Sensory and Insulin-Like Signals Suppress Seizures are present, AWC activity is attenuated. However, under food-deprived conditions, AWC activity is up-regulated and results in release of insulin-like peptides from downstream neurons. Insulin-like peptides activate insulin-like receptors on the male tail, resulting in activation of PLC-c and CaMKII. CaMKII then reduces sex-muscle excitability through the activation of EAG K+ channels. doi:10.1371/journal.pgen.1000117.g005 sexually dimorphic differences in AWC function, both males and hermaphrodites appear to use these neurons to mediate attraction to an odorant source [24,29,32,72]. Based on our AWC-G-CaMP experiment, we propose that, in males, AWC likely mediates attraction to food odorants as well as hermaphrodite pheromones. We hypothesize that chemosensory neurons may coordinate muscle excitability with other physiological responses to food availability. Specifically, a common mechanism could be through regulating the release of neuroendocrine signals from downstream neurons that also regulate metabolism, such as insulin-like signals. Support for a link between chemosensory function and metabo- lism come from studies of the mammalian tubby gene, Tub, which is a highly conserved protein expressed in the central nervous system. Mice with loss-of-function mutations in Tub suffer from late onset obesity and have defects in ciliated neurons including retinal degeneration and hearing loss [73–75]. Similar to the mammalian studies, loss of tub-1 function in C. elegans results in increased fat storage and defects in olfactory behavioral response mediated by the AWA and AWC olfactory neurons [62,64,73,74]. Specifically, control of lipid homeostasis has been proposed to be dependent on the neuroendocrine function of a set of 15 ciliated neurons, including the AWC neurons [62]. Consistent with a role of chemosensory function in the control of fat breakdown in males, we found sensation of food suppresses mobilization of fat stores, independent of ingestion. Interestingly, when we reduced the male’s ability to sense inedible E. coli, we saw a reduction in fat staining. This suggests that sensation of food by chemosensory neurons commonly block the effects of food deprivation on muscle excitability and fat mobilization. Additionally, we found insulin- like signaling, which is known to regulate lipid-homeostasis in hermaphrodites [18,39], regulates sex-muscle excitability. Previous studies suggest that neuroendocrine regulation of fat storage is independent of DAF-2, since FOXO/DAF-16 mutants have no effect on tub-1-increases in fat storage [62]. Therefore, neuroen- docrine regulation of sex-muscle excitability and fat storage may be via different signaling molecules, or commonly through a FOXO/DAF-16-independent insulin-like signaling mechanism. Historically, insulin signaling has been proposed to be active under well-fed conditions and regulate the body’s ability to handle food by internalizing glucose. In C. elegans, a similar paradigm has been proposed with DAF-2/insulin-like signaling. Food depriva- tion has generally been proposed to inhibit DAF-2 activity, which can lead to FOXO/DAF-16-mediated dauer formation and increased longevity [39–43]. In contrast to studies in longevity and dauer formation, our data indicates DAF-2 activity is required under food-deprived conditions to suppress excitable motor output. This difference could possibly be attributed to sexually dimorphic differences, since males have been reported to have a significantly longer lifespan than hermaphrodites [76]. Although DAF-2 is not required for increased male longevity compared to hermaphrodites, daf-2(lf) mutations still significantly increase male lifespan, suggesting that, similar to hermaphrodites, insulin-like signaling also regulates similar pathways in males. Therefore, we propose that the assumption of food deprivation generally leading to decreased DAF-2 activity in all tissues may not hold true in all biological contexts, and insulin-like signaling may be more complicated then originally thought under these conditions. Interestingly, the requirement for active insulin-like signaling under food-deprived conditions has been investigated by groups studying other biological responses to food availability. For example, one characterized response to food deprivation, termed Fasting-Induced Redistribution of Esterase (FIRE), which results in the relocation of intestinal esterase activity from the cytoplasm to the nucleus during fasting, has been reported to require DAF-2 activity [77]. Loss-of-function mutations in daf-2 suppress the FIRE response associated with food deprivation. Additionally, DAF-2 activity is required for salt chemotaxis learning, which allows worms that have previously been exposed to NaCl under starved conditions negatively chemotax away from a NaCl source [45]. Similarly, C. elegans also associate temperature with food deprived conditions by avoiding temperatures at which they were previously food deprived. This behavioral association of food and temperature requires the insulin-like ligand, ins-1, which appears to negatively regulate DAF-2 activity in this context [44]. The observation that ins-1 is required to actively suppress DAF-2 activity under food-deprived conditions suggest that DAF-2 can be active under these conditions and must be inactivated for certain biological responses. Interestingly, ins-1 is thought to act as a DAF- 2 agonist for salt chemotaxis learning. Thus, the action of DAF-2 and insulin-like signals under different environmental conditions is likely more complex than a general activation or reduction in DAF-2 activity, and likely varies depending on the physiological response in question. This could possibly explain the requirement for so many insulin-like ligands [78] and the observations that certain DAF-2 responses such as reduction in sex-muscle output and salt-chemotaxis learning [45] do not require the FOXO/ DAF-16 transcription factor. Given our observations, we propose that sensory perception of food can regulate organismal physiology. In C. elegans males, we have demonstrated that suppression of mutant-induced seizures by food-deprivation requires chemosensory neurons and insulin-like signaling. Specifically, we propose that in low-food environments, AWC-olfactory neuron activity is up-regulated, leading to the release of insulin-like peptides by downstream neurons (Figure 5C- D). These peptides bind insulin-like receptors on the sex muscles and lead to the Ca2+ activation of CaMKII through PLC-c. CaMKII then leads to reduced excitability by activating EAG K+ channels. This regulation of muscle excitability by chemosensory and insulin-like signaling may indicate a general mechanism used by organisms to regulate multiple behavioral and physiological responses to changing environmental conditions. Materials and Methods Strains All strains contain him-5(e1490) (LGV) [79] and were main- tained according to [80]. The following strains were used in this study: LGII: The plc-3(sy698) strain used in this study is PS5109 described previously in [58] and graciously provided by Cheryl Van Buskirk; LGIII: daf-2(e1368) [81], daf-2(m41) [49], unc- 103(n1213) [23], and pha-1(e2123) [82]; LG IV: osm-9(ky10) [83]; LGV: egl-2(n693) [84]; LGX: osm-5(p813) [22]. Behavioral Assays Scoring of the spontaneous spicule-muscle seizure phenotype for the different feeding conditions was done as previously described [1]. Briefly, approximately 20–30 L4 males were separated and PLoS Genetics | www.plosgenetics.org 12 July 2008 | Volume 4 | Issue 7 | e1000117