, 2010). For vector-based RNA interference (RNAi) analysis, we used a BLOCK-iT Pol II miR RNAi Expression Vector Kit (Invitrogen). The engineered miRNA constructs were produced by PCR amplification click here of miRNA region in BLOCK-iT Pol II miR RNAi expression vector followed by subcloning into pCL20c-L7 at 5′- or 3′-side of a fluorescent protein or ChR2 as indicated in the figures. Other details for preparation of viral vector constructs and

methods for virus infection are described in the Supplemental Experimental Procedures. Recordings from PCs in the cocultures were performed as described previously (Uesaka et al., 2012) and are detailed in the Supplemental Experimental Procedures. To stimulate CFs, square voltage pulses (duration, 0.1 ms; amplitude, 0–90 V) were applied between two of the eight tungsten electrodes placed in the medullary explants. All possible combinations of two electrodes were tested, and stimulus

intensity was carefully increased from 0 V to 90 V for each stimulation pair so as not to miss the CFs innervating the recorded PC. Preparation of acute cerebellar slices and recording from PCs were made as described previously (Hashimoto and Kano, 2003 and Hashimoto et al., 2009b) and are detailed in the Supplemental Experimental Procedures. To record CF-EPSCs, stimuli (duration, 0.1 ms; amplitude, PI3K Inhibitor Library solubility dmso 0-90 V) were applied at 0.2 Hz through a patch pipette filled with normal external solution. CFs were stimulated in the granule cell layer 20–100 μm away from the PC soma. For each PC, the pipette for CF stimulation was moved systematically by about 20 μm isothipendyl step around the PC soma, and the stimulus intensity was increased gradually from 0 V to about 90 V at each stimulation site. The number of CFs innervating the recorded PC was estimated by the number of discrete CF-EPSC steps as previously described (Hashimoto and Kano, 2003 and Hashimoto et al., 2009b). All statistical values were presented as mean ± SEM unless

indicated otherwise. The Mann-Whitney U test or Student’s t test was used as indicated in the text when two independent samples were compared. For multiple comparison, Kruskal-Wallis test, Steel-Dwass test, Dunnett test, and two-way ANOVA were used as indicated in the text. Statistical analysis was conducted with JMP Pro. Differences between data sets were judged to be significant at p < 0.05. ∗, ∗∗, ∗∗∗, and ∗∗∗∗ represents p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. The authors thank A. Nienhuis, St. Jude Children’s Research Hospital, and George Washington University for the gifts of the lentiviral backbone vector and the packaging plasmid, T. Nakazawa for helpful advice for real-time PCR, K. Kitamura and K. Hashimoto for helpful discussions, M. Mahoney for critically reading this manuscript, and K. Matsuyama, M. Sekiguchi, S. Tanaka, and A. Koseki for technical assistance.