Near Infrared Genetically Encoded Voltage Indicators (NIR-GEVIs) for All-Optical Electrophysiology (AOE)

获得有效的光学调制和读出神经元回路活动一直是一个长期的目标 这也是BRAIN Initiative的一个关键的近期目标。这种神经技术需要 解读大脑的电信号如何与感知、认知、情感和运动功能相关。 使用光来调节神经元活动的想法在第一次广泛成功的实现中发现了它的存在。 笼状谷氨酸的发展，但只是因为使用遗传编码（光遗传学）执行器 例如通道视紫红质，这种方法已经取得了压倒性成功。利用光来 记录大脑中的电信号是随着第一个电压敏感的发现而概念化的。 半个多世纪前的染料。电压成像方法对我们的 了解大脑生理学，无论是在细胞和系统水平，但广泛的实验使用 这些小分子染料中的大多数受到几种限制包括侵入性染色程序， 药理学副作用和对细胞多样性的盲目性。这三个限制是 最近发明的基因编码电压指示器（GEVI）克服了这一问题。尽管在许多 尽管在上级方面优于经典的电压敏感染料，但GEVI尚未得到令人满意的优化， 它们与光遗传学调节的结合在实践中很难实现。一个主要障碍 是用于激活基于视蛋白的致动器的光的光谱带的重叠， 激发和图像可用GEVI。克服这一障碍所需要的是表现良好的远红 GEVI可以与蓝光激活的视蛋白基致动器正交组合。我们建议 使用新的近红外（NIR）光敏色素为基础的荧光蛋白（FP），以产生一类新的 在近红外光谱中激发并发出荧光的GEVI，基于我们的专业知识生成GEVI 使用类似GFP的FP。我们计划将这些联合收割机与蓝光激活的兴奋和抑制 视蛋白，使光学方法，扩展经典的基于微电极的细胞内单细胞 电流钳记录大量的基因定义的神经元在清醒的小鼠。转基因小鼠 其中该工具可以通过表达Cre重组酶的驱动小鼠系被激活，这将是我们的一项研究。 关键交付成果。

Attaining effective optical modulation and readout of neuronal circuit activities has been a longstanding goal in neuroscience and is a key near-term aim of the BRAIN Initiative. Such neurotechnology is required to decipher how the brain’s electrical signals relate to perceptual, cognitive, emotional and motor functions. The idea to use light to modulate neuronal activities found its first broadly successful realization with the development of caged glutamate, but only since the use of genetically encoded (optogenetic) actuators such as channelrhodopsin, has this approach become overwhelmingly successful. The idea to use light to record electrical signals in the brain was conceptualized with the discovery of the first voltage-sensitive dyes more than half a century ago. Voltage imaging approaches have contributed much to our understanding of brain physiology, both at the cellular and systems levels, but the broad experimental use of these small molecule dyes suffers from several limitations including invasive staining procedures, pharmacological side effects, and blindness towards cellular diversity. These three limitations have been overcome by the recent invention of genetically-encoded voltage indicators (GEVIs). Although in many aspects superior to classical voltage sensitive dyes, GEVIs have not yet been satisfactorily optimized and their combination with optogenetic modulation has been difficult to achieve in practice. One major obstacle is the overlap of the spectral bands of light used to activate opsin-based actuators and at the same time excite and image available GEVIs. What is required to overcome this hurdle are well performing far red GEVIs that can be orthogonally combined with blue light-activated opsin-based actuators. We propose to use novel near-infrared (NIR) phytochrome-based fluorescent proteins (FPs) to generate a new class of GEVIs that are excited and fluoresce in the NIR spectrum, building on our expertise to generate GEVIs using GFP-like FPs. We plan to combine these NIR-GEVIs with blue-light activated excitatory and inhibitory opsins, to enable an optical approach that expands classical microelectrode-based intracellular single cell current-clamp recordings to large numbers of genetically defined neurons in awake mice. Transgenic mice in which this tool can be activated via Cre-recombinase expressing driver mouse lines will be one of our key deliverables.