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神经活动监测方法，作为功能神经科学的新工具。刺激神经活动的光遗传方法正在给体外和体内的神经生物学研究带来革命性的变化。表达通道视紫红质-2(ChR2)的细胞短暂暴露在光中可以引起兴奋性阳离子流(或细菌视紫红质BR的抑制性离子流)。迄今为止，光遗传刺激的影响通常是通过电生理方法监测的，这些方法准确且具有良好的特征性，但在活体动物和/或同时在多个神经元中实施困难且昂贵。光遗传刺激最好是与侵入性较小的方法相结合，以监测许多细胞的活动，例如通过光学方法。不幸的是，目前光学测量神经活动的首选方法是基于与ChR2/BR不匹配的荧光方法，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，荧光可以使探针光漂白，并激发组织自发荧光，从而产生不希望看到的背景。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，这种探针可在基因上编码，并可以针对特定的细胞类型和特定的细胞位置 都与神经活动有关。这些探针将通过改变它们的发光强度和/或发光光谱来响应神经元的活动。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制，从而当神经元被激活时发光发射光谱发生变化。我们的新发光方法将避免电生理学和荧光激发的缺点(特别是。因此，它将与体外和体内刺激的光遗传方法最佳配合。 这些神经活动的发光报告者将结合对海马区初级神经元和在体外重建神经回路的脑片的光遗传刺激来表征。此外，编码这些报告的病毒载体将被用来以微创的方式将探针引入大脑，以监测自由行为啮齿动物的神经活动(I)来自下丘脑的昼夜周期，以及(Ii)体内光遗传大脑刺激皮质前后的神经活动。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，以推进对多种模式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.