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

**组成大脑的1000亿个神经元组成了一个高度复杂且相互关联的网络。支持这些神经元高能量需求的是一个复杂的、分层的血管网络。在这种解剖学的复杂性之上，神经网络和血管网络之间的相互作用具有很大的多样性。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血流的增加。尽管对健康的大脑功能至关重要，但神经血管耦合仍然不完全清楚。迄今为止，神经血管耦合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，最近的方法发展已经大大改进了神经元活动的光学测量和神经元的直接光学刺激。这些新技术有可能使体内神经元活动的非侵入性细胞尺度检查成为可能，从而为在细胞水平上获得神经血管耦合的机制理解提供了前所未有的机会。*本提案的目标是利用这些最先进的光学技术来检查单个神经元和周围大脑微血管之间的相互作用，同时使用我们已建立的成像和计算分析技术，以数学术语来捕获局部神经元和血管网络的结构。在我们先前研究的基础上，我们将采用两种光子荧光显微镜，提供细胞尺度分辨率，在麻醉的活体大鼠中，在涉及处理体感刺激的大脑区域上准备一个颅窗，从而使我们能够评估类似于人类正中神经刺激的简单周围刺激的神经元和血管反应。为了实现对单个神经元的非侵入性刺激和神经元活动的记录，我们将把设计用于感染特定神经元亚群的病毒结构直接注入动物的大脑。一旦被这些病毒感染，神经元将在其膜上表达光激活离子通道，并在其细胞质中表达钙浓度敏感的荧光染料，从而使我们能够利用光来刺激或抑制被感染的神经元，并光学记录它们的活动。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，我们将对动物外周进行刺激，同时将光线照射到体感皮层中特定的受感染神经元上，从而抑制它们的活动，并测量单个神经元的抑制对外周刺激血管反应的影响。*综合起来，这些研究将使我们对神经元和大脑微血管之间的联系有一个机制上的理解，从而为理解神经元和血管网络的复杂变化提供基础，这些变化产生了“大脑可塑性”，是维持健康大脑功能的基础。此外，在这些研究中获得的见解将使功能性MRI的定量解释成为可能，这可以根据血管状态的测量来推断神经元的活动。鉴于功能性MRI在人脑功能研究中的广泛应用，功能性MRI信号的定量模型将对神经科学研究产生广泛的影响，并将大大提高我们研究人脑功能的能力。

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.