activation of the striatum relieves this inhibition [76]. The striatum has a low excitability due to the inward rectifier K+ current [77– 80]. Therefore, the striatum only becomes activated when it receive strong stimulation from the thalamus or the cortex, where relevant sensory inputs are integrated. Using this mechanism, the animal adapts its locomotion properly in response to environmen- tal conditions [75,81]. Materials and Methods Strains All strains were grown at 20uC on nematode growth media (NGM) plates seeded with E. Coli OP50 [58]. Pharmacological and behavioral assays were conducted between 21-23uC. All males contained him-5(e1490) on linkage group V(LGV) [82]. Additional alleles used were: unc-29(e193) [31,33] on LGI; unc-103(n1213) [83], pha-1(e2123) [84], unc-64(e246) [58] on LGIII; acr-16(ok789) on LGV was generated by the C. elegans Gene Knockout Consortium; and unc-9(e101) [58], lite-1(ce314) on LGX [85]. Behavior assays Mating potency assay. Males were separated from hermaphrodites at the L4 stage and put onto a fresh OP50- containing NGM plate. The next day, each male was put onto an individual plate containing a ‘‘mating lawn’’ of OP50 with one free-moving pha-1(e2123) hermaphrodite. 25 ml of a saturated OP50 culture was seeded on each plate the previous night. Males and hermaphrodites were incubated together for 4 hours at 20uC, after which the males were picked off. Hermaphrodites were then incubated at 25uC, and their progeny was scored three days later. Homozygotes of pha-1(e2123) cannot survive beyond the L1 stage at 25uC [84]. Plates with L2 or older progeny indicated that cross fertilization occurred. The percentage of males that can sire progeny within 4 hours was recorded for each strain. To present the data, the wild-type potency was arbitrarily set to 100%, and mating potency of mutants was calculated proportional to the wild type. Vulval contact assay. Males and hermaphrodites were separated from each other at the L4 stage, and put onto separate OP50-containing plates. The next day, 20–30 paralyzed hermaphrodites were put on a mating lawn. One male was then put onto the mating lawn and observed with a Zeiss Stemi SV 11 microscope. The male behavior was video recorded once he started scanning the hermaphrodite with his tail. Recordings were stopped 5 minutes later or when the male inserted his spicules (whichever happened first). From these videos, we quantified how long a male spent performing each step of mating behavior using a time recording Visual Basic Macro described elsewhere [26]. To determine how well a male maintains contact with the hermaphrodite’s vulva, we determined the average (not cumula- tive) duration of vulval contact of each male. 1-day-old paralyzed unc-64(e246) hermaphrodites were used in this assay. The vulvas of hermaphrodites at this age were not dilated due to extended egg-laying, thus, the vast majority of the males tested in this work could not penetrate the vulva for the duration of the first vulval contact (77.3% of wild type, n = 66; 78.9% of unc-29(lf) locomotion-restored males, n = 19; 92.3% of acr-16(0) males, n = 13; 87.5% of unc-29(lf);acr-16(0) locomotion-restored males, n = 32; 100% of the SPC-ablated males, n = 11), and multiple vulval contacts were recorded for these males. Therefore, the average duration of vulval contact of an individual male was averaged from multiple vulval contacts, and the efficiency of spicule insertion was not likely to affect our measurements of vulval contact duration. Schema of the Spicule Insertion Circuit To present the data in figures, the average duration of vulval contact for each male was normalized to the mean of wild type or the mock-ablated males to obtain the ‘‘normalized score for vulval contact duration’’, as a measurement of male’s ability to maintain precise vulval contact. We calculated the ‘‘normalized score for contact duration’’ of an individual male using: Pxxiri (nri is the ( ) number of males in the reference group, xri is the avnerirage duration of contract for an individual reference male (wild type or mock- ablated males), xi is the average duration of contact for an individual male). The height of the bar represents the mean of P xi P xri ) ( nri normalized score for each strain: (ni is the number of ni males tested for a specific strain or treatment). Error bars indicate the standard error of the mean. Multiple males were analyzed for each strain or treatment. Wild-type males/un-operated males were always observed in parallel with mutant males/operated males as control. Statistic comparisons were only conducted between male samples that were observed in parallel, since the wild-type behavior can vary on a day-to-day basis. Comparison between two samples was done by using the Mann-Whitney non-parametric test. Sperm release. The behavior of males was filmed and the duration of sperm ‘‘release’’ was recorded as described earlier. As defined by a previous study, sperm ‘‘release’’ refers to the process when sperm leaves the seminal vesicle and is release from the vas deferens [20]. Measuring velocity of backward locomotion during vulva search behavior. Twenty 48 hrs adult pha-1(e2123); him-5(e1490); lite-1(ce314);rgEx431[Phsp-16:egl-2(n693gf); Punc-103E:mDsRed; pha- 1(+)] hermaphrodites were heat shocked for 30 minutes at 30uC, and then incubated at 20uC. After 3 hrs, the heat shocked-expressed EGL-2(gf) K+ channels induced complete paralysis. The hermaphrodites were placed on a 5 mm diameter lawn of E. coli OP50. Their postures were straightened out using an eyelash pick. 18–24 hrs adult males were then placed with the hermaphrodites and monitored at 606 total magnification on a stereomicroscope mounted with a Hamamatsu ImagEM CCD camera. Digital recordings (containing timestamps) were made at 30 frames per second. To facilitate quantification of the distances that the males moved during the vulval search behavior, images of a stage micrometer were acquired prior to recording the copulations. Velocity was determined as the time required for the male tail (focusing on the spicule tip) to travel 300 to 800 mm along the hermaphrodite’s cuticle. The straight posture of the paralyzed hermaphrodites allowed easy determination of the males’ position along the hermaphrodite’s cuticle. Pharmacology Levamisole (LEV) (ICN Biomedicals, Aurora, OH) was prepared in distilled water and stored at 220uC. 400 ml of LEV solution at various concentrations was added to a Pyrex Spot Plate (nine 1 ml volume depressions), and ,5–10 virgin males were put into the drug. We observed these males for 3 minutes under a Leica MZ 7.5 stereomicroscope. A male was considered sensitive to the drug if he fully protracted his spicules for .5 seconds. Fresh drug was used after ,30 virgin males were assayed. Comparisons were made by using the Fisher’s exact test. Plasmids Expression pattern of unc-29, acr-16, unc-38, unc-63, and acr-18. Primer sequences are provided in Table S1. A 5.3 kb PLoS Genetics | www.plosgenetics.org 14 March 2011 | Volume 7 | Issue 3 | e1001326