Optogenetic Approaches to Restore Vision and to Analyse Neural Circuits in the Retina

微生物视蛋白的引入是恢复视网膜退行性疾病光敏感性的一种很有前途的方法。本项目的目标是通过病毒载体在灵长类动物视网膜的双极细胞中表达红光敏感通道视紫质变异体。在这里，我们将重点放在中央视网膜的双极细胞(上-侏儒BPC)。这些细胞的一个关键特征是它们接受来自单个视锥体光感受器的突触输入，然后这些信号被传输到单个神经节细胞(侏儒RGC)。这种一对一的连接是包括人类在内的灵长类动物中心凹高敏锐度视力的重要解剖学先决条件。通过对侏儒BPC的光遗传刺激，我们将在连接的神经节细胞中触发光反应，这将用电生理学进行分析。特别是，我们将确定它们的感受场特性，这将提供关于这种光遗传学方法所能达到的潜在空间分辨率的重要信息。在一个平行的项目中，我们将开发一个串联结构，由一个氯化物泵/传感器组成，允许同时操作和观察细胞内的氯浓度。这种结构的细胞类型特异性表达将能够以定量的方式(比率传感器)控制这些细胞的细胞内氯浓度，以研究瞬时抑制对局部神经回路的影响。因此，我们将使用定制的设置，允许使用可见光波长的图案化光刺激视网膜锥体，结合双光子诱导的光遗传(全息)刺激和双光子成像。

The introduction of microbial opsins is a promising approach to restore light sensitivity in retinal degenerative diseases. The goal of this project is to express a red-light sensitive channelrhodopsin-variant in ON-bipolar cells of the primate retina, ex vivo by using a viral vector. Here, we will focus on the ON-bipolar cells of the central retina (ON-midget BPCs). A key feature of these cells is that they receive synaptic input from single cone photoreceptors, and these signals are then transmitted to single ganglion cells (midget RGCs). This one-to-one connectivity is an important anatomical prerequisite for the foveal high-acuity vision in primates, including humans. By using optogenetic stimulation of ON-midget BPCs we will trigger light-response in connected ganglion cells which will be analysed with electrophysiology. In particular, we will determine their receptive field properties which will provide important information about the potential spatial resolution that can be achieved with this optogenetic approach. In a parallel project, we will develop a tandem-construct, composed of a chlorid-pump/sensor that will allow for simultaneous manipulation and observation of intracellular chloride concentrations. Cell-type specific expression of this construct will enable to control the intracellular chloride concentration of these cells in a quantitative manner (ratiometric sensor), in order to investigate the impact of transient inhibition on local neuro-circuits. Therefore, we will use a custom-made setup that allows to stimulate retinal cones with patterned light at visible wavelengths, combined with 2-photon induced optogenetic (holographic) stimulation and 2-photon imaging.