All optical control and monitoring of neural activity

项目概述：神经活动的光遗传学干预的成功需要优化 将编码光敏蛋白（视蛋白）的基因递送至特定细胞，并记录 在光遗传学刺激期间细胞和组织的变化。最常用的方法是 递送视蛋白是使用病毒载体，其易于引起意想不到的炎症 反应，免疫反应和不适当的基因整合。此外，病毒方法 限制质粒的大小，可以包装和交付，因此不能携带多个 视蛋白编码基因或大启动子。此外，在一些人类疾病的案例中，例如 色素性视网膜炎（RP），其中光感受器的进行性丧失发生在周边视网膜中， 将有助于定位视蛋白的表达，不仅在特定的细胞类型中， 受限空间区域（RP中的外围）。该提案的第一个目的是优化一个非病毒 递送方法，以减轻病毒递送带来的挑战。我们最近使用了聚焦 近红外超快激光束方法将视蛋白（ChR 2）递送到空间图案化的区域， 神经组织（视网膜）。然而，这种技术需要优化，以便最小化 有害影响。此外，将证明开发无标记光学技术是有用的（相反， 电生理学）以非侵入性地评估光遗传学敏化的 具有高空间分辨率和大吞吐量的神经元。最近，我们展示了使用 相位敏感频域光学相干层析成像（PSFD-OCT）用于检测 光遗传学刺激细胞的波动。PSFD-OCT是一种基于 低相干干涉测量的原理，可以检测几十个量级的位移， 皮米。由于光的低相干长度，检测到的信号必须在 光源的相干长度（~10 μ m）。这使得PSFD-OCT能够研究 纳米的变化在组织体积的非常小的区域内，并且适用于高度 局部检测本研究的总体目标是优化基因编码的光学递送 视蛋白，并开发基于PSFD-OCT的无标记非侵入式光学读出方法， 监测皮层神经元和组织因激活而产生的变化。 1

Project Summary: Success of optogenetic intervention of neural activity requires optimization of delivery of genes encoding light sensitive proteins (opsins) to specific cells, and to record the changes in cells and tissue during optogenetic stimulation. The most-commonly used method for delivering opsin(s) is use of viral vector, which is prone to cause unexpected inflammatory responses, immunological reactions, and improper gene integration. Further, the viral methods limit the size of plasmid that can be packaged and delivered and therefore cannot carry multiple opsin-encoding genes or large promoters. Further, in several cases of human diseases such as retinitis pigmentosa (RP) where progressive loss of photoreceptors happens in peripheral retina, it will be useful to localize the expression of the opsins not only in specific cell types, but in a restricted spatial region (peripheral in RP). The first aim of the proposal is to optimize a non-viral delivery method to mitigate the challenges posed by viral delivery. We have recently used focused near-IR ultrafast laser beam method to deliver opsins (ChR2) into spatially-patterned regions of neural tissue (retina). However, this technique needs to be optimized so as to minimize the deleterious effects. Further, it will prove useful to develop a label-free optical technique (in contrast to electrophysiology) to non-invasively evaluate functional activation of optogenetically-sensitized neurons with high spatial resolution and large throughput. Recently, we demonstrated use of Phase-Sensitive Frequency Domain Optical Coherence Tomography (PSFD-OCT) for detection of fluctuations in optogenetically-stimulated cells. PSFD-OCT is a novel technique based on the principles of low-coherence interferometry that can detect displacements of the order of tens of picometers. Because of the low-coherence length of the light, the detected signal has to be within the coherence length of the light source (~10¿m). This enables PSFD-OCT to investigate sub- nanometer changes within a very small region of the tissue volume and is suitable for highly localized detection. The overall aim of this study is to optimize optical delivery of gene encoding opsins, and develop label-free non-invasive optical readout method based on PSFD-OCT to monitor the changes in cortical neurons and tissue resulting from the activation. 1