We injected the virus for P/Q knockdown together with that for mOrange (P/Q knockdown + mOrange) or with that for Arc overexpression (P/Q knockdown + Arc overexpression) into the mouse cerebellum at P2–P3 (Figure 8C). We found that 51% of PCs with P/Q knockdown + mOrange and 43% of PCs with P/Q knockdown + Arc overexpression were innervated by two or three CFs at P20–P23, and there was no significant difference in CF innervation patterns between the two groups (Figures 8C and 8D; p = 0.4702, Mann-Whitney U test). Again, about 80% of uninfected control PCs were innervated by single CFs in both groups, indicating that

there was no significant learn more experimental bias between the two groups (Figures S7C and S7D; p = 0.9229, Mann-Whitney U test). These results demonstrate that Arc overexpression alone cannot rescue the impaired CF synapse elimination in P/Q knockdown PCs. Thus, whereas Arc activation is essential, Arc may cooperate with other factors induced by P/Q-type VDCC-mediated Ca2+ elevation in PCs to collectively accomplish the late phase of CF synapse elimination. Previous

studies in the neuromuscular junction (Favero et al., 2009 and Thompson, 1983) and the cerebellum (Lorenzetto et al., 2009) have indicated that postsynaptic check details activity is crucial for synapse elimination. However, the mechanisms as to how postsynaptic activity mediates synapse elimination and which activity-dependent mediators are

involved have remained unclear. In this study, we showed that Arc expression increased in the developing cerebellum during the period of CF synapse elimination and its activity-dependent expression in PCs required P/Q-type VDCCs. Then we demonstrated that Arc knockdown in PCs suppressed the enhancement of CF synapse elimination by increasing PC activity in olivo-cerebellar coculture preparations in vitro. Finally, we found that Arc knockdown in PCs in the developing cerebellum in vivo resulted in a significant impairment of CF synapse elimination. These results indicate that Arc is a critical postsynaptic mediator for activity-dependent CF synapse elimination Adenylyl cyclase downstream of P/Q-type VDCCs. Our previous studies indicate that P/Q-type VDCCs mediate most of the Ca2+ influx into PCs during CF activity (Hashimoto et al., 2011) and that VDCCs in PCs are required for selective strengthening of a single “winner” CF in each PC, dendritic translocation of the “winner” CF, and elimination of weak “loser” CF synapses from the PC soma (Hashimoto et al., 2011 and Miyazaki et al., 2004). In the present study, we found that PC-specific Arc knockdown in vivo did not affect the disparity index and disparity ratio, the height of CF synaptic terminals in the molecular layer, and CF innervation patterns at P11–P12.

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