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

项目概要/摘要 了解大脑功能和可塑性需要创新的方法来研究局部（突触） 建立认知和行为的神经回路（连接体）的分子机制。这里 我们提出了一种“分子连接组学”方法，该方法整合了细胞类型特异性转录组学，蛋白质组学， 超分辨率结构成像和光遗传学功能分析，以研究局部蛋白质的作用 连接体发育中的翻译。我们将通过研究活动依赖性 视网膜神经节细胞（RGCs）与突触后神经元之间的联系 背外侧膝状体核（dLGN）中的神经元用于有意识的视觉感知和行为。工作 使用转基因小鼠品系（ET 33-Cre），其中眼睛特异性RGC是遗传可及的，我们将 量化眼睛特异性连接体过程中的分子（1）、结构（2）和功能（3）突触变化 发展眼睛特定通路的出生后发育受视网膜活动的调节， 使用转基因和药理学工具来破坏RGC尖峰，并进一步量化活性依赖性 局部蛋白质合成机制的变化驱动眼睛特异性突触发育和可塑性。 分子分析（1）将使用轴突-TRAP来免疫沉淀眼特异性突触mRNA（局部突触mRNA）。 translatomes）用于下一代测序。局部mRNA丰度/多样性将使用进一步验证 多轮荧光原位杂交和mRNA条形码用于空间转录组成像分析。 我们将使用邻近标记和非规范氨基酸标记来定量局部突触蛋白质组 标记和分离突触蛋白网络的技术，用于定量高分辨率质谱分析。 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retinogeniculate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 方法将用于定量蛋白质和mRNA分布在大，电路水平的组织体积， 突触下分辨率眼睛特异性突触（3）的功能表征将使用 视紫红质通道介导的眼特异性轴突的光学刺激与dLGN细胞中的突触后记录。 对记录的神经元进行事后超分辨率显微镜检查，可以直接相关测量神经元的活动。 结构/功能的关系，潜在的活动依赖性变化的突触强度。 这项工作将建立一种新的方法-分子连接组学-连接局部mRNA翻译 在亚细胞区室与连接体组装和细化机制。 转录组学/蛋白质组学分析将有助于确定未来差异调节的基因/蛋白质候选物 增益/功能丧失实验。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.