Sensory mechanisms in brain capillary endothelial cells that initiate functional hyperemia

神经血管耦联（NVC）是脑循环中的一个独特过程， 血流量（CBF）被精确地引导到活跃的脑区域。NVC是所有大脑功能不可或缺的， 这反映了中枢神经元几乎没有储存能量底物的能力， 传递在突触活动期间快速消耗的代谢物。一种新兴的概念范式 设想人类大脑中数百英里的毛细血管作为一个感觉网络，通过 检测升高的神经元活动并启动传播性扩张信号以产生局部的功能性扩张， 充血反应这项提案的总体目标是确定新的感觉机制固有的， 脑毛细血管内皮细胞（EC），快速检测神经元活动的增加，并阐明如何 这种信号传播并作用于脑微循环以触发功能性充血。我们提出 5型代谢型谷氨酸受体（mGluR 5s）存在于 脑毛细血管内皮细胞，当受到星形胶质细胞终足释放的谷氨酸刺激时， 上游实质小动脉（PA）。目的1是阐明细胞内信号转导 由mGluR 5s在脑毛细血管EC上启动的机制，其触发上游PA的扩张。我们将测试 假设mGluR 5启动Gq/11/PLC信号级联，导致活性氧增加 通过TRPA 1通道的ROS生成和Ca 2+内流触发上游PA的扩张。 拟议的研究将使用Ca 2+和ROS成像，天然脑毛细血管EC的膜片钳电生理学， 和一种新开发的离体小动脉-毛细血管制剂， 评估脑毛细血管在调节上游脉管系统中的作用。目标2的目标是阐明 负责上游PA响应于 刺激mGluR 5s对脑毛细血管EC的作用。我们将检验谷氨酸与mGluR 5结合的假设， 导致TRPA 1的激活和细胞间Ca 2+波的产生，所述Ca 2+波传播到上游PA 来发出扩张的信号拟议的研究将使用脑微循环的高分辨率Ca 2+成像， 超分辨率显微镜来建立脑毛细血管中Ca 2+信号复合物的结构。的 目的3的目的是检验脑毛细血管内皮细胞上的mGluR 5刺激脑缺血后功能性充血的假设。 vivo.这些研究将使用双光子激光扫描显微镜来测量mGluR 5-和TRPA 1-。 红细胞流量和体内毛细血管中Ca 2+信号的依赖性变化，以及激光多普勒血流仪， 测量对触须刺激作出反应的躯体感觉皮层中CBF的变化。使用新型 EC特异性Ca 2+生物传感器小鼠和EC特异性mGluR 5-和TRPA 1-敲除小鼠增强了我们的研究。 在所有目标上。预期的发现将支持一种新的NVC模型，其中神经元活动刺激 谷氨酸在毛细血管附近释放以激活EC上的mGluR 5，这引发血流量的局部增加。

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 our approach in all aims. Anticipated findings will support a new model of NVC in which neuronal activity stimulates glutamate release near capillaries to activate mGluR5s on ECs, which initiate localized increases in blood flow.