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

导致神经发育和神经精神疾病的不成比例的大量突变 疾病的目标是突触蛋白。神经退行性变中突触重塑和丢失先于细胞死亡 疾病，以及上瘾的药物可以改变电路的连接。如此多的大脑疾病汇聚在一起 Synapse表明，正确的突触结构和有效性对正常的大脑功能至关重要。虽然有多个 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体的组装/拆解还没有被 勾勒出来的。在体内解决导致突触形成的步骤一直受到离散困难的限制 标记并同时跟踪单个突触组件的招募和组装，这些组件 需要在生理成像时以可区分的颜色解析多个蛋白质标记的能力 相关的时间尺度。强大的、实时监测体内突触组装/拆解的技术将 不仅对我们理解大脑发育的这一基本特征产生了巨大的影响 可塑性，但它在许多疾病模型中的应用也是如此，这些疾病模型源于突触缺陷。 为了解决这一成像挑战，我们的目标是开发高分辨率、高通量的时间 聚焦双光子显微镜在大鼠体内突触组装/拆解大体积成像中的应用 老鼠的大脑。利用这种新的并行化方法，我们希望实现1-2的活体高分辨率成像 吞吐量比点扫描高几个数量级，但分辨率和信噪比相当 比率(SNR)。这将使整个树突乔木及其驻留的突触在 需要较短的时间间隔来实时观察突触的形成和消除，而不需要 麻醉变得无法忍受。为了试验和验证该方法，我们将成像 兴奋性和抑制性突触后支架蛋白分布于第2/3层的全树突状树枝 (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转铁蛋白 带有蛋白质组放大分析(MAP)的系统，这是一种膨胀显微镜，用于询问 从活体成像中我们有动态历史的突触的蛋白质含量。有了这些的力量 结合方法，我们的数据可以第一次以前所未有的详细信息揭示时间和 作为突触形成和修剪的单个突触前和突触后组件的招募序列 在开发电路方面。

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.