Modulatory Effects of Noradrenergic and Serotonergic Signaling Pathway on Neurovascular Coupling

Summary 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.


Introduction
Neurovascular coupling (NVC) is a critical mechanism that serves to control local cerebral blood flow (CBF) to match metabolic demands. Because the blood supply is finite, ensuring adequate blood delivery to metabolically demanding regions without compromising the broader global network requires heterogeneous regulation of the cerebral circulation to match CBF with local energy requirements. The integrative relationship between astrocytes and vascular cells has attracted considerable attention. The anatomical features of astrocytes, particularly their cerebral microvasculatureensheating endfeet, makes these cells an ideal conduit for relaying neuronal information to vascular cells and ultimately eliciting vascular changes. Because astrocytes are nonexcitable cells, their Ca 2+ dynamics have been used as an index of their activity. These Ca 2+ signals have been linked to synaptic plasticity 1,2 and NVC [3][4][5][6][7][8][9] . Moreover, while the critical role of localized synaptic glutamatergic signaling pathways in initiating the NVCmediated increases in local blood flow have been extensively investigated [10][11][12] , the contributions of other neurotransmitters to NVC have received less attention.
In vivo studies of NVC have traditionally been performed in anesthetized animals 4,13 , and thus have generally discounted contributions of behavior-dependent signaling pathways. Neurotransmitters of the reticular activating system associated with an awake, alert, vigilant, and engaged state of the animal, such as norepinephrine (NE) and serotonin (5hydroxytriptamine ), are robust vasoactivators in the peripheral circulation [14][15][16][17][18] . Although, astroglia are known targets of glutamatergic activation 3,19,20 and increasingly appear to be direct targets of the reticular activation system through noradrenergic and serotonergic signaling [21][22][23][24][25] , surprisingly, data linking these neuromodulators to CBF regulation appear discordant. For example, some studies have reported an increase in cortical blood perfusion following direct stimulation of the locus coeruleus (LC) 26,27 , the primary site of NE synthesis, whereas others reported a reduction in CBF 28 . Importantly, these NVC responses have not been studied in the more physiologically valid context of awake, freely moving animals. Recent technical advances in live imaging of CBF in fully awake, behaving rodents using two-photon laser-scanning microscopy have made it possible to explore how different neuronal networks are integrated [29][30][31] . Taken together with improved genetic tools, these developments have enabled us to gradually uncover details of Ca 2+ signals in different subcellular compartments of astrocytes and elucidate the heterogeneity of these signals [32][33][34] . Because the mechanistic basis of the astrocytevasculature relationship in NVC remains a matter of debate, going beyond the conventional glutamatergic system-based NVC paradigm to understand the contributions of other signaling pathways could provide insight into independent redundant and/or complementary mechanisms.
We and others have previously reported the dependence of astrocyte Ca 2+ signals on behavior 24,35 . However, whether the behavior-related neuromodulators, NE, and 5-HT, contribute to NVC has yet to be studied. Here, we used in vivo two-photon imaging in awake mice to investigate the contributions of NE and 5-HT to NVC in the cerebral cortex. Our results reveal that, while both neuromodulators facilitate astrocyte Ca 2+ elevations in response to whisker stimulation, they differentially affect functional hyperemia. Given recent discoveries showing an association between the degeneration of noradrenergic neurons and Alzheimer's disease 36,37 , as well as the well-known association between serotonergic dysfunction and neuropsychiatric and cardiovascular disorders 38 , our findings may offer insights into how these neuromodulators regulate CBF and potentially provide alternatives for treating these related disorders.

Results
Animal behavior contributes to whisker stimulation-induced astrocyte Ca 2+ elevation and changes in functional hyperemia On the basis of previous work, it was proposed that behavioral states such as arousal and vigilance prime astrocytes to respond to local neuronal activity in the neocortex 23,24 . However, whether behavioral states contribute to NVC has remained uncertain. To examine the dependence of NVC on behavior, we applied a 5-second air puff to the contralateral whiskers of a fully awake mouse and monitored subsequent vascular changes and astrocyte Ca 2+ transients in vivo by two-photon microscopy and tracked changes in animal reactions to the air puff. To facilitate imaging of astrocyte Ca 2+ signals surrounding penetrating arterioles in the barrel cortex (i.e., layer I-III), we utilized mice expressing a genetically encoded Cre-dependent Ca 2+ sensor (GCaMP6f) crossed with mice expressing tamoxifen-inducible Cre recombinase under the control of the Aldh1l1 (aldehyde dehydrogenase 1 family member L1) promoter. Rhodamine B dextran or Texas Red dextran was injected via the tail vein to label the vasculature ( Figure 1A). Trained, head-fixed mice were monitored while running freely on a passive air-supported treadmill. We focused on two patterned behavioral responses to whisker stimulation (5 second air puff): quiet-to-running (QR; 132 trials, 28 mice), in which mice went from a quiescent state to running, and continuous quiet (CQ; 110 trials, 26 mice), where mice remained quiet prior to, during and after whisker stimulation ( Figure 1). We observed significant differences in sensory-induced responses between the two behavioral states, with QR mice exhibiting greater increases in astrocyte Ca 2+ fluorescence signals (F/F, expressed as a percentage) than CQ mice. Increases in Ca 2+ signals were significantly greater in QR mice in all astrocyte subcellular compartments, including the endfoot (F/F = 134.7  12.5%, and 39.7  5.7% in QR vs. CQ, p  0.0001), soma (F/F = 200.1  20.7% and 96.2  22.5% in QR vs. CQ, p  0.05), and arbor (F/F = 66.5  5.7% and 30.6  5.3% in QR vs. CQ, p  0.0001) ( Figure 1B). These observed differences in astrocyte Ca 2+ responses to sensory stimulation between the two behavioral states echo previous observations 24,35,39 . Interestingly, our data also showed that the magnitude of the hyperemic response, measured as the amplitude of the arteriole dilation (A/A, expressed as a percentage) was significantly greater in mice that showed a change from a quiet to a running state than in those that remained quiet (A/A = 42.2  2.2% and 28.9  2.2% for QR vs. CQ, p  0.001), a previously unreported finding. Although the behavioral state did not affect the arteriole dilation onset time (1.5  0.1 s and 1.2  0.1 s for QR vs. CQ, p  0.05), it significantly affected the duration of the response (14.5  1.0 s and 10.6  1.0 s for QR vs. CQ, p  0.05) ( Figure 1C & D). Astrocyte Ca 2+ elevations onset times were similar despite changes in mouse behavioral state (4.8  0.2 s and 4.9  0.3 s for QR vs. CQ, p  0.05) ( Figure 1C), yet the duration of the Ca 2+ signal was significantly longer in QR mice than in CQ mice (9.0  0.5 s and 5.9  0.5 s for QR vs. CQ, p  0.0001) ( Figure 1D). Although there were a few trials where we observed no change in arteriole diameter (QR = 6.8%; CQ = 13.2%) or astrocyte Ca 2+ signals (QR = 4.8%, CQ = 24.6%) or both (QR = 2.7%; CQ = 7.0%), in most trials, mice displayed both functional hyperemic responses and astrocyte Ca 2+ elevations (QR = 85.6%; CQ = 55.3%) to a 5-second whisker stimulation ( Figure 1E).

Long-range neuromodulators contribute to sensory-induced astrocyte Ca 2+ transients and functional hyperemia
Long-range neuromodulators such as NE have been shown to trigger astrocyte Ca 2+ transients associated with locomotion and arousal 24 . As such we focus our attention on the QR behavior onward. To assess the contribution of these neuromodulatory signaling pathways to NVC, we treated mice acutely with trazodone, a broad-spectrum inhibitor of serotonergic, and adrenergic receptors 40 , and measured astrocyte and arteriole response of the same astrocyte and vessel before and after trazodone treatment ( Figure 2A). Although, trazodone (10 mg/kg, i.p.) did not alter resting vasomotor tone or spontaneous astrocyte endfoot Ca 2+ signals ( Figure 2B), it dramatically reduced sensory-induced arteriole cross section/lumen area (A/A = 12.1  3.0% and 25.4  4.0% for trazodone vs. control, p = 0.001; n = 22 trials) and astrocyte Ca 2+ elevations in endfeet (F/F = 11.4  4.2 and 59.0  10.7% for trazodone vs. control, p  0.001; n = 22 trials), soma (F/F = 6.5  5% and 57.8%  11.8% for trazodone vs. control, p = 0.005; n = 10 trials), and arbor (F/F = 5.9  2.2% and 19.4%  3.1% trazodone vs. control, p  0.05; n = 12 trials) ( Figure  2C & D). This indicates that a single or multiple of these neuromodulators contribute to stimulation-evoked astrocyte Ca 2+ transients and functional hyperemia either synergistically or independently of each other. Since NE and 5-HT are both known to be robust vasoconstrictors in peripheral systems, we focused our attention on these two neuromodulators, testing whether they elicit similar effects in the cerebral circulation.
Noradrenergic signaling enhances sensory-induced increases in astrocyte Ca 2+ transients while exerting polarized effects on functional hyperemia NE is a key neuromodulator that plays critical roles in various higher brain functions in the central nervous system (CNS) 41 . Noradrenergic neurons originate in the LC and send their projections diffusely throughout the brain. Their axons target the vasculature as well as astrocytes and neurons in the neocortex. In the brain, the release of NE is associated with increases in arousal, attention, and vigilance 42 . Paukert and colleagues 24 reported that NE released during periods of heightened vigilance enhances the responsiveness of astrocytes to local neuronal activity. The majority of perivascular axon terminals are found to be closer to capillaries than arterioles 43 , suggesting that NE may preferentially act at the capillary level. Recent work by Korte et. al. 44 demonstrated that released NE induces pericyte contraction via 2-adrenergic receptors, an action that was proposed to play a role in regulating vascular tone. Using a chronic in vivo mouse model, we assessed whether chemically ablating noradrenergic (i.e., tyrosine hydroxylase-expressing) neurons altered functional hyperemia and astrocyte Ca 2+ transients in response to a 5-second whisker stimulation at the penetrating arteriole level ( Figure 3A). To this end, we treated mice with N-(2-Chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4; 50mg/kg, i.p.), an adrenergic neurotoxin that exclusively destroys noradrenergic projections from the LC but not those from non-LC neurons 45,46 . An immunohistochemical analysis of the LC using an anti-dopamine beta hydroxylase antibody confirmed that DSP-4 effectively ablated noradrenergic neurons within 2 days ( Figure 3C). DSP-4 treatment predominantly attenuated endfoot Ca 2+ responses to a 5-second whisker stimulation, an observation in agreement with previous work 24 . Surprisingly, DSP-4 treatment initially appeared to have no effect on functional hyperemia ( Figure 3D). However, we noted a bimodal distribution of DSP-4-treated functional hyperemia ( Figure 3E), which prompted us to separate the two clusters, and identified that DSP-4 treatment bidirectionally affected functional hyperemic responses ( Figure 3F). In about 40% of recorded trials, DSP-4 reduced both astrocyte endfoot Ca 2+ elevations (F/F = 56.0  14.4% and 162.4  31.8% for DSP-4 vs. control, p  0.001) and functional hyperemic responses (A/A = 34.2  7.1% and 56.4  8.3% for DSP-4 vs. control, p = 0.0001; n = 17 trials) (Figure 3Fi). In the other 60% of recorded trials, DSP-4 reduced astrocyte Ca 2+ signals in endfeet (F/F = 154.5  37.0% and 232.9  38.3% for DSP-4 vs. control, p = 0.003), but augmented vasodilation (A/A = 78.7  6.8% and 52.6  5.4% for DSP-4 vs. control, p  0.0001; n = 25 trials) ( Figure 3Fii). There were no significant differences in onset times for arteriole dilation and endfoot Ca 2+ elevations between DSP-4 and control in trials with attenuated functional hyperemia (dilation: 1. Serotonergic signaling modulates sensory-induced astrocyte Ca 2+ and functional hyperemia. 5-HT, a monoamine neurotransmitter that acts as a potent vasoconstrictor in the peripheral system, is found in the gastrointestinal tract, blood platelets, and the brain 47 . 5-HT has been implicated in numerous physiological and behavioral functions, including cognition, motor function, sensory function, sleep-wake cycle, and vascular function 48,49 . Serotonergic neurons exhibit steady-state firing during waking and show a decrease in firing during slow-wave sleep 47,50 . In the CNS, serotonergic neurons originating from brainstem raphe nuclei project to almost every part of the brain, including the cerebral cortex. Studies of the functional role of 5-HT in vascular control have predominantly focused on the peripheral system. In the context of the cerebral circulation, the primary focus has been on large cerebral arteries, such as the middle cerebral artery, which are known to be innervated by extrinsic nerves originating in the peripheral nervous system, such as superior cervical, sphenopalatine, otic, or trigeminal ganglia 51 . Reports based on results obtained in isolated cerebral arteries showed that 5-HT mediates contraction via 5-HT1 receptors 52,53 . In another study employing isolated human and bovine brain intracortical arterioles, Elhusseiny and Hamel showed that activation of 5-HT1 receptors induced a biphasic response, with a low concentration of the serotonergic agonist, sumatriptan, causing a small dilation and a higher concentration causing constriction 54 . However, the involvement of serotonergic signaling in NVC has not been studied in awake mice. To assess the role of 5-HT in sensory-induced functional hyperemia and astrocyte Ca 2+ transients, we imaged responses to a 5-second whisker stimulation in a chronic mouse model using two-photon laser-scanning microscopy without any treatment and one week after treatment with 5,7 dihydroxytryptamine (5,7-DHT) (30 g in 1% ascorbic acid, i.c.v.), a neurotoxin used to ablate serotonergic neurons 55 . Damage to noradrenergic neurons by 5,7-DHT was prevented by pretreating mice with desipramine (20 mg/kg i.p.) at least 30 minutes prior to the injection of 5,7-DHT, as reported previously 56,57 . Our protocol is depicted in Figure 4A. One week after 5,7-DHT treatment, we imaged the mouse again and performed immunohistochemistry on the dorsal raphe using an anti-5-HT antibody to verify the extent of serotonergic neuron loss ( Figure

Discussion
In the current study, we assessed the contributions of noradrenergic and serotonergic neurons to NVC using in vivo two-photon imaging in an awake mouse model. Based on the results of these experiments, we propose that NE and 5-HT modulate sensory-induced functional hyperemia and astrocyte Ca 2+ transients. Here, we showed that mice undergoing a change in behavioral state from quiet to running exhibit stronger astrocyte Ca 2+ elevations in response to a 5-second gentle air puff to the whiskers compared with mice that remained quiet throughout. Our data is consistent with previous reports showing that locomotion induces Ca 2+ transients in cortical astrocytes 24,35 . In the absence of locomotion, the only likely source of sensory input is whisker stimulation, which generates minimal astrocyte activity, an observation in line with prior studies suggesting that quiescent astrocytes have a high threshold for activation and require recruitment of other systems, such as noradrenergic neurons, to reach this threshold 24 . Previous studies showed that running behavior (i.e., quiet-to-running) is associated with activation of Bergmann glia in the cerebellum 20 and astrocytes in the visual cortex 24 . Our data also showed that, in cases where whisker stimulation caused a behavioral change from quiet to running, it induced a stronger functional hyperemic response than that observed in cases where the mouse did not physically react to the gentle air puff, an observation that has not been reported previously. A significant difference in functional hyperemic response between the two behaviors obtained in this study could be attributed to a higher n for CQ behavior trials. These findings echo recent observations on the behavioral dependence of hemodynamic responses 58,59 .
As indicated above, noradrenergic neurons, unlike many other neurons, release NE from axonal varicosities through a volume-release mechanism rather than through specific synaptic contacts, allowing NE to simultaneously target closely appose blood vessels, axons, dendrites, and glial processes 43,60,61 . Cortical astrocytes express 1-, 2-, and 1adrenergic receptors 62 . Interestingly, among brain cells, glia appears to express more adrenergic receptors than neurons 63 . Direct application of -adrenergic agonists to the cortex in vivo has been shown to elicit Ca 2+ waves in astrocytes. Furthermore, direct LC stimulation was reported to rapidly induce monophasic astrocyte Ca 2+ transients 21 that were significantly reduced when treated with DSP-4, an observation that is consistent with our data in which sensory-induced astrocyte Ca 2+ elevations were significantly reduced in DSP-4-treated mice.
Non-neural cells of the NVC also express adrenergic receptors. Vascular smooth muscle cells appear to express predominantly 1-adrenergic receptors 64 , whereas endothelial cells show greater 2and 2-adrenergic receptors expression 62 . However, whether NE directly or indirectly activates vascular smooth muscle cells and/or endothelial cells, and how this affects activity-dependent blood flow in the cerebral microcirculation, remain to be determined. In the current study, we uncovered a dichotomy. Surprisingly, ablating noradrenergic neurons with DSP-4 consistently attenuated sensory-induced astrocyte Ca 2+ elevations, but had both enhancing and attenuating effects on sensory-induced functional hyperemia. While this observation is indeed puzzling, the enhancement of functional hyperemia we observed partially reflects a previously reported phenomenon that global NE-mediated vasoconstriction redistributes blood flow to active areas 65 . This finding suggests that NE-mediated vasoconstriction may also be involved in the "centersurround effect", in which a "hot spot" of functional hyperemia occurs in the core region of the neuronal response and gradually dissipates with distance from the center 66 . It has been proposed that inhibitory neurons shape the "surround" effect through the actions of neuropeptide Y acting on Y1 receptors 67 . In the current study, we have not mapped center and surround areas in the whisker barrel, yet our data could be interpreted as a loss of interregional differences at the penetrating arteriole level. Attenuated dilation by DSP-4 treatment could be observed from arterioles that were most likely located in the "hot spot" of an activated column. Conversely, augmented dilation after DSP-4 treatment could be from arterioles that were likely situated in the surround region of an activated column. A role for NE in enhancing sensory-induced functional hyperemia, though seemingly counterintuitive given the well-known robust constricting effects of NE, finds support in a report by Toussay and colleagues, who showed that LC stimulation increases cortical perfusion 27 . Its volume release implies that NE can simultaneously affect different cell types. The resulting selective activation of specific cell types, which depends on which receptor subtypes they express, can set up a competition among different cell types such that the balance between vasodilatory and vasoconstrictive cellular influences ultimately dictates the outcome. Indeed, a widely proposed model posits that direct effects of NE to astrocytes during whisker stimulation optimizes astrocyte Ca 2+ transients, which could consequently elicit vascular responses 3,5,19 . However, this astrocyte-vascular interaction may more likely play a role in "maintaining" functional hyperemia rather than initiating it. This interpretation is supported by our demonstration that DSP-4 did not significantly change the onset time of functional hyperemia but did significantly reduce the duration of attenuated functional hyperemia while prolonged the duration of augmented vasodilation. These observations echo those of a recent report by Institoris and colleagues 9 . The specific mechanism underlying this NE-mediated pathway could be complicated, and the conditions under which either vasoconstriction or vasodilation dominates may depend on the complex behavioral state of the animal (i.e., beyond the transition from quiet to running). Resolving this question will require a more comprehensive investigation beyond the scope of the current study.
Aforementioned, 5-HT release from serotonergic neurons, like NE release from noradrenergic neurons, occurs via volume transmission; this release mechanism delivers 5-HT from axonal varicosities onto the surrounding area where it could potentially impact multiple cell types 47 , an action that could generate complementary or opposing responses. Serotonin receptors are expressed in both neuronal and non-neuronal cells, including specific GABA interneurons, vascular smooth muscle cells, endothelial cells, microglia, and astrocytes (38). Studies on the effects of 5-HT in the cerebral microcirculation are conflicting, with some studies showing a lack of 5-HT-mediated vasomotor response 48 and others showing both vasodilation and vasoconstriction 49,68 . These conflicting reports could be attributable to differences in experimental approaches. For examples, most of these studies were performed in isolated vessels or in brain slices, where vessel tone might dictate the polarity of the 5-HT-induced response. These inconsistent results also reflect the complex and heterogeneous nature of the serotonergic network and its numerous receptor classes, which can generate opposing responses.
Here, we showed that serotonergic neurons modulate sensory-induced elevations in astrocyte Ca 2+ and functional hyperemia. Our demonstration of modulatory effects of 5-HT on astrocytes echoes the previous findings that selective serotonin reuptake inhibitors and 5-HT trigger astrocyte Ca 2+ transients 25 . Such increases in astrocyte Ca 2+ can consequently contribute to the maintenance of functional hyperemia 3,4,[69][70][71] . We propose that 5-HT released during sensory stimulation modulates astrocyte Ca 2+ and functional hyperemia, reflecting the fact that ablation of serotonergic neurons not only attenuated the percent peak response but also reduced the duration of the response. However, we would note that we did not examine how ablating 5-HT neurons affected interneurons, a potentially important caveat given that Perrenoud et al. showed that 5-HT3A-expressing interneurons can induce vasodilation and vasoconstriction 68 . This observation again highlights the complexity of NVC, which can be mediated and modulated by an integrative network of neurons, astrocytes, and vascular cells that are competing or complementing each other.
Collectively, our data show that NVC is behavior dependent and that both noradrenergic and serotonergic neurons modulate sensory-induced astrocyte Ca 2+ elevations and functional hyperemia in an awake in vivo mouse model.

Animals
All animal procedures were approved by the Animal Care and Use Committee of the University of Nevada, Reno. All studies were performed on male FVB-Tg(Aldh1l1 cre/ERT2)1Khakh/J (Jax#029655) x 129S-Gt(ROSA)26Sor tm95.1(CAG-GCaMP6f)Hze /J (Jax#024105) mice between postnatal day 30 (P30) and P60. Animals were injected on five consecutive days with tamoxifen (75 mg/kg, Sigma), prepared as a 10 mg/mL stock in corn oil. Injections started between P19 and P35. Animals were kept on a normal 12hour light/12-hour dark cycle with ad libitum access to food and water.

Acute Awake In Vivo Preparation
All surgical procedures and isoflurane anesthesia were performed as previously described 29 . Briefly, 1 week before the imaging session, a head bar was surgically affixed to the animal, after which the animal was returned to its home cage and allowed to recover. Mice were initially trained on a passive air-supported Styrofoam ball treadmill under head restraint for 45 minutes and habituated to whisker stimulation with an air puff on contralateral vibrissae once every minute for 5 seconds using a picospritzer III (General Valve Corp.) for two consecutive days. After training, the animal was returned to its home cage. On imaging day, bone and dura over the primary somatosensory cortex were removed and a ∼3 x 3-mm cover glass (thickness #0) was installed over the cranial window.

Chronic Awake In Vivo Preparation
All surgical procedures and isoflurane anesthesia for the chronic in vivo model were similar to those for the acute awake in vivo preparation, with two exceptions: 1) head bar installation and craniotomy were performed in one surgical session; and 2) a double cover glass (i.e., 2.6-mm cover glass glued onto a 3.5-mm cover glass) with a smaller cover glass, positioned on top of the brain tissue, was used. After surgery, the animal was returned to its home cage and allowed to recover for at least 3 weeks before the first imaging session. Head-fixed animals underwent training prior to imaging as noted above.

Vessel Indicators
For visualization of blood plasma, a 2.3% (w/v) solution of Rhodamine B isothiocyanate (RhodB)-dextran (MW 70KDa; Sigma) or Texas Red dextran (MW 70KDa; Sigma) in saline (15mg dissolved in 300l lactated Ringer's solution (5%) 200-250 μL total volume) was injected via the tail vein prior to imaging, after which the animal, with its head immobilized, was allowed to recover on the treadmill for 30 minutes. Penetrating arterioles were identified based on their upstream parent pial arterioles, undulations in diameters, thick vessel wall, and the direction of blood flow.

Two-Photon Fluorescence Imaging and Whisker Stimulations
Fluorescence images were obtained using a custom-built in vivo two-photon microscope (Bergamo II, Thorlabs), equipped with a Nikon 16X (0.8NA, 3mm WD) or an Olympus 20X objective lens (1.0NA, 2.5mm WD) and GaAsP PMTs (Hamamatsu) and controlled by ThorImage. GCaMP6f and Rhodamine B dextran or Texas Red dextran were excited at 920 nm using a tunable Ti:sapphire laser (Tiberius, Thorlabs). Green fluorescence signals were obtained using a 525/50-nm band-pass filter, and orange/red signals were obtained using a 605/70-nm band-pass filter. Imaging was performed at a rate of 3.2 or 3.8Hz. Animal behaviors were captured using a near-infrared LED (780 nm) and a camera. A 5second air puff that deflected all whiskers on the contralateral side without impacting the face was applied using a Picospritzer, and vessel surface area and astrocyte Ca 2+ responses were monitored in the barrel cortex (layers I-III).

Whisker Stimulation
A 5-second air puff was applied to the contralateral whiskers using as little air pressure as necessary to deflect all the whiskers. The same protocol was applied during training. The air output was divided into two mounted glass capillary tubes directed at separate groups of vibrissae so as to stimulate as many whiskers as possible without impacting the face.

Confocal Microscopy
Slices were mounted in SlowFade Diamond. Images were acquired on an Olympus FV1000 confocal microscope at 20x (NA, 0.75) at 12-bit depth using the same acquisition settings for all compared images. For each image, five (NE) to nine (5-HT) z-steps from the slice surface inward were acquired at 1.26 m intervals.

Data Analysis and Statistics
All data were processed using FIJI/ImageJ. For in vivo data, movement artifacts in the xy plane were corrected for using the align_slices plugin. Regions of interest (ROIs) corresponding to astrocyte endfeet, soma, and arbor were analyzed separately. Ca 2+ responses were calculated as ΔF/F = (Ft-Frest)/Frest where Ft is the measured fluorescence at any given time and Frest is the average fluorescence obtained over 2 seconds prior to whisker stimulation. Ca 2+ signals with an intensity that crossed a 2-standard deviation (SD) threshold relative to signal fluctuations during a 2-second pre-stimulus baseline and remained above the threshold for at least 0.5 seconds were detected as astrocyte Ca 2+ increases. Cross-sectional areas of penetrating arteriole were analyzed using the threshold feature in ImageJ, after which the area of the lumen filled with either RhodB-dextran or Texas Red-dextran was measured using particle analysis as previously described 35 . Cross-sectional area changes were calculated as ΔA/A = (At-Arest)/Arest where At is the area obtained at any given time and Arest is the average baseline area obtained over 2 seconds prior to whisker stimulation. Areas exhibiting a change in intensity that crossed a 2-standard deviation threshold relative to signal fluctuations during a 2-second pre-stimulus baseline and remained above the threshold for at least 0.5 seconds were detected as vasodilation. Onset corresponds to the first time point at which the signal reached the threshold and remained above it for at least 0.5seconds. Duration was calculated as the difference between response onset and response offset. Statistical analyses used a paired or unpaired t-test or one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test, as appropriate. Statistical "n"-values constituted a single experimental trial or an experimental animal, as indicated. Data are expressed as means  SEM. P-values < 0.05 were considered statistically significant. All statistical analyses were done using GraphPad Prism.
For immunohistochemistry data, objects in images were isolated using the auto-threshold feature, and the average of threshold for the untreated condition was used for subsequent analyses. Cells were identified using the Measure Particles function with Areas > 100 um 2 , a setting that captured most cell soma but excluded most processes. Results (number of particles, Area, Average Intensity) were averaged per image section. Results were analyzed and presented using Graphpad Prism (v. 9.2.0). Statistical comparisons used Student's t-test. Figure 1. Mouse behavior in response to a gentle 5-second air puff to the whiskers affects sensory-induced functional hyperemia and astrocyte Ca 2+ elevations. A. Penetrating arteriole (red) and Ca 2+ response (green) in endfoot and soma immediately prior to (0 s) and after (1s, 3s and 5s) whisker stimulation. B. Summary data showing peak dilation and astrocyte Ca 2+ response (%) in endfoot, soma, and arbor in two behavioral states in response to a 5-second whisker stimulation. C. Summary data showing dilation and astrocyte Ca 2+ onset times and associated behavioral states. D. Summary data showing duration of arteriole dilation and endfoot Ca 2+ in two behavioral states. E. Percentage of trials that generated different combinations of dilation and astrocyte Ca 2+ transients in two behavioral states. Data are means  SEM. (**p  0.05, ***p  0.001****p  0.0001).