We used very young [postnatal day: 1 (P1), 7 (P7), 14 (P14) and 21 (P21), n=12 in each group] male C57BL/6 mice. The day of birth was considered as day 0. Litters were culled to a maximum of eight pups at the day of birth. From any one litter, a maximum of two animals were taken for each age group, ensuring that animals of a given age originated from at least two different litters. The procedures for the handling and care of the animals adhered to the guidelines that comply with the current international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985, revised 1996) and were approved by the Institutional Animal Care and Use Committee at Hallym's Medical Center (Hallym-1-35). All of the experiments were conducted in a way as to minimize the number of animals used and the suffering caused by the procedures used in the present study.

The animals at each age were anesthetized by injecting 30 mg/kg Zoletil 50 (Virbac, Carros, France), and the brain was removed. Thereafter, brains were fixed for 24 hours in 4% paraformaldehyde at 4℃ and dehydrated with graded concentrations of alcohol before being embedded in paraffin. Thereafter, paraffin-embedded tissues were sectioned on a microtome (Leica Microsystems GmbH, Wetzlar, Germany) into 3-µm coronal sections, and they were mounted onto silane-coated slides. The sections were stained with cresyl violet acetate according to a previously published procedure [3].

To ensure that the immunohistochemical data were comparable between groups, the sections were carefully processed under the same conditions. The sections were hydrated and treated with 0.3% hydrogen peroxide (H₂O₂) in phosphate-buffered saline (PBS) for 30 min. For antigen retrieval, the sections were placed in 400-mL jars filled with citrate buffer (pH 6.0) and heated in a microwave oven (Optiquick Compact, Moulinex) operating at a frequency of 2.45 GHz and a 800-W power setting. After three heating cycles of 5 min each, slides were allowed to cool at room temperature and were washed in PBS. After washing, the sections were incubated successively in 10% normal rabbit serum in PBS for 30 min and in diluted goat anti-DCX (diluted 1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-CB (1:5,000, Chemicon International, Temecula, CA, USA) for 48 hours at 4℃. Thereafter, they were exposed to biotinylated rabbit anti-goat IgG or goat anti-rabbit IgG, streptavidin peroxidase complex (diluted 1:200, Vector Laboratories, Burlingame, CA, USA), and then visualized with 3,3-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO, USA) in 0.1 M Tris-HCl buffer (pH 7.4).

In order to confirm the colocalization of DCX and CB in the brain, the sections in the P14 and P21 group were processed by double immunofluorescent staining under the same conditions. Double immunofluorescent staining for goat anti-DCX (diluted 1:25)/rabbit anti-CB (1:500) was performed. The sections were incubated in the mixture of antisera overnight at room temperature. After washing 3 times for 10 min with PBS, they were then incubated in a mixture of both Cy3-conjugated goat anti-rabbit IgG (1:600; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and FITC-conjugated donkey anti-goat IgG (1:600; Jackson ImmunoResearch Laboratories) for 2 hours at room temperature. The immunoreactions were observed under the AxioM1 microscope-attached HBO100 (Carl Zeiss, Inc., Göttingen, Germany). DCX and CB double-labeled cells in the DG were counted using an image analysis system equipped with a computer-based CCD camera (software: Optimas 6.5, CyberMetrics, Scottsdale, AZ, USA) in 10 sections per animal. Cell counts were obtained by averaging the counts from the sections taken from each animal.

In order to confirm the changes in DCX or CB levels in the DG at each age, five animals at each age were sacrificed and used for Western blot analysis. In brief, aliquots containing 20 µg of total protein were boiled in loading buffer containing 150mM Tris (pH 6.8), 3mM DTT, 6% SDS, 0.3% bromophenol blue, and 30% glycerol. The aliquots were then loaded onto a 10% polyacrylamide gel. After electrophoresis, the gels were transferred to nitrocellulose transfer membranes (Pall Co., East Hills, NY, USA). To reduce background staining, the membranes were incubated with 5% nonfat dry milk in PBS containing 0.1% Tween 20℃ for 45 min, which was followed by incubation with rabbit anti-CB (1:2,000) or goat anti-DCX (1:200), peroxidase-conjugated donkey anti-goat IgG (Sigma-Aldrich) or goat anti-rabbit IgG, and an ECL kit (Pierce, Rockford, IL, USA). The results of the Western blot analysis were scanned, and the quantification of the Western blotting was done using Scion Image software (Scion, Frederick, MD, USA), which was used to measure the optical density. The relative optical density as a percentage of the P1 group is shown in the graph.

Data are expressed as mean±SE. The data were evaluated by a one-way ANOVA SPSS program, and the means were assessed using Duncan's multiple-range test in order to elucidate the differences between all age groups. P-values <0.05 were considered statistically significant.

At P1, cells were stained with cresyl violet throughout the DG. Three layers of the DG were not well defined (Figures 1A and 1B). At P7, the DG was divided into three layers: the molecular (ML), the granule cell (GCL), and the polymorphic (PL) layers (Figures 1C and 1D). At P14, the DG was clearly divided into three layers (Figures 1E and 1F). At P21, neuronal cells in all of the layers were clearly stained with cresyl violet (Figures 1G and 1H).

At P1, DCX-positive cells were detected in the DG (Figures 2A and 2B), although three layers of the DG were not well defined. At this time, DCX-positive neuroblasts did not have any processes (Figure 2B). DCX-positive neuroblasts were detected in the PL and GCL from P7 (Figures 2C-2H). At P7, DCX-positive neuroblasts had very short processes (Figure 2D). At P14, DCX-positive neuroblasts were also detected in the GCL and PL (Figures 2E and 2F). At this time, DCX-immunoreactive neuroblasts had well-developed processes, which extended into the upper two-thirds of the ML of the DG (Figures 2E and 2F). At P21, DCX-immunoreactive neuroblasts were mainly located in the subgranular zone, as it is named in the adult brain, and the neuroblasts had well-developed processes that extended into the upper two-thirds of the ML (Figures 2G and 2H), although the density and length of the processes were decreased compared to those in the P14 group.

At P1, CB immunoreactivity was detected in a few neurons that were round in shape in the DG (Figures 3A and 3B). CB immunoreactivity was mainly detected in the GCL and ML from P7 (Figures 3C-3H). At P7, CB-immunoreactive neurons were numerous in the dorsal blade of the GCL; however, CB-immunoreactive neurons in the ventral blade were rarely found (Figures 3C and 3D). In addition, the neuropil of the ML of the dorsal blade was stained with CB immunoreactivity (Figures 3C and 3D). At P14, CB-immunoreactive neurons were distributed in both blades of the GCL, and CB immunoreactivity in the ML was found in both blades (Figures 3E and 3F). At P21, CB immunoreactivity was apparent in granule cells of the GCL and in the neuropil of the ML of both the blades. At this time, the width of the GCL and ML became wider than that at P14 (Figures 3G and 3H).

At P1, a few DCX and CB double-labeled (DCX⁺CB⁺) cells were detected throughout the DG (data not shown). At P7, about 92% of CB-positive cells were DCX⁺CB⁺ cells, and they were distributed throughout the GCL of the dorsal blade only (Figures 4A-4C). At P14, about 66% of CB-positive cells were DCX⁺CB⁺ cells, and they were found in the lower half of the GCL (Figures 4D-4F). In contrast, at P21, about 18% of the CB-positive cells were DCX⁺CB⁺ cells, and they were mainly detected only in the subgranular zone of the DG, which is a neurogenic zone in the adult brain (Figures 4G-4I).

At P1, DCX protein bands were very strong, and DCX protein levels decreased with age. At P21, DCX protein levels were 47% of that in the P1 group (Figure 5A). At P1, the CB protein bands were very weak, and CB protein levels increased with age. At P21, CB protein levels were 10 times compared to that in the P1 group (Figure 5B).