Mice were perfused transcardially with 50 ml ice-chilled 4% paraformaldehyde in PBS; brains were collected and kept in fixation solution at least overnight at 4°C. Transverse hippocampal sections of 150 μm thickness were generated as previously described (Galimberti et al., 2010). To generate coronal sections, brains Dinaciclib nmr from perfused animals were incubated overnight in PBS containing 30% sucrose and sectioned at 50 μm on a cryostat (Microm) at −5°C. Free-floating coronal and transverse sections

were blocked for 1 hr at room temperature in PBS-T containing 3% BSA, then incubated with the primary antibody solution (PBS-T, 3% BSA) over night at 4°C, and subsequently incubated with the secondary antibody solution (PBS-T) for 3 hr at room temperature. Anisomycin (Tocris) at the concentration of 50 mg/ml and pH 7.2 was injected bilaterally into mouse DG at position −2.18 posterior, 0.96 lateral, 1.90 ventral. Chelerythrine (LC laboratories) at the concentration of 0.5 mg/ml in PBS was injected i.p. at a dosis of 5 mg/kg weight. For the anisomycin experiments, chelerythrine injections were either 1 hr before or 12 hr after anisomycin. For the behavioral or immunoblot analyses chelerythrine was injected 12 hr before learning (novel object recognition) or sacrifice (immunoblot

analysis). Transverse hippocampal sections from perfused animals were used for the analysis of mossy fiber projections in CA3, CA3 pyramidal cell thorny excrescences, Y27632 and CA1 pyramidal cell dendrites. High-resolution images were acquired on an LSM510 confocal microscope (Zeiss) using

a 63× (1.4) oil-immersion objective. Microscope images were deconvolved (Huygens) and analyzed using Imaris 7.0.0 (Bitplane AG) software. We defined LMTs as mossy fiber terminal regions of >2.5 μm diameter in CA3a–c that were arranged either en-passant or as side structures connected to the mossy fiber axon or another LMT by an axonal process (satellite) (Gogolla et al., 2009). For the quantification of LMT volumes and surface areas at least three confocal 3D stacks were acquired in CA3b for each preparation (at least two mice per condition) and analyzed using Imaris Bay 11-7085 7.0.0 software by creating an isosurface object corresponding to each LMT. Complexity was defined as a ratio of the maximum volume (i.e., sphere) given the measured surface area to actual measured volume. Protein extracts for immunoblot experiments were obtained as follows. Brains were flash-frozen at −40°C and cut into 1 mm coronal slices on an iced stage. Stratum lucidum fragments were dissected using a tip of a Pasteur pipette, and homogenized by shearing in lysis buffer (25 mM sucrose, 25mM KCl, 1M Tris, COMPLETE protease inhibitors, pH 7.5). For immunoblots, 20 mg total protein of each lysate was run on an SDS-PAGE gel (5%–10%). The blots were scanned and analyzed using ImageJ.