Enhancing the photostimulation kinetics of channelrhodopsin-2 encoded neurons

本提案旨在研究一种用光学手段诱导神经元电活动的技术，该技术比目前的电方法有许多优点。本工作旨在更好地表征和改进光敏剂的动力学。电生理学本身可以追溯到路易吉·伽伐尼的早期工作，他于1766年开始研究电刺激对青蛙腿的影响。从那时起，随着膜片钳技术、体内多位点刺激电极和商业体外微电极阵列的出现，该领域得到了改进。此外，应用已经从基础神经科学扩展到假肢应用。人工耳蜗和心脏起搏器已经问世多年。然而，活体电子刺激也有缺点。在许多情况下，电极会降解，电极会被污染或被死组织层覆盖，从而降低其效率。在基本的神经科学层面上，大的电脉冲会掩盖任何信号在其刺激时刻，使某些类型的实验更加困难。因此，如果我们能在视觉上刺激神经元，就会有很多优势。光学神经刺激的尝试已经有一段时间了，但随着新的视蛋白膜蛋白的发现，该领域取得了重大进展；黑视素在1998年，通道视紫红质在2003年。通道视紫红质（Chr2）显示出特别的前景。它是一个简单的光门控离子通道，当蓝光（峰值470纳米）照射时打开。先前的研究表明，它可以在体外和体内进行工程改造。然而，在这些出版物中有一个统一的障碍，以超过每秒50个尖峰的速度刺激神经元，而不是1000赫兹左右，这是他们能够做到的。本提案旨在通过光谱和光刺激电记录充分实验表征ChR2的动力学。我们通过我们的合作者获得了新的LED阵列技术，我们将使用该技术来研究多个亚细胞组件对电信号的贡献。然后，我们想利用实验结果来建立一个模型，说明ChR2最终能做什么，不能做什么。

This proposal aims to investigate a technique to induce electrical activity in neurons with optical means, which has many advantages over present electrical approaches. This work aims to better characterize and improve the kinetics of the light sensitization agent. Electrophysiology itself dates back to the early work by Luigi Galvani who began investigating the effect of electrical stimuli on frog's legs in 1766. Since then, the field has improved with the advent of patch clamp techniques, in-vivo multi-site stimulation electrodes and commercial in-vitro microelectrode arrays. Also, the application has broadened from basic neuroscience to prosthetic applications. Cochlear implants and heart pacemakers have been available for many years. There are however drawbacks to electronic stimulation in-vivo. The electrodes in many cases can degrade, and the electrodes can get fouled or covered with layers of dead tissue decreasing their efficiency. At a basic neuroscience level, large electrical pulses will mask any signally during their moment of stimulus, making certain types of experiments more difficult. Thus if we can optically stimulate neurons, there could be many advantages. There have been attempts at optical neural stimulation for some time, but the field has been significantly advanced with the discoveries of novel opsin membrane proteins; Melanopsin in 1998 and Channelrhodopsin in 2003. Channelrhodopsin (Chr2) has shown particular promise. It is a simple light gated ion channel that opens when illuminated with blue (470nm peak) light. Previous work has shown it engineered both in-vitro and in-vivo. However, there has been a uniform barrier in these publications to stimulate the neurons beyond a rate of 50 spikes per second compared to around 1000Hz, to which they are capable. This proposal aims to fully experimentally characterize the kinetics of the ChR2 via spectroscopy and photostimulated electrical recordings. We have access to novel LED array technology through our collaborators which we will use to investigate the contribution of multiple subcellular components to the electrical signal. We then want to use the experimental findings to build a model of what ultimately ChR2 is and is not capable of.