Sex-based Differences in C. elegans Responsiveness to Aversive Stimuli

Behavioral differences between sexes are evident across many species. The underlying mechanisms surrounding such differences are not fully elucidated, however, due to the complexities of animal behavior. The nematode Caenorhabditis elegans (C. elegans) is a well-characterized, genetically amenable species with two sexes, hermaphrodites (XX) and males (XO). This makes it an appropriate model system for investigating sex-based behavioral differences. Chemosensation in C. elegans is mediated by exposed ciliated sensory neurons, one of which is ASH. ASH is a polymodal nociceptor that elicits reversal when an animal encounters aversive stimuli. We hypothesized that hermaphrodite and male C. elegans worms respond differently to stimuli detected by ASH such as the bitter tastant quinine, the detergent sodium dodecyl sulfate (SDS), and the heavy metal copper (CuCl2). Wild-type assay-age hermaphrodites and males were picked from a nematode growth media (NGM) plate with E. coli OP50 and kept on an NGM plate without food for 10 minutes prior to assaying. A drop of aversive stimulus was placed in front of a forward-moving animal, and the animal’s response was recorded. A positive response is backwards movement within 4 seconds after contact with the stimulus. Our results reveal a quantifiable difference in how wild-type hermaphrodite and male C. elegans respond to aversive stimuli. Specifically, wild-type males are less responsive than hermaphrodites to quinine, SDS, and CuCl2. Further investigations will be conducted through experiments with C. elegans strains in which hermaphrodites have masculinized, and males have feminized nervous systems or subsets of neurons. Through these experiments, we aim to explore potential sites of difference that lead to these observable differences in responsiveness to aversive stimuli

Using a Robust and Sensitive GFP-Based cGMP Sensor for Real-Time Imaging in Intact Caenorhabditis elegans.

cGMP plays a role in sensory signaling and plasticity by regulating ion channels, phosphodiesterases, and kinases. Studies that primarily used genetic and biochemical tools suggest that cGMP is spatiotemporally regulated in multiple sensory modalities. FRET- and GFP-based cGMP sensors were developed to visualize cGMP in primary cell culture and Caenorhabditis elegans to corroborate these findings. While a FRET-based sensor has been used in an intact animal to visualize cGMP, the requirement of a multiple emission system limits its ability to be used on its own as well as with other fluorophores. Here, we demonstrate that a C. elegans codon-optimized version of the cpEGFP-based cGMP sensor FlincG3 can be used to visualize rapidly changing cGMP levels in living, behaving C. elegans We coexpressed FlincG3 with the blue-light-activated guanylyl cyclases BeCyclOp and bPGC in body wall muscles, and found that the rate of change in FlincG3 fluorescence correlated with the rate of cGMP production by each cyclase. Furthermore, we show that FlincG3 responds to cultivation temperature, NaCl concentration changes, and sodium dodecyl sulfate in the sensory neurons AFD, ASEL/R, and PHB, respectively. Intriguingly, FlincG3 fluorescence in ASEL and ASER decreased in response to a NaCl concentration upstep and downstep, respectively, which is opposite in sign to the coexpressed calcium sensor jRGECO1a and previously published calcium recordings. These results illustrate that FlincG3 can be used to report rapidly changing cGMP levels in an intact animal, and that the reporter can potentially reveal unexpected spatiotemporal landscapes of cGMP in response to stimuli

Aversive Behavior in the Nematode <i>C</i>. <i>elegans</i> Is Modulated by cGMP and a Neuronal Gap Junction Network

<div><p>All animals rely on their ability to sense and respond to their environment to survive. However, the suitability of a behavioral response is context-dependent, and must reflect both an animal’s life history and its present internal state. Based on the integration of these variables, an animal’s needs can be prioritized to optimize survival strategies. Nociceptive sensory systems detect harmful stimuli and allow for the initiation of protective behavioral responses. The polymodal ASH sensory neurons are the primary nociceptors in <i>C</i>. <i>elegans</i>. We show here that the guanylyl cyclase ODR-1 functions non-cell-autonomously to downregulate ASH-mediated aversive behaviors and that ectopic cGMP generation in ASH is sufficient to dampen ASH sensitivity. We define a gap junction neural network that regulates nociception and propose that decentralized regulation of ASH signaling can allow for rapid correlation between an animal’s internal state and its behavioral output, lending modulatory flexibility to this hard-wired nociceptive neural circuit.</p></div

cGMP generation in ASH dampens quinine sensitivity.

<p>The ASH-selective <i>osm-10</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref048" target="_blank">48</a>] and <i>srb-6</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref080" target="_blank">80</a>] promoters were used to drive expression of a blue light-inducible guanylyl cyclase (BlgC) or blue light-inducible adenylyl cyclase (BlaC) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref081" target="_blank">81</a>]. <i>osm-10p</i> drives expression in ASH and weakly in ASI in the head, and in PHA and PHB in the tail. <i>srb-6p</i> drives expression in the ASH, ADL and ADF head sensory neurons, and in PHA and PHB in the tail. Adult animals expressing BlgC or BlaC were tested without blue light exposure (white bars) or after a 30 second exposure (grey bars). (A) While <i>lite-1(lof)</i> animals responded robustly to 5 mM quinine, similar to wild-type animals (p > 0.4), transgenic animals expressing BlgC displayed a significantly diminished response following blue light exposure (p < 0.0001 for each ASH-selective promoter). Transgenic animals expressing BlaC remained sensitive following blue light exposure (p > 0.05 when compared to wild-type animals). (B) While <i>odr-1(lof)</i> animals were hypersensitive in their response to 1 mM quinine, transgenic animals expressing BlgC displayed a significantly diminished response following blue light exposure (p < 0.0001). Transgenic animals expressing BlaC remained hypersensitive following blue light exposure (p > 0.5 when compared to <i>odr-1(lof)</i> animals). The percentage of animals responding is shown. The combined data of ≥ 2 independent lines and n ≥ 95 transgenic animals is shown. Alleles used: <i>lite-1(ce314)</i> loss-of-function, <i>odr-1(n1936)</i> loss-of-function. WT = the N2 wild-type strain. lof = loss-of-function. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.s004" target="_blank">S4 Fig</a>.</p

Model for ODR-1 modulation of ASH-mediated nociceptive signaling.

<p>(A) Our working model is that the transmembrane guanylyl cyclase ODR-1 functions in the AWB, AWC and ASI sensory neurons to decrease <i>C</i>. <i>elegans</i> behavioral sensitivity to the bitter tastant quinine (and possibly the volatile odorant octanol). Removal of food leads to cGMP accumulation, at least in the AWCs, likely by direct/indirect activation of ODR-1 in these neurons. Based on the re-annotated wiring diagram (WormWiring.org), we propose that cGMP then flows via gap junction connections from the site of its production in the ODR-1-expressing AWB/AWC/ASI sensory neurons, through ADF, AFD and AIA, to the ASH nociceptors. Once in ASH, cGMP activates the cGMP-dependent protein kinase EGL-4, which likely directly phosphorylates the regulator of G protein signaling proteins RGS-2 and RGS-3, stimulating their activity [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref058" target="_blank">58</a>]. RGS-2 and RGS-3 downregulate Gα proteins that signal downstream of G protein-coupled receptors (GPCRs) that are activated by ligands such as quinine and octanol. When animals are well-fed, cGMP levels are low and there is only minimal inhibition of G protein-coupled signaling in ASH. Upon food remove, cGMP influx into ASH activates EGL-4, resulting in diminished nociceptive behavioral sensitivity to a subset of ASH-detected aversive stimuli. Decentralized modulation of ASH sensitivity may allow an animal to integrate multiple environmental cues with its internal state to maximize the appropriateness of its response to its surroundings. (B) The same diagram depicted in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.g005" target="_blank">Fig 5A</a> is shown here, but with only those gap junction connections originally reported in White et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref002" target="_blank">2</a>] included. (C) The left column shows a list of the neurons reported by White et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref002" target="_blank">2</a>] to make gap junctions with ASH, while the right column lists the neurons currently annotated at WormWiring.org to make gap junctions with ASH. The neurons shown in bold are those that are common to both lists.</p

ODR-1 functions in adult sensory neurons.

<p>(A) ODR-1 expression in the AWB, AWC and ASI sensory neurons is sufficient to rescue the behavioral hypersensitivity of <i>odr-1(lof)</i> animals. The <i>str-1</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref066" target="_blank">66</a>], <i>ceh-36p3</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref065" target="_blank">65</a>], <i>gpa-4</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref064" target="_blank">64</a>], <i>trx-1</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref063" target="_blank">63</a>] and <i>srbc-66</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref062" target="_blank">62</a>] promoters were used to drive expression of wild-type <i>odr-1</i> (genomic sequence) in <i>odr-1(lof)</i> animals. These promoters drive expression in the following cells: <i>str-1</i> (AWB), <i>ceh-36p3</i> (AWC), <i>gpa-4</i> (ASI), <i>trx-1</i> (ASJ and intestinal cells), <i>srbc-66</i> (ASK). While more <i>odr-1(lof)</i> animal respond to 1 mM quinine than do wild-type animals, co-expressing <i>odr-1</i> in the AWB, AWC and ASI sensory neurons returned response to wild-type levels (p > 0.1). (B) ODR-1 functions in adult animals to regulate behavioral sensitivity. Adult <i>odr-1(lof)</i> animals expressing <i>odr-1</i> (genomic sequence) under the control of a heat shock inducible promoter (hsp) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref068" target="_blank">68</a>] were tested without heat shock (white bars) or 4 hours after heat shock treatment (grey bars). While <i>odr-1(lof)</i> animals have a hypersensitive response to dilute (1 mM) quinine, heat shock induced expression of <i>odr-1</i> in adult <i>odr-1(lof)</i> animals abolished this hypersensitivity and returned quinine response to the degree seen in wild-type animals (p > 0.1 when comparing <i>odr-1(lof)</i> animals with heat shock treatment to wild-type animals either with or without heat shock). (C) The extracellular domain of ODR-1 is not required for regulation of quinine sensitivity. In <i>odr-1(lof)</i> animals, the <i>odr-1</i> promoter was used to drive expression of either wild-type ODR-1 (genomic sequence), ODR-1 lacking its extracellular domain (ΔECD) or ODR-1 with a point mutation (E874A) that abolishes GTP binding in the catalytic domain. ODR-1(ΔECD) rescued the hypersensitivity of <i>odr-1(lof)</i> animals as well as wild-type ODR-1 (p > 0.1). Expression of ODR-1(E874A) had no effect on response sensitivity (p > 0.5 when compared to <i>odr-1(lof)</i> animals). The percentage of animals responding is shown. The combined data of ≥ 3 independent lines and n ≥ 120 transgenic animals is shown in each panel. Allele used: <i>odr-1(n1936)</i> loss-of-function. WT = the N2 wild-type strain. lof = loss-of-function. n.s. = not significant.</p

cGMP generation in ADF dampens quinine sensitivity.

<p>The ADF-specific <i>srh-142</i> promoter [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref079" target="_blank">79</a>] was used to drive expression of a blue light-inducible guanylyl cyclase (BlgC) or blue light-inducible adenylyl cyclase (BlaC) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref081" target="_blank">81</a>] in <i>lite-1(lof)</i> or <i>inx-4(lof);lite-1(lof)</i> animals. <i>srh-142p</i> drives expression only in the two ADF head sensory neurons. Adult <i>lite-1(lof)</i> or <i>inx-4(lof);lite-1(lof)</i> animals expressing BlgC or BlaC were tested without blue light exposure (white bars) or after a 30 second exposure and 30 second recovery (grey bars). While <i>lite-1(lof)</i> animals responded robustly to 5 mM quinine, similar to WT animals (p > 0.1), transgenic animals expressing BlgC in the ADFs displayed a significantly diminished response following blue light exposure (p < 0.0001). Transgenic animals expressing BlaC did not display a diminished response following blue light exposure (p > 0.2 when compared to wild-type animals). <i>inx-4(lof);lite-1(lof)</i> transgenic animals expressing either BlgC or BlaC did not display a diminished response following blue light exposure (p > 0.7 when compared to wild-type animals). The percentage of animals responding is shown. The combined data of ≥ 3 independent lines and n ≥ 120 transgenic animals is shown in each panel. Alleles used: <i>lite-1(ce314)</i> loss-of-function and <i>inx-4(ok2373)</i> loss-of-function. WT = the N2 wild-type strain. lof = loss-of-function. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.s005" target="_blank">S5 Fig</a>.</p

Response to quinine is modulated by feeding status.

<p>The response of wild-type animals to dilute quinine diminishes upon food deprivation (p < 0.00001 for 2.5 mM when comparing animals assayed “off food” versus “on food”). The percentage of animals responding is shown. The combined data of n ≥ 60 animals is shown for each concentration. Animals were assayed 10–20 minutes after transfer to plates with or without food (no bacterial lawn). conc = concentration. n.s. = not significant. WT = the N2 wild-type strain.</p