NMDAR developmental switch monitored by TCN 213 BJP Figure 3 Schild analysis of TCN 213 antagonism of GluN1/GluN2A NMDA receptor-mediated responses. (A) Illustration of a set of TEVC current traces, obtained from an oocyte expressing GluN1/GluN2A NMDA receptors, and used to generate ‘two-point’ dose–response curves in either the absence or presence of TCN 213. (B) Partial, low-concentration dose–response curves obtained from the TEVC current traces illustrated in panel A and used to estimate dose ratios (r). The slope of the ﬁtted line to the control responses (no TCN 213) was used to ﬁt the responses obtained in the presence of 3 mM, 10 mM and 30 mM TCN 213. (C) Schild plot for antagonism by TCN 213 of GluN1/GluN2A NMDA receptors using dose ratios estimated from a series of experiments (n = 5, 5, 4), such as that illustrated in panel B. The dashed line represents a ‘free’ ﬁt of the data and has a slope of 1.13. This was considered not to be signiﬁcantly different from 1 (95% conﬁdence interval: 0.94–1.31) and the solid line is the ﬁt of the data points to the Schild equation (i.e. the slope of this line is unity). The intercept on the abscissa (where the log10 value of the dose ratio equals zero) gives an equilibrium constant (KB) value for TCN 213 of 2.06 0.17 mM. (D) Bar graph summary showing that antagonism of GluN1/GluN2A NMDA receptor-mediated currents is surmountable when the glycine concentration is increased. TCN 213 antagonism of GluN2A-containing NMDA receptors in cortical neurones The experiments illustrated in Figure 4 show that neurones predominantly express GluN2B-containing NMDA receptors at DIV 7–10. Thus, in a ﬁnal series of experiments, we assessed the ability of TCN 213 to block native NMDA receptors con- taining GluN2A subunits. For these experiments, we increased the concentration of glycine (to 3 mM) in our exter- nal recording as we wanted to increase the proportion of GluN2A-containing NMDA receptors that would be activated in this set of experiments. We conﬁrmed, however, that the effect of this increased glycine concentration did not prevent us from blocking selectively GluN1/GluN2A over GluN1/ GluN2B NMDA receptors by measuring the extent of TCN 213 antagonism at recombinantly expressed NMDA receptors (Figure S3A,B). For GluN1/GluN2A NMDA receptors, TCN 213 (30 mM) produced a mean block of 78 4% (n = 5), whereas at GluN1/GluN2B NMDA receptors only 2 0.5% (n = 4) of the NMDA-evoked current was antagonized. To compare TCN 213 block of NMDA-evoked currents from a similar developmental age with those used in the British Journal of Pharmacology (2012) 166 924–937 931