L. Rene Garcia1,2* Abstract A Cholinergic-Regulated Circuit Coordinates the Maintenance and Bi-Stable States of a Sensory-Motor Behavior during Caenorhabditis elegans Male Copulation Yishi Liu1, Brigitte LeBeouf1,2, Xiaoyan Guo 1 , Paola A. Correa1, Daisy G. Gualberto1,2, Robyn Lints1, 1 Department of Biology, Texas A&M University, College Station, Texas, United States of America, 2 Howard Hughes Medical Institute, Texas A&M University, College Station, Texas, United States of America Penetration of a male copulatory organ into a suitable mate is a conserved and necessary behavioral step for most terrestrial matings; however, the detailed molecular and cellular mechanisms for this distinct social interaction have not been elucidated in any animal. During mating, the Caenorhabditis elegans male cloaca is maintained over the hermaphrodite’s vulva as he attempts to insert his copulatory spicules. Rhythmic spicule thrusts cease when insertion is sensed. Circuit components consisting of sensory/motor neurons and sex muscles for these steps have been previously identified, but it was unclear how their outputs are integrated to generate a coordinated behavior pattern. Here, we show that cholinergic signaling between the cloacal sensory/motor neurons and the posterior sex muscles sustains genital contact between the sexes. Simultaneously, via gap junctions, signaling from these muscles is transmitted to the spicule muscles, thus coupling repeated spicule thrusts with vulval contact. To transit from rhythmic to sustained muscle contraction during penetration, the SPC sensory-motor neurons integrate the signal of spicule’s position in the vulva with inputs from the hook and cloacal sensilla. The UNC-103 K+ channel maintains a high excitability threshold in the circuit, so that sustained spicule muscle contraction is not stimulated by fewer inputs. We demonstrate that coordination of sensory inputs and motor outputs used to initiate, maintain, self-monitor, and complete an innate behavior is accomplished via the coupling of a few circuit components. Citation: Liu Y, LeBeouf B, Guo X, Correa PA, Gualberto DG, et al. (2011) A Cholinergic-Regulated Circuit Coordinates the Maintenance and Bi-Stable States of a Sensory-Motor Behavior during Caenorhabditis elegans Male Copulation. PLoS Genet 7(3): e1001326. doi:10.1371/journal.pgen.1001326 Editor: Miriam B. Goodman, Stanford University School of Medicine, United States of America Received July 6, 2010; Accepted February 4, 2011; Published March 10, 2011 Copyright: 2011 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: YL, XG, and PAC were supported by the NIH grant GM-70431. BL, DGG, and LRG were supported by the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rgarcia@mail.bio.tamu.edu Introduction Male copulation is the most complex behavior for C. elegans [1]. The nervous system must integrate sensory cues from the hermaphrodite to generate instructions for each step of mating. In addition, the sex-specific mating circuits monitor the progress, and then determine whether to retain the current motor pattern or to adopt a new action [2]. This is done simultaneously as they convey information to the general locomotion circuit to coordinate the male’s movements with the hermaphrodite’s [3]. The function of the male’s nervous system is also modulated by gender determining factors, nutritional state and the presence of food odors [4–7]. The 302 neurons in Caenorhabditis elegans hermaphrodites and the 383 neurons in males encode a variety of general behaviors like feeding, locomotion, defecation and sensory modalities in both sexes. The circuits controlling these behaviors are comprised of a few neurons and muscles that are multifunctional in sensing, processing and commanding signals [8–17]. In contrast to many C. elegans hermaphrodite-studied sensory and motor actions, the male-executed copulation is a multi-component task-orientated behavior. Under an idealized mating situation, the male presses his sensory laden tail firmly on a receptive hermaphrodite, and adjusts his posture so that he laterally scans the dorsal and ventral cuticle of his mate, searching for the vulva. He would then position his tail over the vulva and extend a pair of copulatory spicules from his cloacal opening to pry apart the vulva lips. Finally, he would transfer sperm into the uterus of his mate [2,18–21]. However in reality, young virgin hermaphrodites attempt to crawl away from the male during mating, and depending on the age of the hermaphrodites, their vulval slits can be too tight for males to breach immediately. Thus, the male adopts an alternative behavior, which is to produce high frequency (7–11 Hz) repetitive spicule thrusts at the vulva while maintaining contact with it, until partial spicule insertion induces the full penetration [22–24]. Essential cellular components for spicule insertion were identified in previous work using aged paralyzed hermaphrodites as mates. The movements of the spicules are caused by contractions of the spicule ‘‘protractor’’ muscles, which are attached to the base of the spicules. Rhythmic contractions of PLoS Genetics | www.plosgenetics.org 1 March 2011 | Volume 7 | Issue 3 | e1001326