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

光遗传学刺激策略，这是一种替代电刺激的方法，目前正在进行临床试验 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携带AAV的 视蛋白使高级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。我们在干性AMD小鼠模型中的光遗传学研究表明， 可以使退化的视网膜高度感光，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，视觉引导行为随着渐进性的 变性，其在光遗传学处理后恢复到更非变性的水平。虽然主要 已知在视网膜变性的受试者中，尽管长时间的视觉刺激， 损失，详细了解高级神经元的光遗传学敏化如何在神经元的恢复中表现出来。 目前缺乏视觉皮层活动。因此，需要绘制视觉皮质的变化 活性作为视网膜变性的进展和随后通过光遗传敏化的视力恢复 视网膜发生。本研究的目标是开发和表征工具的同时光学调制 以及使用光谱分离的激活和检测通道对视网膜和皮层活动进行成像。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， 荧光，以便允许在视觉刺激时同时进行长期皮质成像， 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。的 我们方案的创新之处包括用生物发光膜电压指示器来测量神经活动 在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 包括同时测量皮质活动和行为，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： 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 在视网膜光感受器变性过程中视觉皮层活动的变化 头戴式sCMOS摄像机;以及（iii）评估在重新植入后视觉皮层活动的恢复。 通过玻璃体内注射MCO使变性视网膜光敏化。这项提案的成功将导致 开发一个模块化和可扩展的接口系统，能够为多种 应用于调节和监测神经系统中的大规模活动。长远来说，我们的目标是 证明了临床上可行的光学刺激装置，其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。

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.