Contractile pericytes determine the direction of blood flow at capillary junctions

Significance

Capillaries—the most abundant vessels in the circulatory system—deliver O₂ and nutrients to all cells of the body. In the brain and retina, capillaries also act as a sensory web that detects neuronal activity. Here, we demonstrate that pericytes localized at capillary junctions in a postarteriole transitional region possess unique properties, notably including contractility, that enable them to dynamically manipulate capillary branch diameters and exert fine control over the distribution of blood within the capillary network. In so doing, these contractile junctional pericytes fine tune the delivery of O₂ and nutrients and thus serve to meet the specific needs of neurons. Given these unique properties, pericytes represent a therapeutic target for cardiovascular and neurodegenerative diseases.

Abstract

The essential function of the circulatory system is to continuously and efficiently supply the O₂ and nutrients necessary to meet the metabolic demands of every cell in the body, a function in which vast capillary networks play a key role. Capillary networks serve an additional important function in the central nervous system: acting as a sensory network, they detect neuronal activity in the form of elevated extracellular K⁺ and initiate a retrograde, propagating, hyperpolarizing signal that dilates upstream arterioles to rapidly increase local blood flow. Yet, little is known about how blood entering this network is distributed on a branch-to-branch basis to reach specific neurons in need. Here, we demonstrate that capillary-enwrapping projections of junctional, contractile pericytes within a postarteriole transitional region differentially constrict to structurally and dynamically determine the morphology of capillary junctions and thereby regulate branch-specific blood flow. We further found that these contractile pericytes are capable of receiving propagating K⁺-induced hyperpolarizing signals propagating through the capillary network and dynamically channeling red blood cells toward the initiating signal. By controlling blood flow at junctions, contractile pericytes within a functionally distinct postarteriole transitional region maintain the efficiency and effectiveness of the capillary network, enabling optimal perfusion of the brain.

The fundamental purpose of the circulatory system is to provide an uninterrupted supply of O₂ and nutrients to all cells of the body and to remove CO₂ and other metabolic waste products. Capillaries, which constitute the vast majority of the vasculature in terms of length, are the sites of gas and nutrient exchange between the blood compartment—including O₂-carrying red blood cells (RBCs)—and the surrounding tissue. Despite a general appreciation of the relationship between the requirements of tissues and the microvasculature that serves them, how RBCs and plasma are efficiently distributed throughout capillary networks so as to meet the needs of every cell remains poorly understood. Nowhere is our understanding of the mechanisms that regulate the distribution of blood within capillary networks less complete than in the brain.

The brain vasculature is composed of a network of interconnected surface (pial) vessels that give rise to arterioles that penetrate orthogonally into the brain and feed a vast network of capillaries. Arterioles are composed of an inner layer of endothelial cells (ECs), oriented in the direction of blood flow, surrounded by a single layer of smooth muscle cells that wrap circumferentially around the endothelial cell layer and are separated from it by an internal elastic lamina (IEL) (1). Capillaries, on the other hand, are composed of endothelial cell tubes without a smooth muscle cell layer or IEL; instead, much of their surface is covered over with perivascular mural cells (pericytes), which are embedded in the basement membrane.

Pericytes are ubiquitous in the capillary microcirculation of all vascular beds, reaching their highest densities within retinal and cerebral circulations (2). The defining morphological characteristics of a capillary pericyte, first depicted in meticulous hand drawings in the early 20th century and later described in detail in electron micrographs (3, 4), are a prominent outward protruding nucleus that aligns with the vessel lumen, extensions that span the long axis, and projections that wrap around the endothelial tube. This contrasts with all differentiated smooth muscle cells described in the vasculature, which exhibit a morphology characterized by a long, fusiform cell body that adopts a ring-shaped structure in vivo that encircles the endothelial cell layer in arteries and arterioles, with its long axis oriented perpendicular to the direction of blood flow.

The brain consumes a disproportionate share of the body’s energy resources and is highly sensitive to even brief disruptions in blood flow. Because neurons lack the capacity to store significant energy reserves, the brain has evolved on-demand mechanisms for preferentially allocating blood flow to regions of higher neuronal activity. Such activity-dependent increases in local blood flow (functional hyperemia) are mediated by an ensemble of mechanisms collectively termed neurovascular coupling (NVC). The fact that NVC mechanisms exist suggests that simply oversupplying the entire brain is an inadequate evolutionary solution to the neuronal energy-resupply problem, implying that precision and efficiency are organizing principles that govern the operation of these mechanisms. In fact, such precision is a fundamental assumption underpinning brain-activity mapping based on measurements of blood oxygen level-dependent functional magnetic resonance imaging (BOLD-fMRI). Our recent work has established a mechanistic basis for the neuron-to-microvascular signaling necessary for efficient communication of neuronal metabolic demands to the cerebral vasculature, showing that activation of capillary endothelial cell inwardly rectifying potassium (K_ir) channels by K⁺, a byproduct of neuronal activity, induces a propagating electrical (hyperpolarizing) signal that causes upstream arteriolar dilation and increased blood flow into the capillary network (5). However, although this NVC mechanism provides a means of communicating the need for increased blood flow toward a metabolically active anatomical region, it leaves open the question of how blood flow is regulated within the capillary network.

Whereas pial vessels are interconnected, and thus have considerable capacity for redirecting blood flow, a single parenchymal arteriole and associated capillary networks feed blood to a distinct cylindrical cortical volume (diameter, ∼500 μm) (6). In the absence of control mechanisms downstream of the arteriole, this arrangement predicts a stochastic distribution of blood within a volume of nonuniform neural activity, and thus has implications for the precision of blood delivery. An alternative mechanism tested here is that contractile junctional pericytes dynamically control capillary branch diameters to exert fine control over the distribution of blood. Pericytes within the cerebral microcirculation are predominantly located at capillary junctions (7, 8); this is also the case for the microvasculature of the retina, which shares the same neural crest developmental origin as the brain vasculature (9). Our data support the conclusion that these junctional pericytes, specifically those in a specialized postarteriole transitional region, both structurally and dynamically determine the geometry of capillary junctions and regulate the directional distribution of RBCs through the capillary network. Our findings further suggest that relaxation of proximal pericytes by K⁺-dependent hyperpolarizing signals plays a role in NVC mechanisms, directing blood flow toward the source of the signal. These mechanisms challenge the view that blood flow within the capillary network is essentially a passive process determined by the static architecture of the vasculature and suggest instead that pericytes provide dynamic control of blood perfusion in capillary networks to fine tune the delivery of O₂ and nutrients to the tissue.

Results

Pericytes within CNS Capillary Networks Show Region-Specific Differences in Morphology and Distribution and Contribute to the Asymmetry of Capillary Branch Diameters.

The nature and function of pericytes has been a matter of controversy since these cells were first described by Rouget in 1873 (10). Speculation surrounding their potential role in regulating capillary blood flow, which began in earnest nearly a century ago (11, 12), has simmered to this day, with considerable bodies of literature supporting divergent points of view (for early reviews, see refs. 7, 13–17). There are also differences in opinion on the point at which the arteriolar system ends and the capillary network begins—differences that are intertwined with views on pericyte-related questions. Thus, any meaningful discussion of pericytes in central nervous system (CNS) capillary networks must begin with a shared sense of what is meant by “pericytes” and “capillaries.” In terms of the brain microvaculature, we define vessels furthest removed from surface pial arteries that are completely covered by a single continuous layer of concentric smooth muscle cells as feeding arterioles. The transition from this last arteriole segment to the first segment of the capillary network is marked by a sharp boundary formed by the presence of an IEL on one side (arteriole) and its absence on the other (capillary). From the single capillary segment emerging from the feeding arteriole, capillary networks branch out to form an interconnected web containing both diverging and converging junctions, with segments ultimately reuniting to drain blood back into the venous circulation. In the retina, arterioles radiating from the optical disk, visualized using hydrazide staining to preferentially stain the IEL (present only in arteries and arterioles), are analogous to the penetrating arterioles found in the cerebral circulation (18) (Fig. 1A). Thus, in this formulation, all vascular elements downstream of these radiating arterioles are considered capillaries. Within these so-defined capillary regions, all perivascular cells with a protruding nucleus and cell body located atop the vessel are considered to be pericytes.

Fig. 1.

The morphology of pericytes confers asymmetry on capillary junctions within retinal capillary networks. (A) Representative low-magnification image identifying the sharp boundary (*) between arteriole and capillary vessels of the retinal vascular network stained with FITC-conjugated lectin (green) and Texas Red-hydrazide (red). (Scale bar, 50 µm.) (B) Schematic depiction (Left) and fluorescence images (Right) of the retinal vasculature of NG2-dsRed mice stained with FITC-conjugated lectin showing arterioles and transitioning pericyte morphology from ensheathing to mesh and thin-strand type. (Scale bars, 5 µm.) (C, Left) Representative low-magnification image of the retinal vascular network from an NG2-dsRed mouse stained with FITC-conjugated lectin (green). (Scale bar, 50 µm.) (Right) Summary data showing the percentage of capillary junctions containing cells positive for the pericyte marker NG2 and the ratio of daughter branch diameters, expressed as Diameter_Small/Diameter_Large. The presence of NG2-positive cells was determined from 526 junctions from nine confocal stacks. The ratio of daughter branch diameters was determined from 102 junctions from 11 vascular trees (n = 3 to 4 mice).

In addition to their classic “bump on a log” appearance, pericytes possess cytoplasmic extensions that spread along, and projections that wrap around, the capillary tube (8) (Fig. 1B). These pericyte projections can take different forms, producing a continuum of pericyte morphologies within the capillary network that can be broadly assigned to three subtypes. The most arteriole-proximate segments of the capillary network are occupied by pericytes with short extensions and densely packed projections that wrap around segments of the capillary (4), almost completely encasing it (Fig. 1B). Superficially, this “dense,” or ensheathing, morphology appears similar to that of smooth muscle cells in arteries and arterioles, but instead of individual vascular smooth muscle cells, the dense banding pattern observed in these segments reflects wrapping of bands of tightly packed projections from single pericytes. This region is sometimes referred to as the “precapillary arteriole” (19), a term which gives an impression that the ensheathing pericytes in this region are smooth muscle cells, a distinction addressed in greater detail below. Beyond this region of the capillary tree, dense pericytes transition to a second “mesh” type that shows considerable capillary coverage or longer extensions but lacks a prominent dense banding pattern, and later into a third, “loose” or “thin-strand” morphology (8) with long extensions and fewer and minimal projections wrapping around the capillary tube (Fig. 1B).

To further assess the regional and structural distribution of microvascular pericytes, we quantified pericyte coverage in retinal and brain capillary networks using NG2-DsRed-BAC transgenic mice, which express DsRed (Discosoma red fluorescent protein) under the control of the promoter for the Ng2 gene, encoding the putative pericyte marker neural/glial antigen 2 (NG2) (20). The retinal microvascular network was visualized by confocal imaging of flat-mounted retinal preparations stained with fluorescence-conjugated isolectin B4, which binds to D-galactose residues within the basement membrane (21). In line with previous reports (7, 8), we found that ∼52% of pericytes throughout the capillary network were located at capillary bifurcations (n = 581 pericytes from 10 confocal stacks). Further analysis revealed that ∼93% of junctions in the arteriole-proximate region of the capillary network corresponding roughly to the region occupied by pericytes with ensheathing or mesh morphologies (approximately first through third junctions), referred to hereafter as the “postarteriole transitional region,” contained NG2-positive cell bodies, whereas only ∼55% of junctions in more distal regions (fourth junction or beyond) were occupied by such cells (Fig. 1C). A transcranial two-photon laser-scanning microscopy (2PLSM) examination of the somatosensory cortex microvasculature of NG2-DsRed-BAC transgenic mice, injected with fluorescein isothiocyanate (FITC)-dextran to illuminate the brain vasculature, revealed a similar distribution of pericytes at vascular junctions. In this case, ∼90% of junctions in the transitional region contained NG2-positive cells, whereas ∼45% of more distal capillary bifurcations were occupied by such cells (SI Appendix, Fig. S1).

The common occurrence of pericytes at capillary junctions, particularly the extensive coverage at junctions in the postarteriole transitional region, led us to consider the possibility that these cells might influence the static geometry of capillary branches. As a first step in determining the relationship between pericyte junctional coverage and branch symmetry, we assessed differences in the luminal diameters of downstream (daughter) branches, measured as the ratio of the smaller to larger branch (Dia_Small/Dia_Large), ex vivo in the retinal preparation and in vivo in the brain. Interestingly, branch diameter symmetry increased with increasing distance of junctions from the feeding arteriole in both retinal (Fig. 1C) and brain (SI Appendix, Fig. S1) capillary networks. In the retinal preparation, Dia_Small/Dia_Large values were ∼0.65, 0.73, and 0.87 at first, second, and third junctions, respectively, and plateaued at >0.9 at more distal (fourth and fifth) junctions. The fact that the symmetry of branch diameters increased in parallel with decreasing pericyte occupancy led us to hypothesize that pericytes at junctions in the postarteriole transitional region modulate capillary diameters and contribute to the general structural asymmetry of perijunctional vessel diameters.

The Arteriole-Proximate Region Constitutes a Functionally Distinct Domain of the CNS Capillary Bed.

We next investigated potential molecular correlates of region-dependent differences in pericyte morphology and distribution within CNS capillary networks by analyzing the expression of a panel of structural and marker proteins. Using the mouse retinal preparation, we found that pericytes throughout the capillary tree were positive for filamentous actin, although staining intensity was significantly lower in capillaries compared with that in arterioles (Fig. 2A and SI Appendix, Fig. S2). Notably, only pericytes in the postarteriole transitional region showed immunostaining for α-actin (Acta2), the dynamically contractile isoform of actin (Fig. 2B). Consistent with previous observations (19, 22–24), quantification of immunostaining revealed that α-actin immunofluorescence intensity exhibited a stepwise decrease in pericytes at first, second, and third junctions within the capillary network compared with that in the feeding arteriole (Fig. 2 B, Bottom), reaching undetectable levels at fourth junctions and beyond. Similar fluorescence patterns were observed in vivo and ex vivo using acta2-GCaMP-mCherry as well as acta2-GCaMP-mVermillion mice, in which fluorescence is driven by the Acta2 promoter (SI Appendix, Figs. S3, S4, and S6), which differs from findings of others (24). Perivascular cells within the retinal capillary bed also expressed a number of additional markers in common, including desmin (25), and shared a lack of calponin (Fig. 2C) (25, 26). Curiously, although these cells express tubulin monomers (26, 27), they lacked polymerized tubulin (i.e., microtubules) (Fig. 2D), unlike most cell types studied to date. Notably, many of these attributes, including the absence of large microtubule filaments, are strikingly different from those of smooth muscle cells in the upstream arteriole (summarized in SI Appendix, Table S1). Similar to the case for the retinal preparation, we found that expression of α-actin in pericytes in the cerebral circulation was limited to capillary branches proximal to the feeding arteriole (SI Appendix, Fig. S4A). Intriguingly, all perivascular cells within the retinal and cerebral capillary microcirculation, including those deeper in the capillary bed that are universally recognized as pericytes, expressed the smooth muscle “specific” protein, myosin heavy chain (Myh11) (Fig. 2E and see SI Appendix, Fig. S4B) (26, 28). Although very low level Myh11 promotor activity can lead to a strong fluorescence signal, the fact that fluorescence intensity tended to decrease at successive junctions of capillaries suggests that the observed signals are a meaningful reflection of bona fide Myh11 expression.

Fig. 2.

Cytoskeletal elements within a feeding retinal arteriole and capillary networks. (A to E) Stitched together montage of high resolution fluorescence images (Top) and whisker-box plot (Bottom) showing the retinal vasculature stained with FITC-conjugated lectin (gray) and labeled with phalloidin, a pan-specific stain for filamentous actin (from 5 vascular trees) (A); immunostained for α-actin (n = 11 vascular trees) (B); immunostained for calponin (n = 12 vascular trees) (C); stained with Tubulin Tracker, for filamentous microtubules (n = 5 vascular trees) (D); and imaged in SMMHC (Myh11)-tdTomato mice (n = 5 vascular trees) (E). Capillary junctional fluorescence was calculated as described in Methods. (*P ≤ 0.05 vs. smooth muscle cells [SMC], ^#P ≤ 0.05 vs. first junction, ^$P ≤ 0.05 vs. second junction, and ^ϮP ≤ 0.05 vs. third junction). (Scale bars, 10 µm.)

The fact that expression of the dynamically contractile α-isoform of actin is limited to the subpopulation of pericytes in the postarteriole transitional region (Fig. 1D) (23) predicts that only pericytes within this region would be capable of dynamically constricting or dilating branches of capillary bifurcations. To test this, we performed real-time measurements of changes in capillary branch diameter at proximal (first to third) and distal (fourth or beyond) junctions in fluorescence-conjugated isolectin B4-stained retinal preparations in response to two different stimuli: the G protein-coupled thromboxane A2 receptor agonist, U46619, and membrane depolarization with 60 mM K⁺. All pericytes at the first two to three (2.1 ± 0.2 junctions, 115 capillary trees from 17 confocal stacks) capillary junctions proximal to the feeding arterial rapidly contracted following administration of 100 nM U46619 or membrane potential depolarization with 60 mM KCl (Fig. 3A), resulting in average decreases in diameter of ∼35% (U46619) and ∼15% (60 mM K⁺); they also dilated following exposure to a Ca²⁺-free extracellular solution (Fig. 3 A, Right). Pericytes at capillary bifurcations more than four junctions distal to the feeding arteriole also responded to U46619, albeit much more slowly and to a lesser extent, gradually constricting by ∼5% over 10 to 15 min; however, they did not respond to 60 mM KCl or Ca²⁺-free solution (Fig. 3B and SI Appendix, Fig. S5). Notably, pretreatment with the actin-depolymerizing agents cytocholasin D (5 µM) and latrunculin B (1 µM) or the myosin light chain kinase (MLCK) inhibitor, ML-7 (5 µM), prevented the U46619-induced rapid contraction of junctional pericytes in the transitional region and the slow contraction of distal pericytes (Fig. 3 C and D). Consistent with the absence of detectable microtubule filaments in pericytes (Fig. 2D), microtubule depolymerization with nocodazole (10 µM) had no effect on the relaxation of junctional pericytes following exposure to a Ca²⁺-free extracellular solution but did prevent the relaxation of arterial smooth muscle cells in feeding arterioles (Fig. 3E). Thus, in contrast to observations in every muscle cell examined to date (29, 30), microtubules appear to play no role in the relaxation or morphology of capillary pericytes in the transitional region.

Fig. 3.

U46619-induced dynamic contraction of pericytes in the postarteriole transitional region. (A and B) Representative images (Left), time course (Middle), and summary data (Right) showing capillary constriction following administration of U46619 (100 nM; red) or membrane depolarization with 60 mM KCl (blue), and immediate vessel relaxation to application of zero extracellular Ca²⁺ ([Ca²⁺]_o = 0, green) mediated by pericytes in the postarteriole transitional region (proximal) (A) and at locations deeper in the capillary bed (distal) (B). Average starting baseline diameter indicated by dotted line (n = 30 junctions, n = 5 to 6 mice; *P ≤ 0.05 vs. U56619 and ^#P ≤ 0.05 vs. 60 mM KCl). (Scale bars, 5 µm.) (C and D) Summary data showing U46619 (100 nM)-induced pericyte-mediated capillary constriction in the presence of cytocholasin D (5 µM) and latrunculin B (1 µM; orange) (n = 27 to 38 junctions from n = 5 mice) (C) or the MLCK inhibitor ML-7 (5 µM, purple) (n = 17 to 23 junctions from n = 4 mice) (D). (E) Summary data showing relaxation of arterioles and pericytes in the postarteriole transitional region (proximal) and more distal locations (distal), following microtubule depolymerization with nocodazole (10 µM, light blue) (n = 8 vascular trees from n = 4 mice; *P ≤ 0.05 vs. nocodazole and U46619 treatment).

Collectively, these observations establish vascular segments in the postarteriole transitional region containing dynamically contractile pericytes as a functionally distinct region of the CNS capillary network.

Ca²⁺-Dependent Contraction of Pericytes in the Postarteriole Transitional Region Is Regulated by IP₃R and L-Type Ca²⁺ Channel Activity, but Not by Ryanodine Receptor Activity.

Contraction, initiated by myosin and actin cross-bridge cycling following phosphorylation of the myosin regulatory light chain by MLCK, requires an increase in intracellular Ca²⁺ concentration (31). U46619 acts through the Gα_q/11-coupled thromboxane receptor to activate phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP₂) to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃), the latter of which engages IP₃ receptors (IP₃Rs) to promote release of Ca²⁺ from intracellular stores (32), whereas 60 mM K⁺ causes depolarization through activation of voltage-dependent L-type Ca²⁺ channels, allowing influx of extracellular Ca²⁺. To obtain a better understanding of the Ca²⁺ dynamics that lead to pericyte contraction, we examined the frequency of agonist- and depolarization-induced Ca²⁺ events preceding contractions in three to six projections of pericytes in the first three segments of the postarteriole transitional region (Fig. 4A). These experiments were performed in retinal preparations from acta2-GCaMP-GR BAC transgenic mice, which express a modified GCaMP Ca²⁺ sensor fused to the Ca²⁺-insensitive fluorescent protein mCherry under control of the Acta2 promoter (33). We found that U46619 (100 nM) increased the average frequency of Ca²⁺ events in projections of proximal pericytes from 1.4 ± 0.2 (baseline) to 2.6 ± 0.2 events per 10 s (Fig. 4B). U46619 (100 nM) also stimulated Ca²⁺ events in pericyte projections of distal pericytes, increasing their average frequency from 1.9 ± 0.2 (baseline) to 3.2 ± 0.2 events per 10 s (SI Appendix, Fig. S6). Because these distal pericytes do not express α-actin, we performed these Ca²⁺ imaging experiments using SMMHC-GCaMP6f transgenic mice, which express a modified GCaMP Ca²⁺ sensor under control of the promoter of the Myh11 gene encoding SMMHC (smooth muscle myosin heavy chain). Depolarization with 60 mM K⁺ produced an effect similar to that of U46619 in proximal pericytes, increasing the frequency of Ca²⁺ events from 1.7 ± 0.1 (baseline) to 3.8 ± 0.2 events per 10 s (Fig. 4C). Consistent with previous reports of a functional role for L-type voltage-dependent Ca²⁺ channels in pericytes (34), the selective L-type channel antagonist nimodipine eliminated high K⁺-induced Ca²⁺ events and completely relaxed precontracted (with 60 mM K⁺) pericytes in the postarteriole transitional region (Fig. 4 C and D). However, nimodipine treatment only partially inhibited U46619-induced Ca²⁺ events and did not fully relax pericytes precontracted with U46619 (Fig. 4 E and F), suggesting that other sources of Ca²⁺ besides voltage-dependent Ca²⁺ channels (35) contribute to agonist-induced contraction. To further test this, we examined pericyte-localized Ca²⁺ events following depletion of intracellular Ca²⁺ stores or blocking endoplasmic reticulum (ER) Ca²⁺ release via IP₃Rs and ryanodine receptors (RyRs). Pretreatment with the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor, cyclopiazonic acid (CPA; 10 µM), prevented U46619-induced Ca²⁺ events and constriction (Fig. 4 G and H), implying that intracellular Ca²⁺ stores also contribute to agonist-induced contraction of pericytes. Consistent with this, administration of the membrane permeable IP₃ analog Bt-IP₃ (10 µM) increased the overall frequency of Ca²⁺ events, and inhibition of IP₃Rs by preincubation with xestospongin C (1 µM) blunted the effectiveness of subsequent U46619 treatment, reducing the frequency of U46619-stimulated events by ∼50% (Fig. 4I). Notably, application of the RyR inhibitor tetracaine (100 nM) had no effect on baseline or U46619-stimulated Ca²⁺ events in pericytes in the postarteriole transitional region (Fig. 4J). The RyR activator caffeine (5 mM) similarly had no effect on Ca²⁺ in these pericytes but did increase intracellular Ca²⁺ in neighboring vascular smooth muscle cells in the feeding arteriole (Fig. 4K). These observations imply that RyRs, a defining feature of muscle cells, are functionally absent in pericytes within the transitional region, reinforcing the idea that, despite certain overt similarities, pericytes—including those in the postarteriole transitional region—differ from smooth muscle cells in key functional respects.

Fig. 4.

L-type Ca²⁺ channels and IP₃Rs, but not RyRs, contribute to the contractile dynamics of pericytes in the postarteriole transitional region. (A and B) Representative images and traces (A) and summary data (B) showing Ca²⁺ events in pericytes (P1, P2, and P3) following administration of 100 nM U46619 (n = 40 ROIs from 12 cells, n = 3 mice; *P ≤ 0.05 vs. baseline). (C) Summary data showing the frequency (freq.) of Ca²⁺ events at baseline, and following 60 mM K⁺-induced membrane depolarization in the absence and presence of 100 nM nimodipine (n = 36 ROIs from 12 cells, n = 4 mice; *P ≤ 0.05 vs. baseline and ^#P ≤ 0.05 vs. 60 mM KCl). (D) Summary data showing contraction of projections in response to K⁺-induced membrane depolarization (60 mM KCl) in the absence and presence of 100 nM nimodipine (n = 9 cells, n = 3 mice. *P ≤ 0.05 vs. 60 mM KCl). (E) Summary data showing the frequency of Ca²⁺ events in the presence of nimodipine (n = 30 ROIs from 10 cells, n = 4 mice. *P ≤ 0.05 vs. nimodipine), without and with added U46619. (F) Summary data showing U46619-induced contractions in the absence and presence of nimodapine (100 nM) (n = 14 cells, 4 mice; *P ≤ 0.05 vs. U46619). (G) Summary data showing the frequency of Ca²⁺ events in the presence of cyclopiazonic acid (CPA, 10 µM), without and with added U46619. (H) Summary data showing contraction of projections in the presence of CPA (10 µM), without and with added U46619 (n = 7 cells, n = 3 mice; *P ≤ 0.05 vs. U46619). (I) Summary data showing the contribution of ER Ca²⁺ to Ca²⁺ events. (Left) Ca²⁺ event frequency at baseline and following administration of Bt-IP₃ (10 µM) (n = 28 ROIs from 5 cells, n = 3 mice; *P ≤ 0.05 vs. baseline). (Right) Ca²⁺ event frequency in the presence of 1 µM xestospongin C (Xesto C), without and with added U46619 (n = 25 ROIs from 6 cells, n = 3 mice). (J) Summary data showing Ca²⁺ event frequency in the presence of 100 nM tetracaine, without and with added U46619 (n = 47 ROIs from 12 cells, n = 6 mice; *P ≤ 0.05 vs. baseline). (K) Representative image (Left) and trace (Right) of Ca²⁺ in smooth muscle (arteriole, red) and pericytes in the postarteriole transitional region (blue) following administration of caffeine (5 mM) and ionomycin (10 µM). The trace shows the average and SE of five arteriole/pericyte preparations (n = 5 vascular trees, n = 4 mice).

Projections of Contractile Junctional Pericytes Are Capable of Acting as Individual Functional Units to Differentially Contract Daughter Branches and Direct RBC Flux at Capillary Bifurcations.

We next directly assessed Ca²⁺ signaling in individual pericyte projections in relation to contractile responses under basal (unstimulated) conditions in the retinal preparation from acta2-GCaMP-GR transgenic mice, utilizing Ca²⁺-insensitive mCherry fluorescence to confirm that changes in fluorescence are attributable to changes in Ca²⁺ levels and not to contraction-associated movement artifacts. A 3D reconstruction of a single pericyte revealed projections wrapping around all three junctional branches (Fig. 5 and Movie S1). This morphology is typical of pericytes in the transitional region, which exhibited an average of 8.0 ± 0.4 projections per pericyte (n = 21 pericytes, n = 3 mice). Simultaneous tracking of increases in local Ca²⁺ (Fig. 5, upward deflection) and capillary diameter (Fig. 5, shaded downward deflection) in all projections of an individual branch-covering junctional pericyte revealed that each projection from a single pericyte exhibited distinct Ca²⁺ signaling and contraction profiles (Movie S2), suggesting that individual projections are capable of acting as independent functional units. Additional examples of Ca²⁺ and contractile events in individual projections of proximal pericytes are depicted in Movie S3. Using a Pearson’s correlation analysis, we examined whether local Ca²⁺ events within and between projections of an individual junctional pericyte are correlated. Pairwise linear correlation coefficients between regions of interest (ROIs) were averaged over each projection (Fig. 5C, diagonal elements) or pair of projections (Fig. 5C, nondiagonal elements) to construct a correlation coefficient matrix. Data from a single junctional pericyte revealed that ROIs within the same projection were highly correlated, whereas correlation coefficients between some projection pairs were low (Fig. 5C). We next examined whether the location of projections influences the correlation of Ca²⁺ events by assessing correlations between projections enwrapping the same capillary branch and those enwrapping different branches. This analysis revealed that Ca²⁺ events in ROIs within a projection or between projections enwrapping the same capillary branch were more highly correlated than were ROIs in projections enwrapping different capillary branches (Fig. 5D and SI Appendix, Fig. S7). Localized Ca²⁺ activity between ROIs within a projection or between projections within the same capillary branch was more highly correlated than that between projections located on different capillary branches. Thus, projections enwrapping different capillary branches are capable of exhibiting independent Ca²⁺ events, allowing them to act as independent functional units to differentially constrict capillary branches at bifurcation points.

Fig. 5.

Junctional pericytes are capable of independently controlling Ca²⁺ and contraction of capillary branches. (A) Representative images of a single proximal junctional pericyte from a retina isolated from an acta2-GCaMP-GR transgenic mouse showing projections wrapping around all capillary branches. (Scale bars, 5 µm.) (i) Schematic showing placement of ROIs for recording Ca²⁺ events (boxes) and changes in luminal diameter (dashed lines) for eight cellular projections from a single pericyte. (ii–iv) Representative image of fluorescence intensities relative to baseline (F/F_o) for Ca²⁺ events restricted to one side (ii) or occurring on both sides (iii and iv) of a pericyte projection wrapping around capillary branches. (B) Representative traces of Ca²⁺ fluorescence (upward deflection) and contraction events (shaded downward deflection) from the eight ROIs depicted in i. (C) Pearson’s correlation coefficient (Corr.) matrix of the average correlation coefficients for all possible combinations of ROIs within (diagonal elements) and between projections 1 and 8. Heat map depicts the degree of positive (red) and negative (blue) correlation. (D) Whisker-box plot from junctional pericytes depicting average correlation coefficients for Ca²⁺ events within a projection or between projections located on the same or on different capillary branches. Data from n = 5 pericytes (n = 36 projections; 114 ROIs) revealed a higher correlation for Ca²⁺ events in ROIs within a projection (137 ROI pairs in 36 projections; Corr. = 0.46 ± 0.04) or across projections within the same capillary branch (323 ROI pairs between 36 projection pairs; Corr. = 0.26 ± 0.03) compared to ROIs from different capillary branches (861 ROI pairs between 84 projection pairs; Corr. = 0.07 ± 0.02).

Next, using 2PLSM and 3D reconstructions of z-stack images (Fig. 6A), we examined the relationship between Ca²⁺ signals and contraction of pericyte projections at junctions in the transitional region in an in vivo setting. Consistent with Ca²⁺-driven contractions, we found that the frequency of Ca²⁺ events in pericyte projections was correlated with decreases in the lumen diameter of the corresponding branch (Fig. 6B), such that pericyte projections encircling smaller-diameter (constricted) capillary segments exhibited a higher frequency of Ca²⁺ events than those encircling larger-diameter (dilated) segments. To further determine how differential control of branch diameters ex vivo translates to differential control of blood flow in vivo, we measured the fractional distribution of RBCs at junctions—the proportion of cells flowing down each branch—following application of a contractile stimulus directly to individual junctional pericytes. The fractional distribution of RBCs was obtained by line-scan analysis, in which lines were positioned so as to span the cross-section of both daughter branches (Fig. 6C) and scanned at a frequency of 5 kHz. For these experiments, we used acta2-GCaMP-GR mice i.v. injected with tetramethylrhodamine (TRITC)-dextran (red) or NG2-dsRed mice injected with FITC-dextran (green) to allow visualization of pericytes and capillaries. To deliver a focal stimulus, we inserted a micropipette containing fluorescently labeled dextran (to visualize the administration zone) and U46619 (100 nM), or saline (control), through the cranial window and maneuvered it to within 5 µm of the targeted junctional pericyte. After picospritzing U46619 (or saline) onto a junctional pericyte within the postarteriole transitional region, we tracked the flux of RBCs in both downstream branches by 2PLSM. Local delivery of U46619 onto such pericytes decreased the combined RBC flux through both daughter branches by an average of 21 ± 5.3 cells per second (n = 10), but the degree of decrease between the two branches (d₁ and d₂) varied. For example, in some cases, RBC flux was completely halted (3 of 10 branches) or decreased (4 of 10 branches) to the same degree in each branch (symmetric). Notably, however, in a subset of cases (3 of 10), the decrease in RBC flux was greater in one branch than the other (asymmetric) (Fig. 6 D and E), consistent with differential contraction of individual branches. By comparison, any changes in flux following delivery of saline (control) were generally symmetrical (Fig. 6 F–H). Transient activation of pericytes by focally picospritzing U46619 had no effect on RBC flux at more distal capillary junctions (Fig. 6 I–K), consistent with the absence of the dynamic smooth muscle α-actin in this pericyte subpopulation or slower dynamics that might not be captured with the transient U46619 delivery.

Fig. 6.

Pericyte Ca²⁺ events determine capillary branch diameter in vivo. (A) Cerebral circulation in an anesthetized acta2-GCaMP-GR transgenic mouse injected with TRITC (red)-dextran to illuminate the vasculature, visualized through a cranial window using 2PLSM. (Scale bars, 50 µm.) (B, Left) Representative in vivo images showing the fluorescence intensity of GCaMP (Ca²⁺ events) and mCherry, and the geometry of capillary branches. (i) mCherry fluorescence; arrowheads indicate pericytes. (ii) GCaMP fluorescence intensity relative to baseline (F/Fo). (iii) Branch angle. (iv) Branch diameter. (Middle) Representative GCaMP traces; mCherry trace shown for comparison. (Right) Correlation between Ca²⁺ event frequency (event/s) and branch angle and diameter in the two daughter branches (d₁ and d₂) of individual junctional pericytes. Different daughter branches are denoted by triangles and circles. (C–K) Disruption of blood flow following U46619-induced constriction of junctional pericytes in the postarteriole transitional region in vivo. (C) Representative images showing pipette placement and RBC flux before and after picospritzing U46619 (100 nM) onto the targeted proximal pericyte. (D and E) Representative traces and summary data at 60 s showing the effects of U46619 on RBC flux (cells/s) down daughter branches d₁ (purple) and d₂ (orange). Flow of RBCs was completely, but transiently, halted in both branches (30%) (i), differed between the two branches (30%) (ii), or decreased in both branches (40%) (n = 10, n = 7 mice; *P ≤ 0.05 vs. baseline) (iii). (F–H) Blood flow remained symmetrical following application of aCSF (control) onto junctional pericytes in the postarteriole transitional region in vivo. (F) Representative images showing pipette placement and RBC flux (line scans) through daughter branches d₁ and d₂ of a proximal pericyte, before and after picospritzing aCSF. (G) Representative traces showing the running average of RBC flux (cells/s) down each daughter branch, d₁ (purple) and d₂ (orange). RBC flux remained relatively constant (i and ii) throughout imaging; when changes in flux occurred, they were symmetrical between branches (iii). (H) Summary data showing RBC flux during baseline and 30 s after picospritzing aCSF (n = 10 pericyte junctions, n = 5 mice). (I–K) Blood flow is unaffected by stimulation of distal, noncontractile pericytes with U46619. (I) Representative images showing pipette placement and RBC flux (line scans) through the daughter branches of distal pericytes before and after picospritzing U46619. (J) Representative trace and summary data showing the effects of picospritzing aCSF onto distal pericytes (n = 19 pericyte junctions, n = 10 mice). (K) Representative trace and summary data showing the effects of picospritzing U46619 (100 nM) onto distal pericytes (n = 10 pericyte junctions, n = 6 mice). (Scale bars, 10 µm.)

Junctional Pericytes Serve as Control Elements in K⁺-Mediated Functional Hyperemia.

We recently reported that retrograde hyperpolarizing signals mediated by K⁺-dependent activation of capillary endothelial K_ir channels cause upstream arteriolar dilation and thereby increase blood flow into the capillary network (5). Pericytes and capillary endothelial cells are electrically coupled via gap junctions (36), implying that pericytes are capable of receiving electrical signals from the underlying endothelium. Accordingly, we hypothesized that the retrograde hyperpolarizing signal induced by a K⁺ stimulus applied downstream of the transitional region would be conferred via gap junctions to overlying pericytes along the track of the propagating hyperpolarization, leading to branch-specific changes in capillary diameter in the direction of the stimulus through hyperpolarization-dependent relaxation of pericyte projections (Fig. 7A).

Fig. 7.

Branch-specific dilation in response to retrograde hyperpolarization. (A) Proposed mechanism by which K⁺-induced retrograde hyperpolarization increases cerebral blood flow (5). Increases in local K⁺ around capillaries activates K_IR channels, generating local hyperpolarization that propagates upstream to the feeding arteriole. Membrane hyperpolarization causes arterial and pericyte relaxation, promoting an increase in blood flow into the capillaries. (B) Schematic of a pressurized retina preparation. The ophthalmic artery of an isolated mouse retina is cannulated and the retina is pinned down en face. (C, Left) Representative image of the feeding arteriole and capillary branch most proximate to the arteriole. Picospritzing pipette is targeted downstream of one branch (Stim. branch). (C, Right) Ca²⁺ and branch diameters recorded over time. (D) Representative traces showing average pericyte Ca²⁺ and diameter in stimulated and unstimulated branches following picospritzing 15 mM K⁺ and TRITC-dextran tracer downstream of one branch of the monitored postarteriole transitional segment. (E and F) Summary data showing the increase in branch diameter and decrease in Ca²⁺ in pericyte projections after picospritzing 15 mM K⁺ downstream of one branch (n = 18, n = 5 mice; *P ≤ 0.05 vs. Stim. branch).

To test this hypothesis ex vivo, we used our recently developed pressurized retina preparation to examine changes in daughter branch diameters at pericyte-occupied junctions of the postarteriole transitional region following K⁺ stimulation of a downstream capillary branch. In this retinal preparation, the distal portion of the ophthalmic artery that feeds the retina is isolated and cannulated, and the retina tissue is pinned down en face, allowing visualization of the entire retinal vasculature (Fig. 7B). After pressurizing the preparation, which caused the vascular tree fed by the ophthalmic artery to develop myogenic tone (SI Appendix, Fig. S8), we locally delivered 15 mM K⁺ onto a capillary segment downstream of a junctional pericyte located in the transitional region and measured changes in the diameter of the stimulated branch and unstimulated branch. The retrograde hyperpolarizing signal induced by K⁺ led to a change in pericyte contractility, producing asymmetric responses in branch diameters such that diameter increased preferentially in the branch immediately upstream of the stimulus (Fig. 7 C–E). To further investigate this, we created mice expressing a modified Ca²⁺ sensor with lower maximum brightness and lower Ca²⁺ affinity fused to mVermilion (a modified mCherry protein with twofold higher fluorescence intensity) driven by an Acta2 promoter (Methods). Retinas were prepared from acta2-GCaMP8.1-mVermilion transgenic mice, enabling the simultaneous ratiometric measurement of Ca²⁺-dependent (green) and Ca²⁺-independent (red) fluorescence signals in individual projections. Notably, application of K⁺ onto a distal capillary segment asymmetrically impacted Ca²⁺ signaling in pericyte projections at upstream daughter branches, specifically decreasing Ca²⁺ signals in pericyte projections surrounding the dilated branch immediately upstream of the stimulus (Fig. 7F). To further demonstrate branch-specific dilation, we subsequently moved the picospritzing pipette to a capillary segment downstream of the previously unstimulated branch, and applied a second 15-mM K⁺ stimulus. Similar to our observations in the first experiment, this second experiment yielded a larger upstream dilation in the stimulated branch (SI Appendix, Fig. S9). Collectively, these results are consistent with the idea that junctional pericytes in the postarteriole transitional region can be engaged by retrograde hyperpolarizing signals. Because individual pericyte projections are capable of acting as separate functional units, the arriving vasodilatory signals can preferentially dilate the daughter vessel supplying the stimulated region.

Finally, to test the hypothesis that different projections of a contractile pericyte are capable of receiving and isolating branch-specific stimulation (hyperpolarization) in an in vivo setting, we locally delivered 15 mM K⁺ onto capillary segments downstream (∼40 µm) of one branch of a junctional pericyte in the postarteriole transitional region of NG2-DsRed-BAC transgenic mice and measured RBC flux in each daughter branch (Fig. 8A). As shown in Fig. 8 B and C, this led to an asymmetric shift in RBC flux, such that flux was robustly increased in the stimulated branch (75% ± 21%) in association with a decrease (8 of 12 junctions) or no change (4 of 12 junctions) in flux in the other branch. By contrast, picospritzing 15 mM K⁺ onto a capillary segment downstream of one branch of a distal junctional pericyte caused a symmetrical increase in blood flow (30% ± 5%) in both daughter branches (10 of 11 branches) (Fig. 8 D and E). This latter observation indicates that the increase in blood flow induced upstream (i.e., at the arteriole level) by our previously reported K⁺-induced NVC mechanism is not subject to directional regulation by these noncontractile pericytes, which are incapable of actively facilitating blood flow in the direction of the stimulus. Collectively, these findings suggest that contractile pericytes in the postarteriole transitional region are capable of receiving propagating K⁺-induced hyperpolarizing signals through the capillary network and dynamically modulating blood supply.

Fig. 8.

Branch-specific increases in capillary blood flow in response to retrograde hyperpolarization. (A) Representative images showing pipette placement and RBC flux (line scans) through daughter branches d₁ and d₂ enwrapped by a junctional pericyte in the postarteriole transitional segment, before and after picospritzing 15 mM K⁺ downstream of one branch of the monitored junction. (B) Representative traces showing the running average of RBC flux (cells/s) down each daughter branch, d₁ (purple) and d₂ (orange), following administration of 15 mM K⁺ downstream of one branch. (i–iii) K⁺-dependent increases in RBC flux through the stimulated branch reduced flux (67%; i and ii) or had no effect (33%; iii) on flow in the unstimulated branch. (C) Summary data showing RBC flux at baseline and 30 s after stimulation by picospritzing 15 mM K⁺ downstream of one branch of the monitored junctional pericyte in the postarteriole transitional segment (n = 11 pericyte junctions, n = 6 mice; *P ≤ 0.05 vs. baseline). (D and E) Representative images, trace, and summary data showing the effects of picospritzing 15 mM KCl downstream of a distal pericyte (n = 12 pericyte junctions, n = 6 mice; *P ≤ 0.05 vs. baseline). (Scale bars, 10 µm.)

Discussion

Considerable research effort has been devoted to understanding how active neurons communicate their energy needs to the brain microvasculature to increase the local delivery of blood-borne nutrients and O₂. The focus of much of this research has been on the arteriolar level of the vascular tree, especially the role of astrocytes in translating neuronal activity into parenchymal arteriolar dilation. Recent work from our laboratory (5, 37, 38) and those of others (39–41) has shifted the focus downstream, showing that the expansive network of capillaries serves as a “sensory web” that detects neuronal activity and converts it to an electrical (hyperpolarizing) signal that propagates upstream to cause arteriolar dilation. This process, initiated by neuronal activity-derived K⁺ and mediated by Kir2.1 channels in capillary ECs, provides an efficient mechanism for driving an increase in blood flow toward active regions. But, like proposed astrocyte/arteriole-level signaling processes, this mechanism leaves open the question of how the distribution of blood flow and RBC flux within the capillary network is regulated. In the absence of such a regulatory process, blood distribution within the capillary bed would be governed solely by the static architecture of the microvascular network; this would decrease the efficiency of targeted blood delivery and invariably result in squandering of resources on relatively quiescent areas. Here, we demonstrate that pericytes structurally alter the static symmetry of capillary junctions within the microcirculation. We further show that contractile pericytes at capillary junctions in the transitional region dynamically and differentially regulate daughter branch diameters to control the distribution and directed perfusion of RBCs within brain tissue. Importantly, we provide strong experimental evidence that pericytes play an integral role in our previously established electrical-based NVC mechanism, showing that contractile pericytes are capable of modulating junctional blood flow by dilating in response to K⁺-dependent hyperpolarizing signals initiated at the site of neuronal activity, thereby channeling RBC flux in the direction of the stimulus. Because, in theory (supported by modeling; see SI Appendix, Fig. S10), the fraction of total RBCs decreases nonlinearly at each successive junction in the capillary network, the first few junctions make the bulk of the “decisions” impacting the distribution of blood flow throughout the network. Thus, contractile junctional pericytes in this postarteriole transitional region are well situated to exert an outsized influence on the distribution of RBCs in a local capillary network.

Role of Pericytes in Regulating Blood Flow in the Brain: The Definition Problem.

Much of the controversy surrounding the functional role of capillary pericytes in NVC and regulation of blood flow is definitional, reflecting the fact that some consider the microvascular region containing α-actin-expressing, contractile perivascular cells to be a precapillary arteriole and the cells themselves to be atypical smooth muscle cells. This formulation simply defines away any functional role for capillary pericytes. The persistent view that these contractile cells are smooth muscle cells also finds support in the fact that some of these cells are capable of exhibiting a smooth muscle cell-like banding pattern. However, these limited criteria are not definitive and ignore a large body of evidence to the contrary. First, smooth muscle α-actin expression and contractile behavior are observed in cells that are agreed to be distinct from smooth muscle cells, including myofibroblasts (granulation tissue fibroblasts) (42), lung fibroblasts (43), myoepithelial cells (44), and developing embryonic cardiac myocytes (45, 46). The appearance of a smooth muscle-like banding pattern is an even less compelling criterion, given that this overtly similar morphology likely reflects a convergent phenotype driven by biophysical factors—an enwrapping morphology simply more efficiently imparts a contractile force on a tubular structure than any other morphology, regardless of the origin of the enwrapping cell(s). This banding pattern aside, the general morphology of pericytes is clearly distinct from that of smooth muscle cells, as described above. Moreover, there is a general consensus that perivascular cells deeper in the capillary bed are pericytes; yet these cells, like perivascular cells in the postarteriole transitional region, express the “smooth muscle-specific” protein, Myh11. Perhaps most important, none of these perivascular cells express functional RyRs, a defining feature of all known smooth muscle cells; thus, the absence of RyR activity may in fact represent a definitive, pericyte-specific marker. Much remains to be learned about the ontogenetic relationship between pericytes in the CNS microvasculature and other cell types, as well as how pericytes come to populate the CNS capillary network. However, regardless of the nomenclature ultimately adopted, the unique features and functional significance of the critical cell network in the postcapillary transitional zone are clear.

Control of RBC Flux through Effects of Junctional Pericytes on the Static Symmetry of Capillary Bifurcations.

Vascular network hemodynamics are governed by the applied pressure drop (ΔP; difference between inlet and outlet pressure) and the vascular geometry, which can dynamically change. Blood flow (Q) depends on the radius (r) and length (l) of each vessel and on the effective blood viscosity (n_eff), and can be approximated from Poiseuille’s law (Q = π ΔPr⁴/8η_effl). In the microcirculation, the presence of the endothelial surface layer (47) and the particulate nature of blood introduce nonlinearities and a strong dependence of n_eff on hematocrit and vessel diameter (Fåhræus-Lindqvist effect) (48, 49), as well as uneven partitioning of RBCs at diverging bifurcations (phase separation) (50). Thus, RBCs entering a capillary bed will be nonuniformly distributed in a manner that reflects the arborization of the network and complex non-Newtonian fluid dynamics. The observed flow responses (RBC fluxes) in capillary networks cannot be explained by these physical constraints, suggesting active regulation of blood perfusion by ensheathing pericytes.

In this context, we identified a role for junctional pericytes in structurally modifying the resting geometry of capillary bifurcations. Specifically, we showed that the symmetry of capillary junctions in wild-type mice, both in an ex vivo retinal preparation and in the mouse brain in vivo, is inversely related to pericyte coverage such that junctions in the postarteriole transitional region, where pericytes are more prevalent, are less symmetrical than more distal junctions. This asymmetry of pericyte-associated junctions manifests most clearly as differences in diameters of daughter branches. Thus, by virtue of their potential influence on the structural symmetry of bifurcations, junctional pericytes are uniquely positioned to exert effects on capillary diameter that compensate for the default geometry of the system and the resistance imposed by physical factors.

Dynamic Control of Daughter Branch Diameters and RBC Flux by Junctional Pericytes in the Postarteriole Transitional Region.

An analysis of spontaneous Ca²⁺ signaling in junctional pericytes in the transitional region of ex vivo retinal preparations from acta2-GCaMP-GR transgenic mice revealed different Ca²⁺-signaling profiles in individual pericyte projections enwrapping branches of a junction. Moreover, there were clear cases in which contractile responses were restricted to individual projections in which Ca²⁺-signaling events occurred. Thus, unlike all muscle cells, in which the functional working unit is the entire myocyte (51), an individual pericyte is made up of multiple functional units corresponding to separate capillary-enwrapping projections, each of which constitutes a restricted Ca²⁺-signaling domain that is capable of contracting independently of other projections.

Because pericytes are able to compartmentalize Ca²⁺ and contract individual projections, the prediction is that junctional pericytes should be able to independently regulate the diameter of the daughter branches they enwrap. This prediction was borne out by experiments performed on retinal capillary preparations, which showed that stimulation with U46619 was capable of causing asymmetric constriction of daughter branches. Importantly, this differential control of branch diameters observed ex vivo translated into differences in RBC flux in vivo. In these latter experiments employing the mouse cranial window model and 2PLSM, focal stimulation of a junctional pericyte in the postarteriole transitional region dynamically regulated RBC flux in downstream branches, producing both symmetric and asymmetric changes in flux.

Contributions to NVC.

Neurons are metabolically demanding cells and rely heavily on an on-demand process, termed neurovascular coupling, that translates neural activity into local increases in blood flow and thus delivery of needed nutrients. Recent work from our laboratory has demonstrated the operation of a NVC mechanism, identifying capillaries as a “wiring” system that electrically communicates neuronal demand upstream to promote arteriole dilation (5). Pericytes, which are electrically coupled to the underlying capillary endothelial cells (36), are in a position to receive and potentially manipulate these electrical signals, either as electrical sinks or boosters. Our data suggest that projections of contractile pericytes enwrapping one branch of a capillary junction in the postarteriole transitional region are able to dilate in response to K⁺-induced hyperpolarizing signals propagating along that branch, dynamically manipulating junctional blood flow to favor the flux of RBCs in the direction of the stimulus. This surprising finding implies that membrane potential is not necessarily uniform across all processes of an individual pericyte. This suggests that pericyte processes may be electrically compartmentalized through an as-yet-unidentified mechanism, a behavior reminiscent of neuronal processes and completely unlike that found in smooth muscle cells. With their ability to independently control the diameters of different branches, contractile pericytes are able to receive hyperpolarizing signals from downstream neurons, causing a branch-specific dilation that provides a pathway that guides increased blood flow in the direction of active neurons. By controlling the distribution of RBCs, contractile pericytes located at capillary bifurcations in the postarteriole transitional region provide a mechanism that ensures the preferential delivery of RBCs to cells in need, thereby increasing the efficiency of the system.

Methods

Animals.

All animals were used in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Vermont. Acta2-GCaMP-GR mice were obtained from the CHROMus resource (33) and Acta2-GCaMP8.1-mVermilion BAC transgenic mice were generated as part of this study. Detailed descriptions of transgenic mice have been included in SI Appendix, Methods. Animals were housed on a 12-h light:dark cycle with free access to food, water, and environmental enrichments.

Ex Vivo Imaging of Pericyte Contraction and Ca²⁺ in the Retina.

Animals were deeply anesthetized with pentobarbital sodium (50 mg, i.p.) and killed by exsanguination and decapitation. Retinas were isolated, pinned down in an en face (vitreal side up) orientation, and stored in ice-cold retinal physiological saline solution (SI Appendix, Methods). For immunocytochemistry studies, en face retina preparations were mounted on a silicone block and washed in a magnesium-based physiological saline solution (Mg-PSS) (SI Appendix, Methods). Retinas were fixed by incubating with 4% paraformaldehyde (Mg-PSS) for 15 min, permeabilized with 0.1% Triton X, and blocked with a blocking solution (Mg-PSS) containing 2% bovine serum albumin (BSA) and 2% normal goat serum. Retinas were incubated overnight at 4 °C in blocking solution containing cyanin (Cy)-conjugated mouse monoclonal anti-α-actin (Sigma-Aldrich) or anti-calponin (Abcam, ab46794) antibodies. Filamentous actin and microtubules were stained ex vivo by incubating isolated en face retinal preparations with Alexa Flour 488 Phalloidin (1:200; Thermo Fisher Scientific) and Tubulin Tracker Green (1:50; Thermo Fisher Scientific), respectively. Blood vessels were counterstained with rhodamine (1:50; Vector Laboratories) or FITC-conjugated isolectin B4 (VWR) in Mg-PSS for 20 min at 37 °C. The junctional fluorescence minus background fluorescence was normalized to the smooth muscle fluorescence minus background at each junction and then multiplied by 100 to obtain the percentage. A junctional pericyte was defined as a pericyte whose nucleus was within 8 μm (the size of ∼1 pericyte nucleus) of the center of the junction. For ex vivo pericyte Ca²⁺ and contractility studies, en face retinal preparations were mounted on a silicone block, washed, and stained with rhodamine- or FITC-conjugated isolectin B4 (diluted 1:25 in rPSS bubbled with 95% O₂/5% CO₂) by incubating for 20 min at 37 °C. Care was taken to ensure that proper pH (bicarbonate based) and oxygenation (95% O₂) were maintained throughout isolation, pinning, staining, and experimental procedures. Pericytes were imaged using a Revolution confocal system (Andor Technology) mounted on an upright Nikon microscope equipped with a 60× water-dipping objective (numerical aperture [NA] 1.0). Fluorescence was excited with a 488-nm (FITC) or 560-nm (rhodamine) solid-state laser, and emitted fluorescence collected through a 527.5/49 nm (FITC) or 641.5/117 nm (rhodamine) band-pass filter. For contractility experiments, time-lapse images were acquired at 30 frames/min and z-stack images were obtained at 0.2-µm increments with a 1.2-µm z-resolution. For Ca²⁺-imaging experiments, images were acquired at a resolution of 512 × 512 pixels at 30 frames/s at 36 °C. For ex vivo pressurized-retina studies, the entire orbit containing the eye, optic nerve, ophthalmic artery, and surrounding musculature was isolated and placed in ice-cold, 95% O₂/5% CO₂-bubbled Ca²⁺-free and Mg²⁺-supplemented rPSS (SI Appendix, Methods). Outer muscles, nerves, and surrounding support tissues were removed using fine scissors, leaving the optic nerve, fine musculature, and ophthalmic artery intact. Branches from the ophthalmic artery that did not feed the central retinal artery were ligated using a fine suture. The ophthalmic artery was cannulated using a fine glass cannula attached to a micromanipulator and filled with filtered rPSS. The retina was slit at strategic positions along the edge to allow the spheroidal structure to lay flat on a custom silicon platform, with the ophthalmic artery and surrounding structures positioned under the imaging plane. The entire retina was continuously perfused with 37 °C rPSS buffer at 5 mL/min. The cannula was pressurized via a gravity-fed line attached to a perfusion pressure monitor (PM-4; Living Systems) by moving the pressure column to ∼60 mmHg (corresponding to pressure at the ophthalmic artery) and opening a two-way valve. Rapid removal of blood cells from all retinal vessels confirmed successful cannulation and pressurization. Glass microinjection pipettes filled with 15 mM KCl were guided to targeted pericytes and endothelial cells using a micromanipulator (Sutter Instruments), and a small volume was pressure injected using a Picospritzer III (Parker).

In Vivo Cerebral Imaging.

The cranial window model and in vivo imaging were performed as previously described (5), and further details can be found in SI Appendix, Methods. FITC and TRITC were excited at 820 nm (∼2 to 189 mW of output power at the objective), GCaMP5 was excited at 940 nm (∼5 to 55 mW of output power at the objective), and emitted fluorescence was separated through 500- to 550- and 570- to 610-nm bandpass filters. Vessel diameters were determined using reconstructions of z-stack images (SI Appendix, Methods). Ca²⁺ images were acquired at a resolution of 512 × 100 pixels at ∼40 frames/s. RBC velocity and flux data were collected by line scanning at 5 kHz. Glass microinjection pipettes filled with U46619 or 15 mM KCl were guided to targeted pericytes and endothelial cells, respectively, using a micromanipulator (Sutter Instruments), and a small volume was pressure ejected using a Picospritzer III (Parker), as previously described (5). For in vivo tests of branch-specific responses, downstream capillary segments were chosen at random. Care was taken to ensure that ejection duration and pressure were calibrated to obtain a small solution plume, and that placement of the pipette restricted agent delivery to the capillary under study and caused minimal displacement of the surrounding tissue. Spatial coverage of the ejected solution was monitored by including TRITC- or FITC-labeled dextran solution (150 kDa; 0.2 mg/mL).

Modeling, Calculations, and Statistics.

All data are presented as means ± SE. Values of “n” refer to the number of projections, junctions, or junctional pericytes, and “N” refers to the number of animals. Relationships between local Ca²⁺ activity recorded in a single projection or in different projections of an individual pericyte were evaluated by performing a Pearson’s correlation analysis using MATLAB R2020a (The MathWorks, Inc.). Detailed description of modeling and statistics are provided in SI Appendix, Methods.

Data Availability.

All study data are included in the article and supporting information.