Longitudinal recording of neuronal function using two photon fluorescence microscopy in adult rats co-expressing genetically encoded calcium indicators and channelrhodopsin-2

建议概要**组成大脑的千亿个神经元组织在一个高度复杂和相互连接的网络中。支持这些神经元的高能量需求的是一个复杂的、层次分明的血管网络。在这种解剖复杂性的基础上，神经元和血管网络之间的相互作用具有极大的多样性。为了使该系统发挥作用，神经元活动的增加必须伴随着周围血管中血流量的增加。尽管神经血管偶联对健康的大脑功能至关重要，但人们仍然不完全了解它。到目前为止，神经血管偶联的研究一直受到刺激单个神经元并记录其活动及其对邻近血管的影响的方法困难的阻碍。然而，最近的方法学发展极大地改进了神经元活动的光学测量和对神经元的直接光学刺激。这些新技术有可能实现对活体神经元活动的非侵入性细胞尺度检查，从而提供了在细胞水平上从机制上了解神经血管耦合的前所未有的机会。*这项建议的目标是利用这些最先进的光学技术来研究单个神经元与周围脑微血管之间的相互作用，同时使用我们现有的成像和计算分析技术来用数学术语捕捉局部神经元和血管网络的结构。在我们先前研究的基础上，我们将使用双光子荧光显微镜，它可以在活体麻醉大鼠身上提供细胞尺度的分辨率，在参与躯体感觉刺激处理的大脑区域上准备一个颅窗，从而使我们能够评估神经元和血管对简单的外周刺激的反应，类似于人类的正中神经刺激。为了实现对单个神经元的非侵入性刺激和记录神经元的活动，我们将把旨在感染特定神经元亚群的病毒构建体直接注入动物的大脑。一旦被这些病毒感染，神经元将在其细胞膜上表达光激活的离子通道，在其胞浆中表达钙浓度敏感的荧光染料，从而允许我们利用光来刺激或抑制受感染的神经元以及光学记录其活动。在项目的第一部分，我们将用光刺激单个受感染的神经元，并测量它们增加的活动对周围血管的影响。在项目的第二部分，我们将对动物进行外周刺激，同时将光线照射到体感皮质中特定的受感染神经元，以抑制它们的活动，并测量单个神经元的抑制对血管对外周刺激的反应的影响。*综合这些研究，我们将从机制上了解神经元和大脑微血管之间的联系，从而为了解神经元和血管网络的复杂变化提供基础，这些变化导致“大脑可塑性”，并对维持健康的大脑功能至关重要。此外，在这些研究中获得的洞察力将使对功能磁共振的定量解释成为可能，功能磁共振基于对血管状态的测量来推断神经元的活动。鉴于功能磁共振在人脑功能研究中的广泛应用，功能磁共振信号的定量模型将对神经科学研究产生广泛的影响，并将极大地提高我们检测人脑功能的能力。

Summary of Proposal **The hundred billion neurons that make up the brain are organized in a highly complex and interconnected network. Supporting the high energetic needs of these neurons is an intricate, hierarchical network of vessels. Superimposed on this anatomical complexity, is great diversity in the interaction between neuronal and vascular networks. For the system to function, an increase in neuronal activity must be accompanied by an increase in blood flow in the surrounding vessels. Although central to healthy brain functioning, the neurovascular coupling is still incompletely understood. Hitherto, neurovascular coupling studies have been impeded by the methodological difficulty of stimulating individual neurons and recording their activity and its effects on neighboring vessels. Recent methodological developments, however, have greatly improved optical measurements of neuronal activity and direct optical stimulation of neurons. These novel techniques have the potential to enable non-invasive cellular scale examinations of neuronal activity in vivo and thus offer an unprecedented opportunity to gain mechanistic understanding of neurovascular coupling at the cellular level. *The goal of this proposal is to make use of these state-of-the-art optical techniques to examine the interaction between individual neurons and surrounding brain microvessels while using our established imaging and computational analysis techniques to capture, in mathematical terms, the architecture of local neuronal and vascular networks. Building upon our prior studies, we will employ two photon fluorescence microscopy, which affords cellular scale resolution, in live, anesthetized rats prepared with a cranial window over the brain region involved in processing of somatosensory stimuli, thus allowing us to evaluate neuronal and vascular responses to simple peripheral stimuli akin to the median nerve stimulation in humans. To allow non-invasive stimulation of individual neurons and recording of the neuronal activity, we will inject viral constructs, designed to infect specific subpopulations of neurons, directly into the animals' brains. Once infected by these viruses, neurons will express light activatable ion channels in their membranes and calcium concentration sensitive fluorescent dyes in their cytosol, thus allowing us to use light to stimulate or inhibit the infected neurons as well as record their activity optically. In the first part of the project, we will stimulate individual infected neurons with light and measure the effect of their increased activity on the surrounding vessels. In the second part of the project, we will stimulate the animals peripherally while shining light onto specific infected neurons in the somatosensory cortex so as to inhibit their activity and measure the effects of the individual neuron's inhibition on the vascular response to peripheral stimulation.*Combined, these studies will give us a mechanistic understanding of the link between neurons and brain microvessels, thus providing the foundation for understanding the complex changes in neuronal and vascular networks that give rise to "brain plasticity" and that are fundamental for maintenance of healthy brain function. Moreover, the insight obtained in these studies will enable quantitative interpretation of functional MRI, which makes inferences about neuronal activity based on measurements of vascular state. Given the widespread use of functional MRI for studying human brain function, a quantitative model of functional MRI signal will have a broad impact on neuroscience research and will greatly enhance our ability to examine human brain function.