An Autonomous Biologic Controller for Optogenetic Applications

描述（由申请人提供）：光遗传学最近彻底改变了神经科学领域。该技术包括将光敏离子通道或泵（视蛋白）输送到大脑中基因定义的神经元亚群。在适当波长的光照下，膜定位的视蛋白被激活，并使表达它们的神经元的膜电位去极化或超极化（取决于视蛋白的类型）。因此，光遗传学提供了一种在时间和空间上精确控制神经活动的无与伦比的手段。光遗传学不仅作为研究复杂神经网络的研究工具具有广泛的意义，而且还显示出直接用于治疗癫痫等疾病的巨大潜力。事实上，光遗传学方法最近已被用于在各种动物模型中停止癫痫活动。然而，尽管在未来的临床应用中取得了进展，光遗传技术在体内的使用仍然面临许多需要解决的重要挑战。这些挑战大多与光源有关，目前依赖于将激光或led与植入大脑的光纤相结合。这些光源不仅不适合在长期的体内环境中使用（即硬件依赖性，有限的组织外显率），而且还存在重大的安全风险（例如热致损伤）。该项目的总体目标是通过开发生物发光蛋白作为激活光敏视蛋白的替代光源，解决目前在体内使用光遗传学的技术限制。拟议的研究将开发几种使用生物发光蛋白的光遗传系统。在第一个目标中，我们将证明荧光素酶可以在体外激活视蛋白。在第二个目标中，我们将把生物发光与神经活动联系起来，以创建新的光遗传反馈系统。这些系统将在控制急性脑切片癫痫活动的背景下进行研究。因此，这项研究不仅提供了一种激活视蛋白的替代方法（增加了神经科学研究工具的稳健性），而且还提供了一种控制癫痫发作活动的新方法。

DESCRIPTION (provided by applicant): Optogenetics has recently revolutionized the field of neuroscience. The technique involves the delivery of light- sensitive ion channels or pumps (opsins) to genetically defined subpopulations of neurons in the brain. Upon illumination with the appropriate wavelength of light, membrane localized opsins become activated and either depolarize or hyperpolarize (depending on the type of opsin) the membrane potential of neurons expressing them. Thus, optogenetics offers an unparalleled means of controlling neural activity in a temporally and spatially precise manner. Not only does optogenetics have broad implications as a research tool for studying complex neural networks, but it also shows great potential to be used directly as a treatment for diseases like epilepsy. Indeed, the optogenetic approach has been recently used to halt seizure activity in various animal models. However, despite the progress made towards a future clinical application, the use of optogenetic techniques in vivo still faces many important challenges that need to be addressed. Most of these challenges lie with the light source, which currently relies on using lasers or LEDs coupled to optical fibers implanted into the brain. Not only are these light sources impractical to use in long term in vivo settings (i.e. hardware dependency, limited tissue penetrance), they also pose a significant safety risk (e.g. heat-induced injury). The overall goal of this project is to addres the current technical limitations of using optogenetics in vivo by developing bioluminescent proteins as an alternative light source for activating light-sensitive opsins. The proposed research will develop several optogenetic systems using bioluminescent proteins. In the first aim, we will show that luciferases can be used to activate opsins in vitro. In the second aim, we will tie bioluminescence to neural activity in order to create novel optogenetic feedback systems. These systems will then be studied in the context of controlling epileptic activity in acute brain slices. The proposed research would therefore not only provide an alternative means to activate opsins (adding to the robustness of the tool for neuroscience research), but would also provide a novel approach to controlling seizure activity seen in epilepsy.