Synaptoneurosome preparation

Synaptoneurosomes were prepared as previously described (17) with slight modifications (16). Briefly, four to six hippocampi were dissected from adult Wistar rats and tissue was minced with scissors and homogenized with a KONTES Dounce tissue grinder in a buffer containing 0.32 M sucrose, 10 mM Hepes-tris (pH 7.4), and 0.1 mM EGTA using, first, a pestle with large clearance, 0.889 to 0.165 mm (8 to 10 strokes), followed by a small clearance pestle, 0.025 to 0.076 mm (8 to 10 strokes). After centrifugation for 3 min at 1000g, the supernatant was collected and passed initially through nylon membranes (150 and 50 μm, VWR) and finally through an 8-μm pore size filter (Millipore). The flow-through was centrifuged for 15 min at 10,000g, and the resulting pellet was resuspended in incubation buffer [8 mM KCl, 3 mM CaCl₂, 5 mM Na₂HPO₄, 2 mM MgCl₂, 33 mM tris, 72 mM NaCl, and 100 mM sucrose (pH 7.4)]. All procedures were performed at 4°C. Before stimulation, synaptoneurosomes were prewarmed at 30°C during 5 min in the same buffer. Incubation with BDNF (50 ng/ml; PeproTech) was performed at the same temperature, and a control experiment in the absence of the neurotrophin was also performed for each time point considered. Where indicated, synaptoneurosomes were preincubated with cycloheximide (50 μg/ml; Merck-Millipore) or with the vehicle, DMSO (1:1000 dilution; Sigma-Aldrich), for 15 min at 30°C. Synaptoneurosomes were then briefly centrifuged in a Minispin microcentrifuge (Eppendorf) for 30 s, and the pellet was resuspended in radioimmunoprecipitation assay (RIPA) buffer [150 mM NaCl, 50 mM tris-HCl, 5 mM EGTA, 1% Triton, 0.5% dissolved organic carbon (DOC), and 0.1% SDS (pH 7.5)] supplemented as indicated for the preparation of extracts, followed by sonication and protein quantification using the bicinchoninic acid (BCA) method. For polysome extraction, the pellet was resuspended in mammalian lysis buffer [15 mM tris-HCl (pH 8), 5 mM MgCl₂, 0.3 M NaCl, 0.5 mM dithiothreitol (DTT), cycloheximide (0.1 mg/ml) (Merck-Millipore), and 1% Triton X-100] supplemented as indicated for isolation of polysomes.