In vivo two-photon imaging of brain vascular endothelial cell calcium dynamics

脑血管内皮细胞钙动力学的体内双光子成像

• 批准号: 8449321
• 负责人: Tyler Clark Brown
• 金额: $ 5.77万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8449321
• 关键词: Acetylcholine; Alzheimer' s Disease; Animals; Arteries; Astrocytes; Biological Models; Biological Neural Networks; Blood - brain barrier anatomy; Blood Circulation; Blood Vessels; Blood Volume; Blood flow; Brain; Brain imaging; Brain region; Calcium; Cell physiology; Cells; Communication; Data; Development; Diabetes Mellitus; Distal; Elements; Endothelial Cells; Epilepsy; Event; Exhibits; Failure; Functional disorder; Future; Health; Human; Hyperemia; Image; Imagery; In Vitro; Individual; Laboratories; Lead; Link; Measures; Methods; Molecular; Multiple Sclerosis; Mus; Neocortex; Network-based; Neuraxis; Neurons; Neurosciences; Organism; Pathway interactions; Photons; Physiological; Play; Population; Positioning Attribute; Process; Regulation; Relaxation; Role; Second Messenger Systems; Sensory; Signal Transduction; Site; Smooth Muscle; Stroke; Testing; Time; Tissues; Training; Travel; Vascular Endothelial Cell; Vascular Endothelium; Vascular blood supply; Vasodilation; Vibrissae; arteriole; awake; barrel cortex; base; cell type; cholinergic; constriction; excitatory neuron; extracellular; hemodynamics; improved; in vivo; inflammatory pain; inhibitory neuron; mouse model; neocortical; nervous system disorder; neurovascular unit; novel therapeutic intervention; optogenetics; relating to nervous system; response; second messenger; selective expression; somatosensory; two-photon

DESCRIPTION (provided by applicant): An intimate relationship exists between brain function and the brain vasculature. In the brain, the vasculature is engaged in active, bidirectional communication with neurons and astrocytes, collectively termed the neuro(glio)vascular unit. When local populations of neurons are active, coordinated activity in the neurovascular unit leads to increased blood flow and volume to the activated brain region, a process known as "functional hyperemia". Endothelial cells are a specialized cell-type located within vessel walls that play a crucial role in regulation of the vasculature. Perhaps most prominent is their role as the primary substrate of the blood-brain barrier (BBB). Disrupting vascular endothelial cell (vEC) physiology can lead to breakdown of the BBB and development of several pathological conditions, including diabetes, multiple sclerosis, inflammatory pain, Alzheimer's disease, epilepsy, and stroke. Calcium (Ca2+) is generally recognized as a universal second messenger that contributes to essential cellular signaling events in a broad range of tissues and organisms. Depletion of extracellular Ca2+ levels or conditions that lead to abnormally high or low intracellular Ca2+ concentrations in vECs can lead to alterations in vascular responses and eventually to a breakdown in the BBB. Despite the clear importance of vEC Ca2+ signaling to vascular dynamics, to our knowledge there has never been a study of vEC Ca2+ dynamics in the brain in vivo, nor any direct examination of the natural processes or specific cell types that drive vEC dynamics. The aims of this proposal are to test the hypothesis that Ca2+ dynamics are spontaneously expressed in vECs, driven by natural (sensory) input, and driven by local and distal neuromodulatory activation in vivo. To this end, we optimized two new methods that will allow visualization of vEC Ca2+ dynamics using two- photon imaging of genetically-encoded Ca2+ indicators in neocortical vECs in awake animals. Preliminary data using this approach has led to the characterization of ongoing basal vEC Ca2+ oscillations in the brain. Further, for the first time, we have found that vEC Ca2+ dynamics can be evoked by physiological sensory input to the mouse barrel cortex. We propose to extend these preliminary findings and use cell-type specific optogenetic activation to functionally dissect the precise cellular components within local somatosensory and distal neural networks that contribute to sensory-evoked vEC Ca2+ dynamics in the brain.

描述（由申请人提供）：脑功能与脑血管系统之间存在密切关系。在大脑中，血管系统与神经元和星形胶质细胞进行活跃的双向通信，统称为神经（胶质）血管单位。当神经元的局部群体活跃时，神经血管单元中的协调活动导致到激活的脑区域的血流量和体积增加，这一过程被称为“功能性充血”。内皮细胞是位于血管壁内的特化细胞类型，其在脉管系统的调节中起关键作用。也许最突出的是它们作为血脑屏障（BBB）的主要底物的作用。破坏血管内皮细胞（vEC）生理学可导致BBB的破坏和几种病理状况的发展，包括糖尿病、多发性硬化、炎性疼痛、阿尔茨海默病、癫痫和中风。钙（Ca2+）通常被认为是一种通用的第二信使，有助于在广泛的组织和生物体中的基本细胞信号传导事件。细胞外Ca2+水平的耗竭或导致vEC中异常高或低的细胞内Ca2+浓度的条件可导致血管反应的改变并最终导致BBB的破坏。尽管vEC Ca2+信号对血管动力学的重要性明确，但据我们所知，从未有过体内脑中vEC Ca2+动力学的研究，也没有直接检查驱动vEC动力学的自然过程或特定细胞类型。本提案的目的是测试的假设，即Ca2+动态自发表达的血管内皮细胞，驱动自然（感觉）输入，并在体内驱动本地和远端的神经调节激活。为此，我们优化了两种新方法，其将允许使用清醒动物中新皮层vEC中遗传编码的Ca2+指示剂的双光子成像来可视化vEC Ca2+动力学。使用这种方法的初步数据已经导致了正在进行的基础vEC Ca2+振荡在大脑中的表征。此外，第一次，我们已经发现，vEC的Ca2+动力学可以诱发生理感觉输入到小鼠桶皮质。我们建议扩展这些初步研究结果，并使用细胞类型特异性光遗传学激活功能解剖局部体感和远端神经网络内的精确细胞成分，这些细胞成分有助于大脑中感觉诱发的vEC Ca2+动力学。