Author Summary Animal behaviors are generated when a sequence of muscle movements is coordinated by neural circuits. In complex invertebrates or lab-studied vertebrates, due to the large number of cells in their nervous systems and the complexities of their behaviors, it is difficult to address how circuits process information to direct each step of behavior. Here, we used C. elegans male mating behavior as a model to address how a compact circuit coordinates different behavioral programs. Male copulation is a multi- step innate behavior, where the male senses his mate, searches for her vulva, then maintains contact with it as he repetitively attempts to insert his copulatory spicules. We demonstrate that the cloacal neurons sense the vulval signal, then command the posterior muscles to contract, so that the male tail sustains contact with the vulva. Simultaneously, via low-resistance electrical connections, the activities of the posterior muscles are relayed to the spicule muscles to cause repetitive spicule insertion attempts. This allows two different behavior patterns to be coordinated. Rhythmic spicule insertion attempts cease and full spicule penetration is induced when multiple contingencies are detected by distinct circuit components. the protractors, initiated by the postcloacal sensilla neurons PCA, PCB and PCC, cause the spicules to thrust repeatedly against the vulval slit. Tonic contraction of the protractors causes the spicules to extend fully from the male tail and to penetrate the vulva. This step requires the activity of the cholinergic SPC neurons, which innervate the protractors [2,22,25]. Although copulation is initiated by the postcloacal and SPC sensory-motor neurons, it remains elusive how these cells generate and coordinate each step of the behavior. Understanding the functional cellular/molecular architecture that drives circuit output is a necessity before one can appreciate why certain genes are or are not the evolutionary targets that underlie the diversity of sexual behavior and reproduction. In this study, we addressed how the C. elegans male spicule protraction circuit generates and coordinates different motor outputs to penetrate the hermaphrodite’s vulva. We determined that stimulation of distinct acetylcholine receptors in posterior sex muscles is used to maintain the male’s position over the vulva. This information is simultaneously relayed to the spicule protractors through electrical junctions, thus providing a mechanism for coupling spicule insertion and vulva location behavior. In addition, we identified multiple sensory inputs that control when the protractors should transit from rhythmic to sustained contraction. Results Males require cholinergic signaling to maintain contact with the hermaphrodite’s vulva during mating During their attempts to inseminate the hermaphrodite, C. elegans males must maintain precise contact between their cloacal opening and the vulva. In our previous study, we observed that males lacking the muscarinic acetylcholine receptor (mAChR) GAR-3 do not sustain their position over the vulva during prolonged attempts to insert their spicules. This mAChR is expressed in the cholinergic spicule-related neurons, PCB and SPC, to enhance ACh secretion from these neurons [26]. ACh secreted from the SPC neurons activates ionotropic ACh receptors on the spicule protractor muscles to induce muscle contraction, which results in tonic spicule protraction [22]. We speculated that Schema of the Spicule Insertion Circuit the ionotropic ACh receptors in the cells of the spicule circuit can also be used to sustain precise vulval contact behavior; therefore in the gar-3 deletion mutant, reduced cholinergic synaptic transmis- sion between the spicule-related neurons and the sex muscles resulted in defective vulval contact. To address whether ionotropic ACh receptors are utilized to regulate precise vulval contact, we first asked how effectively males that lack the functional levamisole (LEV)-sensitive receptor (L- AChR) mated. The L-AChR had been the only ionotropic ACh receptor described to be involved in ACh agonist-induced spicule protraction. This receptor contains three a-subunits: UNC-38, UNC-63 and LEV-8, and two non-a subunits: UNC-29 and LEV- 1 [22,27–30]. We used mutants carrying the unc-29(e193) allele, which has a missense mutation that changes a proline to serine at amino acid 258 within the hypothetic first transmembrane domain of the channel [31–33]. LEV induces spicule protraction via activating functional UNC-29-containing L-AChRs, and the effective concentration that induces prolonged protraction for 90% of the males is 2 mM [22]. unc-29(e193) males were resistant to LEV. We observed that none of the mutant males protracted their spicules in 2 mM LEV (n = 47), compared to 87.7% of the wild-type males (n = 57, p,0.0001). Like other unc-29 loss-of- function (lf) alleles, the e193 allele causes worms to have severe uncoordinated locomotion [29,32], which disrupts the males’ ability to move backwards along the hermaphrodite during mating. We restored locomotion to the mutant males by using the upstream region of the acr-8 gene to drive unc-29 cDNA expression (Pacr-8:unc-29cDNA::SL2::GFP) in the body wall muscles, but not in the male sex muscles [34]. Similar to the unc-29(lf) mutant males, the transgenic males were also resistant to LEV; 6% of them protracted their spicules in 2 mM LEV (n = 35, p = 0.18). We assayed the mating potency of the locomotion- restored unc-29(lf) males by pairing each male with a moving hermaphrodite for 4 hours, and observed that these males could sire progeny with 83.3% of wild-type efficiency (n = 38, Figure 1A). This indicates that even though the L-AChR is used to facilitate spicule protraction in response to LEV, there are other receptors compensating for its function during male mating. Next, we asked which other ionotropic ACh receptors could be functioning redundantly with the L-AChR. One possibility was the homomeric receptor formed by the a7-like nicotinic ACh receptor (nAChR) subunit, ACR-16. Since it was previously shown to have an ancillary function with the L-AChR in the C. elegans locomotion circuit [30,35], we asked if ACR-16 was also used during male mating behavior. After 4 hours of contact with hermaphrodites, males that contained the acr-16(ok789) deletion (acr-16(0)) achieved 88% of wild-type mating potency (n = 41, Figure 1A), suggesting that like the L-AChR, ACR-16 is not essential for male potency. We reasoned that if the L-AChR and the ACR-16-containing nAChR were required for mating but were interchangeable, then a severe mating defect would be seen in the unc-29(lf);acr-16(0) double mutant. The unc-29(lf);acr-16(0) males are completely paralyzed. Since the acr-16(0) allele alone does not cause any gross locomotion defect [30,35], we only needed to restore the impaired locomotion of the double mutant using the construct Pacr-8:unc- 29cDNA::SL2::GFP. We quantified the velocity of backward locomotion during vulva search behavior and found that the transgenic double mutant males moved relatively well, albeit slightly slower than wild-type males; 105633 mm/sec for wild type (n = 5 males) and 71635 mm/sec for the double mutant (n = 5 males) (Figure S1). In our mating potency assay, we found that the transgenic unc-29(lf);acr-16(0) males could only achieve 15% of wild-type mating potency (n = 33, p,0.001, Figure 1A). This suggests that both L-AChR and the ACR-16 nAChR are used in PLoS Genetics | www.plosgenetics.org 2 March 2011 | Volume 7 | Issue 3 | e1001326