Dynamic Regulation of Adult-Specific Functions of the Nervous System by Signaling from the Reproductive System

Abstract

Unlike juveniles, adult animals engage in suites of behaviors related to the search for and selection of potential mates and mating, including appropriate responses to sex pheromones. As in other species [1], male sex pheromones modulate several behaviors and physiological processes in C. elegans hermaphrodites [2-5]. In particular, one of these small-molecule signals, an ascaroside ascr#10, causes reduced exploration, more avid mating, and improved reproductive performance (see the accompanying paper by Aprison and Ruvinsky in this issue of Current Biology) [6]. Here, we investigated the mechanism that restricts pheromone response to adult hermaphrodites. Unexpectedly, we found that attainment of developmental adulthood was not alone sufficient for the behavioral response to the pheromone. To modify exploratory behavior in response to male pheromone, adult hermaphrodites also require functional germline and egg-laying apparatus. We show that this dependence of behavior on the reproductive system is due to feedback from the vulva muscles that reports ongoing reproduction to the nervous system. Our results reveal an activity-dependent conduit by which the reproductive system continuously licenses adult behaviors, including appropriate responses to the pheromones of the opposite sex. More broadly, our results suggest that signals from peripheral organs may serve as an important component of assuring age-appropriate functions of the nervous system.

Keywords: C. elegans; adulthood; behavioral maturation; exploratory behavior; neuromodulation; neuronal feedback; reproduction; serotonin; sex pheromone.

Figure 1.. Larvae do not respond to ascr#10.

(A) Exploratory behavior of sexually immature L4 larval hermaphrodites compared to young adults. (B) Latency of mating initiation with sexually immature L4 larval hermaphrodites. (C) Expression of tph-1::YFP in NSM and (D) HSN neurons in L4 larval hermaphrodites. Fluorescence and fluorescence overlaid with differential contrast images. Arrows point to cell bodies. An asterisk marks the position of the developing vulva. Anterior is to the left and ventral is down. In A, each square represents one animal, in C and D, each triangle represents one imaged neuron. ** p<0.01. See Figure S1 for additional related results and Data S1 for primary data and details of statistical analyses.

Figure 2.. Functional egg-laying apparatus regulates behavioral response to ascr#10.

(A) Exploratory behavior of hermaphrodites with compromised reproduction. (B) Exploratory behavior of fertile, but vulvaless animals. (C) Exploratory behavior of fertile animals that have dramatically reduced brood sizes (~3 in cpb-3, ~2 in lin-41 vs. ~13 in N2). (D) Exploratory behavior in mutant hermaphrodites lacking HSN neurons. (E) Exploratory behavior in adult hermaphrodites in which HSN has been chemogenetically silenced. Each square represents one animal. * p<0.05, ** p<0.01. See Figure S2 for additional related results and Data S1 for primary data and details of statistical analyses.

Figure 3.. The role of vm2 vulva muscles in regulating behavioral response to ascr#10.

(A,B) Exploratory behavior of mutant hermaphrodites with defective muscle excitability. (C) Exploratory behavior in a mutant hermaphrodite in which vm2 vulva muscles do not synapse with HSN neurons. (D) Exploratory behavior in hermaphrodites with functional or silenced vm2 vulva muscles. In this experiment silencing commenced prior to initiation of egg-laying. Comparison of the two grey data series demonstrates that silencing of vm2 muscles increases exploratory movement. (E) Expression of tph-1::YFP in NSM and (F) HSN neurons in adults with functional or silenced vm2 vulva muscles. Arrows mark cell bodies. Anterior is to the left and ventral is down. In A-D, each square represents one animal. In E,F, each triangle represents one neuron. * p<0.05, ** p<0.01. See Figure S3 for additional related results and Data S1 for primary data and details of statistical analyses.

Figure 4.. The role of active reproductive state in regulating exploratory behavior.

(A) Exploratory behavior in hermaphrodites before and after chemogenetic silencing of vm2 vulva muscles. Note, top of this panels shows exploratory behavior in hermaphrodites after they have laid their first egg. The experiment in top portion of the panel was carried out on animals that were between 48 and 60h after release from L1 arrest. In the bottom, the same animals between 60 and 72h. Comparing grey data series between animals before and after vm2 silencing (right portion of the panel) demonstrates that silencing of vm2 increases exploratory movement. (B) Expression of tph-1::YFP in NSM and HSN neurons in adults exposed to ascr#10 before they have laid their first egg (48 to 54 hours after release from L1 arrest) and after they have laid their first egg (54 to 60 hours after release from L1 arrest). Arrows point to the cell body. Anterior is to the left and ventral is down. (C) Exploratory behavior of individual hermaphrodites during four episodes (~6 hours each) that encompass key events across the reproductive span. First and second episodes were carried out immediately before and immediately after the onset of egg-laying, respectively. In the third time window hermaphrodites had few remaining self-sperm, while in the fourth they exhausted supplies of self-sperm. The same animals were tested across these four time windows – grey traces connect exploratory movements of the same animal. Age is expressed as hours after release from L1 arrest. (D) A model showing exploratory behavior superimposed on the egg-laying schedule. Purple trace represents average performance from Figure 4C, egg-laying data (represented as grey ovals) are from [38]. In A and C, each square represents one animal. In B, each triangle is one neuron. *p<0.05, ** p<0. 01, *** p<0.001. See Figure S4 for additional related results and Data S1 for primary data and details of statistical analyses.