In Vivo Imaging of Neuronal Plasticity in Mouse Visual Cortex

描述（由申请人提供）：本提案的目标是通过结合创新的体内成像技术和经典的视觉操作来阐明皮质结构可塑性的机制。这种综合方法有可能彻底改变我们对自适应电路修改的理解，这是大脑功能的一个基本方面。表征皮层神经元的动态电位将为未来测试具有促进大脑皮层可塑性的治疗潜力的分子提供基线。这类分子可用于补偿视觉通路多个层次上的损伤或退化。为了研究哺乳动物大脑结构可塑性的机制，我们利用多光子显微镜系统对完整的啮齿动物大脑皮层的神经元形态进行了慢性体内成像。利用该系统对thy1-GFP转基因小鼠视觉皮层神经元树突状树进行了成像和重建。这些小鼠在随机分布的神经元亚群中表达GFP，这些神经元稀疏分布在通过手术植入的颅窗光学可到达的浅表皮质层中。我们将在视觉干扰之前、期间和之后，对对照thy1-GFP小鼠或thy1-GFP小鼠的新皮层浅层神经元进行慢性成像，以解决以下目标：具体目标1：为了阐明界定成人皮层结构可塑性的细胞类型特异性规则，我们将在神经元横截面上进行结构动力学调查，反映视觉皮层浅层内新皮层细胞类型的多样性。对视觉、体感和前额叶皮层的比较分析将使我们能够解决中间神经元重塑是否是一种普遍现象。具体目标2：为了研究视觉经验在2/3层皮层神经元结构动力学中的作用，我们将使用在成年啮齿动物皮层中产生眼优势（OD）可塑性的实验方案来操纵视觉输入：延长单眼眼睑缝合，单眼眼睑缝合之前进行先前的单眼剥夺（MD），或单眼眼睑缝合之前进行黑暗适应。为了比较，我们还将采用两种额外的操作，双目剥夺（BD）和眼内注射TTX单眼封锁。通过比较未治疗的成年thy1-GFP小鼠皮层树突乔木的变化与长期MD后的小鼠皮质树突乔木的变化，经过先前剥夺或暗适应的短暂MD后的小鼠皮质树突乔木的变化，BD或眼内注射TTX后的小鼠皮质树突乔木的变化，我们可以验证只有产生OD变化的活动形式也会增强成像神经元的结构可塑性的假设。主成分分析和聚类分析将用于定量分类和分析成像神经元的形态和细胞特征。聚类分析应该提供关于中间神经元细胞类型是否比其他类型更具有结构可塑性，以及中间神经元子集是否表现出与OD可塑性相关的结构可塑性的见解。

DESCRIPTION (provided by applicant): The goal of this proposal is to elucidate the mechanisms of cortical structural plasticity by combining innovative in vivo imaging technology with classical visual manipulations. This integrative approach holds the potential to revolutionize our understanding of adaptive circuit modification, a fundamental aspect of brain function. Characterizing the dynamic potential of cortical neurons will provide a baseline for future testing of molecules with therapeutic potential for promoting plasticity in the cerebral cortex. Such molecules may be used to compensate for insults or deterioration at multiple levels of the visual pathway. To investigate the mechanisms underlying structural plasticity in the mammalian brain we utilized a multi- photon microscope system for chronic in vivo imaging of neuronal morphology in the intact rodent cerebral cortex. Using this system we have imaged and reconstructed the dendritic trees of neurons in visual cortex of thy1-GFP transgenic mice. These mice express GFP in a random subset of neurons sparsely distributed within the superficial cortical layers that are optically accessible through surgically implanted cranial windows. We will chronically image neurons in the superficial layers of the neocortex in control thy1-GFP mice, or thy1-GFP mice before, during, and after visual perturbations, to address the following aims: Specific aim 1: To clarify cell type-specific rules that delimit structural plasticity in the adult cortex, we will conduct a survey of structural dynamics in a cross section of neurons that reflects the diversity of neocortical cell types within the superficial layers of visual cortex. A comparative analysis of visual, somatosensory, and pre-frontal cortex will allow us to address whether interneuron remodeling is a general phenomenon. Specific aim 2: To investigate the role of visual experience in structural dynamics of layer 2/3 cortical neurons, we will manipulate visual input using experimental protocols that produce ocular dominance (OD) plasticity in the adult rodent cortex: prolonged monocular lid suture, monocular lid suture preceded by a previous monocular deprivation (MD), or monocular lid suture preceded by dark adaptation. For comparison, we will also apply two additional manipulations, binocular deprivation (BD) and monocular blockade by intraocular TTX injection. By comparing dendritic arbor changes in the cortex of untreated adult thy1-GFP mice with those in mice after a long MD, after a brief MD primed by a previous deprivation or by dark adaptation, after BD, or intraocular TTX injection, we can test the hypothesis that only the forms of activity that produce OD shifts will also enhance structural plasticity in the imaged neurons. Principle component analysis and cluster analysis will be used to quantitatively classify and analyze the morphological and cellular characteristics of imaged neurons. Cluster analysis should provide insight as to whether there are interneuron cell types that are more structurally plastic than others, and whether subsets of interneurons exhibit structural plasticity correlated with OD plasticity.