Animals and housing conditions
Fifteen male Wistar rats (25 days old at the start of the experiment) were used (SLC, Hamamatsu, Japan). The rats were housed in SE (2 rats in each laboratory cage, 40 × 23 × 18 cm, n = 8) or EE (7 rats in a large cage, 81 × 51 × 53 cm, n = 7). We have adopted the experimental design with the standard definition of EE, “a combination of complex inanimate and social stimulation” by van Praag et al.. The EE was equipped with horizontal platforms and various toys such as a running wheel, tunnels, a climbing ladder, wooden blocks, a bridge, and a maze, as previously described [24, 30]. The spatial arrangement of the objects was changed and some of toys were replaced with new toys twice a week in the EE cage. The rats were maintained under controlled temperature (22 ± 1°C) and 12-h light/dark cycle (lights on 7:00 AM), and food and water were available ad libitum. All experiments and animal housing adhered to the Guideline for Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (1996). Experimental procedures were approved by the ethical review board for animal experiments at the University of Toyama (approval number; S-2009 MED-29).
Open field test
Five weeks after rearing in each condition, activity in an open field was evaluated. The test was initiated by placing each subject in the center of an open field arena (center arena, 30 cm in diameter) of a circular open field (60 cm in diameter, surrounded by a 40 cm wall). The behavioral field was set on a black plastic sheet to obtain clear image contrast of the animals. Behavior was video recorded for 10 min using a digital video camera (Everio GZ-MG275, Victor, Kanagawa, Japan), and locomotive behavior was analyzed offline using a software program (TopScan ver 1.00, Clever Sys., Inc., VA, USA). We analyzed % distance traveled in the center arena and the time spent in the center of the arena. The % distance traveled in the center arena was defined as the following formula; 100 × [distance traveled in the center arena]/[total distance traveled]. The total distance traveled was defined as the distance traveled in the whole open field.
Beam walking test
Two days after the open field test, beam walking ability [24, 30] was evaluated. In brief, the rats were trained so as to walk on a wooden beam (25 mm wide) elevated 600 mm above the floor three times. After training, walking to traverse the beam (700 mm distance) was video recorded 3 times (Everio GZ-MG275) and walking time was calculated.
Principle staining procedures have been previously described in detail . In brief, the brains of the rats were obtained after perfusion with heparin saline solution under deep anesthesia with pentobarbital (50 mg/kg, i.p.) and fixation with 4% paraformaldehyde in 0.1 M phosphate buffer. After fixation (4% paraformaldehyde solution) and cryoprotection in 20% sucrose solution, coronal sections (40 μm thick) were cut on a freezing microtome. For stereological quantification, 5 sections each were processed for one staining each: one for Nissl staining with cresyl violet, one for PV staining, one for CalB, and two for other staining including double immunolabeling to identify the phenotype of PV-positive cells.
For all staining, free-floating sections were rinsed 3 times in phosphate buffer saline (PBS) between each incubation step. These sections were quenched for 10 min in 3% H2O2/20% methanol in PBS, and incubated in a blocking solution, 3% normal horse serum in PBS-T (0.25% Triton X-100) for 30 min at room temperature. For PV and CalB immunohistochemistry, the sections were incubated overnight at 4°C with mouse anti-PV antibodies (1:10000, Sigma, St. Louis, MO, USA) or mouse anti-CalB antibodies (1:8000, Swant, Bellinzona, Switzerland) in 1% blocking solution. After rinsing, sections were incubated for 1 h at room temperature with biotinylated secondary antibodies (1:500, Vector Laboratories, Burlingame, CA, USA) and then reacted with avidin biotin peroxidase complex (ABC-Elite, Vector Laboratories). The reaction was visualized with a detection solution (0.25 mg/ml 3, 3’-diaminobenzidine, 0.03% H2O2 in PBS).
Cell counting and morphological analysis
For cell counting, the stereological methods (optical dissector method) have been recommended [31, 32]. However, poor penetration of the CalB immunostaining made it quite difficult to apply to the optical dissector method . Therefore, we counted all somatic profiles contained in the optical sections 0–4 μm from the surface . Images of the sections were obtained with a light microscope (BX 61, Olympus, Tokyo, Japan) equipped with a digital camera (DP 70, Olympus) or all-in-one fluorescence microscope system (BZ-9000, Keyence, Osaka, Japan). At the outset, we examined the penetration of the immunostaining for PV and CalB in 40 μm thick sections. Stacks of serial optical sections 1 μm apart were obtained with all-in-one microscope system BZ-9000 under an objective lens (× 20, NA 0.75).
We counted stained cells in the whole area of the basolateral complex of the amygdala in each section, including the lateral (dorsolateral, ventrolateral, and medial divisions), basal (magnocellular, intermediate, and parvicellular divisions), and accessory basal nuclei (magnocellular and parvicellular divisions) of the amygdala. The basolateral complex of the amygdala was divided into the 2 subareas; the lateral amygdala (LA, lateral nuclei of amygdala) and basolateral amygdala (BLA, basal and accessory basal nuclei of amygdala). Cell counting was performed for each animal in 4 anatomically matched-sections that referred to adjacent Nissl staining sections [3, 34]: rostrocaudal location at (1) anterior to posterior (AP) −2.60, (2) AP −2.80, (3) AP −3.00, and (4) −3.20 mm from the bregma for PV-positive cell counting. The sections adjacent to PV-staining were used for CalB-positive cell counting. PV and CalB-positive cells were quantified separately in the SE and EE rats and expressed as mean values ± SEM (standard error of means) per section. PV-immunoreactive cells were differentially evaluated according to diameters of positive cell bodies: the cells with large cell bodies more than 25 μm rectangular diameter (length along the long axis plus those of short axis) were defined as large-positive cells, and the cells with small cell bodies less than 25 μm rectangular diameter were defined as small-positive cells. All differential counts were performed blind in randomized section. Morphological images were adjusted for brightness and contrast using Adobe Photoshop CS (v 8.0, Adobe Systems Incorporated, San Jose, CA, USA).
Phenotype of PV-positive cells
For double-immunolabeling studies, the sections were incubated in the blocking solution, followed by overnight incubation of the sections for 2 days at 4°C with 2 primary antibodies simultaneously—rabbit anti-PV antibodies (1:1000, Calbiochem, La Jolla, CA, USA) and mouse anti-GAD 67 antibodies (1:200, Millipore, Temecula, CA, USA). The secondary antibodies were Alexa Fluor 488 goat anti-rabbit IgG (1:500, Molecular Probes, Eugene, OR, USA) and Alexa Fluor 568 goat anti-mouse IgG (1:200, Molecular Probes). The sections were mounted on MAS-coated glass slides (Matsunami, Osaka, Japan) in mounting medium with DAPI (DAPI: a nuclear counterstain for blue-fluorescence, Vector Laboratories). Immunofluorescent images were obtained using the confocal laser scanning microscope LSM 700 (Carl Zeiss, Oberkochen, Germany) and analyzed to determine whether PV-positive cells colocalize with GAD-67 using software (ZEN 2009 Light Edition, Carl Zeiss). The sections for immunofluorescence examination were located at rostrocaudal levels from AP −2.88 to AP −3.08 mm from the bregma. We confirmed that there was no non-specific labeling of cells when each primary antibody was omitted. Only double immunofluorescence-positive cells that also expressed DAPI-positive fluorescence were identified in order to avoid counting of partial and insufficient somata.
All values are expressed as mean values ± SEM. All statistical analyses were performed using the software package SPSS (v 19, IBM, Somer, NY, USA). Significant differences between the groups in behavioral and immunohistochemical analyses were evaluated using Student’s t-test and two-way repeated measures ANOVA. The correlation between behavioral data and the number of small cells was analyzed using simple linear regression analysis. Differences were considered statistically significant at p < 0.05.