Fast 3D imaging in the auditory cortex of awake mice reveals that astrocytes control neurovascular coupling responses at arteriole-capillary junctions.

Fast 3D imaging in the auditory cortex of awake mice reveals that astrocytes control neurovascular coupling responses at arteriole-capillary junctions. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Fast 3D imaging in the auditory cortex of awake mice reveals that astrocytes control neurovascular coupling responses at arteriole-capillary junctions. Barbara Lind, Andrea Volterra This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6539397/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Neurovascular coupling (NVC) increases blood flow, assuring adequate supply to active cortical regions by local redistribution via penetrating arterioles (PA) and branching capillaries. Astrocyte end-feet enwrapping these vascular structures possess machinery to regulate blood flow, but their participation in NVC is controversial. Via a new 3D + t two-photon imaging approach we visualized PA and capillaries simultaneously during naturally-occurring and tone-evoked dilations in the auditory cortex of awake mice. We observed that dilations occurred bidirectionally, and a fraction of them extended between compartments across the interconnecting sphincter, depending on the animal activity states. These multi-compartment dilations were preceded by rapid astrocyte end-foot Ca2 + signals around the sphincter. Reduction of this astrocytic Ca2 + activity in IP3R2KO mice suppressed multi-compartment dilations, revealing a pivotal role of pre-capillary sphincters in their bidirectional spread between vascular compartments under local control by astrocytes. This novel mechanism contributes to physiological regulation of laminar blood flow during NVC. Biological sciences/Neuroscience/Glial biology/Astrocyte Biological sciences/Neuroscience/Neuro–vascular interactions Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Adequate regulation of cerebral blood flow is the basis of healthy brain function. The neurovascular coupling (NVC) response ensures sufficient oxygen and energy during increased neuronal activity and, when impaired, contributes to pathophysiological alterations in several common cerebral diseases 1 . The blood is recruited to the capillary bed from the brain surface via penetrating arterioles (PAs). The first branch point from the PAs into the capillary bed, the 1st order capillary, also dubbed precapillary arteriole 2 , 3 or post-arteriolar transition zone 4 , is believed to be critically important for blood flow regulation during NVC 5 . Intriguingly, the presence of a gating structure, the pre-capillary sphincter, was recently described precisely at this level 6 . This structure expresses α-smooth muscle actin in its mural cells (αSMA) 6 – 8 , which makes it contractile and capable of modifying vessel diameter. Changes in the sphincter diameter may then control the blood flow to the downstream brain tissue 9 . Astrocytes’ end-feet continuously cover all the blood vessel segments and could, in principle, impose control at each level 10 . Intracellular Ca2 + increases in astrocytes can induce the release of vasoactive factors 11 , yet the role of astrocytes in blood flow regulation remains controversial. Notably, it is unclear if Ca2 + elevations in astrocytes are rapid and prevalent enough to contribute to blood flow recruitment 12 . To this point, different studies produced discrepant results: some reported that astrocytes recruit blood flow, but exclusively at the capillary level 13 , 14 , others that astrocytes instead regulate PA diameter, but only during extended sensory experiences 15 , and others that astrocytes do not exert any Ca2+-dependent regulation on blood flow. The latter conclusion was based on the authors’ inability to observe any changes in the NVC response in mice lacking IP3 receptor type-2 (IP3R2), the IP3R isoform thought to drive most of the intracellular Ca 2+ elevations in astrocytes 16 – 18 . Independent of their different conclusions, all the above studies looked at blood vascular dynamics in a simplistic manner, mainly focusing on individual compartments, and disregarding the heterogenous nature of dilations progressing through the vascular bed 19 – 21 . Moreover, different studies used different imaging conditions to investigate astrocyte intracellular Ca 2+ dynamics with respect to size, timing, and frequency of the recorded Ca2 + signals 15 , 17 , 18 , 22 – 31 . Last but not least, most studies were performed in anesthetized mice in which both astrocytic Ca 2+ 32,33 and blood flow responses 34 are severely dampened. Overall, these several shortcomings did not permit to draw firm conclusions on the role of astrocyte Ca2 + signaling in the control of brain hemodynamics. In the present study, we aimed to overcome those pitfalls. To start, we performed our experiments in awake mice, which permitted us to consider the impact of the mouse activity state on the NVC response and the heterogeneity of the dilation patterns in different inherent conditions. Secondly, we used a 3D + t two-photon imaging approach 32 , to our knowledge for the first time in NVC studies. With this approach, we could monitor entire regions (30–150 µm in the z-axis) at the branching point between penetrating arterioles and capillary bed and investigate simultaneously the vascular responses at the levels of PA, 1st order capillary, and connecting sphincter, while also monitoring the Ca2 + activity in the surrounding astrocyte structures. Thirdly, we imaged these stacks at high speed (10 Hz), to capture the full range of astrocytic Ca2 + dynamics, including fast and local signals. We report the existence of different patterns of natural dilations at the PA-capillary junctions in the mouse auditory cortex, which depends on the inherent activity of the mouse. Part of the observed dilations involved both PA and capillary, whereas others remained confined to only one compartment. Astrocytes occasionally underwent large Ca2 + activations, but they also often displayed smaller Ca2 + rises in the end-feet enwrapping the pre-capillary sphincters. Such local Ca2 + rises preceded spread of the dilations and specifically correlated with multicompartment dilations. Both the astrocyte Ca2 + activity and the spread of dilations across the sphincter were affected in mice lacking IP3R2 (IP3R2KO). When mice were subjected to auditory stimulation, dilation patterns were analogous to those seen in naturally behaving mice and similarly depended on astrocyte Ca2 + activity. We conclude that pre-capillary sphincters play a central role in the bi-directional spread of dilations between vascular compartments and that astrocyte Ca2 + signaling controls their function locally. Results 3D imaging of vessel dilations and astrocyte Ca2+ activity in the auditory cortex of awake mice: observations along the descending PA paths. We investigated the astrocytic contribution to hemodynamic responses in the auditory cortex of awake mice (Figure 1A) focusing at first on the PA tracts. We mapped the surface vessels to identify appropriate positions for imaging, where PAs dive into the tissue, i.e., where in the vascular tree, the freshly oxygenated blood enters the cortex (Figure 1B). By 2-photon imaging, we then studied astrocyte and blood vessel dynamics along the descending PA paths. Astrocyte Ca2+ activity was visualized using the GFAPCreERT2:GCaMP6f mouse strain that enables astrocyte-selective, inducible expression of the green Ca2+ indicator, GCaMP6f, upon tamoxifen (TAM) injections (Methods). In parallel, PA dynamics were revealed by loading blood vessels with red fluorescent dye (Texas Red-dextran) to highlight their lumen. In this first set of experiments, we positioned our three-dimensional fields of view (3D-FOV) along the PAs to cover descending tracts up to 280 µm in the z dimension (Figure 1C). Our fast 3D imaging approach 32 enabled us to monitor vascular and astrocyte dynamics quasi-simultaneously in multiple focal planes along the z-axis (Figure 1C, Sup. figure S1, Video V1). Our investigation of the astrocyte dynamics focused on the Ca2+ activity within the end-feet, as they represent the points in which the astrocytes contact the contractile vascular cells. The end-feet regions of interest (ROIs) were automatically defined as the structures present within 2 µm from the vessel surface in each z-plane and repositioned during vascular dynamics, according to the vessel diameter changes (Methods and Sup. figure S1). In this way, we followed astrocyte end-foot Ca2+ activity along single PAs while tracing the volume changes occurring in the corresponding PA segments (Figure 1D). Some end-foot Ca2+ activities spread far along the PA and covered up to 205 µm in the z-dimension, whereas others stayed local and were seen just in a single focal plane (Figure 1E, Sup. figure S2A). As for the spontaneous PA dilations that we observed, only about half (324/668) were accompanied by Ca2+ activity in the end-feet along the PA. In these cases, the spread of PA dilations was larger than in the absence of the astrocyte Ca2+ activity and reached further down inside the cortex (Figure 1F). Moreover, it correlated with the spread of the end-foot Ca2+ activity (Sup. figure S2B). Since large astrocytic Ca 2+ elevations are seen during mouse locomotion 22,32,35 , we in parallel recorded EMGs with electrodes implanted in the mice’s neck muscles (Figure 1D, bottom). We found that the spread of the astrocytic end-foot Ca2+ activity in the z-direction almost doubled during mouse movements with respect to when the mouse was at rest, changing, on average, from 23±3µm to 50±7µm (Figure 1E). In agreement with previous reports, we found that not all mouse movements triggered an astrocytic Ca2+ activity in the end-feet along the PA 22 . Still, >70% of PA dilations seen during mouse movements (184/262) were associated to increased Ca2+ activity in the astrocyte end-feet, a higher proportion than the one seen in the resting mouse. As during rest, the PA dilations during movement that coincided with Ca2+ increases in the end-feet, also spread significantly further than those occurring solitary (Figure 1G). These data suggest that large end-foot Ca2+ activity influences PA dilations without being a necessary prerequisite for the dilations to occur. 3D imaging of vessel dilations and astrocyte end-foot Ca2+ activity in the auditory cortex of awake mice: observations at the PA-capillary junction. While PA dilations increase blood flow distribution to many cortical layers, a second and more specific level of regulation occurs at the capillary branching points at different cortical depths 36 . An increased blood flow in one of the 1 st order capillary branches enhances the delivery to areas downstream of the branch 5 . Recently, an investigation in the barrel cortex described a pre-capillary sphincter at the junction with the PA 6 . Therefore, we decided to focus our study of NVC in the auditory cortex on this junction (Figure 2A). Via the luminal red fluorescence, we visualized the sphincter as a narrowing of the lumen proximal to the PA. In addition, in 50% of the junctions (n = 20), we noticed the presence of a bulb at the capillary edge, likely generated by a local reduction in the coverage by contractile cells 37 . The shape of the PA-capillary junctions was variable, and some junctions had a less obvious indentation. Nonetheless, nearly all junctions showed some degree of pre-constriction and, irrespective of the shape, it is known that the entire 1 st order capillaries are contractile 6,37 . Therefore, we decided to define all these connections between a PA and the 1 st order capillary as sphincters. Initially, we investigated the position of the astrocytic structures at the vascular junction using GFAP-EGFP mice with green-fluorescent astrocytes 38 . The astrocytes were localized at the branching points (Figure 2A and Video V1), covering most of the 1 st order capillary up to the sphincter and in contact with it (Figure 2B and Video V2). To detect the potential role of these astrocytes, we then performed dynamic studies in TAM-treated GFAPCreERT2:GCaMP6f mice, centering our 3D-FOV at the level of the PA-1 st order capillary junction. In these experiments, we selected 3D-FOVs much smaller than those utilized for studying dilations along the PAs and took focal planes at 1µm z-steps to ensure continuity in the local imaging around the vasculature (Sup. Figure S3). Thanks to fast two-photon scanning, we could investigate quasi-simultaneously the dilation patterns of the two vascular compartments and the rapid Ca 2+ changes in astrocyte end-feet ROIs at both the arteriole and capillary, i.e. 1 st order capillary, levels. This setting enabled us to define location and temporal sequence of the astrocyte Ca2+ elevations relative to the vascular volume changes. In previous studies in awake mice, when recording from 3D-FOVs of dimensions similar to those used here, we had detected two different types of activities in astrocytic end-feet, involving either local or much larger Ca2+ events 32 . The former were asynchronous and small (µm-scale), generally restrained within a single end-foot, whereas the latter expanded widely to occupy several end-feet in different z-planes. In the present recordings around the vascular junction, we similarly observed small and large events. Concerning the large Ca2+ elevations, they were rather infrequent, occurring every few minutes, and invaded ≥75% of the end-foot ROIs present in all z-planes of our 3D-FOV. We defined these events “large Ca2+ activities” (Figure 2C) and found that 71±7% of them overlapped in time with mouse movements (Sup. figure S2C). Since large astrocytic Ca 2+ activities are known to occur during arousal states driven by noradrenaline release 39 , and noradrenaline release is signaled by the animal’s pupil expansions 40-43 , we monitored the mouse pupil diameter during the imaging sessions using an IR-camera. Thereby, we verified if large astrocyte Ca2+ activities coincided with pupil expansions. Indeed, while most pupil expansions occurred without a large astrocytic Ca 2+ activity, when such an astrocyte activity took place, it almost always (92 ± 13% of the cases) coincided (within 1 sec) with a pupil expansion (Sup. figure S2D). However, as observed for the dilations along the PA path, the dilations around the junction were not triggered just by animal movement or by large astrocytic Ca2+ activities. Indeed, in the majority of cases (75.4±5.9%) dilations occurred when the mouse was at rest and without being accompanied by any large astrocytic Ca2+ activity. Looking at the temporal sequence of the events, when a large astrocytic Ca2+ activity was associated with a mouse movement, the Ca2+ rise in the astrocyte started after the onset of the movement (by 1.0 ± 1.25s, Sup. figure S2E and S2F). In some cases, it preceded the onset of the dilation, but, on average, it rose after the vessel dilation (by 2.3 ± 0.84s, Figure 2D). Therefore, this type of large end-foot astrocytic Ca2+ activity appears to be associated with movements and arousal states but not to be responsible for triggering the dilations seen at the PA-1 st order capillary junctions during the mouse movements. Bidirectional spread of dilations at the pre-capillary sphincter: dependence on the activity and arousal state of the awake mouse. During our repeated 3D-imaging sessions which were in most cases of 2 min duration, we detected many spontaneous vessel dilation events at the junction (Sup. Figure S3). Initially we thought that such events were part of the NVC response evoked by the natural sound perception, increasing excitation and metabolic needs of the auditory neurons. However, we noticed that the dilation events varied largely depending on whether they occurred when the mouse was moving or resting, as well as if a large astrocytic Ca 2+ activity took place coincidentally or not (Figure 2E and F). Notably, dilations occurring when the mouse was moving lasted significantly longer (mean duration: 7.57 ± 1.32 s) than those in the resting mouse (3.06 ± 0.27 s, Figure 2E and S2G). Moreover, dilations in the moving mouse were in most cases (77%) multicompartmental, involving both the PA and the capillary branch (Figure 2F). In contrast, in the resting mouse, ~50% of the dilations were mono-compartmental, confined to the vessel tract either upstream or downstream the sphincter. When dilations occurring in the moving mouse were accompanied by a large astrocytic Ca2+ activity, they lasted even longer (Figure 2E) and were almost always (92% of cases) multicompartmental (Figure 2F). Also in the resting mouse, coincidence of a large astrocyte Ca2+ event, increased the duration of dilations (Figure 2E), but without significantly increasing the proportion of multicompartmental dilations (Figure 2F). These data indicate that the animal movements, and to a lesser degree the arousal state, shape the hemodynamic responses in the auditory cortex, prolonging dilations at this junction in time and space. Next, we analyzed the origin and direction of the dilations in the different conditions. Since only 51±5 % of the dilations during rest were multicompartmental, i.e., involved both PA and capillary, we evaluated which part of the vascular bed dilated alone in the remaining cases (Figure 2G). We found that monocompartmental dilations more frequently involved the capillary branch (37%±6% of all the dilations at rest), and only in 12±3% of cases the PA (Figure 2G). This heterogeneity was also reflected in the origin of the multicompartmental dilations: some of them first appeared at the PA, others at the capillary level and others at the junction. The latter ones were the only dilations initiating within our 3D-FOV; they represented about one third of all the multicompartmental dilations, and in most cases started at the sphincter, and just in a few cases at the bulb. The remaining dilations mostly originated outside the 3D-FOV; in 33 ± 5% of cases they were seen coming from the PA, and in 32 ±6% of cases from the capillary bed (Figure 2H). This heterogeneity in the sites of origin of the multicompartmental dilations did not depend on whether the mouse moved or was at rest, or the dilation was accompanied by a large astrocytic Ca2+ event or not. The site of origin did not influence the characteristics (volume and duration) of the dilations in each vascular compartment. In contrast, these were modulated by both the mouse' behavioral state and the occurrence of large Ca2+ activity in astrocyte end-feet (Sup. figure S4). On the other hand, the fact that we observed both monocompartmental dilations stopping at the sphincter and multicompartmental ones crossing it in the resting mouse (Figure 2I), indicated to us that the pre-capillary sphincter is a site of control of the dilations’ spread. Such control was overcome during animal movements or arousal states, notably when these conditions were associated with large astrocytic Ca 2+ activity, leading to the recruitment of both the upstream and the downstream vascular compartments, independent of the initiation site and the direction of the dilation. Based on these findings, we focused our next investigations on the astrocytic activity around the sphincter. Local Ca 2+ activity in astrocytic end-feet at the sphincter precedes the arrival of multicompartment dilations. We considered that, when evaluating the role of astrocytes in the NVC response to auditory activation, the large astrocytic Ca2+ activities described above are likely confounding factors. According to recent data, they would be involved in associating brain states 43 or in integrating past events 44 , functions that are not directly relevant to NVC initiation. Therefore, we decided to focus our next investigations on dilations and astrocyte Ca2+ elevations that are not associated to mouse movements/arousals, i.e. that occur during the animals' resting periods (Figure 3). We hypothesized that astrocytes could exert their NVC regulation at the junction level and focused our attention on the astrocyte Ca2+ activity related to multicompartmental dilations spreading across the sphincter. By looking at time-averaged 3D images, we found heterogeneous local Ca2+ activities in the astrocyte end-feet and processes around the junction, which did not spread widely as for the large Ca2+ activities shown in Figure 2C. Following this initial observation, we decided to develop an automated analysis of the astrocytic end-feet Ca2+ activity in relation to the onset of spontaneous dilations. To establish the spatial-temporal sequence of these local astrocyte Ca2+ events with respect to the dilation events in an unbiased and accurate way, we aligned the 3D astrocyte Ca2+ imaging data to the onset of all the spontaneous dilations. We then identified voxels of interest (VOIs) with increased Ca 2+ activity in the astrocytic end-feet (Methods and Sup. figure S5) and evaluated the probability that a Ca 2+ increase occurred within these VOIs prior to a given dilation pattern (Sup. figure S5). Thereby, we identified VOIs that consistently responded in a defined temporal relation with a dilation (Figure 3A, Sup. figure S5), i.e., we identified the position and timing of pre-dilation Ca 2+ activities in the astrocytic end-feet. Despite a certain variability in the VOIs positions and in the time-course of the related Ca2+ activities (Figure 3A and Videos V3), pre-dilation Ca2+ responses were consistently found primarily in the end-feet covering the sphincter (Figure 3B) and occurred at increased frequency when dilations were multicompartmental and spread across the sphincter (Sup. figure S6A and Video V3). These Ca2+ responses did not depend on a specific origin and direction of the multicompartmental dilations (Figure 3B). Localized Ca2+ activity was also present in end-feet along the capillaries (Figure 3A and B). In contrast, we rarely saw pre-dilation Ca2+ activity in the end-feet on the PAs (Figure 3B). Regardless of their position, all the pre-dilation Ca 2+ signals were local (9.7 ±3.8 µm z-axis) and short-lasting (0.98 ± 0.42 s) (Figure 3C). The interval by which pre-dilation astrocyte Ca2+ elevations at the sphincter preceded onset of dilations was longer when dilations arrived from upstream or downstream locations than when dilations started at the sphincter itself (Figure 3D and E). However, this longer anticipation might just depend on the fact that dilations coming from PA or capillaries initiated outside the FOV, i.e., earlier than observed. This been said, we observed that when a dilation that started at the capillary bed level and arrived at the sphincter was associated with Ca2+ events in both compartments, the astrocyte Ca2+ activity occurred first at the sphincter and then at the capillary (Figure 3D and F). This sequence did not visibly depend on spreading of the Ca2+ from the sphincter along the capillary (sup. Figure S6B). Rather, the two groups of end-foot astrocyte Ca2+ events appeared to be temporally separated and independent (Figure 3A, D-F and Sup. figure S6C), suggesting that Ca2+ activity at the sphincter is the primary pre-dilation event. Overall, the pre-dilation astrocyte Ca2+ activity at the sphincter showed anatomical and temporal characteristics in line with a possible role in controlling spreading of dilations across the sphincter. The astrocytic pre-dilation Ca 2+ activity in end-feet at the sphincter is reduced in IP3R2KO mice. We then determined if the local pre-dilation astrocytic Ca 2+ activity was IP3R2-dependent by repeating experiments in IP3R2KO mice that display reduced astrocyte end-foot Ca2+ activity in the in vivo awake condition 45 . Indeed, in this group of mice, we observed a significant reduction in the number of local pre-dilation Ca2+ signals in astrocyte end-feet compared to wild-type mice. This reduction was seen in particular at the sphincter (Figure 4A-C, Sup. figure S7), while it was not significant at the PA and capillary (Sup. figure S7A and B). The reduction was most evident in association with multicompartmental dilations, independent of whether they arrived from the PA or the capillary (Sup. figure S7C), it involved particularly the earliest pre-dilation Ca 2+ activity, namely the activity starting >1 s before dilation onset (Figure 4D; Sup. figure S7D) and occurring closest to the sphincter (Figure 4E; Sup. figure S7D). Overall, these results support the presence of an early, pre-dilation, IP3R2-dependent, Ca2+ increase in astrocytic end-feet, notably in the end-feet surrounding the pre-capillary sphincter, prior to the spread of dilations beyond the branch point. Spread of dilations across the sphincter is impeded in IP3R2KO mice. We next assessed whether the reduced pre-dilation astrocyte Ca2+ activity at the sphincter in IP3R2KO mice had an impact on the vascular dynamics around the sphincter. In WT mice, we had observed that the pre-dilation Ca2+ activity was associated in particular to multicompartmental dilations, independent if they arrived from the PA or the capillary bed. Supporting a role for the astrocyte activity in controlling those dilation events specifically, we counted a decreased total number of multicompartmental dilations (-7%) and a proportionally increased number of single compartment dilations (+10%) in IP3R2KO compared to WT mice (Sup. figure S8A). In addition, we found that the proportion of multicompartmental dilations arriving at the junction from outside the 3D-FOV (either from the PA or the capillary compartment) was reduced in IP3R2KO mice, while the proportion of those initiated within the 3D-FOV (at either the sphincter or bulb) was increased (Figure 5A). Notably, it was the number of monocompartment dilations arriving from the capillary branch which increased (Sup. figure S8A), suggesting that less of them could spread across the sphincter to the PA in IP3R2KO mice (Figure 5B). We then discovered a second significant effect of IP3R2KO when assessing the speed by which the residual multicompartmental dilations spread from one compartment to the next one (Figure 5C). In WT mice, dilations spread from PA to capillary, on average, in 488 ± 18 ms, and, in the opposite direction, from capillary to PA, in 667 ± 17 ms (Figure 5C). Thus, the speed of compartmental transition was significantly higher when a dilation progressed from PA to capillary (608 ± 57 µm/s) than in the opposite direction (112 ± 5 µm/s, Sup. figure S8B). In IP3R2KO mice, dilations arriving from the PA took on average 260 ms longer in engaging the capillary than in WT mice. Likewise, dilations arriving from the capillaries, were delayed by 140 ms in crossing the sphincter and reaching the PA (Figure 5C, Sup. figure S8B). In the case of dilations initiated at the pre-capillary sphincter, spreading to the capillary was also delayed (by 110 ms) in IP3R2KO compared to WT mice (Figure 5C, Sup. figure S8B). In all cases, the slower spread of dilations was not due to local structural changes occurring in IP3R2KO mice, such as an increased distance separating PA and capillary structures compared to WT mice (Sup. figure S8C). These disturbances in the vascular dynamics of IP3R2KO mice had not been described before. In fact, several publications reported no changes in blood flow regulation in IP3R2KO with respect to WT mice 16-18 . Interestingly, if we performed our analysis without taking into account intercompartmental spread and directionality of the observed dilations, but simply adding all the dilations together, we also obtained that average frequency (Sup. figure S9A) was not modified in IP3R2KO compared to WT mice. While all the above measures were done in the resting mouse, we observed additional effects produced by IP3R2KO during spontaneous movements of the animals. During movements we counted fewer dilation events (Sup. figure S9A) as well as a reduced intercompartmental spread of the dilations compared to WT mice (Sup. figure S9B-C). Thus, the astrocyte Ca2+ regulation of vascular dynamics appears to operate both during resting periods and movements of the mice. Overall, in IP3R2KO mice, both the pre-dilation Ca 2+ activity in astrocytic end-feet surrounding the sphincter and the speed and efficiency of spread of the dilations across the sphincter were reduced. The latter phenomenon is likely responsible for the failed recruitment of additional vessel compartments and the reduced number of multicompartmental dilations, with a qualitative alteration in blood redistribution. Auditory stimulations induce dilations and local astrocytic Ca2+ activity at the sphincter resembling those occurring naturally in resting mice. Our investigations so far focused on naturally-occurring dilations at the pre-capillary sphincter in the resting animal, excluding contributions by movement/arousal-induced mechanisms. However, these investigations did not directly demonstrate that the observed vascular responses belong to NVC, nor excluded that they depend on factors generated by sound perception other than the local neuronal activation 46 . Thus, next we induced vascular responses via direct auditory stimulation and compared their properties to those of the “natural” dilations. The auditory cortex is formed by zones that gradually change sensitivity to different pitches 47 . Therefore, we tested several tones and used intrinsic optical signaling (IOS) detection to determine the tonalities responsible for stimulating the cortical region exposed in our craniotomy. With IOS, we could also establish rough tonality maps and identify where, in a given cortical area, tone frequencies activated maximum blood flow increase (Figure 6A). To relate tone stimulations (10 sec) and associated IOS-blood flow responses (IOS maps) to local dilations at PA-capillary junctions (Figure 6A-B), we used combinations of three tones as stimulus, predicting that they would trigger a response in at least one of the cortical regions present in our cranial window. Initially, we investigated the correlation between wide-field IOS maps and individual vessel regulations at a locus in the auditory cortex containing four PA-capillary junctions. We sampled in parallel the individual vessel responses at the four branch points (one in layer I and three in layer II/III) present on three PAs entering the tissue at different cortical locations. The corresponding dilations differed in origin and directionality, but all had duration in line with the strength of the IOS signal detected in the same area: longer-lasting dilations were associated with tones producing strong local IOS responses, and shorter-lasting ones with tones producing weak IOS responses (Figure 6B). Noteworthy, the layer I junction showed less tone specificity than the layer II/III junctions, possibly because the flow pattern in superficial layers is less restrictive than in the deeper layers downstream. We found fewer junctions in the loci that we selected for the next observations. To incorporate all of them in our study, we tried a classification based on the individual IOS maps, i.e., we tried to link the observed dilation pattern at a given PA-capillary junction to the strength of the related tone-evoked IOS response. In particular, we considered the position in which the PA entered in the cortex with respect to the IOS map, i.e., whether its path and the related PA-capillary junction were at the center of the IOS map or displaced from it (Figure 6C). We found that most of the tones induced IOS responses somewhere in our cranial window, but not all these responses were centered on the region where a PA entered the cortex. When a tone induced a strong IOS response in the area of a junction (Figure 6C left), most of the dilations were long-lasting and quickly progressed across the pre-capillary sphincter to become multicompartmental, independent if they began at the capillary or the PA. In cases when the junction area was not at the center of the tone-evoked IOS response, we could still observe dilations in our 3D-FOV, but they mainly arrived from the capillaries (rarely from the PA) or originated at the sphincter (Figure 6C right). Tone-induced dilations varied largely in duration, ranging from >60 seconds to <1 sec (Sup. figure S10A). If the junction area was located at the center of the IOS response, dilations were at least 5 s-long and progressed beyond the sphincter to an extent proportional to their duration. The shortest ones were primarily restricted to the capillary bed, while the longest ones always propagated to all compartments (Sup. figure S10B), resembling the “natural” dilations in the resting animal (Figure 2). Also, the proportion of tone-evoked dilations that started at each location (respectively upstream of the sphincter, downstream, or at the sphincter) was analogous to that of the “natural” dilations (Sup. figure S10C). These observations strengthen the case that naturally occurring dilations in the resting animal directly reflect the auditory experience and represent the associated NVC response. As a next step, we investigated whether tone-evoked dilations, like “natural” dilations, were preceded by local astrocyte end-foot Ca 2+ activity. For this analysis, we used the same unbiased automated approach used for extracting the Ca2+ dynamics preceding “natural” dilations (Figure 3). In this case, our inclusion criterion was less permissive due to the much lower number of tone evoked dilations compared to the “natural” ones (Sup. figure S5, Methods). Nonetheless, we could consistently identify Ca2+ signals occurring mainly at the pre-capillary sphincter, preceding multicompartmental dilations (Figure 6D, Sup. figure S10D and S10E). The tone-evoked astrocyte Ca2+ responses and the blood vessel dilations are affected in IP3R2KO mice. To directly address the impact of the local astrocyte Ca2+ activity on the progression of tone-evoked dilations, we repeated the experiment in IP3R2KO mice. In WT mice, the large majority (79%) of the dilations arriving from either the PA or the capillary compartments were preceded by an astrocytic Ca2+ activity at the sphincter. This proportion decreased to 48% in IP3R2KO mice and was reflected in fewer pre-dilation events (Figure 6E). Similar to what was observed for the spontaneous dilations, in IP3R2KO mice the fraction of tone-evoked dilations that could not cross the sphincter and remained confined to the capillary bed was more than twice that in WT mice (28.7 ± 10.8% vs. 13.2 ± 7.4% in WT, Figure 6F). In complement, we observed that the progression of tone-evoked dilations across the sphincter in IP3R2KO mice was slowed down. Dilations arriving from the PA were delayed by 416 ± 145ms in reaching the capillary. Dilations from the capillary bed were also delayed by 376 ±208ms, mostly in recruiting the sphincter (Figure 6G). We conclude that IP3R2 deletion has a negative impact on tone-evoked dilations similar to the one seen in naturally occurring dilations. These data indicate that astrocytes, by their local Ca2+ activity at the sphincter, act as physiological regulators of the spreading of dilations between vascular compartments as part of the NVC response to auditory stimulation. Discussion In this study, we identified a new role for astrocytes in NVC as regulators of the spread of dilations between the PA and capillary compartments in response to sound perception in the auditory cortex. The astrocyte control is exerted focally, via end-foot Ca2 + elevations occurring at the pre-capillary sphincters that connect the two compartments. Such local astrocyte activity precedes the arrival of dilations at the sphincters and controls their contractility, determining how much and how far blood is distributed during cortical activation. We obtained this information in the awake mouse thanks to fast volumetric two-photon imaging of vascular and astrocyte Ca2 + dynamics in large FOVs comprising the PA and capillary compartments and the connecting junction. By centering our 3D-FOVs on the vascular tree and the associated astrocytic end-feet and peri-vascular regions, we could capture even the most transient local Ca2 + activities and their interplay with the vascular dilations throughout the imaged vascular tracts. Only a minority of preceding studies were performed in the awake mouse, and none of them used fast volumetric imaging. This new approach enabled us to study all the natural dilation patterns occurring in a behaving mouse, and also to establish their origin, directionality, and level of progression between imaged compartments, revealing a previously unappreciated complexity. Thanks to this methodological advance, we could, on the one hand, overcome several of the shortcomings that affected previous work and alimented multi-year controversies on the role of astrocytes in NVC (see Introduction), and on the other, revisit the few recent studies performed in the awake mouse 15 , 17 , 22 , 31 , 48 , complementing their findings and providing a more comprehensive picture. Among others, we could here dissociate the components of the natural astrocyte and vascular responses in the auditory cortex evoked by sound perception from those depending on the activity state of the mouse during locomotion and arousal. Given the large analogy between natural responses seen in the resting animals and responses evoked by natural tone stimulations, we conclude that the natural responses at rest are directly induced by the sensory experience and most likely represent the physiological NVC response to auditory activation. The dilation patterns observed in our study differ significantly from those reported in previous work in anesthetized mice, characterized by quite stereotyped hemodynamic responses 7 , 49 and less correlation with the neural activity 50 . Here, in our naturally behaving mice, the dilation patterns were highly heterogeneous and incorporated contextual contributions from the animal’s brain state, or inputs from other cortical areas, such as the motor cortex. During our recordings, mice moved and underwent arousals, and these activities were associated with large astrocytic Ca2 + events not seen in anesthetized mice 32 . Such large astrocytic Ca2 + elevations were reminiscent of those recently shown to prolong the duration of PA dilations during sustained whisker stimulation in the barrel cortex of awake mice 15 . Since during arousals, more dilations spread across the pre-capillary sphincters than in the resting mouse, the associated bursts of astrocytic Ca2 + activity might function to ensure an abundance of blood flow to the tissue in the aroused state, keeping the neurons metabolically prepared to handle additional auditory stimulations. An important advantage of our volumetric imaging approach was in its ability to follow the spatial and temporal dynamics of dilations more comprehensively than in past studies. Thereby, we could define sites of origin and direction of the dilations as they progressed along the different vascular compartments. This approach was instrumental to our discovery of the central regulatory role of the pre-capillary sphincters, which was difficult to appreciate in previous work that imaged dilations at either the capillary or the PA level separately. Likewise, fast volumetric imaging was necessary to identify the presence of the local astrocyte Ca2 + activity that precedes the passage of dilations from the sphincters. The spatial dynamics of dilations were investigated in previous work, but without reaching firm conclusions about their origin. In those studies, mostly performed in anesthetized mice, dilations had to be evoked by artificial stimulations. Some authors reported that they initiated at lower cortical levels 51 triggered by activity along the capillaries 5 , 19 , 49 , 52 , whereas others that they initiated at upper levels, with the recruitment of the PA from the surface arteries 53 , 54 . This diversity might reflect the different methods used for evoking the dilatory responses in other studies 21 and the fact that a blood flow increase can be initiated by stimulation at any cortical lamina 55 . In our study focusing on the PA-capillary junctions in awake mice, we observed a natural variety in the sites of origin of the dilations. Thanks to simultaneous fast imaging of all the relevant compartments, we could capture the specific directionality of these natural dilations, finding that some arrived from capillaries, other from PA, and others initiated at the junction level. Interestingly, a similar variety of originations was recently described in the barrel cortex of awake mice 21 , 56 . This is not surprising because the two regions are similarly organized for blood flow regulation, with a large number of branching vessels 57 , relays in the neuronal circuits, and projections from other cortical areas 58 . In brain regions with simpler organization, the observed blood flow responses are more stereotyped 19 , 59 . The different positions in the auditory cortex at which the naturally occurring dilations originated must depend on differences in the modes by which blood flow regulation is recruited, which, in turn, may depend on the diversity of the neuronal circuit relays that project the sensory stimuli to the processing cortical region 60 – 62 . In our analysis of tone-evoked dilations, we propose a classification based on the positioning of the dilations relative to the IOS response, which we considered to represent the core of the neuronal excitation. When the IOS response coincided with the area supplied by the PA, the region of neuronal excitation triggering the dilation most probably was along the PA inflow tract. In contrast, when the IOS response was in the vicinity but not exactly at the PA, the neuronal excitation likely occurred further down in the vascular tree and triggered the dilation around the capillary compartment (Fig. 7 a). We believe that this classification is roughly reliable, although it may not fully grasp the complexity of the phenomena giving rise to the dilation patterns that we observed. Additional factors may need to be considered. For example, the fact that the tonotopy has a more articulated nature than the IOS map, given its heterogeneity at the cellular level 63 , 64 with a diversity of neuronal activation patterns capable of triggering the NVC response. In addition, the fact that the sounds that induce the natural vascular responses are more complex than the three tones that we used here to evoke them, and complex sounds are known to often trigger tonotopic neuron responses in separate fields of the auditory cortex in parallel 65 . In addition to describing the varied origin of natural dilations in awake mice, our study describes, for the first time, the central role of the pre-capillary sphincters in determining by which extent these natural dilations spread between PA and capillary compartments under local control by astrocyte Ca2 + signaling. Pre-capillary sphincters have been well characterized anatomically only in recent years and only in the somatosensory cortex 6 . They have been described as a hemodynamic structural division between capillary and arterial blood flow, acting as a bottleneck opposing high resistance to blood flow 9 . In view of their strategic interposition between PA and capillaries, sphincters in the upper cortical layers likely function to protect capillaries from high arterial pressures under baseline conditions. However, sphincters express α-smooth muscle actin in their mural cells, suggesting that they do not provide only passive flow resistance but can also actively contract. Indeed, previous experiments in anesthetized mice have shown that sphincters respond with significant changes in diameter to whisker stimulation 6 or vasoactive agent infusion 8 and thus could be involved in blood flow regulation during endogenous functional stimulation. The sphincters investigated here were not anatomically homogenous, for instance they differed in their level of indentation. However, contractile mural cells completely cover this part of the vascular inflow tract, thus all sphincters must be contractile 6 , 37 and their morphological differences may reflect mainly the extent of force in that contractility. Here, we provide the first direct evidence that pre-capillary sphincters play a physiological role during the auditory NVC response. Due to their strategic placement at the PA’s branching points, sphincters can control blood supply to larger parenchymal domains than downstream contractile capillary pericytes. Thus, a single PA connects to various capillaries that, in turn, branch out and supply blood flow to distinct sensory areas 53 , 66 . Flow regulation at the PA’s branching points could optimize downstream blood delivery in register with neural activity 67 , 68 and be the base of the laminar activation pattern observed in human fMRI brain imaging 69 . On the other hand, spread of dilations in the opposite direction, from capillaries to PA 19 , 70 , can be equally important for pairing the increased local demands to global activation and contribute to the accurate and sufficient blood distribution essential for optimal brain function and health 5 , 36 . This upstream-directed regulation was reported to depend on endothelial K + signaling 20 triggered by TRPA1 or KIR channels stimulation directly on 1st and higher-order capillaries 71 , supported by another level of upstream-directed regulation at higher-order capillary branch points 72 . While all the above observations align with our current results, they have provided only a fragmentary picture of the NVC-related dilation dynamics. Thanks to our 3D imaging approach and the large number of natural dilations that we recorded, we have been able to put pieces together and can propose the following interpretation of the observed dilation patterns: dilations arriving from the PA compartment may or may not involve the capillaries. The main factor determining their spread beyond the pre-capillary sphincter with increased blood delivery to the downstream capillary bed is most likely the position at which the neuronal activation driving the NVC response occurs (Fig. 7 b). If the activation localizes primarily to cortical layers deeper than the layer II/III junction, the dilation will generally involve just the PA. However, if the activation also concerns the upper cortical lamina, the downstream capillary region will receive additional local blood flow via expansion of the sphincter and 1st order capillary. Likewise, a local neuronal activation can trigger dilations that initiate at the 1st order capillary 5 , 19 , which will only involve the capillary branches. However, if the activation is part of a larger neuronal response, it will recruit additional blood from the surface vessels by spreading across the sphincter to increase delivery via the PA (Fig. 7 b). Our study also describes for the first time the role exerted by astrocytes via local Ca2 + signaling in controlling the spread of dilations at the pre-capillary sphincters. The existence of the astrocyte control is supported by the observation that IP3R2KO mice, which lack part of the astrocyte Ca 2+ signaling, display an altered NVC response, specifically a decreased capacity of dilations to engage the vascular compartments across the sphincters with reduced blood redistribution. 325671 Several past studies rejected a contribution of astrocyte signaling to NVC 16 – 18 because they failed to observe any changes in the blood flow responses in IP3R2KO mice. This may not be very surprising for the early studies in view of their experimental pitfalls, including having been performed in anesthetized animals. However, similar negative conclusions were drawn also in a recent study 17 that was performed in conditions similar to ours’, i.e., in awake mice and considering multiple compartments, from PA to 4th order capillaries. We do not contest the observations by Del Franco et al., as we could reproduce, using their analytical conditions, a lack of changes in some parameters of the dilation patterns in IP3R2KO mice (Sup. figure S9A). Instead, we highlight the important differences in the way experiments were conducted and data analyzed in their study compared to ours’ and which likely explain the discrepant conclusions. First, Del Franco et al., did not image all the vessel compartments at the junction simultaneously; second, they did not consider the pre-capillary sphincter region in their analysis, nor the site of origin of the dilations; third, they evoked dilations by stimulations that elicited widespread Ca2 + elevations in astrocytes not directly comparable to the local endogenous Ca2 + elevations that we observed just before the naturally occurring dilations, and, finally, they did not image the dilations along the z-axis. We could identify vascular abnormalities in IP3R2KO mice because we used a fast 3D imaging approach and focused on an aspect never investigated before, the dynamic progression of dilations across the sphincter. This led us to a twofold discovery: first, the recognition of the pre-capillary sphincter as a locus of physiological blood flow regulation, and second, the identification of a local astrocyte IP3R2-dependent Ca2 + activity controlling the sphincter’s contractility. One of the problematic aspects in studying the functional roles of the astrocyte Ca 2+ activity is its high level of complexity, involving a panoply of signals whose interpretation often remains enigmatic 32 , 73 . Such signals go from highly visible, spatially large, and temporally long Ca2 + elevations, to more elusive, local, and fast Ca2 + transients. Considering that the Ca2 + signals relevant to the control of synaptic activity were identified as part of the local transients occurring at the level of peri-synaptic astrocytic processes 32 , 74 – 76 , we expected that vascular-relevant signals would be mostly seen at astrocytic end-feet. Therefore, we focused attention on the Ca2 + events visible in the perivascular compartment, themselves quite heterogeneous 15 , 77 , 78 . Aiming at identifying among them potential dilation-regulatory events, we recorded and analyzed the perivascular astrocyte Ca2 + dynamics having in mind how the extra blood flow was recruited in our experiments. Thus, we focused on the naturally-occurring vascular changes and 3D-imaged the peri-vascular regions around the pre-capillary sphincters that regulate blood flow to a specific cortical lamina. Since this approach had not been attempted before, we did not apply strict analytical criteria for defining the relevant astrocyte Ca2 + activity, rather defined a number of features that we expected such activity to have, like: (a) occurring in the end-feet within short distance from the vessel surface; (b) being time correlated to the dilations, i.e. shortly preceding them; and (c) appearing in association with all the dilations having a given pattern, i.e. originating at a given site and being able to cross the sphincter. As discussed, distinct dilation patterns reflect distinct types and loci of neuronal activation, suggesting that astrocytic Ca2 + activities related to distinct dilation patterns should also be diverse. Indeed, we observed pre-dilation end-foot astrocyte Ca2 + responses with different onset times and positions along the PA-capillary junction. Nonetheless, our “loose” analytical approach was strong enough to identify the NVC-relevant astrocyte Ca2 + activity, particularly because we sampled only physiological vascular events induced by auditory perception, either occurring naturally in the resting mice, or evoked by natural sounds. Our observation that the frequency of local astrocytic Ca2 + transients as well as the frequency of dilations progressing across the sphincter were both reduced in IP3R2KO mice, provide strong support to the relevance of the identified astrocytic Ca2 + activity to the NVC auditory response. Importantly, IP3R2KO is known to only partially reduce and not abolish astrocyte end-foot Ca2 + activity in the awake mouse 45 , as we also observed for the pre-dilation Ca2 + events preceding natural (Fig. 4 ) and tone-evoked dilations (Fig. 6 ). Thus, it is possible that a more effective interference capable of abolishing any astrocyte end-foot Ca2 + activity might have revealed a stronger control by the astrocytes. Some key aspects of the vascular regulation identified here remain to be addressed in future investigations. To start, the exact functional implications of the local control of blood distribution at pre-capillary junctions, by which dilations are enabled to expand their parenchymal territories of blood supply bidirectionally, as well as the extent of the functional consequences that alterations targeting this physiological mechanism can produce on brain function in pathological conditions. In this context, the fact that during several CNS diseases, astrocytes undergo morphological changes that alter or impede the contact between end-feet and blood vessels surface 79 , implies noxious consequences for the regulatory functions here identified, that could likely contribute to the disturbed blood flow seen in such pathologies 1 , notably in those accompanied by cognitive decline 80 . Another aspect to be clarified in the future is the additional impact of the large peri-vascular astrocytic Ca2 + activities that we saw invading several end-feet during movements or arousal states of the mice. Considering the astrocyte Ca2 + control at the pre-capillary junction in the resting mouse, it is intriguing that when we saw large Ca2 + events during mouse movements, we also observed an increase in multicompartment dilations (Fig. 2 F). It remains to be defined how exactly these astrocyte phenomena influence NVC during motion and/or changes in brain states involving activation of secondary pathways and/or neuromodulator effects that overlap with the primary auditory excitation. 81 , 82 . Understanding these relations will lead to a more comprehensive understanding of how astrocytes contribute to keeping healthy and adequate levels of substrate and oxygen in the brain of behaving mice, and how these levels could be altered under pathological brain conditions. In this context, deciphering the mechanism(s) triggering the relevant Ca2 + activity in astrocyte end-feet and the subsequent changes in sphincter responsivity would be important. Are the local astrocyte Ca2 + elevations secondary to neuronal activation and synaptic transmission 32 , 74 ? How are they related to larger Ca2 + elevations triggered by specific brain states and changes in noradrenaline levels 39 , 43 ? Several mechanisms have been described by which astrocytes can sense synaptic activity locally and generate a Ca2 + increase response 83 . To start, via activation of Gq-GPCRs that signal via IP3 like mGluR5 or P2Y1R 26 , 75 , 84 and whose effects could be attenuated in IP3R2KO. However, also ionotropic receptors like P2X1 and P2X5 can produce direct or indirect intracellular Ca2 + influx in astrocytes 11 , 14 . Other mechanisms capable of producing astrocyte Ca2 + elevations independent of IP3R2 receptors could involve plasma membrane Ca2 + influx channels like TRPA1 85 or TRPV4 86 , or the activity of Na+/Ca2 + exchangers following neurotransmitter uptake 87 . Activation of TRPA1 channels in endothelial cells 71 could also initiate a local Ca2 + response in astrocytes by triggering K + release onto their end-feet. While our study leaves these important questions open, we believe that its identification of the peculiar control exerted by astrocytes at the sphincter, a critical point of blood flow regulation in the vascular tree, significantly advances understanding of the astrocytic contribution to NVC. Materials and Methods Animals Most experiments in this study utilized mice induced to genetically express the Ca2+ sensitive indicator GCaMP6f selectively in the cytosol of astrocytes based on the GFAP promotor. This was obtained by crossing two transgenic mouse lines, a lox-STOP-lox-cytosolic-GCaMP6f (purchased from The Jackson Laboratory http://www.jax.org, JAX 024105 ( B6;-Gt(ROSA)26Sortm95.1(CAG-GCaMP6f)Hze/J ), and hGFAPCreERT2 mice 88 obtained from Prof. F. Kirchhoff, University of Saarland, Germany, as previously described 32 . A subgroup of these GFAPCreERT2xGCAMP6f mice was then crossed with the Itpr2KO mouse strain 89 that carries a constitutive deletion of IP3R2 expressed in the ER and displays conspicuous reduction in astrocytic Ca 2+ activity 16 . We obtained this strain from the laboratory of prof. Ju Chen of University of California San Diego, La Jolla, California, US. We used only IP3R2 ko/ko homozygotes in this study. Though IP3R2 contributes importantly to astrocytic Ca 2+ activity, especially in Ca 2+ mediated Ca2+ release from the ER in soma and large processes, not all the astrocyte Ca 2+ activities depend on this receptor, and only the largest are abolished in the knockout 90 . The mice were treated with Tamoxifen at 8 weeks of age to induce Cre recombination and trigger GCaMP6f expression in astrocytes. Tamoxifen (Sigma-Aldrich) was dissolved in corn oil (10mg/ml) and administered i.p. (0.1 ml/10 g body weight/day) for 5 days prior to the chronic cranial window surgery. The mice had surgery when they were 8 weeks old and, after a 4-week period of recovery and training, were included in the experiments between 12-28 weeks of age. Two 12-16 weeks-old GFAP-EGFP mice 38 were used for morphological investigations. All experiments and procedures were conducted under license and according to regulations of the Cantonal Veterinary Offices of Vaud (Switzerland). Chronic cranial window preparation Chronic cranial windows were established as previously described 32 . To target the auditory cortex, the window was centered around -3mm anterior-posterior (AP) and 5 mm mediolateral (ML) from bregma, right hemisphere. The surgical procedure was done under isoflurane 1.5% anesthesia supplemented with a Carprofen injection (5 mg/kg, s.c.) and local anesthesia in the form of Lidocaine (0.2 %, s.c.) under the scalp. Animals were kept warm on a temperature-controlled heat blanket during the procedure, and their eyes were protected against dehydration with viscotears. Hair was removed, and Betadine used to sterilize the skin prior to the first incision. A square hole 3x3 mm was drilled in the bone and, leaving the dura intact, was covered with a fitting glass coverslip (thickness #1), which was then glued to the cranium. A L-shaped metal plate used to head-restrain the mice during the imaging was fixed to the skull by glue and dental cement. Analgesics were given for three days after surgery (Paracetamol 125 mg dissolved in 250 ml water). Electromyography (EMG) We recorded animal movements by electromyography (EMG) 91,92 . To achieve this, two electrodes were implanted within the neck muscles of the mouse after the chronic cranial window preparation and connected to two micro sockets. During in vivo imaging, the sockets were connected to a preamplifier and a Molecular Devices digitizer (Digidata 1440A). The signal was recorded using pClamp and then filtered (50 Hz filter) using a custom-made Matlab script. The onset of each recording was triggered by a digital signal from the Bruker microscope upon initiation of each two-photon imaging sequence (see “ 3D awake imaging” section). The recorded EMG signal was first filtered using a 50 Hz notch filter. Then the filtered recording was normalized to standard deviation values, using the mean and SD values from the entire 90-120 seconds acquisition period. Timepoints in which the normalized signal reached >5SD were identified, grouped together and considered part of the same movement event if they were <500 ms apart. Fluorophores To visualize the lumen of the blood vessels, just prior to the imaging session we introduced in the circulation by tail-vein injection a dextran conjugate with Texas red 70.000 MW (2 % in 0.9 % NaCl sterile solution, 100 µl bolus I.V.). The vascular dye was of a size that impedes extrusion to the tissue surrounding the vessel. Hence, it was ideally suited to highlight the lumen of the blood vessels and signal diameter changes 14,93 . 3D Awake Imaging Before imaging, the mouse was habituated to human handling for two days and gradually introduced to being head-restraint under the microscope and then thoroughly trained for an additional five days. The head of the animal was restrained by a custom-made system in which a metal plate attached to the head mount of the mouse was fastened with a screw to a matching metal bar. During imaging, the mouse sat on an air-supported, freely floating ball that acted as a spherical treadmill, allowing the animal to run when it wanted. The head of the mouse was slightly tilted, and the objective was at a 40° angle from the vertical position to allow perpendicular imaging through the glass covering the auditory cortex. Two-photon imaging was done with a Bruker in vivo Investigator system (Bruker Nano Surfaces Division, Madison, WI, USA) equipped with an 8kHz resonant galvanometer scanner, coupled to a MaiTai eHP DS laser (Spectra-physics, Milpitas, CA, USA) with 70 fs pulse duration, tuned to 920 nm. Negative dispersion was optimized for each wavelength, and laser power was rapidly modulated by Pockels cells. A 20x LUMPFL60X W/IR-2 NA 0.9 Olympus objective was used. Emission from red and green channels was separated by a dichroic beam splitter (565 nm Long pass) allowing shorter wavelengths to reach a 520/540m band pass filter before a GaAsP detector (520 nm, optimum for green emission) and longer wavelengths to reach a 610/675 nm band pass filter before another GaAsP detector (610 nm, optimum for red emission). The combination of a resonant scanner with a piezoelectric actuator made high-speed 3D imaging possible, while the highly sensitive GaAsP detectors with negative dispersion allowed applying minimal laser dose to the tissue. The laser power varied during experiments depending on the depth of the focus, but it was kept below 7 mW and measured continuously with a power meter. These imaging settings were previously thoroughly tested to ensure their non-toxicity to the tissue, even at high-speed image acquisitions 32 . The anatomical localization of PA and 1 st order branch points defined the depths at which the imaging was performed. All the junctional sites except one were located ≥80 µm below the brain surface, with a maximal depth of 328 µm. Two different volumetric imaging approaches were applied: in one, imaging was performed in small volumes with stacks centered around the PA-capillary junction, and in the other, in larger volumes with stacks following the PA up and down the upper cortical layers. More specifically, in the first approach, the 3D-FOV covered an imaging volume of 56-75 µm x 15-44 µm x 21-35 µm (x,y,z), the acquisition speed was 200 Hz and the optical-zoom 8x, with pixel size of 0.29-0.59 µm lateral resolution and 1 µm axial resolution. In the second approach, the 3D-FOV covered an imaging volume of 75-121 µm x 75-112 µm x 190-280 µm (x,y,z), the acquisition speed was 200 Hz and the optical-zoom 8x, with pixel size of 0.29-0.94 µm lateral resolution and 10 µm (and in one case 20 µm) axial resolution. The scanning rate per 3D stack was 8-10 Hz. A maximum of 20 imaging sequences, 90-120 seconds-long (800-1000 stacks in total), were taken with 1-5 min breaks between them, depending on the mouse behavior. Auditory stimulations In this set of experiments, mice were subjected to a single sound stimulation during each of the imaging sequences. Sounds were produced by an Electrostatic loudspeaker (Tucker-Davis Technologies, Inc.) placed 30cm from the left ear of the mouse, connected to the pClamp digitizer. The latter was programmed to produce a single 10 s-long, 1 Hz auditory stimulation consisting of 10 repetitions of an individual tone, each one lasting 500ms, that was started 30 s after the initiation of the imaging sequence. Three different tone frequencies were used as stimuli: 3 kHz, 20 kHz, or 30 kHz. Each tone stimulation was repeated during three different imaging sessions. Intrinsic optical signaling (IOS) In the auditory stimulation experiments, before performing two-photon imaging, we used intrinsic optical signaling for obtaining a tonicity map of the auditory cortex. Using the 4× objective of our microscope, we could include the entire cranial window in the field of view. The light source was filtered with a green light excitation filter (532 nm). This wavelength is equally scattered by oxygenated and deoxygenated blood, so the reduction in the emitted light reflects changes in total blood volume 94 . We sampled images at 10 Hz and compared 10 s periods before and during tone stimulation, repeating comparisons for all the tone frequencies. We calculated the difference light scattering before and during stimulations 95 to detect the position of the largest hemodynamic response to each of the different tone stimulations. Mouse pupil imaging In a sub-group of the two-photon imaging experiments, we performed pupil imaging using a high-resolution, fast-speed IR camera centered on the eye of the mouse. The camera was a Dalsa Genie Nano, run with Sapera LT and CamExpert software. Pupil imaging was started by a digital signal from the Bruker microscope upon initiation of each imaging sequence. A bandpass filter (850 nm) was placed before the objective to exclude visible light, only permitting visualization of the mouse pupil by the reflected IR light. This enabled us to follow the pupil contractions and dilations during two-photon imaging and to quantify them using a simple custom-made MATLAB script. Periods involving pupil dilations (expansions) were detected based on the data z-score and defined as pupil enlargements >2SD with respect to the average size of the pupil during the whole imaging period. The timings of pupil expansions were compared to those of astrocytic Ca2+ elevations averaged across the entire FOV, roughly measuring large astrocytic Ca2+ events. Periods in which both measurements reached values >2SD were defined as periods of occurrence of the two phenomena in overlap (Suppl. Fig S2D). 3D imaging data analysis : general aspects. The data obtained via 3D two-photon imaging were analyzed with custom-made MATLAB (Matworks, 2019b version) scripts and visualized using both ImageJ and Imaris 8.2 software (Bitplane). The analysis consisted of several steps. First, with the script “VesSegCreateMasks.m”, each z-level (or focal plane) in the 3D stack was considered as an independent 2D image (Sup. Figure S1A and B). In each of these images, the user manually defined the position of the different vessel compartments and drew a rectangular region around each of them (Sup. Figure S1C). In the “VesSegRoiData.m” script, these selections were then applied to the whole 3D timeseries, i.e., the rectangular regions were automatically used in each focal plane, so that each region contained and roughly outlined a specific vessel compartment (Sup. Figure S1D and S3). Then, the vessel compartments and the related astrocyte end-feet structures were automatically detected within each rectangle, their position found throughout the imaging sequence, and the areas calculated at each of the z-levels. Detection of the vessel structures was done first by normalizing all the pixel intensities in the red channel within the selected rectangular region during the entire imaging sequence and by calculating the z-score of each pixel. Then, the area corresponding to the structure within the rectangular region was masked at each time point using a gaussian filter (imgaussfilt.mat, with the variable sigma decreasing from 8 to 5 with the increase of the imaging depth). The mask’s area was defined by the sum of all the red channel pixels in the rectangle that had significant fluorescence level (>2SD) for all the time points in the timeseries (Sup. Figure S1E). Movements were corrected based on the position of the centromere of the specific mask in the rectangular region under analysis compared to the centromere of the average mask from the entire imaging sequence, and the operation repeated to reposition the rectangular region for each time point. To detect whether a vessel structure was contained within the rectangular region, we applied a size threshold and established that the vessel structure within the rectangular region should cover >8% of the region’s area to be retained for further analysis (bwareaopen.mat). In cases in which movements of the mouse caused exit of the vessel structure from the 3D-FOV at a given time point and in which repositioning of the rectangular region could not recover the structure, the timepoint was deleted from the entire 3D+t stack and the data thus excluded from the study. Such a deletion was sometimes necessary during substantial movements or locomotion of the mice. The GCaMP6f signal in the green channel within the rectangular selection was normalized in the same way as done for the red channel. From this data, end-feet structures were defined for each imaging plane as structures departing from the edges of the vessel structure and occupying the first 2 µm external to such edges, which gave a 3D annulus ROI around the vascular structure (Sup. Figure S1F). These ROIs would then comprise all the Ca2+ activity occurring in astrocytic structures within 2 µm from the vessel structure, regardless of the astrocyte to which the end-foot structure belonged. The ROI’s width of 2 µm was chosen based on previous EM descriptions of the continuity and thickness of the end-foot layer around CNS vessels 96 . In fact, the end-feet thickness in EM is ≤1 µm, but we added a conservative +1 µm margin to ensure its complete inclusion taking into account the scattering of the emitted light during two-photon imaging. This ROI was divided into four quadrants that enabled repositioning the ROI in relation to the vessel position and its constriction/dilation activity along the x and y axes (Sup. Figure S1G-I). Thus, first, the position of the rectangular region was adjusted according to the detected movements, and then the end-feet ROIs were moved in relation to the position of the vessel mask. Detection of dilation events and patterns The following analytical approach was utilized both for the imaging experiments in small volumes at PA-capillary junctions and for those in larger volumes along PAs and both for natural and tone-evoked dilations. At first, the vascular structures within the 3D-FOV were classified as PA, sphincter, bulb, or capillary compartments utilizing the 3D stacks reconstructed from the ensemble of the rectangular regions manually drawn for each 2D image at a given z-level (Sup. Figure S1C and S3B). To note, some of the regions in the stacks were empty, i.e. lacked their structure at specific z-levels, especially in stacks covering large volumes with long steps between z-levels. Next, the spatial-temporal pattern of each dilation event was defined using the custom written “VesSegActivitySearch.m” script. For each vessel compartment, the vessel area at each z-level was normalized to SD values (Sup. Figure S3C), which were calculated taking periods without movements as baseline. By combining the relative changes in area at any z and t data point, we created a 3D image of the vascular dynamics in time and depth (Sup. Figure S3D). This z-t rendering of the dilation in 3D was then normalized (A-mean(A) /std(A)) considering all the time points in the imaging sequence (where A = area). Finally, to define dilation periods that extended three-dimensionally along the vessel structures, a 2SD threshold was used to detect dilations that occurred in several z-levels, prior to a gaussian blurring (imgaussfilt.mat, sigma=2) followed by removal of ambiguous dilation events that were either highly local or very short (bwareaopen.mat, filter: ¼ x z-levels x imaging frequency) (Sup. Figure S3D). Via this procedure, the vascular activity (Sup. Figure S3E) was simplified and binarized into periods of either dilation (1) or no dilation (0). Binarization was performed for each vessel compartment during the whole acquisition period (Sup. Figure S3F). By comparing the information from the different compartments, we could evaluate if the onset of each dilation event occurred within or outside the 3D-FOV. Moreover, we could establish which vessel compartment dilated first and when, as well as the number of other vessel compartments that participated in the dilation and the onset time of the dilation in each compartment relative to the initial one (Sup. Figure S3G). Based on this information, dilations involving more than one compartment were considered a single event when they occurred in temporal continuity in the different compartments, whereas they were considered separate events when they were spaced by >500 ms intervals. In tone stimulation experiments, dilations occurring within the tone stimulation period were considered to be evoked. The delay from stimulation to dilations’ detection in the FOV ranged between 1.9 and 3.3 s depending on the dilations’ direction, in line with the commonly reported delay of evoked NVC responses 37 . We excluded movement-related events as components of such dilations but cannot fully exclude contribution by other factors such as coincident natural sounds. 3D imaging at the PA-capillary junction Detection and analysis of pre-dilation astrocytic 3D Ca2+ activity Astrocytic pre-dilation Ca2+ activity was investigated in small volume 3D imaging stacks centered around the sphincter based on the GCaMP6f green fluorescence signals. Once we had quantified the timing and pattern of vessel dilations based on the Texas red fluorescence in the vessel lumen (see “ Detection of dilation events and patterns” section and Sup. Figure S5A-B), the onset of each dilation event displaying a given pattern was used to identify Ca2+ activity in astrocytic end-feet that had the specific feature of appearing recurrently with a defined temporal relation with the dilation event (Sup. Figure S5C). For this, we used the custom written VesSegPreDilCa.m script. To start, we considered the average astrocytic Ca2+ activity present in each end-foot ROI around a vessel compartment at each z-level of the 3D-FOV stack and normalized it to SD values, defining the baseline mean value and the SD values from the periods without movements (F-meanF baseline )/stdF baseline ). As done for the vessel dilation data, this normalized single-plane information was combined in a z-t matrix, obtaining a 3D+t reconstruction of the average Ca2+ changes in the astrocytic end-feet ROIs shown as changes along the z-axis over time at each x-y imaging level. We then temporally aligned this map of the 3D+t astrocyte Ca2+ changes to the onset time of each dilation, considering specifically a 3-seconds period including the 2 seconds preceding the dilation event and the first second after its start, the latter to avoid abrupt cut of Ca2+ dynamics started in the pre-dilation period but still ongoing at dilation onset (Sup. Figure S5D). The 2-seconds pre-dilation period was chosen based on the expected delay of vascular responses from neuronal activation according to past NVC studies 15,19,49 and our own previous observations 23 . Noteworthy, some of the naturally occurring dilations here investigated started outside the 3D-FOV, so for calculating pre-dilation delays we could rely just on their observable timings, i.e. when they entered our 3D-FOV. The astrocytic Ca2+ activity was then normalized along all the z-levels over the entire 3 s selected period. Within this time frame, all the z-axis locations and timings in which Ca2+ events occurred were identified by using a >2SD threshold for defining a Ca2+ increase, gaussian filtering with a 0.5 sigma gaussian blur (imgaussfilt.mat), and excluding events smaller than 3 µm x 100ms (bwareaopen.mat). The result was a binary mask in z-t defining the temporal and spatial position of each of the astrocytic end-foot Ca2+ activities in relation to each dilation event. We then regrouped all the astrocyte Ca2+ masks that were associated to dilations with the same pattern, i.e. same onset location and same vessel compartments involved. The regrouping of the masks from all the imaging sessions of a given experiment allowed their comparative analysis. We found that the pooled Ca2+ masks contained some overlapping Ca2+ activity, i.e. activity that occurred in all of them at the same time and 3D location. We defined the recurrent regions as the VOIs (voxel of interests) putatively involved in the pre-dilation astrocytic Ca2+ activity. We then came back to each specific map of Ca2+ activity associated with each individual dilation event, overlaid the template VOIs map to it, and checked if the individual map showed dilation-related astrocyte end-foot Ca2+ activity at the z-level and time identified by the VOI (Sup. Figure S5E). The size, timing and duration of these average Ca2+ signals were then used to describe the spatial and temporal extent of the pre-dilation Ca2+ signals (Figure 3C). We considered that a dilation was preceded by this type of pre-dilation Ca2+ activity when the Ca2+ activity overlapped with the average z-t VOI at that specific position in 3D-t. Finally, we investigated how recurrently each putative pre-dilation Ca2+ activity was detected prior to dilations regrouped by the same pattern (Sup. Figure S5F). As inclusion criterion we considered the frequency of occurrence, with a margin defined by the number of dilations belonging to a given pool and using a 0.1 confidence limit (1-(((N/2)-((sqrt(N)/2)x1.282)/N)). Hence, for example, if a pre-dilation Ca2+ signal occurred in 59 % of N=50 dilations of the same type, we considered it to be a recurring event related to that dilation pattern because it was within the margin of 41 %. In the case we had only N=10 dilations, the event would have been considered recurring if it was seen in 70 % of the dilations due to an error margin of 30 % (Sup. Figure S5F). This criterion was adopted to avoid positive bias towards dilation patterns that occurred rarely. This means, however, that our criterion was more restrictive in the experiments on tone-evoked dilations because in those experiments we observed a much lower number of dilation events than in experiments on naturally-occurring dilations. Nonetheless, the number of pre-dilation Ca2+ events that we identified in the tone-evoked dilation experiments was in line with the number in experiments on naturally-occurring dilations (Sup. Figure S10D and S10E). Detection of large astrocytic 3D Ca2+ activity. In small volume 3D-FOVs at the PA-capillary junction we also identified periods with large pre-dilation astrocytic Ca2+ activity during resting periods. Analysis was performed on the z-t projection of the end-foot Ca2+ activity ROIs as for the small Ca2+ activity. This was done because our pre-dilation Ca2+ detection approach, described in the previous section, would not distinguish small from large activities. To tease apart the two types of events, we used the z-t matrix plot and defined “large astrocytic Ca2+ activity” as periods of extensive activity where Ca2+ increased >2SD above the baseline value in non-movement periods and spread through the z-layers to include the entire depth of the z-stack in >75 % of the end-feet ROIs. The dilations that occurred within these periods were then excluded in the following analysis of pre-dilation Ca2+ events. 3D imaging along the PA Detection and analysis of astrocytic Ca2+ activity In experiments in which we scanned large volumes along the PA, the astrocytic end-feet structures were imaged in focal planes 10-20 µm apart along the z-axis (Figure 1C). Thus, the astrocyte structures present in the 2 µm annulus created along the vessel for each focal plane were treated as individual entities, and their activities averaged within the corresponding focal plane and analyzed separately. In this type of analysis, astrocytic Ca2+ activities were defined as peaks standing out of the mean baseline fluorescent signal from all the end-feet surrounding the PA at the given z-level. Peaks were detected starting from the normalized ((x-mean)/sd), smoothed (sgolayfilt.mat, order value=10) mean baseline signal using the Matlab command findpeaks.mat.The following parameters were used: MinPeakHeight = 1 SD of the smoothed data; 'MinPeakProminence'= 0.8 SD of the smoothed data, MinPeakWidth =1 second, and widthReference=halfheight. After detection of the start and end times of the peaks, which correspond to the times in which the smoothed fluorescence data went, respectively, above and below the mean baseline value, we used the non-smoothed normalized data to verify that the signal prominence was >2 SD. Finally, we compared the spatial-temporal characteristics of the Ca2+ events determined in each focal plane with those in other focal planes and when we found that they overlapped, we classified the events in different planes as a single 3D Ca2+ activity, quantifying its z-spread (µm) and duration (s). The time periods showing Ca2+ events in end-feet along PAs were analyzed with regards to whether they overlapped with periods of animal movement or with PA dilation events to evaluate if correlations existed between the phenomena. Statistics Data are presented as mean ± 95% confidence limit (C.L., 1.96xSEM), unless otherwise stated. For all statistical analyses, OriginPro 2018b (OriginLab, Northampton, MA), Matlab 2019b, and Excel (Microsoft Office 2016) software was used. Statistical analyses were performed per 3D-FOV. For experiments on naturally-occurring dilations involving imaging small volumes at PA-capillary junctions, we analyzed 20 FOVs in 6 WT mice and 14 FOVs in 4 IP3R2KO mice, respectively. In each individual statistical test, we provided information on the number of dilations analyzed/3D-FOV in terms of mean dilations/experiment ± 95 % C.L.. For experiments involving imaging large volumes along the PA, we analyzed 8 FOVs in 6 WT mice and, in view of the reduced number of FOVs, we gave just the N value corresponding to the total number of dilations observed. In terms of statistical analyses, initially we compared the distribution of two data groups using graphical quantile-quantile plots (Q-Q plots), i.e. comparing the quantiles of the two groups: when the distribution was normal, a parametric two-tailed t-test was used considering whether data were paired or not. When the two data groups could not be considered independent or did not have normal distribution, the data were analyzed using non-parametric tests: the Wilcoxon signed-rank test was used for paired data, and the Mann-Whitney test for non-paired data.­ In data sets with several groups,­ before comparing individual points from two groups, to exclude group effects, we initially performed the Kruskal-Wallis ANOVA (KWA) for non-parametric data, and the two-way ANOVA for normally distributed data. If the data points in a group could not be considered to be independent, as in the case of vessel compartment dilations, we initially used the Friedmans ANOVA test instead. KWA and Friedmans ANOVA tests provide both a p-value and a X 2 value. The latter was used for defining the level of difference between the compared groups. In analyses that required a multiple comparisons test, such as ANOVA or KWA, the values from the final analysis were adjusted with the Holm correction (similar to Holm-Bonferroni) on both parametric and non-parametric data. This correction depends on the number of comparisons in the data group and the adjustment is performed in a ranked way depending on the p values: the smallest p-value gets the strongest adjustment (multiplied by the number of comparisons), while the following increasing p-values are adjusted depending on the number of p-values already adjusted according to: P original x(N comparisons -(counts of P< P original ))=P corrected . Data were considered significantly different when the p-value, corrected or non-corrected, was *<0.05, but additional levels of significance were marked with **<0.01 and *** <0.005. Declarations Animal Ethics statement All experiments and procedures were conducted under license and according to regulations of the Cantonal Veterinary Offices of Vaud (Switzerland). Data availability All scripts forming the code used in this study together with data examples to verify code function are provided with the submitted manuscript in a zipped folder together with a read-me instruction on how to run the analysis. All other data can be made available from the author (BLL) upon reasonable request and will be uploaded to a public repository upon publication of the study. A space in the public repository Zenodo has been reserved for this data under the doi: 10.5281/zenodo.15224578. Acknowledgments We would like to thank Giovanni Carriero and Erika Bindocci for their advice and support during the project; Tania Barkat, for her advice on setting up the auditory stimulations; and Martin Lauritzen and Søren Grubb for valuable scientific discussions. 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Glia 58 , 1094-1103 (2010). https://doi.org:10.1002/glia.20990 Additional Declarations There is NO Competing Interest. Supplementary Files SphinctPaperVideos.zip Supplemental Videos. Video V1: 3D-FOV z-stack along the PA, 90-360 micrometers below cortex surface, showing astrocytes at two capillary junctions with sphincter indentations . The movie is from the auditory cortex of an EGFP-GFAP mouse and depicts a PA with two 1 st order capillary branches . Red: masked vessel lumen; green : EGFP signal showing position of the astrocytes. Scale bar: 50 µm. Video V2: 3D-FOV scan of an astrocyte at PA-capillary junction shows multiple points of contact with capillaries and sphincter . The movie depicts a 3D stack from 250-330 µm below the auditory cortical surface and highlights the astrocyte end-feet coverage of a 1 st order capillary branch junction on the PA. The astrocyte is in contact with the vessel at numerous positions. Red: masked vessel lumen; white : mean cumulative average of GFAP-EGFP fluorescence levels, from which the masks of the astrocyte structures are obtained; green : masked astrocyte process structures (kernel size: 1 µm in Imaris) with the relatively largest and brightest highlighted ( green arrow ); yellow : masked astrocyte structures with end-feet regions touching the capillary surface ( yellow arrow ). Video V3: Comparing a multicompartment dilation to a dilation that does not spread from capillary to PA . The movie depicts a 3D-FOV from a stack acquired 95-125 µm below the auditory cortical surface. The movie shows in sequence the average vascular and Ca2+ responses for, first, a multicompartmental ( left ) and, second, a monocompartmental dilation ( right ) both beginning in the capillary bed. In parallel, during each dilation event, on the side appears a zoom-in of the activity in the region around the sphincter. Towards the end of the movie, the two dilation events are shown comparatively side-by-side. The dilation shown as first, on the left, progresses across the sphincter to the PA to become multicompartmental, whereas the one shown as second, on the right, remains within the 1 st capillary bed. Red : shows the masked average changes in vessel lumen; green : shows the mean GFAP-GCaMP6f fluorescence levels outlining perivascular astrocyte structures; pseudocolor : shows the normalized Ca2+ activity levels representing mean Ca2+ changes per voxel in the two types of dilation events. White circles: appear at the level of the vessel segment that is about to dilate and remain there until the segment is constricted again. A thin cyan line outlines the pre-dilation Ca2+ ROI. Cyan circles: identify pre-dilation Ca2+ activity events at the sphincter in the last second before dilation. Note the presence of Ca2+ activities also during the dilation. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6539397","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":454335185,"identity":"1e8606d2-af22-4180-a9ae-b55ed056f557","order_by":0,"name":"Barbara Lind","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIie2QsQrCQAyGA4V0ibq2nPgMJwctYvFZBKEuDoLg4qAgOImz7+HqEHDoZF0FF13OxRcQHLxbRY66Ody3JIR88CcAHs8fIgHQ1mY9XIDtzQBUJYWQ+Gcl6ldU0vCor899jzC+azGegpIc7KRL6ayHaXutB4RilIhtCYlknPSdwTjHiDiwCoraCjLJpNipnDTGL56bYIWuqJxzFMQHcz4kVkms4gzW2epQNbkwTx6pLpWRig84cZ6fNnK8PXjWaoTF7ULTrL0plrvIpXxiloNf9j0ej8fzlTfirjtl/0rImQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-0027-5226","institution":"University of Copenhagen","correspondingAuthor":true,"prefix":"","firstName":"Barbara","middleName":"","lastName":"Lind","suffix":""},{"id":454335186,"identity":"ebcad172-b393-498e-9711-5ed4c89cefb4","order_by":1,"name":"Andrea Volterra","email":"","orcid":"","institution":"Wyss Center","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Volterra","suffix":""}],"badges":[],"createdAt":"2025-04-27 09:21:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6539397/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6539397/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83016602,"identity":"d78a6e64-7d05-42b1-9f87-6960bd238106","added_by":"auto","created_at":"2025-05-19 06:32:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":471147,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3D imaging of the PA dynamics in the auditory cortex of awake mice: both naturally occurring dilations and astrocytic Ca2+ activity spread along the descending PA path.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Complete setup for our experiments in chronically implanted head-fixed awake mice.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Brightfield image of the vessels at the surface of the auditory cortex seen from the chronic cranial window. Visual inspection of the blood flow direction in vessels allows distinction between arterioles and venules. \u003cem\u003eStars\u003c/em\u003e: PAs.\u0026nbsp; Scale bar: 500 µm.\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Representative cumulative average of the 3D timeseries taken in the auditory cortex of a \u003cem\u003eGFAPCreERT2:GCaMP6f\u003c/em\u003e mouse (green, reporting astrocyte Ca2+ activity) injected in the circulation with Texas red dextran (red, reporting vessels lumen). Imaging was performed in 20 µm z-steps, from 50 to 210 µm below the surface. Scale bar: 20 µm.\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003cem\u003eTop:\u003c/em\u003e Example traces reporting simultaneous astrocyte end-foot Ca2+ activity (mean fluorescence) and PA diameter (area) changes normalized to SD values in the different z-planes of our 3D-FOV, before and during a period of mouse locomotion. The resting periods are used as baseline. \u003cem\u003eBottom\u003c/em\u003e: The movements are visible in the electromyogram (EMG) recorded from the mouse’s neck muscles throughout the imaging session.\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Spread of end-foot astrocyte Ca2+ activity along the length of the PA; \u003cem\u003egreen\u003c/em\u003e: mouse is resting; \u003cem\u003eviolet\u003c/em\u003e: mouse is moving (Mann-Whitney test: p=1.8x10\u003csup\u003e-11\u003c/sup\u003e; N=668 Ca2+ events in the resting mouse and 262 in the moving mouse).\u003c/p\u003e\n\u003cp\u003eF.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Spread of PA dilations along the length of the PA in the resting mouse; \u003cem\u003eblack\u003c/em\u003e: without Ca2+ activity in the astrocyte end-feet;\u003cem\u003e green\u003c/em\u003e: with the astrocytic Ca2+ activity (Mann-Whitney test: p=5.7x10\u003csup\u003e-16\u003c/sup\u003e; N=324 dilations without astrocyte Ca2+ and 344 with Ca2+).\u003c/p\u003e\n\u003cp\u003eG.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Spread of PA dilations along the length of the PA as in F, but in the moving mouse; \u003cem\u003eblack\u003c/em\u003e: without Ca2+ activity in the astrocyte end-feet; \u003cem\u003eviolet\u003c/em\u003e: with the astrocytic Ca2+ activity (Mann-Whitney test: p=1.5x10\u003csup\u003e-9\u003c/sup\u003e; N=78 dilations without astrocyte Ca2+ and 184 with Ca2+).\u003c/p\u003e\n\u003cp\u003eIn E-G, data are from 8 FOVs: x=75-121 µm; y=53-111 µm; z=100-280 µm, in 10-20 µm steps from 6 mice.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6539397/v1/52f8b42afb59ed4e8096fdcf.png"},{"id":83016604,"identity":"24dc91d5-91d5-4dbc-a16d-8d5374b947b1","added_by":"auto","created_at":"2025-05-19 06:32:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":459842,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpread of naturally occurring dilations in the auditory cortex of awake mice is gated at the sphincter between PA and capillary compartments. Correlation with end-foot astrocyte Ca2+ dynamics and animal motor state.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Reconstruction of a 3D stack imaged live from 90 to 360 µm below the surface in the auditory cortex of an \u003cem\u003eEGFP-GFAP\u003c/em\u003e mouse. Shown from the side (\u003cem\u003eleft\u003c/em\u003e) and from above (\u003cem\u003eright\u003c/em\u003e). The reconstruction shows a penetrating arteriole delivering blood into the tissue and two 1\u003csup\u003est\u003c/sup\u003e order capillaries branching at different depths of the cortical layer II/III (a and b white squares on the \u003cem\u003eleft\u003c/em\u003e). \u003cem\u003eRed\u003c/em\u003e: Texas red-dextran highlights vessels lumen; \u003cem\u003ewhite\u003c/em\u003e: EGFP signal shows reconstruction of the astrocyte larger processes. See also Video V1. Scale bar: 20 µm.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Zoom-in on the 3D stack shown in A, depicting the lower branch (b) region (from 250 to 330 µm below the surface). Colors as in A. Scale bar: 20 µm. See also Video V2.\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Representative cumulative average of a 3D time series, taken from a \u003cem\u003eGFAPCreERT2:GCaMP6f\u003c/em\u003e mouse injected with Texas red-dextran in the vessel. The 3D-stack was imaged from 170 to 200 µm below the cortical surface, at 30 levels 1 µm apart. \u003cem\u003eLeft, top\u003c/em\u003e: cumulative signals show in red the vessel lumen and in green the neighboring astrocyte structures. \u003cem\u003eLeft, bottom\u003c/em\u003e: the same picture as above, but with multiple 3D-ROIs of astrocyte Ca2+ activity measuring a large astrocytic Ca2+ event associated with the animal’s locomotion and a dilation initiated at the pre-capillary sphincter. Ca2+ activity ROIs are color-coded to appreciate their temporal-spatial dynamics (Ca2+ onset time) relative to the dilation onset. The red signal is masked to show the 3D structure of the vessel. \u003cem\u003eRight\u003c/em\u003e: Traces of mean astrocyte end-foot Ca2+ activity from each ROI. ROIs and traces are color-coded in relation to the timing of dilation onset. Scale bar: 8 µm.\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Delay between the onset time of all the large astrocytic end-foot Ca2+ activities and the onset time of the vessel dilations seen in the 3D-FOV when the mouse is resting (\u003cem\u003egreen\u003c/em\u003e) or is moving (\u003cem\u003eviolet\u003c/em\u003e). Mean Delay 2.3±0.86 s (Mean: \u003cem\u003eThick black line, \u003c/em\u003e95% C.L. limits: \u003cem\u003ethin black lines\u003c/em\u003e). N=223 dilation events in the moving mouse accompanied by large astrocyte Ca2+ activity.\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Average duration of dilations per 3D-FOV in different conditions. \u003cem\u003eBlack\u003c/em\u003e: in the resting mouse when no astrocyte Ca2+ activity is observed; 162±31.2 dilations/FOV); \u003cem\u003egreen\u003c/em\u003e: in the resting mouse in the presence of large astrocyte Ca2+ activity (4.2±2.0 dilations/FOV); \u003cem\u003eazure\u003c/em\u003e: in the moving mouse when no astrocyte Ca2+ activity is observed (47.6±22.1 dilations/FOV); \u003cem\u003eviolet\u003c/em\u003e: in the moving mouse in the presence of large astrocyte Ca2+ activity (6.5±2.5 dilations/FOV). Dilations occurring in the resting animal were significantly longer when accompanied by an astrocyte Ca2+ event (t-test: without vs. with Ca2+, p=0.046), but significantly shorter than dilations occurring during mouse movements, accompanied or not by astrocyte Ca2+ (t-test: resting vs. moving without Ca2+: p= 6x10\u003csup\u003e-7\u003c/sup\u003e; resting vs moving with Ca2+:\u0026nbsp; p = 3.3x10\u003csup\u003e-6\u003c/sup\u003e). Dilations during mouse movements were significantly longer when accompanied by an astrocyte large Ca2+ activity (t-test: moving vs moving with Ca2+: p= 0.0016). N=20 FOVs in 6 mice; all values were compared with ANOVA before t-test p=1.46x10\u003csup\u003e-9\u003c/sup\u003e. T-tests were paired, two-tailed, and p-values adjusted with a Holm correction for multiple comparisons.\u003c/p\u003e\n\u003cp\u003eF.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Average fraction of dilations per 3D-FOV comprising all the vessel compartments in the FOV in different conditions. Color coding, conditions, number of dilations analyzed/FOV, number of FOV, and number of mice are as in E. The fraction of dilations significantly increased when the mouse moved with respect to when it was at rest, both when a large astrocyte Ca2+ activity accompanied the movement and when it did not (Kruskal Wallis ANOVA (p=2.7 x 10\u003csup\u003e-6\u003c/sup\u003e) followed by Wilcoxon test with Holm correction: resting without vs. with Ca2+: p=0.059; resting vs moving, both without Ca2+: p= 0.00019; resting vs moving with Ca2+: p=0.00016). The fraction of multicompartmental dilations in the moving mouse further increased when movement was accompanied by an astrocytic Ca+ elevation (Wilcoxon test with Holm correction: moving without Ca2+ vs moving with Ca2+: p=0.013).\u003c/p\u003e\n\u003cp\u003eG.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Average fraction of dilations per 3D-FOV in the resting animal according to the involved vessel compartments. \u003cem\u003eTop\u003c/em\u003e: Representation of the vascular compartments present in the 3D-FOV in all our experiments. The different vascular compartments have been subdivided and depicted in different colors: PA (\u003cem\u003ered\u003c/em\u003e), capillary branch, comprising the sphincter, bulb and 1\u003csup\u003est\u003c/sup\u003e order capillary (\u003cem\u003eblue\u003c/em\u003e). \u003cem\u003eBottom\u003c/em\u003e: Average fraction of dilations involving either all the compartments (multicompartmental, \u003cem\u003eblack\u003c/em\u003e) or each of them individually according to color coding. 162±31.2 dilations/FOV, N=20 FOVs, 6 mice. Half the dilations were multicompartmental, while the other half involved each compartment individually, with significantly more dilations occurring in the capillary branch (\u003cem\u003eblue\u003c/em\u003e) compared to the PA (\u003cem\u003ered\u003c/em\u003e). Friedmans ANOVA (p=1.59 x 10\u003csup\u003e-7\u003c/sup\u003e) followed by Wilcoxon test with Holm correction: multicompartment vs. PA: p =1.4 x 10\u003csup\u003e-4\u003c/sup\u003e; multicompartment vs. capillary branch: p= 0.015; capillary branch vs PA: p= 1.0 x 10\u003csup\u003e-4\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eH.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Average fraction of dilations per 3D-FOV in the resting mouse according to the compartment in which they originate: segmented compartments are depicted with different colors and symbols: PA (\u003cem\u003ered, diamond\u003c/em\u003e), sphincter (\u003cem\u003ecyan, triangle\u003c/em\u003e), bulb (\u003cem\u003elight blue, square\u003c/em\u003e), capillary (\u003cem\u003edark blue, circle\u003c/em\u003e); 162±31.2 dilations/FOV, N=20 FOVs, 6 mice. Dilations had heterogeneous origin and direction, with significantly higher fractions originating from PA and capillary than from sphincter and bulb (Friedmans ANOVA (p=1.56x10\u003csup\u003e-7\u003c/sup\u003e) followed by Wilcoxon test with Holm correction: PA vs. sphincter: p = 0.032; PA vs bulb: p = 4.4 x 10\u003csup\u003e-4\u003c/sup\u003e; capillary vs sphincter: p = 0.025; capillary vs bulb: p = 1.3 x 10\u003csup\u003e-3\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003eIllustration of the direction of spreading of the dilations across the sphincter according to the compartment of origination (color-coding as in H). \u003cem\u003eTop\u003c/em\u003e: multi-compartment dilations; \u003cem\u003ebottom\u003c/em\u003e: single-compartment dilations.\u003c/p\u003e\n\u003cp\u003eAll data in C-I of this figure are from N=20 FOVs in 6 mice. 3D imaging coordinates: x=56-75 µm; y=15-44 µm; z=21-35 µm in 1µm steps.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6539397/v1/c8eeddac11048f20ba168d2f.png"},{"id":83016605,"identity":"9970e3db-67ae-4eb0-9cce-ff0a94ad8cfc","added_by":"auto","created_at":"2025-05-19 06:32:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":584903,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLocal Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e activity in astrocytic end-feet at the sphincter precedes multicompartment dilations arriving from distant vascular compartments in the resting mouse.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Representative data showing local astrocyte end-foot Ca2+ activity preceding dilations originating in different compartments in our 3D-FOV (x=56.6 µm; y= 17.5 µm; z: from 194 to 216 µm below the brain surface in 1 µm steps). \u003cem\u003eLeft, top\u003c/em\u003e: cumulative average of the GCaMP6f (end-feet) and Texas-Red (vessel lumen) signals; \u0026nbsp;\u003cem\u003ebottom\u003c/em\u003e: the same vascular structures (\u003cem\u003ered\u003c/em\u003e: PA; \u003cem\u003ecyan\u003c/em\u003e: sphincter; \u003cem\u003eblue\u003c/em\u003e: bulb/1\u003csup\u003est\u003c/sup\u003e order capillary) shown in two different orientations with several astrocytic VOIs at different locations around the junction, corresponding to the end-foot regions displaying pre-dilation Ca2+ activity shown in the traces on the \u003cem\u003eright\u003c/em\u003e. Numbers identify each VOI; color identifies the VOI’s position on the vasculature. \u003cem\u003eRight\u003c/em\u003e: Representative traces showing: \u003cem\u003etop\u003c/em\u003e: time-course of astrocyte Ca2+ dynamics in VOIs numbered on the \u003cem\u003eleft\u003c/em\u003e during three dilations starting either at the PA (left column), the sphincter (middle column), or the capillary (right column); \u003cem\u003ebottom:\u003c/em\u003e for each column, corresponding time-course of the dilation at the specific vessel segment where the dilation began. Pre-dilation Ca2+ activity appeared at several astrocyte end-foot locations, consisted of either individual peaks or more composite patterns, and was seen first in VOIs closest to the sphincter, including dilations arriving from the PA or the capillaries. Scale bars: 5 µm.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Analysis of local pre-dilation end-foot astrocyte Ca2+ elevations associated with multicompartmental dilations. Quantification is by the number of end-foot VOIs with recurrent pre-dilation Ca2+ activity per FOV. Colors indicate the vascular location of the Ca2+ elevation (\u003cem\u003ered\u003c/em\u003e: PA; \u003cem\u003ecyan\u003c/em\u003e: sphincter; \u003cem\u003elight blue\u003c/em\u003e: bulb; \u003cem\u003eblue\u003c/em\u003e: capillary); symbols indicate the compartment of origin of the dilation (\u003cem\u003ediamond\u003c/em\u003e: PA; \u003cem\u003etriangle\u003c/em\u003e: pre-capillary sphincter; \u003cem\u003eempty circle\u003c/em\u003e: capillary). Data are from 20 FOVs in 6 mice. End-foot Ca2+ events occurred more frequently at the sphincters, independent if the dilation initiated at the PA, the sphincter, or the capillaries. Tested with Kruskal Wallis ANOVA (KWA) followed by Wilcoxon test with Holm-correction for multiple comparisons: (a) dilation starting at PA: KWA p=0.019; count of end-foot Ca2+ events at sphincter vs. at PA p= 5.5x10-4; at sphincter vs. at bulb: p=0.062; at sphincter vs. at capillary: p = 0.027; (b) dilation starting at sphincter: KWA p=0.031; count of end-foot Ca2+ events at sphincter vs. at PA: p = 0.006; at sphincter vs. at bulb: p=0.25; at sphincter vs. at capillary: p = 0.41; (c) dilation starting at capillaries: KWA p=0.0033; count of end-foot Ca2+ events at sphincter vs at PA: p = 0.0011; at sphincter vs at bulb: p=0.048; at capillary vs at sphincter: p=0.21. The number of dilations/FOV starting at PA, sphincter and capillary, respectively, was 116.1±24.7, 82.4±19.7, and 67.2±16.2. N=20 FOV.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Properties of the local astrocyte end-foot Ca2+ activities observed at four different vascular locations before multicompartmental dilations. Ca2+ activities are color-coded according to the vascular compartment where they are seen (\u003cem\u003ered\u003c/em\u003e: PA; \u003cem\u003ecyan\u003c/em\u003e: sphincter; \u003cem\u003elight blue\u003c/em\u003e: bulb; \u003cem\u003eblue\u003c/em\u003e: capillary). \u003cem\u003eTop\u003c/em\u003e: spread of the end-foot Ca2+ activity along the z-axis (at PA: 10.1 ±1.9 µm; sphincter: 9.7 ±1.13 µm; bulb: 7.3 ±1.7 µm; capillary: 8.6 ±1.2 µm). \u003cem\u003eBottom\u003c/em\u003e: duration of the Ca2+ activity (at PA: 0.91 ±0.18 s; sphincter: 0.98 ±0.13 s; bulb: 1.5 ±0.58 s; capillary: 0.94 ±0.15 s). Data are from 20 FOVs in 6 mice.\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Timing of the astrocytic end-foot Ca2+ activity associated with multicompartmental dilations with different origin (\u003cem\u003eleft\u003c/em\u003e: PA; \u003cem\u003emiddle\u003c/em\u003e: sphincter; \u003cem\u003eright\u003c/em\u003e: capillary bed). \u003cem\u003eTop\u003c/em\u003e: Representative time-series of 3D-FOV (x= 75.5µm, y= 23.6µm taken from 95 to 125 µm below brain surface). \u0026nbsp;Images are taken 3 sec before, 2 sec before and 2 sec after the dilation start. In \u003cem\u003ered\u003c/em\u003e: Texas red-dextran masked signal highlighting vessel lumen; in \u003cem\u003egreen\u003c/em\u003e: cumulative astrocyte GCaMP6f signal highlighting active perivascular end-feet; \u003cem\u003epseudocolor\u003c/em\u003e scale: normalized Ca2+ activity levels to show Ca2+ changes per voxel. \u003cem\u003eDotted line circles\u003c/em\u003e highlight astrocyte Ca2+ activity at the sphincter (\u003cem\u003ecyan\u003c/em\u003e) and at the capillary (\u003cem\u003eblue\u003c/em\u003e). The activity is most precocious and intense when dilations arrive from PA or capillaries. Scale bar: 7 µm. \u003cem\u003eBottom\u003c/em\u003e: Distribution of the onset times of astrocytic Ca2+ signals seen at the sphincter before multicompartmental dilations starting at different vessel compartments.\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Comparison of the onset time of the different groups of pre-dilation end-foot Ca2+ activities shown in D. \u0026nbsp;Following Kruskal Wallis ANOVA test we looked at individual comparisons. \u003cem\u003eLeft\u003c/em\u003e: timing of the pre-dilation Ca2+ signals for dilations starting at the sphincter vs at the PA. On average, there is no significant difference in onset time, but the number of early events (onset \u0026gt;1 sec before dilation) is significantly higher for dilations starting at PA (Mann-Whitney test; onset time: p=0.15; Wilcoxon test: count of early events: p=0.029). \u003cem\u003eRight\u003c/em\u003e: same comparison but with dilations starting at the sphincter vs at the capillary. In this case, both onset time and number of early events are significantly different: end-foot Ca2+ activity starts earlier when dilations initiate at the capillary, and there are more early events (Mann-Whitney: onset time: p=0.027; Wilcoxon test: count of early events: p=0.043). \u0026nbsp;The number of Ca2+ events detected before dilations starting at PA, sphincter and capillary, respectively, was N= 42, 28 and 31. The number of dilations/FOV analyzed starting at PA, sphincter, and capillary was 116.1±24.7, 82.4±19.7, and 67.2±16.2 respectively. All data are from 20 FOVs in 6 mice.\u003c/p\u003e\n\u003cp\u003eF.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Comparison of the onset times of astrocyte Ca2+ activities seen at the sphincter and at the capillary before arrival of multicompartmental dilations from the capillary bed. \u003cem\u003eLeft\u003c/em\u003e: Distribution of the Ca2+ signals at the capillary (\u003cem\u003eblue\u003c/em\u003e). \u003cem\u003eRight\u003c/em\u003e: The end-foot Ca2+ events around the sphincter started earlier and were more numerous in the early phases than those at the capillary (Onset time: Mann-Whitney test p=0.016 N=31 Ca2+ events; count of early Ca2+ events: Wilcoxon test, p=0.03, 67.2±16.2 dilations/FOV, from 20 FOVs in 6 mice.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6539397/v1/2d1949b413b99ba6d8bcf7b3.png"},{"id":83017500,"identity":"37171917-ad7d-40bd-8425-0ac530628af3","added_by":"auto","created_at":"2025-05-19 06:40:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":410059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePre-dilation astrocytic Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e activity at the sphincter is suppressed in IP2R2KO mice; the most affected events are the earliest ones and those closest to the sphincter.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Pre-dilation astrocyte end-foot local Ca2+ dynamics in wild-type (WT) and IP3R2KO mice. To illustrate the recurrence of the \u0026nbsp;pre-dilation Ca2+ signals, their averaged activity in association with 3 spontaneous dilations is shown in representative 3D-FOVs in the period from 3 sec before to 1 sec after the start of the vasodilations. All vasodilations start at the capillary bed and spread to the PA. Each image is the cumulative average of 1 s interval in a 3D-FOV in: WT (\u003cem\u003eleft\u003c/em\u003e, x=75.5 µm, y=23.6 µm, z=170-200 µm below the cortical surface), or IP3R2KO (\u003cem\u003eright\u003c/em\u003e, x=75.5 µm, y=30.7 µm, z=138-162 µm below the surface). In \u003cem\u003ered\u003c/em\u003e: masked vessel lumen; in \u003cem\u003egreen\u003c/em\u003e: cumulative astrocyte GCaMP6f showing active end-feet; \u003cem\u003epseudocolor scale\u003c/em\u003e: normalized Ca2+ activity. \u003cem\u003eDotted line circles\u003c/em\u003e highlight astrocyte Ca2+ activity at the sphincter. Scale bars: 7 µm.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Comparison of pre-dilation astrocyte Ca2+ signals at the pre-capillary sphincter before multicompartment vasodilations in WT and IP3R2KO mice. \u003cem\u003eTop\u003c/em\u003e: astrocyte Ca2+ signals and vasodilations are measured in two ROIs spatially related to the vascular segments undergoing vasodilation: in the case shown, the vasodilation starts at the 1\u003csup\u003est\u003c/sup\u003e order capillary level (\u003cem\u003eblue\u003c/em\u003e) and spreads to the PA (\u003cem\u003ered\u003c/em\u003e). \u003cem\u003eBottom\u003c/em\u003e: representative astrocyte Ca2+ and vessel dilation traces in the defined ROIs. In the IP3R2KO mouse, the pre-dilation Ca2+ signals are reduced, and the PA dilation is delayed.\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Number of astrocyte Ca2+ events seen at the pre-capillary sphincter before multicompartmental dilations in several experiments in WT (\u003cem\u003eopen symbols\u003c/em\u003e) and IP3R2KO (\u003cem\u003esolid symbols\u003c/em\u003e) mice. \u003cem\u003eBlack lines\u003c/em\u003e are mean values. Separate comparisons are shown for dilations starting at PA (\u003cem\u003ediamonds\u003c/em\u003e), sphincter (\u003cem\u003etriangles\u003c/em\u003e) or capillaries (\u003cem\u003ecircles\u003c/em\u003e). The number of pre-dilation Ca2+ events was significantly reduced in IP3R2KO vs. WT mice for dilations starting at PA or capillary, but not for dilations starting at the sphincter (Mann-Whitney test: WT vs KO: (a) dilations with origin at PA: p = 0.0077; (b) dilations with origin at sphincter: p = 0.45; (c) dilations with origin at capillary: p=0.029. Number of dilations/FOV analyzed with origin at PA, sphincter or capillary was, respectively: in WT: 116.1±24.7, 82.4±19.7 and 67.2±16.2 in N=20 FOVs in 6 mice. In IP3R2KO: 130.3±35.4, 76.9±21.9, and 50.9±14.3 in 14 FOVs in 4 mice.\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Timing of the pre-dilation astrocyte Ca2+ events seen at the sphincter in the 2 sec preceding multicompartment dilations in WT and IP3R2KO mice (\u003cem\u003eopen\u003c/em\u003e and \u003cem\u003esolid symbols\u003c/em\u003e, respectively). Evaluation of the difference in onset time is conducted on the entire population of events and followed up by quantification at the level of the sub-population of earlier-onset events (EO, starting \u0026gt;1 before dilations start). Comparisons are performed separately for dilations starting at PA, sphincter or capillaries (\u003cem\u003esymbols\u003c/em\u003e as in C). Despite no significant difference emerged between WT and IP3R2KO in the onset time of the entire population, the number of EO events was significantly reduced in IP3R2KO mice (\u003cem\u003edashed grey circles\u003c/em\u003e) for dilations starting at PA or capillary (Mann-Whitney test, WT vs KO: Dilations starting at (a) PA: onset time: all events: p=0.71; EO events: p = 0.0049; \u0026nbsp;(b) at sphincter: onset time: all events:\u0026nbsp; p= 0.79; EO events: p = 0.18; (c) at capillary: onset time: all events: p=0.13; EO events: p = 0.0075). The number of dilations/FOV analyzed with origin at PA, sphincter and capillary were, respectively: in WT: 116.1±24.7, 82.4±19.7, and 67.2±16.2 in 20 FOVs in 6 mice and in IP3R2KO: 130.3±35.4, 76.9±21.9 and 50.9±14.3 in N=14 FOVs in 4 mice.\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Distance from the sphincter of the pre-dilation astrocyte Ca2+ events in the 2 sec preceding multicompartmental dilations in WT and IP3R2KO mice (\u003cem\u003eopen\u003c/em\u003e and \u003cem\u003esolid symbols\u003c/em\u003e, respectively). Comparisons are performed for dilations starting at PA, sphincter or capillaries (\u003cem\u003esymbols\u003c/em\u003e as in C). \u003cem\u003eBlack lines\u003c/em\u003e are mean values: no statistical difference emerges between WT and IP3R2KO in any group of dilations. However, this could depend on the wide spatial dispersion of the events. Thus, for further analysis, events were sub-divided in two groups: those occurring at shorter distance (SD) from the sphincter (\u0026lt;5 µm) and those occurring at longer distances (LD, 5-20 µm). SD events were significantly reduced (\u003cem\u003edashed grey circles\u003c/em\u003e) for dilations starting at PA or capillary (Mann-Whitney test: WT vs KO: for dilations starting at (a) PA: SD: p=0.0038; LD: p=0.18; (b) sphincter: SD: p=0.52, LD p=0.18; (c) capillary: SD: p=0.023, LD p=0.15). The number of dilations with origin at PA, sphincter and capillary were, respectively: in WT: 116.1±24.7, 82.4±19.7 and 67.2±16.2 in 20 FOVs in 6 mice; in IP3R2KO: 130.3±35.4, 76.9±21.9 and 50.9±14.3 in 14 FOVs in 4 mice.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6539397/v1/5fb7d7e04fa72601558a464b.png"},{"id":83016610,"identity":"a11efea0-8558-4bf2-9d3f-57346fc39f02","added_by":"auto","created_at":"2025-05-19 06:32:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":259674,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe spread of dilations from one vascular compartment to another is impaired in IP3R2KO mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Fraction of multicompartment dilations according to their initiation site in WT and IP3R2KO mice. WT: \u003cem\u003eopen symbols\u003c/em\u003e; IP3R2KO: \u003cem\u003esolid symbols\u003c/em\u003e. Dilations starting at PA: \u003cem\u003ered diamonds\u003c/em\u003e; at sphincter: \u003cem\u003ecyan triangles\u003c/em\u003e; at bulb, \u003cem\u003elight blue squares\u003c/em\u003e; at capillaries, \u003cem\u003eblue circles\u003c/em\u003e. \u003cem\u003eBlack lines\u003c/em\u003e are mean values. In IP3R2KO mice, the fraction of multicompartment dilations starting at the PA or the capillary bed was reduced compared to WT mice (First level statistical analysis: Friedmans ANOVA: WT: p=2.9x10\u003csup\u003e-7\u003c/sup\u003e, X\u003csup\u003e2\u003c/sup\u003e=33.2; KO: p=0.00022, X\u003csup\u003e2\u003c/sup\u003e=19.5; X\u003csup\u003e2\u003c/sup\u003e test indicates more equal distribution of dilation origin in KO mice. Second level analysis: Mann-Whitney test: WT vs KO: dilations at PA: p = 0.017; at sphincter: p = 0.16; at bulb: p = 0.044; at capillary: p= 0.025). The number of dilations/FOV analyzed in WT was 82.65±20.13 in N=20 FOVs in 6 mice; in IP3R2KO, 75.27 ±16.73 in N=14 FOVs in 4 mice.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Characteristics of the dilations arriving from the capillary compartment in WT and IP3R2KO mice. WT: \u003cem\u003eblue open circles\u003c/em\u003e; IP3R2KO: \u003cem\u003eblue solid circles\u003c/em\u003e. \u003cem\u003eBlack lines\u003c/em\u003e are mean values. In IP3R2KO mice, the fraction of dilations that did not cross the sphincter to become multicompartmental was larger than in WT mice (Mann-Whitney test: WT vs IP3R2KO: p = 0.027). Number of dilations/FOV analyzed: in WT: 166.84±30.41 in N=20 FOVs in 6 mice; in IP3R2KO: 176.2±41.24 in N=14 FOVs in 4 mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Delay in the spread of multicompartmental dilations between vessel segments in WT and IP3R2KO mice. Dilations are presented according to their initiation segment (\u003cem\u003etop\u003c/em\u003e: PA, in \u003cem\u003ered\u003c/em\u003e; \u003cem\u003emiddle\u003c/em\u003e: sphincter, in \u003cem\u003ecyan\u003c/em\u003e; \u003cem\u003ebottom\u003c/em\u003e: capillary, in \u003cem\u003eblue\u003c/em\u003e). Color-coded dots along the x-axis mark the start time of dilation of a given vessel compartment, which is measured as delay from the start time of the initiating compartment. The color-coded rectangles are the corresponding mean values, and the surrounding thin lines show the SEM. In WT mice (20 FOVs from 6 mice), dilations spread faster from PA to capillary than in the opposite direction, from capillary to PA (\u003cem\u003eOpen stars \u003c/em\u003ein vertical; Wilcoxon signed-rank test, p = 4.4 x 10\u003csup\u003e-6\u003c/sup\u003e; n = 366 dilations spreading from PA to capillary and 323 dilations from capillary to PA). In IP3R2KO mice, the spread of dilations across the sphincter is slower than in WT mice for dilations arriving from outside the FOV, regardless of the initiating compartment. Also, dilations starting at the sphincter are slower than in WT mice in reaching the capillary (Mann-Whitney test; WT vs. KO: (\u003cem\u003etop\u003c/em\u003e) initiation at PA: spread: to sphincter: p=0.62; to capillary: p = 0.0000048; n = 366 dilations in WT and 142 in IP3R2KO mice; (\u003cem\u003emiddle\u003c/em\u003e) initiation at sphincter: spread: to capillary: p = 0.0017; to PA: p = 0.62; n = 329 dilations in WT and 239 in IP3R2KO mice; (\u003cem\u003ebottom\u003c/em\u003e) initiation at capillary: spread:\u0026nbsp; to PA: p = 0.0083; to sphincter: p = 0.93; n= 323 dilations in WT and 145 in IP3R2KO mice. Data from 20 FOVs in 6 WT mice and from 14 FOVs in 4 IP3R2KO mice.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6539397/v1/8265c77ce541f1d8b92559ec.png"},{"id":83016614,"identity":"2ea9ec23-9bd4-4629-9980-646055d28103","added_by":"auto","created_at":"2025-05-19 06:32:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":517737,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePre-dilation astrocytic Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e activity at the sphincter and vascular dilation patterns after auditory stimulations resemble those naturally occurring in resting mice and, like them, are altered in IP3R2KO mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003cem\u003eTop\u003c/em\u003e: Brightfield image of the brain surface in a chronic craniotomy over the auditory cortex. \u003cem\u003eBottom\u003c/em\u003e: Intrinsic optical signal (IOS)-based map of the tonality in the same auditory cortex region, with an overlay of three colors, each one marking the response pattern to the stimulation of the corresponding tonality (\u003cem\u003eazure\u003c/em\u003e: 3kHz; \u003cem\u003egreen\u003c/em\u003e: 20 kHz; \u003cem\u003ered\u003c/em\u003e: 30 kHz). The map shows that the three tone frequencies activate distinct but partly overlapping regions. Black circles identify the areas where three PAs enter the tissue (see panel B), indicating the tone-specificity of the blood flow response in each area. Scale bar: 1mm.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Representative dilations at PA-1\u003csup\u003est\u003c/sup\u003e order capillary junctions in response to tone stimulations. Recordings are from 4 3D-FOVs in the 3 auditory cortex areas shown with circles in panel A at the center of which is the entrance point of the PAs. \u003cem\u003eLeft\u003c/em\u003e: images show vascular and astrocytic structures in the 4 3D-FOVs in the 3 areas and are cumulative averages of 2-min-long 3D timeseries; in \u003cem\u003ered\u003c/em\u003e: Texas red-dextran masked signal highlighting vessel lumen; in \u003cem\u003egreen\u003c/em\u003e: astrocyte GCaMP6f signal highlighting the active perivascular end-feet. The 3D-FOVs in Areas 1 and 2 contain a single pre-capillary junction each, with the branching of the capillary from the PA at the level of layer II/III (170-200 µm and 177-202 µm below the cortical surface, respectively). The 3D-FOV in Area 3 contains 2 junctions (circled 1 and 2, respectively): the upper one in layer I (1, 64-86 µm below surface), and the lower one in layer II/III (2, 194-216 µm below surface). The different vascular sections are marked as: P for PA; S for sphincter; B for bulb; and C for capillary. \u003cem\u003eRight\u003c/em\u003e: schematic representation of dilations evoked by tones of different frequencies (tones color-coded as in panel A). The timing of individual vessel segment dilation is depicted on a white-to-dark-red temporal scale (black indicates no dilation). The direction and duration of the dilations are schematized by the direction and thickness of the arrows, respectively (longer duration = thicker arrow). The position of the PA with respect to the IOS response evoked by a given tone frequency stimulation is summarized by color-coded stars: \u003cem\u003eblack\u003c/em\u003e, PA at the center of the IOS response; \u003cem\u003egrey\u003c/em\u003e, PA at the border of the IOS response; \u003cem\u003elight grey\u003c/em\u003e, PA further away from the IOS border. The length of the dilations correlated with the tone frequency that elicited the strongest IOS response in the specific 3D-FOV. Vessel dilation durations: Area 1: 3kHz stimulation: 40.7s; 20kHz: 15.4s; 30kHz: 3s. Area 2: 3kHz: 3.5s; 20kHz: 25.5s; 30kHz: 7.2s. Area 3, upper junction 1: 3kHz: 13.5s; 20kHz: 6.5s; 30kHz: 63.8s; lower junction 2: 3kHz: no dilation; 20kHz: 12.4s; 30kHz: 10.3s. The junction in layer I showed less tone specificity than the three junctions in layer II/III (see color-coded map in A). Scale bars: 10µm.\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Dilation patterns in the average responses to different tones in all the 3D-FOVs included in this study (n=73 tone responses in 20 FOVs in 6 mice). \u003cem\u003eLeft:\u003c/em\u003e patterns associated with tones that evoked a strong IOS response around the PA; \u003cem\u003eright:\u003c/em\u003e patterns associated with tones that evoked weak or no IOS response around the PA. Dilation responses are ordered according to their duration (\u003cem\u003eblue plots\u003c/em\u003e on the right of each column), from bottom (shortest) to top (longest). Timing of dilation of individual vessel compartments is represented in white-to-dark-red temporal scale as in panel B. The different vascular sections are marked as: P for PA; S for sphincter; B for bulb; and C for capillary. The vessel segment where the dilation originated is marked by a rectangular symbol in the following color code, PA: \u003cem\u003ered\u003c/em\u003e; sphincter: \u003cem\u003ecyan\u003c/em\u003e; bulb: \u003cem\u003elight blue\u003c/em\u003e; capillary: \u003cem\u003eblue\u003c/em\u003e. Dilations with the longest duration were evoked by tone stimulations with IOS response centered around the PA (\u003cem\u003eleft\u003c/em\u003e). They began at either the capillary (circled by \u003cem\u003eblue dotted line\u003c/em\u003e) or PA (circled by \u003cem\u003ered dotted line\u003c/em\u003e) and quickly progressed across the sphincter. In contrast, short dilations were evoked in areas with approximately no IOS response and often did not involve all compartments (\u003cem\u003eright\u003c/em\u003e). Those involving all compartments mostly originated from the capillary (circled by \u003cem\u003eblue dotted line\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp;\u0026nbsp;Representative time-series of pre-dilation astrocytic Ca2+ activity associated with tone-evoked multicompartmental dilations. The 3D-FOV, x= 75.5µm, y= 23.6µm, goes in z from 194 to 216 µm below the brain surface. Images are from 1.5 seconds before the spread of a multicompartmental dilation starting at the PA to 0.5 seconds after its start. \u003cem\u003eRed\u003c/em\u003e: Texas red-dextran masked signal highlights vessel lumen; \u003cem\u003egreen\u003c/em\u003e: cumulative astrocyte GCaMP6f signal highlights active perivascular end-feet. \u003cem\u003ePseudocolor\u003c/em\u003e: Normalized astrocytic Ca2+ activity. \u003cem\u003eDotted line circle\u003c/em\u003e shows astrocytic Ca2+ activity at the sphincter prior to dilation onset. Scale bar: 10µm.\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Number of pre-dilation astrocyte end-foot Ca2+ activities seen at the sphincter before tone-evoked multicompartment dilations in WT and IP3R2KO. WT: \u003cem\u003ecyan, open circles\u003c/em\u003e; IP3R2KO: \u003cem\u003ecyan, solid circles\u003c/em\u003e. \u003cem\u003eBlack lines\u003c/em\u003e are mean values. Comparisons are presented in separate columns according to the site of origination of the dilation. The number of pre-dilation Ca2+ signals was significantly reduced in IP3R2KO compared to WT mice both for dilations that began upstream (PA) and downstream (capillary) the sphincter (Mann-Whitney test: WT vs. KO: dilation starting at PA: p = 0.026; at capillary: p=0.0046) but not for those initiated at the sphincter (WT vs KO: p = 0.22). Number of dilations/FOV starting at PA, sphincter, and capillary, respectively, were in WT: 1.98±0.62, 0.85±0.38, and 1.25±0.58, from N=20 FOVs in 6 mice; in IP3R2KO:\u0026nbsp; 1.4±0.68, 1.3±0.83, and 1.13±0.63 from N=14 FOVs in 4 mice.\u003c/p\u003e\n\u003cp\u003eF.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Fraction of multicompartmental and single compartment dilations evoked by auditory stimulations in WT and IP3R2KO mice. WT: \u003cem\u003eopen circles\u003c/em\u003e; IP3R2KO: \u003cem\u003esolid circles\u003c/em\u003e. \u003cem\u003eBlack lines\u003c/em\u003e are mean values. Analysis is restricted to dilations lasting \u0026gt;5 sec. Comparisons are presented for the ensemble of multicompartmental dilations (\u003cem\u003eblack circles\u003c/em\u003e), and separately for single-compartment ones involving the PA (\u003cem\u003ered circles\u003c/em\u003e), or the capillary (\u003cem\u003eblue\u003c/em\u003e \u003cem\u003ecircles\u003c/em\u003e). Multicompartmental dilations were significantly reduced in IP3R2KO mice, whereas single compartment dilations confined to the capillary were increased (First level statistical analysis: Friedmans ANOVA: WT: p=4.7x10\u003csup\u003e-6\u003c/sup\u003e, X\u003csup\u003e2\u003c/sup\u003e=24.5; KO: p=0.00032, X\u003csup\u003e2\u003c/sup\u003e=16.3. Second level analysis: Mann-Whitney test: WT vs KO: multicompartmental dilations: p=0.049; single compartment involving: the PA: p=0.52; the capillary: p = 0.012). The number of dilations/FOV analyzed in WT was 7.55±1.79 in N=20 FOVs in 6 mice; in IP3R2KO: 9.67±2.52 in N=14 FOVs in 4 mice.\u003c/p\u003e\n\u003cp\u003eG.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Temporal progression of tone-induced multicompartmental dilations arriving from outside the FOV in WT and IP3R2KO mice. WT: \u003cem\u003eopen circles\u003c/em\u003e; IP3R2KO: \u003cem\u003esolid circles\u003c/em\u003e. Representation as detailed in Fig. 5C, according to the starting compartment: \u003cem\u003ered\u003c/em\u003e, PA; \u003cem\u003ecyan\u003c/em\u003e, sphincter; \u003cem\u003eblue\u003c/em\u003e, capillary. Data are normalized to the arrival of the dilation in the FOV, but the average start point after the tone stimulation for each compartment is reported on the y-axis. The spread of the dilations to and across the sphincter was delayed in IP3R2KO mice (Kruskal Wallis followed by Mann-Whitney test; WT vs KO: (\u003cem\u003etop\u003c/em\u003e) for dilations starting at PA: onset time at sphincter p = 0.55; at capillary p =0.019. N= 43 dilations in WT and 21 dilations in IP3R2KO; (\u003cem\u003emiddle\u003c/em\u003e) for dilations starting at sphincter: onset time at PA: p=0.82; at capillary p=0.37. N= 17 dilations in WT and 20 dilations in IP3R2KO; (\u003cem\u003ebottom\u003c/em\u003e) for dilations starting at capillary: onset time at sphincter: p = 0.032; at PA: p = 0.19. N= 29 dilations in WT and 17 dilations in IP3R2KO. Data from 20 FOVs in 6 WT mice and form 14 FOVs in 4 IP3R2KO mice.\u0026nbsp;\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6539397/v1/ba5daf513ce81edfe72ba97a.png"},{"id":83016616,"identity":"58b5ac9d-27f3-4389-8c98-1bd89f2524f4","added_by":"auto","created_at":"2025-05-19 06:32:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":199192,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of the mode of progression of vascular dilations at upper cortical junctions under the control of astrocyte Ca2+ activity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Origin and direction of dilations at any given PA-capillary junction with respect to the areas of neuronal excitation.\u003cem\u003e Top\u003c/em\u003e: case in which the excitation (\u003cem\u003egrey regions\u003c/em\u003e) occurs exactly in cortical areas around the PA (\u003cem\u003ered\u003c/em\u003e). In this situation, the NVC is either (a) initiated in the underlying layers by the excitation of layer IV-VI neurons or (b) arrives from the surface vessels due to widespread excitation. Either way, the dilation is perceived to arrive at the junction (\u003cem\u003edashed-line rectangle\u003c/em\u003e) from the PA (\u003cem\u003ered arrows\u003c/em\u003e). \u003cem\u003eBottom\u003c/em\u003e: when the neuronal excitation (\u003cem\u003egrey regions\u003c/em\u003e) occurs in regions close to, but not coincident with, the area directly supplied by the PA (\u003cem\u003ered\u003c/em\u003e), the dilatory response arrives at the junction (\u003cem\u003edashed-line rectangle\u003c/em\u003e) from the capillary compartment (c, \u003cem\u003eblue arrow\u003c/em\u003e) and will first recruit either the capillary (\u003cem\u003eblue\u003c/em\u003e) or the pre-capillary sphincter (\u003cem\u003ecyan\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Illustration of how astrocyte Ca2+ activities at strategic locations regulate the spread of dilations across the sphincter between the PA and the 1\u003csup\u003est\u003c/sup\u003e order capillary. Astrocytes (\u003cem\u003egreen\u003c/em\u003e) regulate the progression of dilations with local and short-lasting Ca2+ activities in the end-feet enwrapping the pre-capillary sphincters (\u003cem\u003eyellow\u003c/em\u003e). This regulation occurs independent if dilations arrive from the PA in deeper cortical layers (a,\u003cem\u003e red arrow\u003c/em\u003e), or superficial layers (b, \u003cem\u003ered arrow),\u003c/em\u003e or from activity along the capillaries (c, \u003cem\u003eblue arrow\u003c/em\u003e), as detailed in panel A.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6539397/v1/042fca48be247b7316ddb789.png"},{"id":85741307,"identity":"bf532e4f-3811-48cf-90fc-5e77b028b188","added_by":"auto","created_at":"2025-07-01 08:54:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4605043,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6539397/v1/1afa283b-0114-4eb4-9af2-182e29882517.pdf"},{"id":83016640,"identity":"b352f669-0f28-4e9b-ae10-3284cb8efb26","added_by":"auto","created_at":"2025-05-19 06:32:42","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":37687738,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Videos.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVideo V1:\u003c/strong\u003e \u003cem\u003e3D-FOV z-stack along the PA, 90-360 micrometers below cortex surface, showing astrocytes at two capillary junctions with sphincter indentations\u003c/em\u003e. The movie is from the auditory cortex of an \u003cem\u003eEGFP-GFAP\u003c/em\u003e mouse and depicts a PA with two 1\u003csup\u003est\u003c/sup\u003e order capillary branches\u003cem\u003e. Red:\u003c/em\u003e masked vessel lumen; \u003cem\u003egreen\u003c/em\u003e: EGFP signal showing position of the astrocytes. Scale bar: 50 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVideo V2:\u003c/strong\u003e \u003cem\u003e3D-FOV scan of an astrocyte at PA-capillary junction shows multiple points of contact with capillaries and sphincter\u003c/em\u003e. The movie depicts a 3D stack from 250-330 µm below the auditory cortical surface and highlights the astrocyte end-feet coverage of a 1\u003csup\u003est\u003c/sup\u003e order capillary branch junction on the PA. The astrocyte is in contact with the vessel at numerous positions. \u003cem\u003eRed:\u003c/em\u003e masked vessel lumen; \u003cem\u003ewhite\u003c/em\u003e: mean cumulative average of GFAP-EGFP fluorescence levels, from which the masks of the astrocyte structures are obtained; \u003cem\u003egreen\u003c/em\u003e: masked astrocyte process structures (kernel size: 1 µm in Imaris) with the relatively largest and brightest highlighted (\u003cem\u003egreen arrow\u003c/em\u003e); \u003cem\u003eyellow\u003c/em\u003e: masked astrocyte structures with end-feet regions touching the capillary surface (\u003cem\u003eyellow arrow\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVideo V3:\u003c/strong\u003e \u003cem\u003eComparing a multicompartment dilation to a dilation that does not spread from capillary to PA\u003c/em\u003e. The movie depicts a 3D-FOV from a stack acquired 95-125 µm below the auditory cortical surface. The movie shows in sequence the average vascular and Ca2+ responses for, first, a multicompartmental (\u003cem\u003eleft\u003c/em\u003e) and, second, a monocompartmental dilation (\u003cem\u003eright\u003c/em\u003e) both beginning in the capillary bed. In parallel, during \u0026nbsp;each dilation event, on the side appears a zoom-in of the activity in the region around the sphincter. \u0026nbsp;\u0026nbsp;Towards the end of the movie, the two dilation events are shown comparatively side-by-side. The dilation shown as first, on the \u003cem\u003eleft, \u003c/em\u003eprogresses across the sphincter to the PA to become multicompartmental, whereas the one shown as second, on the \u003cem\u003eright,\u003c/em\u003e remains within the 1\u003csup\u003est\u003c/sup\u003e capillary bed. \u003cem\u003eRed\u003c/em\u003e: shows the masked average changes in vessel lumen; \u003cem\u003egreen\u003c/em\u003e: shows the mean GFAP-GCaMP6f fluorescence levels outlining perivascular astrocyte structures; \u003cem\u003epseudocolor\u003c/em\u003e: shows the normalized Ca2+ activity levels representing mean Ca2+ changes per voxel in the two types of dilation events. \u003cem\u003eWhite circles:\u003c/em\u003e appear at the level of the vessel segment that is about to dilate and remain there until the segment is constricted again. A thin \u003cem\u003ecyan line\u003c/em\u003e outlines the pre-dilation Ca2+ ROI. \u003cem\u003eCyan circles:\u003c/em\u003e identify pre-dilation Ca2+ activity events at the sphincter in the last second before dilation. Note the presence of Ca2+ activities also during the dilation.\u003c/p\u003e","description":"","filename":"SphinctPaperVideos.zip","url":"https://assets-eu.researchsquare.com/files/rs-6539397/v1/dec7a2cb0663fdce81018c6c.zip"},{"id":83017503,"identity":"1c343228-2946-47f4-a2c8-cc5940fc2273","added_by":"auto","created_at":"2025-05-19 06:40:41","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3104376,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6539397/v1/bb712dc85f12b8668512c072.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Fast 3D imaging in the auditory cortex of awake mice reveals that astrocytes control neurovascular coupling responses at arteriole-capillary junctions.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAdequate regulation of cerebral blood flow is the basis of healthy brain function. The neurovascular coupling (NVC) response ensures sufficient oxygen and energy during increased neuronal activity and, when impaired, contributes to pathophysiological alterations in several common cerebral diseases \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The blood is recruited to the capillary bed from the brain surface via penetrating arterioles (PAs). The first branch point from the PAs into the capillary bed, the 1st order capillary, also dubbed precapillary arteriole \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e or post-arteriolar transition zone \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, is believed to be critically important for blood flow regulation during NVC \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Intriguingly, the presence of a gating structure, the pre-capillary sphincter, was recently described precisely at this level \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. This structure expresses α-smooth muscle actin in its mural cells (αSMA) \u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, which makes it contractile and capable of modifying vessel diameter. Changes in the sphincter diameter may then control the blood flow to the downstream brain tissue \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Astrocytes\u0026rsquo; end-feet continuously cover all the blood vessel segments and could, in principle, impose control at each level \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Intracellular Ca2\u0026thinsp;+\u0026thinsp;increases in astrocytes can induce the release of vasoactive factors \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, yet the role of astrocytes in blood flow regulation remains controversial. Notably, it is unclear if Ca2\u0026thinsp;+\u0026thinsp;elevations in astrocytes are rapid and prevalent enough to contribute to blood flow recruitment \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. To this point, different studies produced discrepant results: some reported that astrocytes recruit blood flow, but exclusively at the capillary level \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, others that astrocytes instead regulate PA diameter, but only during extended sensory experiences \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, and others that astrocytes do not exert any Ca2+-dependent regulation on blood flow. The latter conclusion was based on the authors\u0026rsquo; inability to observe any changes in the NVC response in mice lacking IP3 receptor type-2 (IP3R2), the IP3R isoform thought to drive most of the intracellular Ca\u003csup\u003e2+\u003c/sup\u003e elevations in astrocytes \u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Independent of their different conclusions, all the above studies looked at blood vascular dynamics in a simplistic manner, mainly focusing on individual compartments, and disregarding the heterogenous nature of dilations progressing through the vascular bed \u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Moreover, different studies used different imaging conditions to investigate astrocyte intracellular Ca\u003csup\u003e2+\u003c/sup\u003e dynamics with respect to size, timing, and frequency of the recorded Ca2\u0026thinsp;+\u0026thinsp;signals \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan additionalcitationids=\"CR23 CR24 CR25 CR26 CR27 CR28 CR29 CR30\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Last but not least, most studies were performed in anesthetized mice in which both astrocytic Ca\u003csup\u003e2+ 32,33\u003c/sup\u003e and blood flow responses \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e are severely dampened. Overall, these several shortcomings did not permit to draw firm conclusions on the role of astrocyte Ca2\u0026thinsp;+\u0026thinsp;signaling in the control of brain hemodynamics.\u003c/p\u003e \u003cp\u003eIn the present study, we aimed to overcome those pitfalls. To start, we performed our experiments in awake mice, which permitted us to consider the impact of the mouse activity state on the NVC response and the heterogeneity of the dilation patterns in different inherent conditions. Secondly, we used a 3D\u0026thinsp;+\u0026thinsp;t two-photon imaging approach \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, to our knowledge for the first time in NVC studies. With this approach, we could monitor entire regions (30\u0026ndash;150 \u0026micro;m in the z-axis) at the branching point between penetrating arterioles and capillary bed and investigate simultaneously the vascular responses at the levels of PA, 1st order capillary, and connecting sphincter, while also monitoring the Ca2\u0026thinsp;+\u0026thinsp;activity in the surrounding astrocyte structures. Thirdly, we imaged these stacks at high speed (10 Hz), to capture the full range of astrocytic Ca2\u0026thinsp;+\u0026thinsp;dynamics, including fast and local signals.\u003c/p\u003e \u003cp\u003eWe report the existence of different patterns of natural dilations at the PA-capillary junctions in the mouse auditory cortex, which depends on the inherent activity of the mouse. Part of the observed dilations involved both PA and capillary, whereas others remained confined to only one compartment. Astrocytes occasionally underwent large Ca2\u0026thinsp;+\u0026thinsp;activations, but they also often displayed smaller Ca2\u0026thinsp;+\u0026thinsp;rises in the end-feet enwrapping the pre-capillary sphincters. Such local Ca2\u0026thinsp;+\u0026thinsp;rises preceded spread of the dilations and specifically correlated with multicompartment dilations. Both the astrocyte Ca2\u0026thinsp;+\u0026thinsp;activity and the spread of dilations across the sphincter were affected in mice lacking IP3R2 (IP3R2KO). When mice were subjected to auditory stimulation, dilation patterns were analogous to those seen in naturally behaving mice and similarly depended on astrocyte Ca2\u0026thinsp;+\u0026thinsp;activity. We conclude that pre-capillary sphincters play a central role in the bi-directional spread of dilations between vascular compartments and that astrocyte Ca2\u0026thinsp;+\u0026thinsp;signaling controls their function locally.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e3D imaging of vessel dilations and astrocyte Ca2+ activity in the auditory cortex of awake mice: observations along the descending PA paths.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated the astrocytic contribution to hemodynamic responses in the auditory cortex of awake mice (Figure 1A) focusing at first on the PA tracts. We mapped the surface vessels to identify appropriate positions for imaging, where PAs dive into the tissue, i.e., where in the vascular tree, the freshly oxygenated blood enters the cortex (Figure 1B). By 2-photon imaging, we then studied astrocyte and blood vessel dynamics along the descending PA paths. Astrocyte Ca2+ activity was visualized using the\u0026nbsp;\u003cem\u003eGFAPCreERT2:GCaMP6f\u003c/em\u003e mouse strain that enables astrocyte-selective, inducible expression of the green Ca2+ indicator, GCaMP6f, upon tamoxifen (TAM) injections (Methods). In parallel, PA dynamics were revealed by loading blood vessels with red fluorescent dye (Texas Red-dextran) to highlight their lumen. In this first set of experiments, we positioned our three-dimensional fields of view (3D-FOV) along the PAs to cover descending tracts up to 280 \u0026micro;m in the z dimension (Figure 1C). Our fast 3D imaging approach \u003csup\u003e32\u003c/sup\u003e enabled us to monitor vascular and astrocyte dynamics quasi-simultaneously in multiple focal planes along the z-axis (Figure 1C, Sup. figure S1, Video V1). Our investigation of the astrocyte dynamics focused on the Ca2+ activity within the end-feet, as they represent the points in which the astrocytes contact the contractile vascular cells. The end-feet regions of interest (ROIs) were automatically defined as the structures present within 2 \u0026micro;m from the vessel surface in each z-plane and repositioned during vascular dynamics, according to the vessel diameter changes (Methods and Sup. figure S1). In this way, we followed astrocyte end-foot Ca2+ activity along single PAs while tracing the volume changes occurring in the corresponding PA segments (Figure 1D). Some end-foot Ca2+ activities spread far along the PA and covered up to 205 \u0026micro;m in the z-dimension, whereas others stayed local and were seen just in a single focal plane (Figure 1E, Sup. figure S2A). As for the spontaneous PA dilations that we observed, only about half (324/668) were accompanied by Ca2+ activity in the end-feet along the PA. In these cases, the spread of PA dilations was larger than in the absence of the astrocyte Ca2+ activity and reached further down inside the cortex (Figure 1F). Moreover, it correlated with the spread of the end-foot Ca2+ activity (Sup. figure S2B). Since large astrocytic Ca\u003csup\u003e2+\u003c/sup\u003e elevations are seen during mouse locomotion \u003csup\u003e22,32,35\u003c/sup\u003e, we in parallel recorded EMGs with electrodes implanted in the mice\u0026rsquo;s neck muscles (Figure 1D, bottom). We found that the spread of the astrocytic end-foot Ca2+ activity in the z-direction almost doubled during mouse movements with respect to when the mouse was at rest, changing, on average, from 23\u0026plusmn;3\u0026micro;m to 50\u0026plusmn;7\u0026micro;m (Figure 1E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn agreement with previous reports, we found that not all mouse movements triggered an astrocytic Ca2+ activity in the end-feet along the PA \u003csup\u003e22\u003c/sup\u003e. Still, \u0026gt;70% of PA dilations seen during mouse movements (184/262) were associated to increased Ca2+ activity in the astrocyte end-feet, a higher proportion than the one seen in the resting mouse. As during rest, the PA dilations during movement that coincided with Ca2+ increases in the end-feet, also spread significantly further than those occurring solitary (Figure 1G). These data suggest that large end-foot Ca2+ activity influences PA dilations without being a necessary prerequisite for the dilations to occur.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3D imaging of vessel dilations and astrocyte end-foot Ca2+ activity in the auditory cortex of awake mice: observations at the PA-capillary junction.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhile PA dilations increase blood flow distribution to many cortical layers, a second and more specific level of regulation occurs at the capillary branching points at different cortical depths \u003csup\u003e36\u003c/sup\u003e. An increased blood flow in one of the 1\u003csup\u003est\u003c/sup\u003e order capillary branches enhances the delivery to areas downstream of the branch \u003csup\u003e5\u003c/sup\u003e. Recently, an investigation in the barrel cortex described a pre-capillary sphincter at the junction with the PA \u003csup\u003e6\u003c/sup\u003e. Therefore, we decided to focus our study of NVC in the auditory cortex on this junction (Figure 2A). Via the luminal red fluorescence, we visualized the sphincter as a narrowing of the lumen proximal to the PA. In addition, in 50% of the junctions (n = 20), we noticed the presence of a bulb at the capillary edge, likely generated by a local reduction in the coverage by contractile cells \u003csup\u003e37\u003c/sup\u003e. The shape of the PA-capillary junctions was variable, and some junctions had a less obvious indentation. Nonetheless, nearly all junctions showed some degree of pre-constriction and, irrespective of the shape, it is known that the entire 1\u003csup\u003est\u003c/sup\u003e order capillaries are contractile \u003csup\u003e6,37\u003c/sup\u003e. Therefore, we decided to define all these connections between a PA and the 1\u003csup\u003est\u003c/sup\u003e order capillary as sphincters. Initially, we investigated the position of the astrocytic structures at the vascular junction using \u003cem\u003eGFAP-EGFP\u003c/em\u003e mice with green-fluorescent astrocytes \u003csup\u003e38\u003c/sup\u003e. The astrocytes were localized at the branching points (Figure 2A and Video V1), covering most of the 1\u003csup\u003est\u003c/sup\u003e order capillary up to the sphincter and in contact with it (Figure 2B and Video V2). To detect the potential role of these astrocytes, we then performed dynamic studies in TAM-treated \u003cem\u003eGFAPCreERT2:GCaMP6f\u003c/em\u003e mice, centering our 3D-FOV at the level of the PA-1\u003csup\u003est\u003c/sup\u003e order capillary junction. In these experiments, we selected 3D-FOVs much smaller than those utilized for studying dilations along the PAs and took focal planes at 1\u0026micro;m z-steps to ensure continuity in the local imaging around the vasculature (Sup. Figure S3). Thanks to fast two-photon scanning, we could investigate quasi-simultaneously the dilation patterns of the two vascular compartments and the rapid Ca\u003csup\u003e2+\u003c/sup\u003e changes in astrocyte end-feet ROIs at both the arteriole and capillary, i.e. 1\u003csup\u003est\u003c/sup\u003e order capillary, levels. This setting enabled us to define location and temporal sequence of the astrocyte Ca2+ elevations relative to the vascular volume changes. In previous studies in awake mice, when recording from 3D-FOVs of dimensions similar to those used here, we had detected two different types of activities in astrocytic end-feet, involving either local or much larger Ca2+ events \u003csup\u003e32\u003c/sup\u003e. The former were asynchronous and small (\u0026micro;m-scale), generally restrained within a single end-foot, whereas the latter expanded widely to occupy several end-feet in different z-planes. In the present recordings around the vascular junction, we similarly observed small and large events. Concerning the large Ca2+ elevations, they were rather infrequent, occurring every few minutes, and invaded \u0026ge;75% of the end-foot ROIs present in all z-planes of our 3D-FOV. We defined these events \u0026ldquo;large Ca2+ activities\u0026rdquo; (Figure 2C) and found that 71\u0026plusmn;7% of them overlapped in time with mouse movements (Sup. figure S2C). Since large astrocytic Ca\u003csup\u003e2+\u003c/sup\u003e activities are known to occur during arousal states driven by noradrenaline release \u003csup\u003e39\u003c/sup\u003e, and noradrenaline release is signaled by the animal\u0026rsquo;s pupil expansions \u003csup\u003e40-43\u003c/sup\u003e, we monitored the mouse pupil diameter during the imaging sessions using an IR-camera. Thereby, we verified if large astrocyte Ca2+ activities coincided with pupil expansions. Indeed, while most pupil expansions occurred without a large astrocytic Ca\u003csup\u003e2+\u003c/sup\u003e activity, when such an astrocyte activity took place, it almost always (92 \u0026plusmn; 13% of the cases) coincided (within 1 sec) with a pupil expansion (Sup. figure S2D). However, as observed for the dilations along the PA path, the dilations around the junction were not triggered just by animal movement or by large astrocytic Ca2+ activities. Indeed, in the majority of cases (75.4\u0026plusmn;5.9%) dilations occurred when the mouse was at rest and without being accompanied by any large astrocytic Ca2+ activity. Looking at the temporal sequence of the events, when a large astrocytic Ca2+ activity was associated with a mouse movement, the Ca2+ rise in the astrocyte started after the onset of the movement (by 1.0 \u0026plusmn; 1.25s, Sup. figure S2E and S2F). In some cases, it preceded the onset of the dilation, but, on average, it rose after the vessel dilation (by 2.3 \u0026plusmn; 0.84s, Figure 2D). Therefore, this type of large end-foot astrocytic Ca2+ activity appears to be associated with movements and arousal states but not to be responsible for triggering the dilations seen at the PA-1\u003csup\u003est\u003c/sup\u003e order capillary junctions during the mouse movements. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBidirectional spread of dilations at the pre-capillary sphincter: dependence on the activity and arousal state of the awake mouse.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring our repeated 3D-imaging sessions which were in most cases of 2 min duration, we detected many spontaneous vessel dilation events at the junction (Sup. Figure S3). Initially we thought that such events were part of the NVC response evoked by the natural sound perception, increasing excitation and metabolic needs of the auditory neurons. However, we noticed that the dilation events varied largely depending on whether they occurred when the mouse was moving or resting, as well as if a large astrocytic Ca\u003csup\u003e2+\u003c/sup\u003e activity took place coincidentally or not (Figure 2E and F). Notably, dilations occurring when the mouse was moving lasted significantly longer (mean duration: 7.57 \u0026plusmn; 1.32 s) than those in the resting mouse (3.06 \u0026plusmn; 0.27 s, Figure 2E and S2G). Moreover, dilations in the moving mouse were in most cases (77%) multicompartmental, involving both the PA and the capillary branch (Figure 2F). In contrast, in the resting mouse, ~50% of the dilations were mono-compartmental, confined to the vessel tract either upstream or downstream the sphincter. When dilations occurring in the moving mouse were accompanied by a large astrocytic Ca2+ activity, they lasted even longer (Figure 2E) and were almost always (92% of cases) multicompartmental (Figure 2F). Also in the resting mouse, coincidence of a large astrocyte Ca2+ event, increased the duration of dilations (Figure 2E), but without significantly increasing the proportion of multicompartmental dilations (Figure 2F). These data indicate that the animal movements, and to a lesser degree the arousal state, shape the hemodynamic responses in the auditory cortex, prolonging dilations at this junction in time and space.\u003c/p\u003e\n\u003cp\u003eNext, we analyzed the origin and direction of the dilations in the different conditions. Since only 51\u0026plusmn;5 % of the dilations during rest were multicompartmental, i.e., involved both PA and capillary, we evaluated which part of the vascular bed dilated alone in the remaining cases (Figure 2G). We found that monocompartmental dilations more frequently involved the capillary branch (37%\u0026plusmn;6% of all the dilations at rest), and only in 12\u0026plusmn;3% of cases the PA (Figure 2G). This heterogeneity was also reflected in the origin of the multicompartmental dilations: some of them first appeared at the PA, others at the capillary level and others at the junction. The latter ones were the only dilations initiating within our 3D-FOV; they represented about one third of all the multicompartmental dilations, and in most cases started at the sphincter, and just in a few cases at the bulb. The remaining dilations mostly originated outside the 3D-FOV; in 33 \u0026plusmn; 5% of cases they were seen coming from the PA, and in 32 \u0026plusmn;6% of cases from the capillary bed (Figure 2H). This heterogeneity in the sites of origin of the multicompartmental dilations did not depend on whether the mouse moved or was at rest, or the dilation was accompanied by a large astrocytic Ca2+ event or not. The site of origin did not influence the characteristics (volume and duration) of the dilations in each vascular compartment. In contrast, these were modulated by both the mouse\u0026apos; behavioral state and the occurrence of large Ca2+ activity in astrocyte end-feet (Sup. figure S4). On the other hand, the fact that we observed both monocompartmental dilations stopping at the sphincter and multicompartmental ones crossing it in the resting mouse (Figure 2I), indicated to us that the pre-capillary sphincter is a site of control of the dilations\u0026rsquo; spread. Such control was overcome during animal movements or arousal states, notably when these conditions were associated with large astrocytic Ca\u003csup\u003e2+\u003c/sup\u003e activity, leading to the recruitment of both the upstream and the downstream vascular compartments, independent of the initiation site and the direction of the dilation. Based on these findings, we focused our next investigations on the astrocytic activity around the sphincter.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLocal Ca\u003csup\u003e2+\u003c/sup\u003e activity in astrocytic end-feet at the sphincter precedes the arrival of multicompartment dilations.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe considered that, when evaluating the role of astrocytes in the NVC response to auditory activation, the large astrocytic Ca2+ activities described above are likely confounding factors. According to recent data, they would be involved in associating brain states \u003csup\u003e43\u003c/sup\u003e or in integrating past events \u003csup\u003e44\u003c/sup\u003e, functions that are not directly relevant to NVC initiation. Therefore, we decided to focus our next investigations on dilations and astrocyte Ca2+ elevations that are not associated to mouse movements/arousals, i.e. that occur during the animals\u0026apos; resting periods (Figure 3). We hypothesized that astrocytes could exert their NVC regulation at the junction level and focused our attention on the astrocyte Ca2+ activity related to multicompartmental dilations spreading across the sphincter. By looking at time-averaged 3D images, we found heterogeneous local Ca2+ activities in the astrocyte end-feet and processes around the junction, which did not spread widely as for the large Ca2+ activities shown in Figure 2C. Following this initial observation, we decided to develop an automated analysis of the astrocytic end-feet Ca2+ activity in relation to the onset of spontaneous dilations. To establish the spatial-temporal sequence of these local astrocyte Ca2+ events with respect to the dilation events in an unbiased and accurate way, we aligned the 3D astrocyte Ca2+ imaging data to the onset of all the spontaneous dilations. We then identified voxels of interest (VOIs) with increased Ca\u003csup\u003e2+\u003c/sup\u003e activity in the astrocytic end-feet (Methods and Sup. figure S5) and evaluated the probability that a Ca\u003csup\u003e2+\u003c/sup\u003e increase occurred within these VOIs prior to a given dilation pattern (Sup. figure S5). Thereby, we identified VOIs that consistently responded in a defined temporal relation with a dilation (Figure 3A, Sup. figure S5), i.e., we identified the position and timing of pre-dilation Ca\u003csup\u003e2+\u003c/sup\u003e activities in the astrocytic end-feet. Despite a certain variability in the VOIs positions and in the time-course of the related Ca2+ activities (Figure 3A and Videos V3), pre-dilation Ca2+ responses were consistently found primarily in the end-feet covering the sphincter (Figure 3B) and occurred at increased frequency when dilations were multicompartmental and spread across the sphincter (Sup. figure S6A and Video V3). These Ca2+ responses did not depend on a specific origin and direction of the multicompartmental dilations (Figure 3B). Localized Ca2+ activity was also present in end-feet along the capillaries (Figure 3A and B). In contrast, we rarely saw pre-dilation Ca2+ activity in the end-feet on the PAs (Figure 3B). Regardless of their position, all the pre-dilation Ca\u003csup\u003e2+\u003c/sup\u003e signals were local (9.7 \u0026plusmn;3.8 \u0026micro;m z-axis) and short-lasting (0.98 \u0026plusmn; 0.42 s) (Figure 3C). The interval by which pre-dilation astrocyte Ca2+ elevations at the sphincter preceded onset of dilations was longer when dilations arrived from upstream or downstream locations than when dilations started at the sphincter itself (Figure 3D and E). However, this longer anticipation might just depend on the fact that dilations coming from PA or capillaries initiated outside the FOV, i.e., earlier than observed. This been said, we observed that when a dilation that started at the capillary bed level and arrived at the sphincter was associated with Ca2+ events in both compartments, the astrocyte Ca2+ activity occurred first at the sphincter and then at the capillary (Figure 3D and F). This sequence did not visibly depend on spreading of the Ca2+ from the sphincter along the capillary (sup. Figure S6B). Rather, the two groups of end-foot astrocyte Ca2+ events appeared to be temporally separated and independent (Figure 3A, D-F and Sup. figure S6C), suggesting that Ca2+ activity at the sphincter is the primary pre-dilation event. Overall, the pre-dilation astrocyte Ca2+ activity at the sphincter showed anatomical and temporal characteristics in line with a possible role in controlling spreading of dilations across the sphincter.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe astrocytic pre-dilation Ca\u003csup\u003e2+\u003c/sup\u003e activity in end-feet at the sphincter is reduced in IP3R2KO mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe then determined if the local pre-dilation astrocytic Ca\u003csup\u003e2+\u003c/sup\u003e activity was IP3R2-dependent by repeating experiments in IP3R2KO mice that display reduced astrocyte end-foot Ca2+ activity in the \u003cem\u003ein vivo\u003c/em\u003e awake condition \u003csup\u003e45\u003c/sup\u003e. Indeed, in this group of mice, we observed a significant reduction in the number of local pre-dilation Ca2+ signals in astrocyte end-feet compared to wild-type mice. This reduction was seen in particular at the sphincter (Figure 4A-C, Sup. figure S7), while it was not significant at the PA and capillary (Sup. figure S7A and B). The reduction was most evident in association with multicompartmental dilations, independent of whether they arrived from the PA or the capillary (Sup. figure S7C), it involved particularly the earliest pre-dilation Ca\u003csup\u003e2+\u003c/sup\u003e activity, namely the activity starting \u0026gt;1 s before dilation onset (Figure 4D; Sup. figure S7D) and occurring closest to the sphincter (Figure 4E; Sup. figure S7D). Overall, these results support the presence of an early, pre-dilation, IP3R2-dependent, Ca2+ increase in astrocytic end-feet, notably in the end-feet surrounding the pre-capillary sphincter, prior to the spread of dilations beyond the branch point. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpread of dilations across the sphincter is impeded in IP3R2KO mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next assessed whether the reduced pre-dilation astrocyte Ca2+ activity at the sphincter in IP3R2KO mice had an impact on the vascular dynamics around the sphincter. In WT mice, we had observed that the pre-dilation Ca2+ activity was associated in particular to multicompartmental dilations, independent if they arrived from the PA or the capillary bed. Supporting a role for the astrocyte activity in controlling those dilation events specifically, we counted a decreased total number of multicompartmental dilations (-7%) and a proportionally increased number of single compartment dilations (+10%) in IP3R2KO compared to WT mice (Sup. figure S8A). \u0026nbsp;In addition, we found that the proportion of multicompartmental dilations arriving at the junction from outside the 3D-FOV (either from the PA or the capillary compartment) was reduced in IP3R2KO mice, while the proportion of those initiated within the 3D-FOV (at either the sphincter or bulb) was increased (Figure 5A). Notably, it was the number of monocompartment dilations arriving from the capillary branch which increased (Sup. figure S8A), suggesting that less of them could spread across the sphincter to the PA in IP3R2KO mice (Figure 5B). We then discovered a second significant effect of IP3R2KO when assessing the speed by which the residual multicompartmental dilations spread from one compartment to the next one (Figure 5C). In WT mice, dilations spread from PA to capillary, on average, in 488 \u0026plusmn; 18 ms, and, in the opposite direction, from capillary to PA, in 667 \u0026plusmn; 17 ms (Figure 5C). Thus, the speed of compartmental transition was significantly higher when a dilation progressed from PA to capillary (608 \u0026plusmn; 57 \u0026micro;m/s) than in the opposite direction (112 \u0026plusmn; 5 \u0026micro;m/s, Sup. figure S8B). In IP3R2KO mice, dilations arriving from the PA took on average 260 ms longer in engaging the capillary than in WT mice. Likewise, dilations arriving from the capillaries, were delayed by 140 ms in crossing the sphincter and reaching the PA (Figure 5C, Sup. figure S8B). In the case of dilations initiated at the pre-capillary sphincter, spreading to the capillary was also delayed (by 110 ms) in IP3R2KO compared to WT mice (Figure 5C, Sup. figure S8B). In all cases, the slower spread of dilations was not due to local structural changes occurring in IP3R2KO mice, such as an increased distance separating PA and capillary structures compared to WT mice (Sup. figure S8C). These disturbances in the vascular dynamics of IP3R2KO mice had not been described before. In fact, several publications reported no changes in blood flow regulation in IP3R2KO with respect to WT mice \u003csup\u003e16-18\u003c/sup\u003e. Interestingly, if we performed our analysis without taking into account intercompartmental spread and directionality of the observed dilations, but simply adding all the dilations together, we also obtained that average frequency (Sup. figure S9A) was not modified in IP3R2KO compared to WT mice. While all the above measures were done in the resting mouse, we observed additional effects produced by IP3R2KO during spontaneous movements of the animals. During movements we counted fewer dilation events (Sup. figure S9A) as well as a reduced intercompartmental spread of the dilations compared to WT mice (Sup. figure S9B-C). Thus, the astrocyte Ca2+ regulation of vascular dynamics appears to operate both during resting periods and movements of the mice. Overall, in IP3R2KO mice, both the pre-dilation Ca\u003csup\u003e2+\u003c/sup\u003e activity in astrocytic end-feet surrounding the sphincter and the speed and efficiency of spread of the dilations across the sphincter were reduced. The latter phenomenon is likely responsible for the failed recruitment of additional vessel compartments and the reduced number of multicompartmental dilations, with a qualitative alteration in blood redistribution. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuditory stimulations induce dilations and local astrocytic Ca2+ activity at the sphincter resembling those occurring naturally in resting mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur investigations so far focused on naturally-occurring dilations at the pre-capillary sphincter\u0026nbsp;in the resting animal, excluding contributions by movement/arousal-induced mechanisms. However, these investigations did not directly demonstrate that the observed vascular responses belong to NVC, nor excluded that they depend on factors generated by sound perception other than the local neuronal activation \u003csup\u003e46\u003c/sup\u003e. Thus, next we induced vascular responses via direct auditory stimulation and compared their properties to those of the \u0026ldquo;natural\u0026rdquo; dilations. The auditory cortex is formed by zones that gradually change sensitivity to different pitches \u003csup\u003e47\u003c/sup\u003e. Therefore, we tested several tones and used intrinsic optical signaling (IOS) detection to determine the tonalities responsible for stimulating the cortical region exposed in our craniotomy. With IOS, we could also establish rough tonality maps and identify where, in a given cortical area, tone frequencies activated maximum blood flow increase (Figure 6A). To relate tone stimulations (10 sec) and associated IOS-blood flow responses (IOS maps) to local dilations at PA-capillary junctions (Figure 6A-B), we used combinations of three tones as stimulus, predicting that they would trigger a response in at least one of the cortical regions present in our cranial window. Initially, we investigated the correlation between wide-field IOS maps and individual vessel regulations at a locus in the auditory cortex containing four PA-capillary junctions. We sampled in parallel the individual vessel responses at the four branch points (one in layer I and three in layer II/III) present on three PAs entering the tissue at different cortical locations. The corresponding dilations differed in origin and directionality, but all had duration in line with the strength of the IOS signal detected in the same area: longer-lasting dilations were associated with tones producing strong local IOS responses, and shorter-lasting ones with tones producing weak IOS responses (Figure 6B). Noteworthy, the layer I junction showed less tone specificity than the layer II/III junctions, possibly because the flow pattern in superficial layers is less restrictive than in the deeper layers downstream. We found fewer junctions in the loci that we selected for the next observations. To incorporate all of them in our study, we tried a classification based on the individual IOS maps, i.e., we tried to link the observed dilation pattern at a given PA-capillary junction to the strength of the related tone-evoked IOS response. In particular, we considered the position in which the PA entered in the cortex with respect to the IOS map, i.e., whether its path and the related PA-capillary junction were at the center of the IOS map or displaced from it (Figure 6C). We found that most of the tones induced IOS responses somewhere in our cranial window, but not all these responses were centered on the region where a PA entered the cortex. When a tone induced a strong IOS response in the area of a junction (Figure 6C left), most of the dilations were long-lasting and quickly progressed across the pre-capillary sphincter to become multicompartmental, independent if they began at the capillary or the PA. In cases when the junction area was not at the center of the tone-evoked IOS response, we could still observe dilations in our 3D-FOV, but they mainly arrived from the capillaries (rarely from the PA) or originated at the sphincter (Figure 6C right). Tone-induced dilations varied largely in duration,\u0026nbsp;ranging from \u0026gt;60 seconds to \u0026lt;1 sec (Sup. figure S10A). If the junction area was located at the center of the IOS response, dilations were at least 5 s-long and progressed beyond the sphincter to an extent proportional to their duration. The shortest ones were primarily restricted to the capillary bed, while the longest ones always propagated to all compartments (Sup. figure S10B), resembling the \u0026ldquo;natural\u0026rdquo; dilations in the resting animal (Figure 2). Also, the proportion of tone-evoked dilations that started at each location (respectively upstream of the sphincter, downstream, or at the sphincter) was analogous to that of the \u0026ldquo;natural\u0026rdquo; dilations (Sup. figure S10C). These observations strengthen the case that naturally occurring dilations in the resting animal directly reflect the auditory experience and represent the associated NVC response.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs a next step, we investigated whether tone-evoked dilations, like \u0026ldquo;natural\u0026rdquo; dilations, were preceded by local astrocyte end-foot Ca\u003csup\u003e2+\u003c/sup\u003e activity. For this analysis, we used the same unbiased automated approach used for extracting the Ca2+ dynamics preceding \u0026ldquo;natural\u0026rdquo; dilations (Figure 3). In this case, our inclusion criterion was less permissive due to the much lower number of tone evoked dilations compared to the \u0026ldquo;natural\u0026rdquo; ones (Sup. figure S5, Methods). Nonetheless, we could consistently identify Ca2+ signals occurring mainly at the pre-capillary sphincter, preceding multicompartmental dilations (Figure 6D, Sup. figure S10D and S10E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe tone-evoked astrocyte Ca2+ responses and the blood vessel dilations are affected in IP3R2KO mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo directly address the impact of the local astrocyte Ca2+ activity on the progression of tone-evoked dilations, we repeated the experiment in IP3R2KO mice. In WT mice, the large majority (79%) of the dilations arriving from either the PA or the capillary compartments were preceded by an astrocytic Ca2+ activity at the sphincter. This proportion decreased to 48% in IP3R2KO mice and was reflected in fewer pre-dilation events (Figure 6E). Similar to what was observed for the spontaneous dilations, in IP3R2KO mice the fraction of tone-evoked dilations that could not cross the sphincter and remained confined to the capillary bed was more than twice that in WT mice (28.7 \u0026plusmn; 10.8% vs. 13.2 \u0026plusmn; 7.4% in WT, Figure 6F). In complement, we observed that the progression of tone-evoked dilations across the sphincter in IP3R2KO mice was slowed down. Dilations arriving from the PA were delayed by 416 \u0026plusmn; 145ms in reaching the capillary. Dilations from the capillary bed were also delayed by 376 \u0026plusmn;208ms, mostly in recruiting the sphincter (Figure 6G). We conclude that IP3R2 deletion has a negative impact on tone-evoked dilations similar to the one seen in naturally occurring dilations. These data indicate that astrocytes, by their local Ca2+ activity at the sphincter, act as physiological regulators of the spreading of dilations between vascular compartments as part of the NVC response to auditory stimulation.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we identified a new role for astrocytes in NVC as regulators of the spread of dilations between the PA and capillary compartments in response to sound perception in the auditory cortex. The astrocyte control is exerted focally, via end-foot Ca2\u0026thinsp;+\u0026thinsp;elevations occurring at the pre-capillary sphincters that connect the two compartments. Such local astrocyte activity precedes the arrival of dilations at the sphincters and controls their contractility, determining how much and how far blood is distributed during cortical activation. We obtained this information in the awake mouse thanks to fast volumetric two-photon imaging of vascular and astrocyte Ca2\u0026thinsp;+\u0026thinsp;dynamics in large FOVs comprising the PA and capillary compartments and the connecting junction. By centering our 3D-FOVs on the vascular tree and the associated astrocytic end-feet and peri-vascular regions, we could capture even the most transient local Ca2\u0026thinsp;+\u0026thinsp;activities and their interplay with the vascular dilations throughout the imaged vascular tracts. Only a minority of preceding studies were performed in the awake mouse, and none of them used fast volumetric imaging. This new approach enabled us to study all the natural dilation patterns occurring in a behaving mouse, and also to establish their origin, directionality, and level of progression between imaged compartments, revealing a previously unappreciated complexity. Thanks to this methodological advance, we could, on the one hand, overcome several of the shortcomings that affected previous work and alimented multi-year controversies on the role of astrocytes in NVC (see Introduction), and on the other, revisit the few recent studies performed in the awake mouse \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, complementing their findings and providing a more comprehensive picture. Among others, we could here dissociate the components of the natural astrocyte and vascular responses in the auditory cortex evoked by sound perception from those depending on the activity state of the mouse during locomotion and arousal. Given the large analogy between natural responses seen in the resting animals and responses evoked by natural tone stimulations, we conclude that the natural responses at rest are directly induced by the sensory experience and most likely represent the physiological NVC response to auditory activation.\u003c/p\u003e\n\u003cp\u003eThe dilation patterns observed in our study differ significantly from those reported in previous work in anesthetized mice, characterized by quite stereotyped hemodynamic responses \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e and less correlation with the neural activity \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Here, in our naturally behaving mice, the dilation patterns were highly heterogeneous and incorporated contextual contributions from the animal\u0026rsquo;s brain state, or inputs from other cortical areas, such as the motor cortex. During our recordings, mice moved and underwent arousals, and these activities were associated with large astrocytic Ca2\u0026thinsp;+\u0026thinsp;events not seen in anesthetized mice \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Such large astrocytic Ca2\u0026thinsp;+\u0026thinsp;elevations were reminiscent of those recently shown to prolong the duration of PA dilations during sustained whisker stimulation in the barrel cortex of awake mice \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Since during arousals, more dilations spread across the pre-capillary sphincters than in the resting mouse, the associated bursts of astrocytic Ca2\u0026thinsp;+\u0026thinsp;activity might function to ensure an abundance of blood flow to the tissue in the aroused state, keeping the neurons metabolically prepared to handle additional auditory stimulations.\u003c/p\u003e\n\u003cp\u003eAn important advantage of our volumetric imaging approach was in its ability to follow the spatial and temporal dynamics of dilations more comprehensively than in past studies. Thereby, we could define sites of origin and direction of the dilations as they progressed along the different vascular compartments. This approach was instrumental to our discovery of the central regulatory role of the pre-capillary sphincters, which was difficult to appreciate in previous work that imaged dilations at either the capillary or the PA level separately. Likewise, fast volumetric imaging was necessary to identify the presence of the local astrocyte Ca2\u0026thinsp;+\u0026thinsp;activity that precedes the passage of dilations from the sphincters.\u003c/p\u003e\n\u003cp\u003eThe spatial dynamics of dilations were investigated in previous work, but without reaching firm conclusions about their origin. In those studies, mostly performed in anesthetized mice, dilations had to be evoked by artificial stimulations. Some authors reported that they initiated at lower cortical levels \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e triggered by activity along the capillaries \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, whereas others that they initiated at upper levels, with the recruitment of the PA from the surface arteries \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThis diversity might reflect the different methods used for evoking the dilatory responses in other studies \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and the fact that a blood flow increase can be initiated by stimulation at any cortical lamina \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. In our study focusing on the PA-capillary junctions in awake mice, we observed a natural variety in the sites of origin of the dilations. Thanks to simultaneous fast imaging of all the relevant compartments, we could capture the specific directionality of these natural dilations, finding that some arrived from capillaries, other from PA, and others initiated at the junction level. Interestingly, a similar variety of originations was recently described in the barrel cortex of awake mice \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. This is not surprising because the two regions are similarly organized for blood flow regulation, with a large number of branching vessels \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, relays in the neuronal circuits, and projections from other cortical areas \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. In brain regions with simpler organization, the observed blood flow responses are more stereotyped \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe different positions in the auditory cortex at which the naturally occurring dilations originated must depend on differences in the modes by which blood flow regulation is recruited, which, in turn, may depend on the diversity of the neuronal circuit relays that project the sensory stimuli to the processing cortical region \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. In our analysis of tone-evoked dilations, we propose a classification based on the positioning of the dilations relative to the IOS response, which we considered to represent the core of the neuronal excitation. When the IOS response coincided with the area supplied by the PA, the region of neuronal excitation triggering the dilation most probably was along the PA inflow tract. In contrast, when the IOS response was in the vicinity but not exactly at the PA, the neuronal excitation likely occurred further down in the vascular tree and triggered the dilation around the capillary compartment (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea). We believe that this classification is roughly reliable, although it may not fully grasp the complexity of the phenomena giving rise to the dilation patterns that we observed. Additional factors may need to be considered. For example, the fact that the tonotopy has a more articulated nature than the IOS map, given its heterogeneity at the cellular level \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e with a diversity of neuronal activation patterns capable of triggering the NVC response. In addition, the fact that the sounds that induce the natural vascular responses are more complex than the three tones that we used here to evoke them, and complex sounds are known to often trigger tonotopic neuron responses in separate fields of the auditory cortex in parallel \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn addition to describing the varied origin of natural dilations in awake mice, our study describes, for the first time, the central role of the pre-capillary sphincters in determining by which extent these natural dilations spread between PA and capillary compartments under local control by astrocyte Ca2\u0026thinsp;+\u0026thinsp;signaling. Pre-capillary sphincters have been well characterized anatomically only in recent years and only in the somatosensory cortex\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. They have been described as a hemodynamic structural division between capillary and arterial blood flow, acting as a bottleneck opposing high resistance to blood flow \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In view of their strategic interposition between PA and capillaries, sphincters in the upper cortical layers likely function to protect capillaries from high arterial pressures under baseline conditions. However, sphincters express \u0026alpha;-smooth muscle actin in their mural cells, suggesting that they do not provide only passive flow resistance but can also actively contract. Indeed, previous experiments in anesthetized mice have shown that sphincters respond with significant changes in diameter to whisker stimulation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e or vasoactive agent infusion\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and thus could be involved in blood flow regulation during endogenous functional stimulation. The sphincters investigated here were not anatomically homogenous, for instance they differed in their level of indentation. However, contractile mural cells completely cover this part of the vascular inflow tract, thus all sphincters must be contractile \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and their morphological differences may reflect mainly the extent of force in that contractility. Here, we provide the first direct evidence that pre-capillary sphincters play a physiological role during the auditory NVC response. Due to their strategic placement at the PA\u0026rsquo;s branching points, sphincters can control blood supply to larger parenchymal domains than downstream contractile capillary pericytes. Thus, a single PA connects to various capillaries that, in turn, branch out and supply blood flow to distinct sensory areas \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Flow regulation at the PA\u0026rsquo;s branching points could optimize downstream blood delivery in register with neural activity \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e and be the base of the laminar activation pattern observed in human fMRI brain imaging \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. On the other hand, spread of dilations in the opposite direction, from capillaries to PA \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e, can be equally important for pairing the increased local demands to global activation and contribute to the accurate and sufficient blood distribution essential for optimal brain function and health\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. This upstream-directed regulation was reported to depend on endothelial K\u0026thinsp;+\u0026thinsp;signaling\u003csup\u003e20\u003c/sup\u003e triggered by TRPA1 or KIR channels stimulation directly on 1st and higher-order capillaries \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e, supported by another level of upstream-directed regulation at higher-order capillary branch points \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. While all the above observations align with our current results, they have provided only a fragmentary picture of the NVC-related dilation dynamics. Thanks to our 3D imaging approach and the large number of natural dilations that we recorded, we have been able to put pieces together and can propose the following interpretation of the observed dilation patterns: dilations arriving from the PA compartment may or may not involve the capillaries. The main factor determining their spread beyond the pre-capillary sphincter with increased blood delivery to the downstream capillary bed is most likely the position at which the neuronal activation driving the NVC response occurs (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eb). If the activation localizes primarily to cortical layers deeper than the layer II/III junction, the dilation will generally involve just the PA. However, if the activation also concerns the upper cortical lamina, the downstream capillary region will receive additional local blood flow via expansion of the sphincter and 1st order capillary. Likewise, a local neuronal activation can trigger dilations that initiate at the 1st order capillary\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, which will only involve the capillary branches. However, if the activation is part of a larger neuronal response, it will recruit additional blood from the surface vessels by spreading across the sphincter to increase delivery via the PA (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e\n\u003cp\u003eOur study also describes for the first time the role exerted by astrocytes via local Ca2\u0026thinsp;+\u0026thinsp;signaling in controlling the spread of dilations at the pre-capillary sphincters. The existence of the astrocyte control is supported by the observation that IP3R2KO mice, which lack part of the astrocyte Ca\u003csup\u003e2+\u003c/sup\u003e signaling, display an altered NVC response, specifically a decreased capacity of dilations to engage the vascular compartments across the sphincters with reduced blood redistribution. \u003csup\u003e325671\u003c/sup\u003e Several past studies rejected a contribution of astrocyte signaling to NVC \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e because they failed to observe any changes in the blood flow responses in IP3R2KO mice. This may not be very surprising for the early studies in view of their experimental pitfalls, including having been performed in anesthetized animals. However, similar negative conclusions were drawn also in a recent study\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e that was performed in conditions similar to ours\u0026rsquo;, i.e., in awake mice and considering multiple compartments, from PA to 4th order capillaries. We do not contest the observations by Del Franco et al., as we could reproduce, using their analytical conditions, a lack of changes in some parameters of the dilation patterns in IP3R2KO mice (Sup. figure S9A). Instead, we highlight the important differences in the way experiments were conducted and data analyzed in their study compared to ours\u0026rsquo; and which likely explain the discrepant conclusions. First, Del Franco et al., did not image all the vessel compartments at the junction simultaneously; second, they did not consider the pre-capillary sphincter region in their analysis, nor the site of origin of the dilations; third, they evoked dilations by stimulations that elicited widespread Ca2\u0026thinsp;+\u0026thinsp;elevations in astrocytes not directly comparable to the local endogenous Ca2\u0026thinsp;+\u0026thinsp;elevations that we observed just before the naturally occurring dilations, and, finally, they did not image the dilations along the z-axis. We could identify vascular abnormalities in IP3R2KO mice because we used a fast 3D imaging approach and focused on an aspect never investigated before, the dynamic progression of dilations across the sphincter. This led us to a twofold discovery: first, the recognition of the pre-capillary sphincter as a locus of physiological blood flow regulation, and second, the identification of a local astrocyte IP3R2-dependent Ca2\u0026thinsp;+\u0026thinsp;activity controlling the sphincter\u0026rsquo;s contractility.\u003c/p\u003e\n\u003cp\u003eOne of the problematic aspects in studying the functional roles of the astrocyte Ca\u003csup\u003e2+\u003c/sup\u003e activity is its high level of complexity, involving a panoply of signals whose interpretation often remains enigmatic \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. Such signals go from highly visible, spatially large, and temporally long Ca2\u0026thinsp;+\u0026thinsp;elevations, to more elusive, local, and fast Ca2\u0026thinsp;+\u0026thinsp;transients. Considering that the Ca2\u0026thinsp;+\u0026thinsp;signals relevant to the control of synaptic activity were identified as part of the local transients occurring at the level of peri-synaptic astrocytic processes \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e74\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e, we expected that vascular-relevant signals would be mostly seen at astrocytic end-feet. Therefore, we focused attention on the Ca2\u0026thinsp;+\u0026thinsp;events visible in the perivascular compartment, themselves quite heterogeneous \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e77\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. Aiming at identifying among them potential dilation-regulatory events, we recorded and analyzed the perivascular astrocyte Ca2\u0026thinsp;+\u0026thinsp;dynamics having in mind how the extra blood flow was recruited in our experiments. Thus, we focused on the naturally-occurring vascular changes and 3D-imaged the peri-vascular regions around the pre-capillary sphincters that regulate blood flow to a specific cortical lamina. Since this approach had not been attempted before, we did not apply strict analytical criteria for defining the relevant astrocyte Ca2\u0026thinsp;+\u0026thinsp;activity, rather defined a number of features that we expected such activity to have, like: (a) occurring in the end-feet within short distance from the vessel surface; (b) being time correlated to the dilations, i.e. shortly preceding them; and (c) appearing in association with all the dilations having a given pattern, i.e. originating at a given site and being able to cross the sphincter. As discussed, distinct dilation patterns reflect distinct types and loci of neuronal activation, suggesting that astrocytic Ca2\u0026thinsp;+\u0026thinsp;activities related to distinct dilation patterns should also be diverse. Indeed, we observed pre-dilation end-foot astrocyte Ca2\u0026thinsp;+\u0026thinsp;responses with different onset times and positions along the PA-capillary junction. Nonetheless, our \u0026ldquo;loose\u0026rdquo; analytical approach was strong enough to identify the NVC-relevant astrocyte Ca2\u0026thinsp;+\u0026thinsp;activity, particularly because we sampled only physiological vascular events induced by auditory perception, either occurring naturally in the resting mice, or evoked by natural sounds. Our observation that the frequency of local astrocytic Ca2\u0026thinsp;+\u0026thinsp;transients as well as the frequency of dilations progressing across the sphincter were both reduced in IP3R2KO mice, provide strong support to the relevance of the identified astrocytic Ca2\u0026thinsp;+\u0026thinsp;activity to the NVC auditory response. Importantly, IP3R2KO is known to only partially reduce and not abolish astrocyte end-foot Ca2\u0026thinsp;+\u0026thinsp;activity in the awake mouse \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, as we also observed for the pre-dilation Ca2\u0026thinsp;+\u0026thinsp;events preceding natural (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) and tone-evoked dilations (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). Thus, it is possible that a more effective interference capable of abolishing any astrocyte end-foot Ca2\u0026thinsp;+\u0026thinsp;activity might have revealed a stronger control by the astrocytes.\u003c/p\u003e\n\u003cp\u003eSome key aspects of the vascular regulation identified here remain to be addressed in future investigations. To start, the exact functional implications of the local control of blood distribution at pre-capillary junctions, by which dilations are enabled to expand their parenchymal territories of blood supply bidirectionally, as well as the extent of the functional consequences that alterations targeting this physiological mechanism can produce on brain function in pathological conditions. In this context, the fact that during several CNS diseases, astrocytes undergo morphological changes that alter or impede the contact between end-feet and blood vessels surface \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e, implies noxious consequences for the regulatory functions here identified, that could likely contribute to the disturbed blood flow seen in such pathologies \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, notably in those accompanied by cognitive decline \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e. Another aspect to be clarified in the future is the additional impact of the large peri-vascular astrocytic Ca2\u0026thinsp;+\u0026thinsp;activities that we saw invading several end-feet during movements or arousal states of the mice. Considering the astrocyte Ca2\u0026thinsp;+\u0026thinsp;control at the pre-capillary junction in the resting mouse, it is intriguing that when we saw large Ca2\u0026thinsp;+\u0026thinsp;events during mouse movements, we also observed an increase in multicompartment dilations (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF). It remains to be defined how exactly these astrocyte phenomena influence NVC during motion and/or changes in brain states involving activation of secondary pathways and/or neuromodulator effects that overlap with the primary auditory excitation. \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e81\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. Understanding these relations will lead to a more comprehensive understanding of how astrocytes contribute to keeping healthy and adequate levels of substrate and oxygen in the brain of behaving mice, and how these levels could be altered under pathological brain conditions. In this context, deciphering the mechanism(s) triggering the relevant Ca2\u0026thinsp;+\u0026thinsp;activity in astrocyte end-feet and the subsequent changes in sphincter responsivity would be important. Are the local astrocyte Ca2\u0026thinsp;+\u0026thinsp;elevations secondary to neuronal activation and synaptic transmission \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e? How are they related to larger Ca2\u0026thinsp;+\u0026thinsp;elevations triggered by specific brain states and changes in noradrenaline levels \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e? Several mechanisms have been described by which astrocytes can sense synaptic activity locally and generate a Ca2\u0026thinsp;+\u0026thinsp;increase response \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e. To start, via activation of Gq-GPCRs that signal via IP3 like mGluR5 or P2Y1R \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e75\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e and whose effects could be attenuated in IP3R2KO. However, also ionotropic receptors like P2X1 and P2X5 can produce direct or indirect intracellular Ca2\u0026thinsp;+\u0026thinsp;influx in astrocytes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Other mechanisms capable of producing astrocyte Ca2\u0026thinsp;+\u0026thinsp;elevations independent of IP3R2 receptors could involve plasma membrane Ca2\u0026thinsp;+\u0026thinsp;influx channels like TRPA1 \u003csup\u003e85\u003c/sup\u003e or TRPV4 \u003csup\u003e86\u003c/sup\u003e, or the activity of Na+/Ca2\u0026thinsp;+\u0026thinsp;exchangers following neurotransmitter uptake \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e. Activation of TRPA1 channels in endothelial cells\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e could also initiate a local Ca2\u0026thinsp;+\u0026thinsp;response in astrocytes by triggering K\u0026thinsp;+\u0026thinsp;release onto their end-feet.\u003c/p\u003e\n\u003cp\u003eWhile our study leaves these important questions open, we believe that its identification of the peculiar control exerted by astrocytes at the sphincter, a critical point of blood flow regulation in the vascular tree, significantly advances understanding of the astrocytic contribution to NVC.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003eAnimals\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMost experiments in this study utilized mice induced to genetically express the Ca2+ sensitive indicator GCaMP6f selectively in the cytosol of astrocytes based on the GFAP promotor. This was obtained by crossing two transgenic mouse lines, a lox-STOP-lox-cytosolic-GCaMP6f (purchased from The Jackson Laboratory http://www.jax.org, JAX 024105 (\u003cem\u003eB6;-Gt(ROSA)26Sortm95.1(CAG-GCaMP6f)Hze/J\u003c/em\u003e), and \u003cem\u003ehGFAPCreERT2\u003c/em\u003e mice \u003csup\u003e88\u003c/sup\u003e obtained from Prof. F. Kirchhoff, University of Saarland, Germany, as previously described \u003csup\u003e32\u003c/sup\u003e. A subgroup of these \u003cem\u003eGFAPCreERT2xGCAMP6f\u0026nbsp;\u003c/em\u003emice was then crossed with the \u003cem\u003eItpr2KO\u003c/em\u003e mouse strain \u003csup\u003e89\u003c/sup\u003e that carries a constitutive deletion of IP3R2 expressed in the ER and displays conspicuous reduction in astrocytic Ca\u003csup\u003e2+\u003c/sup\u003e activity \u003csup\u003e16\u003c/sup\u003e. We obtained this strain from the laboratory of prof. Ju Chen of University of California San Diego, La Jolla, California, US. We used only IP3R2 ko/ko homozygotes in this study. Though IP3R2 contributes importantly to astrocytic Ca\u003csup\u003e2+\u003c/sup\u003e activity, especially in Ca\u003csup\u003e2+\u003c/sup\u003e mediated Ca2+ release from the ER in soma and large processes, not all the astrocyte Ca\u003csup\u003e2+\u003c/sup\u003e activities depend on this receptor, and only the largest are abolished in the knockout \u003csup\u003e90\u003c/sup\u003e. The mice were treated with Tamoxifen at 8 weeks of age to induce \u003cem\u003eCre\u003c/em\u003e recombination and trigger GCaMP6f expression in astrocytes. Tamoxifen (Sigma-Aldrich) was dissolved in corn oil (10mg/ml) and administered i.p. (0.1 ml/10 g body weight/day) for 5 days prior to the chronic cranial window surgery. The mice had surgery when they were 8 weeks old and, after a 4-week period of recovery and training, were included in the experiments between 12-28 weeks of age. Two 12-16 weeks-old \u003cem\u003eGFAP-EGFP\u003c/em\u003e mice \u003csup\u003e38\u003c/sup\u003e were used for morphological investigations. All experiments and procedures were conducted under license and according to regulations of the Cantonal Veterinary Offices of Vaud (Switzerland).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eChronic cranial window preparation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eChronic cranial windows were established as previously described \u003csup\u003e32\u003c/sup\u003e. To target the auditory cortex, the window was centered around -3mm anterior-posterior (AP) and 5 mm mediolateral (ML) from bregma, right hemisphere. The surgical procedure was done under isoflurane 1.5% anesthesia supplemented with a Carprofen injection (5 mg/kg, s.c.) and local anesthesia in the form of Lidocaine (0.2 %, s.c.) under the scalp. Animals were kept warm on a temperature-controlled heat blanket during the procedure, and their eyes were protected against dehydration with viscotears. Hair was removed, and Betadine used to sterilize the skin prior to the first incision. A square hole 3x3 mm was drilled in the bone and, leaving the dura intact, was covered with a fitting glass coverslip (thickness #1), which was then glued to the cranium. A L-shaped metal plate used to head-restrain the mice during the imaging was fixed to the skull by glue and dental cement. Analgesics were given for three days after surgery (Paracetamol 125 mg dissolved in 250 ml water).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eElectromyography (EMG)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe recorded animal movements by electromyography (EMG)\u003csup\u003e91,92\u003c/sup\u003e. To achieve this, two electrodes were implanted within the neck muscles of the mouse after the chronic cranial window preparation and connected to two micro sockets. During \u003cem\u003ein vivo\u003c/em\u003e imaging, the sockets were connected to a preamplifier and a Molecular Devices digitizer (Digidata 1440A). The signal was recorded using pClamp and then filtered (50 Hz filter) using a custom-made Matlab script. The onset of each recording was triggered by a digital signal from the Bruker microscope upon initiation of each two-photon imaging sequence (see \u0026ldquo;\u003cem\u003e3D awake imaging\u0026rdquo;\u003c/em\u003e section). The recorded EMG signal was first filtered using a 50 Hz notch filter. Then the filtered recording was normalized to standard deviation values, using the mean and SD values from the entire 90-120 seconds acquisition period. Timepoints in which the normalized signal reached \u0026gt;5SD were identified, grouped together and considered part of the same movement event if they were \u0026lt;500 ms apart. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFluorophores\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo visualize the lumen of the blood vessels, just prior to the imaging session we introduced in the circulation by tail-vein injection a dextran conjugate with Texas red 70.000 MW (2 % in 0.9 % NaCl sterile solution, 100 \u0026micro;l bolus I.V.). The vascular dye was of a size that impedes extrusion to the tissue surrounding the vessel. Hence, it was ideally suited to highlight the lumen of the blood vessels and signal diameter changes \u003csup\u003e14,93\u003c/sup\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3D Awake Imaging\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBefore imaging, the mouse was habituated to human handling for two days and gradually introduced to being head-restraint under the microscope and then thoroughly trained for an additional five days. The head of the animal was restrained by a custom-made system in which a metal plate attached to the head mount of the mouse was fastened with a screw to a matching metal bar. During imaging, the mouse sat on an air-supported, freely floating ball that acted as a spherical treadmill, allowing the animal to run when it wanted. The head of the mouse was slightly tilted, and the objective was at a 40\u0026deg; angle from the vertical position to allow perpendicular imaging through the glass covering the auditory cortex. Two-photon imaging was done with a Bruker \u003cem\u003ein vivo\u003c/em\u003e Investigator system (Bruker Nano Surfaces Division, Madison, WI, USA) equipped with an 8kHz resonant galvanometer scanner, coupled to a MaiTai eHP DS laser (Spectra-physics, Milpitas, CA, USA) with 70 fs pulse duration, tuned to 920 nm. Negative dispersion was optimized for each wavelength, and laser power was rapidly modulated by Pockels cells. A 20x LUMPFL60X W/IR-2 NA 0.9 Olympus objective was used. Emission from red and green channels was separated by a dichroic beam splitter (565 nm Long pass) allowing shorter wavelengths to reach a 520/540m band pass filter before a GaAsP detector (520 nm, optimum for green emission) and longer wavelengths to reach a 610/675 nm band pass filter before another GaAsP detector (610 nm, optimum for red emission). The combination of a resonant scanner with a piezoelectric actuator made high-speed 3D imaging possible, while the highly sensitive GaAsP detectors with negative dispersion allowed applying minimal laser dose to the tissue. The laser power varied during experiments depending on the depth of the focus, but it was kept below 7 mW and measured continuously with a power meter. These imaging settings were previously thoroughly tested to ensure their non-toxicity to the tissue, even at high-speed image acquisitions \u003csup\u003e32\u003c/sup\u003e. The anatomical localization of PA and 1\u003csup\u003est\u003c/sup\u003e order branch points defined the depths at which the imaging was performed. All the junctional sites except one were located \u0026ge;80 \u0026micro;m below the brain surface, with a maximal depth of 328 \u0026micro;m. Two different volumetric imaging approaches were applied: in one, imaging was performed in small volumes with stacks centered around the PA-capillary junction, and in the other, in larger volumes with stacks following the PA up and down the upper cortical layers. More specifically, in the first approach, the 3D-FOV covered an imaging volume of 56-75 \u0026micro;m x 15-44 \u0026micro;m x 21-35 \u0026micro;m (x,y,z), the acquisition speed was 200 Hz and the optical-zoom 8x, with pixel size of 0.29-0.59 \u0026micro;m lateral resolution and 1 \u0026micro;m axial resolution. In the second approach, the 3D-FOV covered an imaging volume of 75-121 \u0026micro;m x 75-112 \u0026micro;m x 190-280 \u0026micro;m (x,y,z), the acquisition speed was 200 Hz and the optical-zoom 8x, with pixel size of 0.29-0.94 \u0026micro;m lateral resolution and 10 \u0026micro;m (and in one case 20 \u0026micro;m) axial resolution. The scanning rate per 3D stack was 8-10 Hz. A maximum of 20 imaging sequences,\u0026nbsp;90-120 seconds-long (800-1000 stacks in total), were taken with 1-5 min breaks between them, depending on the mouse behavior.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuditory stimulations\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn this set of experiments, mice were subjected to a single sound stimulation during each of the imaging sequences. Sounds were produced by an Electrostatic loudspeaker (Tucker-Davis Technologies, Inc.) placed 30cm from the left ear of the mouse, connected to the pClamp digitizer. The latter was programmed to produce a single 10 s-long, 1 Hz auditory stimulation consisting of 10 repetitions of an individual tone, each one lasting 500ms, that was started 30 s after the initiation of the imaging sequence. Three different tone frequencies were used as stimuli: 3 kHz, 20 kHz, or 30 kHz. Each tone stimulation was repeated during three different imaging sessions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIntrinsic optical signaling (IOS)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn the auditory stimulation experiments, before performing two-photon imaging, we used intrinsic optical signaling for obtaining a tonicity map of the auditory cortex. Using the 4\u0026times; objective of our microscope, we could include the entire cranial window in the field of view. The light source was filtered with a green light excitation filter (532 nm). This wavelength is equally scattered by oxygenated and deoxygenated blood, so the reduction in the emitted light reflects changes in total blood volume \u003csup\u003e94\u003c/sup\u003e. We sampled images at 10 Hz and compared 10 s periods before and during tone stimulation, repeating comparisons for all the tone frequencies. We calculated the difference light scattering before and during stimulations \u003csup\u003e95\u003c/sup\u003e to detect the position of the largest hemodynamic response to each of the different tone stimulations.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMouse pupil imaging\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn a sub-group of the two-photon imaging experiments, we performed pupil imaging using a high-resolution, fast-speed IR camera centered on the eye of the mouse. The camera was a Dalsa Genie Nano, run with Sapera LT and CamExpert software. Pupil imaging was started by a digital signal from the Bruker microscope upon initiation of each imaging sequence. A bandpass filter (850 nm) was placed before the objective to exclude visible light, only permitting visualization of the mouse pupil by the reflected IR light. This enabled us to follow the pupil contractions and dilations during two-photon imaging and to quantify them using a simple custom-made MATLAB script. Periods involving pupil dilations (expansions) were detected based on the data z-score and defined as pupil enlargements \u0026gt;2SD with respect to the average size of the pupil during the whole imaging period. The timings of pupil expansions were compared to those of astrocytic Ca2+ elevations averaged across the entire FOV, roughly measuring large astrocytic Ca2+ events. Periods in which both measurements reached values \u0026gt;2SD were defined as periods of occurrence of the two phenomena in overlap (Suppl. Fig S2D).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3D imaging data analysis : general aspects.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe data obtained via 3D two-photon imaging were analyzed with custom-made MATLAB (Matworks, 2019b version) scripts and visualized using both ImageJ and Imaris 8.2 software (Bitplane). The analysis consisted of several steps. First, with the script \u0026ldquo;VesSegCreateMasks.m\u0026rdquo;, each z-level (or focal plane) in the 3D stack was considered as an independent 2D image (Sup. Figure S1A and B). In each of these images, the user manually defined the position of the different vessel compartments and drew a rectangular region around each of them (Sup. Figure S1C). In the \u0026ldquo;VesSegRoiData.m\u0026rdquo; script, these selections were then applied to the whole 3D timeseries, i.e., the rectangular regions were automatically used in each focal plane, so that each region contained and roughly outlined a specific vessel compartment (Sup. Figure S1D and S3). Then, the vessel compartments and the related astrocyte end-feet structures were automatically detected within each rectangle, their position found throughout the imaging sequence, and the areas calculated at each of the z-levels. Detection of the vessel structures was done first by normalizing all the pixel intensities in the red channel within the selected rectangular region during the entire imaging sequence and by calculating the z-score of each pixel. Then, the area corresponding to the structure within the rectangular region was masked at each time point using a gaussian filter (imgaussfilt.mat, with the variable \u003ccode\u003esigma\u003c/code\u003e decreasing from 8 to 5 with the increase of the imaging depth). The mask\u0026rsquo;s area was defined by the sum of all the red channel pixels in the rectangle that had significant fluorescence level (\u0026gt;2SD) for all the time points in the timeseries (Sup. Figure S1E). Movements were corrected based on the position of the centromere of the specific mask in the rectangular region under analysis compared to the centromere of the average mask from the entire imaging sequence, and the operation repeated to reposition the rectangular region for each time point. To detect whether a vessel structure was contained within the rectangular region, we applied a size threshold and established that the vessel structure within the rectangular region should cover \u0026gt;8% of the region\u0026rsquo;s area to be retained for further analysis (bwareaopen.mat). In cases in which movements of the mouse caused exit of the vessel structure from the 3D-FOV at a given time point and in which repositioning of the rectangular region could not recover the structure, the timepoint was deleted from the entire 3D+t stack and the data thus excluded from the study. Such a deletion was sometimes necessary during substantial movements or locomotion of the mice. The GCaMP6f signal in the green channel within the rectangular selection was normalized in the same way as done for the red channel. From this data, end-feet structures were defined for each imaging plane as structures departing from the edges of the vessel structure and occupying the first 2 \u0026micro;m external to such edges, which gave a 3D annulus ROI around the vascular structure (Sup. Figure S1F). These ROIs would then comprise all the Ca2+ activity occurring in astrocytic structures within 2 \u0026micro;m from the vessel structure, regardless of the astrocyte to which the end-foot structure belonged. The ROI\u0026rsquo;s width of 2 \u0026micro;m was chosen based on previous EM descriptions of the continuity and thickness of the end-foot layer around CNS vessels \u003csup\u003e96\u003c/sup\u003e. In fact, the end-feet thickness in EM is \u0026le;1 \u0026micro;m, but we added a conservative +1 \u0026micro;m margin to ensure its complete inclusion taking into account the scattering of the emitted light during two-photon imaging. This ROI was divided into four quadrants that enabled repositioning the ROI in relation to the vessel position and its constriction/dilation activity along the x and y axes (Sup. Figure S1G-I). Thus, first, the position of the rectangular region was adjusted according to the detected movements, and then the end-feet ROIs were moved in relation to the position of the vessel mask. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDetection\u003c/em\u003e\u003cem\u003e\u0026nbsp;of dilation events and patterns\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe following analytical approach was utilized both for the imaging experiments in small volumes at PA-capillary junctions and for those in larger volumes along PAs and both for natural and tone-evoked dilations. At first, the vascular structures within the 3D-FOV were classified as PA, sphincter, bulb, or capillary compartments utilizing the 3D stacks reconstructed from the ensemble of the rectangular regions manually drawn for each 2D image at a given z-level (Sup. Figure S1C and S3B). To note, some of the regions in the stacks were empty, i.e. lacked their structure at specific z-levels, especially in stacks covering large volumes with long steps between z-levels. Next, the spatial-temporal pattern of each dilation event was defined using the custom written \u0026ldquo;VesSegActivitySearch.m\u0026rdquo; script. For each vessel compartment, the vessel area at each z-level was normalized to SD values (Sup. Figure S3C), which were calculated taking periods without movements as baseline. By combining the relative changes in area at any z and t data point, we created a 3D image of the vascular dynamics in time and depth (Sup. Figure S3D). This z-t rendering of the dilation in 3D was then normalized (A-mean(A) /std(A)) considering all the time points in the imaging sequence (where A = area). Finally, to define dilation periods that extended three-dimensionally along the vessel structures, a 2SD threshold was used to detect dilations that occurred in several z-levels, prior to a gaussian blurring (imgaussfilt.mat,\u0026nbsp;sigma=2) followed by removal of ambiguous dilation events that were either highly local or very short (bwareaopen.mat, filter: \u0026frac14; x z-levels x imaging frequency) (Sup. Figure S3D). Via this procedure, the vascular activity (Sup. Figure S3E) was simplified and binarized into periods of either dilation (1) or no dilation (0). Binarization was performed for each vessel compartment during the whole acquisition period (Sup. Figure S3F). By comparing the information from the different compartments, we could evaluate if the onset of each dilation event occurred within or outside the 3D-FOV. Moreover, we could establish which vessel compartment dilated first and when, as well as the number of other vessel compartments that participated in the dilation and the onset time of the dilation in each compartment relative to the initial one (Sup. Figure S3G). Based on this information, dilations involving more than one compartment were considered a single event when they occurred in temporal continuity in the different compartments, whereas they were considered separate events when they were spaced by \u0026gt;500 ms intervals. In tone stimulation experiments, dilations occurring within the tone stimulation period were considered to be evoked. The delay from stimulation to dilations\u0026rsquo; detection in the FOV ranged between 1.9 and 3.3 s depending on the dilations\u0026rsquo; direction, in line with the commonly reported delay of evoked NVC responses\u003csup\u003e37\u003c/sup\u003e. We excluded movement-related events as components of such dilations but cannot fully exclude contribution by other factors such as coincident natural sounds.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3D imaging at the PA-capillary junction\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cem\u003eDetection and analysis of pre-dilation astrocytic 3D Ca2+ activity\u0026nbsp;\u003c/em\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eAstrocytic pre-dilation Ca2+ activity was investigated in small volume 3D imaging stacks centered around the sphincter based on the GCaMP6f green fluorescence signals. Once we had quantified the timing and pattern of vessel dilations based on the Texas red fluorescence in the vessel lumen (see \u0026ldquo;\u003cem\u003eDetection of dilation events and patterns\u0026rdquo;\u0026nbsp;\u003c/em\u003esection and Sup. Figure S5A-B), the onset of each dilation event displaying a given pattern was used to identify Ca2+ activity in astrocytic end-feet that had the specific feature of appearing recurrently with a defined temporal relation with the dilation event (Sup. Figure S5C). For this, we used the custom written VesSegPreDilCa.m script. To start, we considered the average astrocytic Ca2+ activity present in each end-foot ROI around a vessel compartment at each z-level of the 3D-FOV stack and normalized it to SD values, defining the baseline mean value and the SD values from the periods without movements (F-meanF\u003csub\u003ebaseline\u003c/sub\u003e)/stdF\u003csub\u003ebaseline\u003c/sub\u003e). As done for the vessel dilation data, this normalized single-plane information was combined in a z-t matrix, obtaining a 3D+t reconstruction of the average Ca2+ changes in the astrocytic end-feet ROIs shown as changes along the z-axis over time at each x-y imaging level. We then temporally aligned this map of the 3D+t astrocyte Ca2+ changes to the onset time of each dilation, considering specifically a 3-seconds period including the 2 seconds preceding the dilation event and the first second after its start, the latter to avoid abrupt cut of Ca2+ dynamics started in the pre-dilation period but still ongoing at dilation onset (Sup. Figure S5D). The 2-seconds pre-dilation period was chosen based on the expected delay of vascular responses from neuronal activation according to past NVC studies \u003csup\u003e15,19,49\u003c/sup\u003e and our own previous observations \u003csup\u003e23\u003c/sup\u003e. \u0026nbsp;Noteworthy, some of the naturally occurring dilations here investigated started outside the 3D-FOV, so for calculating pre-dilation delays we could rely just on their observable timings, i.e. when they entered our 3D-FOV. The astrocytic Ca2+ activity was then normalized along all the z-levels over the entire 3 s selected period. Within this time frame, all the z-axis locations and timings in which Ca2+ events occurred were identified by using a \u0026gt;2SD threshold for defining a Ca2+ increase, gaussian filtering with a 0.5 sigma gaussian blur (imgaussfilt.mat), and excluding events smaller than 3 \u0026micro;m x 100ms (bwareaopen.mat). The result was a binary mask in z-t defining the temporal and spatial position of each of the astrocytic end-foot Ca2+ activities in relation to each dilation event. We then regrouped all the astrocyte Ca2+ masks that were associated to dilations with the same pattern, i.e. same onset location and same vessel compartments involved. The regrouping of the masks from all the imaging sessions of a given experiment allowed their comparative analysis. We found that the pooled Ca2+ masks contained some overlapping Ca2+ activity, i.e. activity that occurred in all of them at the same time and 3D location. We defined the recurrent regions as the VOIs (voxel of interests) putatively involved in the pre-dilation astrocytic Ca2+ activity. We then came back to each specific map of Ca2+ activity associated with each individual dilation event, overlaid the template VOIs map to it, and checked if the individual map showed dilation-related astrocyte end-foot Ca2+ activity at the z-level and time identified by the VOI (Sup. Figure S5E). The size, timing and duration of these average Ca2+ signals were then used to describe the spatial and temporal extent of the pre-dilation Ca2+ signals (Figure 3C). \u0026nbsp;We considered that a dilation was preceded by this type of pre-dilation Ca2+ activity when the Ca2+ activity overlapped with the average z-t VOI at that specific position in 3D-t. Finally, we investigated how recurrently each putative pre-dilation Ca2+ activity was detected prior to dilations regrouped by the same pattern (Sup. Figure S5F). As inclusion criterion we considered the frequency of occurrence, with a margin defined by the number of dilations belonging to a given pool and using a 0.1 confidence limit (1-(((N/2)-((sqrt(N)/2)x1.282)/N)). Hence, for example, if a pre-dilation Ca2+ signal occurred in 59 % of N=50 dilations of the same type, we considered it to be a recurring event related to that dilation pattern because it was within the margin of 41 %. In the case we had only N=10 dilations, the event would have been considered recurring if it was seen in 70 % of the dilations due to an error margin of 30 % (Sup. Figure S5F). This criterion was adopted to avoid positive bias towards dilation patterns that occurred rarely. This means, however, that our criterion was more restrictive in the experiments on tone-evoked dilations because in those experiments we observed a much lower number of dilation events than in experiments on naturally-occurring dilations. Nonetheless, the number of pre-dilation Ca2+ events that we identified in the tone-evoked dilation experiments was in line with the number in experiments on naturally-occurring dilations (Sup. Figure S10D and S10E). \u0026nbsp;\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cem\u003eDetection of large astrocytic 3D Ca2+ activity.\u0026nbsp;\u003c/em\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eIn small volume 3D-FOVs at the PA-capillary junction we also identified periods with large pre-dilation astrocytic Ca2+ activity during resting periods. Analysis was performed on the z-t projection of the end-foot Ca2+ activity ROIs as for the small Ca2+ activity. This was done because our pre-dilation Ca2+ detection approach, described in the previous section, would not distinguish small from large activities. To tease apart the two types of events, we used the z-t matrix plot and defined \u0026ldquo;large astrocytic Ca2+ activity\u0026rdquo; as periods of extensive activity where Ca2+ increased \u0026gt;2SD above the baseline value in non-movement periods and spread through the z-layers to include the entire depth of the z-stack in \u0026gt;75 % of the end-feet ROIs. The dilations that occurred within these periods were then excluded in the following analysis of pre-dilation Ca2+ events. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3D imaging along the PA\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cem\u003eDetection and analysis of astrocytic Ca2+ activity\u0026nbsp;\u003c/em\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eIn experiments in which we scanned large volumes along the PA, the astrocytic end-feet structures were imaged in focal planes 10-20 \u0026micro;m apart along the z-axis (Figure 1C). Thus, the astrocyte structures present in the 2 \u0026micro;m annulus created along the vessel for each focal plane were treated as individual entities, and their activities averaged within the corresponding focal plane and analyzed separately. In this type of analysis, astrocytic Ca2+ activities were defined as peaks standing out of the mean baseline fluorescent signal from all the end-feet surrounding the PA at the given z-level. Peaks were detected starting from the normalized ((x-mean)/sd), smoothed (sgolayfilt.mat, order value=10) mean baseline signal using the Matlab command findpeaks.mat.The following parameters were used: \u0026nbsp;MinPeakHeight = 1 SD of the smoothed data; \u0026apos;MinPeakProminence\u0026apos;= 0.8 SD of the smoothed data, MinPeakWidth =1 second, and widthReference=halfheight. After detection of the start and end times of the peaks, which correspond to the times in which the smoothed fluorescence data went, respectively, above and below the mean baseline value, we used the non-smoothed normalized data to verify that the signal prominence was \u0026gt;2 SD. \u0026nbsp;Finally, we compared the spatial-temporal characteristics of the Ca2+ events determined in each focal plane with those in other focal planes and when we found that they overlapped, we classified the events in different planes as a single 3D Ca2+ activity, quantifying its z-spread (\u0026micro;m) and duration (s). The time periods showing Ca2+ events in end-feet along PAs were analyzed with regards to whether they overlapped with periods of animal movement or with PA dilation events to evaluate if correlations existed between the phenomena.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistics\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as mean \u0026plusmn; 95% confidence limit (C.L., 1.96xSEM), unless otherwise stated. For all statistical analyses, OriginPro 2018b (OriginLab, Northampton, MA), Matlab 2019b, and Excel (Microsoft Office 2016) software was used. Statistical analyses were performed per 3D-FOV. For experiments on naturally-occurring dilations involving imaging small volumes at PA-capillary junctions, we analyzed 20 FOVs in 6 WT mice and 14 FOVs in 4 IP3R2KO mice, respectively. In each individual statistical test, we provided information on the number of dilations analyzed/3D-FOV in terms of mean dilations/experiment \u0026plusmn; 95 % C.L.. For experiments involving imaging large volumes along the PA, we analyzed 8 FOVs in 6 WT mice and, in view of the reduced number of FOVs, we gave just the N value corresponding to the total number of dilations observed. In terms of statistical analyses, initially we compared the distribution of two data groups using graphical quantile-quantile plots (Q-Q plots), i.e. comparing the quantiles of the two groups: when the distribution was normal, a parametric two-tailed t-test was used considering whether data were paired or not. When the two data groups could not be considered independent or did not have normal distribution, the data were analyzed using non-parametric tests: the Wilcoxon signed-rank test was used for paired data, and the Mann-Whitney test for non-paired data.\u0026shy; In data sets with several groups,\u0026shy; before comparing individual points from two groups, to exclude group effects, we initially performed the Kruskal-Wallis ANOVA (KWA) for non-parametric data, and the two-way ANOVA for normally distributed data. If the data points in a group could not be considered to be independent, as in the case of vessel compartment dilations, we initially used the Friedmans ANOVA test instead. KWA and Friedmans ANOVA tests provide both a p-value and a X\u003csup\u003e2\u003c/sup\u003e value. The latter was used for defining the level of difference between the compared groups. In analyses that required a multiple comparisons test, such as ANOVA or KWA, the values from the final analysis were adjusted with the Holm correction (similar to Holm-Bonferroni) on both parametric and non-parametric data. This correction depends on the number of comparisons in the data group and the adjustment is performed in a ranked way depending on the p values: the smallest p-value gets the strongest adjustment (multiplied by the number of comparisons), while the following increasing p-values are adjusted depending on the number of p-values already adjusted according to: P\u003csub\u003eoriginal\u003c/sub\u003ex(N\u003csub\u003ecomparisons\u003c/sub\u003e-(counts of P\u0026lt; P\u003csub\u003eoriginal\u003c/sub\u003e))=P\u003csub\u003ecorrected\u003c/sub\u003e. Data were considered significantly different when the p-value, corrected or non-corrected, was *\u0026lt;0.05, but additional levels of significance were marked with **\u0026lt;0.01 and *** \u0026lt;0.005.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cspan\u003eAnimal Ethics statement All experiments and procedures were conducted under license and according to regulations of the Cantonal Veterinary Offices of Vaud (Switzerland).\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll scripts forming the code used in this study together with data examples to verify code function are provided with the submitted manuscript in a zipped folder together with a read-me instruction on how to run the analysis. All other data can be made available from the author (BLL) upon reasonable request and will be uploaded to a public repository upon publication of the study. A space in the public repository \u0026nbsp;Zenodo has been reserved for this data under the doi: 10.5281/zenodo.15224578.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Giovanni Carriero and Erika Bindocci for their advice and support during the project; Tania Barkat, for her advice on setting up the auditory stimulations; and Martin Lauritzen and S\u0026oslash;ren Grubb for valuable scientific discussions. This work was supported by ERC advanced \u0026ldquo;Astromnesis\u0026rdquo;, SNSF 31003A-173124, and SNSF 31003B-201276 grants to AV and by postdoc fellowships R210-2015-3320 and R265-2017-4437 to BLL from The Lundbeck Foundation, Denmark.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBLL designed and performed all experiments, custom wrote all matlab analysis tools, performed the analysis and statistics, designed figures and wrote the manuscript. A.V. supervised the project, defined strategy, discussed experimental design and analysis, designed figures and wrote the manuscript. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eIadecola, C. 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The perivascular astroglial sheath provides a complete covering of the brain microvessels: an electron microscopic 3D reconstruction. \u003cem\u003eGlia\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 1094-1103 (2010). https://doi.org:10.1002/glia.20990\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6539397/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6539397/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeurovascular coupling (NVC) increases blood flow, assuring adequate supply to active cortical regions by local redistribution via penetrating arterioles (PA) and branching capillaries. Astrocyte end-feet enwrapping these vascular structures possess machinery to regulate blood flow, but their participation in NVC is controversial. Via a new 3D\u0026thinsp;+\u0026thinsp;t two-photon imaging approach we visualized PA and capillaries simultaneously during naturally-occurring and tone-evoked dilations in the auditory cortex of awake mice. We observed that dilations occurred bidirectionally, and a fraction of them extended between compartments across the interconnecting sphincter, depending on the animal activity states. These multi-compartment dilations were preceded by rapid astrocyte end-foot Ca2\u0026thinsp;+\u0026thinsp;signals around the sphincter. Reduction of this astrocytic Ca2\u0026thinsp;+\u0026thinsp;activity in IP3R2KO mice suppressed multi-compartment dilations, revealing a pivotal role of pre-capillary sphincters in their bidirectional spread between vascular compartments under local control by astrocytes. This novel mechanism contributes to physiological regulation of laminar blood flow during NVC.\u003c/p\u003e","manuscriptTitle":"Fast 3D imaging in the auditory cortex of awake mice reveals that astrocytes control neurovascular coupling responses at arteriole-capillary junctions.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-19 06:32:36","doi":"10.21203/rs.3.rs-6539397/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6befff25-aa8f-475c-90e6-79341677f2b7","owner":[],"postedDate":"May 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48313263,"name":"Biological sciences/Neuroscience/Glial biology/Astrocyte"},{"id":48313264,"name":"Biological sciences/Neuroscience/Neuro\u0026#x2013;vascular interactions"}],"tags":[],"updatedAt":"2025-07-01T08:46:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-19 06:32:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6539397","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6539397","identity":"rs-6539397","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}