A molecular connectomics platform for multi-scale analysis of activity-dependent synapse development and plasticity

项目摘要/摘要 了解大脑功能和可塑性需要研究局部(突触)的创新方法 在认知和行为基础上建立神经回路(连接)的分子机制。这里 我们提出了一种“分子连接学”的方法，它将特定细胞类型的转录，蛋白质组， 超分辨结构成像和光遗传功能分析研究局部蛋白质的作用 连接体发育中的翻译。我们将通过研究依赖于活动的 连接视网膜神经节细胞和突触后的视黄素生成途径的研究进展 背外侧膝状核(DLGN)中的神经元，负责有意识的视觉知觉和行为。劳作 在转基因小鼠品系(ET33-CRE)中，眼睛特异的RGC是可以遗传获得的，我们将 量化眼部特异性连接体中分子(1)、结构(2)和功能(3)突触的变化 发展。眼睛特定通路的出生后发育受视网膜活动的调节，从而使我们 使用转基因和药理学工具干扰RGC尖峰并进一步量化活性依赖 局部蛋白质合成机制的变化，驱动眼部特异性突触的发育和可塑性。 分子分析(1)将使用轴突陷阱来免疫沉淀眼睛特异的突触mRNAs(局部突触 翻译体)，用于下一代测序。将进一步验证本地信使核糖核酸丰度/多样性 用于空间转录成像分析的多轮荧光原位杂交和mRNA码。 我们将使用邻近标记和非规范氨基酸标记来量化局部突触蛋白质组 用于定量高分辨率质谱学的标记和分离突触蛋白网络的技术。 蛋白质组将使用超分辨率结构成像方法进行验证。结构分析(2)将映射 使用两种超分辨率成像技术对视网膜原基连接进行分子细化： 体积随机光学重建显微镜(STORM)和膨胀显微镜(EXM)。这些 方法将用于量化大的电路水平组织体积中的蛋白质和信使核糖核酸的分布 突触下分辨。眼部特定突触的功能特征(3)将使用 视紫红质通道介导的视觉刺激dLGN细胞突触后记录的眼特定轴突。 记录的神经元的后超分辨率显微镜允许直接、相关地测量 突触强度依赖活动变化的结构/功能关系。 这项工作将建立一种新的方法学--分子连接学--来连接本地的mrna翻译。 具有连接组组装和精细化的亚细胞室中的机制。 转录/蛋白质组学分析将有助于识别未来差异调控的基因/蛋白质候选 功能增益/损耗实验。我们的长期目标是将我们的平台应用于识别分子 神经发育障碍和精神疾病动物模型中回路功能障碍的机制。

Project summary/abstract Understanding brain function and plasticity requires innovative approaches for studying local (synaptic) molecular mechanisms that establish neural circuits (connectomes) underlying cognition and behavior. Here we propose a “molecular connectomics” approach that integrates cell-type-specific transcriptomic, proteomic, super-resolution structural imaging, and optogenetic functional analyses to investigate the role of local protein translation in connectome development. We will pilot our approach by studying the activity-dependent development of the retinogeniculate pathway, which links retinal ganglion cells (RGCs) with postsynaptic neurons in the dorsal lateral geniculate nucleus (dLGN) for conscious visual perception and behavior. Working with a transgenic mouse line (ET33-Cre) in which eye-specific RGCs are genetically accessible, we will quantify molecular (1), structural (2), and functional (3) synaptic changes during eye-specific connectome development. The postnatal development of eye-specific pathways is regulated by retinal activity, allowing us to use transgenic and pharmacological tools to disrupt RGC spiking and further quantify activity-dependent changes in local protein synthesis mechanisms driving eye-specific synapse development and plasticity. Molecular analyses (1) will use axon-TRAP to immunoprecipitate eye-specific synaptic mRNAs (local synaptic translatomes) for next-generation sequencing. Local mRNA abundance/diversity will be further validated using multi-round fluorescence in-situ hybridization and mRNA barcoding for spatial transcriptomic imaging analysis. We will quantify the local synaptic proteome using proximity-labeling and non-canonical amino-acid labeling techniques to tag and isolate synaptic protein networks for quantitative high-resolution mass spectrometry. Proteomes will be validated using super-resolution structural imaging methods. Structural analyses (2) will map the molecular refinement of retinogeniculate connections using two super-resolution imaging techniques: volumetric STochastic Optical Reconstruction Microscopy (STORM) and Expansion Microscopy (ExM). These methods will be used to quantify protein and mRNA distributions in large, circuit-level tissue volumes with subsynaptic resolution. Functional characterization eye-specific synapses (3) will be performed using channelrhodopsin-mediated optical stimulation of eye-specific axons with postsynaptic recording in dLGN cells. Post hoc super-resolution microscopy of recorded neurons allows for direct, correlative measurement of structure/function relationships underlying activity-dependent changes in synaptic strength. This work will establish a novel methodology – molecular connectomics – to link local mRNA translation mechanisms in subcellular compartments with connectome assembly and refinement. Transcriptomic/proteomic analyses will help identify differentially-regulated gene/protein candidates for future gain/loss-of-function experiments. Our long-term goal is the application of our platform to identifying molecular mechanisms of circuit dysfunction in animal models of neurodevelopmental disorders and mental illness.