The N-methyl-D-aspartate (NMDA) receptor is a heteromeric ligand-gated ion channel comprising a range of subunits, namely NR1, NR2A-D and NR3A-B. The specific expression profile of the NR2B subunit in the brain, is developmentally and regionally regulated, and is involved in key processes such as learning, memory and motor coordination. There is clear evidence for different types of NR2B-containing receptor subtypes (eg. NR1/2B, NR1/2B/2A, NR1/2B/2D) (Chazot, 2004). Previous studies in our laboratory have provided evidence that there are at least two classes of NR2B-selective compounds (Chazot et al., 2002). Recently, we reported evidence that ifenprodil bound to more than a single [³H] Ro 25,6981 binding site in adult rat forebrain membranes, indicating that ifenprodil may bind to distinct NR2B-containing subtypes expressed later in development (Sheahan et al., 2004). In this present study, we have performed a series of competition binding experiments using [³H] Ro-25,6981 and two membrane preparations derived from P21 and adult rat male Sprague-Dawley forebrain membranes, to determine the pharmacological properties of two further NR2B selective ligands, histamine and haloperidol. The assays were performed as described in Chazot et al., 2002, except non-specific binding was defined by 100 µM Ifenprodil. Data were means ± SD for at least 3 separate experiments, and were analysed by GraphPad Prism 3.

Two structurally distinct molecules, histamine and haloperidol have been previously shown to display selectivity for NR2B containing NMDA receptors (Williams, 1994; Gallagher et al., 1998). Interestingly, histamine (up to 1 mM) displayed no significant displacement or potentiation of [³H] Ro-25,6981 binding, to both P21 and adult forebrain membranes. This suggests that Histamine binding to NR2B-containing receptors is occurring at a distinct site from Ro-25,6981. In contrast to histamine, but as seen with ifenprodil, haloperidol displayed a complete displacement of [³H] Ro-25,6981 binding (at 1mM) to both P21 and adult forebrain membranes. However, in contrast to ifenprodil, haloperidol displayed a shallow competition curve (-0.33 ± 0.12) with an overall pIC₅₀ value = 4.74 ± 0.55 in P21 rat membranes. These data were best-fitted to a two-site binding model, yielding a high affinity site (pIC₅₀ = 6.22 ± 0.14, 46 ± 13%) and a low-affinity site (pIC₅₀ = 3.92 ± 0.43, 44 ± 13%). These results, together with previous data (Sheahan et al., 2004), provide further evidence for heterogeneity of NR2B-containing NMDA receptors in both juvenile and adult rat forebrain.

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This study was funded, in part, by Pfizer Inc (Japan) .