors .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
 in this article , we will review molecular , anatomical , physiological and pharmacological data in an attempt to better understand how excitatory and inhibitory neurons recruited by distinct afferent inputs to the cerebral cortex contribute to the coupled hemodynamic response , and how astrocytes can act as intermediaries to these neuronal populations . we aim at providing the pros and cons to the following statements that , depending on the nature of the afferent input to the neocortex , ( i ) different neuronal or astroglial messengers , likely acting in sequence , mediate the hemodynamic changes , ( ii ) some recruited neurons release messengers that directly alter blood vessel tone , ( iii ) others act by modulating neuronal and astroglial activity , and ( iv ) astrocytes act as intermediaries for both excitatory and inhibitory neurotransmitters . 
 we will stress that a given afferent signal activates a precise neuronal circuitry that determines the mediators of the hemodynamic response as well as the level of interaction with surrounding astrocytes . 
Activity in Neuronal Networks Underlies Activity-Dependent Changes in Hemodynamic Signals
Identification of the Underlying Neuronal Circuitry
Perivascular Astrocytes as Intermediaries to Both Glutamate and GABA Pathways
Neurally-Derived Vasoactive Messengers
Temporal Sequences in the Recruitment of Neurons and Astrocytes
Multiplicity of Vasoactive Messengers: Are They all Required?
Current Understanding, Future Directions and Clinical Implications
Conflict of Interest Statement