The vascular endothelium in intracerebral (parenchymal) arterioles is a critical mediator of normal cerebral function, serving as both a physical barrier and a modulator of blood flow within the brain. Ca2+ signaling and the Ca2+-sensitive K+ channels, IK and SK, and TRPV4 channels in endothelial cells (ECs) activate pathways that transmit vasoregulatory signals to adjacent smooth muscle (SM) and along the endothelial lining of blood vessels. These signals may also communicate to nearby astrocytes and neurons to modulate neurovascular coupling (NVC). Despite the importance of parenchymal arteriolar (PA) endothelium, little is known about its control of vascular tone or potential influence on NVC in the brain. To support the Project, we provide novel data that EC SK, IK and TRPV4 channels have a profound effect on PA tone and cortical cerebral blood flow. Aim 1 will elucidate the properties and roles of endothelial Ca2+ signaling modalities, and IK, SK and TRPV4 channels in PA ECs using a novel mouse model that expresses a Ca2+ biosensor (GCaMP2) in the endothelium. Exploiting these GCaMP2 mice, we have recently discovered a localized, stationary IP3- mediated Ca2+ signal in endothelial projections to the SM. This signal, termed a "pulsar", activates co-localized IK channels to modulate vascular tone. Aim 2 will determine the impact of endothelial function on the SM of PAs, exploring Ca2+ signaling, membrane potential and vascular diameter. Aim 3 builds on Aims 1 and 2 to explore the role of the endothelium in the context of the brain, evaluating its effects on NVC and blood flow using a novel approach based on simultaneous measurement of astrocytic endfoot Ca2+ and vascular responses. The proposed project will provide significant new insight into PA endothelial function and communication to SM. This project should reveal novel targets involved in modulating blood flow in the brain and suggest therapeutic agents that do not require passage through the blood brain barrier. PUBLIC HEALTH RELEVANCE: The cells (endothelial) that line the small blood vessels (arterioles) in the brain are critical mediators of normal cerebral function, serving as both a physical barrier and a modulator of blood flow. Endothelial cell dysfunction is a common feature of blood vessel diseases (hypertension, atherosclerosis) and a likely contributor to neurological disorders (Alzheimer's). This project will elucidate key control mechanisms by which endothelial cells in cerebral arterioles regulate blood flow in the brain.