receptor is required developmentally, then raising the daf-2(ts) mutants at 25uC should block food-deprivation suppression, regardless of the temperature at which they are food-deprived. On the contrary, if DAF-2 is required acutely at the time of food deprivation, then the daf-2(ts) mutations should not block suppression of seizures when food deprived at 15uC. We found that daf-2(e1368) unc-103(0) males raised at 25uC and then food- deprived at 15uC behaved like unc-103(0) single mutants; i.e. they were significantly suppressed for spontaneous muscle seizures (Figure 4D). These results suggest that DAF-2 activity is required acutely to suppress sex-muscle excitability. However, it is possible that daf-2(e1368) is not truly wild-type at 15uC, and the effects observed may be due to temperature changes rather than true wild-type function of daf-2. To rule out this possibility, we rescued daf-2(e1368) in daf-2(e1368) unc-103(0) mutants using the inducible heat-shock promoter, Phsp-16 (Figure 4D). Similar to the temperature shift assay, we found that L4 stage males heat- shocked 30min prior to our 15hr food deprivation assay had a significantly lower probability of spontaneous sex-muscle seizures. Additionally, although not quite statistically significant, the Phsp- 16:daf-2(+) construct appeared to reduce spontaneous spicule protraction in well-fed males as well, suggesting that over- expression of daf-2 may be able to compensate for loss of unc- 103 function. In C. elegans, many daf-2(lf)-mediated behavioral, metabolic and developmental responses require the FOXO transcription factor, DAF-16 [46–48]. Previous studies have shown that DAF-2 regulates longevity and developmental pathways by inhibiting DAF-16 from entering the nucleus and activating downstream genes [49–52]. To determine if DAF-2 acts on DAF-16 to regulate sex-muscle excitability during food deprived conditions, we constructed the triple mutant, daf-16(m26); daf-2(e1368) unc- 103(0) and tested the effects of the mutations under the three different food conditions. We found that mutation of daf-16 did not significantly alter the percentage of males with spontaneous seizures for any of the three-food conditions (Figure 4A). Thus, in contrast to dauer and longevity regulation, DAF-2 is acting through a DAF-16-independent signaling pathway to regulate sex- muscle excitability. Sex-Muscle DAF-2 Suppresses Muscle Seizures in Response to Food Deprivation DAF-2 is broadly expressed in C. elegans and controls multiple facets of physiology, including aging, fat regulation, and Sensory and Insulin-Like Signals Suppress Seizures development [39–43,49,53]. Previous studies have shown that DAF-2 regulates life-span by acting in neurons, while DAF-2 signaling in the muscles mediates intestinal fat levels [54]. To determine where DAF-2 is required to suppress sex-muscle seizures in response to food deprivation, we generated tissue- specific constructs using wild-type DAF-2 cDNA to rescue daf- 2(e1368) unc-103(0) mutants (Table 3). We used the lev-11 body- wall and sex-muscle promoter, the tnt-4 pharyngeal muscle promoter, the pan-neuronal promoter of aex-3, and the intestinal gtl-1 promoter. Only the Plev-11:daf-2(+) construct restored food- deprivation suppression of sex-muscle seizures, suggesting that DAF-2 acts in the muscles. To determine if DAF-2 acts in the body-wall muscles or sex muscles to suppress spontaneous sex-muscle contraction, we expressed daf-2(+) in the sex muscles using the unc-1031E promoter. Promoter Punc-103E is expressed in approximately 7 pairs of neurons in the head, one pair of pharyngeal neurons and the spicule protractor muscles [16]. Interestingly, the sex-muscle specific Punc-1031E:daf-2(+) construct restored suppression of unc- 103(0)-induced seizures. These results suggest that DAF-2 receptors expressed on the sex muscles respond to food- deprivation signals. It is therefore possible that the AWC olfactory neurons signal to the sex muscles directly or indirectly via regulating the release of insulin-like peptides. In addition to acting in the sex muscles, we found that DAF-2/ insulin-like receptor signaling can also function in neurons to regulate sex-muscle excitability. Surprisingly, the Paex-3:daf-2(+) construct significantly suppressed unc-103-induced seizures on the food condition, suggesting that increased DAF-2 signaling in the neurons can compensate for loss of UNC-103 function in the sex muscles (Table 3). However, this regulation is separate from daf-2 regulation of sex-muscle excitability during food-deprived condi- tions, since there is no further decrease in seizure susceptibility in food-deprived males (Table 3). Although neuronal daf-2 signaling may influence sex-muscle excitability on food, our results suggest that sex-muscle insulin-like receptor signaling reduces seizure susceptibility during food-deprived conditions. We have previously demonstrated that in starved males, CaMKII is responsible for suppressing sex-muscle seizures [1]. Since DAF-2 regulation of sex-muscle excitability does not require DAF-16, we looked for other targets of insulin-like receptor signaling that could affect CAMKII activity. Work by others has demonstrated that IGF-1 can activate CaMKII signaling pathways in human neuroblastoma and rat hippocampal neurons, and this Table 3. Tissue-specific rescues of daf-2(e1368) in daf-2(e1368) unc-103(0) males. Rescue constructa None rgEx178 [Plev-11:daf-2(+)] rgEx180 [Paex-3:daf-2(+)] rgEx179 [Punc-1031E:daf-2(+)] rgEx199 [Punc-1031F:daf-2(+)] rgEx236 [Pgtl-1:daf-2(+)] rgEx237 [Ptnt-4:daf-2(+)] Tissue Expression None Body-wall muscles, sex muscles All neurons Sex muscles, anal depressor, AIB, RIM, AIY,AVJ, ALA,AVH,NSM, I5 ALA, ADL, ASK, AVH, AVJ, AIN, AVA, ASJ, SMDD, SIA, ADE, AVD, I2, NSM, IL, IL2, OLL, URA, ASH, AVD, AUA,AUA, SIA, OLQ, RIV, URY, AIN, AIA, SPC, PCA, PCB, HSN, ray 1,2,3,4,6,9 intestines pharyngeal muscles aBackground strain is daf-2(e1368) unc-103(0). bSignificantly different than the non-transgenic no-food control, Fisher’s exact test. cSignificantly different than the non-transgenic food control, Fisher’s exact test. doi:10.1371/journal.pgen.1000117.t003 % Protracted on Food % Protracted on No Food 40 (212) 41 (37) 14c (21) 38 (26) 46 (48) 48 (33) 41 (49) 28 (138) 9b (33) 11 (27) 9b (22) 29 (38) 34 (41) 38 (21) PLoS Genetics | www.plosgenetics.org 9 July 2008 | Volume 4 | Issue 7 | e1000117