In Vivo Imaging of Neuronal Plasticity in Mouse Visual Cortex

描述(申请人提供)：这项建议的目标是通过结合创新的活体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。这种综合的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。表征大脑皮层神经元的动态潜力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。这样的分子可以用来补偿视觉通路多个层面上的侮辱或恶化。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体内慢性成像。利用该系统，我们对TY1-GFP转基因小鼠视皮层神经元的树突树进行了成像和重建。这些小鼠在一组随机的神经元中表达GFP，这些神经元稀疏地分布在皮质层浅层，通过手术植入的颅窗可以通过光学接触到这些层。我们将在视觉干扰之前、期间和之后对对照TY1-GFP小鼠或TY1-GFP小鼠新皮质浅层神经元进行慢性成像，以解决以下目标：特定目标1：为了阐明界定成人皮质结构可塑性的细胞类型特定规则，我们将在反映视觉皮质浅层新皮质细胞类型多样性的神经元横截面上进行结构动力学调查。对视觉、躯体感觉和前额叶皮质的比较分析将使我们能够解决神经元间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第2/3层皮质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质产生眼优势(OD)可塑性的实验方案来操纵视觉输入：延长单眼眼睑缝合，之前单眼剥夺(MD)的单眼眼睑缝合，或在暗适应之前的单眼眼睑缝合。作为比较，我们还将应用两种额外的手法，双眼剥夺(BD)和眼内注射河豚毒素阻断单眼。通过比较未经处理的成年TY1-GFP小鼠与长时间MD后、由先前剥夺或暗适应启动的短暂MD、BD或眼内注射TTX后的小鼠皮质树突状突起的变化，我们可以检验这样的假设，即只有产生OD移位的活动形式才会增强成像神经元的结构可塑性。主成分分析和聚类分析将用于对成像神经元的形态和细胞特征进行定量分类和分析。聚类分析应该提供关于是否存在比其他类型更具结构可塑性的中间神经元细胞类型的洞察，以及中间神经元亚群是否表现出与外径可塑性相关的结构可塑性。

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.