Bioluminescent Multi-Characteristic Opsin for simultaneous optical stimulation and large-scale monitoring of the visual system

生物发光多特征视蛋白，用于同时进行光学刺激和大规模视觉系统监测

• 批准号: 10206147
• 负责人: Samarendra Kumar Mohanty
• 金额: $ 37.59万
• 依托单位: NANOSCOPE TECHNOLOGIES, LLC
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10206147
• 关键词: Address; Behavior; Behavioral; Behavioral Assay; Biological Assay; Bioluminescence; Biomedical Engineering; Biophysics; Blindness; Calcium; Cells; Characteristics; Clinical; Clinical Trials; Collaborations; Degenerative Disorder; Detection; Development; Devices; Disease Progression; Electric Stimulation; Electrophysiology (science); Engineering; Eye; Fluorescence; Goals; Head; Image; Imaging Device; Implant; Individual; Knowledge; Light; Longitudinal Studies; Maps; Measurement; Measures; Membrane; Methods; Molecular Biology; Monitor; Mus; Nervous system structure; Neurons; Nonexudative age-related macular degeneration; Ocular Prosthesis; Ophthalmology; Opsin; Optics; Photic Stimulation; Photophobia; Photoreceptors; Photosensitivity; Photosensitization; Phototoxicity; Research Personnel; Resolution; Retina; Retinal Degeneration; Retinal Photoreceptors; Scientist; Slice; Source; System; Techniques; Technology; Visual; Visual Cortex; Visual system structure; area striata; base; bioluminescence imaging; detector; efficacy evaluation; experience; fluorescence imaging; ganglion cell; implantation; in vivo; innovation; intravitreal injection; mouse model; multidisciplinary; nanoscope; optogenetics; photonics; photoreceptor degeneration; preclinical study; relating to nervous system; response; restoration; retinal imaging; retinal neuron; retinal stimulation; sight restoration; signal processing; stem cells; success; temporal measurement; tool; voltage

The optogenetic stimulation strategy, which is an alternative to electrical stimulation and now under clinical trial for vision restoration in retinal degenerative diseases, involves simple intravitreal injection of safe AAV-carrying opsin to photosensitize higher order retinal neurons. Compared with other strategies, optogenetic method is cell-specific and provides higher resolution. Our optogenetic studies in dry-AMD mice model suggests that degenerated retina can be made highly photosensitive, enabling vision restoration at low light level. Furthermore, our preclinical studies have shown that the visually guided behavior deteriorated with progressive degeneration, which recovered to more non-degenerated level after optogenetic treatment. Though primary visual cortex is known to maintain its retinotopy in subjects with retinal degeneration despite prolonged visual loss, detailed knowledge of how optogenetic sensitization of higher order neurons manifests in restoration of visual cortical activity is currently lacking. Thus, there is a need for mapping changes in the visual cortical activities as progression of retinal degeneration and subsequent vision restoration by optogenetic sensitization of retina occurs. The goal of this study is to develop and characterize tools for simultaneous optical modulation and imaging of retinal and cortical activities using spectrally-separated activation and detection channels. To address this goal, we propose to use newly developed voltage-sensitive bioluminescence assay instead of fluorescence in order to allow simultaneous long-term cortical imaging upon visual stimulation using multi- characteristic opsin (MCO). This will enable us to modulate neural activity in retina and image visual cortex with high temporal and spatial resolution, providing details about disease progression and restoration. The innovativeness of our proposal includes bioluminescent membrane voltage indicator to measure neural activity in long-term studies with high temporal resolution. This new bioluminescent technique does not require an additional potentially phototoxic external excitation source (as used for fluorescence). Further innovativeness includes simultaneous measurements of cortical activities and behavior in response to visual stimulation during retinal degeneration and vision restoration. Towards this goal we will: (i) Demonstrate functioning of Bioluminescent MCO and hardware for stimulation and bioluminescent neural activity monitoring; (ii) Quantify changes in visual cortical activities during progression of retinal photoreceptor degeneration by bMCO using head-mountable sCMOS-camera; and (iii) Evaluate restoration of visual cortical activities upon re- photosensitization of degenerated retina by intravitreal injection of MCO. Success of this proposal will lead to development of a modular and scalable interface system with the capability to serve a multiplicity of applications to modulate and monitor large-scale activity in the nervous system. In the long term, we aim to demonstrate a clinically viable optical stimulation device that can reliably provide stimulation inputs to the visual cortex and simultaneous recording of cortical activities.

光遗传学刺激策略是电刺激的替代方案，目前正在临床试验中 用于视网膜退行性疾病的视力恢复，涉及简单的玻璃体内注射安全的 AAV 携带 视蛋白使高阶视网膜神经元光敏化。与其他策略相比，光遗传学方法 细胞特异性并提供更高的分辨率。我们对干性 AMD 小鼠模型的光遗传学研究表明 退化的视网膜可以变得高度感光，从而能够在低光水平下恢复视力。 此外，我们的临床前研究表明，视觉引导的行为随着渐进性的恶化而恶化。 变性，经过光遗传学治疗后恢复到更非变性的水平。虽然初级 众所周知，尽管长时间视觉，但患有视网膜变性的受试者视觉皮层仍能维持其视网膜位置 损失，详细了解高阶神经元的光遗传学敏化如何在恢复中表现出来 目前缺乏视觉皮层活动。因此，需要绘制视觉皮层的变化图 视网膜变性的进展和随后通过光遗传学敏化恢复视力的活动 视网膜发生。本研究的目标是开发同步光调制工具并对其进行表征 使用光谱分离的激活和检测通道对视网膜和皮质活动进行成像。到 为了实现这一目标，我们建议使用新开发的电压敏感生物发光测定法来代替 荧光，以便在视觉刺激下使用多通道同时进行长期皮层成像 特征视蛋白（MCO）。这将使我们能够调节视网膜和图像视觉皮层的神经活动 具有高时间和空间分辨率，提供有关疾病进展和恢复的详细信息。这 我们提案的创新之处包括用于测量神经活动的生物发光膜电压指示器 在具有高时间分辨率的长期研究中。这种新的生物发光技术不需要 额外的潜在光毒性外部激发源（如用于荧光）。进一步创新 包括同时测量皮层活动和行为以响应视觉刺激 视网膜变性和视力恢复。为实现这一目标，我们将： (i) 展示 用于刺激和生物发光神经活动监测的生物发光 MCO 和硬件； (ii) 量化 bMCO 使用 bMCO 观察视网膜感光细胞变性过程中视觉皮层活动的变化 头戴式 sCMOS 相机； (iii) 评估视觉皮层活动的恢复情况 玻璃体内注射 MCO 对退化视网膜的光敏作用。该提案的成功将导致 开发模块化和可扩展的接口系统，能够服务多种 调节和监测神经系统大规模活动的应用。从长远来看，我们的目标是 展示一种临床上可行的光刺激装置，可以可靠地向患者提供刺激输入 视觉皮层和皮层活动的同步记录。