Sensory and Insulin-Like Signals Suppress Seizures Figure 3. The olfactory AWC neurons regulate sex-muscle excitability in response to environmental conditions. (A) Graph showing the effect of laser ablation of the AWC olfactory neurons. The * denotes a significant difference (Fisher’s exact test) compared to mock ablated no-food control. (B) G-CaMP intensity changes in the left AWC neurons of 10 individual males taken after fed, 5hr and 15hr of food deprivation, and re-fed. Each number (1–10) identifies the same male for each interval. The p-value listed signifies a significant difference between Fed and 5-hr food- deprived males. (C) A representative G-CaMP intensity image of a well-fed and (D) 15hr food-deprived male (scale bar 9 mm). doi:10.1371/journal.pgen.1000117.g003 To test if the insulin-like pathway is used in food-deprivation induced suppression of seizures, we looked at two temperature sensitive daf-2 alleles. Interestingly, similar to the chemosensory mutants, we found that daf-2(e1368) and daf-2(m41) mutants significantly increased unc-103(0)-induced seizures under the no- food condition, while having no significant effect on the food or inedible-food conditions (Figure 4A). These results suggest that the insulin-like receptor, DAF-2, is required to suppress sex-muscle excitability under food-deprived conditions. Additionally, neither daf-2 allele significantly affected the percentage of protracted males on inedible-food. Inedible food is likely suppressing via a non- sensory component, and our data suggests that this component is still intact in daf-2(ts) mutants; instead, DAF-2 likely acts downstream of sensory neurons. Since our results indicate a role for the AWC chemosensory neurons in food-deprivation suppression of sex-muscle output, we hypothesize that the AWC’s likely mediate their effects through controlling release of insulin-like peptides. To test if the AWCs signal through DAF-2, we performed the AWC ablation in daf- 2(e1368) unc-103(0) double mutants. If the AWC’s act through insulin-like signaling to suppress sex-muscle excitability, then we predicted that the AWC ablation should not affect the percentage of food or food-deprived daf-2(e1368) unc-103(0) males with seized muscles. However, if they function via separate mechanisms, we predicted that ablation of AWC in the double mutant background should significantly increase the percentage of food-deprived males with protracted spicules. Interestingly, we found that ablation of the AWC neurons in daf-2(e1368) unc-103(0) males did not significantly affect the percentage of males with seized muscles on either condition assayed (Figure 4B). Additionally, to mimic the inedible food condition which blocks AWC-mediated suppression of sex-muscle excitability, we soaked Sephadex beads (20–80 mM) in odorants previously shown to reduce AWC activity, isoamyl alcohol, benzaldehyde, and 2, 3-pentanedione [38]. We found that beads soaked with AWC-inhibiting odorants increased the percentage of food-deprived unc-103(0) males with protracted spicules, but had no significant effect on daf-2(e1368) unc-103(0) males (Figure 4C). Taken together, these results suggest that DAF- 2 acts downstream of the AWC chemosensory neurons to suppress spontaneous sex-muscle output. DAF-2 could be regulating sex-muscle excitability acutely by acting in excitable cells at the time of food deprivation, or it could be required developmentally to ensure cells are able to respond when the environment changes. To distinguish between these possibilities, we raised daf-2(e1368) unc-103(0) males at the restrictive temperature (25uC) during late L2 through L4 larval stages and then food deprived them as late L4/adult molt at either the permissive (15uC) or restrictive temperature (25uC). If the PLoS Genetics | www.plosgenetics.org 7 July 2008 | Volume 4 | Issue 7 | e1000117