Coupling Optogenetic Neural Stimulation with Novel Reporters of Synaptic Activity

描述(由申请人提供)：成瘾药物改变突触属性，在分子水平和重塑电路方面都是如此。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科学。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)的细胞短暂暴露于光中可引起兴奋性阳离子通量(或细菌视紫红质BR抑制离子通量)。到目前为止，光遗传刺激的影响通常是通过电生理方法来监测的，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自由行为的情况下实施起来困难且昂贵。光遗传刺激最好是与侵入性较小的光学方法相结合，以监测许多细胞之间的活动。不幸的是，目前光学测量突触活性的首选方法是基于与ChR2/BR不匹配的荧光方法，因为监测突触活性所需的荧光激发将触发ChR2和/或BR。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。记者刺激、光漂白和组织自动荧光)，因此将与光遗传方法进行体内刺激的最佳合作伙伴。发光是一种替代光学技术，可以避免与荧光相关的问题。该项目将开发用于突触活动的新型发光探针，这些探针是可遗传编码的，并针对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，该荧光受到pH或Ca++的调节，这样当突触被光遗传激活时，发光发射的光谱就会改变，从而避免与荧光激发相关的问题。这些突触活性的发光报告器将在一个经过充分研究的海马区原代神经元体外培养准备中进行表征，并在内源性大麻素治疗前后结合光遗传刺激。最后，编码这些报告的病毒载体将被用于监测自由行为大鼠在体光遗传刺激后大脑皮质的神经活动。该项目适合NIDA的尖端基础研究奖(CEBRA)R21机制，因为它提议开发将促进药物滥用和相关神经生物学研究的新技术。

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.