Neurovascular coupling (NVC) is the distinctive process within the cerebral circulation by which local cerebral blood flow (CBF) is precisely directed to active brain regions. NVC is indispensible for all brain functions, reflecting the fact that central neurons have little capacity to store energetic substrates and require prompt delivery of metabolites that are rapidly consumed during synaptic activity. An emerging conceptual paradigm envisions the hundreds of miles of capillaries in the human brain as a sensory web that mediates NVC by detecting elevated neuronal activity and initiating a propagating dilatory signal to produce a local, functional hyperemic response. The overall goals of this proposal are to identify novel sensory mechanisms intrinsic to brain capillary endothelial cells (ECs) that rapidly detect increases in neuronal activity and to elucidate how such signals propagate and act on the cerebral microcirculation to trigger functional hyperemia. We propose the novel mechanistic hypothesis that type 5 metabotropic glutamate receptors (mGluR5s) are present on brain capillary ECs and, when stimulated by glutamate released from astrocytic endfeet, initiate dilation of upstream parenchymal arterioles (PAs). The goal of Aim 1 is to elucidate the intracellular signaling mechanisms initiated by mGluR5s on brain capillary EC that trigger dilation of upstream PAs. We will test the hypothesis that mGluR5 initiates a Gq/11/PLC signaling cascade that leads to increased reactive oxygen species (ROS) generation and Ca2+ influx through TRPA1 channels to trigger dilation of upstream PAs. Proposed studies will use Ca2+ and ROS imaging, patch-clamp electrophysiology of native brain capillary ECs, and a newly developed ex vivo arteriolar-capillary preparation that provide an ideal reduced setting for assessing the role of brain capillaries in regulating the upstream vasculature. The goal of Aim 2 is to elucidate the intercellular signaling mechanisms responsible for conducted dilation of upstream PAs in response to stimulation of mGluR5s on brain capillary ECs. We will test the hypothesis that glutamate binds to mGluR5s, leading to activation of TRPA1 and the generation of intercellular Ca2+ waves that propagate to upstream PAs to signal dilation. Proposed studies will use high-resolution Ca2+ imaging of the cerebral microcirculation and super-resolution microscopy to establish the architecture of Ca2+ signaling complexes in brain capillaries. The goal of Aim 3 is to test the hypothesis that mGluR5s on brain capillary ECs stimulate functional hyperemia in vivo. These studies will use two-photon laser-scanning microscopy to measure mGluR5- and TRPA1- dependent changes in RBC flux and Ca2+ signaling in capillaries in vivo, as well as laser Doppler flowmetry to measure changes in CBF in the somatosensory cortex in response to whisker stimulation. The use of novel EC-specific Ca2+ biosensor mice and EC-specific mGluR5- and TRPA1-knockout mice strengthens...