8 ± 5.1%, P = 0.0002)

and GluA3 (35.9 ± 7.0%, P = 0.01) and by 40% for GluA4 (57.6 ± 6.1%, P = 0.002), while no reduction was found for GluA1 (75.6 ± 16.7%, P = 0.24; Fig. 4A and B). These reductions became more remarkable in the synaptosome fraction (Fig. 4A, middle panel) and PSD fraction (Fig. 4A, right panel; Fig. 4C). All four subunits displayed further reductions in DKO cerebellum (Fig. 4C). In the PSD fraction, protein levels relative to those in WT mice were 38.3 ± 7.2% for GluA1, 9.5 ± 4.6% for GluA2, 15.2 ± 3.3% for GluA3 and 37.8 ± 5.4% for GluA4, showing significant differences (P = 0.0011, <0.0001, 0.0001 and 0.0014, respectively). Next, immunohistochemical changes in GluA1–GluA4 were examined using subunit-specific antibodies (supporting Fig. S1) and pepsin pretreatment, an antigen-exposing method particularly effective in detection of postsynaptic molecules (Fukaya & Watanabe, click here 2000). In WT mice,

the molecular layer was stained intensely for all four subunits, while the granular layer was stained weakly for GluA2 and GluA4 (Figs 5 and 6). These patterns of immunohistochemical distribution appeared to reflect cell type-specific subunit expression shown by previous in situ hybridization and single-cell PCR: GluA1–GluA3 mRNAs in Purkinje cells, GluA1 and GluA4 mRNAs in Bergmann glia, and GluA2 and GluA4 mRNAs in granule cells SGI-1776 chemical structure (Keinänen et al., 1990; Pellegrini-Giampietro Clomifene et al., 1991; Lambolez et al., 1992). Brains from WT, γ-2-KO and DKO mice were embedded

in single paraffin blocks, mounted on single glass slides and processed simultaneously for immunoreaction (Fig. 5). Compared to the intensity in WT mice, striking reductions were noted in the cerebellar cortex for GluA2 and GluA3, with intensities in the order WT > γ-2-KO > DKO (Fig. 5B, C, F and G). In particular, GluA2 became almost blank in the granular layer of γ-2-KO mice and in the molecular layer of DKO mice (Fig. 5B and F; supporting Fig. S4A). On the other hand, GluA4 was reduced mildly in the molecular layer and severely in the granular layer of γ-2-KO and DKO mice (Fig. 5D and H; supporting Fig. S4B). GluA1 was reduced mildly in the molecular layer of γ-2-KO and DKO mice (Fig. 5A and E). Likewise, WT and γ-7-KO brains embedded in single paraffin blocks were examined (Fig. 6). In contrast to the staining in γ-2-KO cerebellum, moderate reduction was noted for GluA1 and GluA4 in the molecular layer of γ-7-KO mice (Fig. 6A–C and J–L). GluA4 was also reduced in the granular layer of γ-7-KO mice (Fig. 6J–L; supporting Fig. S4D). On the other hand, the reduction in immunohistochemical intensity was relatively mild for GluA3 (Fig. 6G–I), while no difference was noticed for GluA2 in the molecular and granular layers (Fig. 6D–F; supporting Fig. S4C).