Ambient O₂ levels set CO₂ avoidance.

<p>A. <i>C. elegans</i> avoids shallow gradients of CO₂ more strongly when O₂ levels are low. The CO₂ gradients used are indicated above the graph. B. Artificially high O₂ levels can reduce CO₂ avoidance further. **, <i>p</i><0.01; *, p<0.05, Student's <i>t</i>-test.</p

Shallow and steep CO₂ gradients evoke qualitatively different Ca²⁺ responses in AFD.

<p>A. Ca²⁺ responses evoked in AFD by CO₂ switches indicated at top, involving linear 0–5% and 5%–0% CO₂ gradients occurring over 2 minutes. This corresponds to a rate of change of 0.04% CO₂/second. The upper part of the panel shows traces obtained from 10 randomly selected individual AFD neurons; an average trace is plotted at the bottom. Animals imaged in this panel were acclimated to 22°C. B, C. Ca²⁺ responses evoked in AFD by CO₂ switches indicated at top, involving linear switches from 0–5% and 5%–0% CO₂ occurring over 8 minutes. This corresponds to a change of 0.01% CO₂/second. The upper part of the panels shows traces obtained from 10 randomly selected individual AFD neurons; average traces are plotted at the bottom. Animals imaged in (B) were acclimated to 22°C; those in (C) were acclimated at 15°C. For each panel, individual and average traces are at the same scale. The scale bar in each panel represents 0.4 YFP/CFP ratio unit.</p

TTX-7 acts in RIA interneurons to promote CO₂ avoidance when ambient O₂ levels are low.

<p>A–C. Mutations in <i>ttx-7</i> strongly reduce CO₂ avoidance at 11% O₂ but have relatively weak effects on CO₂ avoidance at 21% O₂. ns, not significant, ** <i>p</i><0.01, Student's <i>t</i> test. D. Expressing <i>ttx-7</i> specifically in RIA neurons, using the <i>glr-3</i> or <i>glr-6</i> promoters, restores strong CO₂ avoidance to <i>ttx-7</i> mutants assayed at 11% O₂. Expressing <i>ttx-7</i> specifically in AFD, using the <i>gcy-8</i> promoter, or in AWB and AWC, using the <i>odr-1</i> promoter does not rescue the <i>ttx-7</i> phenotype. ns, not significant, ** <i>p</i><0.01, Student's <i>t</i> test. E. CO₂ evokes a Ca²⁺ response in RIA neurons. Ca²⁺ responses were measured in immobilized animals cultivated at 22°C using a <i>pglr-6::YC3.60</i> Ca²⁺ reporter. Shading highlights gas switch times. The CO₂/O₂ stimulus train used is indicated above the plot.</p

Acclimation temperature and ambient O₂ levels have additive effects on CO₂ avoidance.

<p>A. Animals cultivated at 22°C but assayed at 15°C avoid CO₂ more strongly when ambient O₂ is low. B–C. Reducing O₂ levels from 21% to 11% increases CO₂ avoidance regardless of acclimation temperature or assay temperature. In A–C, ** <i>p</i><0.01, * <i>p</i><0.05, ns, not significant, Student's <i>t</i> test. D. Coalitions of CO₂ sensors elicit CO₂ escape responses according to O₂ environment, temperature experience, and CO₂ stimulus dynamics. Triangles represent sensory neurons and hexagons interneurons. Black arrows indicate synapses. Several neurons respond to CO₂ (blue arrows), each with distinct kinetics. Each of these neurons also responds to other sensory cues, as indicated. Three of the four identified CO₂ sensors synapse directly onto the RIA interneuron. The fourth, AFD, synapses onto AIY which in turn synapses on RIA. The URX O₂ sensor also synapses onto RIA. Note each neuron makes additional connections besides the ones highlighted here.</p

Re-configuring O₂ sensing circuits by altering the <i>npr-1</i> and <i>glb-5</i> genes alters CO₂ avoidance behavior.

<p>A. Tuning of CO₂ avoidance behavior by different O₂ concentrations in N2 (Bristol), CB4856 (Hawaiian), <i>npr-1(ad609)</i>, <i>glb-5(Haw)</i>; <i>npr-1(ad609)</i>, and <i>glb-5(Haw)</i> animals. All assays used a 1–0% CO₂ gradient. Statistical comparisons are to the N2 response at the corresponding O₂ concentration, *** <i>p</i><0.001; ** <i>p</i><0.01; *<i>p</i><0.05 (Anova, <i>p</i> value protected Fisher's LSD). B. <i>gcy-35</i> and <i>gcy-36</i> mutants strongly avoid CO₂ regardless of genotype at the <i>npr-1</i> locus. Statistical comparisons are to the <i>npr-1</i> response at the corresponding O₂ concentration. ***, <i>p</i><0.001, **, <i>p</i><0.01, Anova, Bonferroni corrected <i>p</i> value). C. CB4856 (Hawaii) animals defective in <i>gcy-36</i> strongly avoid CO₂ regardless of O₂ levels. Statistical comparisons are to the CB4856 response at the corresponding O₂ concentration. ***, <i>p</i><0.001, **, <i>p</i><0.01, Anova, Bonferroni corrected <i>p</i> value). D, E. Tonic Ca²⁺ levels in URX neurons of <i>glb-5(Haw); npr-1</i> animals kept at 21% O₂ and 17% O₂ is lower than Ca²⁺ levels in URX in <i>npr-1</i> animals kept at the corresponding O₂ concentrations. Ca²⁺ measurements were made using cameleon YC2.60.</p

Disrupting <i>gcy-35</i> or <i>gcy-36</i> confers strong CO₂ avoidance regardless of ambient O₂.

<p>A. <i>gcy-35</i> or <i>gcy-36</i> mutants strongly avoid the high CO₂ half of a 1–0% CO₂ gradient regardless of ambient O₂. Statistics refer to comparisons to N2 at 21% O₂. ***, <i>p</i><0.001, Anova, Bonferroni corrected <i>p</i>-value. None of the strains apart from N2 show significant differences between assays carried out at 21% and 11% O₂ (Student's <i>t</i>-test). B. The CO₂-avoidance phenotype of <i>gcy-36</i> mutants can be rescued by expressing <i>gcy-36</i> cDNA in AQR, PQR and URX, using <i>gcy-32</i> or <i>gcy-36</i> promoters, or in URX alone, using the <i>flp-8</i> promoter. **, <i>p</i><0.01, ***, <i>p</i><0.001, Anova, Bonferroni corrected <i>p</i>-value.</p

CO₂ avoidance is modulated by acclimation temperature.

<p>A. Assay for <i>C. elegans</i> CO₂ responses. Animals navigate a defined CO₂ gradient in a microfluidic device. The chemotaxis index is calculated by counting animals in two halves of the device, using the formula shown. B–D. Chemotaxis indices for animals cultivated at either 15°C or 22°C and assayed in different CO₂ gradients at either 15°C or 22°C. **, <i>p</i><0.01; n.s., not significant, Student's <i>t</i>-test. E. A mutation in <i>ttx-1</i>, which is specifically required to confer AFD neural identity, disrupts modulation of CO₂ avoidance by acclimation temperature. Assays were performed in 3%–0% CO₂ gradients. **, <i>p</i><0.01; n.s., not significant, Student's <i>t</i>-test.</p

Acclimation temperature alters CO₂-evoked Ca²⁺ responses in AFD neurons.

<p>In animals cultivated at 22°C a rise and fall in CO₂ evokes a complex Ca²⁺ response in AFD neurons (A). Ca²⁺ initially falls when CO₂ begins to increase, then rises. When CO₂ levels fall, there is a Ca²⁺ spike. By contrast, animals cultivated at 15°C show a simple response to the same stimulus (B). C–D The effect of acclimation temperature on CO₂-evoked Ca²⁺ responses in AFD neurons is unaltered in <i>snb-1</i> mutants defective in synaptobrevin. E. Ca²⁺ responses evoked in AFD by a 0%–1%–3%–5%–3%–1%–0% CO₂ stimulus train in animals acclimated to 22°C. Shading highlights switch times. Acclimation temperature is shown for each panel under genotype.</p