Sensory Perception of Food and Insulin-Like Signals Influence Seizure Susceptibility Todd R. Gruninger, Daisy G. Gualberto, L. Rene Garcia* Department of Biology, Texas A&M University, College Station, Texas, United States of America Abstract Food deprivation is known to affect physiology and behavior. Changes that occur could be the result of the organism’s monitoring of internal and external nutrient availability. In C. elegans, male mating is dependent on food availability; food- deprived males mate with lower efficiency compared to their well-fed counterparts, suggesting that the mating circuit is repressed in low-food environments. This behavioral response could be mediated by sensory neurons exposed to the environment or by internal metabolic cues. We demonstrated that food-deprivation negatively regulates sex-muscle excitability through the activity of chemosensory neurons and insulin-like signaling. Specifically, we found that the repressive effects of food deprivation on the mating circuit can be partially blocked by placing males on inedible food, E. coli that can be sensed but not eaten. We determined that the olfactory AWC neurons actively suppress sex-muscle excitability in response to food deprivation. In addition, we demonstrated that loss of insulin-like receptor (DAF-2) signaling in the sex muscles blocks the ability of food deprivation to suppress the mating circuit. During low-food conditions, we propose that increased activity by specific olfactory neurons (AWCs) leads to the release of neuroendocrine signals, including insulin-like ligands. Insulin-like receptor signaling in the sex muscles then reduces cell excitability via activation of downstream molecules, including PLC-c and CaMKII. Citation: Gruninger TR, Gualberto DG, Garcia LR (2008) Sensory Perception of Food and Insulin-Like Signals Influence Seizure Susceptibility. PLoS Genet 4(7): e1000117. doi:10.1371/journal.pgen.1000117 Editor: Stuart K. Kim, Stanford University Medical Center, United States of America Received March 25, 2008; Accepted June 4, 2008; Published July 4, 2008 Funding: All authors (TRG, DGG, and LRG) were supported by NIH grant GM-70431. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rgarcia@mail.bio.tamu.edu Copyright: 2008 Gruninger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction The feeding status of an organism can alter physiology and motor output leading to changes in health and behavior. For example, food deprivation can improve stress resistance, increase life-span, and alleviate muscle seizures [1–7]. In addition to promoting these phenomena under food-deprived conditions, organisms must also attenuate circuits not involved in food- foraging, such as reproductive behaviors. Although food depriva- tion has been established to modulate the physiology of multiple neuromuscular circuits in different species, the detailed mecha- nisms that integrate these circuits are just beginning to emerge. Since an organism’s experience of food consists of multiple sensory cues such as odor, texture, and temperature, it is likely that physiological responses to food are dependent on both sensory perception and ingestion of food. Here, we use the regulation of C. elegans male sex-muscle excitability under well-fed and food- deprived conditions to dissect these mechanisms. In the laboratory, C. elegans male mating behavior normally occurs on a food source (E. coli OP50) when a well-nourished male encounters a hermaphrodite. Contact with a hermaphrodite by sensilla in the male tail causes the male to stop forward locomotion, and begin scanning the hermaphrodite for its vulva. Once the male has located the vulva, he rapidly contracts sex muscles, which consist of two retractor muscles and two protractor muscles attached to each of his two copulatory spicules. Once his spicules breach the vulva, he maintains a tonic contraction of his sex muscles to keep the spicules inserted while sperm is transferred [1,8–13]. In contrast to food-satiated males, we find that food- deprived males mate with less efficiency, suggesting that the excitability of the muscles and neurons controlling mating is reduced. Loss-of-function mutations in the gene unc-103, which encodes an ERG-like K+ channel, cause well-fed males to display spontaneous spicule-muscle seizures [14]. Interestingly, food- deprivation suppresses the seizures of unc-103(lf) mutants. We have previously demonstrated that suppression of unc-103-induced muscle seizures requires calcium/calmodulin-dependent kinase II (CaMKII) and ether-a-go-go (EAG) -like K+ channel activity in the sex muscles [1]. This pathway could be activated by a sensory response to decreased food in the environment or an internal response to decreased ingestion of food. In this study, we investigate mechanisms that act upstream of the sex-muscle CaMKII/EAG K+ channel pathway that reduce seizure susceptibility under food-deprived conditions. Our results demonstrate that sensation of food, independent of ingestion, can influence muscle excitability. We propose that under low-food conditions, AWC olfactory neuron activity signals to the sex muscles to suppress cell excitability via the insulin-like receptor, DAF-2. Additionally, we find that DAF-2 does not regulate sex- muscle excitability by signaling through the canonical FOXO/ DAF-16 transcription factor. Instead, our observations suggest that DAF-2 signals to CAMKII via the C. elegans phospholipase C-c (PLC- c), PLC-3, to suppress seizures. PLoS Genetics | www.plosgenetics.org 1 July 2008 | Volume 4 | Issue 7 | e1000117