We studied DIV10 cultured cortical neurons from GluN2B+/+ and GluN2B2A(CTR)/2A(CTR) littermates. These cultures exhibited similar levels of basal viability and levels of synaptic connectivity and strength, as measured by mini EPSC frequency/size, spontaneous EPSC frequency, and AMPA receptor currents ( Figures S2A–S2D), as well as unaltered cell capacitance ( Figure S2E). Whole-cell and extrasynaptic NMDAR currents in both GluN2B+/+ DAPT mouse and GluN2B2A(CTR)/2A(CTR) neurons were found to be similarly sensitive to the GluN2B-specific antagonist ifenprodil. In neurons

of both genotypes, we observed a blockade of around 60% ( Figure 2B), indicative of a high (∼80%) level of GluN1/GluN2B heterodimeric receptors. Moreover, the proportion of extrasynaptic NMDARs was found to be the same for GluN2B2A(CTR)/2A(CTR)

and GluN2B+/+ neurons ( Figure 2C). Thus, any differential CTD subtype-specific effects on excitotoxicity could be studied without the potentially confounding factor of altered NMDAR location. We also investigated whether any differences in use-dependent run-down of whole-cell NMDAR currents were observed because this may be relevant to long-term exposure to NMDA. Having measured baseline whole-cell NMDAR currents, ten further 10 s CH5424802 chemical structure applications of NMDA were applied over a 10 min period. We found no difference in run-down of steady-state NMDAR currents in GluN2B+/+ and GluN2B2A(CTR)/2A(CTR) neurons (around 3% per application;

Figure S2F). We also examined NMDAR single-channel properties. We excised outside-out patches from DIV9 GluN2B+/+ and GluN2B2A(CTR)/2A(CTR) neurons and measured NMDA-evoked unitary currents, finding no difference in their mean single-channel conductance of approximately 50 pS, which is typical for GluN2B-containing NMDARs ( Figure S2G). Despite the aforementioned similarities, we found one important difference; whole-cell NMDAR currents in Thymidine kinase GluN2B2A(CTR)/2A(CTR) neurons were around 30% lower than GluN2B+/+ ( Figure 2D). Levels of GluN2B protein were lower in DIV10 GluN2B2A(CTR)/2A(CTR) cortical neurons ( Figure S2H) and in P7 cortical protein extracts ( Figure S2I; ruling out the possibility of an in vitro artifact). An explanation for this difference was found when we looked at GluN2B2A(CTR) mRNA levels, which were lower both in DIV10 GluN2B2A(CTR)/2A(CTR) cortical neurons and in P7 cortical extracts ( Figures S2H and S2I). However, this decrement appeared to be a developmental-stage-dependent effect because by adulthood, levels of forebrain GluN2B mRNA ( Figure 3A) and protein (p = 0.51, n = 5,5) were unaltered in GluN2B+/+ versus GluN2B2A(CTR)/2A(CTR) mice. We hypothesize that GluN2B2A(CTR), compared to wild-type GluN2B, may be transcribed, processed, or exported slightly less efficiently, which manifests itself in a mRNA decrement in development when expression of many genes, including those encoding NMDAR subunits, is changing rapidly.