We found that the disparity index and disparity ratio were identical between control and GAD67+/GFP mice throughout postnatal development and in adulthood (Figure 3F). Taken together, these results indicate that initial CF synapse formation, functional differentiation and maturation of CF synapses, and elimination of surplus CFs until P9 are normal, whereas CF synapse Regorafenib elimination after P10 is specifically impaired, in GAD67+/GFP mice. The late phase of CF synapse elimination after P12 is known to require mGluR1 and its downstream signaling (Ichise

et al., 2000, Kano et al., 1995, Kano et al., 1997, Kano et al., 1998 and Offermanns et al., 1997), which is driven by neural activity along MF-GC-PF pathway involving NMDA receptors at MF-GC synapses (Kakizawa et al., 2000). GluD2 (or glutamate receptor δ2) and CaV2.1, a pore forming component of P/Q-type voltage-dependent Ca2+ LY294002 chemical structure channel (VDCC), are also known to be crucial for CF synapse elimination (Hashimoto et al., 2001, Hashimoto et al., 2011, Ichikawa et al., 2002 and Miyazaki et al., 2004). We therefore examined the expressions of these molecules by immunohistochemistry and found that they

were expressed normally in GAD67+/GFP cerebellum (Figures S3A–S3R). Furthermore, we confirmed that synaptically evoked mGluR1 signaling in PCs (Figure S3S), NMDA receptor-mediated EPSC at MF-GC synapse (Figure S3T), and contribution of P/Q-type VDCC to depolarization-induced Ca2+ transients in PCs (Figure S3U) were normal in GAD67+/GFP cerebellum. Therefore, the impaired CF synapse elimination in GAD67+/GFP mice is not likely to result from altered mGluR1 signaling, reduced GluD2 expression, altered CaV2.1 function or reduced NMDAR-mediated GC activation. Since GAD67 expression is reduced throughout the brain of the GAD67+/GFP mice, it Fossariinae is possible that the impaired CF synapse elimination might result from reduction of GAD in brain regions other than the cerebellum. Therefore, we examined whether chronic local application of the GAD inhibitor 3-MP

into the cerebellum of control mice causes impairment of CF synapse elimination. First, we checked whether 3-MP application affects GABAergic synaptic transmission in cerebellar slices. We recorded mIPSCs from PCs in cerebellar slices from control mice (P10–P13) that had been pre-incubated in ACSF with or without 0.1 mM 3-MP ((+) 3-MP and (−) 3-MP) for 3–5 hr at room temperature (Figures 4A–4C). The mean amplitude of mIPSCs was significantly smaller in PCs from (+) 3-MP slices than those from (−) 3-MP slices ((+) 3-MP: 54 ± 1.0 pA, n = 7; (−) 3-MP: 130 ± 17.4 pA, n = 6, p < 0.001) (Figures 4A and 4B). The mean frequency was identical between the two groups ((+) 3-MP: 4.1 ± 1.0 Hz, n = 7; (−) 3-MP: 6.0 ± 1.0 Hz, n = 6, p = 0.181) (Figure 4C). These results demonstrate that the 3-5 hr of 3-MP treatment significantly attenuated GABAergic transmission in PCs.