La prueba del pragmatismo

For western blotting, samples were prepared as a total volume of 10µl

containing 10µg of protein per lane. The samples were separated (3 repeats/sample) on a 12% NuPage Novex Bis-Tris gel (Invitrogen) by electrophoresis at 200V for 20 minutes at room temperature. Following, proteins were transferred to an activated

PVDF membrane at 20V for 60 minutes. Membranes were blocked for 2 hours in 5% commercial skim milk powder. Membranes were incubated in primary antibodies overnight at 4°C in combinations of rabbit anti-synaptophysin (1:2000, Millipore); mouse anti-VGlut1 (1:1000, Millipore); mouse anti-PSD-95 (1:1000, Abcam) mouse anti-GAD65; mouse anti-GAD67 (1:1000, Millipore), rabbit anti-Gephyrin (1:1000, Abcam) and anti-β-actin (1:5000, Sigma-Aldrich) (Table 2.1). Membranes were washed in Tris-buffered saline with 0.1% Tween-20 (Sigma) and incubated in species-

appropriate secondary antibodies (1:7000, Dako, Table 2.2). Bands were visualised by staining with a chemiluminescent substrate kit (Millipore).

2.8.2 Aβ42 ELISA

Neocortex and hippocampus were homogenized in RIPA buffer (Sigma) containing a protease (Roche diagnostics) and phosphatase inhibitor cocktail (AG Scientific). The samples were centrifuged at 13000 RPM for 15 minutes, rotated for a further 30 minutes, and centrifuged again at 4°C for 15 minutes at 13000 RPM. The resulting supernatant was removed and stored at -80°C for protein analysis. The protein concentrations of samples were determined using the Bradford assay. For the

quantitation of human Aβ42, a sandwich antibody ELISA was performed according to

the manufacturer’s instructions (KHB3441, Invitrogen). APP/PS1 mice (n = 5 per group) were terminally anaesthetized first with gas anaesthesia (isoflurane) followed by sodium pentobarbitone (100 mg/kg delivered intraperitoneally) and perfused

transcardially with PBS (0.1M). Postmortem brains were removed and the cortex and hippocampus were dissected and immediately frozen in liquid nitrogen. Cortex and hippocampal samples were stored at -80°C for later analysis. Soluble human Aβ42 levels

were normalized to total protein levels and expressed as picogram of Aβ42content per

microplate reader (SpectraMax, Molecular Devices), and concentrations of Aβ42 were

determined by comparison to the standard curve using a 4-parameter algorithm.

2.8.3 Corticosterone ELISA

In order to determine if there were differences in levels of stress between the housing conditions and genotypes, blood was collected at time of perfusion by cardiac puncture, performed at the start of the dark period (3 p.m.). Blood samples were centrifuged for 5 minutes at 13 000 RPM and the resulting sera was collected. The sera samples were diluted at 1:100 in the buffer provided, and levels of serum corticosterone were measured by a competitive commercial ELISA kit (ab108821; Abcam) according to manufacturer instructions. Optical densities were read at 450 nm on a microplate reader (SpectraMax, Molecular Devices), and concentrations of corticosterone were determined by comparison to the standard curve using a 4-parameter algorithm.

2.8.4 BDNF ELISA

Each sample was prepared in duplicate and diluted in coating buffer (1:100; 60% NaHCO3, 30% Na2CO3 in distilled water), and 50 µl of diluted sample was added

per well to a 96-well flat-bottomed plate (Costar 5395, Sigma-Aldrich) and incubated at 4°C overnight. Following overnight incubation, the plate was washed five times with washing buffer (0.05% tween-20 in 0.01M PBS). Following, 100 µl of blocking buffer (5% fetal calf serum in 0.01M PBS) was added to each well and incubated at 37°C for 30 minutes. Following five washes, 50 µl of diluted primary BDNF antibody (1:500; Santa Cruz, Table 2.1) was added to each sample well, and incubated at room

temperature for 1 hour. Five washes were undertaken and the secondary HRP antibody was added (1:2000; anti-Rabbit, Dako, Table 2.2) and incubated at room temperature for 45 minutes. Following washing, 100 µl of freshly prepared Tetramethylbenzidine

(TMB; Sigma-Aldrich) substrate was applied to each well for 10 minutes, and 0.1M Sulphuric acid was added to stop the colour reaction. Optical densities were read at 450 nm on a microplate reader (SpectraMax, Molecular Devices), and concentrations of BDNF were determined by comparison to the standard curve using a 4-parameter algorithm. Values were averaged between the duplicate samples, and expressed as a percentage relative to Wt controls.

2.9 Statistical analyses

Analyses were performed using IBM SPSS (Version 20;). Statistical analyses were performed using independent t-tests, two-way analysis of variance (ANOVA), and repeated measures ANOVA. A statistically significant two-way ANOVA was followed up by separate independent t-tests, where the variables considered were genotype (Wt or Tg) and housing condition (SH or EE). When the variables considered were SH, EE, and EE+, a statistically significant two-way ANOVA was followed up with post hoc tests with Bonferroni correction applied for multiple comparisons. Means were reported as ± the standard error of the mean (SEM). The magnitude of differences between the means were reported as Cohen’s d. Values of p < .05 for differences between group means were classified as statistically significant.

Table 2.1. List of primary antibodies Antibody name Host

organism (Clone)

Isotype Immunizing agent Source (catalogue number)

Dilution

MOAB-2 Mouse

monoclonal IgG2b

C-terminal for Aβ40 and Aβ42 (Youmans et al., 2012) Novus Biologicals (NBP2- 13075) 1:2000 (IHC)

Synaptophysin Rabbit _polyclonal IgG

Synthetic peptide of human synaptophysin (313 amino acids) (Rehm, Wiedenmann, & Betz, 1986) Millipore (AB9272) 1:200 (IHC) 1:2000 (WB) VGlut1 Mouse monoclonal IgG1

Recombinant protein from rat VGlut1 (560 amino acids) (Fazzari et al., 2014) Millipore (MAB5502) 1:1000 (WB) PSD-95 Mouse monoclonal (clone 6G6- 1C9) IgG2a

Recombinant protein from rat PSD-95 (Li et al., 2010) Abcam (Ab2723) 1:1000 (WB) Gephyrin Rabbit polyclonal IgG Synthetic peptide of human Gephyrin (amino acids 396-445) (Harvey et al., 2004) Abcam (Ab83401) 1:1000 (WB) GAD65 Mouse monoclonal (clone GAD- 6) IgG2a

Purified GAD65 from rat (585 amino acids) (Besser et al., 2015) Millipore (MAB351) 1:1000 (WB) GAD67 Mouse monoclonal (clone 1G10.2) IgG2a Recombinant GAD67 protein (594 amino acids) (Fong, Stornetta, Foley, & Potts, 2005) Millipore (MAB5406) 1:1000 (WB) β-actin Mouse monoclonal (clone AC- 74) IgG2a Modified β-cytoplasmic actin N-terminal peptide (Arellano, Guadiana, Breunig, Rakic, & Sarkisian, 2012) Sigma- Aldrich (A5316) 1:5000 (WB) BDNF Rabbit polyclonal IgG

Epitope mapping, internal region of Human BDNF (amino acids 128-147) (Flores-Otero & Davis, 2011) Santa Cruz (SC-546) 1:500 (ELISA) Iba1 Rabbit Synthetic peptide corresponding to C- terminus of Iba1from rabbit (Ito et al., 1998)

Wako (019-

Table 2.2. List of secondary antibodies

Emission (nm) Species/Reactivity Dilution Supplier (catalogue

number)

488 Goat/Mouse IgG1 1:1000 Molecular probes (A- 11001)

546 Goat/Mouse IgG2b 1:1000 Molecular probes (A- 21143)

488 Goat/Rabbit IgG 1:1000 Molecular probes (A- 11008

546 Goat/Rabbit 1:1000 Molecular probes (A- 11035)

3. Mid-life environmental enrichment increases synaptic density in