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（由应用程序提供）：将在该项目中开发一种基于发光的新方法，用于监测神经元活动作为功能性神经科学的新工具。刺激神经元活性的光遗传学方法正在革新体外和体内神经生物学研究。迄今为止，短暂暴露于表达通道Ropopsin-2（CHR2）的细胞可以引起兴奋性阳离子通量（或抑制性离子通量，带有细菌近红局部BR）迄今为止，光遗传学刺激的影响通常是通过具有精确和昂贵的动物来及其且在自由上或在Viv中进行的，或者在Viv中进行的，或者在VIV中进行了良好的特征性和昂贵的动物。光遗传学刺激将通过侵入性较小的方法进行最佳合作，以监测许多细胞的活动，例如通过光学方法。不幸的是，目前首选的用于测量神经活动的方法是基于与CHR2/BR匹配不佳的荧光方法，因为监测合成活性所需的荧光兴奋会触发CHR2和/或BR。此外，荧光可以光漂白问题，并激发组织自动荧光，从而产生难以置信的背景。发光是避免与荧光有关的问题的替代光学技术。该项目将为神经元活性带来新的发光问题，该神经元活性具有遗传编码，可以针对特定的细胞类型以及特定的细胞局部局部 这些问题将通过改变其发光强度和/或发光光谱来应对神经元活性。在后来的情况下，基于生物发光谐振能量转移（BRET）的问题将受神经元活性调节，以便当激活神经元时发光发射的光谱会发生变化。我们的新发光方法将避免电生理学和荧光兴奋的缺点（尤其是靶非目标光遗传学刺激，光漂白和组织自荧光），因此将最佳地使用用于体外和体内刺激的光遗传学方法。 这些神经元活性的发光记者将与海马原发性神经元的光遗传学刺激以及在体外重构神经元的脑切片的同时表征。此外，编码这些记者的病毒向量将以微创的方式将问题引入大脑，以监测下丘脑的昼夜节律循环中的神经元活性（i），以及（ii），（ii）（ii）在Vivo中的Opto遗传学脑模拟之前和之后。该项目适用于NIMH的R21探索/发展研究授予机制，因为它将开发新的技术和工具，以推进多种模型动物物种大脑中复杂电路和细胞相互作用的空间时间分析。