, 2005). We developed an NR2B miRNA construct that effectively reduced the level of endogenous NR2B in primary hippocampal neurons (Figures 4Aa–4Af and 4C). NR2A immunoreactivity was remarkably suppressed in neurons in which NR2B was knocked down (Figures 4Ba–4Bf and 4D; Kim et al., 2005). To determine whether the ubiquitin-proteasome pathway is required for the NR2B miRNA-induced loss of NR2A, we treated NR2B miRNA-transfected

cells with the proteasome inhibitor lactacystin and monitored the NR2A level immunocytochemically. Importantly, NR2A localization in neurons was restored in the presence of the proteasome inhibitor (Figures 4Bm–4Br and 4D), suggesting that the downregulation of NR2A following NR2B knockdown is dependent on the ubiquitin-proteasome system. On the other hand, chronic blockade of neuronal activity by ifenprodil (3 μM) or TTX (1 μM) caused Dasatinib mw a significant decrease in NR2A levels in cultured hippocampal neurons, whereas NR2B levels were increased by prolonged activity blockade (Figures 4G and 4H). To test the possibility that ubiquitin-dependent degradation is also involved in the decrease in NR2A level caused by activity blockade, Talazoparib purchase NR2A subunits were immunoprecipitated from lysates of cultures

treated simultaneously with ifenprodil (3 μM) and actinomycin D (10 μg/ml) for 24 hr and probed for ubiquitin. Treatment of neurons with ifenprodil and actinomycin-D increased the

abundance of ubiquitinated NR2A relative to the levels in neurons treated with actinomycin-D alone (Figure 4I). Consistently, examination of total cell extracts showed a loss of NR2A protein following ifenprodil 3-mercaptopyruvate sulfurtransferase and actinomycin D treatment, which was not seen following of neurons with actinomycin D alone (Figures 4J and 4K). These results suggest that the ubiquitin pathway participates in this activity-regulated decrease in the levels of NR2A. To evaluate the functional changes in kif17−/− mouse neurons, we performed electrophysiological analysis of acute slices from the hippocampal CA1 region. The input-output relationships between Schaffer collateral fiber excitability and the slopes of field excitatory postsynaptic potentials (fEPSPs) ( Figure 5A), paired-pulse facilitation (PPF) ( Figure 5B), and the current-voltage relationships of NMDA receptor channel currents ( Figure 5C) were not different between kif17+/+ and kif17−/− slices. Importantly, NMDA receptor-mediated excitatory postsynaptic currents (EPSCs), expressed as the ratio of NMDA to AMPA EPSC amplitudes ( Sakimura et al., 1995), were reduced in kif17−/− slices (49.8% ± 2.3%, n = 12) compared with kif17+/+ slices (74.3% ± 2.3%, n = 12) ( Figures 5D and 5E). To assess the difference in the subunit compositions of NMDA receptors between genotypes, we treated hippocampal slices with ifenprodil (3 μM, a blocker of NMDA receptors containing the NR2B subunit).