NMDAR developmental switch monitored by TCN 213 BJP Introduction NMDA receptors are a subclass of ligand-gated ion channels activated by the CNS excitatory neurotransmitter L-glutamate (receptor nomenclature follows Alexander et al. 2011). In addition to their fundamental role in mediating the slow component of the glutamatergic excitatory postsynaptic potential/current, these receptors play pivotal roles in CNS development, learning and memory and pathophysiology. NMDA receptors are tetrameric proteins, the majority of which comprise two GluN1 and two GluN2 subunits (see Erreger et al., 2004, Chen and Wyllie, 2006; Traynelis et al., 2010). The GluN1 subunit exists in eight isoforms that are generated by alternative splicing of exons 5, 21 and 22 (Sugi- hara et al., 1992) while GluN2A-D subunits are encoded by four separate genes (Monyer et al., 1992). Uniquely among the family of glutamate ionotropic receptors, NMDA recep- tors require both glutamate and glycine (a co-agonist) to bind to the receptor for channel activation to occur (Figure S1A; Johnson and Ascher, 1987; Kleckner and Dingledine, 1988) with the glycine binding site being formed by the S1 and S2 domains of the GluN1 subunit and glutamate binding site being formed from the homologous regions on the GluN2 subunit. GluN2 subunit expression is regulated both spatially and temporally (Monyer et al., 1994), and it is the identity of the GluN2 subunits contained within the tetrameric NMDA receptor complex that determines many of the distinct bio- physical and pharmacological properties of NMDA receptor subtypes (see Erreger et al., 2004, Chen and Wyllie, 2006; Traynelis et al., 2010). The obligatory requirement of agonist and co-agonist binding for NMDA receptor activation means that the func- tion of these receptors can be blocked by antagonists that act either at the glycine (GluN1-located) site or at the glutamate (GluN2-located) site. A few NMDA receptor subtype selective antagonists already exist: non-competitive antagonists such as ifenprodil (Williams, 1993), R-(R*,S*)-a-(4-hydroxyphenyl)- b-methyl-4-(phenylmethyl)-1-piperidine propranol (Ro 25– 6981; Fischer et al., 1997) and (1S,2S)-1-(4-hydroxyphenyl)- 2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP 101 606; Mott et al., 1998) display a selectivity for GluN1/GluN2B NMDA receptors over other heterodimeric NMDA receptor combinations that is enough to allow these antagonists to be used to probe effectively NMDA receptor subunit composition and function in native neurones. However an antagonist that selectively blocks the GluN1/GluN2A receptor com- bination while sparing other NMDA receptor subtypes is lacking. Although (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]- ( 2, 3 - dioxo - 1, 2, 3, 4 - tetrahydroquinoxalin - 5 - yl ) - methyl ] - phosphonic acid (NVP-AAM077; Auberson et al., 2002) shows increased potency at GluN2A- over GluN2B-containing NMDA receptors, the difference in the respective equilibrium constants is not enough for this antagonist to discriminate unambiguously between these two receptor combinations (Frizelle et al., 2006; Wyllie and Chen, 2007). A recent study by Bettini et al. (2010) identiﬁed a series of novel NMDA receptor receptor antagonists from a chemical library of around two million compounds using high- throughput screening methods. One of their ‘hits’, N - (cyclohexylmethyl) - 2 - ({5 - [(phenylmethyl)amino] - 1, 3, 4- thiadiazol-2-yl}thio)acetamide (Figure S1B), referred to as Compound 13 in their study and now named ‘TCN 213’ by Tocris Bioscience, was shown to preferentially block GluN2A- containing over GluN2B-containing NMDA receptors. Their study demonstrated that antagonism by this compound could be reversed by increasing glycine but not glutamate concentrations, suggesting that this antagonist acted at the GluN1 subunit. This observation in itself raises the notion that the nature of the interaction of GluN1 and GluN2 sub- units in tetrameric NMDA receptor complexes may be depen- dent on the identity of the GluN2 subunit and may confer distinct pharmacology to the GluN1 subunit. Indeed, this type of GluN2-dependent pharmacology for agonists acting at the GluN1 subunit has been reported (Chen et al., 2008). However, in a binding assay, TCN 213 failed to displace the GluN1 ligand MDL 105 519, raising the possibility that TCN 213 acts at a site on the GluN1 subunit that is distinct from the glycine binding site itself. In addition no assessment has been made of the ability of TCN 213 to antagonize NMDA receptor-mediated currents using electrophysiological record- ing techniques. Moreover, assessment of the potency of TCN 213 acting at GluN2A- and GluN2B-containing NMDA recep- tors under conditions where both receptors are activated to a similar extent is lacking. Our study assessed the conditions under which TCN 213 displayed selective antagonism of GluN2A-containing NMDA receptors and extended the investigation to the study of native NMDA receptors in cortical neurones to assess the action of TCN 213 on NMDA-evoked currents and NMDA- evoked excitotoxic neuronal cell death. Furthermore, we demonstrated that TCN 213 allowed direct pharmacological assessment of the expression of GluN2A subunits in develop- ing cortical neurones in dissociated culture. Our data indicate that this antagonist and its related sulphonamide derivatives (Bettini et al., 2010) provide promising novel pharmacologi- cal tools to investigate NMDA receptor function and permit new opportunities to monitor developmental changes in native NMDA receptor subunit composition. Methods Plasmid constructs, cRNA synthesis and receptor expression in oocytes Nomenclature of NMDA receptor subunits follows Collin- gridge et al. (2009) and Alexander et al. (2011). The expres- sion plasmids for rat GluN1-1a (excluding exon 5, including exons 21 and 22, and referred to here as ‘GluN1’) and GluN2A subunits have been described previously (Chen et al., 2005). The cDNA construct containing the rat GluN2B subunit was a gift from Dr Stephen Traynelis (Emory University, Atlanta, GA). cRNA for each of the NMDA receptor subunits was synthesized as run-off transcripts as described previously (Chen et al., 2005; 2008; Erreger et al., 2007). cRNA amounts and integrity were estimated by intensity of ﬂuorescence in ethidium bromide-stained agarose gels. GluN1 and GluN2 cRNAs were mixed at a nominal ratio of 1:1 and diluted with nuclease-free water to 5 ng·mL-1, prior to injection. All animal care and experimental procedures complied with current UK Home Ofﬁce regulations. Stage V–VI oocytes were obtained from Xenopus laevis that had been anaesthe- British Journal of Pharmacology (2012) 166 924–937 925