RNAi-mediated temporal knockdown of OPHN1 selectively in CA1 neurons has no detectable effect on presynaptic function and it minimizes the possibility of developmental compensations ( Nadif Kasri et al., 2009); both of these events could affect the induction and expression of mGluR-LTD ( Khelfaoui et al., 2007). CA1 neurons in cultured hippocampal slices were infected with the OPHN1#2 shRNA containing lentivirus, and 8 to

10 days post-infection the magnitude of mGluR-dependent LTD induced in control uninfected and OPHN1#2 shRNA infected cells with Fludarabine research buy bath application of DHPG (100 μM, 5 min) was examined. Consistent with previous studies ( Huber et al., 2000, Huber et al., 2001 and Volk et al., 2006), DHPG caused a depression of AMPAR-mediated Palbociclib supplier synaptic transmission in control cells, which is protein translation dependent, and, notably, is attenuated by blockade of mGluR1 with LY367385 throughout the experiment ( Figure 2B and Figures S3A and S3B). When compared to mGluR-LTD induced in simultaneously recorded control cells, knockdown of OPHN1 greatly reduced the magnitude of mGluR-LTD. A depression in AMPAR-mediated synaptic transmission of approximately 40% was observed in control

cells versus 10% in OPHN1#2 shRNA expressing cells, 30–35 min after DHPG application ( Figure 2B). To ensure that this effect was specifically caused by impaired OPHN1 expression, we performed rescue experiments by using OPHN1 cDNA that is resistant to OPHN1#2 shRNA-mediated RNAi ( Nadif Kasri et al., 2009). The levels of OPHN1 expression in hippocampal neurons coexpressing RNAi-resistant OPHN1WT and OPHN1#2 shRNA were restored below to normal levels ( Figure 2A), and, most importantly, the magnitude of mGluR-LTD was comparable to that of control neurons ( Figure 2C). Thus, knockdown of OPHN1 impairs mGluR-LTD. One possible explanation for the impaired mGluR-LTD is that it is due to reduction in basal synaptic strength, as OPHN1 RNAi depresses glutamatergic synaptic transmission

(Figure 2B, left panel before DHPG application, and see Figure 4A), thereby occluding LTD. Alternatively, however, activity-dependent OPHN1 induction could play a critical role in mediating mGluR-LTD independent of its effects on basal synaptic strength. Distinguishing between these two possibilities requires a dissociation of OPHN1′s role in regulating basal synaptic transmission and mGluR-LTD. To determine whether such dissociation can be achieved, we resorted to OPHN1 mutants and synthetic blocking peptides that selectively disrupt the interaction between OPHN1 and OPHN1-binding partners present in dendritic spines; the synaptic effects of these mutants and peptides were subsequently tested. We previously described an interaction between OPHN1 and the small GTPase RhoA, as well as Homer 1b/c, at the postsynaptic site of hippocampal neurons (Govek et al., 2004).