By skyforbes 
Phenotypes of CD157KO mice
Although there have been fragmentary reports about CD157KO mice8,13,14,29,30, here, we systematically summarized the reports on social interaction behaviour and concentrations of related molecules. Like humans, wild-type (C57BL6/N) and CD157KO mice are social creatures that prefer to spend time with another mouse than with an inanimate object (Fig. 1a,b)31,32. As shown in Fig. 1a, when placed in a box with an unfamiliar mouse (Stranger 1) in the left zone and an inanimate object in the right zone, wild-type males spent nearly all of their time with the mouse (unpaired t-test, t(44) = 10.13, P 1b; t(24) = 4.346. P
Social behaviour of adult wild-type (BL6) or CD157KO male mice. (a–d) Three chamber test. (a) The Sociability stage. Time spent around wire mesh cages with the social target (a conspecific male mouse, stranger 1, str 1) or with a non-social object (usually a plastic test tube, object). (b) Delta sociability (the time difference spent between the social and nonsocial targets calculated from (a)). (c) Social preference (time spent with the familiar mouse, str1, or with new mouse, str2. (d) Delta social preference (the time difference spent between the unfamiliar and familiar social targets calculated from (c)). (e–h) The light-dark transition test. (e) The number of entries from light to dark zone. (f) Latency to the first entry from the light to dark zones. (g) Time spent in the hidden zone. (h) The average speed in the light zone. (i–l) The open field test. (i) The distance traveled in all arenas (i), average speed (j), immobile time (k), and time spent in the center zone (l). Oxytocin concentrations in the cerebrospinal fluid (CSF, m) and the hypothalamus (n). NAD+ concentrations in the cortex (o) and hypothalamus (p). Unpaired t-test. *P P P P
As shown in Fig. 1c, when the same test mice were reintroduced to the box with a second unfamiliar mouse (Stranger 2), wild-type mice spent approximately twice as much time with a new mouse than with the familiar one (Stranger 1; t(44) = 5.755, P 1d; t(24) = 0.8542, P = 0.4015).
Mice are nocturnal and cautious by nature. When placed in the light side of a light/dark box, mice experience some discomfort and typically find their way to the dark side, where they feel less stressed31. Wild-type males behaved this way, as indicated by a number of different parameters, such as the number of entries into the dark side (Fig. 1e), latency to the first entry (Fig. 1f), time in the hidden zone (Fig. 1g) and average speed in the light zone (Fig. 1h). However, CD157KO mice showed fewer entries (Fig. 1e; t(32) = 4.646, P 1f; t(32) = 2.399, P 1g; t(32) = 3.596, P 1h; t(32) = 4.165, P
The third test was the open field test for anxiety-like behaviour and locomotion activity33. Compared with wild-type mice, the distance moved (Fig. 1i; t(11) = 2.530, P 1j; t(11) = 2.504, P 1l; t(11) = 2.283, P 1k; t(11) = 1.11, P = 0.2908).
Next, when we measured OT levels as a biomarker in the cerebrospinal fluid (CSF; Fig. 1m; t(27) = 4.297, P 1n; t(11) = 4.746, P
The basal level of NAD+ was also measured in cortical and hypothalamic tissue (Fig. 1o,p) because the hypothalamus is the region in which OT is produced, and the cortex was used as the OT non-producing control region. CD157KO mice had a significantly lower NAD+ level in the cortex (t(13) = 1.196, P t(17) = 0.1982, P = 0.8452).
The above associations between social behaviour and OT concentrations in CD157KO male mice are in good agreement with those in previously published reports13,14,30.
Behaviours in CD157KO female mice are illustrated in Supplementary Fig. 1. Untreated female mice of both genotypes displayed stronger levels of interest for social objects than nonsocial targets (wild-type female mice in Supplemental Fig. 1a Control; t(30) = 7.521, P = 0.0001; and CD157KO females in Supplemental Fig. 1b Control; t(22) = 3.300, P = 0.0033). However, unlike male mice, both wild-type and CD157KO female mice did not show a preference for novel social objects (Control in Supplementary Fig. 1c; t(30) = 1.287 P = 0.2078; Control in Supplementary Fig. 1d; t(22) = 1.840, P = 0.0794) These results indicate that CD157KO male mice, but not female mice, are useful models for psychiatric disorders with social behavioural impairments, because they display fewer social interactions, anxiety-like behaviours and/or social avoidance. Based on these characteristics of CD157KO male mice, we started to examine the effects of oral NR on social behavioural impairment. Adult wild-type and CD157KO male mice treated daily with either saline or 3–26 mg NR/mouse (approximately 100–1000 mg/kg of body weight) for 12 days did not exhibit any apparent changes, including movement dysfunction.
NR corrects the social preference deficit of CD157KO males
We used three chamber tests to determine affinity for social targets (sociability) and new unfamiliar social targets compared with that for familiar targets (social preference). In control experiments with wild-type males, we performed daily gavage with either a saline placebo or 3 mg of NR for 12 days. These mice have a clear behavioural preference for a social target (Stranger 1) over a nonsocial target in sociability, which was not altered by NR (Figs. 2a,b; unpaired t-test, t(36) = 9.403, P t(32) = 14.98, P 2c; two-way ANOVA: effect of treatment, F1,74 = 0.7065, P = 0.4033; effect of genotype, F1,74 = 1.99, P = 0.1625; interaction, F1,74 = 0.6941, P = 0.4074).
Social interaction of adult male wild-type (C57BL/6 N and BL6) and CD157KO mice in the three-chamber test. The sociability stage (a–c) and social preference stages (d–f). Experimental schemes in the sociability task when a stimulus male mouse (Str 1) was placed in the left chamber (a) or in the social novelty task when a new target male mouse (Str 2) was placed in the right chamber (d). Group occupancy plots of test mice initially place in the center in the sociability stage (a) or social preference (d) by the three-chamber box test after oral administration of saline (PL) or nicotinamide riboside (NR, 3 mg/mouse) treatment for 12 days. (b) Time spent in the wire mesh cage with the social target mouse (a conspecific male mouse, Str 1) or a non-social object (a test tube, Obj). Unpaired t-test shows significace between Str 1 and Obj at ****P c) The delta sociability represents time difference between social and non-social targets. (e) Time spent by test wild-type (BL6) or CD157KO mice in the cage with a familiar (Str 1) or new unfamiliar (Str 2) target mouse is plotted. Note that social preference was observed in wild-type mice under 2 conditions (PL and NR). While no social preference was observed in CD157KO mice treated with saline (PL), preference was obvious in KO mice treated with NR. Unpaired t-test shows significance between Str 1 and Str 2 at *P P P P f) The time by NR-treated CD157 KO mice spent with an unfamiliar mice was significantly higher than that with a familiar mouse. Two-way ANOVA, effect of treatment, F1,74 = 6.412, P post hoc analysis shows P
CD157KO males had a somewhat more variable preference for the social target but still showed a strong preference for social interaction over the nonsocial object (Fig. 2a,b; t(42) = 7.449, P t(38) = 9.206, P
As shown in Fig. 2d,e, CD157KO mice displayed no preference for interaction with the new (Stranger 2) mouse (t(42) = 0.1583, P = 0.8749). Remarkably, however, daily gavage of NR reversed this lack of preference for Stranger 2 to a level indistinguishable from that in wild-type males (Fig. 2e, t(38) = 4.689, P 2f; two-way ANOVA: effect of treatment, F1,74 = 6.412, P P post hoc analysis; effect of genotype, F1,74 = 0.9138, P = 0.3422; interaction between genotypes and treatment, F1,74 = 2.067, P = 0.1547). These data indicate that the brains of CD157KO males retained plasticity and were able to recognize and be interested in new mice when they were given a nutritional intervention.
NR corrects the anxiety-related phenotype of CD157 males with respect to light/dark transitions
Provision of daily NR dramatically relieved behavioural impairments in the light/dark test as measured by the number of entries (Fig. 3a; two-way ANOVA; F1,34 = 5.766, P = 0.022; genotype F1,34 = 2,871, P = 0.0993; interaction between genotype and treatment, F1,34 = 2.109, P = 0.1556). Bonferroni’s post hoc analysis revealed a significant difference between placebo and NR in CD157KO mice, P 3b; two-way ANOVA; effect of genotype F1,34 = 3.106, P = 0.087; treatment, F1,34 = 12.62, P = 0.0011; genotype and treatment interaction, F1,34 = 4.695, P = 0.0374). Bonferroni’s post hoc tests showed a significant difference between placebo and NR in CD157KO mice, P 3c; two-way ANOVA; treatment, F1,34 = 5.363, P = 0.0267; interaction between genotype and treatment, F1,34 = 8.005, P = 0.0078). Bonferroni’s post hoc tests revealed a significant difference between placebo and NR in CD157KO mice, P 3d; two-way ANOVA; treatment F1,34 = 1.855, P = 0.1822; treatment between genotype interaction, F1,34 = 0.6007, P = 0.4437), except for a significant effect of genotype (F1,34 = 4.593, P = 0.0393). These data establish that CD157KO mice are capable of feeling the reward of being in the dark if they can overcome the inertia of being put in an uncomfortable place.
Effects of gavage supplementation of nicotinamide riboside (NR) for 12 days on anxiety-like behaviour in the light-dark transition test in wild-type (BL6) and CD157KO male mice, compared with that with saline treatment (PL). (a) Transition numbers between the two compartments. Two-way ANOVA; F1,34 = 5.766, P = 0.022; Bonferroni’s post hoc analysis revealed a significance between placebo and NR in CD157KO mice, P (b) Latency of the first entry into the dark zone. Two-way ANOVA; effect of treatment, F1,34 = 12.62, P = 0.0011; genotype and treatment interaction, F1,34 = 4.695, P = 0.0374. Bonferroni’s post hoc tests showed significance between placebo and nicotinamide riboside in CD157KO mice, P (c) Time spent in the dark zone. Two-way ANOVA; treatment, F1,34 = 5.363 P = 0.0267; interaction between genotype and treatment, F1,34 = 8.005, P = 0.0078). Bonferroni’s post hoc tests revealed significance between placebo and nicotinamide riboside in CD157KO mice, P d) The average speed in the light compartment.Two-way ANOVA; for genotype (F1,34 = 4.593, P = 0.0393). *P P P
NR does not correct anxiety-like behaviors in the open field test
In the open field, wild-type mice cover more distance at greater speed with less time immobile and more time in the centre than do CD157KO male mice. Daily NR did not rescue these behavioural deficits compared with the placebo treatment in CD157KO mice (Fig. 4; only the genotype effect was significant by two-way ANOVA; Fig. 4a; distance travelled, F1,20 = 11.71, P = 0.0027; Fig. 4b; average speed, F1,20 = 11.69, P = 0.0027; Fig. 4c; time immobile, F1,20 = 10.38, P = 0.0043; Fig. 4d; time spent in the centre of the open field arena, F1,20 = 10.32, P = 0.0044, no effect of treatment F1,20 = 1.506, P = 0.234). A trend toward significance was seen in the interaction between genotype and treatment, however F1,20 = 4.059, P = 0.0576.
The behaviour of male mice for 10 min in the open field test. Daily gavage supplementation of nicotinamide riboside (NR, 3 mg/mouse) for 12 days induced no effect on any behavior by wild-type (BL6) nor the behavior deficits by CD157KO mice, compared with those of placebo treatment (PL). Two way ANOVA reveals significant effects only in the genotype: (a) distance traveled, F1,20 = 11.71, P = 0.0027; (b) average speed, F1,20 = 11.69, P = 0.0027; (c) time immobile, F1,20 = 10.38, P = 0.0043; (d) time spent in the centre of the open field *P P
Social preference defecit and anxiety of CD157KO males are best corrected at a relatively low dose of NR
We examined the dose-dependence of oral NR on the restoration of social behavioural impairments observed in the three-chamber test (Fig. 5a,b) and light/dark box (Fig. 5c–f). Each of the following four metrics was the most substantially ameliorated at 3 mg per day: Fig. 5b, delta social preference to new mice (one-way ANOVA, F3, 69 = 3.873, P = 0.0128); Fig. 5c, latency of the first entry into the hidden (dark) compartment (one-way ANOVA, F3,41 = 3.698, P = 0.0191); Fig. 5d, number of transitions into the dark zone (one-way ANOVA, F3,41 = 3.272, P = 0.0306); and Fig. 5f, time spent in the hidden (dark) zone (one-way ANOVA, F3,41 = 3.357, P = 0.0278). However, no dose effect was detected for delta sociability (time to social target; Fig. 5a; one-way ANOVA, F3,69 = 0.03477, P = 0.9912) or average speed of locomotion in the light zone (Fig. 5e; one-way ANOVA, F3,41 = 1.142, P = 0.3435). Beneficial effects were still seen at 13 mg per day (P 5b), latency (Fig. 5c) and time in the dark (Fig. 5f) but were nearly eliminated at the highest dose of 26 mg/day (Fig. 5b–f).
Effects of different doses of nicotinamide riboside (NR) on CD157KO mice in the three-chamber and light-dark transition tests. Different doses (3–26 mg/mouse) of gavage NR was administered for 12 days. Sociability (a) and social preference (b) are shown as time with the Stranger 1 subtracted by that with the non-social target or as time with Stranger 2 subtracted that of Stranger 1, respectively, in the three chamber test. One-way ANOVA followed by Bonferroni’s post hoc comparison: F3,69 = 3.873, P = 0.0128: *P c-f) Three chamber box test. (c) Latency in first entry into the hidden (dark) compartment (One way ANOVA followed by Bonferroni’s post hoc comparison F3,41 = 3.698, P = 0.0191). (d) Transition numbers (One way ANOVA followed by Bonferroni’s post hoc comparison F3,41 = 3.272, P = 0.0306). Bonferroni’s post hoc comparison revealed *P e). No dose-dependent effect was found in the average speed. (f) Time spent in the hidden zone (One way ANOVA followed by Bonferroni’s post hoc comparison F3,41 = 3.357, P = 0.0278). Bonferroni’s post hoc comparison revealed *P
NR elevates brain NAD+ and cerebrospinal OT
NAD+ levels were determined in the cortex and hypothalamus of mice treated with NR. As shown in Fig. 6a,b, both wild-type and CD157KO mice showed that oral NR increased NAD+ in both brain regions. In the cortex, two-way ANOVA revealed an effect of treatment (F1,27 = 28.33, P F1,27 = 4.74, P = 0.0384), but there was no interaction between them (F1,27 = 0.05429, P = 0.8175). In hypothalamic tissues, two-way ANOVA indicated an effect of treatment (F1,42 = 36.58, P F1,42 = 0.5062, P = 0.4807) or interaction (F1,42 = 0.9283, P = 0.3408).
NAD+ and OT levels were determined in the cortex and hypothalamus or cerebrospinal fluid (CSF) of wild-type (BL6) and CD157KO mice treated with NR. (a,b) NR gavage at 3 mg/mouse for 12 days significantly elevated NAD+ concentrations in mice of both genotypes in the cortex (two-way ANOVA; the effect of treatment (F1,27 = 28.33, P F1,27 = 4.74, P = 0.0384) and the hypothalamus (two-way ANOVA; the effect of treatment (F1,42 = 36.58, P post hoc comparison shows a significant difference between placebo and NR in CD157KO mice (P c) The identical treatment of NR significantly increased OT concentrations in the CSF of CD157KO mice (Two-way ANOVA revealed a significant effect of interaction between treatment and genotypes (F1,60 = 7.341, P = 0.0088). Bonferroni’s post hoc comparison shows a significant difference between placebo and NR in CD157KO mice (P d) OT concentrations in the hypothalamus in wild-type and CD157KO mice showed no significant difference after treatment with NR (Two-way ANOVA showed the only effect of genotype (F1,36 = 12.59, P = 0.0011). *P P P P
OT plays important roles in sociability and the ability to overcome irrational fears33. Moreover, cADPR formation has been linked to OT release11,12,15, and OT administration has been used to correct ASD-like phenotypes in CD157KO mice13,29. To test the hypothesis that the NAD+-elevating and ASD phenotype-reverting effects of NR are accompanied by increased OT circulation, we assayed the levels of OT in the CSF and hypothalamus in male wild-type and CD157KO mice (Fig. 6c,d). As shown in Fig. 6c, CD157KO mice have depressed CSF OT levels that are reversed by oral NR. Two-way ANOVA revealed no effect of treatment (F1,60 = 0.2141, P = 0.6453) or genotype (F1,60 = 2.865, P = 0.0957) but a significant effect of their interaction (F1,60 = 7.341, P = 0.0088). Bonferroni’s post hoc comparison shows a significant difference between placebo and NR in CD157KO mice (P 6D). Two-way ANOVA showed an effect of genotype (F1,36 = 12.59, P = 0.0011) but no effect of treatment (F1,36 = 3.115, P = 0.0861) or their interaction (F1,36 = 0.02442, P = 0.8767).