Characterizing excitatory synapse in vivo structural dynamics

表征兴奋性突触体内结构动力学

• 批准号: 10708899
• 负责人: Elly Nedivi
• 金额: $ 47.5万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10708899
• 关键词: Acids; Affect; Anatomy; Architecture; Binding; Brain; Brain Diseases; Cell Nucleus; Cells; Color; Development; Electroporation; Elements; Event; Excitatory Synapse; Extracellular Protein; Failure; Genes; Glutamate Receptor; Glycosylphosphatidylinositols; Goals; Imaging technology; Impairment; Individual; Knockout Mice; Label; Lateral Geniculate Body; Lateral posterior nucleus of thalamus; Lead; Learning; Link; Maps; Mediator; Memory; Methods; Microscopy; Molecular; Monitor; Mus; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neurons; Physiological; Proteins; Proteome; Proteomics; Recording of previous events; Sensory; Specificity; Synapses; Synaptophysin; Testing; Tissues; Transgenic Mice; Transmembrane Domain; Vertebral column; Visual; Visual Cortex; Visual Perception; Visual System; Visualization; area striata; experience; extracellular; fluorophore; functional adaptation; gene product; hippocampal pyramidal neuron; in utero; in vitro Assay; in vivo; in vivo imaging; in vivo monitoring; innovation; mouse genetics; neural circuit; operation; postsynaptic; postsynaptic density protein; presynaptic; prevent; protein complex; pup; receptor; recruit; response; stargazin; synaptic function; synaptogenesis; two photon microscopy; ultra high resolution; visual deprivation

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.

许多脑部疾病表现出综合完整性，稳定性和经验依赖性选择的受损， 导致接线定义和扰动功能。不幸的是，我们监视突触或电路故障的能力 随着它们的发生，在体内可视化突触的困难阻碍了它们。在这里，我们在体内建议 监视兴奋性突触形成和消除的“操作顺序”，并确定步骤 和控制经验依赖性突触选择的分子。我们专注于在那里的视觉系统 是一种适合操纵体验的特定工具包。我们假设突触的动态 组装和拆卸及其对重塑的承诺与其连接身份密切相关， 蛋白质组学含量。为了测试这一点，我们提出以下目的：AIM1：跟踪结构重塑 兴奋性突触及其与其传入的输入特异性和蛋白质组学含量的关系。我们将 标签LGN或LP丘脑输入到小鼠视觉中的单个L2/3锥体神经元的整个树突状乔木 皮层，跟踪他们的日常动态及其对视觉剥夺的反应，并分析其蛋白质组织含量 与动态历史和传入身份有关。为此，我们将实现三色二亮 显微镜简单地跟踪兴奋性突触的体内和突触后元素，然后 通过放大蛋白质组（MAP）的分析，即组织清除和膨胀显微镜的组合，用于超级 整个神经元中突触蛋白含量的分辨率分析。目标2：在分子上剖析 水平，依赖经验的选择和兴奋性突触的稳定。 CPG15/神经蛋白是一个 活性调节的基因产物对于突触稳定和成熟至关重要。在wt中的体内成像 CPG15敲除小鼠发现，虽然脊柱形成通常在没有视觉体验的情况下发生 或CPG15，在这两种情况下，PSD95募集到新生的刺都是不足的。在不存在的情况下CPG15表达 活性足以恢复正常的PSD95募集和脊柱稳定，这表明它充当 活动依赖性突触选择器。在这种情况下，一个拼图器方面是CPG15是细胞外的 PSD95是细胞内的，并且都不具有跨膜结构域。有趣的是，以前已经确定了CPG15 作为AMPA型谷氨酸受体（AMPAR）蛋白质组的一部分。然而，CPG15的作用机理仍然存在 不清楚。为了探测CPG15的突触函数，我们将在AMPAR上绘制最小CPG15结合域， 并测试是否防止其与CPG15的相互作用效应AMPAR与stargazin（适配器）的相互作用 对于在PSD上输送，插入和保留功能受体至关重要的分子。探测如何 CPG15结合影响突触时的AMPAR稳定性，而这又如何影响其突触的存在 下游相互作用的蛋白质，Stargazin和PSD95，我们将开发用于突触AMPAR的体外测定 机动性。最后，我们将询问CPG15的损失如何作为经验的替代物影响分子序列 使用两个光子显微镜，然后是MAP，在体内突触形成，稳定和成熟。