oblique muscles (Figure 3B, 3C; Male Wiring Project, Albert Einstein College of Medicine, http://worms.aecom.yu.edu/ pages/male_wiring_project.htm). The PCB and PCC neurons are cholinergic [22]. The neurotransmitter assignment for PCA has not yet been determined, but the cell can express a reporter construct for the eat-4 vesicular glutamate transporter, suggesting it might secrete glutamate (Garcia, unpublished observation). Ablation of the oblique muscles or reduction in ACR-16 and UNC-29-mediated muscular cholinergic signaling causes males to behave similarly to those that have only two pairs of p.c.s. neurons. The oblique muscles are located slightly posterior to the cloacal opening, and their single sarcomeres, oriented dorsal-ventrally, are attached to the lateral body wall [25]. When they contract, the posterior tail region curls ventrally. This modification in tail curvature might redirect the force, which the male applies laterally to the hermaphrodite cuticle during backing behavior, to a downward force immediately upon vulval contact (Figure 3C). We suggest that this would stabilize the male at the vulva during his repetitive attempts to insert his spicules. The same cells and molecules that stabilize the male at the vulva also promote the insertion of the copulatory spicules. High frequency and sustained muscle contractions of the spicule- associated muscles can be stimulated by acetylcholine (ACh) secretions from the postcloacal sensilla PCB, PCC neurons and the SPC neurons, respectively. It was hypothesized in earlier studies that the cholinergic PCB, PCC and SPC neurons differentially regulate the two types of protractor contractions via secreting different amounts or rates of ACh to activate ionotropic and muscarinic ACh receptors on the protractors [22]. The SPC neurons were known to innervate the protractors [25]. However, electron micrograph reconstruction of the male tail (Male Wiring Project) indicates that the p.c.s. neurons do not form chemical synapses to the spicule muscles. Instead, the p.c.s. neurons make multiple chemical connections to each other, to the SPC neurons and to the oblique-gubernacular muscle group (Figure 3B). Communication between the p.c.s. neurons and the protractors are relayed through electrical junctions between the gubernacular muscles and the protractor muscles, mainly through an interme- diate accessory spicule muscle called the anal depressor (Figure 3B; Male Wiring Project). In adult C. elegans, gap junctions are distributed extensively throughout the nervous system. Based on serial transmission electronic microscopy images and innexin expression patterns, the gap junctions are not limited between neurons or between muscles, but also connect neuron and muscle cells, where the processes penetrate the basement membrane (Male Wiring Project) [25,55,71]. These electrical connections probably play important roles in regulating animal behaviors. The worm gap junctions have been reported to mediate Ca2+ transient propagation throughout the intestinal cells and body wall muscles. A few studies also indicate their role in synchronizing neuron activities [49,59,72,73]. Artificially stimulating the oblique and gubernacular muscles can instantaneously induce Ca2+ transients and subsequent contractions in the protractor muscles. Thus, by utilizing gap junctions in the circuit, the p.c.s. neurons can coordinate both vulval contact and spicule penetration attempts simultaneously. Although connected by gap junctions, different motor outputs are generated by the gubernacular-oblique muscle group and the protractor muscles. While males rhythmically prod their spicules at the vulva, they maintain a stable tail curvature. This suggests that the oblique muscles do not undergo high frequency contractions like the protractors. Direct p.c.s. stimulation of the oblique- gubernacular muscle group causes tonic contraction in these muscles. But signals transmitted from the oblique-gubernacular Schema of the Spicule Insertion Circuit muscle group to the spicule muscles through gap junctions facilitate rhythmic contractions of the protractors (Figure 3B, 3C). This provides a scenario that unpatterned signal input elicits patterned motor output, and this process requires intracellular regenerative Ca2+ transients through the UNC-68 ryanodine receptor channel [22]. The pattern generating mechanism could either be present in the protractor muscles themselves, by utilizing intracellular oscillating Ca2+ transients; or it can be a result of the interplay between contractions of the protractor and the retractor muscles, since the spicules are attached by both types of muscles that cause opposite directions of spicule movements. The core spicule circuit (the p.c.s. neurons, the gubernacular- oblique muscle group, the SPC neurons, the anal depressor muscle and the spicule protractor muscles) is connected to other parts of the nervous system via extensive synapses and gap junctions (Male Wiring Project). Thus, it is likely that activity in other behavioral circuits can cause non-specific activity (noise) in the spicule circuit. Therefore, the circuit must contain contingency mechanisms to reduce the males from fully protracting their spicules into non- vulval orifices or to random environmental stimuli. The light-gated channelrhodopsin (ChR2) was used to activate different compo- nents of the core spicule circuit. Exciting cells via ChR2 is not equivalent to natural activation caused by ligand stimulation, synaptic transmission or electrical junctions in behaving animals, but these experiments provide an idea on how activated circuit components promote differential behavioral outputs. At least three inputs must be integrated by components of the core spicule circuit for the protractors to contract completely with the highest probability via ChR2 stimulation: removal of the negative input from cells derived from the progenitor cell P10.p, possibly from the hook sensillum neurons HOA and HOB; stimulation of rhythmic contractions in the protractors via the p.c.s. sensory-motor neurons; and the addition of positive input from the SPC proprioceptive sensory-motor neurons. During mating, contact with vulval tissue would simultaneously stimulate the p.c.s. neurons and release negative regulation from the P10.p descendants. Finally, partial spicule penetration would trigger the SPC sensory-motor neurons, through their proprioceptive sensory endings, to secrete ACh onto the rhythmically contracting protractor muscles. The ACh signals from the SPC neurons could act additively with the intracellular regenerative Ca2+ transients to transform the oscillating contractions into a sustained one. The final motor output is the result of integrating the activities of different components in the behavioral circuit. A similar observation has been reported in other reproductive behavior systems, such as in the leech, where the oscillation frequencies of ganglia M5 and M6 are integrated to determine the rhythmic twisting behavior performed during mating [74]. We propose that the circuit uses the ERG-like K+ channel UNC-103 to set up a high threshold for stimulating the circuit, so it responds maximally when all inputs are detected ‘‘coincidently’’. The ‘‘coincidence detector’’ mechanism collapses when UNC-103 is removed, which causes the circuit to respond to stimuli that are not normally sufficient to induce spicule protraction in normal animals. This explains why unc-103 mutant males spontane- ously display a fictive mating behavior in the absence of hermaphrodites and prematurely protract their spicules at the hermaphrodite’s vulva before they partially breach the vulval slit [60]. Similar mechanisms are used in nervous systems of other organisms. For example in vertebrates, the low excitability of the striatal neurons maintained by K+ channels has been implied to play an important role in preventing unintended locomotion conditions [75]. The central pattern generators in the spinal cord generate the locomotion. These networks remain inhibited, until PLoS Genetics | www.plosgenetics.org 13 March 2011 | Volume 7 | Issue 3 | e1001326