Optical Silencing of C. elegans Cells with Arch Figure 1. Expression of Arch::GFP in C. elegans driven by various promoters. (A) A fluorescent micrograph of an nc3031Ex[myo-3p::Arch::gfp] animal. Arch::GFP is expressed in longitudinal bands composed of body wall muscles (arrow). (B) An enlarged view of body wall muscle cells. GFP signal is observed along the outline of muscle cells (arrow). Vesicular structures visualized with GFP are localized close to the cell membrane (arrow head). Weak GFP signal is detected in the cytoplasm (asterisk). (C) Expression of Arch::GFP in an nc3034Ex[F25B3.3p::Arch::gfp] animal. Arch::GFP is expressed in head neurons (arrow head), tail neurons (open arrow head) and the ventral nerve cord (arrow). (D) The head of an animal carrying F25B3.3p::Arch::gfp. Arch::GFP is expressed in the axon (arrow) and the cell body (arrow head) of head neurons. (E) Expression of Arch::GFP in an nc3026Ex[aex-3p::Arch::gfp] animal. Arch::GFP is expressed in head neurons (arrow), tail neurons (arrow head), and the ventral nerve cord (open arrow head). (F) The head of an animal carrying aex-3p::Arch::gfp. Arch::GFP is expressed in the axon of head neurons in a punctured pattern (arrow). GFP is seen on the cell membrane of a cell body (arrow head). (G) Expression of Arch::GFP in an nc3003Ex[hsp-16.2p::Arch::gfp] animal. Arch::GFP is expressed everywhere in the body. (H) The head of an animal carrying hsp-16.2p:: Arch::gfp. Arch::GFP is expressed in neurons (arrow). (I) Body wall muscle of an hsp-16.2p::Arch::gfp animal. GFP is clearly localized at the cell membrane (arrow). Scale bar: A, C, E, G = 100 mm; B, D, F, H I = 10 mm. Anterior is toward the right except for (G). doi:10.1371/journal.pone.0035370.g001 backward movements. Worms can also be directed to move backward or forward by tactile stimulation. We have examined how locomotory behaviors are affected by silencing different subsets of motor neurons in the ventral nerve cord. The locomotion is executed by body wall muscles whose contraction and relaxation is regulated by the cholinergic A- and B-type motor neurons, and the GABAergic D-type motor neurons, respectively [13,14,15]. Previous studies also showed that A-type motor neurons (VA, DA) direct backward locomotion while B-type motor neurons (VB, DB) direct forward locomotion [13,16]. First, to achieve silencing of D-type motor neurons (VD, DD) selectively, Arch::GFP was expressed under the control of the promoter of the unc-47 gene encoding a vesicular GABA transporter [17] (Fig. S1A). Without illumination, ncEx2351[unc- 47p::Arch::gfp] animals moved in a normal sinusoidal motion. When ncEx2351 animals moving freely were illuminated with green light, they exhibited loopy sinusoidal forward movement (Fig. 3A), and movement of some animals appeared to slow down in either direction. In wild type animals, gentle touch to the anterior part of the body elicits backward movement, whereas posterior touch elicits forward movement. Under green light illumination, the extent of backward movement elicited by gentle anterior touch was smaller in ncEx2351 worms (Fig. 3C). Likewise, forward movement elicited by gentle posterior touch was affected to various extents. We observed that the onset of touch elicited movement was delayed about 1 second in 2 out of 30 animals, PLoS ONE | www.plosone.org 3 May 2012 | Volume 7 | Issue 5 | e35370