Figure 2. Dependence of locomotion paralysis on light intensity. Animals expressing Arch::GFP under muscle-specific (myo- 3p) and pan-neuronal (aex-3p and F25B3.3p) promoters were illuminated with green light at varying intensities. Animals with nc3034Ex[F25B3.3- p::Arch::gfp] (squares) and nc3026Ex[aex-3p::Arch::gfp] (diamonds) exhib- ited higher responsiveness at lower light intensities than nc3031Ex[myo- 3p::Arch::gfp] (triangles) animals did. (mean6SEM; n = 3. Five animals were examined for each trial.). doi:10.1371/journal.pone.0035370.g002 suggesting that the GABAergic system might play a role in switching promptly the direction of movement. We also examined the effect of silencing of GABAergic neurons in the background of Roller phenotype in ncEx2311[unc-47p::Arch::gfp; rol-6d] animals. Green light illumination retarded or arrested the locomotion, which lasted over 60 seconds of illumination (Fig. S2). Slow locomotion and defective responses to gentle touch are the phenotypes observed in mutants with defects in GABA neurons (Fig. 3B, C). A previous study also showed that killing the four GABAergic RME neurons in the head of wild type worms resulted in an abnormal loopy foraging behavior [14,18]. Thus, green light illumination to ncEx2351 animals appears to lead to phenotypes expected from silencing GABAergic neurons. Next, to achieve silencing of A-type motor neurons specifically, Arch::GFP was expressed under the control of the promoter of the unc-4 gene [19,20] (Fig. S1B). When illuminated with green light, forward movement of freely moving ncEx3068[unc-4p::Arch::gfp] animals was normal, but backward movement was abnormal: Whereas normal worms move backward a distance of about one body length, ncEx3068[unc-4p::Arch::gfp] animals often moved backward only as far as a half the body length, and sometimes did not move at all (Fig. 3B). Under green light illumination, anterior gentle touch to ncEx3068[unc-4p::Arch::gfp] worms did not elicit backward movement, or did it only incompletely (Fig. 3C). In contrast, the posterior touch elicited normal forward movement. Sometimes worms stopped moving for a while after a harsh touch. To achieve silencing of B-type motor neurons specifically, the promoter of the acr-5 gene was used [20] (Fig. S1C). When illuminated with green light, forward movement of ncEx2371[acr- 5p::Arch::gfp] animals slowed down, and about half of the animals stopped locomotion completely within 10 seconds (Fig. 3B). The locomotion was arrested under continuous illumination over 20 minutes. The posterior half of the body was often motionless, while the anterior part of the body connected to the head moved. Backward movement was also affected, though to a lesser extent, and it often stopped halfway. Posterior gentle touch to ncEx2371[acr- 5p::Arch::gfp] animals under green light illumination failed to elicit forward movement almost completely (Fig. 3C). Although in most cases animals responded to anterior gentle touch, they retreated a shorter distance and their movement was loopy. When ncEx2365[del-1p::Arch::gfp] animals, which expressed Arch::GFP in VA and VB motor neurons under the control of the del-1 gene promoter [20,21] (Fig. S1D), were illuminated with Optical Silencing of C. elegans Cells with Arch green light, locomotion appeared to slow down in either direction (Fig. 3B). Response to gentle touch was sometimes affected in either direction (Fig. 3C). Backward movement was often incomplete. Arch::GFP Expressed by Heat-shock Promoter Next, to investigate the timing of the acquisition of the silencing function and the stability of Arch following its expression in C. elegans cells, we exploited a gene induction system using the promoter of the heat-shock protein gene hsp-16.2 [22]. In ncEx3003[hsp-16.2p::Arch::gfp] animals, GFP expression was detected 3 hours after heat-shock. The level of GFP expression then increased gradually, reached a stable plateau 9–12 hours after heat shock, remained constant for as long as 24 hours and started to decay slowly thereafter. The level of GFP expression in most animals 48 hours after heat shock was lower compared with the level 24 hours after heat shock, but higher compared with the level 3 hours after heat shock. In some animals, GFP expression was detected even 72 hours after heat shock. The GFP signal was observed in the entire region of the body (Fig. 1G). In neurons, the GFP signal was localized to the cell membrane and in the cytoplasm, but not in the nucleus (Fig. 1H). In muscle cells, the GFP signal was high at the cell surface (Fig. 1I). An intense GFP signal was sometimes detected in granular or vesicular structures in the cytoplasm. To help clarify the localization of Arch in cells, we compared Arch expression with the GFP signals in ncIs17[hsp-16.2p::gfp] transgenic worms, which only express GFP under the control of the same heat-shock promoter [23]. The GFP signal in ncIs17[hsp-16.2p::gfp] worms was distributed diffusely in the cytoplasm, without visualizing granular or vesicular structures or demarcating cell boundaries. These differences further support the conclusion that Arch::GFP is localized to intracellular vesicles and the cell membrane. Next, we investigated the function of Arch expressed by heat shock (Fig. 4A(i), B). Three hours after heat shock, green light illumination at 19.6 mW/mm2 had no effect on the locomotion of heat-shock-induced ncEx3003[hsp-16.2p::Arch::GFP] worms, al- though a GFP signal was clearly observed. Six hours after heat shock, 5% of the animals stopped locomotion in response to green light illumination. In response to green light illumination, half of the animals stopped locomotion 9 hours after heat shock, and 90% of the animals stopped 12 hours after heat-shock. Up to 48 hours after heat shock, green light illumination still caused locomotion arrest in most animals. Some animals stopped locomotion in response to green light illumination even 72 hours after heat shock, although the fraction of paralyzed worms (20%, n = 10) was much smaller than the fraction 48 hours after heat shock. To summarize the temporal changes of the Arch silencing function and expression, the worms became fully responsive to green light illumination 12 hours after heat shock, while Arch expression monitored with GFP reached the maximal level 9–12 hours after heat shock. Thereafter, the responsiveness of worms to green light illumination appeared to correlate with the expression level of Arch::GFP, which sometimes persisted for longer than 48 hours. Since heat-shock-mediated expression of GFP is induced only temporarily, the long-lasting perdurance of GFP expression, as well as the responsiveness to light observed in some worms, indicate that Arch can be stably retained in a functional form for a long period. We also investigated the light sensitivity of Ex3003[hsp- 16.2p::Arch::GFP] worms 24 hours after heat shock. A small fraction of animals stopped locomotion when illuminated at 2.2 mW/mm2, half of the animals stopped at 4.9 mW/mm2, and 2 all the animals stopped when illuminated at 11.1 mW/mm . PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e35370