Development of Line-Scan Temporal Focusing for fast structural imaging of synapse assembly/disassembly in vivo

不成比例的大量突变导致神经发育和神经精神 疾病靶向突触蛋白。神经退行性变中突触重塑和丧失先于细胞死亡 疾病和成瘾药物可以改变电路连接。如此多的大脑疾病的集合 突触的研究表明，适当的突触结构和功效对正常的脑功能至关重要。虽然多个 蛋白质与突触的形成和消除有关，这一系列事件导致 位于突触两侧的复杂蛋白质复合物的组装/分解尚未完成 划定的解决导致体内突触形成的步骤一直受到难以分离的限制。 标记并同时跟踪单个突触成分的募集和组装， 需要能够分辨出可辨别颜色的多个蛋白质标记，同时在生理上成像。 相关时间表。用于体内突触组装/拆卸的鲁棒的实时监测技术将 不仅对我们理解大脑发育的基本特征有巨大的影响， 可塑性，但也在其应用于许多疾病模型，从突触缺陷。 为了应对这一成像挑战，我们的目标是开发高分辨率，高通量的时间 聚焦（TF）双光子显微镜用于体内突触组装/拆卸的大体积成像 老鼠的大脑有了这种新颖的并行化方法，我们希望在体内实现高分辨率成像， 吞吐量比点扫描高几个数量级，但分辨率和信噪比相当 信噪比（SNR）。这将使整个树突状乔木和他们的居民突触在整个大脑的重复成像成为可能。 在真实的时间内观察突触形成和消除所需的短间隔，而没有 麻醉变得难以忍受。为了试验和验证该方法，我们将对 兴奋性和抑制性突触后支架蛋白分布在第2/3层的整个树突状动脉中 (L2/3）发育中的小鼠视皮层中的锥体神经元。此外，我们建议将我们的体内TF联合收割机 系统与蛋白质组的磁分析（MAP），一种形式的扩展显微镜，以询问 突触的蛋白质含量，我们有一个动态的历史，从体内成像。有了这些的力量 结合这些方法，我们的数据可以首次以前所未有的细节揭示， 当突触形成和修剪时，单个突触前和突触后成分的募集顺序 开发电路。

A disproportionately large number of mutations resulting in neurodevelopmental and neuropsychiatric disorders target synaptic proteins. Synapse remodeling and loss precede cell death in neurodegenerative disorders, and addictive drugs can alter circuit connectivity. The convergence of so many brain disorders at the synapse indicates that proper synapse structure and efficacy are critical to normal brain function. While multiple proteins have been implicated in synapse formation and elimination, the sequence of events leading to assembly/disassembly of the elaborate protein complexes that reside on both sides of the synapse has not been delineated. Resolving the steps leading to synapse formation in vivo has been limited by the difficulty of discretely labeling and simultaneously tracking the recruitment and assembly of individual synaptic components, which requires the ability to resolve multiple protein labels in discriminable colors while imaging over physiologically relevant timescales. Technology for robust, real-time monitoring of synapse assembly/disassembly in vivo would have enormous impact not only on our understanding of this fundamental feature of brain development and plasticity, but also in its application to the many disease models that derive from synaptic deficits. To tackle this imaging challenge, our goal is to develop high-resolution, high-throughput Temporal Focusing (TF) two-photon microscopy for large-volume imaging of synapse assembly/disassembly in vivo in the mouse brain. With this novel parallelized approach, we hope to achieve high-resolution imaging in vivo with 1-2 orders of magnitude higher throughput than point scanning, but with comparable resolution and signal-to-noise ratio (SNR). This would enable repeated imaging of entire dendritic arbors and their resident synapses over the short intervals necessary to observe synapse formation and elimination in real time, without the burden of anesthesia becoming intolerable. To pilot and validate the method, we will image the structural dynamics of excitatory and inhibitory post-synaptic scaffolding proteins distributed across the full dendritic arbors of Layer 2/3 (L2/3) pyramidal neurons in the developing mouse visual cortex. Further, we propose to combine our in vivo TF system with Magnified Analysis of the Proteome (MAP), a form of expansion microscopy, to interrogate the protein content of synapses for which we have a dynamic history from in vivo imaging. With the power of these combined approaches, our data can reveal, for the first time and in unprecedented detail, the timing and sequence of recruitment of individual pre- and post-synaptic components as synapses are formed and pruned in developing circuits.