Figure 3. The same set of interneurons contributes to CO2 avoidance and attraction.

(A-B) In animals raised at high (2.5%) CO₂, silencing of AIZ and ablation of RIA enhances CO₂ attraction, while ablation of AIY reduces CO₂ attraction. Graphs show responses to 0.1% CO₂ (A) or 0.25% CO₂ (B). *p<0.05, ***p<0.001, one-way ANOVA with Dunnett’s post-test (A) or Kruskal-Wallis test with Dunn’s post-test (B). n=6–20 trials per genotype and condition.

(C) Ablation of AIY reduces CO₂ attraction. By contrast, animals with more active AIY neurons due to AIY-specific expression of pkc-1(gf) show enhanced CO₂ attraction. Animals were raised at high (2.5%) CO₂. **p<0.01, ***p<0.001, two-way ANOVA with Dunnett’s post-test. n=6–20 trials per genotype and condition.

(D) Ablation of RIA enhances CO₂ attraction. By contrast, animals with more active RIA neurons due to RIA-specific expression of pkc-1(gf) show reduced CO₂ attraction. Animals were raised at high (2.5%) CO₂. **p<0.01, ***p<0.001, two-way ANOVA with Dunnett’s post-test. n=8–24 trials per genotype and condition.

(E) Silencing of AIZ enhances CO₂ attraction. Animals were raised at high (2.5%) CO₂. *p<0.05, two-way ANOVA with Sidak’s post-test. n=8–20 trials per genotype and condition.

(F-G) Animals with RIA neurons transiently silenced by expression of the histamine-gated chloride channel HisCl1 in RIA show enhanced CO₂ attraction (F). By contrast, animals with AIY neurons transiently silenced using the same approach show reduced CO2 attraction (G). Responses to 0.1% CO₂ (F) and 0.25% CO₂ (G) were tested for wild-type animals and animals expressing HisCl1 in either RIA or AIY without histamine (negative control) and with histamine (neuronal silencing); changes in CO₂ response were observed only in the presence of histamine. Animals were raised at high (2.5%) CO₂. *p<0.05, **p<0.01, one-way ANOVA with Sidak’s post-test (F) or Kruskal-Wallis test with Dunn’s post-test (G). n=8–14 trials per genotype and condition.

(H-I) Ablation of RIG delays the shift from CO₂ attraction to repulsion in animals raised at high (2.5%) CO₂ and transferred to ambient CO₂. Animals were tested for their response to 2.5% CO₂ after 3, 6, or 9 h at ambient CO₂. Responses were compared to those of animals of the same genotype raised at ambient CO₂ and high CO₂ to determine the percent change in valence (see Methods). Graphs show the percent change in valence after 6 h (H) or as a function of time (I). **p<0.01, ***p<0.001, Kruskal-Wallis test with Dunn’s post-test (H) or two-way ANOVA with Sidak’s post-test (I). n=8–26 trials per genotype, time point, and condition.

(J-K) Ablation of AIY delays the shift, and ablation of RIA accelerates the shift, from CO₂ repulsion to attraction in animals raised at ambient CO₂ and transferred to high (2.5%) CO₂. Animals were tested for their response to 2.5% CO₂ after 1, 3, 6, or 9 h at high CO₂. Responses were compared to those of animals of the same genotype raised at ambient CO₂ and high CO₂ to determine the percent change in valence (see Methods). Graphs show the percent change in valence after 3 h

(J) or as a function of time (K). *p<0.05, ***p<0.001, Kruskal-Wallis test with Dunn’s post-test (J) or two-way ANOVA with Dunnett’s post-test (K). n=8–16 trials per genotype, time point, and condition.

For A-K, graphs show medians and interquartile ranges. See also Figure S3.