Characterizing excitatory synapse in vivo structural dynamics

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 监测兴奋性突触形成和消除的“操作顺序”，并确定步骤 以及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 是一个具有良好特性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 兴奋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 标记小鼠L2/3锥体神经元全树突支的LGN或LP丘脑传入 大脑皮质，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 显微镜在活体内同时追踪兴奋性突触的突触前和突触后成分 通过蛋白质组放大分析(MAP)，一种组织透明和扩张显微镜的组合，用于超级 跨整个神经元的突触蛋白含量的分辨率分析。目标2：对分子进行解剖 兴奋性突触的水平、经验依赖的选择和稳定。Cpg15/神经氨酸是一种 活性调节基因产物对突触的稳定和成熟至关重要。WT和WT中的活体成像 Cpg15基因敲除的小鼠显示，虽然脊柱的形成在缺乏视觉经验的情况下正常发生 或cpg15，在这两种情况下，PSD95对新生脊椎的招募都是不足的。Cpg15在缺失时的表达 活动足以恢复正常的PSD95募集和脊柱稳定，这表明它作为一种 依赖活动的突触选择器。这个场景中一个令人费解的方面是cpg15是细胞外的，而 PSD95是细胞内的，两者都没有跨膜结构域。有趣的是，cpg15先前被鉴定出来。 作为AMPA型谷氨酸受体(AMPAR)蛋白质组的一部分。然而，Cpg15的S作用机制仍然存在 不清楚。为了探讨cpg15‘S的突触功能，我们将cpg15的最小结合结构域定位在AMPAR上， 并测试阻止其与cpg15的相互作用是否会影响AMPAR与适配器Stargazin的相互作用 PSD中传递、插入和保留功能性受体所必需的分子。要调查如何 Cpg15结合影响突触的AMPAR稳定性，进而影响其突触的存在 下游相互作用蛋白Stargazin和PSD95，我们将建立突触AMPAR的体外检测方法 机动性。最后，我们将询问cpg15的缺失，作为经验的替代，如何影响分子序列。 利用双光子显微镜和MAP对体内突触的形成、稳定和成熟进行了研究。

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.