Optical Silencing of C. elegans Cells with Arch Proton Pump Ayako Okazaki, Yuki Sudo, Shin Takagi* Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan Abstract Background: Optogenetic techniques using light-driven ion channels or ion pumps for controlling excitable cells have greatly facilitated the investigation of nervous systems in vivo. A model organism, C. elegans, with its small transparent body and well-characterized neural circuits, is especially suitable for optogenetic analyses. Methodology/Principal Findings: We describe the application of archaerhodopsin-3 (Arch), a recently reported optical neuronal silencer, to C. elegans. Arch::GFP expressed either in all neurons or body wall muscles of the entire body by means of transgenes were localized, at least partially, to the cell membrane without adverse effects, and caused locomotory paralysis of worms when illuminated by green light (550 nm). Pan-neuronal expression of Arch endowed worms with quick and sustained responsiveness to such light. Worms reliably responded to repeated periods of illumination and non- illumination, and remained paralyzed under continuous illumination for 30 seconds. Worms expressing Arch in different subsets of motor neurons exhibited distinct defects in the locomotory behavior under green light: selective silencing of A- type motor neurons affected backward movement while silencing of B-type motor neurons affected forward movement more severely. Our experiments using a heat-shock-mediated induction system also indicate that Arch becomes fully functional only 12 hours after induction and remains functional for more than 24 hour. Conclusions/Sgnificance: Arch can be used for silencing neurons and muscles, and may be a useful alternative to currently widely used halorhodopsin (NpHR) in optogenetic studies of C. elegans. Citation: Okazaki A, Sudo Y, Takagi S (2012) Optical Silencing of C. elegans Cells with Arch Proton Pump. PLoS ONE 7(5): e35370. doi:10.1371/ journal.pone.0035370 Editor: Hitoshi Okazawa, Tokyo Medical and Dental University, Japan Received November 29, 2011; Accepted March 16, 2012; Published May 21, 2012 Copyright: 2012 Okazaki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (ST), and from Japan Society for the Promotion of Science (AO). AO is a JSPS DC1 Fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: i45116a@nucc.cc.nagoya-u.ac.jp Introduction Optogenetics is a novel technique for controlling excitable cells such as neurons, muscles and neuroendocrine cells. By exploiting light-driven ion channels or ion pumps derived from microbes and lower eukaryotes, optogenetics enables repression and activation of cells when illuminated with a particular wavelength of light. By enabling temporal manipulation of neuronal activity through an easy-to-induce and non-invasive treatment, this technique has the potential to revolutionize research on the subject of excitable cells in vivo [1–3]. A nematode, Caenorhabditis elegans (C. elegans) has a well-character- ized neural anatomy: the complete structure as well as the wiring patterns of each neuron of this organism have been determined by reconstruction from serial electron micrograph sections [4]. Together with the availability of various easy-to-handle genetic tools, these characteristics make this model organism an attractive one for studying the functions of single targeted cells in neural networks [5]. On the other hand, the small size of C. elegans neurons and their poor accessibility with respect to the application of microelectrodes has hindered conventional electrophysiological approaches. Methods for investigating neural functions in C. elegans have thus been almost entirely limited to laser killing of neurons, administration of neuroactive drugs, or use of neural mutants. As neither temporary activation nor repression of neuronal activity can be achieved with traditional techniques such as laser killing or drug treatment, optogenetic methods will be of great benefit to the study of C. elegans neurons. Optogenetics allows the investigation of neuronal functions in freely moving animals, increasing the prospects for behavioral studies using C. elegans. Optogenetic approaches applied to C. elegans, using channelrhodopsin-2 for activation of cells, and halorhodopsin (NpHR) for silencing of cells, have been reported [6–8]. NpHR is a light-driven inward chloride pump present in Natronomonas pharaonis [9], which inhibits electrical excitation of genetically targeted cells under illumination by yellow light (580 nm) with millisecond temporal resolution. NpHR, however, is capable of only modest hyperpolarizing currents in response to light, and once activated, NpHR enters long-lasting inactive states [10]. Recently, an optogenetical application of another light- driven pump, Archaerhodopsin-3 (Arch), derived from halobac- teria halorubrum sodomense, was reported [11]. Arch is an outward proton pump driven by green light (550 nm), and generates a neural silencing current at a lower level of illumination in vitro compared with NpHR. Additionally, Arch recovers spontaneously from light-dependent inactivation. It has been reported that Arch PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e35370