The Spatial Scale and Cellular Mechanisms of Neurovascular Coupling in vivo

描述(由申请人提供)：本项目的总体目标是确定棘波、突触和星形胶质细胞活动在形成感觉新皮质血管特征选择性方面的贡献。这项工作对促进我们对大脑功能的理解很重要，因为血管(血液动力学)信号现在被广泛用于推断健康和疾病的神经功能。然而，驱动感觉诱发血流动力学信号的许多空间和时间方面的机制还知之甚少。我们将对猫初级视皮层中的神经元、星形胶质细胞和血管进行双光子功能成像。这种动物模型与灵长类动物分享了许多复杂的视觉能力和大脑皮层回路组织原理，包括用于编码刺激方向的空间精确的大脑皮层地图。我们将使用刺激方向图作为探针来确定突触、棘波和星形胶质细胞在塑造单个血管中感觉诱发的血流动力学反应中的作用。我们将结合亚微米分辨率成像和细胞特定的基因编码荧光传感器来检测尖峰活动(通过gCaMP6成像)和突触活动(通过iGluSnFr成像)。我们还包括动脉特异性荧光标记、星形胶质细胞的细胞内记录，以及对星形胶质细胞特异性谷氨酸转运体的药物阻断。在目标1中，我们验证了一种假设，即单个血管感觉诱发的扩张的选择性是由紧邻血管周围的突触活动(具体地说，谷氨酸释放)的空间模式预测的，而不是尖峰活动的空间整合。在目标2中，我们测试了谷氨酸驱动的星形胶质细胞信号是快速感觉诱发的血管激活所必需的假说。据我们所知，这是第一次在任何大脑区域中提取感官诱发的选择性 (调谐曲线)来自单个血管，并将这些单血管调谐曲线与局部突触、尖峰和星形胶质细胞活动联系起来。

DESCRIPTION (provided by applicant): The overall goal of this project is to determine the contributions from spiking, synaptic and astrocytic activity in shaping the feature selectivity of blood vessels in the sensory neocortex. This work is important to advance our understanding of brain function because vascular (hemodynamic) signals are now widely used to infer neural function in health and disease. Yet the mechanisms driving many of the spatial and temporal aspects of sensory-evoked hemodynamic signaling are poorly understood. We will perform two-photon functional imaging of neurons, astrocytes and blood vessels in the primary visual cortex of the cat. This animal model shares many sophisticated visual abilities and cortical circuit organizing principles with primates, including spatially precise cortical maps for encoding stimulus orientation. We will use the mapping of stimulus orientation as the probe to determine the contribution of synapses, spikes, and astrocytes in shaping sensory-evoked hemodynamic responses in individual blood vessels. We will combine sub-micron resolution imaging with cell-specific genetically encoded fluorescent sensors for detecting spiking activity (via gCaMP6 imaging) and synaptic activity (via iGluSnFr imaging). We also include artery-specific fluorescent labeling, intracellular recording from astrocytes, and pharmacological blockade of the astrocyte- specific glutamate transporter. In Aim 1, we test the hypothesis that the selectivit of sensory-evoked dilation of an individual blood vessel is predicted by the spatial pattern of synaptic activity (specifically, glutamate release) immediately surrounding the vessel, not the spatial integration of spiking activity. In Aim 2 we test the hypothesis that glutamate-driven astrocyte signaling is required for the rapid sensory-evoked activation of blood vessels. To our knowledge, this is the first study in any brain region that will extract sensory-evoked selectivity (tuning curves) from individual blood vessels and relate these single-vessel tuning curves to the local synaptic, spiking and astrocytic activity.