Imaging the effects of microvascular network morphology on neurovascular coupling

组成大脑的数千亿个神经元组成了一个高度复杂和相互连接的网络。由于大脑的能量储存能力非常有限，这些神经元的高能量需求由复杂的、分层的脑血管网络支持，这些脑血管网络携带非常高的静息血流量。尽管如此，当局部神经元活动增加时，周围血管的血流总是高于静息水平：这种协调是通过神经元和血管网络之间的电化学相互作用的巨大多样性来实现的。尽管这种现象--称为神经血管耦合--对健康的大脑功能至关重要，但仍然没有完全理解。神经血管偶联的早期研究一直受到方法学困难的阻碍，刺激特定的神经元和记录其活动水平和随后对邻近血管的影响。然而，最近的方法学发展大大改善了刺激单个神经元、记录其活动和测量单个脑血管中血流变化的光学手段。本提案的目标是将这些最先进的光学方法与电生理技术联合收割机相结合，以研究网络中跨脑血管的静息血流水平的异质性如何影响神经血管耦合。使用我们建立的成像和计算分析技术，我们将同时捕获，在数学方面，局部神经元和血管网络的架构。我们将采用双光子荧光显微镜，它提供了细胞尺度的分辨率，在活的，麻醉小鼠准备与颅窗，以允许高分辨率成像的底层脑组织。为了允许对单个神经元的非侵入性刺激，我们将利用转基因小鼠，其神经元在其膜中表达光激活离子通道。线性阵列电极将用于记录局部神经元活动。通过实验调节微血管网络中静息血流的分布，以不可逆或可逆地改变皮质血管中静息血流的变异性。在每一种情况下，我们将检查调制对神经血管耦合的影响。因此，我们将确定最佳的静息状态分散在血管流速方面，以确保有效的血管支持的神经元。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“脑可塑性”的基础过程提供基础，这些过程通常在衰老中逐渐受到损害。在这些研究中获得的神经血管耦合的静息血管状态依赖性的见解也将使定量解释功能性MRI，最广泛使用的神经成像方法研究人脑功能。

The hundred billion neurons that make up the brain are organized in a highly complex and interconnected network. As brain has very limited capacity for energy storage, the high energetic need of these neurons is supported by an intricate, hierarchical network of brain vessels that carry a very high resting blood flow. Notwithstanding, upon an increase in local neuronal activity, the blood flow of the surrounding vessels invariably rises above the resting levels: this coordination is achieved through a great diversity of electrochemical interactions between neuronal and vascular networks. Although central to healthy brain functioning, this phenomenon - termed neurovascular coupling - is still incompletely understood. Hitherto investigations of neurovascular coupling have been impeded by the methodological difficulty of stimulating specific neurons and recording their activity levels and the ensuing effects on neighboring vessels. Recent methodological developments, however, have greatly improved optical means of stimulating individual neurons, recording their activity and measuring the blood flow changes in individual brain vessels. The goal of this proposal is to combine these state-of-the-art optical methods with electrophysiological techniques so as to examine how the heterogeneity in the resting blood flow levels across brain vessels in the network affects the neurovascular coupling. Using our established imaging and computational analysis techniques, we will concomitantly capture, in mathematical terms, the architecture of local neuronal and vascular networks. We will employ two photon fluorescence microscopy, which affords cellular scale resolution, in live, anesthetized mice prepared with cranial windows to permit high resolution imaging of the underlying brain tisuse. To allow non-invasive stimulation of individual neurons, we will make use of transgenic mice whose neurons express light activatable ion channels in their membranes. Linear array electrodes will be employed to record local neuronal activity. The distribution of resting flow across the microvascular network will be modulated experimentally so as to either irreversibly or reversibly change the variability of resting blood flow across cortical vessels. In each instance, we will examine the effect of the modulation on the neurovascular coupling. We will thus identify the optimal resting state dispersion in the vascular flow rates with respect to ensuring effective vascular support of the neurons. Combined, these studies will give us a mechanistic understanding of the link between neurons and brain microvessels, thus providing the foundation for processes that underlie “brain plasticity” and that are often progressively compromised in aging. The insight on resting vascular state dependence of neurovascular coupling obtained in these studies will also enable quantitative interpretation of functional MRI, the most widely used neuroimaging method for studying human brain function.