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

用于全光电生理学 (AOE) 的近红外基因编码电压指示器 (NIR-GEVI)

• 批准号: 9229649
• 负责人: SRDJAN D ANTIC
• 金额: $ 104.78万
• 依托单位: IMPERIAL COLLEGE OF SCIENCE, TECHNOLOGY AND MEDICINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-30 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9229649
• 关键词: Accelerometer; Adverse effects; Anions; Automobile Driving; BRAIN initiative; Benchmarking; Biological; Biological Models; Blindness; Brain; Calcium; Cations; Cell membrane; Cells; Cognitive; DNA cassette; Development; Dyes; Electrophysiology (science); Electroporation; Emerging Technologies; Emotional; Event; Exhibits; Face; Fluorescence; Fluorescence Resonance Energy Transfer; Gene Delivery; Gene Targeting; Genetic; Glutamates; Goals; Image; Individual; Laboratories; Life; Light; Microelectrodes; Molecular; Monitor; Motor; Mus; Neurons; Neurosciences; Opsin; Optics; Performance; Photons; Physiology; Phytochrome; Positioning Attribute; Procedures; Protein Engineering; Proteins; Resolution; Scanning; Series; Signal Transduction; Slice; Staining method; Stains; System; Tail; Technology; Time; Tissue imaging; Transgenic Mice; Variant; Work; absorption; awake; base; blind; comparative; design; improved; in utero; in vivo; in vivo imaging; instrumentation; interest; invention; light emission; mouse model; neuronal cell body; neuronal circuitry; neurotechnology; new technology; next generation; novel; optical imaging; optogenetics; photoacoustic imaging; promoter; protein structure function; quantum; recombinase; research study; sensor; small molecule; stoichiometry; success; tool; two-photon; voltage

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.

实现有效的光学调制和神经元回路活动的读出一直是一个长期目标 神经科学领域的研究，是 BRAIN Initiative 的近期关键目标。需要这样的神经技术 破译大脑的电信号如何与知觉、认知、情感和运动功能相关。 利用光调节神经元活动的想法首次得到广泛成功的实现 笼状谷氨酸的发展，但只是因为使用了基因编码（光遗传学）执行器 例如视紫红质通道，这种方法是否取得了压倒性的成功？利用光的想法 随着第一个电压敏感器件的发现，记录大脑中的电信号被概念化 半个多世纪前的染料。电压成像方法对我们的研究做出了很大贡献 在细胞和系统水平上理解大脑生理学，但广泛的实验用途 这些小分子染料存在一些局限性，包括侵入性染色程序， 药理副作用和对细胞多样性的盲目性。这三个限制 最近发明的基因编码电压指示器（GEVI）克服了这一问题。虽然在很多 在优于经典电压敏感染料的方面，GEVI 尚未得到令人满意的优化和 它们与光遗传学调节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的光谱带的重叠，同时 激发和成像可用的 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视蛋白的执行器正交组合。我们建议 使用新型近红外 (NIR) 光敏色素荧光蛋白 (FP) 生成一类新的 基于我们生成 GEVI 的专业知识，在近红外光谱中激发并发出荧光的 GEVI 使用类似 GFP 的 FP。我们计划将这些 NIR-GEVI 与蓝光激活的兴奋性和抑制性结合起来 视蛋白，以实现扩展基于经典微电极的细胞内单细胞的光学方法 对清醒小鼠中大量基因定义的神经元进行电流钳记录。转基因小鼠 其中该工具可以通过 Cre 重组酶激活，表达驱动小鼠系将是我们的之一 关键交付成果。