BJP S McKay et al. Figure 4 Activity of TCN 213 at native NMDA receptor-mediated responses in rat cortical cultures (DIV 7–9). (A) Whole-cell current recording made from a rat cortical pyramidal cell (7 DIV) and voltage-clamped at -70 mV. TCN 213 (10 mM) does not antagonize the NMDA (50 mM) + glycine (1.5 mM) evoked current, whereas ifenprodil (3 mM) reduces the current by around 75% indicating the presence of a large population of GluN1/GluN2B NMDA receptors in this neurone. (B) Bar graph summary (n = 12 cells) illustrating the mean TCN 213 and ifenprodil block of NMDA/glycine evoked currents. (C) Typical micrographs of neurones used to determine the extent of cell death elicited by each of the treatments. Note the high ratio of pyknotic nuclei compared with non-pyknotic nuclei when neurones are exposed to NMDA (40 mM) in the presence of TCN 213 (10 mM). Calibration bar 20 mm. (D) Summary of the percentage cell death observed in response to 1 h exposure to NMDA at the concentrations indicated (+1.5 mM glycine). Note the neuroprotective effects of the GluN1/GluN2B NMDA receptor selective antagonist, ifenprodil (3 mM) and the non-selective GluN1-site antagonist, 5,7 DCKA (10 mM). TCN 213 (10 mM) is, however, not neuroprotective. experiments illustrated in Figure 4 but where GluN2A sub- units were now present, we transfected cortical cultures (DIV 5–9) with a plasmids containing either GluN2A cDNA or cDNA encoding globin (to act as a control; see Methods). In a separate series of experiments, we recorded form cortical neurones at a later developmental stage (DIV 14–17) when one would anticipate that expression levels of GluN2A sub- units are increased (Monyer et al., 1994). The results of these experiments are illustrated in Figure 5. Figure 5A shows typical whole-cell currents recorded from either a globin- transfected (upper trace), a GluN2A-transfected (middle trace) or an older non-transfected (lower trace) cortical neurone. Control responses to NMDA (50 mM) and glycine (3 mM) 932 British Journal of Pharmacology (2012) 166 924–937 established the magnitude of NMDA receptor-mediated response in each neurone prior to the application of NMDA and glycine together with the GluN2B-selective antagonist, ifenprodil (3 mM). The extent of the ifenprodil block (Figure 5B) was greatest in globin-transfected neurones and lower in both GluN2A-transfected neurones and non- transfected neurones. Once a steady-state response was obtained in response, we applied TCN 213 (30 mM) to deter- mine the amount of the remaining current that was sensitive to this GluN2A-selective antagonist. For TCN 213 antagonism (Figure 5C), we observed that the mean block was greatest at GluN2A-transfected neurones and lower at both non- transfected neurones and globin-transfected neurones.