Blake A. Rowe

[3H]Methoxymethyl-3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine Binding to Metabotropic Glutamate Receptor Subtype 5 in Rodent Brain: In Vitro and in Vivo Characterization

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.

Group II mGlu Receptor Activation Suppresses Norepinephrine Release in the Ventral Hippocampus and Locomotor Responses to Acute Ketamine Challenge

Group II mGlu receptor agonists (eg LY379268 and LY354740) have been shown to reverse many of the behavioral responses to PCP as well as glutamate release elicited by PCP and ketamine. In the present set of experiments, we used in vivo microdialysis to show that, in addition to reversing PCP- and ketamine-evoked glutamate release, group II mGlu receptor stimulation also prevents ketamine-evoked norepinephrine (NE) release. Pretreating animals with the mixed 2/3 metabotropic glutamate (mGlu2/3) receptor agonist LY379268 (0.3–10 mg/kg) dose-dependently inhibited ketamine (25 mg/kg)-evoked NE release in the ventral hippocampus (VHipp). Ketamine hyperactivity was also reduced in a similar dose range. Following our initial observation on NE release, we conducted a series of microinjection experiments to reveal that the inhibitory effects of LY379268 on VHipp NE release may be linked to glutamate transmission within the medial prefrontal cortex. Finally, we were able to mimic the inhibitory effects of LY379268 on ketamine-evoked NE release by using a novel mGlu2 receptor selective positive modulator. (+/−) 2,2,2-Trifluoroethyl [3-(1-methyl-butoxy)-phenyl]-pyridin-3-ylmethyl-sulfonamide (2,2,2-TEMPS, characterized through in vitro GTPγS binding) at a dose of 100 mg/kg significantly reduced the NE response. Together, these results demonstrate a novel means to suppress noradrenergic neurotransmission (ie by activating mGlu2 receptors) and may, therefore, have important implications for neuropsychiatric disorders in which aberrant activation of the noradrenergic system is thought to be involved.

Transposition of Three Amino Acids Transforms the Human Metabotropic Glutamate Receptor (mGluR)-3-Positive Allosteric Modulation Site to mGluR2, and Additional Characterization of the mGluR2-Positive Allosteric Modulation Site

Glutamate is a major neurotransmitter in the central nervous system, and abnormal glutamate neurotransmission has been implicated in many neurological disorders, including schizophrenia, Alzheimers disease, Parkinsons disease, addiction, anxiety, depression, epilepsy, and pain. Metabotropic glutamate receptors (mGluRs) activate intracellular signaling cascades in a G protein-dependent manner, which offer the opportunity for developing drugs that regulate glutamate neurotransmission in a functionally selective manner. In the present study, we further characterize the human mGluR2 (hmGluR2) potentiator binding site by showing that the substitution of the three amino acids found to be required for hmGluR2 potentiation, specifically Ser688, Gly689, and Asn735, with the homologous hmGluR3 amino acids, inactivates the positive allosteric modulator activity of several structurally unique mGluR2 potentiators. Based on the characterization of the hmGluR2 potentiator binding site, we developed a novel scintillation proximity assay that was able to discriminate between compounds that were hmGluR2-specific potentiators, and those that were active on both hmGluR2 and hmGluR3. In addition, we substituted Ser688, Gly689, and Asn735 into hmGluR3 and created an active hmGluR2 allosteric modulation site on the hmGluR3 receptor.

MK-7128, a novel CB1 receptor inverse agonist, improves scopolamine-induced learning and memory deficits in mice.

Cannabinoid receptors (CBRs) play an important role in a variety of physiological functions and have been considered drug targets for obesity and psychiatric disorders. In particular, the CB1R is highly expressed in brain regions crucial to learning and memory processes, and several lines of evidence indicate that pharmacological blockade of this receptor could have therapeutic applications in the treatment of cognitive disorders. In this study, we investigated whether MK-7128 (0.1, 0.3, and 1 mg/kg, orally), a novel and selective CB1R inverse agonist, could improve learning and memory deficits induced by scopolamine (1 mg/kg, subcutaneously) in mice. The investigators also assessed CB1R occupancy in the brain to ensure target engagement of MK-7128, and showed that MK-7128 significantly improved both Y-maze spontaneous alternation and object habituation performance in scopolamine-treated mice and inhibits the binding of radioiodinated AM251 in murine cortex and hippocampus. These data indicate that MK-7128 improves cognitive performance in a model of cholinergic hypofunction and suggest that efficacy is achieved at relatively low levels of CB1R occupancy in the brain. Our results extend earlier findings suggesting a role of CB1Rs in the modulation of memory processes and a potential therapeutic application for CB1R inverse agonists in cognitive disorders.