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

描述（申请人提供）：脑功能与脑血管系统之间存在着密切的关系。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交流，这些细胞统称为神经（胶质）血管单位。当局部神经元群活跃时，神经血管单元的协调活动导致激活脑区的血流量和容量增加，这一过程被称为“功能性充血”。内皮细胞是一种位于血管壁内的特殊细胞类型，在血管系统的调节中起着至关重要的作用。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）的生理机能可导致血脑屏障的破坏和多种病理状况的发展，包括糖尿病、多发性硬化症、炎症性疼痛、阿尔茨海默病、癫痫和中风。钙（Ca2+）通常被认为是一种普遍的第二信使，在广泛的组织和生物体中参与必需的细胞信号事件。细胞外Ca2+水平的消耗或导致vECs细胞内Ca2+浓度异常高或低的条件可导致血管反应的改变，最终导致血脑屏障的破坏。尽管vEC Ca2+信号对血管动力学具有明显的重要性，但据我们所知，从未对体内大脑中的vEC Ca2+动力学进行过研究，也没有对驱动vEC动力学的自然过程或特定细胞类型进行过任何直接检查。本提案的目的是验证Ca2+动力学在vECs中自发表达的假设，由自然（感觉）输入驱动，并由体内局部和远端神经调节激活驱动。为此，我们优化了两种新方法，这两种方法将允许在清醒动物的新皮质vECs中使用遗传编码的Ca2+指标的双光子成像来可视化vEC Ca2+动态。使用这种方法的初步数据导致了大脑中正在进行的基础vEC Ca2+振荡的表征。此外，我们首次发现vEC Ca2+动态可以通过生理感觉输入到小鼠桶状皮质中来激活。我们建议扩展这些初步发现，并使用细胞类型特异性光遗传激活来功能解剖局部体感觉和远端神经网络中的精确细胞成分，这些细胞成分有助于大脑中感觉诱发的vEC Ca2+动态。

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.