enables the silencing of neurons in the awake brain of a mouse [11]. Therefore, Arch may serve as another useful optogenetic tool for neural silencing experiments in vivo. In this study, we examined the applicability of Arch for optogenetic manipulation of C. elegans cells in vivo. We found that worms forced to express Arch::GFP in neurons or muscle cells of the entire body stopped their locomotion and became paralyzed when illuminated with green light. Worms expressing Arch::GFP in different subsets of motor neurons exhibited distinct behavioral defects under green light illumination. We also examined the acquisition of Arch function and its stability in C. elegans cells by using a heat-shock gene induction system. We found that Arch becomes fully functional 12 hours after induction of expression, and remains functional for at least 24 hours. Results Arch::GFP Expression in C. elegans Cells To examine whether Arch, a light-driven proton pump, can be used to repress the electrical activity of neurons and muscle cells in Caenorhabditis elegans (C. elegans), we first tried to express C- terminally GFP-tagged Arch (Arch::GFP) in worms by using the myo-3 gene promoter, which encodes a myosin heavy chain expressed specifically in the body wall and vulva muscles of the organism, and the promoters of the F25B3.3 and aex-3 genes, both of which drive pan-neuronal expression. Then, we analyzed the expression pattern of GFP and the locomotion behavior of transgenic worms under green light illumination. In worms carrying the transgene myo-3p::Arch::gfp, nc3031Ex[myo- 3p::Arch::gfp], an intense GFP signal was observed in granular or vesicular structures close to the cell membrane and in myofibril-like structures of the body wall muscles. The signal was also detected at muscle cell boundaries, suggesting localization of Arch to the cell membrane. A weak GFP signal was seen in the cytoplasm, but not in the nucleus (Fig. 1A, B). In worms carrying the transgene F25B3.3p::Arch::gfp, ncEx3034[F25B3.3p::Arch::gfp], and in worms carrying the trans- gene aex-3p::Arch::gfp, ncEx3026[aex-3p::Arch::gfp], GFP signals were observed in the soma and the axon of many neurons in the head, mid body and tail (Fig. 1C, E). The signal often demarcated the cell soma. An intense granular or vesicular GFP signal was sometimes detected in the soma and in axons (Fig. 1 D, F). All transgenic strains carrying Arch::gfp stopped locomotion and became paralyzed under green light illumination to the whole body. C. elegans worms do not have inherent rhodopsin genes [12], and it is presumed that the level of endogenous retinoids, if these even exist, is insufficient for the proper functioning of rhodopsins [6]. Accordingly, when the transgenic animals were raised in the absence of all-trans-retinal (ATR), an essential cofactor for rhodopsin, illumination with green light failed to stop their locomotion, proving that the paralysis elicited in the presence of ATR is indeed driven by Arch. Light Intensity Dependence of Arch Neuronal Silencing Activity We then examined the response of each transgenic line expressing Arch::GFP in neurons to green light of different intensities. With ncEx3034[F25B3.3p::Arch::gfp] animals expressing Arch::GFP pan-neuronally, 25% of the worms were paralyzed when illuminated with green light (542.5667.5 nm) with an intensity of 0.2 mW/mm2 at 550 nm, the Arch absorption maximum. Eighty-five percent of the animals stopped locomotion at 0.3 mW/mm2, and all animals stopped locomotion at 0.4 mW/ mm2 (Fig. 2). Twenty-five percent of the ncEx3026[aex-3p::Arch::gfp] animals, another pan-neuronally Arch::GFP-expressing line, Optical Silencing of C. elegans Cells with Arch stopped locomotion at 0.2 mW/mm2, and 50% of the animals 2 2 stopped locomotion at 0.3 mW/mm . At 2.7 mW/mm , all animals stopped locomotion (Fig. 2). ncEx3034[F25B3.3p::Arch::gfp] worms stopped locomotion im- 2 mediately upon exposure to green light at 14.7 mW/mm , remained paralyzed during the illumination for 5 seconds, and resumed normal locomotion immediately after the end of such illumination, suggesting that Arch activation and inactivation both occur very rapidly in response to illumination conditions in this case. The observed quick response to conditions of illumination and non-illumination was almost fully retained even when worms were subject to 10 cycles consisting of a 1 second period of illumination followed by 1 second without illumination. When subjected to 30 cycles in this manner, however, the recovery of locomotion after illumination was turned off gradually required more time for most worms. When subjected to continuous illumination for 30 seconds, most ncEx3034[F25B3.3p::Arch::gfp] worms (80%, n = 60) remained paralyzed throughout the illumi- nation, but the others started movement, though without forward or backward locomotion, while under illumination. Thus, contin- uous illumination appears to have a relatively minor inactivating effect on Arch. Post-illumination recovery was delayed in the majority of worms (83%, n = 60), but they eventually resumed locomotion within 15 seconds after illumination was stopped. Light Intensity Dependence of Arch Muscle Silencing Activity In a manner similar to that for the observed Arch neuronal silencing activity, the locomotion of ncEx3031[myo-3p::Arch::gfp] animals expressing Arch in body wall muscle cells was profoundly affected by green light illumination. With ncEx3031[myo-3p::Arch::gfp] animals, however, illumination did not lead to immediate arrest of locomotion; worm locomotion first slowed down, and paralysis 2 gradually occurred. At an illumination intensity of 14.7 mW/mm , complete arrest of locomotion required illumination for 8 seconds (n = 9). This is in contrast to worms expressing Arch in neurons, which stopped locomotion immediately after the onset of illumina- tion. Therefore, we evaluated the effect of illumination on the locomotion of ncEx3031[myo-3p::Arch::gfp] animals over 30 seconds of illumination. At 0.7 mW/mm2, only a fraction of the animals had stopped locomotion. At 8.3 mW/mm2, approximately half of the animals stopped locomotion, whereas all animals appeared to be paralyzed when subjected to 14.7 mW/mm2 illumination (Fig. 2). When continuously exposed to green light at an intensity of 14.7 mW/mm2 for a period of 30 seconds, some ncEx3031[myo- 3p::Arch::gfp] animals remained paralyzed during illumination (48% n = 55), but others recovered partially and started to move while still under illumination, albeit at a much slower rate than usual. With an illumination period longer than 30 seconds, or when subjected to intermittent illumination cycles consisting of 1 second of illumination followed by 1 second of non-illumination, repeated 30 times, locomotion recovery required more time. Some worms failed to resume normal fast locomotion even after a 10- second period of non-illumination. Given a sufficient time following light cessation, however, all worms eventually resumed normal locomotion. Light-mediated Silencing of Subsets of Ventral Cord Motor Neurons In order to utilize Arch for analyzing the function of neuron- circuits of worms, we then tried to express Arch in subsets of neurons. Freely moving C. elegans worms crawl forward on agar in a sinusoidal motion, which was occasionally interrupted by brief PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e35370