To inhibit the function of P/Q-type VDCCs in PCs, we constructed lentiviruses containing engineered microRNA (miRNA) targeting the P/Q-type VDCC (P/Q miRNA) and a fluorescent protein under the control of L7 promoter (see

the Supplemental Text and Figures S2A–S2C). Using this construct, we confirmed that the P/Q-type VDCC was required for CF synapse elimination in cocultures as well as in vivo (Hashimoto et al., 2011 and Miyazaki et al., 2004) (see the Supplemental Text and Figures S2D and S2E). We examined whether the P/Q-type VDCC plays a role in the acceleration of CF synapse elimination by the 2-day photostimulation. There was no significant difference in the amplitude of inward currents evoked by 1 s blue light stimulation between PCs with ChR2 expression + P/Q miRNA expression (P/Q knockdown) and those with ChR2 expression SCR7 purchase alone (Figures S2F and S2G; p = 0.4450, Mann-Whitney U test), indicating a similar expression level of ChR2. Dasatinib price We applied the 2-day blue light illumination to three groups of coculture, namely, cocultures containing PCs with ChR2 expression (yellow), those with ChR2 expression + P/Q knockdown (red), and those with EGFP expression + P/Q knockdown (green), from 10 or 11 DIV, when the majority

of PCs are innervated by four or more CFs (Uesaka et al., 2012). Uninfected (control) PCs sampled from the three groups of coculture exhibited similar CF innervation patterns (Figure 2C; p = 0.8505, Kruskal-Wallis test), which enabled us to compare CF innervation patterns within the three groups of infected PCs. We found that the CF innervation patterns within the three groups significantly differed from each other (Figure 2B; p < 0.0001, Kruskal-Wallis test). We found that a significantly higher number of CFs innervated PCs with ChR2 expression + P/Q knockdown (red) when compared to those with ChR2 expression alone (yellow) (Figure 2B; p = 0.0412, Steel-Dwass test). This observation demonstrates that the acceleration nearly of CF synapse elimination by the 2-day excitation of PCs is significantly attenuated by P/Q knockdown. Thus, Ca2+ influx through P/Q-type VDCCs

is an important factor for the acceleration of CF synapse elimination. On the other hand, a significantly higher number of CFs innervated PCs with EGFP expression + P/Q knockdown (green) when compared to those with ChR2 expression + P/Q knockdown (red) (Figure 2B; p = 0.0461, Steel-Dwass test), suggesting that residual P/Q-type VDCCs after knockdown, and/or other voltage-dependent mechanisms, might contribute to the acceleration of CF synapse elimination. Neural activity induces a number of Ca2+-dependent genes that are involved in synapse development, maturation, and refinement (Greer and Greenberg, 2008). Previous studies demonstrate that CF synapse elimination is an activity-dependent process mediated by P/Q-type VDCCs (Hashimoto and Kano, 2005, Hashimoto et al.