5056, Mann-Whitney U test). The paired-pulse ratio of Proteasome inhibitors in cancer therapy CF-EPSCs (Table S2; p = 0.2568, Mann-Whitney U test), the disparity index and disparity ratio (Table S2; disparity index: p = 0.1829, disparity ratio: p = 0.2100, Mann-Whitney U test), and the 10%–90% rise time of CF-EPSCs (Table S3)

were similar between Arc knockdown PCs and control PCs. These results indicate that basic properties of CF-PC synapses and functional differentiation of CF inputs were not affected by Arc knockdown in PCs. To further confirm the specificity of the effect of Arc knockdown on CF synapse elimination in vivo, we carried out additional experiments using Arc miRNA-2. We found that Arc miRNA-2 impaired CF synapse elimination and increased the total amplitude of CF-EPSCs exactly in the same way as Arc miRNA (see the Supplemental Text and Figures S5C–S5J). Because Arc miRNA and Arc miRNA-2 had the same effects on CF synapse elimination, we used Arc miRNA in the Selleck Inhibitor Library following experiments. We then examined whether Arc influences PF-PC synapses. The paired-pulse ratio of PF-mediated EPSCs (PF-EPSCs) was similar between Arc knockdown PCs and control PCs (Figures S5K and S5L; p =

0.3030, two-way ANOVA). The input-output curves of PF-EPSC amplitude relative to stimulation strength were not significantly different between Arc knockdown and control PCs (Figures S5K and S5M; p = 0.3910, two-way ANOVA). These observations suggest that the persistent innervation of multiple CFs observed in Arc knockdown PCs is not due to malformation or malfunction of PF-PC synapses. Previous studies indicate that there are four distinct phases in postnatal development of CF-PC synapses

(Hashimoto et al., 2009b, Kano and Hashimoto, 2009 and Watanabe and Kano, 2011). Elimination of surplus CFs proceeds in two phases, the early phase from P7 to around P11, which is independent of PF-PC synapse formation, no and the late phase from around P12 to P17, which requires normal PF-PC synapse formation. Notably, CF synapses remaining on the PC soma are eliminated in the late phase and a monoinnervation pattern is attained (Hashimoto et al., 2009b, Kano and Hashimoto, 2009 and Watanabe and Kano, 2011). To examine whether loss of Arc influences the early phase of CF synapse elimination, we compared CF innervation patterns in Arc knockdown PCs and control PCs at P11–P12. We found that there was no significant difference in CF innervation patterns between Arc knockdown PCs and control PCs, suggesting that the late phase rather than the early phase was affected by Arc knockdown (Figures 7A and 7B; p = 0.5538, Mann-Whitney U test).