Receptor-type guanylate cyclase is required for carbon dioxide sensation by Caenorhabditis elegans

Abstract

CO(2) is both a critical regulator of animal physiology and an important sensory cue for many animals for host detection, food location, and mate finding. The free-living soil nematode Caenorhabditis elegans shows CO(2) avoidance behavior, which requires a pair of ciliated sensory neurons, the BAG neurons. Using in vivo calcium imaging, we show that CO(2) specifically activates the BAG neurons and that the CO(2)-sensing function of BAG neurons requires TAX-2/TAX-4 cyclic nucleotide-gated ion channels and the receptor-type guanylate cyclase GCY-9. Our results delineate a molecular pathway for CO(2) sensing and suggest that activation of a receptor-type guanylate cyclase is an evolutionarily conserved mechanism by which animals detect environmental CO(2).

Fig. 1.

BAG neurons are activated by CO₂. (A Left) BAG neuron cell bodies respond to CO₂. R/R₀ is the YFP to CFP ratio (R) divided by the average YFP to CFP ratio of the first 10 frames (R₀). (A Right) Dose-response curve for CO₂. ΔR/ΔR_max is the maximal ratio change caused by presentation of a given CO₂ stimulus normalized to the maximal ratio change measured (evoked by 10% CO₂). (B) ASH, ADL, and AWB do not respond to 10% CO₂. The yellow trace is the average response of the BAG neurons to 10% CO₂. The white area around each trace represents the SEM; the lower black traces indicate the stimulus onset and duration (n = 5–18 animals for each condition or genotype). For all graphs, error bars represent SEMs.

Fig. 2.

Prolonged CO₂ exposure desensitizes BAG neurons. (A and B) Acute CO₂ avoidance after either a (A) 1-min or (B) 5-min exposure to CO₂. For each treatment condition, the stimulus used during the preexposure is indicated followed by the stimulus used for the acute avoidance assay (n = 11–15 trials for each treatment). (C and D) Prolonged pulses of either (C) 10% or (D) 5% CO₂ results in BAG neuron desensitization (n = 7 animals for each condition). (E) Prior exposure to 5% CO₂ blocks the response to 10% CO₂ (n = 7 animals). The white area around each trace represents the SEM; the lower black traces indicate the stimulus onset and duration.

Fig. 3.

A subset of genes required for acute CO₂ avoidance is required for activation of BAG neurons by CO₂. tax-2 and tax-4 mutations eliminate and an rgs-3 mutation reduces CO₂-evoked calcium transients in the BAG neurons. npr-1, daf-11, daf-2, daf-7, and gcy-33; gcy-31 mutations do not affect BAG neuron responses to CO₂ (n = 6–18 animals for each genotype). Dashed line indicates the mean maximum response of WT animals. The white area around each trace represents the SEM; the lower black traces indicate the stimulus onset and duration. (Lower Right) Summary of CO₂ responses for each genotype. Mean responses were calculated as the mean response after CO₂ exposure in the time interval 19.0–19.9 s, normalized to the mean response before CO₂ exposure in the time interval 0.4–1.3 s. Error bars represent average SEMs after CO₂ exposure (19.0–19.9 s).

Fig. 4.

Transcriptional profiling of embryonic BAG neurons. BAG neuron-enriched genes were organized according to gene ontology (GO) terms referring to biological process. The chart depicts the 15 most frequent GO terms, with the number of genes in each listed in parentheses. Shown below are the most abundant transcripts in the largest category (signaling), with fold change (FC) indicating relative enrichment in the BAG neuron profile.

Fig. 5.

gcy-9 is required for CO₂ sensing by BAG neurons. (A) gcy-9 mutants do not respond to CO₂ (n = 10–32 trials for each genotype). (B) gcy-9 mutants do not show CO₂-evoked activity in the BAG neurons (n = 9 animals). (C) Expression of gcy-9 specifically in the BAG neurons completely rescues CO₂ avoidance behavior (Left) and partially rescues CO₂-evoked calcium transients of BAG neurons (Right). Left has n = 22–39 trials for each genotype, and Right has n = 8 animals. The yellow dashed line shows the mean response of WT animals, and the green dashed line shows the mean response of gcy-9(tm2816) animals. The white area around each trace represents the SEM; the lower black traces indicated the stimulus onset and duration. For this experiment, CO₂ avoidance behavioral assays were performed on all three genotypes in parallel.