Local IP3 receptor–mediated Ca2+ signals compound to direct blood flow in brain capillaries

Abstract

Healthy brain function depends on the finely tuned spatial and temporal delivery of blood-borne nutrients to active neurons via the vast, dense capillary network. Here, using in vivo imaging in anesthetized mice, we reveal that brain capillary endothelial cells control blood flow through a hierarchy of IP₃ receptor–mediated Ca²⁺ events, ranging from small, subsecond protoevents, reflecting Ca²⁺ release through a small number of channels, to high-amplitude, sustained (up to ~1 min) compound events mediated by large clusters of channels. These frequent (~5000 events/s per microliter of cortex) Ca²⁺ signals are driven by neuronal activity, which engages G_q protein–coupled receptor signaling, and are enhanced by Ca²⁺ entry through TRPV4 channels. The resulting Ca²⁺-dependent synthesis of nitric oxide increases local blood flow selectively through affected capillary branches, providing a mechanism for high-resolution control of blood flow to small clusters of neurons.

INTRODUCTION

Capillaries are composed of endothelial cells (ECs), which also line all arteries and veins. Collectively, these vessels form a continuous vascular network that permeates all tissues of the body. Intracellular Ca²⁺ elevations in arteriolar ECs exert control over the release of potent vasodilators, such as nitric oxide (NO), which are critical for the control of blood flow and pressure (1). Ca²⁺ release from the endoplasmic reticulum (ER) membrane through ubiquitous inositol trisphosphate receptors (IP₃Rs) is a major pathway for increasing cytosolic Ca²⁺ levels in arteriolar ECs (2), an increase that can be augmented by Ca²⁺ influx across the plasmalemma through TRPV4 (transient receptor vanilloid member 4) nonselective cation channels (3). Although the central role of Ca²⁺ in arteriolar ECs is well established, the nature of capillary EC (cEC) Ca²⁺ signaling and its regulation in the brain and potential contribution to blood flow control are unexplored.

RESULTS

To establish an unambiguous framework for our analyses, we first defined a nomenclature for referring to the brain vasculature. We classify vessels as arterioles, venules, or capillaries according to branch order and orientation. The penetrating arteriole branching from pial arteries on the brain’s surface is defined as the 0-order (0°) parenchymal arteriole, characterized by its coverage by concentric smooth muscle cells (SMCs), which secrete elastin to form an elastic lamina (4). We define elastin-negative vessels branching from this arteriole as capillaries (fig. S1) and further distinguish the initial ~4 branches of the capillary bed as a transition zone (5, 6), reflecting the fact that these vessels have features common to both arterioles and deeper capillaries. Specifically, the narrow, proximal branches (1st- to ~4th-order; 1° to 4°) of the transition zone through which red blood cells (RBCs) pass in single file are heavily covered with processes of α-actin–positive contractile cells whose cell bodies display a characteristic “bump-on-a-log” morphology (7). These cells have been variably referred to as SMCs (8, 9), contractile pericytes (10), or a hybrid cell type (11, 12). Here, we use the term “contractile pericyte.” Deeper capillaries (≥5th order; ≥5°) are in contact with the thin-strand processes of noncontractile pericytes, which express little or no smooth muscle α-actin (5–7). The first large-diameter (>7 μm), vertically oriented vessel carrying blood back to the brain’s surface is designated as a venule.

To study Ca²⁺ signaling in the capillary network, we developed a transgenic mouse line in which GCaMP8, a high signal-to-noise Ca²⁺ indicator, is expressed under transcriptional control of the pan-endothelial Cdh5 (cadherin 5) promoter as part of a bacterial artificial chromosome (13). The resulting Cdh5^BAC-GCaMP8 mice express this indicator exclusively in vascular ECs throughout all observed vascular beds (fig. S2), allowing us to isolate cEC Ca²⁺ signals. To study these signals in the cortex, we created a cranial window by surgically removing the skull overlying the barrel cortex, resecting the dura, and attaching a head plate for immobilization. We then illuminated plasma by delivering TRITC-dextran (tetramethylrhodamine isothiocyanate–dextran) to the bloodstream to enable simultaneous visualization of blood flow and endothelial Ca²⁺ activity using two-photon laser scanning microscopy. To analyze Ca²⁺ signals in vascular structures, we mapped wide areas of the somatosensory cortical vasculature down to approximately layer III before selecting a volume of interest and then performed nested “four-dimensional” (4D) imaging, consisting of repeated temporal sweeps through cuboidal volumes measuring ~425 μm by 425 μm by 50 μm. This volumetric approach provides information on EC Ca²⁺ activity over a broad spatial region over time. To permit temporal analysis of individual Ca²⁺ events, we followed this by imaging a ~425 μm–by–425 μm single xy plane from within the same region with subsecond resolution (Fig. 1A). Brain ECs in all segments of the vasculature displayed dynamic local Ca²⁺ events under basal conditions in vivo (movie S1) that were notable for their striking amplitudes and long durations.