NMDAR developmental switch monitored by TCN 213 BJP nical reasons, the dose ratio estimate for the highest concen- tration of TCN 213 (30 mM) had to be determined in a separate set of experiments from those carried out for the lower two concentrations (3 and 10 mM). NMDA-induced excitotoxicity assay To assess NMDA-induced cell death, neurones were exposed to NMDA (40 mM + glycine, 1.5 mM) for 1 h in the absence or presence of either ifenprodil (3 mM), 5,7 DCKA (10 mM) or TCN 213 (10 mM). Exposure to excitotoxic concentrations of NMDA leads to neurones displaying swollen cell bodies and pyknotic nuclei with small irregular chromatin inclusions. Such characteristics are indicative of necrotic, as opposed to apopotic, cell death (Fujikawa et al., 2000; Hardingham et al., 2002). Assessment of cell death was made 24 h after exposure to NMDA by calculating the ratio of 4′, 6-diamidino-2-phenyl indole-stained pyknotic nuclei as a percentage of the total nuclei. Statistical analysis Results are presented as mean SEM, and statistical compari- son between data sets was assessed using either Student’s t-test (paired where appropriate) or ANOVA tests to determine whether differences between mean values were signiﬁcant (P < 0.05). Microcal Origin v6.0 software (Microcal, Northampton, MA, USA) was used for graphical presentation. Materials Glutamate and glycine were purchased from Sigma-Aldrich. N - (cyclohexylmethyl) - 2 - ({5 - [(phenylmethyl)amino] - 1, 3, 4- thiadiazol-2-yl}thio)acetamide (TCN 213) was originally purchased from Enamine (Kiev, Ukraine) and subsequently from Tocris Bioscience (Bristol, UK). 5,7 DCKA, N-methyl-D- aspartate, ifenprodil, picrotoxin and tetrodotoxin were pur- chased from Tocris Bioscience. Stock solutions of TCN 213 were made by dissolving the antagonist in DMSO. We con- ﬁrmed in a series of preliminary experiments that DMSO at the highest ﬁnal concentration present in our experiments (0.3%) did not have any effect on the magnitude of NMDA receptor-mediated responses (data not shown). Results Antagonism of NMDA receptor-mediated responses by TCN 213 is dependent on glycine concentration The study of Bettini et al. (2010) suggested that the antago- nist action of TCN 213 was in part mediated by inhibition of glycine binding at NMDA receptors. In order to assess this, we conducted a series of initial experiments to determine whether the extent of the antagonism produced by TCN 213 was dependent on the concentration of glycine and/or glutamate used to evoke NMDA receptor-mediated currents. Figure 1A (upper panel) shows a series of representative TEVC current traces obtained from an oocyte expressing GluN1/ GluN2A NMDA receptors in response to application of glutamate (100 mM) and three different concentrations of glycine (0.1, 1 and 10 times its EC50 value; Chen et al., 2008). As can be seen from the traces, the extent of the block pro- duced by TCN 213 (10 mM) was dependent on the glycine concentration. Almost complete block of the current was seen when glycine is present at 150 nM (0.1 ¥ EC50), whereas substantially less block was seen when glycine is present at 15 mM (10 ¥ EC50). Mean data, indicating the extent of the block of steady-state responses, from a series of similar experi- ments (n = 6) are illustrated in Figure 1C. In contrast and as illustrated in the lower panel of Figure 1A, TCN 213 antago- nism was not dependent on the glutamate concentration used to evoke NMDA receptor-mediated currents. In these experiments, glutamate was applied at concentrations again corresponding to 0.1, 1 and 10 times its EC50 value (Erreger et al., 2007), while glycine (50 mM) was present at a maximal concentration. Mean data from a series of experiments (n = 6) examining the effect of glutamate concentration are illus- trated in Figure 1D. The extent of the inhibition produced by TCN 213 was not signiﬁcantly different for any of the glutamate concentrations (F2,15 = 0.76, P = 0.48; one-way ANOVA). Next we carried out the same two sets of experiments as described above but recording from oocytes expressing GluN1/GluN2B NMDA receptors. As it exempliﬁed by the upper panel in Figure 1B, TCN 213 (10 mM) only modestly antagonized responses even when the glycine concentration was low (0.1 ¥ EC50 value) with the mean data from a series of similar experiments (n = 6) plotted next to the corresponding GluN1/GluN2A dataset in the bar graph illustrated in Figure 1C. For GluN2B-containing NMDA receptors, the extent of the inhibition produced by TCN 213 is not signiﬁ- cantly different for any of these glycine concentrations (F2,15 = 0.74, P = 0.49; one-way ANOVA). In addition, in the presence of 50 mM glycine, no antagonism by TCN 213 was observed at three different glutamate concentrations (Figure 1B, lower panel, Figure 1D). IC50 values for TCN 213 and 5,7 DCKA acting at GluN1/GluN2A and GluN1/GluN2B NMDA receptors The experiments illustrated in Figure 1 each examined the effects of TCN 213 at a ﬁxed concentration (10 mM). In order to quantify its antagonist potency at GluN2A- and GluN2B- containing NMDA receptors, we carried out a series of experi- ments to determine concentrations of TCN 213 that produced 50% inhibition (IC50 values) of glutamate/glycine evoked currents. For comparison, we calculated comparable values for the prototypical glycine-site antagonist, 5,7 DCKA. As IC50 values are dependent on the agonist concentration (see Wyllie and Chen, 2007), we again used equipotent con- centrations of glycine based on its EC50 value when acting at GluN1/GluN2A and GluN1/GluN2B NMDA receptors (Chen et al., 2008). Thus, for GluN2A-containing NMDA receptors, IC50 values were determined at 0.15, 1.5 and 15 mM glycine (+100 mM glutamate) representing 0.1, 1 and 10 times its EC50 value respectively. Mean inhibition curves for TCN 213 acting at GluN1/GluN2A NMDA receptors are illustrated in Figure 2A. As is to be expected, the IC50 values for TCN 213 (Table 1) increased with increasing glycine concentrations for GluN1/GluN2A NMDA receptors. However, for GluN1/ GluN2B NMDA receptors (Figure 2B), we observed negligible inhibition of currents when glycine was applied at 1 and 10 British Journal of Pharmacology (2012) 166 924–937 927