the finding that Ex[hsp-16.2p::Arch::GFP] worms exhibited higher sensitivity to green light illumination than Ex[myo-3::Arch::GFP] worms suggest that locomotory paralysis in Ex[hsp-16.2p::Arch::GFP] worms is mainly attributed to the silencing of neurons rather than muscle cells. Arch-expressing C. elegans Responds to Light Precisely By employing various illumination patterns, we showed that worms expressing Arch respond to light quickly and precisely. This finding is consistent with a previous report showing that Arch- mediated neuronal silencing occurs within a millisecond time range [11]. We also showed that continuous illumination for as long as 30 seconds leads to sustained paralysis in most worms expressing Arch in neurons, which is also consistent with a previous finding that prolonged activation does not significantly affect Arch activity. Since Arch is an outward proton pump [11], prolonged activation of Arch may cause crucial changes in intra- and extracellular pH levels. The failure of Arch-expressing worms to immediately recovery locomotion after the cessation of illumination following prolonged illumination might be due to disturbances of cellular pH conditions. Nevertheless, the absence of long-lasting effects on neurons or muscle cells is notable, since worms eventually resumed locomotion at normal rates in the absence of illumination. Silencing of different subsets of neurons elicited distinct behavioral defects. To investigate the neuron-circuits governing the locomotory behavior of worms, we have used several promoters that drive expression of Arch in selected subsets of motor neurons of the ventral nerve cord. We found that worms expressing Arch by different promoters exhibited distinct behavioral phenotypes specific for each promoter when illuminated with green light. Responses of worms expressing Arch in GABAergic neurons including D-type motor neurons generally mimic the phenotype of genetic mutants defective in functioning of GABAergic neurons, supporting the notion that specific silencing of GABAergic neurons is achieved successfully. We have, however, observed several differences in the effects between acute silencing and chronic inactivation in mutants. Under a freely moving condition, animals expressing Arch in GABAergic neurons moved more actively than unc-47 mutants. In unc-47 mutants, movement of the head and tail are usually repressed, while green light-mediated silencing of GABAergic neurons elicited enhanced head move- ment, which resulted in loopy forward movement. We also noticed that light-mediated silencing of GABAergic neurons affected touch responses more strongly than a mutation in the unc-47 gene. The differences may reflect in part incomplete silencing of the GABAergic system due to the mosaic expression of Arch, which is characteristic for transgenes carried by an extrachromosomal array. It may be also due to the properties of the particular mutant allele used in this study. Alternatively, although the finding that acute silencing and chronic inactivation generally has similar effects suggests the absence of developmental mechanisms acting to compensate for locomotory defects of the mutants, neuron- circuits relevant to certain behaviors might be modified through chronic inactivation of GABAergic neurons. We found that silencing subsets of cholinergic motor neurons had neuron subset-specific effects. At hatching, only three types of motor neurons are present, DA, DB and DD. A classic study using the laser ablation method showed that killing most of DA in newly hatched larvae resulted in animals that could not move backward but could move forward normally, whereas killing most of DB neurons resulted in defective forward movement without affecting backward movement [16]. Light-mediated activation of Arch Optical Silencing of C. elegans Cells with Arch expressed in DA and VA motor neurons by using the unc-4 promoter caused the direction-specific defect in locomotion, confirming the essential role of A-type neurons in backward movement. The results of the Arch expression experiment using the arc-5 promoter, which drives gene expression in DB and VB motor neurons, but not in A-type motor neurons, also confirmed the essential role of B-type motor neurons in forward movement. Interestingly, we have found that backward movement was often performed incompletely by silencing B-type neurons. While B-type neurons may not play a significant role in triggering backward movement, they might be critically required during the later phase of backward movement. By silencing both VA and VB neurons in ncEx2365[del-1p::Arch::gfp] animals, we found that movement in either direction was affected. Accordingly, a previous study showed that mec-4(gf)-mediated genetic cell ablation under the control of the del-1 promoter resulted in uncoordinated movement, in which either the forward or backward was irregular and appeared to be executed with difficulty [21]. To summarize, behavioral phenotypes elicited by silencing each subset of motor neurons are generally consistent with known functions of the corresponding neuron subset. It should be noted that we have used the transgenes carried by extrachromosomal arrays, which lead to mosaic expression of Arch among target neurons, indicating that complete silencing of a targeted neuron subset would result in a stronger and clearer phenotype. It is also noted that the behavioral changes were observed throughout the period of continuous illumination, which was sometimes longer than 20 minutes, further confirming the utility of Arch as an optogenetic tool in C. elegans. Time Lag Between Induction and Functioning of Arch The induction of Arch by heat shock seems to occur only transiently. It was reported that the level of hsp16-2 mRNA reached a maximum 1 hour after onset of heat shock, and decreased to the normal level within 2 hours after heat shock was terminated [22]. We found that the expression level of Arch::GPF did not increase markedly later than 9 hours after heat shock, indicating that synthesis of Arch persisted only for a limited period. This transient induction system enabled us to reveal hitherto unnoticed properties of Arch in vivo. We found that Arch remains functional for a relatively long time in C. elegans cells, sometimes for several days, which is one advantage that Arch has as an experimental tool. An unexpected finding is that Arch becomes fully functional only 12 hours after its induction. We also found that the expression of Arch::GPF first detected 3 hours after heat shock and the expression level increased up to 9 hours after heat shock. This relatively slow onset and gradual increase in Arch::GPF expression is likely to account for a major part of the observed delay in acquiring a silencing function after Arch induction. It is, however, possible that other processes, such as translocation to the cell membrane and molecular modification, may also partly contribute to the delay. To affect membrane potential effectively, Arch must be recruited to the cell membrane. We found that Arch::GFP is localized to the cell membrane, but a very intense GFP signal also occurs in intracellular vesicular structures. Translocation of Arch from such vesicles to the plasma membrane might be involved in creating the delay in acquiring the silencing function. Although the GFP segment, by promoting cytoplasmic localization, might affect incorporation of tagged-proteins into the plasma membrane, we found that the removal of GFP from Arch::GFP did not drastically alter either the time course or the dependence on light intensity concerning the responsiveness of Arch-induced worms. PLoS ONE | www.plosone.org 8 May 2012 | Volume 7 | Issue 5 | e35370