01). Next, we delivered Nedd4-1 or Fbx2 shRNA lentivirus

to rat frontal cortex via a stereotaxic injection (Liu et al., 2011) and tested the involvement of these E3 ligases in the action of repeated stress. As shown in Figures 7G and 7H, buy Ivacaftor the effects of repeated restraint stress on AMPAR-EPSC or NMDAR-EPSC were significantly different in animals with different viral infections (AMPA: p < 0.01, ANOVA, n = 13–15 per group; NMDA: p < 0.01, ANOVA, n = 13–19 per group). Post hoc analysis showed that repeated stress caused a substantial downregulation of the eEPSC amplitude in GFP lentivirus-injected animals (AMPA: 48%–58% decrease; NMDA: 38%–52% decrease, p < 0.01) but had little effect on AMPAR-EPSC in Nedd4 shRNA lentivirus-injected animals (7%–10% decrease, p > 0.05) or on NMDAR-EPSC in Fbx2 shRNA lentivirus-injected

animals (5%–7% decrease, p > 0.05). These electrophysiological results suggest that Nedd4-1 and Fbx2 mediate the long-term CORT or repeated stress-induced downregulation of AMPAR and NMDAR responses in PFC, respectively. We further examined the involvement of Nedd4-1 and Fbx2 in the stress-induced glutamate GSK1210151A receptor ubiquitination by in vivo delivery of the shRNA lentivirus against these E3 ligases to PFC. As shown in Figures 8A and 8B, Nedd4-1 shRNA or Fbx2 shRNA lentivirus-injected rats failed to show the increased level of ubiquitinated GluR1 or NR1 after being exposed to 7 day restraint stress (Ub-GluR1: 5.0% ± 4.5% increase; Ub-NR1: 6.4% ± 9.3% increase, n = 4 pairs for each, p > 0.05), which was significantly different from the effects seen in GFP lentivirus-injected rats after repeated stress (Ub-GluR1: 115.0% ± 24.6% increase; NR1: 136.4% ± 31.3% increase, n = 6 pairs, p < 0.01). Moreover, in contrast to the significantly lower level of GluR1 and NR1 expression in GFP lentivirus-injected rats following stress (GluR1: 46.8% ± 8.3% decrease; NR1: 57.2% ± 8.8% decrease, n = 6 pairs, p < 0.01), Nedd4-1 shRNA or Fbx2 shRNA lentivirus-injected rats exhibited the normal level of GluR1 or NR1 after repeated stress (GluR1: 7.3% ± 8.7% decrease; NR1: 5.5% ± 8.8% decrease, n = 4 pairs for each, p > 0.05). These biochemical results suggest

that Nedd4-1 and Fbx2 mediate the repeated stress-induced ubiquitination and degradation of GluR1 and NR1 subunits in PFC, respectively. To find out the role also of Nedd4-1 and Fbx2 in the stress-induced detrimental effect on cognitive processes, we examined the temporal order recognition memory in animals with in vivo knockdown of both E3 ligases in PFC. As shown in Figure 8C, repeated stress caused a significant deficit in the recognition of novel (less recent) object in GFP lentivirus-injected animals (DR in control: 43.6% ± 7.3%, n = 7; DR in stressed: −5.2% ± 4.1%, n = 8, p < 0.001), whereas the deficit was blocked in animals injected with both Nedd4-1 and Fbx2 shRNA lentiviruses into PFC (DR in control: 29.7% ± 10.7%, n = 7; DR in stressed: 33.7% ± 7.1%, n = 8, p > 0.05).