Kurt Lingenhöhl

2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor antagonist.

In the present paper we describe 2-methyl-6-(phenylethynyl)-pyridine (MPEP) as a potent, selective and systemically active antagonist for the metabotropic glutamate receptor subtype 5 (mGlu5). At the human mGlu5a receptor expressed in recombinant cells, MPEP completely inhibited quisqualate-stimulated phosphoinositide (PI) hydrolysis with an IC50 value of 36 nM while having no agonist or antagonist activities at cells expressing the human mGlu1b receptor at concentrations up to 30 microM. When tested at group II and III receptors, MPEP did not show agonist or antagonist activity at 100 microM on human mGlu2, -3, -4a, -7b, and -8a receptors nor at 10 microM on the human mGlu6 receptor. Electrophysiological recordings in Xenopus laevis oocytes demonstrated no significant effect at 100 microM on human NMDA (NMDA1A/2A), rat AMPA (Glu3-(flop)) and human kainate (Glu6-(IYQ)) receptor subtypes nor at 10 microM on the human NMDA1A/2B receptor. In rat neonatal brain slices, MPEP inhibited DHPG-stimulated PI hydrolysis with a potency and selectivity similar to that observed on human mGlu receptors. Furthermore, in extracellular recordings in the CA1 area of the hippocampus in anesthetized rats, the microiontophoretic application of DHPG induced neuronal firing that was blocked when MPEP was administered by iontophoretic or intravenous routes. Excitations induced by microiontophoretic application of AMPA were not affected.

The metabotropic glutamate receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) blocks fear conditioning in rats

Glutamate receptors play an essential role in fear-related learning and memory. The present study was designed to assess the role of the group I metabotropic glutamate receptor (mGluR) subtype 5 in the acquisition and retrieval of conditioned fear in rats. The selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) was applied systemically (0.0, 0.3, 3.0, 30.0 mg/kg per os) 60 min before the acquisition training and before the expression of conditioned fear, respectively, in the fear-potentiated startle paradigm. MPEP dose-dependently blocked the acquisition of fear. This effect was not due to state-dependent learning. MPEP also prevented the expression of fear at a dose of 30.0 mg/kg. As a positive control for these effects, we showed that the benzodiazepine anxiolytic compound diazepam (1.25 mg/kg intraperitoneally) also blocked acquisition and expression of fear potentiated startle. MPEP did not affect the baseline startle magnitude, short-term habituation of startle, sensitisation of startle by footshocks or prepulse inhibition of startle. These data indicate a crucial role for mGluR5 in the regulation of fear conditioning. In the highest dose MPEP might exert anxiolytic properties.

[3H]-M-MPEP, a Potent, Subtype-Selective Radioligand for the Metabotropic Glutamate Receptor Subtype 5

The synthesis of a new potent, subtype-selective radioligand [(3)H]-M-MPEP (2-methyl-6-((3-methoxyphenyl)ethynyl)-pyridine) and its in vitro pharmacological characteristics are described. Science Ltd.

In vitro and in vivo characterization of MPEP, an allosteric modulator of the metabotropic glutamate receptor subtype 5: Review article

Summary. There is a need to identify subtype-specific ligands for mGlu receptors to elucidate the potential of these receptors for the treatment of nervous system disorders. To date, most mGlu receptor antagonists are amino acid-like compounds acting as competitive antagonists at the glutamate binding site located in the large extracellular N-terminal domain.We have characterized novel subtype-selective mGlu5 receptor antagonists which are structurally unrelated to competitive mGlu receptor ligands. Using a series of chimeric receptors and point mutations we demonstrate that these antagonists act as inverse agonists with a novel allosteric binding site in the seven-transmembrane domain. Recent studies in animal models implicate mGlu5 receptors as a potentially important therapeutic target particularly for the treatment of pain and anxiety.

Fear-reducing effects of intra-amygdala neuropeptide Y infusion in animal models of conditioned fear: an NPY Y1 receptor independent effect

RationaleNeuropeptide Y (NPY) and its receptors are densely localized in brain regions involved in the mediation and modulation of fear, including the amygdala. Several studies showed that central NPY is involved in the modulation of fear and anxiety.ObjectivesIn the present study, we investigated (1) whether intra-amygdala injections of NPY affect the expression of conditioned fear and (2) whether NPY Y1 receptors (Y1R) mediates the effects of these intra-amygdaloid NPY injections.ResultsIntra-amygdala NPY injections robustly decreased the expression of conditioned fear measured by conditioned freezing and fear-potentiated startle. These NPY effects were not mimicked by intra-amygdala injections of the Y1R agonists Y-28 or Y-36, and co-infusion of the Y1R antagonist BIBO 3304 did not block the NPY effects. Furthermore, we tested Y1R-deficient mice in conditioned freezing and found no differences between wild type and mutant littermates. Finally, we injected NPY into the amygdala of Y1R-deficient mice. Y1R deficiency had no effect on the fear-reducing effects of intra-amygdala NPY.ConclusionsThese data show an important role of the transmitter NPY within the amygdala for the expression of conditioned fear. Y1R do not appear to be involved in the mediation of the observed intra-amygdala NPY effects suggesting that these effects are mediated via other NPY receptors.

Phenylglycine derivatives antagonize the excitatory response of Purkinje cells to 1S,3R-ACPD : an in vivo and in vitro study

The interactions of the phenylglycine derivatives (S)-4-carboxyphenylglycine (S-4CPG) and (R,S)-alpha-methyl-4-carboxyphenylglycine (MCPG) with responses of rat cerebellar Purkinje cells to (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) were examined by intracellular recordings in acute cerebellar slices and extracellular recordings in vivo, using multibarrel electrodes. In vitro, both S-4CPG (100 microM to 1 mM) and MCPG (250 microM to 1 mM) reversibly and dose-dependently reduced an inward current induced by bath-applied 1S,3R-ACPD, an agonist at metabotropic glutamate receptors (mGluRs), in Purkinje cells voltage-clamped at -60 to -65 mV. S-4CPG applied at a concentration of 1 mM reduced the 1S,3R-ACPD induced current to 17% of control values but when applied alone also produced an inward current amounting to 26.8% of that induced by 1S,3R-ACPD. MCPG bath-applied at 250 microM, 500 microM, or 1 mM reduced the 1S,3R-ACPD-induced current to 85%, 56% or 3% of control values, respectively, and did not cause any current when applied alone even at a concentration of 1 mM. In vivo, iontophoretic application of 1S,3R-ACPD induced a transient increase followed by a decrease in the firing rate of Purkinje cells. The excitatory response of Purkinje cells to 1S,3R-ACPD was suppressed during ejection of either one of the phenylglycine derivatives, while the mechanism resulting in the decreased firing rate was not affected. Our observations demonstrate that both S-4CPG and MCPG antagonized the excitatory response of cerebellar Purkinje cells to 1S,3R-ACPD.(ABSTRACT TRUNCATED AT 250 WORDS)

Reevaluation of ACEA 1021 as an antagonist at the strychnine-insensitive glycine site of the N-methyl-D-aspartate receptor

Electrophysiological experiments were performed in vitro and in vivo to characterize the inhibitory effects of 6,7-dichloro-5-nitro-1,4-dihydro-2,3-quinoxalinedione (ACEA 1021; licostinel) on rat brain glutamate receptors. In vitro, ACEA 1021 was tested on N-methyl-D-aspartate (NMDA)-induced depolarizations in the neocortical slice preparation and on epileptiform activity in Mg2+-free hippocampal slices, which is known to be NMDA receptor mediated. In both in vitro models, ACEA 1021 exhibited antagonistic effects on the NMDA receptor-mediated responses. Selectivity tests in the neocortical slice preparation, using NMDA, kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) showed that 10 microM ACEA 1021 reduced NMDA and kainate responses to 27.9 and 79.9% of the control value, respectively, whereas responses to AMPA were increased by 2.4% above the control value, thus showing that at this concentration ACEA 1021 acts preferentially at NMDA receptors. However, at 30 microM, all the NMDA-, AMPA- and kainate-induced responses were reduced. In vivo, ACEA 1021 was tested on NMDA-induced excitation in the CA1 region. After systemic administration of ACEA 1021, central effects were observed at 10 mg/kg i.v. in the CA1 region. These results indicate that ACEA 1021 is centrally active and inhibits NMDA receptor-mediated responses. Interestingly, selectivity tests in the CA1 region did not show clear differences in the action of ACEA 1021 on NMDA- and AMPA-induced excitations. Furthermore, ACh-induced excitations were also reduced. Thus, at low concentrations, ACEA 1021 seems to be a selective antagonist at the strychnine-insensitive glycine site of the NMDA receptor. However, at 30 microM in vitro and at 10 mg/kg in vivo, non-NMDA receptor-mediated actions of ACEA 1021 are observed. Our results suggest that these additional effects of ACEA 1021 may contribute to its anticonvulsive properties in mice as well as to its neuroprotective properties in animal models of cerebral ischemia.

Electrophysiological characterization of CGP6873OA a N-methyl-D-aspartate antagonist acting at the strychnine-insensitive glycine site

1. Electrophysiological experiments were performed in vitro and in vivo. Voltage clamp recordings were done in Xenopus oocytes. Extracellular recordings were done in vitro in the neocortical slice and in the CA1 region of the hippocampal slice and in vivo in the CA1 region of the hippocampus of the anaesthetized rat. 2. In oocytes expressing either the human NMDAR1A/2A or 1A/2B subunit combinations, CGP68730A [sodium (-)-9-bromo-2,3,6,7-tetrahydro-5,6-dioxo-5H-pyrazino[1,2,3-de]-1,4-benzo thiazine-3-acetic acid] antagonized L-glutamate / glycine induced currents with calculated IC50s of 20.5 and 81.6 nM, respectively. 3. In vitro, CGP68730A was tested on NMDA induced depolarizations in the neocortical slice preparation and on epileptiform activity in hippocampal slices bathed in Mg2+-free-medium, which is known to be NMDA mediated. In both in vitro models CGP68730A exhibited antagonistic effects on the NMDA receptor mediated responses. 4. In vivo CGP68730A was tested on NMDA induced excitations in the CA1 region. CGP68730A abolished NMDA induced excitations when applied microiontophoretically. However, only weak effects on NMDA induced excitation were observed after systemic administration at 100 mg/kg i.v.. These results indicate that CGP68730A has poor central nervous system bioavailability. 5. In oocytes, an increase in the glycine concentration from the EC80 to the EC95.99 shifted the inhibition curves for CGP68730A to the right. Furthermore, in neocortical slices and in anaesthetized rats CGP68730A inhibited NMDA mediated depolarizations, and this effect could be reversed by the addition of the glycine mimetic D-serine. This indicates that these effects of CGP68730A are mediated by an action on the strychnine-insensitive glycine site. 6. Selectivity tests in oocytes and in the neocortical slice preparation, using NMDA, kainate and AMPA showed that CGP68730A was selective in antagonizing NMDA receptor mediated responses. In oocytes, the compound was about 1000 times less potent on the rat GluR3 and the human GluR6 receptors than on the human NMDAR1A/2A subunit combination. In the neocortical slice preparationCGP68730A had no effects on AMPA or kainate induced depolarizations at concentrations of 3 and 10 microM. At 30 microM CGP68730A reduced the effects of each of the three agonists tested. 7. Thus, CGP68730A seems to be a selective antagonist at the strychnine-insensitive glycine coagonist site of the NMDA receptor. However, the compound showed no obvious central NMDA antagonistic effects following intravenous application.

Multiphasic responses of cerebellar Purkinje cells to 1S,3R-ACPD: an in vivo study

The effects of iontophoretically applied (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), an agonist of metabotropic glutamate receptors, were examined in rat cerebellar Purkinje cells in vivo. Multibarrel electrodes were used for extracellular recordings of spontaneous single unit discharges and iontophoretic ejection of 1S,3R-ACPD. The effect of 1S,3R-ACPD depended on both the strength and the duration of the iontophoretic current. Application of the agonist with ejection currents at or slightly above the response threshold for up to 60 s resulted in an increased rate of action potential firing. With larger ejection currents of the same duration the initial increase in activity was followed by a depression and eventually a cessation of activity. In the transition phase between low frequency firing and firing arrest, Purkinje cells generated almost exclusively complex spikes. When the drug application was continued for longer durations (1-10 min) the initial response was followed by a characteristic cyclic firing pattern. These cycles consisted of alternating phases of mainly simple spike activity, predominantly complex spike activity and silent intervals. At the end of drug applications using large ejection currents, a prolonged period (on average 66 s) with almost no spiking activity was observed. This period ended with an abrupt onset of simple spike firing. These findings point to an important function of cerebellar metabotropic glutamate receptors in the regulation of Purkinje cell activity.