Novel Luminescence Reporters of Neural Activity Partnered with Optogenetics

与光遗传学合作的新型神经活​​动发光记者

• 批准号: 8952655
• 负责人: CARL Hirschie JOHNSON
• 金额: $ 22.97万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8952655
• 关键词: Animal Model; Animals; Bacteriorhodopsins; Behavior; Biological Assay; Biological Models; Bioluminescence; Brain; Brain region; Cations; Cells; Circadian Rhythms; Complex; Corpus striatum structure; Development; Electrophysiology (science); Energy Transfer; Exposure to; Family; Fluorescence; Fluorescent Probes; Hippocampus (Brain); Hypothalamic structure; In Vitro; Ions; Kinetics; Laboratories; Light; Light Cell; Measurement; Measures; Mental Health; Methodology; Methods; Monitor; Mus; National Institute of Mental Health; Neurobiology; Neurons; Neurosciences; Optical Methods; Optics; Penetration; Pharmaceutical Preparations; Photobleaching; Phototoxicity; Preparation; Rattus; Recording of previous events; Reporter; Research; Research Project Grants; Rodent; Slice; Synapses; Techniques; Technology; Tissues; Viral Vector; base; cell type; cost effective; in vivo; in vivo imaging; innovation; luminescence; luminescence resonance energy transfer; minimally invasive; neural circuit; neural model; neural stimulation; new technology; novel; optogenetics; physical conditioning; presynaptic; protein protein interaction; public health relevance; ratiometric; reconstitution; relating to nervous system; response; screening; sensor; spatiotemporal; tool; vector; voltage

 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.

 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发光方法，作为功能神经科学的新工具，用于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) 的细胞发出的光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）。通常通过准确且特征明确的电生理学方法来监测刺激，但在体内和/或多个神经元中同时实施自由行为的动物是困难且昂贵的，光遗传学刺激最好与侵入性较小的方法配合以监测神经元之间的活动。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细胞类型。以及特定的细胞位点 这些探针将通过改变其发光强度和发光光谱来响应神经活动，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，从而使发光光谱发生变化。当神经元被激活时，我们的新发光方法将避免电生理学和荧光激发的缺点（特别是脱靶光遗传学刺激、光漂白和组织自发荧光），因此将最佳地发挥作用。与光遗传学方法配合进行体外和体内刺激。 这些神经活动的发光产生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，编码这些产生的病毒载体将用于以最低限度将探针引入大脑。侵入性方式，以便监测自由行为的啮齿动物的神经活动（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。 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，以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。