Immunoreactivities of calbindin‑D28k, calretinin and parvalbumin in the somatosensory cortex of rodents during normal aging

Abstract

Calbindin‑D28k (CB), calretinin (CR) and parvalbumin (PV), which regulate cytosolic free Ca2+ concentrations in neurons, are chemically expressed in γ‑aminobutyric acid (GABA)ergic neurons that regulate the degree of glutamatergic excitation and output of projection neurons. The present study investigated age‑associated differences in CB, CR and PV immunoreactivities in the somatosensory cortex in three species (mice, rats and gerbils) of young (1 month), adult (6 months) and aged (24 months) rodents, using immunohistochemistry and western blotting. Abundant CB‑immunoreactive neurons were distributed in layers II and III, and age‑associated alterations in their number were different according to the species. CR‑immunoreactive neurons were not abundant in all layers; however, the number of CR‑immunoreactive neurons was the highest in all adult species. Many PV‑immunoreactive neurons were identified in all layers, particularly in layers II and III, and they increased in all layers with age in all species. The present study demonstrated that the distribution pattern of CB‑, CR‑ and PV‑containing neurons in the somatosensory cortex were apparently altered in number with normal aging, and that CB and CR exhibited a tendency to decrease in aged rodents, whereas PV tended to increase with age. These results indicate that CB, CR and PV are markedly altered in the somatosensory cortex, and this change may be associated with normal aging. These findings may aid the elucidation of the mechanisms of aging and geriatric disease.

Figure 1.

CB immunohistochemistry in the somatosensory cortex in the (A-C) mice, (D-F) rats and (G-I) gerbils. In all species, CB-immunoreactive neurons were primarily observed in layers II and III. The age-associated alterations were different amongst the species. The neuronal number in the aged was generally lower than that of the adults. Scale bars: (Aa-Ia), 200 µm and (Ab-Ic), 25 µm. (J) The percentage mean number of CB-immunoreactive neurons in whole layers in the somatosensory cortex. *P<0.05 vs. the young group; ^†P<0.05 vs. the adult group. Data are presented as the mean ± the standard error of the mean (n=7/group). CB, calbindin-D28k.

Figure 2.

CR immunohistochemistry in the somatosensory cortex in the (A-C) mice, (D-F) rats and (G-I) gerbils. In all species, CR-immunoreactive neurons were primarily observed in layers II and III, and their number was the highest in the adults. In all species, the pattern of the differing numbers of CR-immunoreactive neurons was similar. Scale bars: (Aa-Ia), 200 µm and (Ab-Ic), 25 µm. (J) The percentage mean number of CR-immunoreactive neurons in whole layers in the somatosensory cortex. *P<0.05 vs. the young group; ^†P<0.05 vs. the adult group. Data are presented as the mean ± the standard error of the mean (n=7/group). CR, calretinin.

Figure 3.

PV immunohistochemistry in the somatosensory cortex in the (A-C) mice, (D-F) rats and (G-I) gerbils. In all species, PV-immunoreactive neurons were primarily observed in layers II, III, V and VI. The number of PV-immunoreactive neurons increased with age. Scale bars: (Aa-Ia), 200 µm and (Ab-Ic), 25 µm. (J) The percentage mean number of PV-immunoreactive neurons in whole layers in the somatosensory cortex. *P<0.05 vs. the young group; ^†P<0.05 vs. the adult group. Data are presented as the mean ± the standard error of the mean (n=7/group). PV, parvalbumin.

Figure 4.

Western blot analysis of CB, CR and PV in the mouse cortex of the young, adult and aged groups. ROD is expressed as the percentage of the immunoblot band values in the young, adult and aged groups. Bands were normalized to β-actin expression. *P<0.05 vs. the young group; ^#P<0.05 vs. the adult group. Data are presented as the mean ± the standard error of the mean (n=5/group). CB, calbindin-D28k; CR, calretinin; PV, parvalbumin; ROD, relative optical density.