we hypothesize that microvascular tone may be defined as a function of the local concentration of specific signaling molecules , such as eet , 20-hete , no , npy , pge , som , and vip . the driving term is neuronal activity , which may be a one - to - one function of the neuronal stimulus for primary sensory areas . formally , we seek a relation of the form where the functionalities , denoted f , are yet to be determined . nonetheless , this general formalism emphasizes the need to study muscle tension on a cell type - by - type or transmitter - by - transmitter basis . it further reinforces the need to map the geometry of the vasculature ( figure 1 ) and the location of neuronal control regions ( figure 2 ) . the greatest uncertainty in the hypothesis that the control of cortical blood flow depends on the balance of neurotransmitters that lead to vasoconstriction versus dilation is that much of our understanding of signaling comes from experiments with brain slice preparations . not only is vascular pressurization absent , but responses take place on tens of seconds ( cauli et al . , 2004 ; gordon et al . , 2008 ) , while the vasculature responds to changes in neuronal activity on the 0.5-s time - scale in vivo . it remains an open issue if signaling is faster in vivo , although such long times are observed when astrocytic ca levels are directly excited in vivo ( wang et al . , 2006 ; two - photon laser scanning microscopy with arbitrary scan pathways to record from functionally labeled cells and vessels . ( a ) neurons and glia are labeled with the ca reporter oregon green bapta-1 am and glia have sulforhodamine 101 as an additional label . ( b ) intensity along the scan path records ca signals and blood cell movement . ( c ) traces of functional changes in intracellular ca and flood flow ; note flow changes concurrent with stimulation . ( 2011 ) . a first approach to define the inputs and output to a model of neurovascular coupling is to measure the activity of identified cell types concurrent with blood flow in a neighboring microvessel . the activity of neurons and glia in cortex may be established by measuring their internal calcium levels with optical contrast agents ( garaschuk et al . , 2006 ; figure 3 ) and in vivo two - photon laser scanning microscopy ( tplsm ; svoboda et al . similarly , both the speed of blood cells and vascular tone may be concurrently established with two - photon microscopy ( helmchen and kleinfeld , 2008 ; figure 3 ) . the clarity of these measurements depends on the ability to record from specific cell types and the specificity of co - release of different neuropeptides by interneurons . this implies the need to use transgenic animals that either express a functional indicator in specific cell types or , as a more general approach , to use mice that express cre recombinase in specific cell types ( luo et al . , 2008 ; bernard et al . , 2009 ; table 1 ) . cre recombinase drives the labeling of these cells types by crossing these mouse lines with fluorescent reporter mice ( madisen et al . , 2010 ) , in which a member of the fluorescent protein ( fp ) family ( tsien , 1998 ; shaner et al . , 2004 ) and/or a genetically encoded functional indicator is coded between lox - sites ( wallace et al . , 2008 ; table 2 ) ; cre recombinase activates sequences between these sites ( mallo , 2006 ) . a complementary and common method is to infect cells that express cre recombinase with a virus whose genetic material is modified to code a fluorescent indicator between lox - sites ( table 2 ) . as a technical issue , a construct with double lox - sites improves specificity of cre recombinase targeting strategies ( atasoy et al . , 2008 ) . mice that express fluorescent proteins or cre recombinase in cell - types relevant to neurovascular regulation . viral expression cassettes for in vivo manipulation and detection of astrocyte and interneuronal a second , albeit related approach is to manipulate the output of the neurons and astrocytes that release signaling factors and measure the volume concentration of signaling molecules , particularly under conditions of changing vascular dilation versus constriction . the activity of neurons and glia may be manipulated with photoexcitation of caged molecules , with ectopic expression of chemical receptors modified to have unnatural affinities to a specific drug ( alexander et al . , 2009 ) , with ectopic expression of receptors with native binding sites ( arenkiel et al . , 2008 ) , and with optogenetic agents that are targeted to specific cell types ( cardin et al . , 2009 , 2010 ; the latter strategy includes the use of channelrhodopsin ( chr2 ) to depolarize cells ( boyden et al . , 2005 ; arenkiel et al . , 2007 ; 2009 ) and halorhodopsin ( nphr ; zhang et al . , 2007 ) or archaerhodopsin-3 ( arch ; chow et al . , 2010 ) to inactivate cells . other agents act directly on specific g - protein coupled pathways ( airan et al . , 2009 ) . all of these agents are typically delivered by viral injection in combination with cre recombinase labeled animals , as described above ( tables 1 and 2 ) . a complementary strategy is the use of short hairpin rna ( shrnas ) to silence specific cell signaling pathways labeled animals , as described above ( table 3 ) . the presence of multiple sources for the different signaling molecules suggests the additional need to measure receptor activation by these molecules directly . receptor activation via the volume conduction of vasoactive signaling molecules may be observed with new cell - based indicators , cnifers , that can be made sensitive to any molecule that has a g - coupled protein receptor ( figures 4a , b ) . in a first realization with ach sensing as the task , hek cells were transfected with the muscarinic ( m1 ) gq - protein receptor along with the [ ca]int reporter tn - xxl ( heim and griesbeck , 2004 ) . these show a strong response to the release of endogenous ach ( figures 4c , d ) . cnifer technology may be extended , by the use of gi / o- and gs - protein receptors and chimeric g-q proteins ( coward et al . , 1999 ) , to sense neuropeptides such as vip and som . ( a ) schematic of the cell - based sensors with a gq family , g - protein coupled receptor for acetylcholine and the tn - xxl genetically expressible calcium reporter . cartoon of the injection of cnifers and the placement of microelectrodes in rat frontal cortex . ( c ) in vivo response of m1-cnifers to cholinergic input after electrical activation of nucleus basalis ( nbm ) . the x  z scan was obtained with tplsm through the depth of cortex before and after activation of cholinergic neurons in nbm . ( d ) time dependence of the m1-cnifer response , together with control data from cnifers without the m1 receptor , and the electrocorticogram ( ecog ) . the decrease in amplitude of the ecog after stimulation , as expected for cholinergic activation of cortex . adapted from nguyen et al . the combination of many in vivo tools , including electrophysiology , in vivo two - photon imaging , intracellular ion measurements ( figure 3 ) , neurotransmitter receptor activation measurements ( figure 4 ) , microdialysis measurements , single vessel blood flow measurements ( figure 3 ) as well as direct measurement of the activation of smooth muscle ( figure 5 ) , and measurement of tissue oxygen levels ( sakadzic et al . , 2010 ) , can allow one to assess the influence of single neurons and networks of neurons on vascular control . these can be further supported by optical and chemical activation of specific cell types , as illustrated by the optical activation of astrocytes to increase blood flow in neighboring capillaries ( figure 6 ) in a manner similar to that seen with caged compounds ( takano et al . , 2006 ) . lastly , automated reconstruction techniques allow one to map local architectonics ( figure 1 ) . in all such endeavors , one anticipated need is a calcium - clamp to control smooth muscle tension , possibly realized with caged molecules and optical indicators , as a means to fix the flow in a region and observe changes in signaling molecule concentration . in vivo measurements of arteriole smooth muscle activation we made use of -actin - bac - gcamp2 mice that expressed the genetically encoded ca indicator gcamp2 in smooth muscle ( ji et al . two - photon laser scanning microscopy was used to measure both calcium concentration and blood flow . the change in vessel diameter in the bottom line - scan data is a vasomotor event . in these transgenic animals the observable changes in [ ca ] are limited to large contractile events ; thus the present data must be considered as preliminary . we used gfap - cre+/ mice with an injection of adeno - associated virus serotype 2/5 with a flex - chr2-tdtomato construct ( zhuo et al . histological analysis shows that these animals have weak , non - specific expression of cre recombinase in cortical neurons during adulthood , when the viral injection was made ; thus the present data must be considered as preliminary . ( ( b ) planar image from layer 2/3 , obtained with tplsm , that includes a scanned capillary . ( c ) change in capillary red blood cell speed upon activation of chr2 ; the black line is a running average to remove heart rate contributions . somatosensory input leads to neuronal activation in ipsilateral parietal cortex as well as contralateral cortex , albeit with a reduced amplitude ( figures 7a , b ) . unexpectedly , contralateral sensory input leads to net dilation of vessels , while ipsilateral input has the paradoxical effect of net constriction ( devor et al . , 2008 ; one possible explanation is that contralateral input leads to rapid release of vip while ipsilateral input preferentially excites som+ interneurons more slowly . one can make use of microdialysis , or for greater spatial localization an extension of cnifer technology to form vpac1-cnifers to detect vasoactive intestinal peptide and sst1-cnifers to detect somatostatin . we predict that the ratio of vpac1-cnifer to sst1-cnifer signals will be greater for contralateral versus ipsilateral stimulation ( figure 8) . ( b ) measured multiunit responses to electrical stimulation of the fore - limb ; 500 stimulation trials were averaged . net vasodilation in surface cortical arterioles in response to somatotopic stimulation on the contralateral side ( upper traces in each pair ) but vasoconstriction in response to ipsilateral stimulation ( lower traces ) . the stimulus - induced ecog ( red ) identifies the locus of the electrical response . this proposal builds on the results in figure 6 , where the same sensory input leads to vasodilation at the epicenter of cortical activation in the contralateral hemisphere by vasoconstriction in the ipsilateral hemisphere . we predict that co - release of the dilator vip dominates in the contralateral hemisphere while the constrictor som dominates in the ipsilateral hemisphere . the conventional view is that neuronal spiking leads to an increase in local metabolism , and that metabolism leads to increased oxygen extraction from the blood as well as an increase in blood speed ( fox and raichle , 1986 ; leybaert , 2005 ) . this paradigm is the accepted view for the interpretation of bold fmri ( logothetis et al . , 2001 ) . but this causal relation breaks down , as shown by a multiplicity of recent experiments ( metea and newman , 2006 ; devor et al . , 2008 ; gordon et al . , 2008 ; sirotin and das , 2008 ; girouard et al . , 2010 ; we envision a model of neurovascular control that maps the activity of different neuronal subtypes to changes in vascular tone ( eqs 1 and 2 ) . in our hypothesized paradigm , neuronal activity forms the inputs , the dynamics of the underlying signaling molecules form the internal state variables , and the vascular tone is the output state variable . this is not unlike the case of neuronal dynamics , where the input is derived from synaptic activity , the opening probabilities of channels form the internal state variables , and voltage is the output . 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 .
the neurovascular system may be viewed as a distributed nervous system within the brain . 
 it transforms local neuronal activity into a change in the tone of smooth muscle that lines the walls of arterioles and microvessels . 
 we review the current state of neurovascular coupling , with an emphasis on signaling molecules that convey information from neurons to neighboring vessels . at the level of neocortex 
 , this coupling is mediated by : ( i ) a likely direct interaction with inhibitory neurons , ( ii ) indirect interaction , via astrocytes , with excitatory neurons , and ( iii ) fiber tracts from subcortical layers . 
 substantial evidence shows that control involves competition between signals that promote vasoconstriction versus vasodilation . 
 consistent with this picture is evidence that , under certain circumstances , increased neuronal activity can lead to vasoconstriction rather than vasodilation . 
 this confounds nave interpretations of functional brain images . 
 we discuss experimental approaches to detect signaling molecules in vivo with the goal of formulating an empirical basis for the observed logic of neurovascular control .