The number of times an

The number of times an animal reared was the only emotional behavior that was significantly higher in the young mice than in the middle-aged group. This may be explained by diminished curiosity or augmented ambulation with aging (Lalonde and Strazielle, 2008). A recent study showed that middle-aged and old male mice showed decreased exploratory behavior, and old mice showed decreased motor activity, anxiety behavior and learning (Lalonde and Badescu, 1995, Gupta et al., 2012), which suggests that emotion decreases with aging in mice, but also that the middle-aged mice used in the current study were not too old to change most of their emotional behaviors. The present study confirmed that some of the free amino acids in the brain increased or decreased with aging. The varied influence of aging on amino NAD/NADH Quantitation Colorimetric Kit synthesis metabolism in different brain regions was also determined. The number of changed amino acids was highest in the hippocampus, followed by the hypothalamus. Accordingly, a specific amino acid may be involved in brain aging. Among amino acids, L-Arg is supposed to be related to aging. A recent study showed that in the case of dogs, plasma L-Arg decreased with aging (Murakami et al., 2011). The present study also showed that it is not only L-Arg that decreases, but that ornithine as a metabolite of L-Arg decreases too. L-Arg is metabolized to nitric oxide (NO) and l-citrulline by nitric oxide synthase, to l-ornithine and urea by arginase, and to agmatine and carbon dioxide by arginine decarboxylase (Wu and Morris, 1998). The relationship between L-Arg metabolites and aging needs to be further elucidated. Among these metabolites, central L-Arg (Suenaga et al., 2008b) and l-ornithine (Suenaga et al., 2008a, Miyake et al., 2014) are known to induce hypnosis. In our study, L-Arg levels in the hippocampus were decreased by aging. This might be a result of the increased metabolism of L-Arg to NO, which is a negative regulator of neurogenesis (Packer et al., 2003). Liu et al. (2009) found that aging-related alterations in L-Arg and two of its metabolites—glutamine and GABA—occurred in memory-associated brain structures in old rats. Rushaidhi et al. (2012) also found these changes that occur in these amino acids with increasing age in the brain tissue and synaptoneurosome. Thus, aging might affect neurogenesis via NO, because in middle-aged mice (10 months old), a decline of neurogenesis in the hippocampus has been found to affect emotional behavior (Jinno, 2003). On the other hand, L-Ser was found to be lower in the hypothalamus and cerebellum of middle-aged mice. Central L-Ser also has a hypnotic effect (Asechi et al., 2006, Ito et al., 2014). The reduction of these amino acids may be one of the reasons length of sleep decreases in aged people. Some amino acids have been revealed to have an effect on pregnancy. For example, dietary arginine supplementation during pregnancy enhances the utero placental blood flow and improves fetal growth (Wu et al., 2013). Therefore, the amino acid levels of the uterus (Table 5) and ovary (Table 6) were measured in the present study, in which some amino acids were found to be higher in the middle-aged groups than they were in the young groups. Recently, we found that plasma free amino acids were not correlated with the free amino acids found in genital organs in mice (data unpublished). Although there is a possibility that our present results were due to the difference that exists between mice that have experienced pregnancy and virgin mice, it could be assumed that the metabolism of some proteins or protein synthesis, at least in part, may not function well with aging. Kermack et al. (2015) compared the total concentration of amino acids in human uterine fluid in women under 38 years with that in women older than 38 years of age, which is when a decline in fertility is thought to occur, and found that the total concentration of amino acids did not differ between the two groups. However, it may be difficult to compare the results of mice and humans, because the amino acid levels in the uterus and uterine fluid may be different. In human uterine fluid, some amino acid concentrations are modified by diet (Kermack et al., 2015). Thus, the aging of the uterus could be slowed by certain dietary regimens. On the other hand, there is only limited information about amino acid concentrations in the ovary. Oocytes have amino acid transport systems that utilize amino acids. For example, Gln, Asp, and Val inhibited the polyspermic fertilization of pig oocytes (Hong and Lee, 2007). In the current study, some of the amino acids that changed with aging differed between the uterus and the ovary. Thus, it could be predicted that protein and amino acid metabolisms in the uterus and the ovary are independently regulated, as sex hormones regulate ovarian and uterine functions in a different fashion. Moreover, in this study, we used different sets of mice in order to know how the stress response differs between young and middle-age. If young mice were allowed to grow old in order to compare the differences in behaviors and amino acid concentrations in the brain and genital organs that exist between young and aged mice, we could have more clear data to support the hypothesis of the current study. A further study will be conducted in relation to this matter.