BJP S McKay et al. ing the concentration of glycine but not glutamate present in the external recording solution. Over the concentration range examined, Schild analysis of TCN 213 antagonism gave a Schild plot with unity slope, which is indicative of com- petitive antagonism (but see below for further discussion). Our observations are not only consistent with some of those made in the study of Bettini et al. (2010) but also extend these earlier ﬁndings as we have calculated for the ﬁrst time a KB value for TCN 213 acting at GluN1/GluN2A NMDA receptors. In addition, we show that TCN 213 permits direct pharma- cological identiﬁcation of functional NMDA receptors con- taining GluN2A subunits and thus will have considerable utility for the study of, and changes in, NMDA receptor composition. TCN 213 permits pharmacological proﬁling of developmental changes in native NMDA receptor subunit composition Early studies describing the in situ expression of mRNA levels of GluN1 and GluN2 subunits indicated both spatial and temporal control of NMDA receptor subtypes (Monyer et al., 1994). Indeed, developmental changes in the subunit com- position of native NMDA receptors are well-documented in, for example, cortical neurones (Carmignoto and Vicini, 1992; Hestrin, 1992; Sheng et al., 1994; Crair and Malenka, 1995; Flint et al., 1997; Stocca and Vicini, 1998) cerebellar neurones (Farrant et al., 1994) and striatal neurones (Logan et al., 2007; Brothwell et al., 2008). For cortical neurones, NMDA receptor composition switches from a population made up from pre- dominantly GluN1 and GluN2B subunits to ones that are heterodimeric combinations of GluN1 and GluN2A, and GluN1 and GluN2B subunits as well as heterotrimeric com- binations of each of these three subunits. The evidence for such changes in subunit composition comes from changes in the kinetics of glutamatergic synaptic currents, protein and mRNA expression levels, co-immunoprecipitation with subunit-speciﬁc antibodies and the sensitivity of NMDA receptor-mediated currents to selective GluN2B antagonists (see van Zundert et al., 2004). What has been lacking, to date, is the ability to block selectively NMDA receptors comprising of GluN1 and GluN2A subunits. Although competitive antagonists such as NVP-AAM077 show some selectivity for GluN1/GluN2A over GluN1/GluN2B NMDA receptors (Aub- erson et al., 2002), this selectivity is not enough to allow unequivocal NMDA receptor subunit identiﬁcation (Frizelle et al., 2006). Therefore, novel ligands such as TCN 213 and 3-chloro-4-ﬂuoro-N-[4-[[2-(phenylcarbonyl)hydrazino] carbonyl]benzyl] benzenesulphonamide (Bettini et al., 2010), with their greater selectivity, offer greater potential for eluci- dation of NMDA receptor composition. Our data clearly show that for cortical neurones at early developmental stages (DIV 7–10), the vast majority of NMDA receptors are comprised of only GluN1 and GluN2B subunits since their sensitivity to ifenprodil is very similar to that seen with recombinant GluN1/GluN2B NMDA receptors. The range of ifenprodil and TCN 213 block in older cultures (DIV 14–17) indicates that this population of neurones express both GluN2A- and GluN2B-containing NMDA receptors. Our data do not address directly the proportion of NMDA receptors that are triheteromeric complexes of GluN1, GluN2A and GluN2B subunits (Chazot and Stephenson, 1997; Rauner and Köhr, 934 British Journal of Pharmacology (2012) 166 924–937 2011) as we do not know how effective TCN 213 is at block- ing such receptor combinations. Nevertheless, comparison with our data from recombinant NMDA receptors indicates, for recordings where TCN 213 blocked around 50% of the current remaining following the application of ifenprodil, that heterotrimeric complexes are unlikely to represent the majority of NMDA receptors in the total population. The nature of TCN 213 subtype selective NMDA receptor antagonism of GluN1/GluN2A NMDA receptors Our data show that TCN 213 can produce complete block of GluN1/GluN2A NMDA receptor-mediated responses, while sparing responses mediated by GluN1/GluN2B NMDA recep- tors. However, this was only achieved when the glycine con- centration was low (0.1 ¥ EC50; Figure 3A,B). At higher glycine concentrations, complete block of GluN1/GluN2A NMDA receptors was not achieved, although the selectivity of TCN 213 is still sufﬁcient to discriminate very effectively between GluN1/GluN2A and GluN1/GluN2B NMDA receptors (Fig- ure S3). It could be argued that one need only increase the concentration of antagonist to overcome higher concentra- tions of glycine. However, extrapolation of our data (Figure 2A) shows that for a glycine concentration that is 10 times its EC50 value, it is predicted that a concentration of around 1 mM TCN 213 would be required to block fully a GluN1/GluN2A NMDA receptor-mediated response. It remains unclear whether at such high concentration if TCN 213 would still retain selectivity of GluN1/GluN2A NMDA receptor-mediated responses over those mediated by GluN2B- containing NMDA receptors, notwithstanding that the solu- bility of TCN 213 would make it unlikely that such high concentrations could be used effectively. Let us now consider how the selectivity of TCN 213 for GluN1/GluN2A NMDA receptors may come about. The glycine dependency of the IC50 values for TCN 213, the Schild analysis giving a unity slope and the fact that the antagonism can be surmounted and a maximum response retained by increasing the glycine concentration are consistent with the notion that TCN 213 is a competitive, reversible antagonist that acts at the GluN1 glycine binding site. Why therefore is TCN 213 selective for GluN1/GluN2A over GluN1/GluN2B NMDA receptors as they both contain the same GluN1 subunit? A potential explanation for this could be that the conformation or accessibility of the site to which TCN 213 binds is different when GluN1 is associated with GluN2A or GluN2B NMDA receptor subunits. Our previous work (Chen et al., 2008) has documented the relative potencies of a series of GluN1 (glycine site) full and partial agonists for each of the four NMDA receptor subtypes. These data showed that for all agonists the rank order of potency was GluN1/GluN2D > GluN1/GluN2C > GluN1/GluN2B > GluN1/GluN2A. These data refer to potencies of agonist where the EC50 value reﬂects all the rate constants governing ‘binding’ and ‘gating’ steps in the reaction scheme that describes the activation of the recep- tor. Indeed, our data concerning the actions of agonists at the GluN1 NMDA receptor subunit suggested that differences in the potencies of glycine-site agonists were due to interactions between amino acids at the interface of GluN1 and GluN2 NMDA receptor subunits, and that this most likely contrib- uted to altered ‘gating’ of GluN1-dependent reactions when