BJP Table 1 S McKay et al. IC50 values for TCN 213 and 5,7 DCKA acting at GluN1/GluN2A or GluN1/GluN2B NMDA receptors, in the presence of different con- centrations of glycine (gly) Antagonist IC50 (mM) TCN 213 5,7 DCKA GluN1/GluN2A [gly] = 0.1 EC50 [gly] = EC50 [gly] = 10 EC50 GluN1/GluN2B [gly] = 0.1 EC50 [gly] = EC50 [gly] = 10 EC50 Values are given as mean ments given in parentheses. n.d., not determined. 0.55 3.5 40 n.d. n.d. n.d. 0.05 (7) 0.05 (7) 3.5 (6) 0.13 0.42 4.1 0.074 0.49 4.1 0.01 (6) 0.03 (9) 0.3 (4) 0.01 (6) 0.01 (6) 0.4 (6) SEM with the number of experi- antagonism is surmountable as would be anticipated for a competitive antagonist. We therefore carried out a further set of experiments to show that antagonism by TCN 213 of a response evoked by maximal concentrations of both glutamate (100 mM) and glycine (50 mM) could be overcome by increasing the glycine concentration. The results from these experiments are summarized in Figure 3D. TCN 213 and native GluN2B-containing NMDA receptors The experiments described so far have only examined the effect of TCN 213 on heterologously expressed recombinant NMDA receptors where the subunit composition of the popu- lation of receptors is known. We next wanted to assess the ability of TCN 213 to antagonize native NMDA receptors. NMDA receptor subunit expression displays both temporal and spatial regulation (Monyer et al., 1994) where in early post-natal forebrain development the GluN2B subunit expres- sion is highest and most ubiquitous. We therefore ﬁrst chose to study native NMDA receptors in cultures of rat cortical neurones at a stage (DIV 7–10) where it would be anticipated that the most prominent NMDA receptor combination is that formed by GluN1 and GluN2B subunits. Figure 4A (trace on left) shows a whole-cell current recording from a cortical pyramidal cell, voltage-clamped at -70 mV, where application of NMDA (50 mM) in the presence of glycine (1.5 mM) gives an inward current that was not antagonized by TCN 213 (10 mM). This glycine concentration was chosen as a priori we did not know the composition of the NMDA receptor population in these neurones and considered it better to use a glycine con- centration that was equivalent to the higher of the EC50 values for GluN2A- and GluN2B-containing NMDA receptors. The average inhibition of NMDA receptor-mediated currents recorded from cortical neurones at this stage of development was only 2 3% (n = 12 cells from ﬁve separate cultures; Figure 4B). The strong ifenprodil block together with the lack of TCN 213 block of NMDA receptor-mediated currents in 930 British Journal of Pharmacology (2012) 166 924–937 these neurones is indicative that the majority of NMDA recep- tors present in these cells are made up of only GluN1 and GluN2B subunits since with the glycine and TCN 213 concen- trations used, we would have expected around 75% block of the current if the population had been made up entirely of GluN2A-containing NMDA receptors (Figure 2A). Indeed, to conﬁrm this, we measured the amount of antagonism pro- duced by ifenprodil (3 mM), the GluN2B-selective antagonist. Our data indicate that NMDA receptor-mediated currents in these neurones were strongly inhibited by this blocker (Figure 4A, trace on right). On average, ifenprodil (3 mM) blocked NMDA receptor-mediated currents by 72 2% (n = 12 cells from ﬁve separate cultures; Figure 4B). This conﬁrms that the majority of NMDA receptors present in these cells are made up of only GluN1 and GluN2B subunits (note that ifenprodil is a non-competitive antagonist that at most blocks 75–80% of the current mediated by GluN1/GluN2B NMDA receptors). Furthermore, if we restrict our analysis to a subset of recordings that showed the greatest block by ifenprodil (78 1%; n = 7 cells from four separate cultures), which will presumably identify cells with the largest GluN1/GluN2B NMDA receptor content, the extent of the TCN 213 block is 4 2% (n = 7). Ifenprodil also antagonizes heterotrimeric NMDA receptors containing GluN1, GluN2A and GluN2B subunits (Kew et al., 1998; Tovar and Westbrook, 1999; Hatton and Paoletti, 2005), and this combination is considered to form a signiﬁcant population of native NMDA receptors (Chazot and Stephenson, 1997; Rauner and Köhr, 2011). Nev- ertheless, the concentration of ifenprodil required to block such heterotrimeric receptors combination is higher than that used here. In addition, the extent of the block produced by ifenprodil (72%) is very similar to the block produced by this concentration when applied to recombinant NMDA receptors containing only GluN1 and GluN2B subunits. Thus, in the conditions studied here (rat cortical neurones, 7–10 DIV), the vast majority of NMDA receptors are heterodimers of the GluN1/GluN2B subtype. In a separate series of experiments, we assessed the ability of TCN 213 to block NMDA-induced excitoxicity in cortical cultures (DIV 7–10). We have previously demonstrated that NMDA-induced cell death in hippocampal neurones at this stage of development in vitro can be completely attenuated by ifenprodil indicating that this process is mediated by GluN2B-containing NMDA receptors (Martel et al., 2009). Figure 4C shows example images of neuronal cell bodies from untreated and NMDA (40 mM)-treated cultures. In the absence of neurotoxic insult, neurones display regularly sized cell bodies (Figure 4Ci). This is also seen when NMDA is applied in the presence of ifenprodil (Figure 4Ciii) or the non-selective GluN1-site antagonist, 5,7 DCKA (Figure 4Civ). However in the absence of any NMDA receptor antagonists (Figure 4Cii) or TCN 213 (Figure 4Cv), the presence of a high percentage of pyknotic cell nuclei is evident. Figure 4D sum- marizes a series of experiments where we have quantiﬁed the neuroprotective effects of ifenprodil, 5,7 DCKA and TCN 213. The extent of cell death seen in the presence of TCN 213 was not signiﬁcantly different from that seen in cultures treated with NMDA alone. Both ifenprodil (3 mM) and 5,7 DCKA (10 mM) reduced signiﬁcantly the percentage of neurones dis- playing pyknotic nuclei (P < 0.05 with Bonferroni correction; n = 3–4 repeats for each condition).