Polymer-Free Nanosensors To Visualize Biochemical Dynamics in Dendritic Spines

描述(申请人提供)：树突棘对于整合中枢神经系统的兴奋性和抑制性输入是必不可少的。棘突的精细结构，颈部小至100 nm，头部小至400 nm，使它们能够形成局部控制信号和生化微环境的超结构隔间。脊椎的超微结构不适合采用标准的表征方法，如共聚焦成像和电生理学。双光子和STED成像的最新进展对于解析结构和位置至关重要，但对生化环境的洞察仍然难以捉摸。我们的目标是使用一种新型的无聚合物纳米传感器(PFN)与双光子激光扫描显微镜相结合来表征树突棘的生化环境。具体地说，我们建议评估脊椎中的离子动力学，从钠开始，然后很容易将平台扩展到其他离子，如氯、钙和钾。由于通过树突棘的颈部加载纳米传感器可能被证明是困难的，即使是小到20 nm的纳米传感器，我们建议开发并表征一种新型的纳米传感器：一种具有油而不是珠子的机械性能的无聚合物配方。这将使我们能够将PFN装载到甚至很小的结构中，然后作为分析物的实时、可逆指示器来监测荧光强度 集中精神。由于脊柱颈部形成了扩散隔离突触的屏障，人们预测这些改变的形态会扰乱突触和脊柱的生物化学和电学分区。我们的建议将确定脊柱颈部在塑造突触诱发信号方面的作用，并将为揭示疾病状态下形态变化的功能后果奠定必要的基础。

DESCRIPTION (provided by applicant): Dendritic spines are essential for integrating excitatory and inhibitory inputs in the central nervous system. The fine structure of the spines, with necks as small as 100 nm and heads as small as 400 nm, allow them to form ultra-structural compartments with localized control of signaling and biochemical microenvironment. The ultra-structure of spines does not readily allow for standard methods of characterization, such as confocal imaging and electrophysiology. Recent progress in two-photon and STED imaging has been essential to resolving structure and location, but obtaining insight to the biochemical environment remains elusive. Our goal is to use a novel Polymer-Free Nanosensors (PFN) combined with 2-photon laser scanning microscopy to characterize the biochemical environment of dendritic spines. Specifically, we propose assessing ion dynamics in the spines, starting with sodium and then easily extending the platform to other ions such as chloride, calcium, and potassium. Since loading nanosensors, even ones as small as 20 nm, through the necks of dendritic spines might prove difficult, we propose developing and characterizing a novel type of nanosensor: a polymer-less formulation that has the mechanical properties of an oil rather than a bead. This would allow us to load PFNs into even small structures, and then monitor fluorescence intensity as a real-time, reversible indicator of analyte concentration. Since the spine neck forms the barrier that diffusionally isolates the synapse, one predicts that these altered morphologies perturb the biochemical and electrical compartmentalization of the synapse and spine. Our proposal will determine the role of the spine neck in shaping synaptically-evoked signals and will set the foundation necessary for revealing the functional consequences of the morphological changes seen in disease states.