All optical control and monitoring of neural activity

描述(申请人提供)：成功的光遗传神经活动干预需要优化将编码光敏蛋白(Opsins)的基因传递到特定细胞，并记录在光遗传刺激过程中细胞和组织的变化。S使用病毒载体传递视蛋白最常用的方法是使用病毒载体，这容易引起意想不到的炎症反应、免疫反应和基因整合不当。此外，病毒方法限制了可包装和递送的质粒的大小，因此不能携带多个视蛋白编码基因或大启动子。此外，在一些人类疾病中，如视网膜色素变性(RP)，光感受器的进行性丧失发生在外围视网膜，定位Opsins的表达不仅在特定的细胞类型中，而且在有限的空间区域(RP中的外围)将是有用的。这个 该提案的第一个目标是优化非病毒递送方法，以减轻病毒递送带来的挑战。我们最近使用聚焦近红外超快激光束方法将视黄素(ChR2)输送到神经组织(视网膜)的空间图案化区域。然而，这项技术需要优化，以最大限度地减少有害影响。此外，开发一种无标记的光学技术(与电生理学相反)，以高空间分辨率和大吞吐量非侵入性地评估光遗传敏感神经元的功能激活将被证明是有用的。最近，我们展示了使用相敏频域光学相干层析成像(PSFD-OCT)来检测光遗传刺激细胞中的波动。PSFD-OCT是一种基于低相干干涉原理的新技术，可以探测几十皮米量级的位移。由于光的低相干长度，检测信号必须在光源的相干长度(~10微米)内。这使得PSFD-OCT能够在非常小的组织体积区域内研究亚纳米级的变化，并且适合于高度局部化的检测。本研究的总体目标是优化基因编码素Opsins的光学传递，并建立基于PSFD-OCT的无标记非侵入性光学读出方法，以监测激活引起的皮质神经元和组织的变化。

DESCRIPTION (provided by applicant): 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 (~10um). 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 encodin 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.