intestinal cells. Adjacent cells that are connected by gap junctions showed Ca2+ spikes that were not complete synchronous [49]. We therefore suggest that activities of the gubernacular-oblique muscle group, initiated by the p.c.s. neurons, can be transmitted to the spicule protractor muscles directly, probably through gap junctions. Gap junctions are required for information transmission from the gubernacular-oblique muscle group to the protractor muscles To determine if gap junctions mediate the activity transmission between the gubernacular-oblique muscles and the protractor-anal depressor muscles, we asked if the application of a gap junction inhibitor could affect Ca2+ transients in the protractor-anal depressor muscles during gubernacular-oblique muscles stimula- tion. Carbenoxolone (CBX) has been shown to inhibit innexin function in invertebrates [50-52]. We found that application of this drug reduced the light-induced Ca2+ transients in the protractor- anal depressor muscles (Figure 5C; Figure S7). To the contrary, application of tubocurarine, which blocks both nicotine-sensitive and LEV-sensitive AChRs, [27,29,30,53,54] and cause the males to be paralyzed, did not affect either repetitive protractor contractions or Ca2+ transients in response to light stimulation (Figure 5C). Although it is possible that ChR2-stimulation of the gubernacular-oblique muscles could indirectly cause some non- specific neuron to secrete neurotransmitters other than ACh to induce Ca2+ transients in the spicule protractor-anal depressor muscles, the tubocurarine result in conjunction with the CBX experiment indicates that functional gap junctions, instead of cholinergic synaptic transmission, are required for activities of the gubernacular-oblique muscles to be relayed to the spicule protractor muscles. The C. elegans genome encodes 25 innexin proteins. To identify the innexin(s) that is important to the coupling of male sex muscles, we first determined innexin genes that are expressed in these muscles. A high-resolution expression map of all innexins has been reported in the C. elegans hermaphrodite, and five innexins are expressed in the hermaphrodite sex muscles: inx-3, inx-8, inx-9 inx-14 and unc-9 [55]. In addition, inx-11 was reported to be expressed in these muscles as well in another study [56]. The hermaphrodite sex muscles are derived from the M-lineage, same as the male sex muscles [57]; hence, it is possible that these genes are also expressed in the male sex muscles. We inspected these genes’ expression patterns in males by checking GFP expression under the control of their promoter regions [55]. We found that unc-9 and inx-14 are expressed in multiple male-specific sex muscles and the sexually dimorphic anal depressor muscle. In addition, unc-9 is also expressed in the SPC and the PCB neurons in the male tail (Figure S8). To test if these innexins are used to transmit signals among the male sex muscles, we asked if the light-induced rapid thrusts of the spicules are affected when these genes are mutated or knocked- down by RNA interference (RNAi). Males that carry the unc- 9(e101) loss-of-function allele have uncoordinated locomotion [58], probably due to inhibited electrical coupling among body wall muscles, as well as neuronal UNC-9 deficiency [59]. We found that when ChR2 was expressed in the gubernacular-oblique muscles in these males, in the presence of all-trans retinal, only 47.1% of the males responded to the blue light with repetitive spicule thrusts (n = 51, p,0.0001), compared to 100% of the wild type (n = 19; Figure 5F). To the contrary, light-induced spicule thrusts were not affected by RNAi of inx-3, inx-9, inx-11 or inx-14. In addition, RNAi of these genes in the unc-9(e101) genetic background did not result in further inhibition of the behavior (Figure 5F). In theory, the reduced spicule thrusts can be resulted Schema of the Spicule Insertion Circuit from a deficiency of UNC-9 in the SPC and PCB neurons. We addressed this possibility by first asking if these neurons are required for blue light-induced spicule thrusts. We reasoned that the PCB neurons do not make synapse or gap junction to the protractors; as a result, for the PCBs to mediate the ‘‘muscle- induced muscle contraction’’ phenomenon, they have to send signals through the SPC neurons, which innervate the protractors. Therefore, we can test whether the PCB and SPC neurons are required for the ‘‘muscle-induced muscle contraction’’ phenom- enon by asking if light-stimulated gubernacular-oblique muscle activity can still induce spicule thrusts when the SPC neurons are ablated. We found that ablation of the SPC neurons did not have an effect (Figure 5F), suggesting that neither pair of these neurons is required and therefore a deficiency of UNC-9 in these neurons is not likely to cause reduced light-induced spicule thrusts. Taken together, these data suggest that the UNC-9 innexin is used in some of the gap junctions that mediate the direct signal transmission from the gubernacular-oblique muscles to the protractor-anal depressor muscles. Identification of additional gap junction subunits awaits further study. The SPC neurons are necessary, but not sufficient to trigger sustained protractor contraction Repetitive protractor contractions eventually transform into a tonic contraction, which causes the spicules to penetrate the vulva. Premature tonic protractor contraction, prior to or during the various sub-steps of mating, rarely occurs in wild-type males [60], suggesting that the triggering of tonic protractor contraction is under strict regulation. We favored the hypothesis that multiple signals are integrated by the spicule circuit to induce sustained protractor contraction at the proper moment of mating. We used ChR2 to activate subsets of spicule circuit components, in males that were not engaged in mating [41,61-63], and then asked if these cells could induce sustained spicule protraction (Figure 6A). To our surprise, when we expressed ChR2 in all muscles, using the tropomyosin Plev-11 promoter (Plev- 11:ChR2::YFP), we observed that blue light induced strong body wall muscle contraction in all males, but only 5% of them displayed tonic contraction of the protractors. The remaining males responded to blue light with shallow repetitive protractor contractions with the tips of their spicules slightly protruding from the cloacal opening (n = 20, Figure 6B, Video S9). Consistent with this result, expressing ChR2 in sex muscles (the spicule protractors, the gubernacular erector and retractor muscles, the anterior and posterior oblique muscles, the sphincter and the diagonal muscles) and the sexually dimorphic anal depressor muscle, using the Punc- 103E promoter (Punc-103E:ChR2::YFP), also induced repetitive shallow protractor contractions. Approximately 18% of males fully protracted their spicules (n = 17, Figure 6B, Video S10). Since light-activated ChR2 channels should depolarize the muscle cells, we speculated that some negative regulatory molecules on the spicule muscles might be keeping them from contracting tonically. To address if spicule activity is controlled by negative regulator molecules, we introduced a allele of the unc-103 gene (n1213) into the Punc-103E:ChR2::YFP-expressing animals. The unc-103 gene encodes an ERG-like K+ channel. The unc-103 deletion males (unc-103(0)) occasionally protract their spicules in the absence of mating stimulation. Previous studies suggested that the K+ channel suppresses premature spicule protraction by reducing the excitability of the spicule muscles [44,60]. In unc- 103(0) males that expressed ChR2 on their sex muscles, blue light induced tonic spicule protraction in 93.8% of the population (n = 16, Figure 6B; see Materials and Methods). In contrast, the transgenic male population that grew in the absence of all-trans PLoS Genetics | www.plosgenetics.org 10 March 2011 | Volume 7 | Issue 3 | e1001326