Abstract

The binding of [³H]methoxymethyl-3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (methoxymethyl-MTEP), a potent and selective antagonist for metabotropic glutamate (mGlu)5 receptors, was characterized in rat brain both in vitro and in vivo. Nonspecific binding, as defined with 10 μM 2-methyl-6-(phenylethynyl)-pyridine (MPEP), was less than 10% of total binding in rat brain membranes. The binding of [³H]methoxymethyl-MTEP was of high affinity (K _d = 20 ± 2.7 nM), saturable (B _max = 487 ± 48 fmol/mg protein), and to a single site. The mGlu5 antagonists methoxymethyl-MTEP and MPEP displaced [³H]methoxymethyl-MTEP binding with IC₅₀values of 30 and 15 nM, respectively. In vivo administration of [³H]methoxymethyl-MTEP (50 μCi/kg i.v.) revealed 12-fold higher binding in hippocampus (an area enriched in mGlu5 receptors) relative to cerebellum (an area with few mGlu5 receptors) in rats. Similarly, administration of [³H]methoxymethyl-MTEP to mGlu5-deficient mice demonstrated binding at background levels in forebrain, whereas wild-type littermates exhibited 17-fold higher binding in forebrain relative to cerebellum. Systemic administration of unlabeled mGlu5 antagonists methoxymethyl-MTEP and MPEP to rats reduced the binding of [³H]methoxymethyl-MTEP with ID₅₀ values of 0.8 and 2 mg/kg i.p., respectively, 1 h post-treatment. The mGlu5 agonist 2-chloro-5-hydroxyphenylglycine (CHPG) (0.3, 1, and 3 μmol) dose-dependently increased phosphoinositide (PI) hydrolysis in the hippocampus after i.c.v. administration in rats. CHPG-evoked increases in PI hydrolysis were blocked with MPEP at a dose (10 mg/kg i.p.) that markedly reduced [³H]methoxymethyl-MTEP binding in vivo. These results indicate that [³H]methoxymethyl-MTEP is a selective radioligand for labeling mGlu5 and is useful for studying the binding of mGlu5 receptors in rat brain in vitro and in vivo.