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Adult male Wistar rats (2–3 months old, weighing 200–300 g) were housed in groups of four per cage and kept in a room with controlled temperature (22 ± 2°C) and a 12-h light/dark cycle (lights on at 07:00 a.m.) with free access to food and water, except during the experiments. Rats were allowed to adapt to the laboratory conditions for one week before the experiments. Behavioral experiments and hormonal dosages were carried out during the light phase of the cycle (between 13:00 and 18:00). All experiments were conducted in accordance with international standards of animal welfare recommended by the Brazilian Law (#11.794–10/08/2008) and Animals (Scientific Procedures) Act 1986, with experimental protocols approved by the Committee for Ethics in Animal Research of the Federal University of Santa Catarina (CEUA-UFSC #23080.025621/2009-03) and Home Office License 30/2512 (UK). The minimum number of animals and duration of observation required to obtain consistent data was used.
Pilocarpine hydrochloride (a non-selective muscarinic receptor agonist; Sigma-Aldrich Co., St. Louis, USA, 150mg/kg, i.p.) and memantine hydrochloride (an NMDA receptor antagonist; Ebix®, Merz Pharma GmbH & Co. KgaA, Frankfurt, Germany, 4 mg/kg, i.p.) were injected intraperitoneally whereas methyl-scopolamine bromide (a muscarinic receptor antagonist; RBI, USA, 1 mg/kg, s.c.) was given subcutaneously and used to prevent the peripheral cholinomimetic effects elicited by pilocarpine. All drugs were freshly dissolved in saline solution (NaCl 0.9%), which was used as control solution as well, in a volume injection of 1 ml/kg. All doses used here were taken from previous studies [5–7, 33].
The EPM apparatus (EP151, Insight Ltda., Ribeirão Preto, Brazil) was made of wood and consisted of two opposing open arms (50 cm × 10 cm) and two opposing enclosed arms (50 cm × 10 cm × 30 cm) mounted at an angle of 90°, all facing a central platform (10 cm × 10 cm), elevated 40 cm from the floor. To prevent falls, the open arms were surrounded by a 1-cm high acrylic rim. The apparatus was placed in a small closed room lit by a 15-W red light that provided 3 lux in both the open and closed arms. Each rat was used only once and was placed individually on the central platform facing an enclosed arm. The frequency of entries into either open or enclosed arms as well as the time spent in each arm type were recorded (in seconds) for 5 min [39]. Ethological parameters such as protected stretch-attend postures, unprotected head-dipping, open-arms end activity and rearing were also recorded to increase the sensitivity of the test [40, 41]. The apparatus was cleaned with 10% ethanol solution between sessions.
Experiments were performed in two main designs: In experiment 1, aiming to better understand hormonal properties of rats treated with pilocarpine, ex vivo biochemical assays were performed on blood samples (n = 3–6 per group) for CORT and adrenocorticotropic hormone (ACTH) quantification and samples from hippocampus (n = 6 per group) were extracted to analyze GR expression short (24 h) and long after treatment (1 month). In experiment 2, taking into account the pivotal role of NMDARs in the modulation of anxiety [26] and neural plasticity [42] following mAChR activation [43], samples from hippocampus were also extracted to analyze NMDARs expression in rats injected with saline or pilocarpine (n = 5–6 per group). Further, additional groups were previously treated with saline or memantine following 30 min later by a single systemic injection of saline or pilocarpine. Behavioral evaluation of these groups (n = 12 per group) was carried out in the EPM test 24 h or 1 month after treatments and the brain randomically extracted (n = 3–6 per group) immediately after the tests. Rats were euthanized and the brain was removed and washed with saline solution (NaCl 0.9%) at 4°C, dissected over a cooled Petri dish. The samples were stored in Eppendorfs and kept in a freezer at -80°C.
Blood sampling and hormone measures: Twenty four hours or 1 month after the injection of pilocarpine, animals were housed in a sound-proof room for up to 48 h, prior to be euthanized with isoflurane and decapitated using a guillotine. Trunk blood was collected on ice into tubes containing 50 μl of EDTA (0.5 m; pH 7.4) and 50 μl of aprotinin (5000 KIU/ml, Trasylol; Bayer, EDTA, Newbury, UK). Plasma was separated by centrifugation and then stored at −80°C until processed for CORT and ACTH measurement. CORT levels were determined in triplicate by a double antibody radioimmunoassay method as previously described [44]. Antisera was kindly supplied by Prof. G. Makara (Institute of Experimental Medicine, Budapest, Hungary), and [125I] CORT was purchased from Izotop (Budapest, Hungary). The intra- and interassay coefficients of variation of the CORT assay were 16.7% and 13.3%, respectively. ACTH in plasma was measured using an immunoradiometric (IRMA) assay (DiaSorin, Stillwater, MN, USA) in accordance with the manufacturer’s instructions. The intra- and inter-assay coefficients of variation of the ACTH assay were 2.8% and 6.4%, respectively.
Rats were killed by decapitation 24 h or 1 month after treatment (immediately after the behavioral tests) and the hippocampus were removed and homogenized 1:10 (w/v) in HEPES 20 mM, pH 7.4 buffer, as previously described by Dutra et al., [45]. For Western blotting assays, the hippocampal tissue was homogenized in complete radioimmunoprecipitation lysis buffer (RIPA) containing 50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 2 mM EDTA, 1% Igepal CA-630, 1 mM Na 3 VO 4, 50 mM NaF, 1 mM PMSF, 20 μg/mL pepstatin A, 20 μg/mL leupeptin and 20 μg/mL aprotinin. The lysate was centrifuged twice at 13,000 g for 10 min at 4°C, and the supernatant was collected for protein concentration determination and preparation for electrophoresis. Equal amounts of protein extract (20 μg) were loaded per lane and electrophoretically separated using 10% denaturing polyacrylamide gel electrophoresis (SDS-PAGE). Afterward, the proteins were transferred to nitrocellulose membranes using a Mini Trans-Blot Cell System (Bio-Rad Laboratories Inc., Hercules, CA, USA) following the manufacturer’s protocol. The membrane was blocked with 5% BSA in 0.05% TBST for 1 h at room temperature and then immunoblotted with the following antibodies: anti-β-actin (#3700, 1:1000, Cell Signaling Technology, Danvers, USA), anti-NMDAR1 (#4204, 1:1000, Cell Signaling Technology, Danvers, USA), anti-NMDAR2B (#MAB5220, 1:1000, EMD Millipore Corporation, Billerica, USA) and anti-GR (#SAB4501310, 1:500, Sigma-Aldrich) in blocking buffer at 4°C overnight. Following washing, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (1:25,000, Cell Signaling Technology, Danvers, MA, USA). The immunocomplexes were visualized using SuperSignal West Femto Chemiluminescent Substrate Detection System (Thermo Fischer Scientific, Rockford, IL, USA) and densitometric values were normalized using β-actin densitometric values. Protein levels were quantified by optical density using Image-J Software® and expressed as the ratio to β-actin represented by arbitrary units.
All values are expressed as means ± S.E.M. Data of experiments were analyzed by unpaired two-tailed Student´s t test when only the treatment factor was the grouping variable or ANOVA when two grouping variables–pretreatment and treatment–were used as factors, followed by the Student Newman–Keuls’ post hoc test for multiple comparisons when appropriate. Differences were considered significant at p ≤ 0.05. All tests were performed using the software Statistica® (StaSoft Inc.,Tulsa, USA), version 8.0 and graphs were drawn with the software GraphPad Prism®, version 5.0.
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