Glial Cell Calcium Signaling Mediates Capillary Regulation of Blood Flow in the Retina.
ABSTRACT: UNLABELLED:The brain is critically dependent on the regulation of blood flow to nourish active neurons. One widely held hypothesis of blood flow regulation holds that active neurons stimulate Ca(2+) increases in glial cells, triggering glial release of vasodilating agents. This hypothesis has been challenged, as arteriole dilation can occur in the absence of glial Ca(2+) signaling. We address this controversy by imaging glial Ca(2+) signaling and vessel dilation in the mouse retina. We find that sensory stimulation results in Ca(2+) increases in the glial endfeet contacting capillaries, but not arterioles, and that capillary dilations often follow spontaneous Ca(2+) signaling. In IP3R2(-/-) mice, where glial Ca(2+) signaling is reduced, light-evoked capillary, but not arteriole, dilation is abolished. The results show that, independent of arterioles, capillaries actively dilate and regulate blood flow. Furthermore, the results demonstrate that glial Ca(2+) signaling regulates capillary but not arteriole blood flow. SIGNIFICANCE STATEMENT:We show that a Ca(2+)-dependent glial cell signaling mechanism is responsible for regulating capillary but not arteriole diameter. This finding resolves a long-standing controversy regarding the role of glial cells in regulating blood flow, demonstrating that glial Ca(2+) signaling is both necessary and sufficient to dilate capillaries. While the relative contributions of capillaries and arterioles to blood flow regulation remain unclear, elucidating the mechanisms that regulate capillary blood flow may ultimately lead to the development of therapies for treating diseases where blood flow regulation is disrupted, including Alzheimer's disease, stroke, and diabetic retinopathy. This finding may also aid in revealing the underlying neuronal activity that generates BOLD fMRI signals.
Project description:Occlusions of penetrating arterioles, which plunge into cortex and feed capillary beds, cause severe decreases in blood flow and are potential causes of ischemic microlesions. However, surrounding arterioles and capillary beds remain flowing and might provide collateral flow around the occlusion. We used femtosecond laser ablation to trigger clotting in single penetrating arterioles in rat cortex and two-photon microscopy to measure changes in microvessel diameter and red blood cell speed after the clot. We found that after occlusion of a single penetrating arteriole, nearby penetrating and surface arterioles did not dilate, suggesting that alternate blood flow routes are not actively recruited. In contrast, capillaries showed two types of reactions. Capillaries directly downstream from the occluded arteriole dilated after the clot, but other capillaries in the same vicinity did not dilate. This heterogeneity in capillary response suggests that signals for vasodilation are vascular rather than parenchymal in origin. Although both neighboring arterioles and capillaries dilated in response to topically applied acetylcholine after the occlusion, the flow in the territory of the occluded arteriole did not improve. Collateral flow from neighboring penetrating arterioles is neither actively recruited nor effective in improving blood flow after the occlusion of a single penetrating arteriole.
Project description:Active neurons increase their energy supply by dilating nearby arterioles and capillaries. This neurovascular coupling underlies blood oxygen level-dependent functional imaging signals, but its mechanism is controversial. Canonically, neurons release glutamate to activate metabotropic glutamate receptor 5 (mGluR5) on astrocytes, evoking Ca2+ release from internal stores, activating phospholipase A2 and generating vasodilatory arachidonic acid derivatives. However, adult astrocytes lack mGluR5, and knockout of the inositol 1,4,5-trisphosphate receptors that release Ca2+ from stores does not affect neurovascular coupling. We now show that buffering astrocyte Ca2+ inhibits neuronally evoked capillary dilation, that astrocyte [Ca2+]i is raised not by release from stores but by entry through ATP-gated channels, and that Ca2+ generates arachidonic acid via phospholipase D2 and diacylglycerol lipase rather than phospholipase A2. In contrast, dilation of arterioles depends on NMDA receptor activation and Ca2+-dependent NO generation by interneurons. These results reveal that different signaling cascades regulate cerebral blood flow at the capillary and arteriole levels.
Project description:Increases in brain blood flow, evoked by neuronal activity, power neural computation and form the basis of BOLD (blood-oxygen-level-dependent) functional imaging. Whether blood flow is controlled solely by arteriole smooth muscle, or also by capillary pericytes, is controversial. We demonstrate that neuronal activity and the neurotransmitter glutamate evoke the release of messengers that dilate capillaries by actively relaxing pericytes. Dilation is mediated by prostaglandin E2, but requires nitric oxide release to suppress vasoconstricting 20-HETE synthesis. In vivo, when sensory input increases blood flow, capillaries dilate before arterioles and are estimated to produce 84% of the blood flow increase. In pathology, ischaemia evokes capillary constriction by pericytes. We show that this is followed by pericyte death in rigor, which may irreversibly constrict capillaries and damage the blood-brain barrier. Thus, pericytes are major regulators of cerebral blood flow and initiators of functional imaging signals. Prevention of pericyte constriction and death may reduce the long-lasting blood flow decrease that damages neurons after stroke.
Project description:Brain capillaries play a critical role in sensing neural activity and translating it into dynamic changes in cerebral blood flow to serve the metabolic needs of the brain. The molecular cornerstone of this mechanism is the capillary endothelial cell inward rectifier K+ (Kir2.1) channel, which is activated by neuronal activity-dependent increases in external K+ concentration, producing a propagating hyperpolarizing electrical signal that dilates upstream arterioles. Here, we identify a key regulator of this process, demonstrating that phosphatidylinositol 4,5-bisphosphate (PIP2) is an intrinsic modulator of capillary Kir2.1-mediated signaling. We further show that PIP2 depletion through activation of Gq protein-coupled receptors (GqPCRs) cripples capillary-to-arteriole signal transduction in vitro and in vivo, highlighting the potential regulatory linkage between GqPCR-dependent and electrical neurovascular-coupling mechanisms. These results collectively show that PIP2 sets the gain of capillary-initiated electrical signaling by modulating Kir2.1 channels. Endothelial PIP2 levels would therefore shape the extent of retrograde signaling and modulate cerebral blood flow.
Project description:Blood flow into the brain is dynamically regulated to satisfy the changing metabolic requirements of neurons, but how this is accomplished has remained unclear. Here we demonstrate a central role for capillary endothelial cells in sensing neural activity and communicating it to upstream arterioles in the form of an electrical vasodilatory signal. We further demonstrate that this signal is initiated by extracellular K+ -a byproduct of neural activity-which activates capillary endothelial cell inward-rectifier K+ (KIR2.1) channels to produce a rapidly propagating retrograde hyperpolarization that causes upstream arteriolar dilation, increasing blood flow into the capillary bed. Our results establish brain capillaries as an active sensory web that converts changes in external K+ into rapid, 'inside-out' electrical signaling to direct blood flow to active brain regions.
Project description:Functional neuroimaging, such as fMRI, is based on coupling neuronal activity and accompanying changes in cerebral blood flow (CBF) and metabolism. However, the relationship between CBF and events at the level of the penetrating arterioles and capillaries is not well established. Recent findings suggest an active role of capillaries in CBF control, and pericytes on capillaries may be major regulators of CBF and initiators of functional imaging signals. Here, using two-photon microscopy of brains in living mice, we demonstrate that stimulation-evoked increases in synaptic activity in the mouse somatosensory cortex evokes capillary dilation starting mostly at the first- or second-order capillary, propagating upstream and downstream at 5-20 µm/s. Therefore, our data support an active role of pericytes in cerebrovascular control. The gliotransmitter ATP applied to first- and second-order capillaries by micropipette puffing induced dilation, followed by constriction, which also propagated at 5-20 µm/s. ATP-induced capillary constriction was blocked by purinergic P2 receptors. Thus, conducted vascular responses in capillaries may be a previously unidentified modulator of cerebrovascular function and functional neuroimaging signals.
Project description:Spreading depolarization is assumed to be the mechanism of migraine with aura, which is accompanied by an initial predominant hyperaemic response followed by persistent vasoconstriction. Cerebral blood flow responses are impaired in patients and in experimental animals after spreading depolarization. Understanding the regulation of cortical blood vessels during and after spreading depolarization could help patients with migraine attacks, but our knowledge of these vascular mechanisms is still incomplete. Recent findings show that control of cerebral blood flow does not only occur at the arteriole level but also at capillaries. Pericytes are vascular mural cells that can constrict or relax around capillaries, mediating local cerebral blood flow control. They participate in the constriction observed during brain ischaemia and might be involved the disruption of the microcirculation during spreading depolarization. To further understand the regulation of cerebral blood flow in spreading depolarization, we examined penetrating arterioles and capillaries with respect to vascular branching order, pericyte location and pericyte calcium responses during somatosensory stimulation and spreading depolarization. Mice expressing a red fluorescent indicator and intravenous injections of FITC-dextran were used to visualize pericytes and vessels, respectively, under two-photon microscopy. By engineering a genetically encoded calcium indicator we could record calcium changes in both pericytes around capillaries and vascular smooth muscle cells around arterioles. We show that somatosensory stimulation evoked a decrease in cytosolic calcium in pericytes located on dilating capillaries, up to the second order capillaries. Furthermore, we show that prolonged vasoconstriction following spreading depolarization is strongest in first order capillaries, with a persistent increase in pericyte calcium. We suggest that the persistence of the 'spreading cortical oligaemia' in migraine could be caused by this constriction of cortical capillaries. After spreading depolarization, somatosensory stimulation no longer evoked changes in capillary diameter and pericyte calcium. Thus, calcium changes in pericytes located on first order capillaries may be a key determinant in local blood flow control and a novel vascular mechanism in migraine. We suggest that prevention or treatment of capillary constriction in migraine with aura, which is an independent risk factor for stroke, may be clinically useful.
Project description:We demonstrate that Alexa Fluor 633 hydrazide (Alexa Fluor 633) selectively labels neocortical arteries and arterioles by binding to elastin fibers. We measured sensory stimulus-evoked arteriole dilation dynamics in mouse, rat and cat visual cortex using Alexa Fluor 633 together with neuronal activity using calcium indicators or blood flow using fluorescein dextran. Arteriole dilation decreased fluorescence recorded from immediately underlying neurons, representing a potential artifact during neuronal functional imaging experiments.
Project description:The accumulation of small strokes has been linked to cognitive dysfunction. Although most animal models have focused on the impact of arteriole occlusions, clinical evidence indicates that venule occlusions may also be important. We used two-photon excited fluorescence microscopy to quantify changes in blood flow and vessel diameter in capillaries after occlusion of single ascending or surface cortical venules as a function of the connectivity between the measured capillary and the occluded venule. Clotting was induced by injuring the target vessel wall with femtosecond laser pulses. After an ascending venule (AV) occlusion, upstream capillaries showed decreases in blood flow speed, high rates of reversal in flow direction, and increases in vessel diameter. Surface venule occlusions produced similar effects, unless a collateral venule provided a new drain. Finally, we showed that AVs and penetrating arterioles have different nearest-neighbor spacing but capillaries branching from them have similar topology, which together predicted the severity and spatial extent of blood flow reduction after occlusion of either one. These results provide detailed insights into the widespread hemodynamic changes produced by cortical venule occlusions and may help elucidate the role of venule occlusions in the development of cognitive disorders and other brain diseases.
Project description:This study was aimed to characterize the geometric arrangement of hamster skeletal muscle arteriolar networks and to assess the in vivo rhythmic diameter changes of arterioles to clarify regulatory mechanisms of the capillary perfusion. The experimental study was carried out in male Syrian hamsters implanted with a plastic chamber in the dorsum skin under pentobarbital anesthesia. The skeletal muscle microvessels were visualized by fluorescence microscopy. The vessel diameters, lengths and the rhythmic diameter changes of arterioles were analyzed with computer-assisted techniques. The arterioles were classified according to a centripetal ordering scheme. In hamster skeletal muscle microvasculature the terminal branchings, differentiated in long and short terminal arteriolar trees (TATs), originated from anastomotic vessels, defined "arcading" arterioles. The long TATs presented different frequencies along the branching vessels; order 4 arterioles had frequencies lower than those observed in the order 3, 2, and 1 vessels. The short TAT order 3 arterioles, directly originating from "arcading" parent vessels, showed a frequency dominating all daughter arterioles. The amplitude of diameter variations in larger vessels was in the range 30-40% of mean diameter, while it was 80-100% in order 3, 2, and 1 vessels. Therefore, the complete constriction of arterioles, caused an intermittent capillary blood perfusion. L-arginine or papaverine infusion caused dilation of arterioles and transient disappearing of vasomotion waves and induced perfusion of all capillaries spreading from short and long TAT arrangements. Therefore, the capillary blood flow was modulated by changes in diameter of terminal arterioles penetrating within the skeletal muscle fibers, facilitating redistribution of blood flow according to the metabolic demands of tissues.