Lab Anim Res.  2011 Jun;27(2):153-159. 10.5625/lar.2011.27.2.153.

Detailed Differentiation of Calbindin D-28k-Immunoreactive Cells in the Dentate Gyrus in C57BL/6 Mice at Early Postnatal Stages

• Yoo DY
• Yoo KY
• Park JH
• Choi JW
• Kim W
• Hwang IK
• Won MH

• KMID: 2053665
• DOI: http://doi.org/10.5625/lar.2011.27.2.153

Abstract

The hippocampus makes new memories and is involved in mental cognition, and the hippocampal dentate gyrus (DG) is critical because neurogenesis, which occurs throughout life, occurs in the DG. We observed the differentiation of neuroblasts into mature neurons (granule cells) in the DG of C57BL/6 mice at various early postnatal (P) ages: P1, P7, P14, and P21 using doublecortin (DCX) immunohistochemistry (IHC) for neuroblasts and calbindin D-28k (CB) IHC for granule cells. DCX-positive cells decreased in the DG with age; however, CB+ cells increased over time. At P1, DCX and CB double-labeled (DCX+CB+) cells were scattered throughout the DG. At P7, DCX+CB+ cells (about 92% of CB+ cells) were seen only in the granule cell layer (GCL) of the dorsal blade. At P14, DCX+CB+ cells (about 66% of CB+ cells) were found in the lower half of the GCL of both blades. In contrast, at P21, about 18% of CB+ cells were DCX+CB+ cells, and they were mainly located only in the subgranular zone of the DG. These results suggest that the developmental pattern of DCX+CB+ cells changes with time in the early postnatal stages.

Keyword

Early postnatal development; subgranular zone; neurogenesis; doublecortin; double immunostaining

MeSH Terms

Animals
Calcium-Binding Protein, Vitamin D-Dependent
Cognition
Dentate Gyrus
Hippocampus
Immunohistochemistry
Mice
Neurogenesis
Neurons
Calcium-Binding Protein, Vitamin D-Dependent

Figure

• 

  Figure 1 Cresyl violet staining in the mouse dentate gyrus (DG) at postnatal day 1 (P1, A and B), P7 (C and D), P14 (E and F), and P21 (G and H). The DG is well laminated into three layers at P14, and cells in each layer are well distinguished from P14. GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer. Bar=25 µm.

• 

  Figure 2 Immunohistochemical staining for doublecortin (DCX) in the DG at P1 (A and B), P7 (C and D), P14 (E and F), and P21 (G and H). At P1, many DCX-positive neuroblasts (arrows) are detected. At P7 and P21, DCX-positive neuroblasts (arrows) are detected in the granule cell layer (GCL). Note that DCX-positive neuroblasts in the GCL are markedly decreased with age, and they are localized at the subgranular zone of the DG. ML, molecular layer; PL, polymorphic layer. Bar=25 µm.

• 

  Figure 3 Immunohistochemical staining for calbindin (CB) in the DG at P1 (A and B), P7 (C and D), P14 (E and F), and P21 (G and H). At P1, CB immunoreactivity is detected in a few neurons (arrows). At P7, CB immunoreactivity is mainly detected in the dorsal blade (arrows) of the GCL. CB immunoreactivity is shown in both blades from P14. ML, molecular layer; PL, polymorphic layer. Bar=50 µm.

• 

  Figure 4 Double immunofluorescent labeling for DCX (A, D and G, green), CB (B, E and H, red), and merged images (C, F and I, yellow) in the DG at P7, P14, and P21. DCX and CB double-labeled (DCX+CB+) cells (arrows) are found in the granule cells layer (GCL): Their location is somewhat different from each group. ML, molecular layer; PL, polymorphic layer. Bar=25 µm. J: The mean number of DCX+CB+ cells per section (n=7 per group; *P<0.05, significantly different from the P7 group, #P<0.05, significantly different from the P14 group). The bars indicate the means±SE.

• 

  Figure 5 Western blot analyses of DCX (A) and CB (B) in the DG at P1, P7, P14, and P21. The relative optical densities (ROD) of the immunoblot bands are demonstrated as percent values (n=5 per group; *P<0.05, significantly different from the P1 group, #P<0.05, significantly different from the P7 group, †P<0.05, significantly different from the P14 group). The bars indicate the means±SE.

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