in vivo imaging of circuit remodeling in mouse visual cortex

许多大脑疾病表现为突触完整性、稳定性和经验依赖性选择受损， 导致接线缺陷和功能受扰。不幸的是，我们监控突触或回路故障的能力 当它们发生时，由于很难在体内可视化突触，这一点一直受到阻碍。在这里我们建议在活体内 监测突触形成和消除的“操作顺序”，并确定步骤和 控制经验依赖的突触选择的分子。我们专注于视觉系统，在那里有 一个特点良好的操控经验的工具包。我们假设突触的动力学 组装和拆卸，以及它的改型倾向，与它的连接身份和 蛋白质组含量。为了验证这一点，我们提出了以下目标：AIM1：跟踪 抑制性突触及其与传入输入的特异性和蛋白质组含量的关系。我们会 小鼠单个L2/3锥体神经元全树突支上生长抑素和小白蛋白的标记 视觉皮质，跟踪他们的日常动态和他们对单眼剥夺的反应，并分析他们的蛋白质组 与动态历史和传入身份相关的内容。为此，我们将实施三色两色- 光子显微镜在体内同时追踪抑制性突触的突触前和突触后成分， 然后是蛋白质组放大分析(MAP)，这是组织透明和扩张显微镜的组合， 用于对整个神经元的突触蛋白质含量进行超分辨率分析。目标2：跟踪结构 兴奋性突触的重塑及其与传入输入特异性和蛋白质组学的关系 内容。使用与目标1中类似的策略，我们将区分一般丘脑、LGN和LP输入，以 横跨L2/3锥体神经元束的兴奋性突触，追踪它们的日常动态和对黑暗的反应 适应，并分析它们的蛋白质组含量与动态历史和传入身份的关系。目标3：实现 在分子水平上剖析兴奋性突触的经验选择和稳定化。 Cpg15/Neuritin是一种活性调控的基因产物，对突触的稳定和成熟至关重要。活体内 WT和cpg15基因敲除小鼠的成像显示，虽然在没有 视觉体验或cpg15，在这两种情况下，PSD95对新生脊椎的招募都是不足的。Cpg15表达 在缺乏活动的情况下足以恢复正常的PSD95募集和脊柱稳定，这表明 充当依赖活动的突触选择器。我们问cpg15缺失如何影响突触中的分子事件 形成和成熟。目标4：发展和实现光谱分辨双光子显微镜 用于同时跟踪标记不同细胞的四个不同的基因编码荧光团 蛋白质。我们将开发新的标记和双光子显微镜配置，用于体内监测多达 一次四个突触组件，具有解决各种实验问题的选项。

Many brain disorders manifest impaired synaptic integrity, stability, and experience-dependent selection, resulting in wiring deficits and perturbed function. Unfortunately, our ability to monitor synaptic or circuit failures as they occur has been hindered by the difficulty of visualizing synapses in vivo. Here we propose in vivo monitoring of the ‘order of operations’ in synapse formation and elimination, and identifying the steps and molecules controlling experience-dependent synapse selection. We focus on the visual system, where there is a well-characterized toolkit for manipulating experience. We hypothesize that the dynamics of a synapse's assembly and disassembly, and its propensity to remodel, are intimately linked to its connection identity and proteomic content. To test this, we propose the following aims: Aim1: To track the structural remodeling of inhibitory synapses and how it relates to their afferent input specificity and proteomic content. We will label Somatostatin and Parvalbumin inputs onto the full dendritic arbor of single L2/3 pyramidal neurons in mouse visual cortex, track their daily dynamics and their response to monocular deprivation, and analyze their proteomic content in relation to dynamic history and afferent identity. To this purpose, we will implement triple color two- photon microscopy to simultaneously track, in vivo, both pre- and postsynaptic elements of inhibitory synapses, followed by Magnified Analysis of Proteome (MAP), a combination of tissue clearing and expansion microscopy, for super resolution analysis of synaptic protein content across the entire neuron. Aim 2: To track the structural remodeling of excitatory synapses and how it relates to their afferent input specificity and proteomic content. Using a similar strategy as in Aim 1, we will discriminate general thalamic, LGN, and LP inputs to excitatory synapses across the arbor of L2/3 pyramidal neurons, track their daily dynamics and response to dark adaptation, and analyze their proteomic content in relation to dynamic history and afferent identity. Aim 3: To dissect, at a molecular level, experience-dependent selection and stabilization of excitatory synapses. CPG15/neuritin is an activity-regulated gene product critical for synapse stabilization and maturation. In vivo imaging in WT and CPG15 knockout mice revealed that while spine formation occurs normally in the absence of visual experience or CPG15, in both cases PSD95 recruitment to nascent spines is deficient. CPG15 expression in the absence of activity is sufficient to restore normal PSD95 recruitment and spine stabilization, suggesting it acts as an activity-dependent synapse selector. We ask how CPG15 loss impacts molecular events in synapse formation and maturation. Aim 4: To develop and implement spectrally resolved two-photon microscopy for simultaneous tracking of four distinct genetically encoded fluorophores marking different cellular proteins. We will develop new labeling and two-photon microscope configurations for in vivo monitoring of up to four synaptic components at once, with options for addressing a variety of experimental questions.