Novel Luminescence Reporters of Neural Activity Partnered with Optogenetics

 描述（由申请人提供）：本项目将开发一种用于监测神经活动的新型基于发光的方法，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 (ChR2) 的细胞短暂暴露在光下可以引发兴奋性阳离子通量（或细菌视紫红质 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进行监测，这些方法准确且特征良好，但在体内自由行为的动物和/或多个神经元中同时实施是困难且昂贵的。光遗传学刺激最好与侵入性较小的方法配合使用，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 ChR2/bR 匹配不佳的荧光方法，因为监测突触活动所需的荧光激发可以触发 ChR2 和/或 bR。此外，荧光可以光漂白探针并激发组织自发荧光，从而产生不需要的背景。发光是一种替代光学技术，可以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，这些探针是可遗传编码的，并且可以针对特定的细胞类型和特定的细胞位点 参与神经活动。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）的探针将受到神经活动的调节，以便当神经元被激活时发光光谱发生变化。我们的新发光方法将避免电生理学和荧光激发（特别是脱靶光遗传学刺激、光漂白和组织自发荧光）的缺点，因此将与体外和体内刺激的光遗传学方法最佳配合。 这些神经活动的发光报告器将结合海马初级神经元和体外重建神经回路的脑切片的光遗传学刺激来表征。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，以便监测自由行为的啮齿类动物的神经活动（i）下丘脑的昼夜节律周期，以及（ii）体内皮层光遗传学脑刺激之前和之后。该项目适合 NIMH 的 R21 探索性/发展研究资助机制，因为它将开发新技术和工具来推进对多种模型动物物种大脑中复杂回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.