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

实现神经元回路活动的有效光学调制和读出一直是一个长期的目标 在神经科学方面，这是大脑倡议的一个关键的近期目标。这种神经技术被要求 破译大脑的电信号如何与感知、认知、情感和运动功能相关。 利用光来调节神经元活动的想法第一次广泛成功地实现是在 笼养谷氨酸的发展，但只有在使用遗传编码(光遗传)执行器之后 例如通道视紫红质，这种方法是否取得了压倒性的成功。利用光来 大脑中有记录的电信号是随着第一个电压敏感电信号的发现而被概念化的 半个多世纪前的染料。电压成像方法对我们的 对大脑生理学的理解，无论是在细胞和系统层面，但广泛的实验用途 这些小分子染料中有几个受到限制，包括侵入性染色程序， 药理副作用，以及对细胞多样性的盲目性。这三个限制一直是 被最近发明的遗传编码电压指示器(GEVI)所克服。尽管在许多情况下 与经典的电压敏感染料相比，GEVI的性能还没有得到令人满意的优化和 它们与光遗传调制的结合在实践中很难实现。一大障碍 是用于激活基于视蛋白的致动器的光谱带的重叠，同时 激发和想象可用的GEVI。克服这一障碍所需要的是表现良好的远红 GEVI可以与蓝光激活的基于光学的致动器垂直组合。我们建议 利用新型近红外(NIR)光敏色素荧光蛋白(FP)生成一类新的 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来生成GEVI 使用类似GFP的FP。我们计划将这些近红外GEVI与蓝光激活的兴奋和抑制相结合 OPTINS，以实现一种光学方法，扩展经典的基于微电极的细胞内单细胞 对清醒小鼠大量基因定义神经元的电流钳记录。转基因小鼠 其中这个工具可以通过Cre重组酶来激活，表达驱动鼠标的品系将是我们的 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.