A Distributed Chemosensory Circuit for Oxygen Preference in C. elegans

The nematode Caenorhabditis elegans has complex, naturally variable behavioral responses to environmental oxygen, food, and other animals. C. elegans detects oxygen through soluble guanylate cyclase homologs (sGCs) and responds to it differently depending on the activity of the neuropeptide receptor NPR-1: npr-1(lf) and naturally isolated npr-1(215F) animals avoid high oxygen and aggregate in the presence of food; npr-1(215V) animals do not. We show here that hyperoxia avoidance integrates food with npr-1 activity through neuromodulation of a distributed oxygen-sensing network. Hyperoxia avoidance is stimulated by sGC-expressing oxygen-sensing neurons, nociceptive neurons, and ADF sensory neurons. In npr-1(215V) animals, the switch from weak aerotaxis on food to strong aerotaxis in its absence requires close regulation of the neurotransmitter serotonin in the ADF neurons; high levels of ADF serotonin promote hyperoxia avoidance. In npr-1(lf) animals, food regulation is masked by increased activity of the oxygen-sensing neurons. Hyperoxia avoidance is also regulated by the neuronal TGF-β homolog DAF-7, a secreted mediator of crowding and stress responses. DAF-7 inhibits serotonin synthesis in ADF, suggesting that ADF serotonin is a convergence point for regulation of hyperoxia avoidance. Coalitions of neurons that promote and repress hyperoxia avoidance generate a subtle and flexible response to environmental oxygen

sGC and <i>ocr-2</i> TRPV Mutations Restore Food Regulation of Hyperoxia Avoidance to <i>npr-1</i>

<p>(A–J) In all panels, dotted lines indicate aerotaxis in the presence of a small amount of bacterial food. (A) Aerotaxis of wild-type N2 animals. (B) Aerotaxis of <i>npr-1</i> mutants. (C) Aerotaxis of <i>qaIs2241</i> strain (URX, AQR, PQR killed). (D) Aerotaxis of <i>npr-1 qaIs2241</i> strain. (E) Aerotaxis of <i>gcy-35</i> mutants. (F) Aerotaxis of <i>gcy-35; npr-1</i> double mutants. (G) Aerotaxis of <i>osm-9</i> mutants. (H) Aerotaxis of <i>osm-9; npr-1</i> double mutants. (I) Aerotaxis of <i>ocr-2</i> mutants. (J) Aerotaxis of <i>ocr-2; npr-1</i> double mutants. For (A–J), asterisks denote distributions different by chi-square analysis at <i>p</i> < 0.01 from the same genotype without food. <i>n</i> ≥ 3 assays per genotype and condition, 80–100 animals/assay. Error bars denote SEM. (K) Hyperoxia avoidance index as defined in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040274#pbio-0040274-g001" target="_blank">Figure 1</a>. Asterisks denote values significantly different from the same genotype without food at <i>p <</i> 0.05 by <i>t</i> test. Error bars denote SEM. Double crosses indicate that <i>ocr-2</i> mutants are significantly regulated by food using chi-square analysis of the entire distribution (<i>p <</i> 0.01), and that <i>ocr-2; npr-1</i> on food is significantly different from <i>ocr-2; npr-1</i> off food and <i>npr-1</i> on food (<i>p <</i> 0.01 by chi-square analysis) and not significantly different from <i>ocr-2</i> mutants on food.</p

Multiple TRPV-Expressing Neurons Contribute to Hyperoxia Avoidance

<div><p>(A) Aerotaxis of <i>TRPV</i> single mutants. The median preferred oxygen concentration of <i>ocr-2</i> mutants was significantly higher than N2 (<i>p <</i> 0.05 by Dunnett test).</p> <p>(B) Aerotaxis of <i>osm-9</i> mutants in which ASH, PHA, and PHB were rescued with an <i>osm-10::osm-9</i> transgene [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040274#pbio-0040274-b026" target="_blank">26</a>]. Animals rescued for ASH function were preselected for avoidance of high osmolarity, an ASH behavior (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040274#s4" target="_blank">Materials and Methods</a>).</p> <p>(C) Aerotaxis of <i>osm-9</i> mutants in which ADF was rescued by expression from a <i>cat-1::osm-9</i> transgene [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040274#pbio-0040274-b026" target="_blank">26</a>].</p> <p>(D) Aerotaxis of <i>tph-1</i> mutants.</p> <p>(E) Aerotaxis of <i>tph-1</i> mutants rescued in ADF neurons using a <i>srh-142::tph-1::gfp</i> transgene [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040274#pbio-0040274-b028" target="_blank">28</a>].</p> <p>(F) Aerotaxis of <i>tph-1</i> mutants rescued in NSM neurons using a <i>ceh-2::tph-1::gfp</i> transgene [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040274#pbio-0040274-b028" target="_blank">28</a>].</p> <p>(G) Aerotaxis of <i>TRPV; qaIs2241</i> double mutants.</p> <p>(H) Aerotaxis of <i>tph-1; qaIs2241</i> double mutants.</p> <p>For (A–H), asterisks denote distributions different by chi-square analysis at <i>p</i> < 0.01 from the first distribution in the panel, unless otherwise noted. <i>n</i> ≥ 3 assays per genotype, 80–100 animals/assay. Error bars denote SEM.</p> <p>(I) Hyperoxia avoidance index as defined in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040274#pbio-0040274-g001" target="_blank">Figure 1</a>. Asterisks, values different from N2 controls at <i>p</i> < 0.01 by Dunnett test. Single crosses, values different from the <i>osm-9</i> or <i>tph-1</i> control at <i>p <</i> 0.05 by Bonferroni <i>t</i> test with N2 and mutant controls. Double crosses, values different from <i>qaIs2241</i> controls at <i>p <</i> 0.01 by Dunnett test. NS, not significant. Error bars denote SEM.</p> <p>(J) ASH and ADF sensory neurons promote hyperoxia avoidance through the activity of TRPV channels <i>osm-9</i> and <i>ocr-2.</i> Serotonin production in ADF by <i>tph-1,</i> which is regulated by TRPV channels, also drives this behavior.</p></div

TGF-β Signaling Mediates Food Suppression of Hyperoxia Avoidance

<p>(A–E), (H), (I) In all panels, dotted lines indicate aerotaxis in the presence of a small amount of bacterial food. (A) Aerotaxis of wild-type N2 animals. (B) Aerotaxis of <i>daf-7</i> mutants. (C) Aerotaxis of <i>daf-3</i> mutants. (D) Aerotaxis of <i>daf-7; daf-3</i> double mutants. (E) Aerotaxis of <i>daf-3 npr-1</i> double mutants. (F–G) <i>daf-7::GFP</i> expression in ASI was reduced in a <i>tax-4</i> mutant. Anterior is to the left. (H) Aerotaxis of <i>tax-4; kyIs342</i> animals, which bear a transgene that rescues <i>tax-4</i> in URX, AQR, and PQR, but not in ASI or other neurons. (I) Aerotaxis of <i>tph-1; daf-3</i> double mutants. For (A–E), (H), and (I), asterisks denote distributions different by chi-square analysis at <i>p</i> < 0.01 from the same genotype without food. <i>n</i> ≥ 3 assays per genotype and condition, 80–100 animals/assay. Error bars denote SEM. (J) Hyperoxia avoidance index as defined in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040274#pbio-0040274-g001" target="_blank">Figure 1</a>. Asterisks, values significantly different from the same genotype without food at <i>p</i> < 0.05 by <i>t</i> test. In the absence of food, no strain is different from N2 controls by Dunnett test. In the presence of food, <i>daf-7, daf-3 npr-1,</i> and <i>tph-1; daf-3</i> are different from N2 at <i>p <</i> 0.01 and <i>tax-4; kyIs342</i> different from N2 at <i>p</i> < 0.05 by Dunnett test. Error bars denote SEM.</p

Levels of Serotonin in ADF Neurons Affect Food Regulation of Hyperoxia Avoidance

<p>(A–E) In all panels, dotted lines indicate aerotaxis in the presence of a small amount of bacterial food. (A) Aerotaxis of wild-type N2 animals. (B) Aerotaxis of <i>tph-1</i> mutants. (C) Aerotaxis of <i>tph-1; npr-1</i> double mutants. (D) Aerotaxis of <i>tph-1</i> animals rescued in ADF with a <i>srh-142::tph-1::gfp</i> transgene [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040274#pbio-0040274-b028" target="_blank">28</a>]. (E) Aerotaxis of wild-type animals expressing <i>tph-1</i> in ADF from a <i>srh-142::tph-1::gfp</i> transgene. For (A–E), asterisks denote distributions different by chi-square analysis at <i>p</i> < 0.01 from the same genotype without food. <i>n</i> ≥ 3 assays per genotype and condition, 80–100 animals/assay. Error bars denote SEM. (F) Hyperoxia avoidance index as defined in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040274#pbio-0040274-g001" target="_blank">Figure 1</a>. Asterisks, values significantly different from the same genotype without food at <i>p</i> < 0.05 by <i>t</i> test. Crosses, values significantly different from N2 on food at <i>p <</i> 0.01 by Dunnett test. NS, not significant. Error bars denote SEM.</p

Hyperoxia Avoidance by <i>npr-1</i> Mutants Requires sGC and TRPV Activity, but not Serotonin

<p>(A) Aerotaxis of <i>npr-1</i> mutants. (B) Aerotaxis of <i>npr-1; qaIs2241</i> strain. (C) Aerotaxis of <i>gcy-35; npr-1</i> double mutants. (D) Aerotaxis of <i>osm-9; npr-1</i> double mutants. (E) Aerotaxis of <i>ocr-2; npr-1</i> double mutants. (F) Aerotaxis of <i>tph-1; npr-1</i> double mutants. For (A–F), asterisks denote distributions different by chi-square analysis at <i>p</i> < 0.01 from the first distribution in the panel. <i>n</i> ≥ 3 assays per genotype, 80–100 animals/assay. Error bars denote SEM. For (B–E), all double mutants are significantly different from <i>npr-1</i> controls, but not significantly different from animals carrying the other mutation (<i>p</i> < 0.01 by chi-square analysis of the complete distribution). (G) Hyperoxia avoidance index as defined in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040274#pbio-0040274-g001" target="_blank">Figure 1</a>. Asterisks and double asterisks, values different from <i>npr-1</i> controls at <i>p</i> < 0.05 and <i>p</i> < 0.01, respectively, by Dunnett test. Error bars denote SEM.</p

Serotonin Production in ADF Neurons is Regulated by TGF-β Signaling

<div><p>(A–B) <i>tph-1::GFP</i> expression in wild-type (A) and <i>daf-7</i> (B) adults. Anterior is to the left. Two NSM neurons and two ADF neurons are visible in each animal.</p> <p>(C) Quantitation of <i>tph-1::GFP</i> fluorescence in ADF neurons. Asterisks, values different from N2 controls at <i>p</i> < 0.01 by Dunnett test. <i>daf-7</i> mutants were different from N2, <i>daf-3</i>, and <i>daf-7; daf-3</i> at <i>p</i> < 0.05 by Bonferroni <i>t</i> test. <i>n</i> ≥ 18 animals per genotype. Error bars denote SEM.</p> <p>(D) Model for food regulation of aerotaxis, combining genetic results from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040274#pbio-0040274-g006" target="_blank">Figure 6</a> with molecular results from this Figure.</p></div

Second and Third Principal Components of Aerotaxis Data (PC2, PC3)

<div><p>(A) Assays with the highest (blue) and lowest (red) values for the second principal component (PC2), out of 36 sets of assays examined. Wild-type N2 animals off food are included for comparison (black, thick lines).</p> <p>(B) Assays with the highest (blue) and lowest (red) values for the third principal component (PC3), out of 36 sets of assays examined. Wild-type N2 animals off food are included for comparison (black, thick lines).</p></div