The impact of age on number and distribution of proliferating cells in subgranular zone in adult mouse brain

Abstract

The mouse brain retains an ability to produce hippocampal granule neurons during the mouse's entire lifespan. The neurons are produced in the subgranular zone (SGZ) located on the inner surface of the granule cell layer in the dentate gyrus (DG). In our study, we used a point cloud approach to characterize how the production and distribution of neural precursors for new hippocampal neurons change in the mouse brain relative to age. We found that the production of neural precursors decreases 64 fold from the age of 30 days to the age of 2.5 years. Within the SGZ the decline of cell proliferation continues during entire mouse life. We reconstructed the distribution of proliferating cells along the longitudinal axis of the SGZ and found that the highest number of proliferating cells are located approximately 0.75 mm from the dorsomedial end of the SGZ that corresponds to the most dorsal part of the DG in the mouse brain. The distribution of proliferating cells in the SGZ showed no apparent aggregations, periodicity or any other readily identifiable spatial characteristics. Proliferating cells in the SGZ tended to be located separately from other proliferating cells. About two thirds of them have no closely located other proliferating cells, and the remaining third is located in small clusters comprised of 2 or 3 proliferating cells. Based on our measurements, we calculated that from the age of 30 days to the age of 2.5 years 1.5 million neural precursors are produced in the SGZ.

Keywords: Adult mouse brain; Adult neurogenesis; Aging; Dentate gyrus; Mouse; Neural precursors; Point cloud; Subgranular zone.

Fig. 1

More proliferating cells are located in the dorsal part SGZ than in the ventral part. (A). The distribution of EdU-labelled nuclei in three consecutive sections #60, #61, and #62 of the mouse brain 120B-A. The fragment of three consecutive registered sections includes the DG on the right side of the brain that could be seen as a dark silhouette. EdU-labelled nuclei in section #60 are shown in red, in section #61 in green, and in section #62 in blue. EdU-labelled nuclei located on these three sections at different locations and no overlaps are observed amongst them. (B). More proliferating cells are located in the dorsal part SGZ than in the ventral part. Coronal view. Each EdU-labelled nucleus is shown as a black dot. The granule cell layer is shown schematically in blue. The brain shape is outlined with a black line.

Fig. 2

Distribution of proliferating cells in the SGZ. Each EdU-labelled nucleus located in the SGZ is shown as a black dot. The brain shape is outlined with a black line. Frontal, lateral, and dorsal views of the brain are shown. Mouse age is shown on the left.

Fig. 3

Age-related decrease in the number and average density of proliferating cells located in the whole mouse brain and the SGZ. (A). The number of proliferating cells in the SGZ decreases with mouse age. The means are significantly heterogeneous (one-way ANOVA, F_{5, 6} = 2459, P = 7.61E-10). The post-hoc t-test shows that all means are significantly different from each other (P < 0.05). (B). The average density of proliferating cells in the SGZ decreases with mouse age. The average volume number density is calculated as the average number of neighbouring EdU-labelled nuclei located closer than 200 μm to each EdU-labelled nucleus. The means are significantly heterogeneous (one-way ANOVA, F_{5, 6} = 230, P = 9.08E-7). The post-hoc t-test shows that all means are significantly different from each other (P < 0.05). Error bars show standard deviation.

Fig. 4

Distribution of proliferating cells in the SGZ along the dorso-ventral, antero-posterior, and left-right axis in a mouse brain. (A, B, C). Distribution of proliferating cells. The number of EdU-labeled nuclei in a 50 μm brain section is shown along the vertical axis of the charts. (D, E, F). Distribution of average volume number density of proliferating cells. The average number of neighboring EdU-labeled nuclei located closer than 200 μm to each EdU-labeled nucleus in a 50 μm brain section is shown along the vertical axis of the charts. (A, D). Distribution along the dorso-ventral axis. The distance from the furthest dorsal part of the SGZ is shown along the horizontal axis of the charts. (B, E). Distribution along the antero-posterior axis. The distance from the furthest anterior part of the SGZ is shown along the horizontal axis of the charts. (C, F). Distribution along the left-right axis. The distance from the furthest right part of the SGZ is shown along the horizontal axis of the charts.

Fig. 5

The number and average density of proliferating cells in four 1-millimeter thick sections of the SGZ along the dorso-ventral axis decreases with mouse age. (A). The number of EdU-labelled nuclei in four 1-millimeter thick sections of the SGZ along the dorso-ventral axis in the brain of a 30 day old mouse. The section position is shown along the horizontal axis of the charts. The means are significantly heterogeneous (one-way ANOVA, F_{3, 4} = 2418, P = 5.69E-7). The post-hoc t-test shows that all means are significantly different from each other (P < 0.05). (B). The average volume number density of EdU-labelled nuclei in four 1-millimeter thick sections of the SGZ along the dorso-ventral axis in brain of a 30 day old mouse. The section position is shown along the horizontal axis of the charts. The means are significantly heterogeneous (one-way ANOVA, F_{3, 4} = 171, P = 1.12E-4). The post-hoc t-test shows that all means are significantly different from each other (P < 0.05) except the mean for the 1–2 mm section and the 2–3 mm section (P = 0.10). (C). The number of EdU-labelled nuclei in all 1-millimeter thick sections of the SGZ along the dorso-ventral axis decreases with mouse age. The number of EdU-labelled nuclei in each section is normalized to the number of EdU-labelled nuclei in the same section in the brain of a 30 day old mouse. (D). The average volume number density of EdU-labelled nuclei in all 1-millimeter thick sections of the SGZ along the dorso-ventral axis decreases with mouse age. The average volume number density of EdU-labelled nuclei in each section is normalized to the average volume number density of EdU-labelled nuclei in the same section of the brain of a 30 day old mouse. Error bars show standard deviation. The distance is measured from the furthest dorsal part of the SGZ.

Fig. 6

Distribution of proliferating cells in the flattened SGZ of a 60 day old mouse. (A). Schematic drawing of a coronal section of the dorsal part of DG showing the location of the external and internal SGZ arms and the apex of DG hilus. (B). Distribution of proliferating cells in the flattened SGZ of a 60 day old mouse. The line that traces the apex of DG hilus was established for the left and right DG in the 3D reconstruction of a 60 day old mouse brain, and the perpendicular (shortest) distances from this line to all EdU labelled cell nuclei in the SGZ was calculated using the line and nuclei coordinates. To generate the flattened image, we straightened the apex lines and showed EdU labelled nuclei as black dots according to the position of these nuclei along the apex lines and the distance from the apex lines. EdU labelled nuclei located in the internal and external arms of the SGZ are shown on different sides of straightened apex lines as indicated in the figure. The distance from the dorsomedial end of the SGZ is shown at the bottom. The distance from the apex lines is shown on the left.

Fig. 7

Distribution of proliferating cells along and across the flattened SGZ of a 60 day old mouse. (A). Distribution of EdU labelled nuclei in the left and right SGZ along the straightened apex of the DG hilus line. (B). Distribution of EdU labelled nuclei in the left and right internal arm of SGZ along the straightened apex of the DG hilus line. (C). Distribution of EdU labelled nuclei in the left and right external arm of the SGZ along the straightened apex of DG hilus line. (D). Location of the highest number of proliferating cells in the SGZ is shown by arrowheads along traces of the apex of the DG hilus. Coronal view. (E). Distribution of EdU labelled nuclei across the flattened left and right SGZ. (F). Number of EdU labelled nuclei in the internal and external arm of SGZ. Error bars show standard deviation. The t-test shows that means are significantly different from each other (P < 0.05).

Fig. 8

Characterization of proliferating cells distribution in the SGZ. (A). Distribution of volume number density of proliferating cells in the SGZ. The number of neighbouring EdU labelled nuclei located closer than 200 μm is shown along the horizontal axis of the chart. The number of EdU labelled nuclei with the indicated number of neighbouring EdU labelled nuclei is shown along the vertical axis. (B). Randomness of proliferating cells distribution in the SGZ. The number of neighbouring EdU labelled nuclei located closer than 200 μm is shown along the horizontal axis of the chart. The average number of EdU labelled nuclei located closed than 50 μm to each EdU labelled nuclei with the same number of neighbouring EdU labelled nuclei located closer than 200 μm is shown along the vertical axis of the chart. The graph also shows 1/16 and 1/64 lines. (C). Presence of tight clusters of proliferating cells in the SGZ. The number of neighbouring EdU labelled nuclei located closer than 10 μm is shown along the horizontal axis of the chart. Percentage of EdU labelled nuclei that have the indicated number of neighbouring EdU labelled nuclei is shown along the vertical axis of the chart. Error bars show standard deviation.