Characterizing excitatory synapse in vivo structural dynamics

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 监测兴奋性突触形成和消除中的“操作顺序”，并确定步骤 以及控制经验依赖性突触选择的分子。我们专注于视觉系统， 是一个很好的工具箱，用于操纵经验。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 兴奋性突触以及它如何与它们的传入输入特异性和蛋白质组内容相关。我们将 将LGN或LP丘脑输入标记到小鼠视觉中单个L2/3锥体神经元的完整树突上 皮层，跟踪他们的日常动态和他们对视觉剥夺的反应，并分析他们的蛋白质组内容 与动态历史和传入身份有关。为此，我们将实现三色双光子 显微镜同时跟踪，在体内，兴奋性突触的突触前和突触后元素， 通过蛋白质组的磁共振分析（MAP），组织清除和扩张显微镜的组合， 整个神经元中突触蛋白含量的分辨率分析。目的2：解剖，在分子 水平、经验依赖性选择和兴奋性突触的稳定。CPG 15/neuritin是一种 活性调节基因产物，对突触稳定和成熟至关重要。在WT中的体内成像和 CPG 15基因敲除小鼠显示，虽然在缺乏视觉经验的情况下， 或CPG 15，在这两种情况下，PSD 95向新生棘的募集都是缺陷的。CPG 15表达缺失 的活性足以恢复正常的PSD 95募集和脊柱稳定，这表明它作为一种 活动依赖性突触选择器。在这种情况下，一个令人困惑的方面是CPG 15是细胞外的， PSD 95是细胞内的，并且两者都没有跨膜结构域。有趣的是，CPG 15以前被鉴定为 作为AMPA型谷氨酸受体（AMPAR）蛋白质组的一部分。然而，CPG 15的作用机制仍然存在， 不清楚为了探测CPG 15的突触功能，我们将在AMPAR上定位最小的CPG 15结合结构域， 并测试阻止其与CPG 15的相互作用是否影响AMPAR与衔接子stargazin的相互作用 在PSD处递送、插入和保留功能性受体所必需的分子。为了探究 CPG 15结合影响突触处AMPAR的稳定性，以及这反过来如何影响其在突触中的存在。 下游相互作用蛋白，stargazin和PSD 95，我们将开发一种体外测定突触AMPAR 迁移率最后，我们将询问作为经验替代物的CPG 15的缺失如何影响分子序列。 突触的形成，稳定和成熟在体内，使用双光子显微镜，然后MAP。

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 excitatory 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 excitatory synapses and how it relates to their afferent input specificity and proteomic content. We will label LGN or LP thalamic inputs onto the full dendritic arbor of single L2/3 pyramidal neurons in mouse visual cortex, track their daily dynamics and their response to visual 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 excitatory 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 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. A puzzling aspect in this scenario is that CPG15 is extracellular while PSD95 is intracellular, and neither has a transmembrane domain. Interestingly, CPG15 was previously identified as part of the AMPA-type glutamate receptor (AMPAR) proteome. Yet, CPG15's mechanism of action remains unclear. To probe CPG15's synaptic function, we will map the minimal CPG15 binding domain on the AMPAR, and test whether preventing its interaction with CPG15 effects AMPAR interaction with stargazin, an adaptor molecule that is essential for delivering, inserting, and retaining functional receptors at the PSD. To probe how CPG15 binding influences AMPAR stability at the synapse and how this, in turn, effects synaptic presence of its downstream interacting proteins, stargazin and PSD95, we will develop an in vitro assay for synaptic AMPAR mobility. Finally, we will ask how loss of CPG15, as a surrogate of experience, impacts the molecular sequence of synapse formation, stabilization, and maturation in vivo, using two photon microscopy followed by MAP.