McKay S , Griffiths NH , Butters PA , Thubron EB , Hardingham GE , Wyllie DJ .

Wellcome Trust , 088156 Wellcome Trust , MRC_G0902044 Medical Research Council , WT088156 Wellcome Trust , G0902044 Medical Research Council

	Figure 1. TCN 213 antagonism of NMDA receptor-mediated responses is both subtype- and glycine-dependent. (A) Upper panel, TEVC currents recorded from an oocyte expressing GluN1/GluN2A NMDA receptors in response to application of glutamate (100 µM) and glycine, which was applied at a concentration indicated as a multiple of its EC50 value (1.5 µM for GluN1/GluN2A NMDA receptors). TCN 213 (10 µM) was applied as indicated and inhibited the glutamate/glycine evoked response, but the extent of the inhibition was dependent on the glycine concentration. Lower panel, a series of similar TEVC current traces but recorded in the presence of a saturating concentration of glycine (50 µM) and variable concentrations of glutamate. The extent of the inhibition produced by TCN 213 is similar in each case. (B) A series of representative TEVC current traces illustrating similar experiments to those shown in panel A but for recordings made from oocytes expressing GluN1/GluN2B NMDA receptors. Note here the modest inhibition produced by TCN 213. (C) Bar graphs summarizing the mean data obtained from a series of experiments (n= 6) that investigated the glycine dependency of TCN 213 antagonism of steady-state responses at both GluN1/GluN2A and GluN1/GluN2B NMDA receptors. (D) As in panel C but for experiments where the glycine concentration was fixed (50 µM) and glutamate was applied at 0.1, 1 and 10 times its EC50 concentration. Calibration bars for TEVC traces illustrated in panels A and B: 750 nA, 50 s.
	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 µM) 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.
	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 µM, 10 µM and 30 µM 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 µM. (D) Bar graph summary showing that antagonism of GluN1/GluN2A NMDA receptor-mediated currents is surmountable when the glycine concentration is increased.
	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 µM) does not antagonize the NMDA (50 µM) + glycine (1.5 µM) evoked current, whereas ifenprodil (3 µM) 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 µM) in the presence of TCN 213 (10 µM). Calibration bar 20 µm. (D) Summary of the percentage cell death observed in response to 1 h exposure to NMDA at the concentrations indicated (+1.5 µM glycine). Note the neuroprotective effects of the GluN1/GluN2B NMDA receptor selective antagonist, ifenprodil (3 µM) and the non-selective GluN1-site antagonist, 5,7 DCKA (10 µM). TCN 213 (10 µM) is, however, not neuroprotective.
	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 µM) in the presence of glycine (3 µM) 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 µM). Once a steady-state response was established, TCN 213 (30 µM) 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.

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