By light microscopic immunohistochemistry, the granular and molec

By light microscopic immunohistochemistry, the granular and molecular layers of

the cerebellum were labeled most intensely for both γ-2 and γ-7 in the brain. Clustered labeling in the granular layer probably reflects their synaptic distribution in granule cells, while punctate labeling in the molecular layer probably represents synaptic distribution in Purkinje cells and molecular layer interneurons, and putative glial expression. Of these elements, postembedding immunogold microscopy revealed robust labeling of γ-2 and γ-7 at the mossy fiber–granule cell synapse, parallel fiber–Purkinje cell synapse, climbing fiber–Purkinje cell synapse and parallel fiber–interneuron synapse. All these synapses are classified as asymmetrical (or type I) synapses, a neuroanatomical feature of excitatory synapses BI 2536 molecular weight (Llinas et al., 2004). However, they were absent at the interneuron–Purkinje cell synapse, a GABAergic symmetrical (or type II) synapse. Moreover, immunogold labeling of γ-2 or γ-7 was preferentially localized to the postsynaptic membrane at all these asymmetrical synapses. This distribution pattern is identical to that of γ-8, which is highly concentrated at various asymmetrical synapses in the hippocampus

(Fukaya et al., 2006; Inamura et al., 2006). As γ-2 and γ-7 mRNAs are expressed in deep cerebellar nucleus neurons and Golgi cells as well (Fukaya et al., 2005; Kato et al., 2007), they may be also expressed at asymmetrical synapses of these neurons. Taken together, γ-2 and γ-7 are the major TARPs at various excitatory synapses in the cerebellum. Using quantitative Western blot analysis Trichostatin A cost and immunohistochemical techniques, we found that protein contents and immunohistochemical signal intensities of AMPA receptor subunits were decreased in γ-2-KO and γ-7-KO cerebella, and further reduced in DKO cerebellum. Importantly, the extent of reduction was apparently larger in the PSD fraction than in the homogenate. For example,

in DKO cerebellum, GluA2 levels were reduced to 30% of the WT level in the homogenate, whereas Uroporphyrinogen III synthase it was reduced to approximately 10% in the PSD fraction. This suggests that the ablation of γ-2 and γ-7 severely affected expression of synaptic AMPA receptors. Indeed, in DKO mice the density of GluA2 immunogold labeling was reduced to 11.6% of the WT level at the parallel fiber–Purkinje cell synapse, the most prevalent synapse in the molecular layer. Furthermore, AMPA receptor-mediated EPSCs also reduced to 9.5% at the climbing fiber–Purkinje synapse. Previous experiments using heterologous cells (Chen et al., 2000; Tomita et al., 2004; Vandenberghe et al., 2005; Kato et al., 2007) and brain extracts (Fukata et al., 2005; Nakagawa et al., 2005; Inamura et al., 2006) demonstrate that γ-2 and γ-7 tightly interact with AMPA receptors and regulate their proper folding, trafficking and stability.

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