Invivo Imaging of Neuronal Structure in Visual Cortex

描述(申请人提供)：这项提案的目标是开发一种系统，用于在完整的啮齿动物视皮层中对神经元形态进行慢性活体成像。一种系统，可以用来直接测试关于每天在视觉输入的基础上发生在视觉皮质中的结构动态的位置和程度的预测。通过将这种创新的成像技术与最先进的小鼠遗传学相结合，我们可以开始解决皮质结构可塑性的分子基础。这种综合的方法不仅将彻底改变我们理解大脑功能这一基本方面的能力，还可以识别具有治疗潜力的分子，这些分子可以促进视觉皮质的可塑性，并可能用于补偿视觉通路较低水平的伤害。 为了监测哺乳动物视皮层神经元的活体结构动力学，我们将制作一种定制的双光子显微镜，用于高保真和短时间尺度地成像小鼠视皮层三维体积内的单个神经元。多光子显微镜允许以亚细胞分辨率深入散射组织的重要成像，光损伤或光毒性最小。我们将使用这台显微镜在体内成像并重建转基因小鼠视皮层中表达增强型绿色荧光蛋白(EGFP)神经元的完整树突树和轴突侧支。这些小鼠在TY1启动子驱动的随机细胞亚群中表达EGFP，这些细胞稀疏分布在皮质层浅层。绿色荧光蛋白填充细胞，使它们从末端到末端都是明亮的荧光，从而可以可视化它们的整个结构，包括丝状足突和树突。随着时间的推移，我们将通过一个允许重复访问相同细胞的颅窗，在麻醉动物身上成像这些神经元。 为了确定在视觉皮质可塑性基础上的结构重塑中发挥作用的分子，我们将把敲除或转基因候选可塑性基因(CPGs)的小鼠品系与thyl-EGFP转基因品系进行杂交。这将创造出缺乏或过度表达特定基因的小鼠，并且它们的皮质中有荧光细胞。监测这些小鼠皮质神经元的结构动力学，并将它们与野生型动物的EGFP标记的皮质神经元进行比较，将揭示这些被操纵的基因在哺乳动物皮质结构可塑性中的作用程度。也可以通过病毒介导的基因转移将基因瞬时导入大脑皮层。第一批测试的一些基因将是我们在筛选涉及突触可塑性的活动调节基因时分离出来的CpGs。

DESCRIPTION (provided by applicant): The goal of this proposal is to develop a system for chronic in vivo imaging of neuronal morphology in the intact rodent visual cortex. A system that can be used to directly test predictions regarding the site and extent of structural dynamics that occur in visual cortex on a day to day basis and in response to visual input. By combining this innovative imaging technology with state of the art mouse genetics we can begin to address the molecular basis of cortical structural plasticity. This integrative approach will not only revolutionize our ability to understand this fundamental aspect of brain function, but can identify molecules with therapeutic potential that can promote plasticity in visual cortex and may be used to compensate for insults at lower levels of the visual pathway. To monitor in vivo structural dynamics of neurons in the mammalian visual cortex, we will fabricate a custom designed two-photon microscope for imaging single neurons within a three-dimensional volume of the mouse visual cortex with high fidelity and at short time scales. Multi-photon microscopy allows for vital imaging deep into scattering tissue at sub-cellular resolution with minimal photodamage or phototoxicity. We will use this microscope to image in vivo and reconstruct entire dendritic trees and axon collaterals of EGFP (enhanced green fluorescent protein) expressing neurons in the visual cortex of transgenic mice. These mice express EGFP driven by the thy1 promoter in a random subset of cells sparsely distributed within the superficial cortical layers. The EGFP fills the cells so that they are brightly fluorescent all the way to the tips of their terminals, allowing visualization of their entire structure including filopodia and dendritic spines. We will image these neurons over time in anesthetized animals through a cranial window that allows repeated access to the same cells. To identify molecules that play a role in the structural remodeling that underlies visual cortical plasticity, we will cross mouse lines that are knockouts or transgenic for candidate plasticity genes (CPGs) with the thyl-EGFP transgenic line. This will create mice that are deficient or overexpressing a specific gene and have fluorescent cells in their cortex. Monitoring the structural dynamics of cortical neurons in these mice and comparing them to those of EGFP labeled cortical neurons from wild-type animals, will reveal to what extent the manipulated genes play a role in structural plasticity of the mammalian cortex. Genes could also be introduced into the cortex transiently by virally mediated gene transfer. Some of the first genes tested will be CPGs that we isolated in a screen for activity-regulated genes involved in synaptic plasticity.