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

组成大脑的数千亿神经元组成了一个高度复杂和相互连接的网络。支持这些神经元的高能量需求是一个复杂的，分层的血管网络。叠加在这种解剖复杂性上的是神经元和血管网络之间相互作用的巨大多样性。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的增加。虽然中枢健康的大脑功能，神经血管耦合仍然没有完全理解。然而，神经血管耦合研究一直受到方法学上的困难，刺激单个神经元和记录其活动及其对邻近血管的影响。然而，最近的方法的发展，大大提高了光学测量的神经元活动和直接光学刺激的神经元。这些新技术有可能使非侵入性的细胞规模的检查体内神经元的活动，从而提供了一个前所未有的机会，以获得在细胞水平上的神经血管耦合的机制的理解。该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脑微血管之间的相互作用，同时使用我们建立的成像和计算分析技术来捕获局部神经元和血管网络的数学结构。基于我们先前的研究，我们将采用双光子荧光显微镜，它提供细胞尺度的分辨率，在活的，麻醉大鼠准备与颅窗在大脑区域参与处理的躯体感觉刺激，从而使我们能够评估神经元和血管的反应，简单的外周刺激类似于正中神经刺激在人类。为了允许非侵入性刺激单个神经元并记录神经元活动，我们将直接将病毒构建体注射到动物的大脑中，该病毒构建体旨在感染特定的神经元亚群。一旦被这些病毒感染，神经元将在其膜中表达光激活离子通道，并在其胞质溶胶中表达钙浓度敏感的荧光染料，从而允许我们使用光来刺激或抑制感染的神经元以及光学记录它们的活动。在该项目的第一部分，我们将用光刺激单个受感染的神经元，并测量其活动增加对周围血管的影响。在项目的第二部分，我们将刺激动物的外周，同时将光线照射到躯体感觉皮层中特定的受感染神经元上，以抑制它们的活动，并测量单个神经元对外周刺激血管反应的抑制作用。结合起来，这些研究将使我们对神经元和脑微血管之间的联系有一个机械的理解，从而为理解神经元和血管网络的复杂变化提供基础，这些变化引起“脑可塑性”，并且是维持健康脑功能的基础。此外，在这些研究中获得的洞察力将使功能性MRI的定量解释成为可能，功能性MRI基于血管状态的测量来推断神经元活动。鉴于功能性MRI在研究人脑功能中的广泛应用，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，并将大大提高我们检查人脑功能的能力。

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.