NMDAR developmental switch monitored by TCN 213 BJP Figure 5 TCN 213 antagonism of GluN2A-containing cortical neurones. (A) Example whole-cell currents recorded from cortical pyramidal cells voltage- clamped at -70 mV. Upper left trace: example of an NMDA receptor-mediated current elicited by NMDA (50 mM) in the presence of glycine (3 mM) and recorded from a globin-transfected (control) pyramidal neurone (DIV 7). The trace to the right illustrates the NMDA receptor-mediated current recorded from the same cell and to the same concentrations of NMDA and glycine but also in the presence of the GluN2B-selective antagonist, ifenprodil (3 mM). Once a steady-state response was established, TCN 213 (30 mM) was applied to determine the amount of the ifenprodil-unblocked current that was sensitive to this GluN2A-selective antagonist. Middle and lower traces: examples of whole-cell currents recorded from either a GluN2A-transfected (DIV 7; middle trace) or non-transfected (DIV 14; lower trace) pyramidal neurone using the same drug application protocol as described above. (B) Bar graph summaries illustrating the mean ifenprodil block of NMDA receptor-mediated currents recorded from neurones in each of the three categories described in panel A. Ifenprodil block is greatest in young (DIV 7–11) neurones transfected with globin (n = 8), and this is signiﬁcantly greater (P < 0.01, t-test with Bonferroni correction) than the ifenprodil block of NMDA-induced currents in either GluN2A-transfected (n = 7) or older (DIV 14–17; n = 13) pyramidal neurones. (C) Equivalent bar graph summaries to those illustrated in panel B, but for TCN 213 block of NMDA receptor-mediated currents. In this case TCN 213 antagonism is observed to be the greatest at GluN2A-transfected neurones and weakest at globin-transfected neurones. (D) Plot illustrating the extent of ifenprodil and TCN 213 antagonism (in the same cell) of NMDA-evoked currents. While a wide range in the amount of block produced by either ifenprodil or TCN 213 is observed (particularly for recordings from GluN2A-transfected and from neurones in older cultures), there is a strong (negative) correlation (R2 = 0.87) between the amount of block produced by ifenprodil and TCN 213. However, these mean values for ifenprodil and TCN 213 block of NMDA-evoked currents in each of these three cat- egories of neurones obscure the fact that there was a highly correlated (R2 = 0.87) relationship between the magnitude of ifenprodil block and TCN 213 block in each cell we examined (Figure 5D). Thus, an NMDA-evoked current that showed high ifenprodil sensitivity was only weakly antagonized by TCN 213, whereas strong block of currents by TCN 213 was observed in cells, which showed lower sensitivity to ifen- prodil. This reciprocal relationship in antagonism by two NMDA receptor subtype-selective blockers is a key observa- tion and allows pharmacological proﬁling of NMDA receptor subunit composition. Discussion Our data demonstrate clearly that TCN 213 showed selective antagonism of GluN1/GluN2A over GluN1/GluN2B NMDA receptors. This antagonism could be surmounted by increas- British Journal of Pharmacology (2012) 166 924–937 933