We examined age-dependent body weight gain in male CD157 and CD38 KO mice (Fig. 1). Twelve-month-old (designated as middle-aged) CD157 KO mice had a significantly higher body weight (Fig. 1A) and body mass index (BMI; Fig. 1B) than 8-week-old mice (young adult): t(12) = 10.37, p < 0.0001 for body weight; t(12) = 9.369, p < 0.0001 for BMI.
In sharp contrast, middle-aged (12 month-old) CD38 KO male mice had a similar range of body weight to that of young adult mice (8 weeks old) (Fig. 1C; t(7) = 0.4878, p = 0.6406). The BMI of middle-aged CD38 KO mice was slightly but significantly lower than that of young adult mice (Fig. 1D; t(7) = 2.417 p = 0.0463). Therefore, middle-aged CD38 KO mice did not gain body mass but rather exhibited a decrease in body mass, while middle-aged CD157 KO mice became obese.
3.1. Locomotion and anxiety-related behavior in a new environment in the open field test
Next, we measured locomotor activity and social behavior in an open field test (Supplementary Figure 1). Anxiety-related behavior was examined in the habituation stage of the open field test, in which mice were exposed to a new environment. The distance traveled (Fig. 2A) and average speed (Fig. 2B) were significantly lower in middle-aged male CD157 KO mice than in young adult male CD157 KO mice (t(14) = 3.637, p = 0.0027 for distance; t(14) = 3.683, p = 0.0025 for average speed). The immobilization time was also longer in middle-aged mice (Fig. 2C; t(14) = 5.006, p = 0.0002). Middle-aged CD157 KO mice remained near the wall of the open field and did not cross the central area. Thus, the average time in the inner zone was significantly lower in middle-aged mice (Fig. 2D; t(14) = 2.8370, p = 0.0132).
CD38 KO male mice displayed a similar pattern of locomotion with aging to CD157 KO mice, including less distance traveled in the arena (Fig. 2E, (t(12) = 2.300, p = 0.0402), a lower average speed (Fig. 2F, (t(12) = 2.260, p = 0.0432), and a longer immobility time (Fig. 3G, (t(12) = 2.979, p = 0.0115). Middle-aged and young adult CD38 KO mice remained in the center area for a similar amount of time, as evidenced by the lack of a significant difference between the two groups (Fig. 2H, t(12) = 0.7930, p = 0.4432). The results showed that middle-aged CD157 KO mice displayed decreased locomotor activity and severe anxiety-related behavior, while middle-aged CD38 KO mice displayed reduced locomotion with less anxiety-related behavior.
3.2. Anxiety-related behavior in the open field test with a non-social object
When a non-social object (empty wire cage) was placed in the center area of the open field, the locomotor activity of middle-aged CD157 KO mice was similar to that observed in the habituation stage: distance traveled (Fig. 3A, (t(14) = 2.611, p = 0.0205), average speed (Fig. 3B, (t(14) = 2.652, p = 0.0189), and time spent immobile (Fig. 3C, (t(14) = 3.027, p = 0.0091). Additionally, middle-aged CD157 KO mice tended to spend less time in the center zone of the arena (Fig. 3D, (t(14) = 1.609, p = 0.1299).
In contrast, when a non-social object was placed in the center of the open field arena, middle-aged CD38 KO mice exhibited a locomotor activity level similar to that of young adult KO mice: distance traveled (Fig. 3E, (t(12) = 0.6049, p = 0.5565), average speed (Fig. 3F, (t(12) = 0.5904, p = 0.5659), time spent immobile (Fig. 4G, (t(12) = 0.3589, p = 0.7259), and time spent in the inner zone (Fig. 3H, (t(12) = 0.4551, p = 0.6571). Additionally, in the presence of the non-social object, age-dependent changes were observed in middle-aged CD157 KO mice, but such changes were not present in middle-aged CD38 KO mice.
3.3. Anxiety-related behavior in the open field test with a social object
Social behavior was assayed in the third stage of the open field test. The social object (an unfamiliar WT mouse of the same sex) was placed in a wire cage at the center of the arena (Fig. 4). Middle-aged CD157 KO mice displayed a significantly decreased distance traveled (Fig. 4A; t(14) = 2.827, p = 0.0135), lower average speed (Fig. 4B; t(14) = 2.793, p = 0.0144), and increased time spent immobile (Fig. 4C; t(14) = 3.088, p = 0.0080). Moreover, middle-aged CD157 KO mice remained for a markedly shorter time in the inner zone of the arena but without significance (Fig. 4D; t(14) = 1.973, p = 0.0686).
Surprisingly, middle-aged male CD38 KO mice displayed nearly the same level of activity as young adult KO mice in the social stage of the open field test. The parameters in Fig. 4E–H were indistinguishable between young and middle-aged mice: distance traveled (Fig. 4E; t(12) = 0.4042, p = 0.6931), average speed (Fig. 4F; t(12) = 0.3755, p = 0.7138), time immobile (Fig. 4: t(12) = 0.7427, p = 0.4720), and time spent in the inner zone (Fig. 4H, t(12) = 0.3348, p = 0.7436).
It was clear that in all stages in the open field test, anxiety-related behavior was more severe in middle-aged CD157 KO male mice than in young adult CD157 KO mice. However, although middle-aged male CD38 KO mice displayed anxiety-related behavior during the habituation stage, no age-associated changes were observed in middle-aged CD38 KO mice in the open field test with the non-social and social targets.
3.4. Sociability test in a three-chamber apparatus
A three-chamber test was performed to evaluate social behavior (Fig. 5). In the sociability stage, mice usually choose to stay with a social target rather than a non-social target [43]. In CD157 KO mice (Fig. 5A), two-way ANOVA showed a significant effect of age (F1,22 = 10.19, p = 0.0042), effect of object (F1,22 = 10.38, p = 0.0039) and age × object interaction (F1,22 = 9.097, p = 0.0064). Young adult male CD157 KO mice spent significantly more time with the mouse (Str1) than with the object (Bonferroni’s post hoc comparison p < 0.001). Middle-aged CD157 KO mice spent equal time with the social and non-social objects. Moreover, the time spent with Str1 by middle-aged CD157 KO mice was significantly shorter than that for young adult mice (Bonferroni’s post hoc test p < 0.001).
For CD38 KO mice (Fig. 5D), two-way ANOVA revealed a significant effect of age (F1,14 = 13.65, p = 0.0024), effect of object (F1,14 = 42.77, p < 0.0001), and age × object interaction (F1,14 = 5.721, p = 0.0314). CD38 KO mice of both ages spent more time with the social object than with the non-social object (Bonferroni’s post hoc p < 0.0001 for young adult mice, p < 0.05 for middle-aged mice). The interaction time with Str1 by middle-aged CD38 KO mice was shorter than that for young adult KO mice (Bonferroni’s post hoc p < 0.01). The results showed that in male CD38 KO mice, interest in both social and non-social targets seems to decrease in middle age, but sociability was not lost, even at middle age.
3.5. Social novelty preference in the three-chamber test
Mice usually show interest in new social targets, which is termed a social novelty preference. Both young adult and middle-aged CD157 KO mice spent nearly equal time with Str1 and Str2, although the time spent in the cage with a new target mouse (Str2) was slightly longer than that spent with the familiar mouse (Str1), without significance (Fig. 5B; two-way ANOVA revealed a significant effect of age (F1,22 = 9.647, p = 0.0052) but not an effect of object (F1,22 = 0.5934, p = 0.4493), or an object × age interaction (F1,22 = 0.0007, p = 0.9793). Regarding the total time spent in the social target zone, the time spent by middle-aged CD157 KO mice was significantly shorter than that of young adult mice (Fig. 5C; t(11) = 2.601, p = 0.0246), indicating that social interaction significantly decreased with aging.
In CD38 KO mice, two-way ANOVA showed a significant effect of age (F1,14 = 9.244, p = 0.0088) and object (F1,14 = 10.29, p = 0.0063) but not an age × object interaction (F1,14 = 0.1537 p = 0.7009). Both age groups of CD38 KO mice spent the same time with Str1 and Str2. Nevertheless, less total time was spent in the social contact zone by middle-aged mice (Fig. 5F; t(7) = 3.182, p = 0.0154).
The results of the three-chamber test indicate that middle-aged CD157 KO mice did not display sociability and social novelty preference; however, middle-aged CD38 KO mice remained sociable and showed a tendency of social novelty preference, similar to WT mice.