in vivo imaging of circuit remodeling in mouse visual cortex

许多脑部疾病表现为突触完整性、稳定性和经验依赖性选择受损， 导致线路缺陷和功能紊乱。不幸的是，我们监测突触或电路故障的能力 由于难以在体内观察突触而受到阻碍。在这里，我们建议在体内 监测突触形成和消除中的“操作顺序”，并确定步骤和 控制经验依赖性突触选择的分子我们专注于视觉系统， 这是一个很好的操纵经验的工具包。我们假设突触的动力学 组装和拆卸，以及它的改造倾向，与它的连接身份密切相关， 蛋白质组含量为了验证这一点，我们提出了以下目标：目标1：跟踪结构重塑 抑制性突触以及它如何与它们的传入输入特异性和蛋白质组含量相关。我们将 小鼠L2/3锥体神经元树突状细胞标记生长抑素和小清蛋白 视觉皮层，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有关的内容。为此，我们将实施三色二- 光子显微术，以在体内同时跟踪抑制性突触的突触前和突触后元件， 随后是蛋白质组的磁共振分析（MAP），组织清除和扩张显微镜的组合， 用于对整个神经元的突触蛋白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， 兴奋性突触穿过L2/3锥体神经元的乔木，跟踪它们的日常动力学和对黑暗的反应。 适应，并分析其蛋白质组内容的动态历史和传入身份。目标3： 在分子水平上剖析兴奋性突触的经验依赖性选择和稳定性。 CPG 15/neuritin是一种活性调节的基因产物，对突触稳定和成熟至关重要。体内 WT和CPG 15基因敲除小鼠的成像显示，尽管在缺乏CPG 15基因的情况下， 视觉经验或CPG 15，在这两种情况下，新生脊柱的PSD 95募集都是不足的。CPG 15表达 在缺乏活动的情况下足以恢复正常的PSD 95募集和脊柱稳定，这表明 充当活动依赖性突触选择器。我们问CPG 15损失如何影响突触中的分子事件 形成和成熟。目标4：开发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种不同的遗传编码荧光团 proteins.我们将开发新的标记和双光子显微镜配置，用于体内监测高达 一次四个突触组件，有解决各种实验问题的选择。

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.