Experimental Designs and Tissue Preparation
Sprague-Dawley rats (Charles River Laboratories, Inc., Wilmington, MA, USA) weighing between 200 and 240g were housed in cages and maintained in environmentally controlled rooms (22–24 °C) with a 12h light/dark cycle. After acclimatization for 1 week on standard rat chow, one set of rats was exposed to a DHA-enriched diet (1.25% DHA), while another set were exposed to regular diet. The rats were then maintained on the diets with or without voluntary exercise for one week. The rats were divided into 4 groups: (1) RD-Sed (regular diet - sedentary), (2) RD-Exc (RD - exercise), (3) DHA-Sed, and (4) DHA-Exc; RD-Sed group was regarded as control. After one week, the rats (n=6 within each group) were tested in the Morris Water maze for learning ability. The rats, still on the diet and exercise, were then killed by decapitation; the fresh tissues including hippocampus were dissected, frozen in dry ice and stored at −70°C until use for biochemical analyses. The diets, fed ad libitum, were provided in powder. The control diet was the standard chow with a ratio of n-6/n-3 at 6:1 (#5001, PMI Nutrition, USA); total fat: 4.5%; arachidonic acid: < 0.01%; Calorie: 4.07kcal/gm. The animals were initially omega-3 sufficient by being maintained on the standard chow, and DHA was supplemented in the control diet. Nordic Naturals, Inc provided this relative pure DHA product. The DHA content was 1.25% in the experimental diet, while the EPA was 0.25%. The DHA content in the diets and n-6/n-3 ratios were in . All experiments were performed in accordance with the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals, and animal suffering was minimized.
Morris Water Maze for Cognitive testing
Briefly, the rats were trained in the water maze with 2 consecutive trials per day for 5 days. The rats were placed into the tank facing the wall from one of the equally spaced start locations that were randomly changed every trial. Each trial lasted until the rat found the platform or for a max of 1 min. 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 rats were allowed to rest on the platform for 1 min, and the escape latencies were recorded.
Hippocampal tissue was homogenized in a lysis buffer containing 137 mM NaCl, 20 mM Tris–HCl pH 8.0, 1% NP40, 10% glycerol, 1 mM PMSF, 10 μg/ml aprotinin, 0.1 mM benzethonium chloride, 0.5 mM sodium vanadate. The homogenates were then centrifuged; the supernatants collected and total protein concentration was determined according to MicroBCA procedure (Pierce, Rockford, IL), using bovine serum albumin as standard. Levels of pro-BDNF, mature BDNF, phospho-synapsin I (p-syn I), total-synapsin I (t-Syn I), phospho-CREB (p-CREB), total-CREB (t-CREB), phospho-Akt (p-Akt), total Akt (t-Akt), phospho-CaMKII (p-CaMKII), and total-CaMKII (t-CaMKII) were analyzed by western blot. Briefly, protein samples were separated by electrophoresis on an 8% polyacrylamide gel and electrotransferred to a nitrocellulose membrane. Non-specific binding sites were blocked in TBS, overnight at 4°C, with 2% BSA and 0.1% Tween-20. Membranes were rinsed for 10 min in buffer (0.1% Tween-20 in TBS) and then incubated with anti-actin or anti-BDNF, p-syn I, t-Syn I, p-CREB, t-CREB, p-Akt, t-Akt, p-CaMKII, t-CaMKII (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), followed by anti-rabbit IgG horseradish peroxidase-conjugate (Santa Cruz Biotechnology). After rinsing with buffer, the immunocomplexes were visualized by chemiluminescence using the ECL kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ) according to the manufacturer’s instructions. The film signals were digitally scanned and then quantified using NIH Image software. Actin was used as an internal control for western blot such that data were standardized according to actin values.
Measurement of oxidized proteins
The amounts of oxidized proteins containing carbonyl groups were measured by using an Oxyblot kit (Intergen, Purchase, NY). Briefly, the protein sample (10 μg) was reacted with 1× dinitrophenylhydrazine (DNPH) for 15 min, followed by neutralization with a solution containing glycerol and β
-mercaptoethanol. These samples were electrophoresed on an 8% polyacrylamide gel and electrotransferred to a nitrocellulose membrane. After blocking, membranes were incubated overnight with a rabbit anti-DNPH antibody (1:150) at 4°C, followed by incubation in goat anti-rabbit (1:300) for 1 hr at room temperature. After rinsing with buffer, the immunocomplexes were visualized by chemiluminescence using the ECL kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ) according to the manufacturer’s instructions. The Oxyblot bands were all grouped together in each group and then analyzed by NIH image software as previous described (Wu, et al., 2004
Actin was employed as the internal standard for normalizing western blot results. The RD-Sed group was included in each gel to normalize the results of different gels. The RD-Sed group was also used as a control to make comparisons between different groups. The total and activated forms of synapsin I, CREB, Akt, and CaMKII were expressed as a ratio of their phosphorylated forms to their respective total forms. The values were then presented in bar figures and represented the mean ± SEM. The data were analyzed by ANOVA followed by Fisher’s protected least significance post hoc test. Statistical differences were considered significant at P < 0.05.