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  • Male C57BL/6JRccHsd mice (Harlan Laboratories, Netherlands), trkB.T1−/− [24], Bdnf+/−/Bdnf−/− [19] or BDNF2L/2LCk-cre [20] and their wild-type littermates were used for the studies. The animals were kept under standard laboratory conditions (21°C, 12 h light-dark cycle, lights on at 6 A.M.). All the experiments were carried out according to the guidelines of the Society for Neuroscience and were specifically approved by the County Administrative Board of Southern Finland (Permit: ESLH-2007- 09085/Ym-23). Dams with their litters were housed individually. For postnatal acute antidepressant treatments, age-matched litters (P5-21) were randomly assigned to receive i.p. injection of saline (SAL) (NaCl 0.9%, 5 ml/kg), imipramine (IMI) (HCl salt, dissolved in SAL, 30 mg/kg, 5 ml/kg; Sigma-Aldrich Finland Oy, Helsinki), or clomipramine (HCl salt, dissolved in SAL, 20 mg/kg, 5 ml/kg; Sigma-Aldrich) and following indicated lag-time (30–60 min) hippocampus and medial prefrontal cortex were collected as described in [9]. Briefly, mice were stunned with CO2, the brains quickly removed and bilateral hippocampus and prefrontal cortex were dissected out on a dish cooled on dry-ice. Samples were homogenized in a NP++ buffer (300 µl/sample; composition: 137 mM NaCl, 20 mM Tris, 1% NP-40, 10% glycerol, 48 mM NaF, H2O, 2× Complete inhibitor mix (Roche) and 2 mM Na3VO4). After incubation on ice for 15 min, samples were centrifuged (16100 g, 15 min, +4°C) and the supernatant collected for further analysis. Dams with their litters were housed individually. The litters were randomly assigned to 4 groups: saline injected (SAL) (NaCl 0.9%, 5 ml/kg), and clomipramine injected (CLO) (dissolved in SAL, 20 mg/kg, 5 ml/kg) [16], starting at postnatal day 4 (P4) until P9 (Early-Postnatal Stage, E-PS), and from P16 to P21 (Late-Postnatal Stage, L-PS). Each pup was weighted and injected once daily (between 9 A.M.–11 A.M.). During the treatment the litter was removed from its home cage and placed in a bucket with some shavings of their home cage. All the pups belonging to a single litter were injected randomly in less than 3 minutes. After the injection the pups were immediately placed back in their home cage. CLO treatments did not have any significant effect on the weight gain of the pups and later in adulthood (Figure S4). Mice were weaned on P22 and males were housed together (4–6 mice/cage) until the behavioural experiments. For biochemical analyses the animals were killed after 2 weeks of the last behavioural test and their tissues were collected and processed as described before. For the acute AD treatments, adult (∼P90) mice received a single i.p. injection of imipramine (dissolved in SAL, 30 mg/kg, 5 ml/kg) or SAL and were killed 30 minutes after. For chronic AD treatment, adult (∼P90) mice had free access to either tap water or to 0.08 mg/ml solution of fluoxetine (FLX; HCl salt; Orion Pharma, Turku, Finland) for 21 days [9], [28]. On the final day of treatment the animals were killed. Hippocampus and prefrontal cortex samples were rapidly dissected out and processed as described before. The ex vivo BDNF stimulation assay was performed according to Knüsel et al [13] with slight modifications. Mouse hippocampi and medial prefrontal cortex at different ages were dissected. After dissection the samples were placed on a filter paper wet with cold Neurobasal Medium (NBM) (Neurobasal medium (Gibco), 2% B27 supplement (Gibco), 0.5 mM Glutamine (Gibco) and penicillin/streptomycin (Sigma), and then the tissues were sliced into equally sized pieces. The slices were transferred in fresh tubes and washed twice with NBM +10% heat-inactivated Fetal Calf Serum (FCS) (Gibco). Tissues were gently re-suspended with a Pasteur pipette. The medium was removed and 600 µl of NBM +10% FCS with or without BDNF (Peprotech) or NGF (Promega) were added. The tubes were closed and incubated at +37°C for 5 or 15 minutes gently shaking. Finally the tubes were put on ice, spun down, the medium removed, the pellet was rinsed once with PBS and then the samples were homogenized in NP++ buffer. A set of samples were pre-incubated with cAMP analog sp-cAMP (10 µM; Sigma-Aldrich) for 15 minutes at +37°C before BDNF stimulation. Assays were performed according to the procedures described by Angeles et al [42] with some modifications. Each assay was performed in a final volume of 20 µL of 50 mM Hepes pH 7.4, 140 mM NaCl, 10 mM MnCl2, 0.05% BSA, 2% DMSO with or without 100 µM ATP and/or 50 ng/mL BDNF. The reaction was initiated adding 40 µg of NP++ lyzed protein extract to the mix and the incubation was allowed to proceed for 10 min at 37°C. For a subset of samples, the reaction was quenched by adding an equal volume of 4× Laemmli sample buffer and proteins were separated by SDS-PAGE and TrkB phosphorylation status analyzed with western blotting as described below. Rest of the samples we immediately transferred to Trk antibody containing ELISA (enzyme-linked immunosorbent assay) plates, 3% BSA/PBS-T (+2 mM Na3VO4) added ad 200 µl, and phospho-TrkB ELISA proceeded as previously described [18]. Sample protein concentrations were measured using the Lowry Method (Bio-Rad DC protein assay). Lectin precipitation was carried out essentially as described [9] using Wheat Germ Agglutinin (EY Laboratories, San Matteo, CA, USA). TrkB immunoprecipitation was carried out using a TrkB specific antibody (5 µl/sample; #AF1494, R&D Systems Europe, Abingdon, UK) in conditions described in [9]. Proteins were separated in a SDS-PAGE under reducing conditions and blotted to a PDVF membrane (300 mA for 1 h at 4°C). Membranes were incubated with the following primary antibodies: anti-p-TrkBY816 (1∶5000; kind gift from Dr. M. Chao, Skirball Institute, NY, USA), anti-p-TrkA/BY490/Y515 (#9141; 1∶2000; Cell Signalling Technology (CST), MA, USA), anti-p-TrkA/BY674-5/Y705-6 (1∶1000; CST), anti-TrkBout (#610102; 1∶2000; RD Transduction Laboratories, Franklin Lakes, NJ USA), anti-p-CREBS133 (#9198; 1∶1000; CST), anti-CREB (sc-186; 1∶100, Santa Cruz Biotechnology (SCB), CA, USA), anti-p-AktThr308 (#9275; 1∶2000, CST), anti-AKT (#4691; 1∶1000, CST), anti-p-PLCγ1Y783 (#2821, 1∶1000, CST) or anti-Trk (#sc-11 (rabbit), 1∶1000, SCB). Further the membranes were washed with TBS/0.01% Tween® (TBST) and incubated with horseradish peroxidase conjugated secondary antibodies (1∶10000 in non fat dry milk, 1 h, RT, Bio-Rad Laboratories, Hercules, CA). After subsequent TBST washes, secondary antibodies were visualized using electrochemiluminescence kits (Amersham Biosciences) followed by an exposure to Fuji LAS-3000 Camera (Tamro Medlabs, Vantaa, Finland) for ECL detection. Battery of behavioural tests [43] were performed on adult male E-PS, L-PS and control mice, starting from P90. The tests were done between 9 A.M.-3 P.M. with at least 3 days of interval between each test. Exploratory locomotion, depression- and anxiety-like behaviours were assessed by the following tests: light-dark test, elevated plus maze, open-field test, forced swim test and novelty-suppressed feeding test. The tests were performed in this order in a period of 3 weeks [43]. Among all these tests, only the light-dark test and the novelty-suppressed feeding test showed statistically significant differences between the drug and saline treated groups, and only these two tests are described in more detail in this manuscript. Testing was performed for 10 min in an acrylic cage (28.5×28.5×20 cm) (TSE, Bad Homburg, Germany) divided into two equal sized compartments: one part with transparent walls, open topped and brightly illuminated (∼450 lx by a 40 W light bulb fixed 55 cm above the floor), the other part made from black plastic (passing infrared light) and covered by a lid. The two compartments were separated by a partition containing an opening (7×5 cm) in its centre at floor level. The animal was placed in the centre of the light compartment facing away from the opening, and the latency to enter dark area, time spent in the compartments, total distance travelled, immobility time and the number of entries to dark were measured over 10 min. In addition, rearing time was also calculated. Testing apparatus was thoroughly cleaned before each animal using 70% ethanol. Novelty-Suppressed Feeding: The Novelty-Suppressed Feeding (NSF) was performed as previously described [30]. Briefly, the test was performed in brightly lit (800–900 lux) open arena (51×35 cm). A small piece of filter paper with a pre-weighted food pellet was placed in the middle of the arena. The animals were deprived of food for 24 h with water available ad libitum. On testing day each animal was removed from its home cage and placed in a holding cage for 30 min before the test and then put in one corner of the arena. The latency to beginning of a feeding episode was measured (maximum over a period of 5 min). Immediately, after starting of feeding the mouse was removed from the arena and placed with the food pellet in its home cage and allowed to feed ad libitum over a period of 5 min. The amount of food consumed was quantified by weighting the pellet. Immunoblot bands were quantified using NIH ImageJ. All the data are expressed as mean ± SEM (Standard Error of Mean) and as percentage of control. Statistical analyses were performed using GraphPad Prism 4.0 for Windows (GraphPad Software, San Diego California USA). For comparison between two groups Two-way Student's t-test was used. Two-way ANOVA or two-way ANOVA for repeated measures was used to reveal main effect and interaction between the factors followed by Bonferroni post hoc test. The criterion for significance was set to p<0.05.
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