2.1. Animals
To perform this study, 6-month-old PPARβ/δ-null (PPARβ/δ-/-)(20) and wild type (WT) littermates with the same genetic background (C57BL/6X129/SV) were used. Exceptionally, to perform open field test, C57BL/6 mice also were used. In all cases, animals were obtained from established breeding couples in the animal facility (Animal facility from the Pharmacy and Food Sciences Faculty from the University of Barcelona; approval number C-0032). After the weaning (at 21 days-old), and throughout their growth, animals were fed either with conventional chow (control diet, CT) (ENVIGO, Madison, Wt 53744-4220) or with a palmitic acid-enriched diet containing 45% of fat mainly from hydrogenated coconut oil (HFD) (Research Diets Inc., New Brunswick, USA). Thus, four groups were defined: WT CT, WT HFD, PPARβ/δ-/- CT and PPARβ/δ-/- HFD.
All animals were kept under stable conditions of humidity and temperature, standard light-dark cycle (12-h light/dark cycle) and food and water ad libitum, following the ethics guidelines defined by the European Committee (European Communities Council Directive 2010/63/EU). Manipulation protocols were previously approved by the ethics committee from the University of Barcelona and, at all times it was made sure that animal numbers, their stress and pain were kept under a necessary minimum following the appropriate animal manipulation ethical methodologies.
Glucose and insulin tolerance tests
For both tests, mice were fasted for 6 h and the tests were performed in a quiet room, preheated to +28 ºC. In the glucose tolerance test (GTT), glucose was administered at a dose of 1g/kg intraperitoneally (i.p). On the other hand, in the insulin tolerance test (ITT), the dosage of insulin used was 0.75 IU/Kg and it was also administered i.p. Next, samples from the tail vein were extracted in consecutive periods of time. In GTT, measurements were made at 5, 15, 30, 60 and 120 min after the administration of glucose. In the ITT case, samples were extracted at 15, 30, 45, 60 and 90 min after the insulin administration.
The animals were monitored in every moment, and in those cases where glucose levels dropped under a concentration of 20 mg/dL, a dosage of 1 mg/Kg of glucose was administered i.p. and they were kept in observation until blood glucose concentrations were stable and the animal behavior was normal. Twelve animals per group were analyzed.
Open field behavior
The open field behavioral test was performed to evaluate the anxiety levels of C57BL/6X129/SV and C57BL/6 mice. Animals were placed individually for 10 min in a circular open-field arena of 40 cm in diameter surrounded by black curtains where the light intensity in the middle of the field was 30 lux. This area was divided virtually into two circular zones placed one above the other, center of 12 cm of diameter and periphery of 28 cm of diameter and once animals placed in the center, their tracking was monitored (Smart 3.0; Panlab). The time spent in these defined zones was recorded and results were measured in seconds and expressed in percentage. At least 10 animals per group were analyzed.
All spaces were properly cleaned with 96% ethanol between animals, in order to eliminate odor or other cues.
Hippocampal spine density analysis
To carry out the spine density analysis, five animals per group were used which were sacrificed by cervical dislocation. After, the brain was isolated, it was processed following the instructions of the GolgiStainTM Kit purchased from FD Neurotechnologies, Inc. (FD Rapid GolgiStainTM Kit; Cat #PK401). Images were obtained with a BX61 Laboratory Microscope (Melville NY-Olympus America Inc.). The quantification was carried out by selecting 5 neurons per animal in the dentate gyrus (DG) of the hippocampus. Measurement was done at least 50 μm from the soma along consecutive 10 μm on secondary branches starting 10 μm after branching from the primary dendrite. Spine density was calculated by dividing the number of spines per segment by the length of the segment and was expressed as the number of spines per 10 μm of dendrite.
Immunoblot blot analysis
Fresh brains of at least 4 mice per group were extracted right after euthanasia (cervical dislocation) and the hippocampus were dissected and kept frozen at -80ºC until use. Next, samples were homogenized in lysis buffer (Tris HCl 1M pH 7.4, NaCl 5M, EDTA 0.5M pH 8, Triton, distilled H20) containing protease and phosphatase inhibitor cocktails (Complete Mini, EDTA-free; Protease Inhibitor cocktail tablets, 11836170001, Roche Diagnostics GmbH, Germany ; Phosphatase Inhibitor Cocktail 3, P0044, Sigma-Aldrich, USA). The samples were centrifuged at 14,000 rpm for 10 min at 4ºC after a 30-min incubation at the same temperature. The supernatant was recovered and frozen at -80ºC until use.
Sample protein concentration was determined using the PierceTM BCA Protein Assay Kit (Thermo ScientificTM). For immunoblot assays, 10 µg per sample were used and denatured at 95ºC for 5-min in a sample buffer [0.5 M Tris HCl, pH 6.8, 10% glycerol, 2%(w/v) SDS, 5% (v/v) 2-mercaptoethanol, 0.05% bromophenol blue]. Electrophoresis was performed on acrylamide gels of 7, 10, and 12% concentration at constant 120 V and transferred to polyvinylidene difluoride sheets (Immobilon®-P Transfer Membrane; IPVH00010; Merk Millipore Ltd., USA) at constant 200 mA for 120 min. Then, membranes were blocked for 1-h with 5% non-fat milk dissolved in TBS-T buffer (0.5 mM Tris; NaCl, Tween® 20 (P1379, Sigma-Aldrich, USA), pH 7.5), washed with TBS-T 3 times for 5-min and incubated with the appropriate primary antibody, detailed in Table 1, overnight (O/N) at 4ºC. Subsequently, blots were washed in TBS-T buffer and incubated at room temperature for 1-h with the appropriate secondary antibody (Table 1). Finally, results were obtained through chemiluminescence detection using the Pierce® ECL Western Blotting Substrate (#32106, Thermo Scientific, USA), a Bio-Rad Universal Hood II Molecular Imager and the Image Lab v5.2.1 software (Bio-Rad laboratories). Measurements were expressed in arbitrary units and all results were normalized with the corresponding loading control (Glyceraldehyde-3-phosphate dehydrogenase; GAPDH).
Immunofluorescence
At least 4 animals per group were previously anesthetized by the i.p. injection of ketamine (100mg/Kg) and xylazine (10mg/Kg). When they were in the no-pain sleep phase, they were intracardially perfused with 4% paraformaldehyde (PFA) diluted in 0.1 M phosphate buffer (PB). After perfusion, brains were removed and stored in 4% PFA O/N at 4ºC. The next day, the solution was changed into 4% PFA+30% sucrose. Coronal sections of 20 µm were obtained by a cryostat (Leica Microsystems), and kept in a cryoprotectant solution at -20ºC until their use.
On the first day of the assay, free-floating sections were washed three times with 0.1 M PBS pH 7.35 and after, five times with PBS-T (0.1M PBS; 0.2% Triton X-100). Then, they were blocked in a solution containing 10% fetal bovine serum (FBS) and 1% Triton X-100 diluted with PBS-T five times for 5 min each and incubated with the primary antibody (Table 2) O/N. On the second day, slices were washed with PBS-T 5 times for 5 min and incubated with the pertinent secondary antibody (Table 2) for 2 h at room temperature. Finally, sections were treated with 0.1 µg/mL Hoechst (Sigma-Aldrich, St Louis, MO, USA) for 8 min in the dark at room temperature and washed with 0.1 M PBS. All reagents, containers and materials exposed to Hoechst were properly managed and processed to avoid any cytotoxic contamination. Finally, brain slices were mounted in gelatin-coated slides using Fluoromount G (EMS) and were left to dry O/N. Image acquisition was obtained using an epifluorescence microscope (BX61 Laboratory Microscope, Melville, NY-Olympus America Inc.) and quantified by ImageJ.
Statistical analysis
All results were represented as MEAN ± SD. Groups were compared against each other using two-way ANOVA. When variables independently were significant denote # p<0.05, ## p<0.01, ### p<0.001 and #### p<0.0001. When the overall ANOVA yielded significant effects, Tukey’s post hoc test was performed for comparison between groups (* p<0.05. ** p<0.01, *** p<0.001 and **** p<0.0001). Student’s t-test was used when the experiment was performed with 2 experimental groups (* p<0.05. ** p<0.01, *** p<0.001 and **** p<0.0001). All analyses and GAPDH representations were obtained using Graph Pad Prism software for Mac version 6.01; Graph Pad Software, Inc.