Schema of the Spicule Insertion Circuit Figure 6. Stimulation of ChR2 in different spicule circuit components. (A) Left panel, a male that expresses Pgar-3:ChR2::YFP holds his spicules inside of his tail prior to illumination. Right panel, the blue light induces sustained spicule protraction of this male. (B) Sustained spicule protraction induced by stimulation of ChR2 in different circuit components. The numbers on the vertical axis represent the percentage of males that protracted their spicules when different circuit components were activated by light-stimulated ChR2. The numbers above the bars show the percentage of males that protracted their spicules. The number of males assayed for each strain is listed within the bar. Asterisks (***) indicate the p value ,0.0001, (**) indicate the p value ,0.001, using the Fisher’s exact test. doi:10.1371/journal.pgen.1001326.g006 retinal did not protract their spicules under the blue light (n = 20). This suggests that in wild-type males, the UNC-103 K+ channel functions on the spicule muscles to counteract the ChR2-mediated depolarization. The transition between protractor contractile states is presumed to be facilitated by the cholinergic SPC neurons. These neurons have proprioceptive sensory endings that are attached to the protractor muscles and are suggested to sense the spicule position in the vulval channel. Removal of these cells results in operated males that rhythmically prod the vulva with their spicules, but never completely insert them during mating [2,22]. Since these neurons make synapses to the muscles, their secretions might be sufficient to evoke tonic muscle contraction. Alternatively, the ACh signals from the SPC neurons might need to act in conjunction with the rhythmically contracting protractors to induce the state change in the muscles. To test these hypotheses, we used the promoter region of the gar-3 gene (Pgar-3B:ChR2::YFP) to express ChR2 in the SPC neurons and two of the p.c.s. neurons (PCA and PCB), in addition to the spicule protractors and the anal depressor [26]. 75% of the males responded to the fluorescent light with sustained spicule protraction (n = 64, Figure 6A and 6B; Video S11). To verify that this was due to SPC neuron activity, we laser ablated these cells, and found that blue light only caused sustained protraction in 20% of the males (n = 20, Figure 6B). In addition, ablation of the PCA and PCB neurons did not affect the light-induced behavior (n = 23, Figure 6B), presumably since the spicule muscles were already stimulated via Pgar-3B-expressing ChR2 and did not require further p.c.s. signaling. If the p.c.s. neurons induce rhythmic spicule protractor contractions, and activation of the SPC neurons switches rhythmic contractions to tonic contraction, then artificially activating these neurons independent of the spicule muscles should cause sustained spicule protraction. To test this, we used one of the unc-103 gene promoter regions (Punc-103F) to express ChR2 in the PCA, PCB and SPC neurons [44]. Only 19.4% of males, the ones that highly PLoS Genetics | www.plosgenetics.org 11 March 2011 | Volume 7 | Issue 3 | e1001326