Nuclear size and soma position are positively correlated with structural maturation and age of newborn DGCs.

<p>(A) Examples of a 21-day-old stage 1 DCX/Prox1+ cell that is located in the subgranular zone (SGZ) and has no dendritic processes (arrowheads, upper panel) and a 21-day-old stage 6 DCX/Prox1+ cell (arrows, lower panel) with a dendrite extending into the outer molecular layer (OML; arrowheads, lower panel). Asterisk denotes the soma of an intensively labeled Thy1-GFP+ cell. (B) Nuclear size (determined with the nuclear marker Prox1, green) increased with structural maturity. There were significant differences in nuclear size between stages 1 and 6, as well as between stages 1 and 5, stages 2 and 6, and stages 3 and 6 (Kruskal-Wallis Dunn's multiple comparison test between animals, *P < 0.05; stage 1: n = 5 animals, stage 2: n = 7, stage 3: n = 4, stage 4: n = 6, stage 5: n = 9, stage 6: n = 11). (C) In BrdU/Prox1+ DGCs, nuclear size increased gradually with age until it reached a plateau at 35 dpi (n = 3 per group). (D, E) The majority of newborn DGCs was positioned in the SGZ and the inner half of the granule cell layer (GCL), regardless of structural stage and age. Error bars represent SEM. Scale bars in (A): 10μm. IML, inner molecular layer.</p

Doublecortin-labeling does not co-localize with Thy1-GFP expression.

<p>(A) Frontal section of the dorsal hippocampal formation from a Thy-1-GFP mouse. Thy1-GFP expression was observed in a subpopulation of dentate granule cells (DGCs) and was expressed throughout dendritic processes of DGCs which extend into the inner molecular layer (IML) and outer molecular layer (OML) toward the hippocampal fissure (hif). Prospero homeobox protein 1 (Prox1, magenta), a specific nuclear marker of granule cells, was confined to granule cell nuclei of the granule cell layer (GCL). Doublecortin (DCX, cyan) labeled young maturing cells that are positioned in the subgranular zone (SGZ). (B) There was no co-localization of DCX and Thy1-GFP which suggests that Thy1-GFP is generally expressed in more mature (DCX-) DGCs. Both DCX+ and Thy1-GFP+ granule cells co-localized with Prox1 even during early stages of DCX expression (see small DCX+ cells in the SGZ). Scale bars: (A) 100 μm; (B) 20 μm. CA1, Cornu Ammonis area 1; H, hilus.</p

Nuclear size measurement as a valuable tool to discriminate between early and late stages in structural maturation and age of newborn DGCs.

<p>(A, D) The correlation of nuclear size with structural stage and cell age is illustrated as a dot plot (each dot represents a single cell, pooled from all animals per group) to highlight the variability. (B, E) Newborn DGCs were pooled in an early (DCX stage 1–3, cell age 7–14 dpi) and a late phase (DCX stage 4–6, cell age 21–77 dpi). The mean nuclear sizes of each group were determined and used to calculate the equidistance between early and late phases, which was then used as a threshold to discriminate and assign newborn DGCs to the early or the late phase of development (shown as red dashed line). (C, F). Based on that threshold, cells were categorized into true positive, false positive and false negative predictive values. True positive classifications were found with a reliability of about 70% across all stages. Number of animals: (A, D) DCX stage 1: n = 5, stage 2: n = 7, stage 3: n = 4, stage 4: n = 6, stage 5: n = 9, stage 6: n = 11; cell age: n = 3 per group. (B, E) DCX stage 1–3: n = 8, stage 4–6: n = 12; cell age 7–14 dpi: n = 6, age 21–77 dpi: n = 18. Error bars represent SEM.</p

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<div><p>Adult neurogenesis is frequently studied in the mouse hippocampus. We examined the morphological development of adult-born, immature granule cells in the suprapyramidal blade of the septal dentate gyrus over the period of 7–77 days after mitosis with BrdU-labeling in 6-weeks-old male Thy1-GFP mice. As Thy1-GFP expression was restricted to maturated granule cells, it was combined with doublecortin-immunolabeling of immature granule cells. We developed a novel classification system that is easily applicable and enables objective and direct categorization of newborn granule cells based on the degree of dendritic development in relation to the layer specificity of the dentate gyrus. The structural development of adult-generated granule cells was correlated with age, albeit with notable differences in the time course of development between individual cells. In addition, the size of the nucleus, immunolabeled with the granule cell specific marker Prospero-related homeobox 1 gene, was a stable indicator of the degree of a cell's structural maturation and could be used as a straightforward parameter of granule cell development. Therefore, further studies could employ our doublecortin-staging system and nuclear size measurement to perform investigations of morphological development in combination with functional studies of adult-born granule cells. Furthermore, the Thy1-GFP transgenic mouse model can be used as an additional investigation tool because the reporter gene labels granule cells that are 4 weeks or older, while very young cells could be visualized through the immature marker doublecortin. This will enable comparison studies regarding the structure and function between young immature and older matured granule cells.</p></div

Survival rate of newborn dentate granule cells decreases over the first 4 weeks.

<p>(A, B) Newly born DGCs labeled with the mitosis marker BrdU (cyan) frequently displayed DCX-expression (magenta) at 7 days post BrdU injection (7 dpi; A), while there was no co-localization of BrdU and DCX at 77 dpi (B). All of the counted BrdU+ cells were Prox1+ (green; A, B). Arrows in (A) point to BrdU/DCX/Prox1+ cells. Arrowheads in (B) point to a BrdU/Thy1-GFP+ cell. Due to their intense somato-dendritic labeling, Thy1-GFP+ cells could be easily distinguished from the green nuclear Prox1 immunostaining. (C) Quantification of BrdU/Prox1+ cells revealed a decline in survival of newborn DGCs between 7 and 35 dpi, whereas the total number of BrdU+ cells did not change between 35 and 77 dpi. Compared to the first week post BrdU injection, 14% of BrdU/Prox1+ cells were retained at 35 dpi, after which there was no further cell loss. (D) The number of BrdU/Prox1+ DGCs that expressed DCX also decreased between 7 and 35 dpi. No DCX/BrdU/Prox1+ cells could be detected between 35 and 77 dpi. All analyses were performed in the suprapyramidal blade of the right dorsal dentate gyrus (n = 3 animals for each group, 3 sections per animal). Error bars represent SEM. Scale bars in (A, B): 10 μm. GCL, granule cell layer; SGZ, subgranular zone.</p

Nuclear size measurement as a valuable tool to discriminate between early and late stages in structural maturation and age of newborn DGCs.

<p>(A, D) The correlation of nuclear size with structural stage and cell age is illustrated as a dot plot (each dot represents a single cell, pooled from all animals per group) to highlight the variability. (B, E) Newborn DGCs were pooled in an early (DCX stage 1–3, cell age 7–14 dpi) and a late phase (DCX stage 4–6, cell age 21–77 dpi). The mean nuclear sizes of each group were determined and used to calculate the equidistance between early and late phases, which was then used as a threshold to discriminate and assign newborn DGCs to the early or the late phase of development (shown as red dashed line). (C, F). Based on that threshold, cells were categorized into true positive, false positive and false negative predictive values. True positive classifications were found with a reliability of about 70% across all stages. Number of animals: (A, D) DCX stage 1: n = 5, stage 2: n = 7, stage 3: n = 4, stage 4: n = 6, stage 5: n = 9, stage 6: n = 11; cell age: n = 3 per group. (B, E) DCX stage 1–3: n = 8, stage 4–6: n = 12; cell age 7–14 dpi: n = 6, age 21–77 dpi: n = 18. Error bars represent SEM.</p

Structural maturation of DCX-expressing newborn DGCs is correlated with cell age.

<p>(A) DCX+ cells were categorized into six stages according to the degree of their structural maturation. Cells were considered to be in stage 1 when the soma was positioned in the subgranular zone (SGZ) and no dendritic processes were visible; stage 2 when the cell displayed short processes that were located within the SGZ; stage 3 when the principal dendritic process projected into the inner half of the granule cell layer (GCL); stage 4 when the leading dendrite reached the outer half of the GCL; stage 5 when the leading dendrite extended into the inner molecular layer (IML); and stage 6 when the leading dendrite reached the outer molecular layer (OML). (B, C) Staging of newborn DCX+ DGCs at different time points revealed a marked shift in stage distribution according to cell age. At 7 dpi, the majority of newborn DCX+ DGCs were classified as stage 1 or 2, while at 14 dpi, the majority of cells were classified as stage 5 or 6. At 28 dpi, about 92% of the DCX+ cells were classified as stage 6, but a small percentage of DCX+ cells were classified as stage 3. (D, E) The distribution of DCX+ cell ages according to each stage illustrates the prevalence of DCX stages 1–4 at 7 dpi and DCX stages 5–6 at 14 to 28 dpi. No BrdU/DCX+ DGCs were observed after 28 dpi. Notably, DCX+ cells of stages 1–6 co-existed at the same time points (7–21 dpi), suggesting a variability in the maturation time course of individual neurons. All data were obtained from 3 animals (n = 3) per group, and 3 sections per animal. Error bars represent SEM. Scale bar in (A): 20μm.</p