Timetable of the behavioral tests.

<p>The behavioral tests were started according to the given timetable (−11d) until sacrifice of animals (0d).</p

Spatial learning in morris water maze.

<p>(A–C) The escape latencies (in ms) of the training were displayed in five blocks (x-axis 1–5). One block consisted of four consecutive trials. The escape latencies decreased throughout the training days. (A–B) For the 4 (4w) and 20 weeks (20w) AL and CR fed groups two-way ANOVA for repeated measures (group vs. block) revealed no significance for the main factor group (4 weeks: F_1,17 = 0.934, P = 0.347; 20 weeks: F_1,18 = 1.523, P = 0.233), but a significant effect for the main factor block (F_1,4 = 29.753, P<0.001; F_1,4 = 62.468, P<0.001). The interaction between these variables is significant between 4 weeks AL and CR fed mice (F_1,4 = 4.807, P = 0.002), but no significance could be shown for interaction of 20 weeks fed groups (F_1,4 = 0.918, P = 0.458). Two-way ANOVA for repeated measures (group vs. block) of the 74 weeks (74w) AL and CR fed mice indicated a significant effect for the main factors (group: F_1,15 = 4.816, P = 0.044; block: F_1,4 = 25.078, P<0.001), but there was no statistically significant interaction between group and block (F_1,4 = 2.312, P = 0.068). Subsequent Holm-Sidak tests with an overall significance level of P = 0.05 showed only significant differences of escape latency in block 2 respectively between 4 and 20 weeks AL and CR fed mice (Fig. 6A–B). (C) Between 74 weeks AL and CR fed groups there were significant differences of escape latency in blocks 4 and 5. Values are given as mean±SEM; ANOVA, post-hoc pairwise comparison tests: ^§P<0.05 vs. AL. (D–F In this test trial, the number of platform crossings during 60 s was measured. (D and E) Two-way ANOVA of the 4 and 20 weeks fed groups (group vs. platform) showed no significances for the main factor group and for interaction between group and platform (4 weeks: group: F_1,72 = 0.536, P = 0.466; interaction: F_1,3 = 2.249, P = 0.090; 20 weeks: group: F_1,72 = 1.188, P = 0.279; interaction: F_1,3 = 0.644, P = 0.589). Values are given as mean±SEM; ANOVA, post-hoc pairwise comparison tests: ^#P<0.05 vs. PF4; ^§P<0.05 vs. AL. (F) The 74 weeks AL fed mice (n = 7) did not show significantly more crossings of PF4. Values are given as mean±SEM; ANOVA, post-hoc pairwise comparison tests: ^#P<0.05 vs. PF4; ^§P<0.05 vs. AL.</p

Locomotor activity and anxiety-like behavior in the open field (OF) and elevated plus maze (EPM).

<p>(A) Anxiety-like behavior was measured by the ratio of center distance to total distance in the OF. The ratio did not detect significant differences between ad libitum (AL) and caloric-restricted (CR) fed mice (AL: n_{4w, 20w, 74w} = 10, 10, 8; CR: n_{4w, 20w, 74w} = 9, 10, 10). However, 74 weeks (74w) vs. 4 weeks (4w) CR fed mice revealed a decreased ratio indicating a raised level of anxiety-like behaviour (F_2,26 = 6.551, P = 0.005). Values are given as mean±SEM; ANOVA, post-hoc pairwise comparison tests: ^*P<0.05 vs. 74w. (B) The defecation rate in OF, a parameter of anxiety-like behavior, was significantly higher in all ad libitum (AL) fed mice when compared to age-related caloric-restricted (CR) fed mice (4 weeks: F_1,17 = 4.729, P = 0.044; 20 weeks: F_1,18 = 9.175, P = 0.007; 74 weeks: F_1,16 = 5.854, P = 0.028. Values are given as mean ± SEM; ANOVA, post-hoc pairwise comparison tests: ^§P<0.05 vs. AL. (C) The ratio of open arms visits to total visits as a parameter of anxiety-like behavior was significantly increased only in 4 weeks CR fed mice when compared to age-matched AL fed mice (F_1,16 = 6.115, P = 0.025) and significantly decreased upon lifelong CR (F_2,24 = 10,713, P<0.001). Values are given as mean±SEM; ANOVA, post-hoc pairwise comparison tests: ^§P<0.05 vs. AL, ^$P<0.05 vs. 4w.</p

Experimental workflow.

<p>4 weeks old AL mice were fed either ad libitum (AL) or caloric restricted (CR, 60% of ad libitum) for 4 weeks, 20 weeks or 74 weeks. An additional group of mice underwent a late-onset CR which started at the age of 66 weeks.</p

Appearance and body weight of mice.

<p>(A) Image of one 74 weeks ad libitum (AL) and one caloric-restricted (CR) fed C57BL/6J mouse. These mice exemplarily showed that in general CR mice were smaller in body size and had fewer external age-related signs as showing denser coat and barely greyed hair than the AL mice. (B) Starting weight (n_{4w, 20w, 74w} = 20, 20, 17) and body weight (g) of the 4 (4w), 20 (20w) and 74 (74w) weeks ad libitum (AL) and caloric-restricted fed mice were shown (AL: n_{4w, 20w, 74w} = 10, 10, 7; CR: n_{4w, 20w, 74w} = 10, 10, 10). In all AL fed mice, 20 and 74 weeks CR fed mice body weight was significantly increased when compared to their starting weight (4 weeks: F_1,28 = 197.69, P<0.001; 20 weeks: F_1,28 = 364.85, P<0.001; 74 weeks: F_1,22 = 346.88, P<0.001). Compared with the age-matched AL fed mice the CR fed mice had significantly lower weights (4 weeks: F_1,18 = 176.14, P<0.001; 20 weeks: F_1,18 = 442.02, P<0.001; 74 weeks: F_1,15 = 156.76, P<0.001). Values are given as mean±SEM; ANOVA, post-hoc pairwise comparison tests: ^&P<0.05 vs. starting weight; ^§P<0.001 vs. AL.</p

Motor coordination on the rotarod and accelerod test.

<p>(A–C) The slip offs of the mice (AL: n_{4w, 20w, 74w} = 10, 10, 8; CR: n_{4w, 20w, 74w} = 10, 10, 10) from the rotarod were measured on 8 training trials (x-axis 1–8). (A) There were no significant differences in falling downs from the rotarod detected between 4 weeks (4w) ad libitum (AL) and caloric-restricted (CR) fed mice (group: F_1,18 = 2.373, P = 0.141; trial: F_1,7 = 1.961, P = 0.066; interaction: F_1,7 = 1.919, P = 0.072). (B) Two-way repeated measures ANOVA (group vs. trial) of 20 weeks groups slip offs showed only a statistical significance for the main factor trial, but not for main factor group and for interaction (group: F_1,18 = 1.301, P = 0.269; trial: F_1,7 = 8.210, P<0.001; interaction: F_1,7 = 1.102, P = 0.366). (C) There are significant differences between 74 weeks AL and CR fed mice for training trial 1–3, but 74 weeks AL fed mice showed a fast improvement of their rotarod performance. 74 weeks CR fed mice exhibited no significant differences between the slip offs of trial 1–8. Two-way repeated measures ANOVA (group vs. trial) of 74 weeks groups slip offs revealed for the main variables group and trial as well as for interaction between these factors statistically significant differences (group: F_1,16 = 20.453, P<0.001; trial: F_1,7 = 24.527, P<0.001; interaction: F_1,7 = 11.732, P<0.001; Fig. 4C). Values are given as mean±SEM; ANOVA, post-hoc pairwise comparison tests: ^§P<0.05 vs. AL; ⁱP<0.05 vs. trial 5–8; ⁱⁱP<0.05 vs. trial 4–8; ⁱⁱⁱP<0.05 vs. trial 2–8. (D–F) In the accelerod test the maximum speed (rpm) was measured in 8 test trials (x-axis 1–8) (AL: n_{4w, 20w, 74w} = 10, 10, 8; CR: n_{4w, 20w, 74w} = 10, 10, 10). Values are given as mean±SEM; ANOVA, post-hoc pairwise comparison tests: ^§P<0.05 vs. AL; ^∧P<0.05 vs. trial 7–8.</p