Sensory mechanisms in brain capillary endothelial cells that initiate functional hyperemia

脑毛细血管内皮细胞启动功能性充血的感觉机制

• 批准号: 9812787
• 负责人: Scott Earley
• 金额: $ 5.24万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9812787
• 关键词: Address; Ankyrins; Architecture; Area; Astrocytes; Binding; Biosensor; Blood Vessels; Blood capillaries; Blood flow; Brain; Brain region; Capillary Endothelial Cell; Cell membrane; Cerebrovascular Circulation; Cerebrum; Complex; Consumption; Coupled; Data; Development; Disease; Electrophysiology (science); Endothelial Cells; Ensure; Gap Junctions; Generations; Glucose; Glutamates; Goals; Human; Hyperemia; Image; Internet; Knockout Mice; Laser Scanning Microscopy; Laser-Doppler Flowmetry; Lead; Measurement; Measures; Mediating; Metabotropic Glutamate Receptors; Microcirculation; Microscopy; Modeling; Molecular; Mus; Nature; Neurons; Oxygen; Oxygen Consumption; Pathway interactions; Phospholipase C; Phospholipases A; Physiological; Preparation; Process; Proteins; Reactive Oxygen Species; Regulation; Resolution; Role; Sensory; Signal Pathway; Signal Transduction; Smooth Muscle Myocytes; Somatosensory Cortex; Stroke; Synapses; TRPA1 Channel; Techniques; Testing; Vascular Cognitive Impairment; Vasodilator Agents; Vibrissae; barrel cortex; cerebral capillary; improved; in vivo; metabotropic glutamate receptor 5; neurovascular coupling; novel; parenchymal arterioles; patch clamp; receptor; response; sensor; sensory mechanism; somatosensory; two-photon

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.

神经血管偶联(NVC)是脑循环内局部脑循环的独特过程。 血流量(CBF)被精确地引导到大脑活跃的区域。NVC是所有大脑功能不可缺少的， 反映了这样一个事实，即中枢神经元几乎没有储存能量底物的能力，需要迅速 传递在突触活动期间迅速消耗的代谢物。一个新兴的概念范式 设想人脑中数百英里的毛细血管是一个感官网络，通过以下方式调节NVC 检测神经元活性升高并启动传播的扩张信号以产生局部的、功能性的 充血反应。这项提议的总体目标是确定新的内在感觉机制 大脑毛细血管内皮细胞(ECs)，快速检测神经元活动的增加并阐明如何 这样的信号传播并作用于大脑微循环，触发功能性充血。我们建议 5型代谢型谷氨酸受体(MGluR5s)存在的新机制假说 大脑毛细血管内皮细胞，当受到星形细胞终足释放的谷氨酸刺激时，启动 上游实质小动脉(PAS)。目标1的目标是阐明细胞内信号转导 MGluR5在脑毛细血管内皮细胞上启动的机制，触发上游PA的扩张。我们将测试 假设mGluR5启动GQ/11/PLC信号级联导致活性氧增加 物种(ROS)的产生和Ca~(2+)通过TRPA1通道内流触发上游PA的扩张。 拟议的研究将使用钙离子和ROS成像，膜片钳天然脑毛细血管内皮细胞的电生理学， 和一种新开发的体外微动脉-毛细血管制剂，为 评估大脑毛细血管在调节上游血管系统中的作用。目标2的目标是阐明 导致上游PA传导扩张的细胞间信号机制 MGluR5对脑毛细血管内皮细胞的刺激作用我们将测试谷氨酸与mGluR5结合的假设， 导致TRPA1的激活和细胞间钙波的产生并传播到上游的PAS 发出扩张的信号。拟议的研究将使用脑微循环的高分辨率钙成像和 用超分辨显微镜建立脑毛细血管内钙信号复合体的结构。这个 目标3的目标是验证脑毛细血管内皮细胞上的mGluR5刺激功能性充血的假设。 活着。这些研究将使用双光子激光扫描显微镜来测量mGluR5-和TRPA1-。 体内毛细血管内RBC流量和钙信号的依赖性变化，以及激光多普勒血流仪 测量体感皮质对胡须刺激的脑血流量变化。小说的使用 EC特异的钙离子生物传感器小鼠和EC特异的mGluR5和TRPA1基因敲除小鼠增强我们的 在所有目标上都要接近。预期的发现将支持一种新的NVC模型，在该模型中，神经元活动刺激 在毛细血管附近释放谷氨酸，激活内皮细胞上的mGluR5，从而启动局部的血流量增加。