expressed Punc-103F:ChR2::YFP, displayed prolonged protractor contraction in response to blue light (n = 36, Figure 6B). When we preselected males that displayed very high expression of Punc- 103F:ChR2::YFP, 65% of these biased males displayed the behavior after light stimulation (n = 37, Figure 6B). Laser ablation of the PCA and PCB sensory neurons reduced the percentage of light-stimulated protraction in preselected biased males to 20% (n = 20, p = 0.002), indicating that stimulation of the SPC neurons in the absence of PCA and PCB activity does not result in efficient tonic muscle contraction. The low efficiency of Punc-103F:ChR2::YFP-induced spicule protraction in unbiased males suggested that the spicule-prod- ding/protraction neurons have intrinsic properties, which to overcome, require high levels of ChR2 stimulation. We reasoned that, similar to the spicule muscles, the UNC-103 K+ channel might be offsetting ChR2 activity in the spicule neurons. When the Punc- 103F:ChR2::YFP construct was introduced into the unc-103(0) males, 77.6% of the population fully protracted their spicules in response to the blue light (n = 94, Figure 6B). The unc-103 gene is expressed in both spicule-associated neurons and the male sex muscles [6,44]. We found that restoring functional UNC-103 in the sex muscles could reestablish these males’ resistance to light-stimulation; however, expressing unc-103 in the spicule-associated neurons can also restore some resistance to light stimulation (Figure 6B). Thus in wild type, the UNC-103 channel likely regulates the excitability threshold of neurons, in addition to muscles. Why do the p.c.s. and SPC neurons promote spicule protraction at the vulva, but not efficiently under artificial non-mating conditions? We hypothesized that in addition to UNC-103, the activities of the p.c.s. and SPC neurons might be attenuated by other neurons. In intact males, spicule prodding and full protraction only occur when the males are at the vulva, which is sensed by both the p.c.s. and the hook sensillum. When the hook sensillum precursor cell, P10.p is laser-ablated, the operated males no longer sense the general area of the vulva but instead randomly prod the cuticle of the hermaphrodite with their spicules [2]. This suggests that in intact males, the cells derived from P10.p might attenuate spicule protractor activity at areas outside the vulva. The P10.p precursor cell gives rise to the hook structural cells, HOA and HOB sensory neurons and the PVZ motor neuron. The PVZ does not make synapses to the spicule-relevant neurons, but the HOA and HOB hook sensillum neurons do make direct chemical and electrical connections to the p.c.s. and SPC neurons (Figure 3A; Male Wiring Project). Thus, it is possible that these connections might negatively modulate spicule activity until the vulva is sensed. We ablated the precursor cell P10.p in Punc-103F:ChR2::YFP- expressing males, and found that in 54% of unbiased operated males (n = 27, Figure 6B) and 87% of high-expressing operated males (n = 23, Figure 6B), the light induced prolonged protraction. In both cases, the incidence of ChR2-induced spicule protraction was higher in operated animals compared to mock-ablated. However, for unbiased males, the difference was statistically significant (p = 0.004), whereas for biased Punc-103F:ChR2::YFP high-expressing males, the increase was not statistically different over the already high protracting non-ablated males (p = 0.08). We conclude that the lineage descendants of P10.p, most likely the hook neurons, can reduce the output of ChR2-activated p.c.s. and SPC neurons, but this reduction can be partly circumvented by expressing more ChR2. Discussion C. elegans male mating behavior is a multi-step goal-directed behavior that utilizes several active sensing processes, as the male Schema of the Spicule Insertion Circuit worm actively acquires sensory inputs using different types of ‘‘motor sampling routines’’ [64]. For example, sensation of the hermaphrodite during male locomotion initiates the backward vulval searching behavior; recognition of the vulva promotes the repetitive spicule thrusts as an attempt to penetrate the vulval slit; and as a result, proprioceptive signals can detect partial penetration and subsequently trigger prolonged spicule extension. The transition from one behavioral step to another is directed by one or more sensory feedback signals, and the consequential motor output is objectively purposeful and intended to obtain further sensory inputs in order to achieve the behavioral goal – vulval penetration. During this process, maintaining a precise contact with the hermaphrodite’s vulva while she is moving is exception- ally challenging, as the hermaphrodite can constantly shift her direction of locomotion and can crawl on average at speeds up to ,200 mm/second [65]. Although feedback signals must be involved in these motor behaviors, sensory feedback can be delayed and can result in motor output errors. A heuristic model called the forward internal model, which potentially involves heavy computation, is proposed as a solution to this problem in motor control. A neural ‘‘controller’’ region generates a command to the motor unit, while simultaneously creating a copy of the command to a hypothetical region called the ‘‘forward model’’. The ‘‘forward model’’ then predicts the sensory consequences of the motor commands. The predicted sensory consequences are then compared with the actual sensory feedback in the ‘‘comparator’’ region, and the integrated information is sent back to the controller region to modify subsequent motor commands [66]. In neural systems of higher invertebrates and vertebrates, their complex brains can theoretically incorporate the computa- tional components predicted in the forward internal model. However, for a simple organism like C. elegans that has a highly compact nervous system, we suggest that coordinating and modifying motor outputs to facilitate goal-oriented tasks can be solved by simpler, yet sufficient mechanisms. For wild-type males, contact with the vulva and contractions of the protractor muscles take place simultaneously and rarely occur independently. This suggests that their mechanisms of execution and regulation are integrated. Efficient sensing of the vulva requires the combined functions of the bilateral set of postcloacal sensilla (p.c.s.) PCA, PCB and PCC sensory-motor neurons and the hook sensillum sensory neurons HOA and HOB. The sensory endings of the p.c.s. neurons are encased in the cuticle, suggesting that they might be mechanosensory; whereas the sensory endings of HOA and HOB neurons are opened to the environment, indicating that these neurons might sense chemical as well as mechano-signals [25]. Laser ablations of the hook sensillum cells or mutations that decrease the hook neurons’ sensory functions severely reduce the ability of the male to recognize the vulva; however at low efficiency, the males can recognize the precise location of the vulva with the p.c.s. and insert their spicules [2,18,67-70]. In contrast, removal of all p.c.s. neurons results in males that can sense the general area of the vulva, but are incapable of positioning their tails over the vulval slit to insert their spicules [2,22]. Males can utilize any two of the three bilateral pairs of the p.c.s. neurons to position the cloacal opening over the vulva. But if a male lacking any single pair of the p.c.s. neurons does not quickly insert his spicules, he will move off sooner than if he had the complete sensilla. This indicates that after the initial sensory contact, the p.c.s. neurons are continually required to maintain the male’s position over the vulval slit. The maintenance of the male’s position over the vulva is partly regulated by direct signaling between the p.c.s. neurons and the male anterior and posterior PLoS Genetics | www.plosgenetics.org 12 March 2011 | Volume 7 | Issue 3 | e1001326