These experiments used adult male C57BL6 mice (24-32 gm; Jackson Laboratory, Bar Harbor, ME) aged 3 months, were allowed one week to habituate in an environment controlled for temperature, lighting, and food and water administration. Animals were housed four per cage.
Animals were anesthetized using isofluorane and implanted subcutaneously either with two 5 mg corticosterone (CORT group, n=14) or two placebo pellets (Placebo group, n=14), 21 day release formulation (Innovative Research of America, Sarasota, FL), in accordance with the manufacturer’s instructions. Accordingly, each corticosterone-implanted animal received a dose of 20mg/kg/day.
After 20 days, the animals were euthanized, their brains removed and bisected, with one hemisphere stored at −80 °C, and the other fixed in 4% paraformaldehyde. All procedures were conducted according to IACUC-approved protocols.
Synaptosomes were prepared using a one-step synaptosome preparation based on the method of Phillips and colleagues,(Phillips et al., 2001
) as described previously.(Louneva et al., 2008
) In brief, synaptosomes were isolated using a sucrose gradient, in which the synaptosomal fraction forms a band at the 1.25/1.0 mol/L sucrose interface. This fraction was collected and stored at −80 °C. Half of this material was used for Western blotting and another half for further fractionation.
Synaptosomes were further fractionated to separate pre- and postsynaptic elements according to previously described methods.(Louneva et al., 2008
) Briefly, synaptosomes were solubilized in 20 mmol/L Tris-HCl, pH 6.0, 1% Triton X-100, 0.1 mmol/L CaCl2
, incubated on ice for 30 minutes, and centrifuged at 40,000 × g
, yielding the supernatant as the synaptic vesicle fraction (SV). The pellet contains the pre-and postsynaptic membranes, and this material was solubilized in 20 mmol/L Tris-HCl, pH 8.0, 1% Triton X-100, 0.1 mmol/L CaCl2
, incubated on ice for 30 minutes, and centrifuged at 40,000 × g
resulting in the supernatant containing the presynaptic fraction (PrS) and the pellet containing the post synaptic density fraction (PSD). Ultimately, all subsynaptic fractions (synaptic vesicle, presynaptic, and PSD fractions) were dissolved in 5% SDS. Protein concentrations were determined by the BCA method (Pierce, Rockford, IL).
For Western blot analysis of synaptosomes, one ml of synaptosome material was washed twice in 0.1 mmol/L CaCl2 and twenty μg of each protein sample in Laemly loading buffer were separated on 12% Tris-glycine gels (Novex Invitrogen, Carlsbad, CA). All 28 samples were run at one time on two gels with seven placebo and seven treated mice brains on one gel and the same design on the other gel. Fresh Western blots were run for PSD-95, NR1 and synaptopodin.
The gels were transferred to PVDF membranes (Bio-Rad, Hercules, CA). Membranes were blocked with 5% milk and incubated with primary antibody overnight at 4 °C. In order to be in linear range primary antibodies were used at the following dilutions in 3% milk in Tris-buffered saline with 0.1% Tween-20: PSD-95, 1:10,000 (MAB1598; Chemicon/Millipore, Billerica, MA); NR1, 1:500 (sc-9058; Santa Cruz Biotechnology, Santa Cruz, CA), synaptopodin, 1:1,500 (S9567; Sigma, St. Louis, MO) and synaptophysin, 1:20,000 (MSB5258; Chemicon, Millipore, Billerica, MA). After primary antibody incubation, membranes were incubated with a horseradish peroxidase-coupled secondary antibody (HRP-conjugated mouse or rabbit IgG, NA931 or NA934 [Amersham, Piscataway, NJ]) for one hour at room temperature and processed with the ECL or ECL Plus chemiluminescence system (Amersham, Piscataway, NJ). All membranes were stripped and re-probed with β-actin, 1:4,000 (A3853; Sigma, St. Louis, MO) for normalization purposes. Band densities were quantified by densitometric analysis with the GS-800 calibrated densitometer and Quantity One 1-D analysis software (Bio-Rad, Hercules, CA). All data is presented in relative units (ratio protein of interest to β-actin). The same Western blot analysis was used for measurement of the 28 PSD fraction samples.
Fixed, paraffin-embedded tissues were cut in the coronal plane at 6 μm on a rotary microtome that had been tested to assure invariant section thickness and then mounted on APES-coated slides. De-waxed sections were immersed in 5% hydrogen peroxide dissolved in absolute methanol for 30 minutes to quench endogenous peroxidase activity. For antigen retrieval, the sections were boiled in 1 mmol/L ethylenediaminetetraacetic acid in 0.1 mol/L Tris buffer, pH 8.0, for 10 minutes. After cooling for 20 minutes and rinsing in water, followed by two changes of Tris-Triton (0.01% Triton X-100 in 0.1 mol/L Tris-HCl buffer, pH 7.6), sections were blocked for 45 minutes in 2% horse serum dissolved in Tris-Triton and incubated in the primary antibody, PSD-95, 1:200 (MAB1598; Chemicon/Millipore, Billerica, MA), NR1, 1:500 (sc-9058; Santa Cruz Biotechnology, Santa Cruz, CA), and synaptopodin, (1:1,000-(S9567; Sigma, St. Louis, MO); 1:4,000[163 002; Synaptic Systems, Goettingen, Germany]) overnight at 4 °C. After Tris-Triton rinses, sections were incubated in a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) for an hour at room temperature. Sections were then treated for another hour at room temperature with an avidin-biotin-peroxidase complex made from a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) and developed for 10 minutes in a solution containing 0.05% diaminobenzidine (Biogenex, San Ramon, CA) and 0.03% hydrogen peroxidase in Tris-Triton, supplemented with 0.25% NiSO4·H2O to amplify the immunohistochemical signal. After clearing in xylenes, all tissue sections were coverslipped under Cytoseal 60 (Richard-Allan Scientific, Kalamazoo, MI).
Immunolabeled slides were first qualitatively examined, and then immunoreactivity was semiquantitatively measured in the molecular layer of the dentate gyrus and the stratum lucidum of the CA3 region of the hippocampal formation by net optical density (OD), defined as the OD of the region of interest minus the OD of the background (obtained from blood vessel walls in the regions of interest). OD analyses were performed on high resolution, gray-scale photomontages acquired on a Leica DMRBE microscope equipped with a motorized microscope stage using Image-Pro Plus software, version 6.2 (Media Cybernetics, Silver Spring, MD). OD comparisons were performed on sections photographed at the same, verified light intensity. The operator was blind to any identifying information throughout data accrual and analysis.
Statistical significance of differences between diagnostic groups was assessed with two-tailed Student’s t-test, using JMP 7.0.2 software (SAS, Cary, NC). Statistical significance was defined as P < 0.05.