Male Mongolian gerbils (n=5-; 3-month-old; 50–60 g) were purchased from Japan SLC, Inc. (Shizuoka, Japan). They were housed under standard conditions at a constant temperature (22°C) and humidity (60%), with a 12-h light/dark cycle, and free access to food and water. The handling and care of the animals conformed to guidelines compliant with the current international laws and policies [National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals; NIH Publication no. 85-23, 1985, revised 1996] (19). Ethical approval was obtained from the Institutional Animal Care and Use Committee of Kangwon National University (Chuncheon, Gangwon, Republic of Korea) for all animal procedures in the present study (no. KW-160802-2). Animals were firstly divided into 3 groups, and were then further divided into 5 subgroups (n=10 in each subgroup): Adolescent [postnatal month (PM) 1], adult (PM6 and PM12) and aged (PM18 and PM24) groups. All experiments were conducted with an effort to minimize the number of animals required and the suffering caused by the procedures employed in the present study.

For histology experiments, the animals (n=5/group) at PM1, 6, 12, 18 and 24 were anesthetized with 1 g/kg urethane by intraperitoneal injection (Sigma-Aldrich; Merck KGaA), then perfused transcardially with 0.1 M of phosphate-buffered saline (PBS; pH 7.4) followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). Brains were then removed and postfixed in the same fixative for 12 h at 25°C prior to cryopreservation via overnight storage in 30% sucrose at 4°C. Serial coronal brain sections (30 µm) were generated using a cryostat (Leica Microsystems GmbH, Wetzlar, Germany) at −25°C and maintained in 6-well plates containing PBS until further processing.

To ensure that the immunohistochemical data were comparable between groups, sections were carefully processed under parallel conditions. Tissue sections located 90 µm apart from each other were selected from within an area between 1.4–2.0 mm posterior to the bregma, as defined by a gerbil atlas (20). Five sections, (30 µm) from each of the tissue sections collected, located 90 µm apart from each other were sequentially incubated with 0.3% hydrogen peroxide (H₂O₂) in PBS for 30 min and 10% normal goat serum (cat no. S1000; Vector Laboratories, Inc., Burlingame, CA, USA) in 0.05 M PBS for 30 min at 25°C. Sections were then incubated with the guinea pig anti-VGLUT1 antibody (dilution 1:5,000; cat no. AB5905; Chemicon, Temecula, CA, USA) and guinea pig anti-VGLUT2 (dilution 1:10,000; cat no. AB2251; Chemicon) overnight at room temperature. Sections were then incubated with a biotinylated goat anti-guinea pig IgG (1:200; cat no. BA-7000; Vector Laboratories, Inc.) for 2 h at room temperature, followed by a streptavidin-peroxidase complex (ABC kit; cat no. PK-6100; Vector Laboratories, Inc.) for 1 h at room temperature. Immunostaining was visualized via detection with DAB in 0.1 M Tris-HCl buffer (pH 7.2). Sections were then dehydrated in graded ethanol (70, 80, 90, 95, 100, 100 or 70–100%) and mounted on gelatin-coated slides in Canada balsam (Kanto Chemical Co., Inc., Tokyo, Japan).

In order to establish the specificity of the VGLUT1 and VGLUT2 antibodies, the procedure included the omission of the VGLUT1 and VGLUT2 antibodies, goat anti-guinea pig IgG, and the substitution of normal goat serum for the primary antibody. In addition, for positive control test, immunohistochemistry was conducted with VGLUT1 and VGLUT2 antibodies in the cerebellum of gerbils as VGLUT1 and VGLUT2 are specifically detected in cerebellum (21).

Analysis of the hippocampal CA1 and CA2/3 regions, and dentate gyrus was performed using an ImageJ software v.1.5 (NIH, Bethesda, MD, USA). Digital images of the mid-point of each region were captured using a BX51 light microscope (Olympus Corporation, Tokyo, Japan) equipped with a digital camera (DP72; Olympus Corporation) connected to a computer monitor. Images were calibrated into an array of 512×512 pixels corresponding to a tissue area of 1,200×900 µm (magnification, ×100). Each pixel resolution was 256 gray levels, and the area was divided into the strata oriens, pyramidale and radiatum. The intensity of VGLUT1 and VGLUT2 immunoreactivity was evaluated by relative optical density (ROD), which was obtained following transformation of the mean gray level using the formula: ROD=log (256/mean gray level). The ROD of background staining was determined in unlabeled portions of the sections using Photoshop CC 2015 software (Adobe Systems, Inc., San Jose, CA, USA); this value was subtracted to correct for nonspecific staining, using ImageJ v.1.50 software (NIH). Data are expressed as a percentage of the PM1 group values (set to 100%).

To confirm the age-associated changes of VGLUT1 and VGLUT2 in the hippocampus, animals at PM1, 6, 12, 18, and 24 (n=5/subgroup) were sacrificed and used for western blot analysis. Following euthanasia and the removal of brains, tissues were cut to 500 µm thick sections using a vibratome (Leica Microsystems GmbH) and the hippocampus was dissected out the using a surgical blade. Hippocampal tissues were homogenized in 50 mM PBS (pH 7.4) at 4°C for 30 sec containing 0.1 mM ethylene glycol bis (2-aminoethyl ether)-N,N,N,N tetraacetic acid (pH 8.0), 0.2% nonidet P-40, 10 mM ethylenediamine tetraacetic acid (pH 8.0), 15 mM sodium pyrophosphate, 100 mM β-glycerophosphate, 50 mM NaF, 150 mM NaCl, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride and 1 mM dithiothreitol (DTT). Following centrifugation for 5 min at 16,000 × g at 4°C, the protein level was determined in the supernatants using a Micro Bicinchoninic Acid protein assay kit with bovine serum albumin as the standard according to the manufacturer's instructions (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Aliquots containing 20 µg of total protein were boiled in loading buffer containing 150 mM Tris (pH 6.8), 3 mM DTT, 6% SDS, 0.3% bromophenol blue and 30% glycerol. Then, each aliquot was loaded onto a 12% polyacrylamide gel. Following electrophoresis, proteins were transferred to nitrocellulose membranes (Pall Corporation, East Hills, NY, USA). The membranes were then incubated with 5% non-fat dry milk in PBS containing 0.1% Tween-20 for 45 min at 25°C, followed by incubation with guinea pig anti-VGLUT1 (dilution 1:10,000; cat no. AB5905; EMD Millipore, Billerica, MA, USA) and guinea pig anti-VGUT2 (dilution 1:20,000; cat no. AB2251; EMD Millipore) for 12 h at 4°C. Then, the membrane was incubated with peroxidase-conjugated anti-guinea pig antibody (1:400; cat no. BA-7000; Vector Laboratories, Inc.). Visualization was performed using an enhanced luminol-based chemiluminescent kit (Pierce; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The blots were scanned and densitometry was performed for the quantification of ROD of each band using Scion Image software (version 4.0.3; Scion Corp., Frederick, MD, USA). These data were normalized against β-actin (dilution 1:500; cat no. ab8229; Abcam, Cambridge, UK).

The data are presented as the mean ± standard error mean of triplicate measurement. Differences among the groups were statistically analyzed by one-way analysis of variance followed by a Bonferroni's post hoc test, using GraphPad Prism v5.01 software (GraphPad Software, Inc., La Jolla, CA). P<0.05 was considered to indicate a statistically significant difference.

VGLUT1 immunoreactivity was identified in the molecular and granular layer of cerebellum, while VGLUT2 immunoreactivity was mainly detected in the Purkinje cell and granular layer of cerebellum. In the molecular layer, VGLUT1 immunoreactivity was diffusely observed, while VGLUT2 immunoreactivity was found in some coarse fibers (Fig. 1A and B). Negative control test with pre-immune serum did not show any marked staining of VGLUT1 and VGLUT2 in the cerebellum (Fig. 1C and D).

In all groups, VGLUT1 immunoreactivity was observed in the mossy fibers of the dentate gyrus, as well as the Schaffer collaterals and stratum lacunosum-moleculare in the hippocampal CA1 region (Fig. 2A-E). There were some variations in the VGLUT1 expression pattern in the inner molecular layer of the dentate gyrus; VGLUT1 immunoreactivity was clearly visible in the PM6 and PM24 groups (Fig. 2B and E). VGLUT1 immunoreactivity was altered in the dentate gyrus, however, not in the hippocampal CA1-3 regions, with increasing age. VGLUT1 immunoreactivity was markedly increased in the dentate gyrus of the PM18 and PM24 groups when compared with the PM1 group (Fig. 2F).

In all groups, VGLUT2 immunoreactivity was observed in the granule cell layer and outer molecular layer of the dentate gyrus. In addition, VGLUT2 immunoreactivity was also detected in the stratum pyramidale, Schaffer collaterals and stratum lacunosum-moleculare in the hippocampal CA1-3 (Fig. 3A-E). VGLUT2 immunoreactivity gradually decreased in the dentate gyrus with age; however, VGLUT2 immunoreactivity in the PM18 group increased significantly in the CA1-3 regions when compared with the PM1 and PM12 groups (Fig. 3F). In addition, VGLUT2 immunoreactivity was also markedly detected in the non-pyramidal cells of the stratum oriens and radiatum of the CA1-3 regions (Fig. 3D). However, VGLUT2 immunoreactivity in the PM24 group was significantly decreased in the hippocampal CA1-3 regions when compared with the PM1 group (Fig. 3E and F).

VGLUT1 protein levels were higher in the PM6 and PM24 groups when compared with the PM1 group; however, statistically significant differences were not identified between the different groups (Fig. 4). VGLUT2 protein levels increased in the PM18 group when compared with the PM12 group; however, no statistically significant difference was detected. VGLUT2 protein levels were significantly decreased in the PM24 group when compared with the PM1 and PM18 groups (Fig. 4).