Sensory perception of food and insulin-like signals influence seizure susceptibility

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-gamma and CaMKII.

Figure 1. Effects of food deprivation on male sex-muscle excitability.

(A) Mating success for fed and 15hr food-deprived males. Males were scored as successful if they sired at least 1 progeny. p-value determined by Fisher's exact test (B) Graph of male muscle arecoline (ARE) sensitivity. For each concentration assayed, 20–30 males were assayed. (C) Mean sex-muscle G-CaMP responses to 10mM ARE for fed (n = 4) and food-deprived (n = 4) males. The * denotes well-fed time points that are significantly different (p≤0.05, Bonferroni posttest) then the food-deprived control. For fed males, the mean dsRed intensity trace is shown as a control. Error bars represent standard error of the mean. (D) Three representative frames displaying ARE-induced calcium changes in the sex muscles of a well-fed male (time between each frame is approximately 0.7 seconds). Anterior is to the right and the dorsal protractor muscles are labeled. Scale bar 6 µm.

Figure 2. Food deprivation suppresses sex-muscle excitability via both internal and external sensory responses.

(A) Adult male on aztreonam-treated E.coli OP50. Scale bar 20 µm. (B) Effects of food, inedible food, and no food on wild-type mating efficiency. p-value listed using Fisher's exact test. (C) Effects of food, inedible food, and no food on unc-103(0)-induced seizures. p-value listed using Fisher's exact test. (D) Bar graph representing the effects of food, inedible food and no food on Nile Red fluorescent intensity for wild-type males (E) Representative Nile Red-stained wild-type males under the three different feeding conditions (scale bar and 23.5 µm). (F) Bar graph representing the effects of different feeding conditions on the mean number of Nile Red-stained fat droplets for wild-type males. (G) Representative Nile Red-stained Posm-12:unc-103(gf) males under the three different feeding conditions (scale bar and 23.5 µm). (H) Bar graph representing the effects of different feeding conditions on the mean number of Nile Red-stained fat droplets in Posm-12:inc-103(gf) males. For each genotype and condition, 10 males were analyzed for Nile Red staining and the p-values were determined using the Student's t test.

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 µm).

Figure 4. The DAF-2/Insulin-like receptor suppresses sex-muscle excitability under food-deprived conditions.

(A) Graph showing the effects of daf-2(lf) mutations on unc-103(0)-seizure susceptibility in food, inedible food, and no food conditions. The * indicates that the p-value is significantly different (p<0.05) compared to the unc-103(0) control condition (Fisher's exact test). (B) Graph displaying the effect of ablating the AWC neurons in daf-2(e1368) unc-103(0) mutant males (p-value Fisher's exact test). (C) Graph displaying the effect of AWC odorants (10⁻⁴ isoamyl alcohol, 10⁻⁷ 2,3-pentanedione, and 10⁻⁴ benzaldehyde) on unc-103(0)-induced seizures in unc-103(0) and daf-2(e1368) unc-103(0) males. Sephadex G-50 beads were used as a vehicle for the odorants (p-value Fisher's exact test). (D) Temperature shift assay and heat-shock rescue for daf-2(e1368ts) unc-103(0). Animals were grown at the restrictive temperature (25°C) and then food-deprived at either the restrictive or permissive temperature (15°C). For heat-shock rescue, L4 males were heat-shocked for 35 minutes prior to the assay. The p-values listed were calculated using Fisher's exact test.

Figure 5. Phospholipase C-gamma (PLC-3) is required for food-deprivation suppression of unc-103(0)-induced muscle seizures.

(A) Graph displaying the effects of PLC-3, PLC-2 RNAi, and plc-3(sy698) on unc-103(0)-seizure susceptibility on food and no-food conditions. p-value for PLC-3 RNAi relative to unc-103(0) Mock RNAi no-food control, p-value for unc-103(0); plc-3(sy698) relative to unc-103(0) no-food control (Fisher's exact test). (B) Pplc-3:YFP expression pattern in young adult male. The ventral protractors are labeled. Scale bar 6 µm. (C) Cartoon displaying the relevant structures in the head and male tail. (D) Proposed model for sensory regulation of sex-muscle excitability during food-deprivation. When food odors 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-γ and CaMKII. CaMKII then reduces sex-muscle excitability through the activation of EAG K⁺ channels.