LV-shNGL2 caused a significant reduction in the level of NGL-2 mRNA. LV-shNGL2 also caused a small increase in the level of NGL-1 mRNA and no change in the level of EphB2, a non-NGL family transsynaptic protein ( Figure 4B). Since shNGL2 does not directly AZD2014 mw affect

NGL-1 levels ( Figure 4A), the increase in NGL-1 levels may be a homeostatic response to the reduction in levels of NGL-2. To further confirm the specificity of the shRNA, we performed postnatal injections of LV-shNGL2 into the CA1 region of NGL-2 KO mice such that the intended target of the shRNA was not present. In this case, if the shRNA only acts on NGL-2 mRNA, there should be no effect on excitatory synaptic transmission. To test this, we performed simultaneous whole-cell recordings from CA1 pyramidal cells that were infected with LV-shNGL2 and neighboring control cells in the NGL-2 knockout background. We measured the amplitudes of both AMPAR- and NMDAR-mediated EPSCs while stimulating shared inputs in SR. We found that the shRNA had no effect on the

amplitudes of AMPAR-mediated ( Figure S2A) or selleck inhibitor NMDAR-mediated ( Figure S2B) EPSCs in the NGL-2 KO, confirming that our shRNA does not cause off-target effects that lead to changes in excitatory synaptic transmission in CA1. To examine the consequences of postsynaptic NGL-2 knockdown on excitatory synaptic transmission, we used in utero electroporation to deliver an shNGL2 plasmid to a subset of CA1 pyramidal cells (Figure 4C) and prepared acute slices from electroporated mice at P12–P16. Electroporated neurons were

identified by GFP epifluorescence. We performed whole-cell recordings from neighboring electroporated and unelectroporated neurons while stimulating SR and SLM synapses in an alternating manner (Figures 4D and 4E). Again, cells were voltage clamped at −70mV to measure AMPAR-mediated EPSCs Histamine H2 receptor and then depolarized to +40mV to measure the NMDAR-mediated EPSCs 50 ms after the stimulus onset. NGL-2 knockdown caused a decrease in AMPAR-mediated currents (Figure 4F) and a similar decrease in NMDAR-mediated currents (Figure 4G) in the stratum radiatum. In contrast, NGL-2 knockdown had no effect on AMPAR- or NMDAR-mediated currents in the SLM (Figures 4H and 4I), suggesting that NGL-2 acts postsynaptically to specifically regulate Schaffer collateral synapses in CA1. Expression of shNGL2 had no effect on the ratio of AMPAR- to NMDAR-mediated currents in either SR or SLM (Figures S2C and S2D), further indicating that NGL-2 does not preferentially regulate AMPA- or NMDA-type glutamate receptors. Furthermore, a control plasmid expressing only GFP had no effect on AMPAR- or NMDAR-mediated currents or on the AMPA/NMDA ratio in stratum radiatum (AMPA: control 61.68 ± 18.16 pA, n = 5; GFP 61.00 ± 18.13 pA, n = 5; p = 0.69; NMDA: control 44.85 ± 13.94 pA, n = 6; GFP 63.85 ± 23.32 pA, n = 6; p = 0.119; AMPA/NMDA: control 1.70 ± 0.30, n = 5; GFP 1.61 ± 0.39, n = 5, p = 0.