NMDAR developmental switch monitored by TCN 213 BJP Figure 2 Inhibition curves for TCN 213 and 5,7 DCKA antagonism of GluN1/GluN2A and GluN1/GluN2B NMDA receptor-mediated responses. (A) Mean inhibition curves for TCN 213 block of GluN1/GluN2A NMDA receptor-mediated currents. Currents were evoked by glutamate (100 mM) and glycine (0.1 ¥ EC50; EC50,or 10 ¥ EC50). The data points were ﬁtted with the Hill equation (see Methods), and the mean IC50 values are indicated by the vertical dashed lines. (B) Summary of the mean inhibition of GluN1/GluN2B NMDA receptor-mediated currents by TCN 213. The low potency of TCN 213 at this receptor combination prevented the estimation of an IC50 value at any of the glycine concentrations used. (C, D) as in panel A but for 5,7 DCKA antagonism of GluN1/GluN2A or GluN1/GluN2B NMDA receptor-mediated currents respectively. The mean IC50 values for TCN 213 and 5,7 DCKA antagonism are reported in Table 1. the agonist used to evoked the response to be antagonized (see Wyllie and Chen, 2007). Schild analysis (Arunlakshana and Schild, 1959) does not have such limitations, and we have used this method to determine the equilibrium constant (KB) for TCN 213 acting at GluN1/GluN2A NMDA receptors. Figure 3A shows a series of pairs of TEVC current traces recorded in the absence or the presence of TCN 213 (3, 10 or 30 mM). In all recordings, the glutamate concentration was ﬁxed at 100 mM to ensure full occupancy of its binding site on the GluN2A NMDA receptor subunit, and we attempted to match the magnitude of the responses recorded in the absence of TCN 213 by increasing the concentration of glycine that was co-applied with TCN 213. Figure 3B shows the ‘two-point’ concentration–response curves obtained from the TEVC current traces like those illustrated in Figure 3A and from which we estimated the dose ratio (r) for each antago- nist concentration. Pooling data from a series of similar experiments gave mean r-values of 1.31 0.17, 5.20 0.79 and 17.22 1.69 for 3, 10 and 30 mM TCN 213 respectively (n = 5, 5 and 4). The resulting Schild plot obtained from these data is shown in Figure 3C, and a linear ﬁt of the data points with the slope constrained to be unity gave a KB value of 2.06 0.17 mM. We also estimated the KB value by simply calcu- lating the value from the Schild equation using the individual r-values obtained from individual experiments – this gave a mean value of 2.2 0.2 mM, whilst conﬁrming that the individual estimates of such KB values were independent of the antagonist concentration (F2,11 = 1.13, P = 0.36; one-way ANOVA). A potential limitation of our use of low agonist con- centrations to generate ‘two-point’ dose–response curves is that while these allow us to assess parallel shifts in the dose– response curves, it does not allow us to determine whether British Journal of Pharmacology (2012) 166 924–937 929