Figure 1. Inhaling CO₂ during a retrieval tone augments the effect of extinction.

(A) Schematic of protocol for auditory aversive conditioning (also called fear conditioning), memory modification, and testing. The protocol contained four main components. (1) On day 1, mice underwent aversive conditioning (Av. Cond.) during which they received 6 tones paired with 6 foot shocks in context X. (2) On day 2, mice were placed in a modified chamber (context Y) and were randomly assigned to one of four groups. After 5 min, one group was presented with a single tone (Ret) and then remained in the chamber for two more minutes. One group breathed 10% CO₂ for 7 min. One group breathed 10% CO₂ and was presented with the tone. And one group received neither CO₂ nor retrieval. (3) Thirty minutes later in context Y, all mice were treated with two blocks of extinction protocols (Ext 1 and Ext 2), each consisting of 20 tones; mice in the retrieval groups received 19 instead of 20 tones in the first block of extinction tones. (4) On day 7, mice were tested for freezing in response to 4 tones delivered in context Y (Spontaneous Recovery). Thirty minutes later they were tested for freezing in response to 4 tones delivered in context X (Renewal). (B–E) Data are the percentage of time mice were freezing in response to tones during aversive conditioning (B), the Ret/CO₂ intervention (C), extinction (D), and during the two memory tests (E). (F) Schematic of protocol for auditory aversive conditioning, memory modification, and testing. The protocol was identical to that shown in panel A with the exception that the retrieval tone was presented and then 100 s later mice inhaled 10% CO₂ for 7 min. (G–J) Data are the percentage of time mice were freezing during aversive conditioning (G), retrieval (H), extinction (I), and the two memory tests (J). Data are mean±SEM. n = 16 mice in each group for panels B-E and n = 8 mice in each group for panels G-J. * indicates p<0.05 by one-way ANOVA with Tukey’s post hoc multiple comparison for panels B-E. For panel E, No ret vs Ret, p=0.0001; No ret vs No ret + CO₂, p=0.9683; No ret vs Ret + CO₂, p<0.0001; Ret vs No ret + CO₂, p<0.0001; Ret vs Ret + CO₂, p=0.0300; No ret + CO₂ vs Ret + CO₂, p<0.0001. For panel E right, No ret vs Ret, p<0.0001; No ret vs No ret + CO₂, p=0.9974; No ret vs Ret + CO₂, p<0.0001; Ret vs No ret + CO₂, p<0.0001; Ret vs Ret + CO₂, p<0.0001; No ret + CO₂ vs Ret + CO₂, p<0.0001. There were no statistically significant differences between groups in panels G-J by unpaired Student’s t-test.

DOI: http://dx.doi.org/10.7554/eLife.22564.002

Figure 1—figure supplement 1. Inhaling CO₂ during a retrieval tone augments the effect of extinction.

In Figure 1, we assayed memory 5 days after extinction. Previous studies also tested memory 1 day after extinction. With that shorter interval, a retrieval cue did not produce a decrease in freezing in the spontaneous recovery test (Monfils et al., 2009; Clem and Huganir, 2010). But, retrieval did decrease freezing in the renewal test. We performed a study like that in Figure 1, except we waited only 1 day after extinction to test memory. Consistent with earlier studies (Monfils et al., 2009; Clem and Huganir, 2010), 1 day after extinction, retrieval did not significantly reduce freezing in the spontaneous recovery test, but it did reduce freezing in the renewal test. Moreover, CO₂ inhalation enhanced the effects of retrieval in the renewal test. (A) Schematic of protocol for auditory aversive conditioning, memory modification, and testing. The protocol was identical to that shown in Figure 1A and described in its legend with the exception that the interval between extinction and testing was 24 hr instead of 5 days. (B–E) Data are the percentage of time mice were freezing in response to tones during aversive conditioning (B), the retrieval/CO₂ intervention (C), extinction (D), and the two memory tests (E). Data are mean±SEM. n = 16 mice in each group. * indicates p<0.05 by ANOVA with Tukey’s post hoc multiple comparison. There were no statistically significant differences between groups during the spontaneous recovery test. In the Renewal test: No ret vs Ret, p<0.0001; No ret vs No ret + CO₂, p=0.5080; No ret vs Ret + CO₂, p<0.0001; Ret vs No ret + CO₂, p=0.0027; Ret vs Ret + CO₂, p=0.0318; No ret + CO₂ vs Ret + CO₂, p<0.0001.

DOI: http://dx.doi.org/10.7554/eLife.22564.004

Figure 1—figure supplement 2. The effect of CO₂ on memory retrieval is concentration dependent.

We tested the effect of varying the CO₂ concentration (5%, 10%, and 20%) and found that inhaling 10% CO₂ during retrieval caused the greatest effect. Therefore, in subsequent experiments we used that concentration. (A) Schematic of protocol for auditory aversive conditioning, memory modification, and testing. The protocol was identical to that shown in Figure 1—figure supplement 1A with the exception that the CO₂ concentration was 5%, 10% or 20%. (B–E) Data are the percentage of time mice were freezing in response to tones during aversive conditioning (B), the Ret/CO₂ intervention (C), extinction (D), and during the two memory tests (E). Data are mean±SEM. n = 8–10 mice in each group. * indicates p<0.05 by ANOVA with Tukey’s post hoc multiple comparison. There were no statistically significant differences between groups during the spontaneous recovery test. In the Renewal test: No ret vs Ret +5% CO₂, p=0.0058; No ret vs Ret +10% CO₂, p<0.0001; No ret vs Ret +20% CO₂, p=0.0461; Ret +10% CO₂ vs Ret +20% CO₂, p=0.4693.

DOI: http://dx.doi.org/10.7554/eLife.22564.005