High-Performance Genetically Targetable Optical Neural Silencing via Light-Driven Proton Pumps-猫眼课题宝

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2010

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B. Chow;Xue Han;A. Dobry;Xiaofeng Qian;A. Chuong;Mingjie Li;Michael A. Henninger;Gabriel M. Belfort-Gabriel-M

以时间上精确的方式沉默遗传上指定的神经元的活动的能力将开启研究特定细胞类别在神经计算、行为和病理中的因果作用的能力。在这里，我们表明，成员的类光驱动外向质子泵可以调解非常强大的，安全的，多色沉默的神经活动。来自Halorubrum sodomense的基因archaerhodopsin-31（Arch）当在小鼠皮层中病毒表达并用黄光照射时，能够使清醒大脑中的神经元接近100%沉默。Arch在低光功率下介导数百皮安的电流，并在体内容易实现的光功率下支持接近900 pA的神经沉默电流。此外，Arch自发地从光依赖性失活中恢复，不像光驱动的氯离子泵在响应光时进入持久的失活状态。Arch的这些特性适合于在行为相关的时间尺度上介导显著脑容量的光学沉默。神经元中的Arch功能耐受良好，因为Arch照明产生的pH值偏移通过自限机制最小化至与通道视紫红质2，3或自然尖峰放电介导的水平相当的水平。为了突出质子泵生态和基因组多样性如何支持新的创新，我们表明，来自真菌Leptosphaeria maculans 4（Mac）的蓝绿光驱动质子泵在神经元中表达时，可以通过蓝光实现神经沉默，从而与其他开发的试剂一起，用户可以查看，打印，复制，下载和文本和数据挖掘这些文件中的内容，用于学术研究，必须遵守完整的用途：http://www.nature.com/authors/editorial_policies/license.html#terms有关材料的通信和请求应发送至E.S.B. (esb@media.mit.edu）..作者贡献B.Y.C.，许氏，和欧洲安全局。设计实验，分析数据，写论文。B.Y.C.和X.H.进行实验。AD辅助电生理记录。X.Q. M. L.，和A.S.C.辅助分子生物学、病毒制造和转染。M.A.H.进行蒙特卡罗建模。体育，通用汽车，和Y. L.创造了海马和皮层神经培养物。（†）这些作者对这项工作作出了同样的贡献。补充信息随附于www.nature.com/nature GENBANK（http：//www.ncbi.nlm.nih.gov/）上的论文，登录号为：哺乳动物密码子优化的Arch，GU 045593;哺乳动物密码子优化的Arch与GFP融合，GU 045594;哺乳动物密码子优化的Mac，GU 045595;哺乳动物密码子优化的Mac与GFP融合，GU 045596; ss-Prl-Arch，GU 045597; ss-Arch-GFP-ER 2，GU 045598; ss-Prl-Arch-GFP，GU 045599。转载和许可信息可在npg.nature.com/reprintsandpermissions上获得。作者声明没有竞争的经济利益。HHS公共访问作者手稿自然。作者手稿;可在PMC 2010年9月15日。以最终编辑形式发表：自然。2010年1月7日; 463（7277）：98-102。doi：10.1038/nature08652。A uhor M anscript

The ability to silence the activity of genetically specified neurons in a temporally precise fashion would open up the ability to investigate the causal role of specific cell classes in neural computations, behaviors, and pathologies. Here we show that members of the class of light-driven outward proton pumps can mediate very powerful, safe, multiple-color silencing of neural activity. The gene archaerhodopsin-31 (Arch) from Halorubrum sodomense enables near-100% silencing of neurons in the awake brain when virally expressed in mouse cortex and illuminated with yellow light. Arch mediates currents of several hundred picoamps at low light powers, and supports neural silencing currents approaching 900 pA at light powers easily achievable in vivo. In addition, Arch spontaneously recovers from light-dependent inactivation, unlike light-driven chloride pumps that enter long-lasting inactive states in response to light. These properties of Arch are appropriate to mediate the optical silencing of significant brain volumes over behaviourallyrelevant timescales. Arch function in neurons is well tolerated because pH excursions created by Arch illumination are minimized by self-limiting mechanisms to levels comparable to those mediated by channelrhodopsins2,3 or natural spike firing. To highlight how proton pump ecological and genomic diversity may support new innovation, we show that the blue-green lightdrivable proton pump from the fungus Leptosphaeria maculans4 (Mac) can, when expressed in neurons, enable neural silencing by blue light, thus enabling alongside other developed reagents Users may view, print, copy, download and text and datamine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms Correspondence and requests for materials should be addressed to E.S.B. (esb@media.mit.edu).. Authors' Contributions B.Y.C., X.H., and E.S.B. designed experiments, analyzed data, and wrote the paper. B.Y.C. and X.H. carried out experiments. A.S.D. assisted with electrophysiological recording. X.Q., M.L., and A.S.C. assisted with molecular biology, virus making, and transfections. M.A.H. performed Monte Carlo modelling. P.E.M., G.M.B., and Y.L. created hippocampal and cortical neural cultures. (†)These authors contributed equally to this work. Supplementary Information accompanies the paper on www.nature.com/nature GENBANK (http://www.ncbi.nlm.nih.gov/) accession numbers: mammalian codon-optimized Arch, GU045593; mammalian codonoptimized Arch fused to GFP, GU045594; mammalian codon-optimized Mac, GU045595; mammalian codon-optimized Mac fused to GFP, GU045596; ss-Prl-Arch, GU045597; ss-Arch-GFP-ER2, GU045598; ss-Prl-Arch-GFP, GU045599. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions The authors declare no competing financial interests. HHS Public Access Author manuscript Nature. Author manuscript; available in PMC 2010 September 15. Published in final edited form as: Nature. 2010 January 7; 463(7277): 98–102. doi:10.1038/nature08652. A uhor M anscript

High-Performance Genetically Targetable Optical Neural Silencing via Light-Driven Proton Pumps

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