Coupling Optogenetic Neural Stimulation with Novel Reporters of Synaptic Activity

描述（由申请人提供）：成瘾药物在分子水平和重塑回路方面改变突触特性。我们将开发一种新的基于发光的方法来监测突触活动，作为功能神经科学的新工具，可以应用于药物滥用的科学。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下可引起兴奋性阳离子通量（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来监测，所述电生理学方法是准确的和良好表征的，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光学方法合作，以监测许多细胞之间的活动。不幸的是，目前优选的用于光学测量突触活动的方法是基于与ChR 2/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将触发ChR 2和/或bR。我们的新发光方法将避免电生理学和荧光激发（特别是报告刺激，光漂白和组织自发荧光）的缺点，因此将与光遗传学方法进行体内刺激。发光是一种替代光学技术，可避免与荧光相关的问题。该项目将开发用于突触活动的新型发光探针，该探针可遗传编码并靶向参与神经活动的特定细胞位点。这将通过在荧光素酶和金星荧光团之间使用生物发光共振能量转移（BRET）来实现，该荧光素酶和金星荧光团受pH或Ca++调节，以便当突触被光遗传学激活时，发光发射光谱发生变化，从而避免与荧光相关的问题激发。突触活性的这些发光报告者将在充分研究的海马原代神经元培养物制备物中在体外结合内源性大麻素治疗前后的光遗传学刺激进行表征。最后，编码这些报告基因的病毒载体将用于监测自由行为大鼠体内光遗传学刺激后皮层的神经活性。该项目适用于NIDA的尖端基础研究奖（CEBRA）R21机制，因为它提出开发新技术，以促进药物滥用和相关的神经生物学研究。

DESCRIPTION (provided by applicant): Addictive drugs change synaptic properties, both at the molecular level and in terms of remodeling circuits. We will develop a novel luminescence-based methodology for monitoring synaptic activity as a new tool for functional neuroscience that can be applied to the science of drug abuse. Optogenetic methods for stimulating neural activity are revolutionizing neurobiological research in vitro and in vivo. Brief exposure to lightof 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 optical methods to monitor activity among many cells. Unfortunately, the currently preferred methods for optically measuring synaptic activity are based on fluorescence methods that are poorly matched with ChR2/bR because the fluorescence excitation needed to monitor synaptic activity will trigger ChR2 and/or bR. Our new luminescence methodology will avoid the drawbacks of electrophysiology and fluorescence excitation (esp. reporter stimulation, photo bleaching & tissue auto fluorescence), and will therefore optimally partner with optogenetic methods for in vivo stimulation. Luminescence is an alternate optical technology that avoids problems associated with fluorescence. This project will develop novel luminescence probes for synaptic activity that are genetically encodable and targeted to specific cellular loci that are involved in neural activity. This will be accomplished by using Bioluminescence Resonance Energy Transfer (BRET) between a luciferase and the Venus fluorophore that is modulated by pH or Ca++ so that the spectrum of luminescent emission changes when synapses are activated optogenetically, thereby avoiding the problems associated with fluorescence excitation. These luminescence reporters of synaptic activity will be characterized in a well-studied hippocampal primary neuron culture preparation in vitro in conjunction with optogenetic stimulation before and after treatment with endocannabinoids. Finally, a viral vector encoding these reporters will be used to monitor neural activity of cortex after optogenetic stimulation in freely behaving rats in vivo. This project is appropriate for the Cutting-Edge Basic Research Award (CEBRA) R21 mechanism of the NIDA because it proposes to develop new technologies that will advance drug abuse and related neurobiological research.