The Conducted Vasomotor Response: Function, Biophysical Basis, and Pharmacological Control.

Arterial tone is coordinated among vessel segments to optimize nutrient transport and organ function. Coordinated vasomotor activity is remarkable to observe and depends on stimuli, sparsely generated in tissue, eliciting electrical responses that conduct lengthwise among electrically coupled vascular cells. The conducted response is the focus of this topical review, and in this regard, the authors highlight literature that advances an appreciation of functional significance, cellular mechanisms, and biophysical principles. Of particular note, this review stresses that conduction is enabled by a defined pattern of charge movement along the arterial wall as set by three key parameters (tissue structure, gap junctional resistivity, and ion channel activity). The impact of disease on conduction is carefully discussed, as are potential strategies to restore this key biological response and, along with it, the match of blood flow delivery with tissue energetic demand.

Intercellular Conduction Optimizes Arterial Network Function and Conserves Blood Flow Homeostasis During Cerebrovascular Challenges.

OBJECTIVE: Cerebral arterial networks match blood flow delivery with neural activity. Neurovascular response begins with a stimulus and a focal change in vessel diameter, which by themselves is inconsequential to blood flow magnitude, until they spread and alter the contractile status of neighboring arterial segments. We sought to define the mechanisms underlying integrated vascular behavior and considered the role of intercellular electrical signaling in this phenomenon. Approach and Results: Electron microscopic and histochemical analysis revealed the structural coupling of cerebrovascular cells and the expression of gap junctional subunits at the cell interfaces, enabling intercellular signaling among vascular cells. Indeed, robust vasomotor conduction was detected in human and mice cerebral arteries after focal vessel stimulation: a response attributed to endothelial gap junctional communication, as its genetic alteration attenuated this behavior. Conducted responses were observed to ascend from the penetrating arterioles, influencing the contractile status of cortical surface vessels, in a simulated model of cerebral arterial network. Ascending responses recognized in vivo after whisker stimulation were significantly attenuated in mice with altered endothelial gap junctional signaling confirming that gap junctional communication drives integrated vessel responses. The diminishment in vascular communication also impaired the critical ability of the cerebral vasculature to maintain blood flow homeostasis and hence tissue viability after stroke. CONCLUSIONS: Our findings highlight the integral role of intercellular electrical signaling in transcribing focal stimuli into coordinated changes in cerebrovascular contractile activity and expose, a hitherto unknown mechanism for flow regulation after stroke.

Tonic Local Brain Blood Flow Control by Astrocytes Independent of Phasic Neurovascular Coupling.

According to the current model of neurovascular coupling, blood flow is controlled regionally through phasic changes in the activity of neurons and astrocytes that signal to alter arteriole diameter. Absent in this model, however, is how brain blood flow is tonically regulated independent of regional changes in activity. This is important because a large fraction of brain blood flow is required to maintain basal metabolic needs. Using two-photon fluorescence imaging combined with patch-clamp in acute rat brain slices of sensory-motor cortex, we demonstrate that reducing resting Ca(2+) in astrocytes with intracellular BAPTA causes vasoconstriction in adjacent arterioles. BAPTA-induced vasoconstriction was eliminated by a general COX blocker and the effect is mimicked by a COX-1, but not COX-2, antagonist, suggesting that astrocytes provide tonic, steady-state vasodilation by releasing prostaglandin messengers. Tonic vasodilation was insensitive to TTX, as well as a variety of synaptic and extrasynaptic receptor antagonists, indicating that the phenomenon operates largely independent of neural activity. Using in vivo two-photon fluorescence imaging of the barrel cortex in fully awake mice, we reveal that acute COX-1 inhibition reduces resting arteriole diameter but fails to affect vasodilation in response to vibrissae stimulation. Our findings demonstrate that astrocytes provide tonic regulation of arterioles using resting intracellular Ca(2+) in a manner that is independent of phasic, neuronal-evoked vasodilation. Significance statement: The brain requires both phasic and tonic regulation of its blood supply to service energy needs over various temporal windows. While many mechanisms have been described for phasic blood flow regulation, how the brain accomplishes tonic control is largely unknown. Here we describe a way in which astrocytes contribute to the management of basal brain blood flow by providing steady-state vasodilation to arterioles via resting astrocyte Ca(2+) and the continuous release of prostaglandin messengers. This phenomenon may be important for understanding the declines in basal brain blood flow that occur in aging and dementia, as well as for the interpretation of fMRI data.

Astrocyte and microvascular imaging in awake animals using two-photon microscopy.

Neurovascular coupling is an important control mechanism in CBF regulation. New insights into the integrated relationship between synaptic activity, astrocytes Ca(2+) , and cerebral blood vessels using two-photon fluorescence imaging are slowly emerging. Here, we provide a brief overview of the current understandings and controversies regarding astrocytes in activity-dependent vasodilation. We highlight the key advantages and disadvantages of the in vitro and in vivo methodologies used to study this topic. In particular, we emphasize some of the drawbacks of acute brain slices as well as the confounding effects of anesthesia in in vivo preparations. To overcome these limitations, we discuss an emerging and important trend in imaging cell Ca(2+) and blood flow control in awake and behaving animals. This new approach may help resolve existing controversies on astrocyte control of arteriole diameter by providing a more physiologically relevant preparation to study CBF regulation.

Modulatory effects of noradrenergic and serotonergic signaling pathway on neurovascular coupling.

Dynamic changes in astrocyte Ca2+ are recognized as contributors to functional hyperemia, a critical response to increased neuronal activity mediated by a process known as neurovascular coupling (NVC). Although the critical role of glutamatergic signaling in this process has been extensively investigated, the impact of behavioral state, and the release of behavior-associated neurotransmitters, such as norepinephrine and serotonin, on astrocyte Ca2+ dynamics and functional hyperemia have received less attention. We used two-photon imaging of the barrel cortex in awake mice to examine the role of noradrenergic and serotonergic projections in NVC. We found that both neurotransmitters facilitated sensory stimulation-induced increases in astrocyte Ca2+. Interestingly, while ablation of serotonergic neurons reduced sensory stimulation-induced functional hyperemia, ablation of noradrenergic neurons caused both attenuation and potentiation of functional hyperemia. Our study demonstrates that norepinephrine and serotonin are involved in modulating sensory stimulation-induced astrocyte Ca2+ elevations and identifies their differential effects in regulating functional hyperemia.

Modulatory Effects of Noradrenergic and Serotonergic Signaling Pathway on Neurovascular Coupling.

Dynamic changes in astrocyte Ca2+ are recognized as contributors to functional hyperemia, a critical response to increased neuronal activity mediated by a process known as neurovascular coupling (NVC). Although the critical role of glutamatergic signaling in this process has been extensively investigated, the impact of behavioral state, and the release of behavior-associated neurotransmitters, such as norepinephrine and serotonin, on astrocyte Ca2+ dynamics and functional hyperemia have received less attention. We used two-photon imaging of the barrel cortex in awake mice to examine the role of noradrenergic and serotonergic projections in NVC. We found that both neurotransmitters facilitated sensory-induced increases in astrocyte Ca2+. Interestingly, while ablation of serotonergic neurons reduced sensory-induced functional hyperemia, ablation of noradrenergic neurons caused both attenuation and potentiation of functional hyperemia. Our study demonstrates that norepinephrine and serotonin are involved in modulating sensory-induced astrocyte Ca2+ elevations and identifies their differential effects in regulating functional hyperemia.

Endothelial Ca2+ wavelets and the induction of myoendothelial feedback.

When arteries constrict to agonists, the endothelium inversely responds, attenuating the initial vasomotor response. The basis of this feedback mechanism remains uncertain, although past studies suggest a key role for myoendothelial communication in the signaling process. The present study examined whether second messenger flux through myoendothelial gap junctions initiates a negative-feedback response in hamster retractor muscle feed arteries. We specifically hypothesized that when agonists elicit depolarization and a rise in second messenger concentration, inositol trisphosphate (IP(3)) flux activates a discrete pool of IP(3) receptors (IP(3)Rs), elicits localized endothelial Ca(2+) transients, and activates downstream effectors to moderate constriction. With use of integrated experimental techniques, this study provided three sets of supporting observations. Beginning at the functional level, we showed that blocking intermediate-conductance Ca(2+)-activated K(+) channels (IK) and Ca(2+) mobilization from the endoplasmic reticulum (ER) enhanced the contractile/electrical responsiveness of feed arteries to phenylephrine. Next, structural analysis confirmed that endothelial projections make contact with the overlying smooth muscle. These projections retained membranous ER networks, and IP(3)Rs and IK channels localized in or near this structure. Finally, Ca(2+) imaging revealed that phenylephrine induced discrete endothelial Ca(2+) events through IP(3)R activation. These events were termed recruitable Ca(2+) wavelets on the basis of their spatiotemporal characteristics. From these findings, we conclude that IP(3) flux across myoendothelial gap junctions is sufficient to induce focal Ca(2+) release from IP(3)Rs and activate a discrete pool of IK channels within or near endothelial projections. The resulting hyperpolarization feeds back on smooth muscle to moderate agonist-induced depolarization and constriction.

Electrical communication in branching arterial networks.

Electrical communication and its role in blood flow regulation are built on an examination of charge movement in single, isolated vessels. How this process behaves in broader arterial networks remains unclear. This study examined the nature of electrical communication in arterial structures where vessel length and branching were varied. Analysis began with the deployment of an existing computational model expanded to form a variable range of vessel structures. Initial simulations revealed that focal endothelial stimulation generated electrical responses that conducted robustly along short unbranched vessels and to a lesser degree lengthened arteries or branching structures retaining a single branch point. These predictions matched functional observations from hamster mesenteric arteries and support the idea that an increased number of vascular cells attenuate conduction by augmenting electrical load. Expanding the virtual network to 31 branches revealed that electrical responses increasingly ascended from fifth- to first-order arteries when the number of stimulated distal vessels rose. This property enabled the vascular network to grade vasodilation and network perfusion as revealed through blood flow modeling. An elevation in endothelial-endothelial coupling resistance, akin to those in sepsis models, compromised this ascension of vasomotor/perfusion responses. A comparable change was not observed when the endothelium was focally disrupted to mimic disease states including atherosclerosis. In closing, this study highlights that vessel length and branching play a role in setting the conduction of electrical phenomenon along resistance arteries and within networks. It also emphasizes that modest changes in endothelial function can, under certain scenarios, impinge on network responsiveness and blood flow control.

Endothelial feedback and the myoendothelial projection.

The endothelium plays a critical role in controlling resistance artery diameter, and thus blood flow and blood pressure. Circulating chemical mediators and physical forces act directly on the endothelium to release diffusible relaxing factors, such as NO, and elicit hyperpolarization of the endothelial cell membrane potential, which spreads to the underlying smooth muscle cells via gap junctions (EDH). It has long been known that arterial vasoconstriction in response to agonists is limited by the endothelium, but the question of how contraction of smooth muscle cells leads to activation of the endothelium (myoendothelial feedback) has, until recently, received little attention. Initial studies proposed the permissive movement of Ca(2+) ions from smooth muscle to endothelial cells to elicit release of NO. However, more recent evidence supports the notion that flux of IP(3) leading to localized Ca(2+) events within spatially restricted myoendothelial projections and activation of EDH may underlie myoendothelial feedback. In this perspective, we review recent data which supports the functional role of myoendothelial projections in smooth muscle to endothelial communication. We also discuss the functional evidence supporting the notion that EDH, as opposed to NO, is the primary mediator of myoendothelial feedback in resistance arteries.

Toolbox for studying neurovascular coupling in vivo, with a focus on vascular activity and calcium dynamics in astrocytes.

Significance: Insights into the cellular activity of each member of the neurovascular unit (NVU) is critical for understanding their contributions to neurovascular coupling (NVC)-one of the key control mechanisms in cerebral blood flow regulation. Advances in imaging and genetic tools have enhanced our ability to observe, manipulate and understand the cellular activity of NVU components, namely neurons, astrocytes, microglia, endothelial cells, vascular smooth muscle cells, and pericytes. However, there are still many unresolved questions. Since astrocytes are considered electrically unexcitable, Ca 2 + signaling is the main parameter used to monitor their activity. It is therefore imperative to study astrocytic Ca 2 + dynamics simultaneously with vascular activity using tools appropriate for the question of interest. Aim: To highlight currently available genetic and imaging tools for studying the NVU-and thus NVC-with a focus on astrocyte Ca 2 + dynamics and vascular activity, and discuss the utility, technical advantages, and limitations of these tools for elucidating NVC mechanisms. Approach: We draw attention to some outstanding questions regarding the mechanistic basis of NVC and emphasize the role of astrocytic Ca 2 + elevations in functional hyperemia. We further discuss commonly used genetic, and optical imaging tools, as well as some newly developed imaging modalities for studying NVC at the cellular level, highlighting their advantages and limitations. Results: We provide an overview of the current state of NVC research, focusing on the role of astrocytic Ca 2 + elevations in functional hyperemia; summarize recent advances in genetically engineered Ca 2 + indicators, fluorescence microscopy techniques for studying NVC; and discuss the unmet challenges for future imaging development. Conclusions: Advances in imaging techniques together with improvements in genetic tools have significantly contributed to our understanding of NVC. Many pieces of the puzzle have been revealed, but many more remain to be discovered. Ultimately, optimizing NVC research will require a concerted effort to improve imaging techniques, available genetic tools, and analytical software.

Brain endothelial cell TRPA1 channels initiate neurovascular coupling.

Cerebral blood flow is dynamically regulated by neurovascular coupling to meet the dynamic metabolic demands of the brain. We hypothesized that TRPA1 channels in capillary endothelial cells are stimulated by neuronal activity and instigate a propagating retrograde signal that dilates upstream parenchymal arterioles to initiate functional hyperemia. We find that activation of TRPA1 in capillary beds and post-arteriole transitional segments with mural cell coverage initiates retrograde signals that dilate upstream arterioles. These signals exhibit a unique mode of biphasic propagation. Slow, short-range intercellular Ca2+ signals in the capillary network are converted to rapid electrical signals in transitional segments that propagate to and dilate upstream arterioles. We further demonstrate that TRPA1 is necessary for functional hyperemia and neurovascular coupling within the somatosensory cortex of mice in vivo. These data establish endothelial cell TRPA1 channels as neuronal activity sensors that initiate microvascular vasodilatory responses to redirect blood to regions of metabolic demand.

Intravascular pressure augments cerebral arterial constriction by inducing voltage-insensitive Ca2+ waves.

This study examined whether elevated intravascular pressure stimulates asynchronous Ca(2+) waves in cerebral arterial smooth muscle cells and if their generation contributes to myogenic tone development. The endothelium was removed from rat cerebral arteries, which were then mounted in an arteriograph, pressurized (20-100 mmHg) and examined under a variety of experimental conditions. Diameter and membrane potential (V(M)) were monitored using conventional techniques; Ca(2+) wave generation and myosin light chain (MLC(20))/MYPT1 (myosin phosphatase targeting subunit) phosphorylation were assessed by confocal microscopy and Western blot analysis, respectively. Elevating intravascular pressure increased the proportion of smooth muscle cells firing asynchronous Ca(2+) waves as well as event frequency. Ca(2+) wave augmentation occurred primarily at lower intravascular pressures (<60 mmHg) and ryanodine, a plant alkaloid that depletes the sarcoplasmic reticulum (SR) of Ca(2+), eliminated these events. Ca(2+) wave generation was voltage insensitive as Ca(2+) channel blockade and perturbations in extracellular [K(+)] had little effect on measured parameters. Ryanodine-induced inhibition of Ca(2+) waves attenuated myogenic tone and MLC(20) phosphorylation without altering arterial V(M). Thapsigargin, an SR Ca(2+)-ATPase inhibitor also attenuated Ca(2+) waves, pressure-induced constriction and MLC(20) phosphorylation. The SR-driven component of the myogenic response was proportionally greater at lower intravascular pressures and subsequent MYPT1 phosphorylation measures revealed that SR Ca(2+) waves facilitated pressure-induced MLC(20) phosphorylation through mechanisms that include myosin light chain phosphatase inhibition. Cumulatively, our findings show that mechanical stimuli augment Ca(2+) wave generation in arterial smooth muscle and that these transient events facilitate tone development particularly at lower intravascular pressures by providing a proportion of the Ca(2+) required to directly control MLC(20) phosphorylation.

The differential hypothesis: a provocative rationalization of the conducted vasomotor response.

Tissue blood flow is controlled by a branching network of resistance arteries coupled in series and parallel with one another. To alter organ perfusion during periods of elevated metabolic demand, the arterial segments comprising these networks must dilate in a coordinated manner. Gap junctions are intercellular pores that facilitate arterial coordination by enabling electrical stimuli to conduct among and between endothelial and/or smooth muscle cells. Through this novel perspective, readers will be introduced to the vascular communication field, the process of intercellular conduction, and how key cellular properties influence charge flow. This overview will begin with a brief historical review and then introduce two differing theories on how electrical phenomena moves among and between vascular cells. The basis of the "syncytium" and "differential" hypothesis will be critically discussed within a framework of biophysical and experimental observations. This foundational understanding will be used to extend our mechanistic insight of: (i) "local" and "global" blood flow control; and (ii) debilitating disorders such as arterial vasospasm.

Heterogeneity of Sensory-Induced Astrocytic Ca2+ Dynamics During Functional Hyperemia.

Astrocytic Ca2+ fluctuations associated with functional hyperemia have typically been measured from large cellular compartments such as the soma, the whole arbor and the endfoot. The most prominent Ca2+ event is a large magnitude, delayed signal that follows vasodilation. However, previous work has provided little information about the spatio-temporal properties of such Ca2+ transients or their heterogeneity. Here, using an awake, in vivo two-photon fluorescence-imaging model, we performed detailed profiling of delayed astrocytic Ca2+ signals across astrocytes or within individual astrocyte compartments using small regions of interest next to penetrating arterioles and capillaries along with vasomotor responses to vibrissae stimulation. We demonstrated that while a 5-s air puff that stimulates all whiskers predominantly generated reproducible functional hyperemia in the presence or absence of astrocytic Ca2+ changes, whisker stimulation inconsistently produced astrocytic Ca2+ responses. More importantly, these Ca2+ responses were heterogeneous among subcellular structures of the astrocyte and across different astrocytes that resided within the same field of view. Furthermore, we found that whisker stimulation induced discrete Ca2+ "hot spots" that spread regionally within the endfoot. These data reveal that astrocytic Ca2+ dynamics associated with the microvasculature are more complex than previously thought, and highlight the importance of considering the heterogeneity of astrocytic Ca2+ activity to fully understanding neurovascular coupling.

Seizures elevate gliovascular unit Ca2+ and cause sustained vasoconstriction.

Seizures can result in a severe hypoperfusion/hypoxic attack that causes postictal memory and behavioral impairments. However, neither postictal changes to microvasculature nor Ca2+ changes in key cell types controlling blood perfusion have been visualized in vivo, leaving essential components of the underlying cellular mechanisms unclear. Here, we use 2-photon microvascular and Ca2+ imaging in awake mice to show that seizures result in a robust vasoconstriction of cortical penetrating arterioles, which temporally mirrors the prolonged postictal hypoxia. The vascular effect was dependent on cyclooxygenase 2, as pretreatment with ibuprofen prevented postictal vasoconstriction. Moreover, seizures caused a rapid elevation in astrocyte endfoot Ca2+ that was confined to the seizure period, and vascular smooth muscle cells displayed a significant increase in Ca2+ both during and following seizures, lasting up to 75 minutes. Our data show enduring postictal vasoconstriction and temporal activities of 2 cell types within the neurovascular unit that are associated with seizure-induced hypoperfusion/hypoxia. These findings support prevention of this event may be a novel and tractable treatment strategy in patients with epilepsy who experience extended postseizure impairments.

Mechanistic basis of differential conduction in skeletal muscle arteries.

The goal of this investigation was to probe intercellular conduction in skeletal muscle feed arteries and to address why smooth muscle-initiated responses fail to robustly spread like their endothelial counterpart. Using computational and experimental approaches, two interrelated rationales were developed to explain this apparent discrepancy in cell-to-cell communication. The first rationale stressed that smooth muscle electrical responses, if initiated, will be actively dissipated as they spread from cell-to-cell along the arterial wall. Charge dissipation is promoted within arteries by the structural and connectivity properties of vascular cells. The second rationale centred on the idea that when agents other than KCl stimulate a limited number of smooth muscle cells, they fail to generate the currents required to elicit a localized membrane potential (V(M)) response. This insufficiency results in part from charge loss, via gap junctions, to neighbouring unstimulated cells. Experiments confirmed the latter rationale by showing that focal phenylephrine application: (1) elicited a localized constriction insensitive to L-type Ca(2+) channel blockade; and (2) failed to substantially depolarize vascular smooth muscle cells. Further investigation revealed that while focal phenylephrine-induced constriction was V(M) independent, it was reliant on internal Ca(2+) mobilization and the activation of inositol 1,4,5-trisphosphate (IP(3)) receptors. The preceding findings illustrate that by using computational modelling and experimentation in a complementary manner, one can isolate key cellular properties and rationally examine their role in limiting the conduction of smooth muscle-initiated responses. Functionally, these observations enable investigators to assign the concept of 'local and global' blood flow control to the electrical and/or non-electrical behaviour of specific cell types.

Astrocytes Integrate Behavioral State and Vascular Signals during Functional Hyperemia.

Dynamic changes in astrocyte free Ca2+ regulate synaptic signaling and local blood flow. Although astrocytes are poised to integrate signals from synapses and the vasculature to perform their functional roles, it remains unclear what dictates astrocyte responses during neurovascular coupling under realistic conditions. We examined peri-arteriole and peri-capillary astrocytes in the barrel cortex of active mice in response to sensory stimulation or volitional behaviors. We observed an AMPA and NMDA receptor-dependent elevation in astrocyte endfoot Ca2+ that followed functional hyperemia onset. This delayed astrocyte Ca2+ signal was dependent on the animal's action at the time of measurement as well as a neurovascular pathway that linked to endothelial-derived nitric oxide. A similar elevation in endfoot Ca2+ was evoked using vascular chemogenetics or optogenetics, and opto-stimulated dilation recruited the same nitric oxide pathway as functional hyperemia. These data show that behavioral state and microvasculature influence astrocyte Ca2+ in active mice. VIDEO ABSTRACT.

Impaired neurovascular coupling in aging and Alzheimer's disease: Contribution of astrocyte dysfunction and endothelial impairment to cognitive decline.

The importance of (micro)vascular contributions to cognitive impairment and dementia (VCID) in aging cannot be overemphasized, and the pathogenesis and prevention of age-related cerebromicrovascular pathologies are a subject of intensive research. In particular, aging impairs the increase in cerebral blood flow triggered by neural activation (termed neurovascular coupling or functional hyperemia), a critical mechanism that matches oxygen and nutrient delivery with the increased demands in active brain regions. From epidemiological, clinical and experimental studies the picture emerges of a complex functional impairment of cerebral microvessels and astrocytes, which likely contribute to neurovascular dysfunction and cognitive decline in aging and in age-related neurodegenerative diseases. This overview discusses age-related alterations in neurovascular coupling responses responsible for impaired functional hyperemia. The mechanisms and consequences of astrocyte dysfunction (including potential alteration of astrocytic endfeet calcium signaling, dysregulation of eicosanoid gliotransmitters and astrocyte energetics) and functional impairment of the microvascular endothelium are explored. Age-related mechanisms (cellular oxidative stress, senescence, circulating IGF-1 deficiency) impairing the function of cells of the neurovascular unit are discussed and the evidence for the causal role of neurovascular uncoupling in cognitive decline is critically examined.

Acute two-photon imaging of the neurovascular unit in the cortex of active mice.

In vivo two-photon scanning fluorescence imaging is a powerful technique to observe physiological processes from the millimeter to the micron scale in the intact animal. In neuroscience research, a common approach is to install an acute cranial window and head bar to explore neocortical function under anesthesia before inflammation peaks from the surgery. However, there are few detailed acute protocols for head-restrained and fully awake animal imaging of the neurovascular unit during activity. This is because acutely performed awake experiments are typically untenable when the animal is naïve to the imaging apparatus. Here we detail a method that achieves acute, deep-tissue two-photon imaging of neocortical astrocytes and microvasculature in behaving mice. A week prior to experimentation, implantation of the head bar alone allows mice to train for head-immobilization on an easy-to-learn air-supported ball treadmill. Following just two brief familiarization sessions to the treadmill on separate days, an acute cranial window can subsequently be installed for immediate imaging. We demonstrate how running and whisking data can be captured simultaneously with two-photon fluorescence signals with acceptable movement artifacts during active motion. We also show possible applications of this technique by (1) monitoring dynamic changes to microvascular diameter and red blood cells in response to vibrissa sensory stimulation, (2) examining responses of the cerebral microcirculation to the systemic delivery of pharmacological agents using a tail artery cannula during awake imaging, and (3) measuring Ca(2+) signals from synthetic and genetically encoded Ca(2+) indicators in astrocytes. This method will facilitate acute two-photon fluorescence imaging in awake, active mice and help link cellular events within the neurovascular unit to behavior.

Activation of endothelial IKCa channels underlies NO-dependent myoendothelial feedback.

Agonist-induced vasoconstriction triggers a negative feedback response whereby movement of charged ions through gap junctions and/or release of endothelium-derived (NO) limit further reductions in diameter, a mechanism termed myoendothelial feedback. Recent studies indicate that electrical myoendothelial feedback can be accounted for by flux of inositol trisphosphate (IP3) through myoendothelial gap junctions resulting in localized increases in endothelial Ca(2+) to activate intermediate conductance calcium-activated potassium (IKCa) channels, the resultant hyperpolarization then conducting back to the smooth muscle to attenuate agonist-induced depolarization and tone. In the present study we tested the hypothesis that activation of IKCa channels underlies NO-mediated myoendothelial feedback. Functional experiments showed that block of IP3 receptors, IKCa channels, gap junctions and transient receptor potential canonical type-3 (TRPC3) channels caused endothelium-dependent potentiation of agonist-induced increase in tone which was not additive with that caused by inhibition of NO synthase supporting a role for these proteins in NO-mediated myoendothelial feedback. Localized densities of IKCa and TRPC3 channels occurred at the internal elastic lamina/endothelial-smooth muscle interface in rat basilar arteries, potential communication sites between the two cell layers. Smooth muscle depolarization to contractile agonists was accompanied by IKCa channel-mediated endothelial hyperpolarization providing the first demonstration of IKCa channel-mediated hyperpolarization of the endothelium in response to contractile agonists. Inhibition of IKCa channels, gap junctions, TRPC3 channels or NO synthase potentiated smooth muscle depolarization to agonists in a non-additive manner. Together these data indicate that rather being distinct pathways for the modulation of smooth muscle tone, NO and endothelial IKCa channels are involved in an integrated mechanism for the regulation of agonist-induced vasoconstriction.