Imaging the effects of microvascular network morphology on neurovascular coupling

组成大脑的千亿个神经元被组织在一个高度复杂和相互关联的网络中。由于大脑的能量存储能力非常有限，这些神经元的高能量需求由复杂的、分级的脑血管网络支持，这些血管携带着非常高的静息血流量。尽管如此，当局部神经元活动增加时，周围血管的血流量总是高于静息水平：这种协调是通过神经元和血管网络之间的大量不同的电化学相互作用实现的。尽管这种被称为神经血管耦合的现象对健康的大脑功能至关重要，但人们仍然不完全了解这种现象。到目前为止，神经血管偶联的研究一直受到刺激特定神经元并记录其活动水平和随后对邻近血管的影响的方法学困难的阻碍。然而，最近的方法学发展极大地改进了刺激单个神经元、记录它们的活动和测量单个脑血管中血流变化的光学手段。该方案的目的是将这些最先进的光学方法与电生理技术相结合，以检查网络中脑血管静息血流水平的不均一性如何影响神经血管耦合。使用我们已建立的成像和计算分析技术，我们将同时用数学术语捕捉局部神经元和血管网络的结构。我们将使用双光子荧光显微镜，它提供细胞规模的分辨率，在活体，麻醉小鼠准备与颅窗，以允许高分辨率成像潜在的脑组织。为了允许对单个神经元进行非侵入性刺激，我们将利用转基因小鼠，这些小鼠的神经元在其膜上表达光可激活离子通道。将使用线性阵列电极来记录局部神经元的活动。静息血流在微血管网络中的分布将通过实验进行调节，从而不可逆转或可逆地改变穿过皮质血管的静息血流的可变性。在每一种情况下，我们都将检查调制对神经血管耦合的影响。因此，我们将确定血管流速中的最佳静息状态离散度，以确保对神经元的有效血管支持。总而言之，这些研究将让我们从机制上了解神经元和脑微血管之间的联系，从而为构成“大脑可塑性”的过程提供基础，而这些过程往往会随着年龄的增长而逐渐受损。在这些研究中获得的关于神经血管偶联的静息血管状态依赖的洞察力也将使对功能磁共振成像的定量解释成为可能，功能磁共振成像是研究人类大脑功能最广泛使用的神经成像方法。

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.