A syp-GFP construct (generated from a mouse complementary DNA clone) was provided by Dr. Niwa (our laboratory). KIF1A and KIF5B expression vectors were generated

by standard molecular methods. Detailed information is provided in the Supplemental Experimental Procedures. Astrocyte cultures were prepared as previously described (Suzuki et al., 2007). The cultures were or were not treated with BDNF (100 ng/ml) for 3 days. Detailed information is provided in the Supplemental Experimental Dinaciclib ic50 Procedures. Neurons were transfected with syp-GFP at 7 DIV and were incubated with or without BDNF (100 ng/ml) for 3 days. At 10 DIV, time-lapse recordings were performed with an LSM710 confocal laser-scanning microscope (Zeiss). We selected the middle part of axons of transfected neurons for live imaging. Images were acquired every 1 s, and syp-GFP containing vesicles moving across the center line of the imaged area were counted. Images were analyzed using ImageJ software. Neurons at 7 DIV were or were not treated with BDNF (100 ng/ml) for 3 days. At 10 DIV, neurons were fixed and immunostained as previously described (Niwa et al., 2008). Cells were fixed with 4% paraformaldehyde in PBS for 10 min, Abiraterone research buy permeabilized

with 0.1% Triton X-100 in PBS, and blocked with 5% bovine serum albumin in PBS. Cells were incubated with primary antibodies overnight at 4°C, followed by incubation with the appropriate Alexa-labeled secondary antibodies for 1 hr. Images were acquired using an LSM510 confocal laser-scanning microscope (Zeiss). Immunopositive puncta

along MAP2-labeled dendrites and synaptophysin/PSD-95-double-positive puncta were counted. For immunocytochemistry, anti-synaptophysin (mouse monoclonal, Chemicon, 1:1000; rabbit monoclonal, Abcam, 1:2000), anti-PSD-95 (mouse monoclonal, ABR, 1:200), and anti-MAP2 (chicken very polyclonal, Abcam, 1:2000) antibodies were used. Neurons were transfected with syp-GFP alone or cotransfected with syp-GFP and KIF1A or KIF5B at 7 DIV. At 10 DIV, neurons were fixed and immunostained for MAP2 and PSD-95, and images were acquired as described above. Synaptophysin-GFP puncta along MAP2-labeled dendrites and colocalized with PSD-95 were counted. Data were analyzed by the two-tailed t test or one-way ANOVA with a post hoc Dunnett’s test. For analysis of water maze test data, one-way ANOVA and two-tailed t test were used in the probe test, and two-way repeated-measures ANOVA with a post hoc Bonferroni’s test was used to compare differences between groups at several time points. We thank H. Sato, H. Fukuda, N. Onouchi, T. Akamatsu, T. Aizawa, and all other members of the Hirokawa laboratory for technical assistance and discussions. This work was supported by a grant-in-aid for specially promoted research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to N.H.).