BJP S McKay et al. tized by immersion in a solution of 3-amino-benzoic acid ethylester (0.5%) and then killed by injection of an overdose of pentobarbital (0.4 mL of 20% solution) followed by decapi- tation and exsanguination after the conﬁrmation of loss of cardiac output. Before injection with cRNA mixtures of inter- est, the follicular membranes of the oocytes were removed. After injection oocytes were placed in separate wells of 24-well plates containing a modiﬁed Barth’s solution with the following composition (in mM): NaCl 88, KCl 1, NaHCO3 2.4, MgCl2 0.82, CaCl2 0.44, Ca(NO3)2 0.33, Tris–Cl 15, adjusted to pH 7.35 with NaOH (Sigma-Aldrich, Poole, UK). This solution was supplemented with 50 IU·mL-1 penicillin, 50 mg·mL-1 streptomycin (Invitrogen, Paisley, UK) and. 50 mg·mL-1 tetracycline (Sigma-Aldrich). Oocytes were placed in an incubator (19°C) for 24–48 h to allow for receptor expression and then stored at 4°C until required for electro- physiological measurements. Culture of rat cortical neurones Cortical neurones from E21 Sprague–Dawley rat embryos were cultured as described previously (Bading and Greenberg, 1991; Papadia et al., 2008; Martel et al., 2009), except that the Neurobasal-A growth medium contained B27 (Invitrogen), 1% rat serum (Harlan UK Ltd., Oxon, UK) and 1 mM glutamine. On the 4th day of culture (DIV 4), 1 mL growth medium containing 9.6 mM cytosine b-D-arabinofuranoside hydrochloride (AraC) was added to each well to inhibit glial cell proliferation. Culture media were replenished every 2 days after DIV 9 by replacing 1 mL of the conditioned media with 1 mL of fresh growth medium that lacked rat serum but was supplemented with glucose (10 mM). Prior to the excito- toxicity assay the cultures were transferred from the growth medium to a non-trophic medium that lacked glycine. This non-trophic medium contained 10% MEM (Invitrogen) and a glycine-free salt-glucose medium (in mM): NaCl 114, KCl 5.3, MgCl2 1, CaCl2 2, HEPES 10, glucose 30, Na-pyruvate 0.5 with NaHCO3 (0.2%) and phenol red (0.1%). Transfection of cortical neurones Neurones were transfected between DIV 5–9 using Lipo- fectamine 2000 (Invitrogen) according to the manufacturer’s suggested protocol. b-globin or pCis-GluN2A (Rutter and Stephenson, 2000) were co-transfected with enhanced green ﬂuorescent protein (eGFP), to identify transfected cells, in a ratio of 2:1. Transfection efﬁciency was approximately 5% with >99% of eGFP-expressing cells being identiﬁed as posi- tive for the neuronal nuclear antigen (NeuN), while <1% were positive for glial ﬁbrillary acidic protein (GFAP) (Soriano et al., 2008). Electrophysiological recordings were made from transfected neurones 48 h post transfection. Electrophysiological recordings and solutions Two-electrode voltage-clamp (TEVC) recordings were made at room temperature (18–21°C) using a GeneClamp 500 (Molecular Devices, Sunnyvale, CA) from oocytes that were placed in a solution that contained (in mM) NaCl 115, KCl 2.5, HEPES 10, BaCl2 1.8, EDTA 0.01; pH 7.3 with NaOH. EDTA was included to chelate contaminating low nanomolar levels of Zn2+ present in our solutions, which cause a potent but voltage-independent block of GluN2A-containing NMDA 926 British Journal of Pharmacology (2012) 166 924–937 receptors. Current and voltage electrodes were made from thin-walled borosilicate glass (GC150TF-7.5, Harvard Appara- tus, Kent, UK) using a PP-830 electrode puller (Narashige Instruments, Tokyo, Japan) and, when ﬁlled with 0.3 M KCl, possessed resistances of between 1 and 2 MW. Oocytes were voltage-clamped at -40 mV. Application of solutions was con- trolled manually, and data were ﬁltered at 10 Hz and digitized at 100 Hz via a Digidata 1200 A/D interface (Molecular Devices) using WinEDR software (Strathclyde Electrophysiol- ogy Software, Strathclyde University, UK). Whole-cell NMDA-evoked currents in cultured rat corti- cal neurones were recorded using an Axopatch 200B ampli- ﬁer (Molecular Devices) using patch pipettes made from thick-walled borosilicate glass with a tip resistance of 4–8 MW, which were ﬁlled with an ‘internal’ solution that contained (in mM) potassium gluconate 141, NaCl 2.5, HEPES 10, EGTA 11; pH 7.3 with KOH. Experiments were conducted at room temperature (18–21°C) in an ‘external’ solution containing (in mM): NaCl 150, KCl 2.8, HEPES 10, CaCl2 2, glucose 10, EDTA 0.01; pH to 7.3 with NaOH. Picro- toxin (50 mM) and tetrodotoxin (300 nM) were included to block GABAA receptor-mediated responses and action potential-driven excitatory/inhibitory postsynaptic events respectively. Access resistances (17.9 1.1 MW) were moni- tored and, recordings where this changed by >20% were dis- carded. Currents were ﬁltered at 2 kHz and digitized at 5 kHz via a BNC-2090A/PCI-6251 DAQ board interface (National Instruments, Austin, TX) and analysed using WinEDR soft- ware (Dr John Dempster, University of Strathclyde, Glasgow, UK). Assessment of antagonist potencies Concentrations of TCN 213 and 5,7 dichlorokynurenic acid (5,7 DCKA) required to inhibit 50% (IC50) of agonist-evoked responses were determined by ﬁtting inhibition curves with the following equation: I = I[B]0 (1 + ([B] IC50 )nH ), where nH is the Hill coefﬁcient, I[B]0 is the predicted current in the absence of antagonist and [B] is the concentration of the antagonist. Each data point was then normalized to the pre- dicted maximum of the dose–response curve. These normal- ized values were then pooled and averaged for each construct and ﬁtted again with this same equation, with the maximum and minimum for each curve being constrained to asymptote to 1 and 0 respectively (Wrighton et al., 2008; Otton et al., 2011). We estimated the equilibrium constant (KB) for TCN 213 acting at GluN1/GluN2A NMDA receptors using the Schild method (Arunlakshana and Schild, 1959; Wyllie and Chen, 2007). Brieﬂy, dose ratios (r) from individual oocytes were determined at low agonist concentrations by construct- ing a partial concentration–response curve generated in the absence of antagonist and in the presence of a series of increasing antagonist concentrations. Each series of two- point concentration–response curves were plotted on a log– log scale, and each data set was ﬁtted with a straight line with the same slope. These parallel ﬁts were used to calculate an overall mean r-value for each antagonist concentration (3, 10 and 30 mM), which were then used to construct a Schild plot (Frizelle et al., 2006; Otton et al., 2011; Figure S2). For tech-