Mouse primary hippocampal neurons were isolated as follows. Briefly, hippocampal tissue was isolated from embryonic day 16 (E16) mouse pups. Then, gentle dissociation of neuronal cells was performed with trypsin (0.25%) and mechanical disruption was done to separate cells from connective tissue while providing minimum damage to individual neuronal cells [19 ]. The isolated neurons were cultured essentially as described [3 (link), 6 (link)]. Transient transfection was carried out using Lipofectamine 2000 (Life Technologies). Immunofluorescence analysis was done as described [6 (link)]. Briefly, cells grown on 13-mm coverslips were fixed in 3.7% formaldehyde in PBS for 15 min, then treated with 0.2% Triton-X 100 for 5 min. 4',6-diamidino-2-phenylindole (DAPI; Nichirei Bioscience, Tokyo, Japan) and anti-ARHGEF9 were used for staining of DNA and ARHGEF9, respectively. Alexa Fluor 488-labeled IgG (Life Technologies) was used as a secondary antibody. Images were captured using FV-1000 confocal laser microscope (Olympus, Tokyo, Japan).