Contribution of the cyclic nucleotide gated channel subunit, CNG-3, to olfactory plasticity in Caenorhabditis elegans.

In Caenorhabditis elegans, the AWC neurons are thought to deploy a cGMP signaling cascade in the detection of and response to AWC sensed odors. Prolonged exposure to an AWC sensed odor in the absence of food leads to reversible decreases in the animal's attraction to that odor. This adaptation exhibits two stages referred to as short-term and long-term adaptation. Previously, the protein kinase G (PKG), EGL-4/PKG-1, was shown necessary for both stages of adaptation and phosphorylation of its target, the beta-type cyclic nucleotide gated (CNG) channel subunit, TAX-2, was implicated in the short term stage. Here we uncover a novel role for the CNG channel subunit, CNG-3, in short term adaptation. We demonstrate that CNG-3 is required in the AWC for adaptation to short (thirty minute) exposures of odor, and contains a candidate PKG phosphorylation site required to tune odor sensitivity. We also provide in vivo data suggesting that CNG-3 forms a complex with both TAX-2 and TAX-4 CNG channel subunits in AWC. Finally, we examine the physiology of different CNG channel subunit combinations

The SUN protein UNC-84 is required only in force-bearing cells to maintain nuclear envelope architecture

The nuclear envelope (NE) consists of two evenly spaced bilayers, the inner and outer nuclear membranes. The Sad1p and UNC-84 (SUN) proteins and Klarsicht, ANC-1, and Syne homology (KASH) proteins that interact to form LINC (linker of nucleoskeleton and cytoskeleton) complexes connecting the nucleoskeleton to the cytoskeleton have been implicated in maintaining NE spacing. Surprisingly, the NE morphology of most Caenorhabditis elegans nuclei was normal in the absence of functional SUN proteins. Distortions of the perinuclear space observed in unc-84 mutant muscle nuclei resembled those previously observed in HeLa cells, suggesting that SUN proteins are required to maintain NE architecture in cells under high mechanical strain. The UNC-84 protein with large deletions in its luminal domain was able to form functional NE bridges but had no observable effect on NE architecture. Therefore, SUN-KASH bridges are only required to maintain NE spacing in cells subjected to increased mechanical forces. Furthermore, SUN proteins do not dictate the width of the NE

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

Gap junctions regulate quinine sensitivity.

<p>(A) Loss-of-function mutations in the innexin gap junction genes <i>inx-4</i> and <i>inx-20</i> resulted in behavioral hypersensitivity to 1 mM quinine (p < 0.001 for each when compared to wild-type animals). (B) The <i>inx-4</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref035" target="_blank">35</a>], <i>osm-10</i> (ASH, ASI, PHA and PHB) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref048" target="_blank">48</a>], <i>srd-10</i> (ASH, PHA and PHB), <i>srh-142</i> (ADF) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref079" target="_blank">79</a>] and <i>odr-1</i> promoters were used to drive expression of wild-type <i>inx-4</i> cDNA in <i>inx-4(lof)</i> animals. ASH-selective expression significantly dampened the <i>inx-4(lof)</i> hypersensitive response (p < 0.0001), while expression in the ODR-1-expressing neurons had no effect (p > 0.4). (C) INX-4 functions in adult animals to regulate behavioral sensitivity. Adult <i>inx-4(lof)</i> animals expressing <i>inx-4</i> cDNA 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>inx-4(lof)</i> animals had a hypersensitive response to dilute (1 mM) quinine, heat shock induced expression of <i>inx-4</i> in adult <i>inx-4(lof)</i> animals dampened this hypersensitivity (p < 0.0001 when comparing <i>inx-4(lof);hsp</i>::<i>inx-4</i> animals with heat shock treatment to <i>inx-4(lof)</i> animals either with or without heat shock). (D) INX-4 gap junctions are required for ODR-1 modulation of quinine sensitivity. Expression of only <i>odr-1</i> or <i>inx-4</i> in <i>odr-1(lof);inx-4(lof)</i> animals had no effect on quinine hypersensitivity (p > 0.2 when comparing rescue of either to <i>odr-1(lof);inx-4(lof)</i> animals). Simultaneous expression of <i>inx-4</i> in ASH using the <i>osm-10p</i> promoter [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref048" target="_blank">48</a>] and expression of <i>odr-1</i> either behind its native promoter or using AWB, AWC and ASI cell-selective promoters (<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>], and <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>], respectively), rescued hypersensitivity (p < 0.0001). 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>inx-4(ok2373)</i> loss-of-function, <i>inx-20(ok426)</i> loss-of-function and <i>odr-1(n1936)</i> loss-of-function. WT = the N2 wild-type strain. lof = loss-of-function. n.s. = not significant. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.s002" target="_blank">S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.s003" target="_blank">S3</a> Figs.</p

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

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

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

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

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