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将创新的体内成像技术与经典的视觉操作相结合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。此类分子可用于补偿视觉通路多个水平的损伤或退化。为了研究哺乳动物大脑中结构可塑性的潜在机制，我们利用多光子显微镜系统对完整啮齿动物大脑皮层中的神经元形态进行慢性体内成像。使用该系统，我们成像和重建的神经元树突树的thy 1-GFP转基因小鼠的视觉皮层。这些小鼠在稀疏分布在表面皮质层内的神经元的随机子集中表达GFP，所述表面皮质层可通过手术植入的颅窗光学访问。我们将对对照thy 1-GFP小鼠或thy 1-GFP小鼠在视觉扰动之前、期间和之后的新皮层浅层中的神经元进行长期成像，以解决以下目标：具体目标1：为了阐明区分成人大脑皮层结构可塑性的细胞类型特异性规则，我们将在神经元的横截面中进行结构动力学的调查，该横截面反映了视觉皮层浅层内新皮层细胞类型的多样性。视觉，体感和前额叶皮层的比较分析将使我们能够解决是否中间神经元重塑是一种普遍现象。具体目标2：为了研究视觉体验在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，单眼眼睑缝合之前的单眼剥夺（MD），或单眼眼睑缝合之前的暗适应。为了比较，我们还将应用两种额外的操作，双眼剥夺（BD）和通过眼内注射TTX的单眼阻断。通过比较未处理的成年thy 1-GFP小鼠的皮质中的树突状乔木的变化与那些在长MD后的小鼠中的树突状乔木的变化，在由先前的剥夺或暗适应引发的短暂MD后，在BD或眼内TTX注射后，我们可以测试这一假设，即仅产生OD变化的活动形式也将增强成像神经元中的结构可塑性。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。聚类分析应提供洞察力，是否有interneuron细胞类型的结构比别人更可塑性，以及是否interneuron的子集表现出与OD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.