An Autonomous Biologic Controller for Optogenetic Applications

描述(申请人提供)：光遗传学最近给神经科学领域带来了革命性的变化。这项技术涉及将光敏离子通道或泵(Opsins)输送到大脑中由基因定义的神经元亚群。在适当波长的光照射下，膜上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无与伦比的手段，可以在时间和空间上精确地控制神经活动。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癫痫等疾病治疗的巨大潜力。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发作活动。然而，尽管在未来的临床应用方面取得了进展，体内光基因技术的使用仍然面临着许多需要解决的重要挑战。这些挑战大多来自光源，目前光源依赖于使用激光或LED与植入大脑的光纤耦合。这些光源不仅不适合在活体环境中长期使用(即对硬件的依赖、有限的组织透过率)，而且还构成了重大的安全风险(例如，热诱导损伤)。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来弥补目前在活体中使用光遗传学的技术限制。这项拟议的研究将开发几个使用生物发光蛋白的光遗传系统。在第一个目标中，我们将展示荧光素酶可以在体外用来激活视蛋白。在第二个目标中，我们将把生物发光与神经活动联系起来，以创造新的光遗传反馈系统。这些系统随后将在控制急性脑片癫痫活动的背景下进行研究。因此，拟议的研究不仅将提供一种激活Opsins的替代方法(增加神经科学研究工具的健壮性)，而且还将提供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.