Novel Luminescence Reporters of Neural Activity Partnered with Optogenetics

 描述（由申请人提供）：本项目将开发一种新的基于发光的方法，用于监测神经活动，作为功能神经科学的新工具。刺激神经活动的光遗传学方法正在彻底改变体外和体内的神经生物学研究。表达通道视紫红质-2（ChR 2）的细胞短暂暴露于光可以引起兴奋性阳离子通量（或与细菌视紫红质bR的抑制性离子通量）。迄今为止，光遗传学刺激的影响通常通过电生理学方法来监测，所述电生理学方法是准确的和充分表征的，但是在自由行为的动物体内和/或同时在多个神经元中实施是困难和昂贵的。光遗传刺激最好与侵入性较小的方法相结合，以监测许多细胞之间的活动，例如通过光学方法。不幸的是，目前用于光学测量神经活动的优选方法是基于与ChR 2/bR匹配不良的荧光方法，因为监测突触活动所需的荧光激发可以触发ChR 2和/或bR。此外，荧光可以光漂白探针并激发组织自发荧光，产生不期望的背景。发光是一种替代光学技术，可避免与荧光相关的问题。该项目将开发用于神经元活动的新型发光探针，这些探针是遗传编码的，可以靶向特定的细胞类型和特定的细胞位点， 参与了神经活动这些探针将通过改变其发光强度和/或发光光谱来响应神经元活动。在后一种情况下，基于生物发光共振能量转移（BRET）的探针将被神经活动调制，使得当神经元被激活时发光发射的光谱改变。我们的新发光方法将避免电生理学和荧光激发（尤其是脱靶光遗传学刺激、光漂白和组织自体荧光）的缺点，并且因此将与用于体外和体内刺激的光遗传学方法最佳地合作。 这些发光报告的神经活动将结合海马原代神经元和脑切片，在体外重建神经回路的光遗传学刺激的特点。此外，编码这些报告基因的病毒载体将用于以微创方式将探针引入大脑，以便监测自由行为啮齿动物中的神经活动（i）在来自下丘脑的昼夜节律周期内，和（ii）在体内皮质的光遗传学脑刺激之前和之后。该项目适用于NIMH的R21探索/发展研究资助机制，因为它将开发新的技术和工具，以推进对多个模型动物物种大脑中复杂电路和细胞相互作用的时空分析。

 DESCRIPTION (provided by applicant): A novel luminescence-based methodology for monitoring neural activity as a new tool for functional neuroscience will be developed in this project. Optogenetic methods for stimulating neural activity are revolutionizing neurobiological research in vitro and in vivo. Brief exposure to light of cells expressing channelrhodopsin-2 (ChR2) can elicit excitatory cation fluxes (or inhibitory ion fluxes with the bacteriorhodopsin bR) To date, the impact of optogenetic stimulation has usually been monitored by electrophysiological methods that are accurate and well characterized, but are difficult and expensive to implement in freely behaving animals in vivo and/or in multiple neurons simultaneously. Optogenetic stimulation would optimally be partnered with less invasive methods to monitor activity among many cells, such as by optical methods. Unfortunately, the currently preferred methods for optically measuring neural activity are based on fluorescence methods that are poorly matched with ChR2/bR because the fluorescence excitation needed to monitor synaptic activity can trigger ChR2 and/or bR. Moreover, fluorescence can photobleach probes and excite tissue autofluorescence that generates undesirable background. Luminescence is an alternate optical technology that avoids problems associated with fluorescence. This project will develop novel luminescence probes for neuronal activity that are genetically encodable and can be targeted to specific cell types and to specific cellular loci that are involved in neural activity. These probes will respond to neuronal activity by changing their luminescence intensity and/or luminescence spectrum. In the latter case, probes based on Bioluminescence Resonance Energy Transfer (BRET) will be modulated by neural activity so that the spectrum of luminescent emission changes when neurons are activated. Our new luminescence methodology will avoid the drawbacks of electrophysiology and fluorescence excitation (esp. off- target optogenetic stimulation, photobleaching & tissue autofluorescence), and will therefore optimally partner with optogenetic methods for in vitro and in vivo stimulation. These luminescence reporters of neural activity will be characterized in conjunction with optogenetic stimulation of hippocampal primary neurons and of brain slices that reconstitute neural circuits in vitro. In addition, viral vectors encoding these reporters will be used to introduce the probes to the brain in a minimally invasive manner so as to monitor neural activity in freely behaving rodents (i) over the circadian cycle from the hypothalamus, and (ii) before and after optogenetic brain stimulation of the cortex in vivo. This project is appropriate for the R21 Exploratory/Developmental Research Grant mechanism of the NIMH because it will develop new technologies and tools to advance spatiotemporal analyses of complex circuits and cellular interactions in the brains of multiple model animal species.