Optical Silencing of C. elegans Cells with Arch all neurons of an ncEx3034 [F25B3.3p::Arch::gfp] animal or an ncEx3026 [aex-3p::Arch::gfp] animal. incubator at 20uC, and before the next observation, plates were left for 30 minutes at 25uC. Heat-shock Induction of Arch All worms tested were F1 roller progeny of P0 roller adults picked onto NGM plates with or without ATR three days before experiments. Late L4 larvae were transferred to NGM plates with or without ATR. The culture plates containing worms were placed in a bacteria culture incubator at 37uC for 30 minutes, and then were returned to the worm incubator at 20uC. The room was maintained at 25uC. Microscopic Observation of GFP-expressing Worms For observation of GFP fluorescence, a fluorescence stereo microscope SZX12 (Olympus, Tokyo, Japan) with a 100W HBO mercury lamp (Osram, Munich, Germany), and a fluorescence microscope Axioplan 2 (Zeiss, Oberkochen, Germany) with filter set #10 for GFP with a 100W HBO mercury lamp (Osram, Munich, Germany) were used. Images were acquired with a digital CCD camera Cool SNAP HQ2 (Photometrics, Tucson, AZ, USA) using PM Capture Pro software (Nippon Roper, Japan), and processed using ImageJ public domain software. Locomotion Assay Under Illumination For locomotion assays to examine dependence on light intensity, 5 animals were transferred to a new 30mm NGM plate supplemented with ATR. Worms exhibiting the Roller phenotype or expressing mCherry- in the pharynx were chosen as transgenic animals. With strains in which the Arch::GFP expression was driven by tissue-specific promoters, animals with intense GFP signals were further selected for the assay. Animals were observed under a fluorescence stereo microscope (SZX12), and were illuminated with a 100W HBO mercury lamp through an excitation filter for Red Fluorescent Protein DsRed (545610 nm, Olympus, Tokyo, Japan). Images of moving animals were recorded using a digital video camera G2-HM570B (JVC, Yokohama, Japan) mounted on the microscope. In most experiments, worms were used only once for a single series of illumination experiments with varying light 2 intensities (from 0.1 to 17 mW/mm ), and were then discarded unless otherwise indicated. For locomotion assays of animals expressing Arch::GFP in motor neuron subsets, animals were observed under a fluorescence stereo microscope (MVX10), and were illuminated with a 100W HBO mercury lamp through the filter set U-MRFPHQ (Olympus, Tokyo, Japan) with the light intensity of 14.7 mW/mm2 at 550 nm. Locomotory defects of freely moving animals were scored through observation of each animal for 5 min. At least 30 animals were examined for each transgenic line. The response to gentle touch was assayed as described by Hart [31]. Briefly, for examining worm’s responses to gentle touch, animals were touched by stroking a hair across the worm’s body at different positions: just behind the pharynx for anterior touch, and just anterior to the anus for posterior touch. For examining the response to harsh touch, animals were prodded with a platinum wire. At least 30 animals were examined for each transgenic line, and each animal was examined 5 times. In time course analyses of silencing activity of ncEx3003 after heat-shock induction, animals were used only once for each time point. In experiments comparing the time course for heat-shock- induced Arch::GFP in ncEx3002, ncEx3003 and ncEx3004 animals, and Arch in ncEx3039, ncEx3040 and ncEx3041 animals, worms were used for a single series of illumination experiments at various time points (3,6,9,12,24 and 48 hours after heat shock). After observation at each time point, plates were returned to the worm Measurement of Light Intensity Light intensity at 550 nm, corresponding to the Arch absorption maximum, was measured at the object plane using an optical power meter (#3664, Hioki, Ueda, Japan) with an optical sensor (#9742, Hioki, Ueda, Japan). Various intensities of illumination were implemented by changing the magnification of the objective lens of the microscope. To calculate the intensity of illumination, we measured the radius of the illuminated area, calculated the area, and then divided the total light intensity by the area. Supporting Information Figure S1 Expression of Arch::GFP in C. elegans driven by various promoters. (A) A fluorescent micrograph of an nc2351Ex[unc-47p::Arch::gfp] animal. Arch::GFP is expressed in D- type motor neurons (VD, DD) (arrow). (B) Expression of Arch::GFP in an nc3068Ex[unc-4p::Arch::gfp] animal. Arch::GFP is expressed in A-type motor neurons (arrow). (C) Expression of Arch::GFP in an nc2371Ex[acr-5p::Arch::gfp] animal. Arch::GFP is expressed in B-type motor neurons (arrow). (D) Expression of Arch::GFP in an nc2365Ex[del-1p::Arch::gfp] animal. Arch::GFP is expressed in VA and VB motor neurons (arrow). The fluorescence of mCherry expressed in the pharynx is also detected. Scale bar = 100 mm. Anterior is toward the right and dorsal is up. (TIF) Figure S2 Defects in the locomotory behavior caused by silencing subsets of motor neurons in strains carrying the rol-6d gene. (A-C) Locomotory behavior of worms expressing Arch in motor neurons subsets. Arch::GFP was expressed in D-type (VD, DD) and VA +VB motor neurons in ncEx2311[unc-47p::Arch::gfp; rol-6d] and ncEx2322 [del-1p::Arch::gfp; rol-6d] animals exhibiting the Roller phenotype. To evaluate differences of animal’s behavior statistically, we used Fisher’s exact test for the locomotion assay of freely moving animals (A), and Student’s t test for the touch response assay (B, C). For all transgenic strains, animals behaviors under green light illumination (ON = open box) and those without illumination (OFF = filled box) differed significantly (p,0.001). Error bars indicate 6SEM. (A) Forward (F) and backward movement (B) was scored in worms moving freely. Percentage of animals exhibiting the ‘‘Class 3 (severe)’’, ‘‘Class2 (mild)’’ and ‘‘Class1 (no)’’ phenotype in locomotory behaviors when they were illuminated with green light is shown. (B) Forward movement (F) to gentle posterior touch and backward (B) movement to gentle anterior touch were scored. Responses out of five touches are shown. (C) Forward (F) and backward movement (B) to harsh touch was scored, and was shown additively in each bar. Responses out of five touches are shown. (TIF) Acknowledgments The authors would like to thank Yoichi Oda and members of the Oda Laboratory for their discussions, comments and encouragement; Hirohito Kuroyanagi for supplying pDEST-myo-3p, pDEST-F25B3.3p, pDEST-aex- 3p, pDEST-hsp-16.2p, pDEST-unc-4p and pDEST-PL plasmids; Andrew Fire for the pPD49.78 plasmid; and Kotaro Kimura, Motoshi Suzuki and Hiroki Tanaka for helpful advice. Author Contributions Conceived and designed the experiments: AO YS ST. Performed the experiments: AO. Analyzed the data: AO ST. Contributed reagents/ materials/analysis tools: YS ST. Wrote the paper: AO ST. PLoS ONE | www.plosone.org 10 May 2012 | Volume 7 | Issue 5 | e35370