occurs due to increased cystolic Ca2+ from the PLC-c/IP3 activity [55–57]. To test if PLC-c is required to suppress sex-muscle seizures under food-deprived conditions, we knocked-down the worm homologue of PLC-c, PLC-3, via RNAi (Figure 5A). RNAi of PLC-3 significantly increased the percentage of males with spontaneous seizures on the no-food condition, whereas RNAi of another PLC-homologue, PLC-2 (PLC-b) had no significant change. To test this more rigorously, we constructed a unc- 103(0); plc-3(sy698) [58] double mutant (Figure 5A). On plates with no E. coli, we found that 25% of unc-103(0); plc-3(sy698) double mutant males had spontaneous muscle seizures, compared to 5% of unc-103(0) males. These results are similar to the effects of the daf-2(lf) mutations, suggesting that daf-2 may act through plc-3 to regulate sex-muscle excitability. In males, PLC-3 was previously reported to be expressed in the seminal vesicle valve cell and the vas deferens [59]. Using a larger upstream promoter region, we found that our 4.4 kb plc-3 promoter construct also expressed in the ALA neuron (data not shown), the male retractor muscles, and the ventral protractor muscles (Figure 5B). By analogy with the mammalian cell-culture studies, these results suggest that under food-deprived conditions, DAF-2 signaling might stimulate PLC-3 activation of IP3 and the resulting increase in sex-muscle cystolic Ca2+ leads to activation of CaMKII, thereby reducing sex-muscle excitability. We have previously reported that CaMKII activates the EAG- + like K channel (EGL-2) in the sex muscles, suggesting that EGL-2 acts downstream of DAF-2 [1]. To test this directly, we generated a daf-2(e1368) unc-103(0); egl-2(gf) triple mutant and assayed the number of food-deprived males with seized muscles. We hypothesized that a constitutively active EGL-2 K+ channel should suppress daf-2(lf) mutants if the channel acts downstream of DAF-2. We found that this was indeed the case, as daf-2(e1368) unc-103(0); egl-2(gf) males were suppressed from 38% (n = 45) to 8% (n = 45) by food deprivation, which was significantly different than the 28% (n = 43) observed of food-deprived daf-2(e1368) unc- 103(0) males (p = 0.03, Fisher’s exact test). Discussion Many animals encounter environments where food availability fluctuates and therefore they must endure periods of food limitation. As a result, animals have evolved adaptive physiological responses to ensure they survive until food becomes available again. The most well studied physiological responses to food availability are life-span and lipid homeostasis. In C. elegans, longevity and lipid homeostasis are regulated by chemosensory neurons, suggesting that perception of food in the environment, and not just intake of nutrients, can directly regulate physiology [60–62]. In mammals, while no direct link between sensory function and lifespan has been made, there is much evidence supporting the connection between chemosensory function and lipid metabolism [63–65]. In addition to promoting longevity and fat breakdown under food-stressed conditions, organisms alter their behavior by activating food seeking behaviors and suppress- ing other behaviors not involved in locating food. Specifically, in circuits not involved in food foraging, food deprivation likely activates signaling molecules that reduce cell-excitability. This natural physiological response could be a possible mechanism for why food deprivation has been observed to reduce seizure susceptibility in rodents [5–7,66]. Understanding the pathways that regulate these adaptive physiological responses could lead to better health treatments for abnormal excitable cell output without the need of food deprivation. Sensory and Insulin-Like Signals Suppress Seizures We found that food availability regulates C. elegans sex-muscle excitability via two inputs, through sensation of food and ingestion of food. Loss of unc-103, which encodes an ERG-like voltage-gated K+ channel, causes about 40% of well-fed C. elegans males to suffer seizures of their sex muscles, whereas only ,8% of food-deprived unc-103 mutants display spontaneous seizures [1,13]. We have previously identified that non-chemosensory neurons and organs, including the neuromuscular organ controlling food ingestion, the pharynx, can regulate sex-muscle excitability. Specifically, in tropomyosin/lev-11 mutants, which phenocopy the effects of food deprivation on mating behavior, the pharyngeal neurosecretory motor neurons (NSMs) suppress unc-103-induced sex-muscle seizures [13]. However, it is still possible that lack of nourishment from food contributes to suppressed muscle seizures. In addition to affecting sensory neurons, food-deprivation induces cellular stress and/or damage. One way to measure this is to visualize the levels of autophagy, or the catabolism of cellular components, which increases during nutrient deprivation [67]. LGG-1 is the C. elegans ortholog of yeast Apg8/Aut7p and mammalian MAP-LC3, and can be used to mark preautophagosmal and autophagosomal membranes [68–71]. However, we found that cellular damage is not a likely mechanism, since the starvation period was not long enough to induce a significant increase in autophagy measured by a GFP-tagged LGG-1 (Figure S2A-B). Additionally, we used phalloidin to stain filamentous actin in fed and 15-hr food- deprived males and found no obvious defects in the muscle structure of food-deprived males (Figure S2C). Based on these results, we propose that reduction in male-mating efficiency and sex-muscle excitability by food deprivation is a regulated and reversible process, rather than the result of muscle damage or dysfunction. In addition to food ingestion, food availability can regulate sex- muscle excitability via a chemosensory component. Interestingly, we found that food-deprivation reduction in unc-103-seizure susceptibility can be partially blocked by placing males in inedible food or by disrupting chemosensory function. We identified one pair of ciliated sensory neurons in the head, the AWCs, which can down-regulate sex-muscle excitability under food-deprived condi- tions. Ablation of these neurons in unc-103(0) males blocks the suppressing effects of food deprivation. Chalsani et al [38] demonstrated that the AWC neurons are activated upon odor removal, suggesting that these neurons are active when no food is being sensed in the environment. We found similar results while measuring AWC activity during our food-deprivation assays. Prior to food deprivation, males showed lower intracellular calcium levels that markedly increased during the food-deprivation interval and then decreased again when the males were placed on food. Thus, in males, AWC activity could also be initiating signals that down-regulate sex-muscle excitability. In hermaphrodites, the AWC neurons mediate attraction to at least 5 attractive volatile odors, and also function to increase turning probability during local search behavior. Local search behavior allows worms that have been removed from food to explore limited areas by initiating a series of reversals and omega turns [24,29,72]. In males, the AWCs have recently been implicated in regulating male-sexual attraction behavior to hermaphrodite pheromones [32]. It is possible that in males, AWC-mediated attraction to hermaphrodites shares a similar mechanism to attractant chemotaxis in hermaphrodites. The AWCs likely regulate turning probability through glutamate- mediated synapses to the AIB and AIY interneurons, which can signal to the SMD and RIV head motor-neurons [38,72]. However, in our studies, it is not exactly clear how the AWCs communicate to the male genitalia. Although there are clear PLoS Genetics | www.plosgenetics.org 10 July 2008 | Volume 4 | Issue 7 | e1000117