Microbial Light-Activatable Proton Pumps as Neuronal Inhibitors to Functionally Dissect Neuronal Networks in C. elegans
Microbial Light-Activatable Proton Pumps as Neuronal Inhibitors to Functionally Dissect Neuronal Networks in C. elegans
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DOI：
10.1371/journal.pone.0040937
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发表时间：
2012-07-16
期刊：
影响因子：
3.7
通讯作者：
Gottschalk, Alexander
中科院分区：
文献类型：
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作者：
Husson, Steven J.;Liewald, Jana F.;Gottschalk, Alexander
基本上，简单和复杂动物的任何行为都取决于神经元网络功能。目前，研究神经元回路的最好定义的系统是线虫秀丽隐杆线虫，因为它的302个神经元的连接是完全已知的。单个神经元可以通过使用蓝光对视紫红质-2（ChR 2）进行光刺激来激活，从而直接探测特定神经元对于所研究的网络的相应行为输出的重要性。类似地，其他可兴奋细胞可以通过表达来自嗜盐单胞菌（NpHR）的盐视紫红质并随后用黄光照射来抑制。但抑制C. elegans神经元使用NpHR是困难的。最近，来自各种来源的质子泵被确立为有价值的替代超极化器。在这里，我们表明，从Halorubrum sodomense的古紫质-3（Arch）和从真菌Leptosphaeria maculans（Mac）的质子泵可以有效地抑制C。优雅的通过光诱发行为和电生理学分析，Arch在黄光或绿色光照射下是最强的超极化器，而Mac的动作光谱蓝移更多。这使得这些工具可以以各种方式与ChR 2相结合，以分析电路中不同的神经元子集。我们通过多模态厌恶感觉ASH神经元和下游命令中间神经元来实现这一点，ASH神经元向其发出信号以触发逆转，然后是方向性转向。光刺激ASH和随后使用不同身体部分的双色照明抑制命令中间神经元，允许调查ASH下游信号传导的时间方面。
Essentially any behavior in simple and complex animals depends on neuronal network function. Currently, the best-defined system to study neuronal circuits is the nematode Caenorhabditis elegans, as the connectivity of its 302 neurons is exactly known. Individual neurons can be activated by photostimulation of Channelrhodopsin-2 (ChR2) using blue light, allowing to directly probe the importance of a particular neuron for the respective behavioral output of the network under study. In analogy, other excitable cells can be inhibited by expressing Halorhodopsin from Natronomonas pharaonis (NpHR) and subsequent illumination with yellow light. However, inhibiting C. elegans neurons using NpHR is difficult. Recently, proton pumps from various sources were established as valuable alternative hyperpolarizers. Here we show that archaerhodopsin-3 (Arch) from Halorubrum sodomense and a proton pump from the fungus Leptosphaeria maculans (Mac) can be utilized to effectively inhibit excitable cells in C. elegans. Arch is the most powerful hyperpolarizer when illuminated with yellow or green light while the action spectrum of Mac is more blue-shifted, as analyzed by light-evoked behaviors and electrophysiology. This allows these tools to be combined in various ways with ChR2 to analyze different subsets of neurons within a circuit. We exemplify this by means of the polymodal aversive sensory ASH neurons, and the downstream command interneurons to which ASH neurons signal to trigger a reversal followed by a directional turn. Photostimulating ASH and subsequently inhibiting command interneurons using two-color illumination of different body segments, allows investigating temporal aspects of signaling downstream of ASH.