Close relationships among the seven grey matters of the limbic system and basal ganglia with regard to the phosphorus content

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.


Introduction
To elucidate compositional changes of the brain with aging, we previously investigated age-related changes of elements in the corpus callosum 1 , anterior commissure 2 and fornix 3 of the white matter, and the hippocampus 3 , dentate gyrus 3 , pineal body 4 , olfactory bulb and tract 5 , mammillary body 6 , lateral geniculate body 7 , superior colliculus 7 , caudate nucleus 8 , putamen 8 , globuspallidus 8 and amygdala 9 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 regions 10,11 .
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 commissure 12 , among the caudate nucleus, putamen and globuspallidus 8 , and between the amygdala and the hippocampus, dentate gyrus, mammillary body, caudate nucleus, putamen or globuspallidus 9 .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.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 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) for the putamen and 0.709 (p < 0.0001) for the globuspallidus.Extremely significant, direct correlations were found between the P contents of the hippocampus and all the grey matters of the dentate gyrus, mammillary body, amygdala, caudate nucleus, putamen and globuspallidus.

Relationships
were examined between the P contents of the dentate gyrus and other grey matters.The correlation coefficients between the dentate gyrus and other grey matters were estimated to be 0.802 (p < 0.0001) for the mammillary body, 0.656 (p < 0.0001) for the amygdala, 0.608 (p < 0.0001) for the caudate nucleus, 0.860 (p < 0.0001) for the putamen and 0.698 (p < 0.0001) for the globuspallidus (Table 2).Extremely significant, direct correlations were found between the P contents of the dentate gyrus and all the grey matters of the mammillary body, amygdala, caudate nucleus, putamen and globuspallidus.

Relationships
were examined between the P contents of the mammillary body and other grey matters.The correlation coefficients between the mammillary body and other grey matters were estimated to be 0.764 (p < 0.0001) for the amygdala, 0.621 (p < 0.0001) for the caudate nucleus, 0.759 (p < 0.0001) for the putamen and 0.687 (p < 0.0001) for the globuspallidus (Table 2).Extremely significant, direct correlations were found between the P contents of the mammillary body and all the grey matters of the amygdala, caudate nucleus, putamen and globuspallidus.

Relationships
were examined between the P contents of the amygdala and other grey matters.The correlation coefficients between the amygdala and other grey matters were estimated to be 0.509 (p = 0.0004) for the caudate nucleus, 0.572 (p < 0.0001) for the putamen and 0.550 (p < 0.0001) for the globuspallidus (Table 2).Extremely significant, direct correlations were found between the P contents of the amygdala and all the grey matters of the caudate nucleus, putamen and globuspallidus.

Relationships
were examined between the P contents of the caudate nucleus and other grey matters.The correlation coefficients between the caudate nucleus and other grey matters were estimated to be 0.641 (p < 0.0001) for the putamen and 0.505 (p = 0.0004) for the globuspallidus (Table 2).Extremely significant, direct correlations were found between the P contents of the caudate nucleus and either the putamen or the globuspallidus.

Relationship between the putamen and globuspallidus
The relationship between the P contents of the putamen and the globuspallidus was examined.The correlation coefficient was estimated to be 0.768 (p < 0.0001) between the putamen and globuspallidus (Table 2).An extremely significant, direct correlation was found between the P contents of the putamen and globuspallidus.
Therefore, extremely significant, direct correlations were found among all the hippocampus, dentate gyrus, mammillary body, amygdala, caudate nucleus, putamen and globuspallidus, with regard to the P content.

Discussion
We previously investigated the relationships among the hippocampus, dentate gyrus, mammillary body, fornix and anterior commissure 12 , between the caudate nucleus, putamen and globuspallidus 8 , and between the amygdala and other limbic system or basal ganglia 9 , from a viewpoint of the elements.We found that regarding the P content, there were extremely significant, direct correlations among all the hippocampus, dentate gyrus, mammillary body, fornix and anterior commissure 12 , and between all the caudate nucleus, putamen and globuspallidus 8 .In addition, it was found that the hippocampus, dentate gyrus, mammillary body, caudate nucleus, putamen and globuspallidus, had extremely significant, direct correlations with the amygdala with regard to the P content 9 .
The present study revealed that extremely significant direct correla-tions existed among the P contents of all the hippocampus, dentate gyrus, mammillary body, amygdala, caudate nucleus, putamen and globuspallidus of the grey matter.
It is well known that P-containing phosphate has five major functions: (1) it is a part of large molecules or molecular assemblies, e.g.DNA, RNA and membranes, (2) it acts as a carrier of substrates, e.g. in glucose phosphate and coenzyme, and as a carrier of chemical energy, e.g.adenosine triphosphate (3) it acts as a signalling device in the cytoplasm, e.g. in cyclic adenosine monophosphate, (4) it acts as a reversible chemical modification of proteins and (5) it is a constituent of biominerals 15 .
The P content of tissues is mostly determined by the nucleic acid content (DNA and RNA) and the phospholipid content of the tissue.Nucleic acids in the cell nucleus and the cytosol and phospholipids in the cell membrane, are indicators of metabolically active cells 16 .Taking these into consideration, it is reasonable to presume that the P content in the grey matter of the brain indicates the active cell density, i.e. the number of active cells per volume.
Because there were extremely significant, direct correlations among the P contents of all the hippocampus, dentate gyrus, mammillary body, amygdala, caudate nucleus, putamen and globuspallidus of the grey matter, in the present study, it is likely that the active cell density correlates well and directly among all the seven grey matters.Consequently, active cell density increases in the hippocampus; the active cell density also increases in the other six grey matters.
The cellular composition of the grey matter basically consists of three cell populations: neurons, glia and endothelial cells.There are numerous reports on age-related changes of the neuronal number in the hippocampus [17][18][19][20] , mammillary body 21,22 , amygdala 23,24 and basal ganglia 25,26 .Simic et al. 17 reported that in the human hippocampus, the neuronal number declined in specific hippocampal areas (CA1 and subiculum) with aging, but not in other areas.Begega et al. 21eported that aging did not provoke any change in the neuronal number of the human medial mammillary nucleus.Regarding the human amygdala, Schumann and Amaral 24 reported that the neuronal number did not correlate with age.However, Garcia-Amado and Prensa 23 reported that the neuronal number tended to decrease in the human amygdala with age.Schmitt et al. 25 reported that the total neuronal number of the human putamen did not correlate significantly with age.Therefore, it is thought that the neuronal number may not change significantly with aging in the human grey matters of the examined limbic system and basal ganglia, except for the hippocampus.
With regard to the glial cells, there are a few reports [21][22][23] of age-related changes in the neuronal number in the limbic system and basal ganglia.Garcia-Amado and Prensa 23 reported that aging did not affect regional size or the amount of total glial cells in the human amygdala.Begega et al. 21reported that the total number of glial cells was not affected in the human medial mammillary nucleus by age.Roberts et al. 22 reported that the total glial number in the hypothalamus of monkeys showed no significant correlation with age.
There is one report 23 on an agerelated change of the endothelial cell number in the limbic system and basal ganglia.Garcia-Amado and Prensa 23 reported that the number of endothelial cells tended to increase in the human amygdala with aging.
With regard to the limbic system and basal ganglia, there were a few reports on age-related changes of total cell density consisting of neurons, glia and endothelial cells.In the present study, the P content increased significantly in all the hippocampus, dentate gyrus and putamen, with aging.These results suggested that the total cell density might increase significantly in all the hippocampus, dentate gyrus and putamen, with aging.There are two possibilities: 1) the age-dependent increase of the P content in the hippocampus, dentate gyrus and putamen, is due to an increase in the endothelial or/and glial cells but not neurons, without any change in volume of the grey matter or 2) the age-dependent increase of the P content is caused due to shrinkage of the grey matter, without any change in the total cell number.Taking the scenarios described above into consideration, the latter possibility seems to be more probable.
Rajan et al. 33 determined the concentrations of 15 elements, Na, K, P, Ca, Mg, Si, Cr, Cu, Ni, Zn, Fe, Al, Cd, Pb and As, in 12 regions, of eight normal human brains, by inductively coupled plasma-atomic emission spectrometry.He reported that there were significant, direct correlations in all 15 trace element concentrations, including P, among the frontal cerebrum, temporal cerebrum, parietal cerebrum, somatosensory cortex, occipital cerebrum, hippocampus, thalamus, hypothalamus, mid-brain, pons, cerebellum and medulla oblongata.It was implied that although the result was not shown, there were significant, direct correlations among the 12 brain regions with regard to the P concentration.It should be noted that although the examined brain regions were different except for the hippocampus, significant, direct correlations were found among the P contents of all the 12 brain regions by Rajan et al. 33 and of all the seven grey matters analysed in the present study.

Conclusion
The present study suggests that as far as the seven grey matters of the limbic system and basal ganglia are concerned, the active cell densities of these grey matters may concomitantly change one another.
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.

Table 1 . Subjects used in the present study. Age (Years) Sex Cause of Death
Note: M, men; W, women.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.

Table 2 . Rela onships among the P contents of the seven grey ma ers. Grey Ma er Correla on Coeffi cient and p Value
Note: p values are indicated in parentheses.Am, amygdala; CN, caudate nucleus; DG, dentate gyrus; GP, globuspallidus; H, hippocampus; MB, mammillary body; P, phosphorous; Pu, putamen.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.