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Introduction Our aim was to elucidate compositional changes of the brain with aging; we investigated the relationships among the seven grey matters belonging to the limbic system and basal ganglia with regard to their phosphorous content. Materials and methods After ordinary dissections were completed, the hippocampus, dentate gyrus, mammillary body, amygdala, caudate nucleus, putamen and globuspallidus of the grey matter, were removed from the identical brains of the subjects. The subjects consisted of 22 men and 23 women. After the brain samples were incinerated with nitric acid and perchloric acid, the phosphorous content was determined by inductively coupled plasma-atomic emission spectrometry. Results We found that there were extremely significant, direct correlations among the phosphorous contents of all the seven grey matters: the hippocampus, dentate gyrus, mammillary body, amygdala, caudate nucleus, putamen and globuspallidus. Conclusion It is reasonable to presume that the phosphorous content in the grey matter of the brain indicates the active cell density, i.e. the number of active cells per volume. Therefore, there is a possibility that the active cell densities correlate well and directly among all the seven grey matters of the hippocampus, dentate gyrus, mammillary body, amygdala, caudate nucleus, putamen and globuspallidus. Introduction To elucidate compositional changes of the brain with aging, we previously investigated age-related changes of elements in the corpus callosum1, anterior commissure2 and fornix3 of the white matter, and the hippocampus3, dentate gyrus3, pineal body4, olfactory bulb and tract5, mammillary body6, lateral geniculate body7, superior colliculus7, caudate nucleus8, putamen8, globuspallidus8 and amygdala9 of the grey matter. Elements changing significantly with aging were different among the various brain regions. In addition, there were significant gender differences in the elements of the five brain regions10,11. Relationships between different brain regions have been previously investigated. Relationships among the hippocampus, dentate gyrus, mammillary body, fornix and anterior commissure, among the caudate nucleus, putamen and globuspallidus, between the amygdala and the limbic system or basal ganglia, and relationships of elements with regard to the P content, have been evaluated in previous studies. There were extremely significant, direct correlations among the hippocampus, dentate gyrus, mammillary body, fornix and anterior commissure12, among the caudate nucleus, putamen and globuspallidus8, and between the amygdala and the hippocampus, dentate gyrus, mammillary body, caudate nucleus, putamen or globuspallidus9. However, it was ambiguous whether there were significant relationships between the phosphorous (P) contents of the limbic system and basal ganglia. Therefore, in this study, the authors investigated the relationships among the hippocampus, dentate gyrus, mammillary body, amygdala, caudate nucleus, putamen and globuspallidus of the grey matter, with regard to the P content. The present study revealed that there were extremely significant, direct correlations between the limbic system and basal ganglia with regard to the P content. Materials and methods This work conforms to the values laid down in the Declaration of Helsinki (1964). The protocol of this study has been approved by the relevant ethical committee related to our institution in which the study was performed. Sampling Japanese cadavers were treated by injection of a mixture of 36% ethanol, Cl in ic al A na to m y * Corresponding author Email: ytohno@med.cmu.ac.th 1 Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand 2 Department of Anatomy, Nara Medical University School of Medicine, Kashihara, Nara 634-8521, Japan 3 Laboratory of Environmental Biology, Department of Life Science, Faculty of Science and Engineering, Kinki University, HigashiOsaka, Osaka 577-8502, Japan 4 Department of Anatomy, Histology, and Embryology, Fujian Medical University, Fuzhou, Fujian 350004, China Original research study Page 2 of 7 Co m pe n g in te re st s: n on e de cl ar ed . C on fl i ct o f i nt er es ts : n on e de cl ar ed . A ll au th or s co nt rib ut ed to th e co nc ep o n, d es ig n, a nd p re pa ra o n of th e m an us cr ip t, a s w el l a s re ad a nd a pp ro ve d th e fi n al m an us cr ip t. A ll au th or s ab id e by th e A ss oc ia o n fo r M ed ic al E th ic s (A M E) e th ic al ru le s of d is cl os ur e. Licensee OA Publishing London 2013. Creative Commons Attribution Licence (CC-BY) F : Tohno Y, Tohno S, Azuma C, Ongkana N, Minami T, Ke L, et al. Close relationships among the seven grey matters of the limbic system and basal ganglia with regard to the phosphorus content. OA Anatomy 2013 Feb 06;1(1):6. 13% glycerine, 6% phenol and 6% formalin, through the femoral artery 13. After ordinary dissections at Nara Medical University were completed, the hippocampus, dentate gyrus, mammillary body, amygdala, caudate nucleus, putamen and globuspallidus of the grey matter, were removed from the identical brains of the subjects. The subjects consisted of 22 men and 23 women, ranging in age from 70 years to 101 years (average age = 83.3 ± 7.5 years). Determination of elements After the brain samples were treated three times with 99.5% ethanol to remove lipids, they were washed thoroughly with distilled water and were dried at 80°C for 16 hours. One mL of concentrated nitric acid was added to the dry samples (about 20 mg), and the mixtures were heated at 100°C for two hours. After the addition of 0.5 mL concentrated perchloric acid, they were heated at 100°C for an additional two hours. The samples were adjusted to a volume of 10 mL by adding ultrapure water and were filtered through filter paper (no. 7; Toyo Roshi, Osaka, Japan). The resulting filtrates were analysed by inductively coupled plasma-atomic emission spectrometry (ICPS-7500; Shimadzu, Kyoto, Japan)14. The conditions were as follows: 1.2 kW of power from a radio frequency generator, a plasma argon flow rate of 1.2 L/min, a cooling gas flow of 14 L/min, a carrier gas flow of 1.0 L/min, an entrance slit of 20 μm, an exit slit of 30 μm, a height of observation of 15 mm and an integration time lapse of 5 s. Specially prepared, standard solution of phosphate for ion chromatography was purchased from Wako Pure Chem. Ind. (Osaka, Japan) and was used as a standard solution. The detection limit of P was determined to be 50 ng/mL from the standard amount. The P amount was expressed on a dry-weight basis. Statistical analysis Statistical analyses were performed using the GraphPad Prism version 5.0 (GraphPad Software, San Diego, California, USA). Pearson’s correlation was used to investigate the association between parameters. A p value of less than 0.05 was considered to be significant. Data were expressed as the mean ± standard deviation. Results Table 1 shows the ages, sexes and causes of deaths of the 45 subjects used in the present study. The average Figure 1: Age-related changes of the P content in the hippocampus (a), dentate gyrus (b), mammillary body (c), amygdala (d), caudate nucleus (e), putamen (f) and globuspallidus (g). Original research study Page 3 of 7 Co m pe n g in te re st s: n on e de cl ar ed . C on fl i ct o f I nt er es ts : n on e de cl ar ed . A ll au th or s co nt rib ut ed to th e co nc ep o n, d es ig n, a nd p re pa ra o n of th e m an us cr ip t, a s w el l a s re ad a nd a pp ro ve d th e fi n al m an us cr ip t. A ll au th or s ab id e by th e A ss oc ia o n fo r M ed ic al E th ic s (A M E) e th ic al ru le s of d is cl os ur e. Licensee OA Publishing London 2013. Creative Commons Attribution Licence (CC-BY) F : Tohno Y, Tohno S, Azuma C, Ongkana N, Minami T, Ke L, et al. Close relationships among the seven grey matters of the limbic system and basal ganglia with regard to the phosphorus content. OA Anatomy 2013 Feb 06;1(1):6. age (83.3 ± 7.5 years) of the subjects at their deaths was similar to the mean lifespan (82.6 years old) of the Japanese at present. One subject for the dentate gyrus and two subjects for the mammillary body were not analysed in the present study. Age-related changes of the P content in the seven grey matters Figure 1 shows the age-related changes of the P content in the seven grey matters. The correlation coefficients between age and P content were estimated to be 0.298 (p = 0.049) for the hippocampus, 0.418 (p = 0.006) for the dentate gyrus, 0.275 (p = 0.079) for the mammillary body, 0.177 (p = 0.246) for the amygdala, 0.230 (p = 0.128) for the caudate nucleus, 0.317 (p = 0.034) for the putamen and 0.268 (p = 0.075) for the globuspallidus. A very significant, direct correlation between age and P content was found in the dentate gyrus and significant, direct correlations were found in both the hippocampus and putamen. In the mammillary body, globuspallidus and caudate nucleus, the P content tended to increase with aging in all the subjects. Relationships between the hippocampus and other grey matters To examine whether there were close relationships among the grey matters, the relationships among the P contents of the hippocampus, dentate gyrus, mammillary body, amygdala, caudate nucleus, putamen and globuspallidus, were analysed with Pearson’s correlation. Figure 2 shows the relationships between the P contents of the hippocampus and other grey matters. The correlation coefficients between the hippocampus and other grey matters were estimated to be 0.832 (p < 0.0001) for the dentate gyrus, 0.903 (p < 0.0001) for the mammillary body, 0.719 (p < 0.0001) for the amygdala, 0.576 (p < 0.0001) for the caudate nucleus, 0.846 (p < 0.0001) Table 1. Subjects used in the present study.