Fig. 7
Fear behavior in endothelial-CHRM3 knockout and 3xTgAD mice. (a) Schematic diagram of fear conditioning test. Freezing response in the 3–6- and 9–12-month-old Chrm3f/f, VECre:Chrm3f/f, and 3xTgAD mice measured as percent time spent freezing during (b i–ii) Habituation period (average of first 300 s) of Day 1. 1-Way ANOVA with Bonferroni’s post hoc test, F(2,29) = 15.19, P < 0.0001 for 3–6-month-old group, and F(2,20) = 7.259, P = 0.0043 for 9–12-month-old group, (c i–iv) Fear acquisition during the first 5 time points of Day 1 and average fear acquisition used as a proxy for learning memory rates between mouse genotypes. Repeated measures ANOVA with Bonferroni’s post hoc test, F(2,29) = 0.8840, P = 0.4240 for 3–6-month-old group, and F(2,19) = 20.75, P < 0.0001 for 9–12-month-old group, (d i,ii) Average fear recall/memory of the first 5 timepoints from Day 2 used as a proxy for fear memory. 1-Way ANOVA with Bonferroni’s post hoc test, F(2,29) = 1.067, P = 0.3573 for 3–6-month-old group, and F(2,20) = 0.8737, P = 0.4328 for 9–12-month-old group, (e i,ii) Fear extinction in response to all 30 tones of Days 2 and 3 used as a proxy for extinction learning. Repeated measures ANOVA with Bonferroni’s post hoc test, F(2,26) = 4.017, P = 0.030 for 3–6-month-old group, and F(2,17) = 3.209, P = 0.066 for 9–12-month-old group. #P < 0.022 significantly different from corresponding values in VECre:Chrm3f/f mice, (f i,ii) Fear extinction post hoc analysis representing the average of the last 5 timepoints of Day 3. 1-Way ANOVA with Bonferroni’s post hoc test, F(2,29) = 5.501, P = 0.0094 for 3–6-month-old group, and F(2,20) = 3.932, P = 0.0363 for 9–12-month-old group, and (g i,ii) Average fear extinction retention assessed by comparing freezing during the first 5 tones of Day 3 to freezing during the last 5 tones of Day 2. 1-Way ANOVA with Bonferroni’s post hoc test, F(2,29) = 0.8604, P = 0.4335 for 3–6-month-old group, and F(2,20) = 0.03907, P = 0.9618 for 9–12-month-old group. N = 8 for VECre:Chrm3f/f and 3xTgAD, and N = 16 for Chrm3f/f male and female mice. Means are ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 significantly different from corresponding values in Chrm3f/f mice and #P < 0.05, ##P < 0.01, and ###P < 0.001 significantly different from corresponding values in VECre:Chrm3f/f mice.
