Experiments were carried out in accordance with the Guidelines laid down by the NIH in the USA regarding

the care and use of animals for experimental procedures. Pregnant ICR mice (SLC, Shizuoka, Japan) were briefly anesthetised with ether, and then killed by cervical dislocation. The preparation of hippocampal cultures from 17-day-old embryonic mice has been described previously (Okabe et al., 1999). The transfection of hippocampal neurons was performed by a Ca2+-phosphate transfection selleck inhibitor method at 5–7 days in vitro (DIV; Jiang & Chen, 2006). Hippocampal neurons were fixed in 2% paraformaldehyde in phosphate-buffered saline for 25 min, permeabilised with 0.2% Triton X-100 for 5 min, blocked with 5% normal goat serum for 30 min and reacted with mouse monoclonal antibody to cytochrome c (Promega, Madison, WI, USA). The first antibody was visualised by secondary antibody staining using goat anti-mouse IgG conjugated to Alexa 647 (Molecular Probes, Eugene, OR, USA). All procedures Selleck 17-AAG were performed at room temperature (set at 24 °C). FM1-43 (Molecular Probes) loading was performed by exposing neurons to the dye (15 μm) in high-K+ saline solution (75 mm NaCl, 70 mm KCl, 2 mm CaCl2, 2 mm MgCl2, 5 mm HEPES and 20 mm glucose, pH 7.4) for 2 min followed by washing in low-Ca2+ saline solution (140 mm NaCl, 5 mm KCl, 0.1 mm CaCl2, 4 mm MgCl2, 5 mm HEPES and 20 mm glucose, pH 7.4) three times for 2 min.

After taking the first images in low-Ca2+ saline

solution, neurons were exposed to high-K+ saline solution for 2 min and then switched to low-Ca2+ saline solution again for washing. Second images were taken at the same axonal regions. The difference selleck kinase inhibitor of fluorescence intensity between the first and second images was used for analysis as FM1-43(Δ). Images were obtained by using a Fluoview confocal laser-scanning microscope with ×60 1.4 NA oil-immersion lenses (Olympus, Tokyo, Japan). A confocal aperture was set at a diameter of 600–700 μm. For some images, multiple optical sections (3–7 sections and z-spacing of 1.0 μm) were collected, and these images were recombined using a maximum-brightness operation. The axons were identified morphologically and we selected imaging areas at least 100 μm away from the soma. For time-lapse imaging, live cells were mounted in a chamber at 37 °C with a water bath and continuous flow of humidified 5% CO2 to maintain the osmolality and pH of the medium during prolonged time-lapse experiments. For time-lapse imaging with tetrodotoxin (TTX; Wako, Tokyo, Japan), the first frame was imaged at least 30 min after adding TTX to the medium (final concentration, 1 μm). For time-lapse imaging at intervals of 1 day, the duration of single imaging sessions was restricted within 30 min. For an analysis of transport properties, mCherry-OMP was imaged at intervals of 3 s and APP-mCherry was imaged at intervals of 1 s.