BJP British Journal of Pharmacology RESEARCH PAPER bph_1748 924.937 Direct pharmacological monitoring of the developmental switch in NMDA receptor subunit composition using TCN 213, a GluN2A-selective, glycine- dependent antagonist S McKay*, NH Grifﬁths*, PA Butters*, EB Thubron, GE Hardingham and DJA Wyllie Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, UK. DOI:10.1111/j.1476-5381.2011.01748.x www.brjpharmacol.org Correspondence DJA Wyllie, Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK. Email: dwyllie1@staffmail.ed.ac.uk ---------------------------------------------------------------- *SMc, NHG and PAB contributed equally to this study. ---------------------------------------------------------------- Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://wileyonlinelibrary.com/ onlineopen#OnlineOpen_Terms ---------------------------------------------------------------- Keywords NMDA; glutamate; glycine; antagonism; oocyte; two-electrode voltage clamp; electrophysiology; neurotoxicity; development ---------------------------------------------------------------- Received 17 June 2011 Revised 26 September 2011 Accepted 10 October 2011 BACKGROUND AND PURPOSE Developmental switches in NMDA receptor subunit expression have been inferred from studies of GluN2 expression levels, changes in kinetics of glutamatergic synaptic currents and sensitivity of NMDA receptor-mediated currents to selective GluN2B antagonists. Here we use TCN 213, a novel GluN2A-selective antagonist to identify the presence of this subunit in functional NMDA receptors in developing cortical neurones. EXPERIMENTAL APPROACH Two-electrode voltage-clamp (TEVC) recordings were made from Xenopus laevis oocytes to determine the pharmacological activity of TCN 213 at recombinant NMDA receptors. TCN 213 antagonism was studied in cultures of primary cortical neurones, assessing the NMDA receptor dependency of NMDA-induced excitotoxicity and monitoring developmental switches in NMDA receptor subunit composition. KEY RESULTS TCN 213 antagonism of GluN1/GluN2A NMDA receptors was dependent on glycine but independent of glutamate concentrations in external recording solutions. Antagonism by TCN 213 was surmountable and gave a Schild plot with unity slope. TCN 213 block of GluN1/GluN2B NMDA receptor-mediated currents was negligible. In cortical neurones, at a early developmental stage predominantly expressing GluN2B-containing NMDA receptors, TCN 213 failed to antagonize NMDA receptor-mediated currents or to prevent GluN2B-dependent, NMDA-induced excitoxicity. In older cultures (DIV 14) or in neurones transfected with GluN2A subunits, TCN 213 antagonized NMDA-evoked currents. Block by TCN 213 of NMDA currents inversely correlated with block by ifenprodil, a selective GluN2B antagonist. CONCLUSIONS AND IMPLICATIONS TCN 213 selectively blocked GluN1/GluN2A over GluN1/GluN2B NMDA receptors allowing direct dissection of functional NMDA receptors and pharmacological proﬁling of developmental changes in native NMDA receptor subunit composition. Abbreviations 5,7 DCKA, 5,7 dichlorokynurenic acid; DIV, days in vitro; TCN 213, N-(cyclohexylmethyl)-2-({5-[(phenylmethyl)amino]- 1,3,4-thiadiazol-2-yl}thio)acetamide; TEVC, two-electrode voltage clamp 924 British Journal of Pharmacology (2012) 166 924–937 © 2011 The Authors British Journal of Pharmacology © 2011 The British Pharmacological Society