The experiments were performed in accordance with the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals. All animals were continually monitored and all procedures were approved by the UCLA Chancellor's Animal Research Committee.
4.1. DHA diet and exercise paradigms
Adult male Sprague–Dawley rats (n=24, Charles River Laboratories, Inc., Wilmington, MA, USA), approximately 2 months of age (200–240 g), were housed in standard polyethylene cages in an environmentally controlled room (22–24 °C) with a 12 h light/dark cycle. The rats were randomly divided into 4 groups: (1) RD (regular diet)-Sed (sedentary), (2) RD-Exc (exercise), (3) DHA-Sed, and (4) DHA-Exc; RD-Sed group was regarded as a control. After acclimatization for 1 week on standard rat chow, the rats were exposed to voluntary exercise or a sedentary condition, while subgroups of each were exposed to a DHA-enriched diet (1.25% DHA) or a regular diet for 12 days. The diets, fed ad libitum, were provided in powder. The regular diet was the standard rat chow with a ratio of omega-6/omega-3 at 6:1 (#5001, PMI Nutrition), total fat: 4.5%; arachidonic acid: <0.01%; calorie: 4.07 kcal/gm. The rats were initially omega-3 sufficient by being maintained on the standard rat chow (0.05% DHA), and DHA was supplemented in the regular diet with a ratio of omega-6/omega-3 at 1:1 (1.25% DHA, 0.25% EPA, Nordic Naturals). The rats were allowed to exercise ad libitum in individual cages with an unlimited access to a running wheel (diameter=31.8 cm, width=10.0 cm). The running wheel rotated freely against a resistance of 100 g attached to a receiver that monitored revolutions every hour (VitalViewer Data Acquisition System software, Mini Mitter, Sunriver, OR, USA). Animals were sacrificed by decapitation the morning after the last running period. The fresh tissue containing the hippocampus was dissected, frozen on dry ice and stored at −70 °C until further use.
4.2. Tissue preparation and protein determination
The hippocampi were rapidly dissected out upon decapitation. Tissue was collected into 1.5 mL Eppendorf tubes, immediately frozen on dry ice and stored at −70 °C. Hippocampi from the left side of the brain were homogenized in a freshly prepared lysis buffer (137 mM NaCl, 20 mM Tris–HCl pH 8.0, 1% NP-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin, 1 μg/mL leupeptin and 0.5 mM sodium vanadate). Homogenates were centrifuged at 12,000 rpm for 20 min to remove insoluble material. The supernatants were collected into clean 1.5 mL tubes, frozen on dry ice and stored at −70 °C. The total protein concentration of hippocampal homogenates was determined with a MicroBCA kit (Pierce, Rockford, IL, USA), using BSA as a standard.
4.3. Western blot
Relative levels of STX-3, STX-1, NR2B, and GAP-43 were analyzed by Western blot. Equal amounts (25 μg) of protein samples were separated by electrophoresis on 8–15% polyacrylamide gels and electrotransferred to an Immobilon-P transfer membrane (Millipore, Bedford, MA, USA). Nonspecific binding sites were blocked with 2% BSA in TBS buffer with 0.1% Tween-20 (pH 7.6). Membranes were incubated with the following primary antibodies: anti-STX-3 (1:5000, Abcam), anti-STX-1 (1:2000, Santa Cruz Biotechnology), anti-NR2B (1:1000; Upstate), anti-GAP-43 (1:2000; Santa Cruz Biotechnology), and anti-actin (1:2000; Santa Cruz Biotechnology) followed by anti-rabbit IgG horseradish peroxidase conjugate for STX-3, STX-1 and NR2B, or anti-goat IgG horseradish peroxidase conjugate for GAP-43 and actin (Santa Cruz Biotechnology). After rinsing with buffer (0.1% Tween-20 in TBS), the immunocomplexes were visualized by chemiluminescence using the Amersham ECL Plus Western Blotting Detection kit (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) according to the manufacturer's instructions. The film signals were digitally scanned using a HP Scanner (HP Scanjet 3970) and quantified with NIH Image software, normalized for actin levels.
Additional rats from all of the four groups (n=8) were injected with a lethal dose of Nembutal (75 mg/kg i.p.), then intracardially perfused with PBS (pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and 20% sucrose in 4% paraformaldehyde. Serial coronal brain sections (25 μμm) were cut on a cryostat, collected free-floating in anti-freeze solution and stored at −20 °C before processing for immunofluorescence. Tissue sections were rinsed three times for 10 min with PBS buffer on a plate shaker and nonspecific binding sites were blocked for 1 h at room temperature in PBS with 1% BSA and 0.3% Triton X-100. The sections were incubated overnight at 4 °C and then for 1 h at room temperature in a solution containing the primary antibody (rabbit-anti-STX-3, 1:150, Sigma; mouse-anti-MAG, 1:500, Chemicon International) diluted in PBS with 1% BSA and 0.1% Triton X-100. After washing three times for 10 min with PBS buffer and 0.1% Triton X-100, the sections were kept in dark and incubated for 1 h in a solution containing the secondary antibody (Cy 3-conjugated goat-anti-rabbit IgG, 1:500, Jackson ImmunoResearch; Fluorescein (FITC)-conjugated goat-anti-mouse IgG, 1:500, Jackson ImmunoResearch) diluted in PBS with 1% BSA and 0.1% Triton X-100. The sections were then rinsed in the dark three times for 10 min in a washing solution, mounted on microscope slides, coverslipped with fluorescence protecting solution and stored in the dark at 4 °C. The immunofluorescence analysis was performed using a Zeiss microscope (Zeiss Imager.Z1, Carl MicroImaging). Control sections were incubated in PBS with 1% BSA and 0.1% Triton X-100 without the primary antibody. No staining was observed in cell structures of control sections.
4.5. Morris water maze (MWM)
After one week on the diet or exercise, the rats were tested in the MWM for their learning ability as previously described (Wu et al., 2004
). Briefly, the rats were trained in the water maze for 5 days with 2 consecutive trials per day. The animals were placed into the tank facing the wall from one of the equally spaced starting locations that were randomly changed every trial. Each trial lasted until the rat found the platform or for a max of 60 s. If the rat failed to find the platform in the allocated time, it was gently placed on the platform. At the end of each trial, the animals were allowed to rest on the platform for 60 s. The escape latencies to find the platform were recorded.
4.6. Statistical analyses
The mean protein levels were calculated for each group (n=6 rats per group). All statistical analyses were done by statistic software SPSS 16.0. A level of 5% probability was considered significant. The results were expressed as the mean percent of control values and represent the mean ±standard error of the mean (SEM). MWM and protein data were analyzed by two-way ANOVA [(behavior: Sedentary vs. Exercise) and (diet: Regular Diet vs. DHA)]. Interaction effects were further analyzed by performing means comparisons, and desired contrast weights were specified. Post hoc analyses were conducted using Bonferroni comparisons.
A linear regression analysis was performed on individual samples to evaluate association between variables (running distance or water maze times with protein levels).