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 receptors (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, one class which binds all NR1/NR2B-containing receptors (Ro 25,6981) and one which binds to just the dimeric NR1/NR2B receptor subtype (CP-101,606) (Chazot et al., 2002). The aims of this study are to map the distinct NR1/NR2B-containing subtypes and in the murine and human brain using ligand autoradiography (Mutel et al, 1998).

Both [³H] Ro 25,6981 and [³H] CP-101,606 specifically labelled mouse and human hippocampus, thalamus, striatum and cortex, consistent with our previous immunohistochemical studies (Thompson et al., 2002). Notably, the number of specific [³H] Ro 25,6981 binding sites were consistently higher than [³H] CP-101,606 binding sites in both murine (not shown) and human brain structures (n = 16-26, p <0.0001) (Figure 1). These data provide further evidence that CP-101,606 labels a subset of NR2B-containing receptors.

Figure 1 Control human brain Ligand autoradiography (mean ± SD for 16-26 samples)

Competition binding studies were used to determine the pharmacological properties of another NR2B ligand, ifenprodil using [³H] Ro 25,6981 binding to adult or P21 male Sprague-Dawley rat forebrain membranes. The assay was performed as described in Chazot et al., 2002, except non-specific binding was defined by 100 µM Ifenprodil. The competiton curve for ifenprodil binding, using P21 membranes, displayed a pseudo Hill slope coefficient close to unity (nH = -0.87 ± 0.20), with an IC₅₀ value of 8.69 ± 1.32µM. In contrast, the curve for ifenprodil binding using adult membranes was best fitted to a two-site fit model, with the nH = 0.65 ± 0.23 (high affinity site, EC₅₀ = 0.25µM, 35 ± 5%; low affinity site, EC₅₀ = 10.1 µM, 63 ± 5%). Therefore, we provide preliminary evidence that ifenprodil binds to more than one subtype of NR2B-containing receptors present in the adult forebrain, and that distinct populations of NR2B-containing receptors exist in young and mature rat forebrain.

Chazot, P.L. et al. (2002) Neuropharmacol. 42, p319-324
Chazot, P.L. (2004) Current Medicinal Chemistry, 10, p1241-1253
Mutel, V. et al. (1998) Journal of Neurochemistry, 70, 5, p2147-2155
Thompson, C.L et al (2002) Molecular Brain Research, 102, p55-61