ly-341495 and eglumetad

Much hope in drug development comes from the discovery of positive allosteric modulators (PAM) that display target subtype selectivity and act by increasing agonist potency and efficacy. How such compounds can allosterically influence agonist action remains unclear. Metabotropic glutamate receptors (mGlu) are G protein-coupled receptors that represent promising targets for brain diseases, and for which PAMs acting in the transmembrane domain have been developed. Here, we explore the effect of a PAM on the structural dynamics of mGlu2 in optimized detergent micelles using single molecule FRET at submillisecond timescales. We show that glutamate only partially stabilizes the extracellular domains in the active state. Full activation is only observed in the presence of a PAM or the G

Topics: Allosteric Regulation; Allosteric Site; Amino Acids; Biphenyl Compounds; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Catalytic Domain; Cell Membrane; Cholesterol Esters; Diosgenin; Disaccharides; Fluorescence Resonance Energy Transfer; Gene Expression; Glucosides; Glutamic Acid; Glycolipids; HEK293 Cells; Humans; Indans; Micelles; Octoxynol; Protein Binding; Protein Conformation; Protein Multimerization; Receptors, Metabotropic Glutamate; Recombinant Proteins; Single Molecule Imaging; Xanthenes

The superfamily of G protein-coupled receptors (GPCRs) represents the largest group of cell surface receptors in the human body. It is estimated that around 40% of the drugs currently on the market target GPCRs. As only a very small number of GPCRs is targeted by these marketed drugs, the potential of GPCRs as novel drug targets remains enormous. As opposed to conventional in vitro assays, label-free cellular assays using a biosensor provide new opportunities for studying GPCRs. Integrated receptor-mediated responses are measured in real-time rather than a single downstream signaling pathway, without the need for the use of any label (e.g., fluorescent or radioactive). Here, we describe a protocol to study GPCR pharmacology using the label-free whole cell impedance-based biosensor system xCELLigence. This assay allows quantification of compound-induced GPCR-mediated responses in real-time. Finally, we have also discussed the analysis and interpretation of the results obtained with this assay using the mGlu

Topics: Amino Acids; Animals; Biological Assay; Biosensing Techniques; Bridged Bicyclo Compounds; Cell Count; CHO Cells; Cricetinae; Cricetulus; Electric Impedance; Humans; Ligands; Receptors, G-Protein-Coupled; Receptors, Metabotropic Glutamate; Signal Transduction; Staining and Labeling; Xanthenes

While many orthosteric ligands have been developed for the mGlu

Topics: Allosteric Regulation; Amino Acids; Animals; Binding Sites; Bridged Bicyclo Compounds; CHO Cells; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Ligands; Receptors, Metabotropic Glutamate; Xanthenes

Xanthurenic acid (XA), a molecule arising from tryptophan metabolism by transamination of 3-hydroxykynurenine, has recently been identified as an endogenous Group II (mGlu2 and mGlu3) metabotropic glutamate (mGlu) receptor ligand in vitro. Impairments in Group II mGlu receptor expression and function have been implicated in the pathophysiology of schizophrenia, as have multiple steps in the kynurenine metabolism pathway. Therefore, we examined XA in vivo to further investigate its potential as a Group II mGlu receptor ligand using a preparation that has been previously demonstrated to efficiently reveal the action of other Group II mGlu receptor ligands in vivo. Extracellular single-neurone recordings were made in the rat ventrobasal thalamus (VB) in conjunction with iontophoresis of agonists, an antagonist and a positive allosteric modulator and/or intravenous (i.v.) injection of XA. We found the XA effect on sensory inhibition, when applied iontophoretically and i.v., was similar to that of other Group II mGlu receptor agonists in reducing inhibition evoked in the VB from the thalamic reticular nucleus upon physiological sensory stimulation. Furthermore, we postulate that XA may be the first potential endogenous allosteric agonist (termed 'endocoid') for the mGlu receptors. As the Group II receptors and kynurenine metabolism pathway have both been heavily implicated in the pathophysiology of schizophrenia, XA could play a pivotal role in antipsychotic research as this potential endocoid represents both a convergence within these two biological parameters and a novel class of Group II mGlu receptor ligand. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Topics: Action Potentials; Administration, Intravenous; Afferent Pathways; Allosteric Regulation; Amino Acids; Animals; Bridged Bicyclo Compounds; Drug Interactions; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Iontophoresis; Male; Neural Inhibition; Neurons; Rats; Rats, Wistar; Ventral Thalamic Nuclei; Vibrissae; Xanthenes; Xanthurenates

Glutamate carboxypeptidase II (GCP II) is a glial enzyme responsible for the hydrolysis of N-acetylaspartylglutamate (NAAG) into glutamate and N-acetylaspartate (NAA). Abnormalities in glutamate neurotransmission are implicated in the pathophysiology of schizophrenia. In this study, we examined the effects of a novel, orally active GCP II inhibitor, 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA), on the prepulse inhibition (PPI) deficits after administration of the N-methyl-d-aspartate (NMDA) receptor antagonist dizocilpine. Oral administration of 2-MPPA (10, 30 or 100mg/kg) significantly attenuated dizocilpine (0.1mg/kg)-induced PPI deficits in mice, in a dose dependent manner. Furthermore, the efficacy of 2-MPPA on dizocilpine-induced PPI deficits was significantly antagonized by pretreatment with the selective group II metabotropic glutamate receptor (mGluR) antagonist LY341495 (1.0mg/kg). In the same model, however, the selective group II mGluR agonist LY354740 (3, 10 or 30 mg/kg) significantly attenuated dizocilpine-induced PPI deficits at only one dose and prepulse intensity. Our findings suggest that GCP II inhibition may be useful therapeutic strategy for schizophrenia. From a mechanistic perspective, while increased NAAG and activation of group II mGluRs may contribute to the therapeutic efficacy of 2-MPPA, it is likely that additional pharmacological activities are also involved.

Topics: Administration, Oral; Amino Acids; Animals; Antipsychotic Agents; Bridged Bicyclo Compounds; Dipeptides; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glutamate Carboxypeptidase II; Glutarates; Male; Mice; Neural Inhibition; Schizophrenia; Sensory Gating; Sulfhydryl Compounds; Xanthenes

Activation and blockade of prefrontal cortical 5-hydroxytryptamine2A (5-HT2A) receptors have been linked to the action of hallucinogenic and antidepressant/antipsychotic drugs; these effects may involve modulation of glutamate release from thalamocortical afferents. Although activation of metabotropic glutamate 2 (mGlu2) receptors may suppress 5-HT-induced excitatory postsynaptic currents (EPSCs), group III mGlu receptors (mGlu4/7/8) also are expressed in the thalamus and may suppress 5-HT-induced EPSCs. We have found by intracellular recordings from layer V pyramidal cells of the medial prefrontal cortex (mPFC) that group III mGlu receptor agonists (R,S)-4-phosphonophenylglycine (PPG), L-4-phosphono-2-aminobutyric acid (L-AP4), L-serine-O-phosphate (L-SOP), and (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4) preferentially suppress 5-HT-induced EPSCs compared with excitatory postsynaptic potentials evoked by electrical stimulation of the white matter. A number of pharmacological features [e.g., the rank order of agonist potency; MAP4 partial agonist action; differential potency for the group III mGlu receptor antagonist (R,S)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) in blocking the suppressant action of PPG or MAP4; and a relatively low potency of 2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)-3(xanthy-9-yl)propanoic acid (LY341495) in blocking the suppressant action of PPG or L-SOP] suggest that activation of both mGlu4 and mGlu8 receptors may play a role in suppressing 5-HT-induced EPSCs. Furthermore, L-SOP did not alter the synaptic currents or steady-state inward current induced by alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid. Thus, although both group III and group II mGlu receptor agonists suppress the frequency of 5-HT-induced EPSCs in the mPFC, they differ in that the group III mGlu receptor agonists appear to have relatively minimal effects on glutamate released by sources other than thalamocortical afferents.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Amino Acids; Animals; Bridged Bicyclo Compounds; Glycine; Male; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Receptor, Serotonin, 5-HT2A; Receptors, Metabotropic Glutamate; Serotonin; Synaptic Transmission; Xanthenes

(1S,2S,5R,6S)-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740) is a potent and selective agonist for group II metabotropic glutamate (mGlu2 and mGlu3) receptors, with anxiolytic-like activity in animal and human models, and efficacy in anxiety patients. However, the lack of mGlu2 or mGlu3 receptor specific agonists has prevented in vivo characterization of individual functions of these two receptors in mediating the anxiolytic-like effects of LY354740.. To utilize mGlu2 receptor and mGlu3 receptor knockout animals and the mGlu2/3 selective antagonist (2S,1'S,2'S)-2-(9-xanthylmethyl)-2-(2'-carboxycyclopropyl)glycine (LY341495) to further investigate the roles of mGlu2 and mGlu3 receptors in mediating the anxiolytic-like actions of LY354740 in a mouse model of anxiety [elevated plus maze (EPM) test].. To confirm that mGlu2/3 receptors are responsible for anxiolytic-like activity in the EPM under these test conditions, mice were pretreated with LY341495 at 30 min prior to s.c. administered LY354740. Subsequently, saline vehicle or LY354740 was administered (s.c.) 30 min before the EPM testing in wild-type, mGlu2 receptor knockout, and mGlu3 receptor knockout mice.. LY354740 reduced in a dose-dependent manner anxiety-related behavior on the EPM in wild-type mice with a maximally effective dose of 10--20 mg/kg s.c. Pretreatment with LY341495 potently prevented the anxiolytic-like effects of LY354740 (20 mg/kg, s.c.) in mice. Although the mGlu2 receptor knockout and mGlu3 receptor knockout mice were grossly normal, the anxiolytic-like activity of LY354740 (20 mg/kg, s.c.) was not evident in either mGlu2 or mGlu3 receptor knockout mice, when compared to their wild-type controls.. The activation of both mGlu2 and mGlu3 receptors by LY354740 appears to be required for anxiolytic-like activity in the EPM test in mice. These studies serve as a foundation for additional studies on underlying circuits, brain structures, and receptor subtypes involved in the anxiolytic-like actions of mGlu receptor active agents, and the design of future drugs for anxiety disorders in humans.

Topics: Amino Acids; Amygdala; Animals; Anti-Anxiety Agents; Bridged Bicyclo Compounds; Excitatory Amino Acid Agonists; Female; Male; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Receptors, Metabotropic Glutamate; Xanthenes

We have studied the effects of groups II and III metabotropic glutamate receptor (mGluR) activation on excitatory responses recorded from hippocampal interneurons of CA1 stratum lacunosum moleculare (SLM). Excitatory postsynaptic currents (EPSCs) evoked by stimulation of the perforant pathway were reduced either by the group II mGluR agonist LY354740 (50-100 nM, 49.1+/-5.7% of control) or by the group III mGluR agonist l-2-amino-4-phosphonobutyric acid (l-AP4) (50 microM, 36.8+/-4.4% of control). Both drugs significantly enhanced paired-pulse facilitation of the EPSCs. Furthermore, both 100 nM LY354740 and 50 microM l-AP4 reduced the frequency, but not the amplitude, of miniature excitatory synaptic currents (mEPSCs), recorded in the presence of 1 microM TTX and 50 microM picrotoxin, or EPSCs evoked by perforant pathway stimulation in the presence of 2.5 mM Sr2+. The broad-spectrum mGluR antagonist LY341495 (10-50 microM) did not affect test EPSCs elicited 210 ms after stimulation at 100 Hz. At network level, 1-5 microM LY354740 significantly reduced the power of gamma frequency oscillations induced by 20 microM carbachol, 600 nM kainate and 5 mM K+ in hippocampal CA1 area. Our results show powerful modulation of excitatory transmission impinging on interneurons of CA1 SLM by presynaptic group II or III mGluRs.

Topics: Amino Acids; Aminobutyrates; Analysis of Variance; Animals; Animals, Newborn; Bridged Bicyclo Compounds; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hippocampus; In Vitro Techniques; Interneurons; Neural Inhibition; Perforant Pathway; Presynaptic Terminals; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Strontium; Tetrodotoxin; Time Factors; Xanthenes

LY341495 and LY354740 are potent and selective antagonist and agonist, respectively, for Group II metabotropic glutamate (mGlu2/3) receptors. Here we demonstrate that LY341495 (3 mg/kg) significantly increased c-Fos expression in almost all brain regions analyzed (44 out of 52 regions) in animals that were prehandled and kept in home-cage environment to minimize stress. Robust c-Fos induction was observed in all cortical regions, hippocampal CA1 and CA3 subregions, amygdala and several other subcortical nuclei. In contrast to LY341495, changes in c-Fos expression following LY354740 were more modest and not generally widespread (decreased in 1 region, dentate gyrus; and increased in 13 out of 52 regions). Interestingly, although LY354740 is anxiolytic in animals, LY341495 did not increase c-Fos expression in the paraventricular nucleus of the hypothalamus which is usually activated by stress/fear and several anxiogenic compounds. To further investigate the behavioral consequences of mGlu2/3 receptor antagonism, LY341495 was administered to prehandled animals that were placed in the elevated plus maze test under low light (low stress) conditions. Here LY341495 increased mouse elevated plus maze (EPM)-anxiety in a dose-dependent manner, significantly decreasing the time spent in open arms, but not affecting total ambulations. The behavioral consequences and associated widespread pattern of brain neuronal activations following blockade of mGlu2/3 receptors suggest that there is considerable endogenous glutamate tone throughout the brain at negative feedback peri-synaptic mGlu2/3 receptors, even under low stress conditions where synaptic glutamate release spillover would be expected to be minimized.

Topics: Amino Acids; Analysis of Variance; Animals; Brain; Bridged Bicyclo Compounds; Cell Count; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Immunohistochemistry; Male; Mice; Mice, Inbred ICR; Neurons; Proto-Oncogene Proteins c-fos; Xanthenes

The metabotropic glutamate (mGlu2/3) receptor agonist, LY354740, exhibits anxiolytic-like properties in a number of rodent models. The present study utilized in vivo microdialysis to examine the effects of LY354740 on extracellular monoamine levels in the medial prefrontal cortex (mPFC) of animals subjected to 30 min immobilization stress. Immobilization stress significantly elevated extracellular levels of noradrenaline (NA) and dopamine (DA) in the mPFC, while systemic administration of LY354740 (30 mg/kg, s.c.) significantly attenuated immobilization-induced increases in both NA and DA. Reverse-dialysis of LY354740 (30 microm) into the mPFC significantly attenuated immobilization-induced increases in NA, but not DA without affecting basal levels of either amine. In separate studies in the presence of citalopram (1 microm; reverse dialysis into the mPFC), systemic administration of LY354740 attenuated immobilization-induced increases in NA and DA, but had no effect on serotonin (5-HT) levels. Co-administration of the selective mGlu2/3 receptor antagonist, LY341495, partially or fully reversed the attenuation in NA and DA levels produced by LY354740, respectively. Taken together, these data suggest that LY354740 may produce anti-stress actions, in part, by blocking stress-related increases in catecholamines in the mPFC via mGlu2/3 receptor stimulation.

Topics: Amino Acids; Animals; Bridged Bicyclo Compounds; Catecholamines; Citalopram; Dopamine; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Extracellular Fluid; Male; Microdialysis; Norepinephrine; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Restraint, Physical; Selective Serotonin Reuptake Inhibitors; Stress, Physiological; Xanthenes

Atrophy of the medial temporal lobes, including the glutamatergic cortical-hippocampal circuitry, is an early event in Alzheimer's disease (AD) and probably contributes to the characteristic short-term mnemonic decline. Pharmacological strategies directly targeted to ameliorating this functional decline may represent a novel approach for the symptomatic treatment of AD. Presynaptic group II metabotropic glutamate receptors (i.e. mGlu2 and mGlu3) exert a powerful modulatory influence on the function of these pathways, in particular the perforant pathway. Using a combination of mGlu2 receptor knockout mice and the group II agonist LY354740, we show that activation of mGlu2 receptors produces a cognitive impairment, i.e. a delay-dependent deficit in delayed matching and non-matching to position, and impaired spatial learning in a Morris water maze. Conversely, a group II antagonist, LY341495, improved acquisition of spatial learning. LY354740 potently reduced field excitatory postsynaptic potentials in hippocampal slices from wild type but not mGlu2 receptor knockout mice. Taken together, these results suggest that activation of mGlu2 receptors evokes a powerful inhibitory effect on hippocampal synaptic transmission and mGlu2 agonists produce a cognitive deficit consistent with this change. Conversely, mGlu2 receptor antagonists may improve certain aspects of cognition and thus represent a novel approach for the symptomatic treatment of AD.

Topics: Amino Acids; Animals; Bridged Bicyclo Compounds; Cognition; Dose-Response Relationship, Drug; Excitatory Postsynaptic Potentials; Male; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Knockout; Psychomotor Performance; Rats; Reaction Time; Receptors, Metabotropic Glutamate; Xanthenes

I have compared the effects of group II or III metabotropic glutamate receptor (mGluR) activation on monosynaptic excitatory responses recorded intracellularly from CA1 pyramidal neurons of rat hippocampus and evoked by perforant pathway stimulation in vitro. The excitatory postsynaptic currents (EPSCs) were reduced either by the group II mGluR agonist LY354740 (500 nM, 31 +/- 6% of control) or by the group III agonist L-AP4 (400 microM, 53 +/- 5% of control). Both drugs enhanced EPSC paired-pulse facilitation (range 125-189% of control). These effects were blocked by the broad-spectrum mGluR antagonist LY341495 (1 or 20 microM) which when applied alone did not significantly change the EPSCs elicited at low (0.1-0.2 Hz) or higher (1-100 Hz) frequency of stimulation. Prior reduction of the EPSCs induced by L-AP4 did not occlude the subsequent inhibition elicited by LY354740. The effect of LY354740, but not that of L-AP4, was blocked in the presence of the cAMP analogue Sp-cAMPS (20 microM) and with the K(+) channel antagonist alpha-dendrotoxin (125 nM). In contrast, the effect of L-AP4, but not that of LY354740, was prevented by the calmodulin inhibitor ophiobolin A (25 microM) and with the N-type Ca(2+) channel antagonist omega-conotoxin-GVIA (1 microM). In the presence of the P/Q type Ca(2+) channel antagonist omega-agatoxin-IVA (400 nM), the EPSCs were depressed either by LY354740 or by L-AP4. Groups II and III mGluRs are segregated at the presynaptic terminal, and there are distinct differences between the properties of the presynaptic inhibition mediated by these two groups of receptors.

Topics: Amino Acids; Aminobutyrates; Animals; Bridged Bicyclo Compounds; Calcium Channel Blockers; Cyclic AMP; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Drug Interactions; Elapid Venoms; Electric Stimulation; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Hippocampus; In Vitro Techniques; Membrane Potentials; Neural Inhibition; omega-Conotoxin GVIA; Perforant Pathway; Phosphinic Acids; Propanolamines; Quinoxalines; Rats; Receptors, Metabotropic Glutamate; Statistics, Nonparametric; Synaptic Transmission; Thionucleotides; Time Factors; Xanthenes

Recent studies have demonstrated that the hallucinogen 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) enhances glutamatergic transmission in the prefrontal cortex. This increase can be suppressed by metabotropic glutamate2/3 (mGlu2/3) receptor activation. In addition to enhancing glutamatergic transmission, DOI increases cortical c-fos expression. We tested if a reduction in glutamate release produced by mGlu2/3 receptor activation attenuates DOI-induced c-fos expression in the cortex. Similar to previous studies, DOI produced a robust increase in c-fos mRNA throughout the cortex, including the prefrontal, frontoparietal, and somatosensory regions. Pretreatment with the mGlu2/3 agonist LY379268 attenuated the DOI-induced increase in the prefrontal cortex. This suppression was blocked by the mGlu2/3 antagonist LY341495. In contrast, the DOI-induced increase in c-fos mRNA in the frontoparietal and somatosensory cortex was unaffected by the mGlu2/3 agents. These findings suggest that Group II metabotropic glutamate receptor agonists are capable of modulating postsynaptic function preferentially in the limbic cortex under conditions of enhanced glutamate release.

Topics: Amino Acids; Amphetamines; Animals; Autoradiography; Bridged Bicyclo Compounds; Cerebral Cortex; DNA; Electrophysiology; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Agonists; Image Interpretation, Computer-Assisted; In Situ Hybridization; Male; Prefrontal Cortex; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; RNA, Messenger; Serotonin Receptor Agonists; Xanthenes

Previous studies have shown that 5-hydroxytryptamine(2A) (5-HT(2A)) receptor activation induces changes in the pattern of brain-derived neurotrophic factor (BDNF) mRNA expression in the neocortex and hippocampus, and that 5-HT(2A) receptor blockade interferes with the induction of BDNF mRNA by stress. Recent studies have also shown that activation of metabotropic glutamate group II (mGlu2/3) receptors suppresses 5-HT(2A) receptor-stimulated excitatory postsynaptic potentials/currents (EPSP/Cs) in pyramidal neurons in medial prefrontal cortex. Conversely, blockade of mGlu2/3 receptors enhances 5-HT-induced EPSCs. The current study examined the effects of the highly selective mGlu2/3 agonist (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate (LY354740) and the mGlu2/3 antagonist 2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)-3(xanthy-9-yl)propanoic acid (LY341495) on BDNF mRNA expression in medial prefrontal cortex induced by the hallucinogen and 5-HT(2A/2B/2C) agonist 1-(2,5-dimethoxy-4-iodophenethyl)-2-aminopropane (DOI). LY354740 (0.1-10 mg/kg) dose-dependently suppressed DOI-induced BDNF mRNA levels in medial prefrontal cortex. In contrast, the mGlu2/3 antagonist LY341495 (1 mg/kg) enhanced DOI-induced BDNF mRNA levels. BDNF mRNA expression was not altered by administration of the mGlu agonist or the antagonist alone. These results are discussed with respect to a potential role for group II mGlu agonists in the treatment of depression and schizophrenia.

Topics: Amino Acids; Amphetamines; Animals; Brain-Derived Neurotrophic Factor; Bridged Bicyclo Compounds; Cerebral Cortex; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; In Situ Hybridization; Male; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; RNA, Messenger; Xanthenes

The fear-potentiated startle paradigm has been characterized for drugs that act via ionotropic (NMDA and AMPA/kainate receptor) glutamate receptor mechanisms. Previous studies have shown that the potent systemically active mGlu2/3 receptor agonist, LY354740, effectively reduced the expression of fear-potentiated startle responses in rats. The present study examined the effects of LY354740 in a pre- versus post-fear conditioning paradigm and compared the effects to diazepam. Diazepam (0.3, 0.6, and 1.0 mg/kg ip) attenuated both pre- and post-fear conditioning startle responses in a dose-related manner. In contrast, LY354740 (0.03, 0.3, and 3.0 mg/kg ip) did not disrupt preconditioning startle responses at doses that attenuated post-fear conditioning responses. The benzodiazepine antagonist, flumazenil, at a dose (2 mg/kg sc) that did not alter fear-potentiated startle per se, selectively reversed suppression of fear responses to diazepam (0.6 mg/kg ip) while not affecting fear suppression induced by LY354740 (0.3 mg/kg ip). At a dose of 1 mg/kg ip, the mGlu2/3 receptor antagonist, LY341495, did not disrupt fear-enhanced startle per se, but completely reversed the postconditioning anxiolytic effects of LY354740 in this model. This dose of LY341495 had no effect on fear suppression by diazepam. These results demonstrate that fear suppression by diazepam and LY354740 involves different neuronal mechanisms. While diazepam acts via the facilitation of GABAergic transmission, LY354740 induces its actions via the glutamatergic system, specifically mGlu2/3 receptor activation. Furthermore, in contrast to disruption of fear conditioning as well as fear suppression by diazepam, LY354740 had selective effects on fear expression, suggesting anxiolytic actions without the associated memory impairment.

Topics: Acoustic Stimulation; Amino Acids; Animals; Anti-Anxiety Agents; Bridged Bicyclo Compounds; Diazepam; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Fear; Flumazenil; GABA Modulators; Male; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Reflex, Startle; Xanthenes

The nucleus accumbens (NAc) is an important cerebral area involved in reward and spatial memory (Pennartz et al., 1994), but little is known about synaptic plasticity in this region. Here, electron microscopy revealed that, in the NAc, metabotropic glutamate receptors 2/3 (mGlu2/3) immunostaining was essentially associated with axonal terminals and glial processes, whereas postsynaptic dendrites and neuronal cell bodies were unstained. Electrophysiological techniques in the NAc slice preparation demonstrated that activation of mGlu2/3 with synaptically released glutamate or specific exogenous agonist, such as LY354740 (200 nm, 10 min), induced long-term depression of excitatory synaptic transmission (mGlu2/3-LTD). Tetanic-LTD and pharmacological mGlu2/3-LTD occluded each other, suggesting common mechanisms. The mGlu2/3-LTD did not require synaptic activity but depended on the cAMP-protein kinase A cascade. Selective inhibition of P/Q-type Ca(2+) channels with omega-agatoxin-IVA occluded the expression of mGlu2/3-LTD, and, conversely, the inhibitory effects of omega-agatoxin-IVA were abolished during mGlu2/3-LTD. Thus, mGlu2/3 play an important role in the control of use-dependent synaptic plasticity at prelimbic cortex-NAc synapses: their activation causes a form of LTD mediated by the long-lasting reduction of P/Q-type Ca(2+)channels contribution to transmitter release.

Topics: Amino Acids; Animals; Bridged Bicyclo Compounds; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Channels, P-Type; Calcium Channels, Q-Type; Cyclic AMP-Dependent Protein Kinases; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Neural Inhibition; Neuronal Plasticity; Nucleus Accumbens; Patch-Clamp Techniques; Presynaptic Terminals; Receptors, Metabotropic Glutamate; Signal Transduction; Synapses; Synaptic Transmission; Xanthenes

1. Neurones in the superior colliculus (SC) respond to novel sensory stimuli and response habituation is a key feature of this. It is known that both ionotropic and metabotropic glutamate (mGlu) receptors participate in visual responses of superficial SC neurones. A feature of Group II and Group III mGlu receptors is that they may modulate specific neural pathways, possibly via presynaptic mechanisms. However, less is known about how this may relate to functions of systems in whole animals. We have therefore investigated whether these receptors affect specific attributes of visual responses in the superficial SC. 2. Recordings were made from visually responsive neurones in anaesthetised rats, and agonists and antagonists of Group II and III mGlu receptors were applied iontophoretically at the recording site. 3. We found that application of the Group III metabotropic glutamate receptor agonist L-2-amino-4-phosphonobutyric acid (L-AP4) produced an increase in visual response habituation, whilst Group III antagonists decreased habituation. These effects were independent of the response habituation mediated via GABA(B) receptors. In contrast, modulation of Group II mGlu receptors with the specific agonist LY354740 or the antagonist LY341495 did not affect response habituation, although these compounds did modulate visual responses. This suggests a specific role for Group III mGlu receptors in visual response habituation. 4. The magnitude of Group II effects was smaller during presentation of low contrast stimuli compared with high contrast stimuli. This suggests that activation of Group II receptors may be activity dependent and that these receptors can translate this into a functional effect in adapting to high contrast stimuli.

Topics: Amino Acids; Animals; Bridged Bicyclo Compounds; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Habituation, Psychophysiologic; Rats; Rats, Inbred Strains; Receptors, Metabotropic Glutamate; Superior Colliculi; Visual Perception; Xanthenes

From previous work, it appears that glutamate can activate the hypothalamic-pituitary-adrenocortical (HPA) axis by an interaction at either ionotopic or metabotropic (G-protein coupled) receptors. For example, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD), a metabotropic glutamate (mGlu) receptor agonist, has been shown to increase the levels of serum corticosterone in rats. The present study was undertaken to further characterize which of the mGlu receptors are substantially involved in control of the HPA axis. The group I mGlu receptor agonists, 3,5-dihydroxyphenylglycine (DHPG), 1S,3R-ACPD, and 2-chloro-5-hydroxyphenylglycine (CHPG) but not the inactive isomer 1R,3S-ACPD were found to dose-dependently increase serum corticosterone 1 h after intracerebroventricular (i.c.v.) injection in male rats. The relative potency, DHPG (EC50 = 520 nmol) > 1S,3R-ACPD (1.4 micromol) = CHPG (2.7 micromol) >> 1R,3S-ACPD (>> 3 micromol) is consistent with activation of group I (mGlu1/5) receptors. The effects of DHPG were long lasting with substantial elevations in corticosterone remaining for at least 3 h. In a similar manner, the group III mGlu receptor agonists, L-AP4 (4-phosphono-2-aminobutyric acid) and L-SOP (serine-O-phosphate), were found to increase serum corticosterone levels at 1 h. In contrast, the mGlu group II selective agonists LY354740 (10 mg/kg, i.p.) and subtype-selective doses of the group II antagonist LY341495 (1 mg/kg, i.p.) did not significantly elevate serum corticosterone. Given the group I agonists results, it was surprising to find that group I selective and mGlu1 selective antagonists given alone also increased serum corticosterone. As with the agonists, the rise in serum corticosterone with LY393675 (an mGlu1/5 antagonist, EC50 = 20 nmol, i.c.v.) and LY367385 (an mGlu1 antagonist, 325 nmol, i.c.v.) were dose-dependent and consistent with their relative affinity for the group I mGlu receptors. The selective mGlu5 antagonist MPEP [2-methyl-6-(phenylethylnyl)pyridine] increased serum corticosterone but only at high doses (> 30 mg/kg, i.p.). A model involving the high glutamatergic tone on GABAergic interneurons in the paraventricular nucleus of the hypothalamus is discussed as a possible explanation for these results.

Topics: Adrenalectomy; Adrenocorticotropic Hormone; Amino Acids; Animals; Benzoates; Bridged Bicyclo Compounds; Corticosterone; Cycloleucine; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Glycine; Male; Neuroprotective Agents; Paraventricular Hypothalamic Nucleus; Phenylacetates; Propionates; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Resorcinols; Xanthenes

The binding properties of [(3)H]-LY354740 were characterized on rat metabotropic glutamate receptors mGlu2 and mGlu3 expressed in Chinese hamster ovary (CHO) cells using Semliki Forest virus vectors. The saturation isotherm gave K(D) values of 20+/-5 and 53+/-8 nM and B(max) values of 474+/-161 and 667+/-89 fmol/mg protein for mGlu2 and mGlu3 receptors, respectively. NMDA, CaCl(2), DHPG and kainate were inactive up to 1 mM, whereas LY341495, DCG IV and ibotenate inhibited [(3)H]-LY354740 binding with similar potencies on both receptors. L-CCG I, L-AP4, L-AP5, LY354740 and 1S,3R-ACPD were 2- to 4-fold more potent inhibitors of [(3)H]-LY354740 binding to mGlu2 than mGlu3 receptors. However, MPPG and L-AP3 had a 6-fold and DTT a 28-fold preference for mGlu2 over mGlu3. ZnCl(2), at 10 mM, inhibited more than 70% of [(3)H]-LY354740 binding to mGlu2 receptors. At the same concentration it did not affect significantly [(3)H]-LY354740 binding to mGlu3 receptors. On the contrary, glutamate, quisqualate, EGLU and NAAG showed a 3-, 5-, 7- and 12-fold preference for mGlu3 over mGlu2. Finally, GTPgammaS, which partially inhibited the binding on mGlu2 receptors, was inactive to inhibit [(3)H]-LY354740 binding on mGlu3 receptors.

Topics: Amino Acids; Animals; Binding, Competitive; Bridged Bicyclo Compounds; Cell Membrane; Chlorides; CHO Cells; Cricetinae; DNA, Recombinant; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Gene Expression; Genetic Vectors; Glutamic Acid; Guanosine 5'-O-(3-Thiotriphosphate); Kinetics; Rats; Receptors, Metabotropic Glutamate; Semliki forest virus; Tritium; Xanthenes; Zinc Compounds

In prefrontal cortex, 5-hydroxytryptamine(2A) (5-HT(2A)) receptors have been linked to the action of hallucinogens and atypical antidepressant/antipsychotic drugs. Previously, we have shown in cortical layer V pyramidal cells that a nonselective metabotropic glutamate (mGlu) receptor agonist suppresses the induction of excitatory postsynaptic potentials/currents (EPSPs/EPSCs) via activation of 5-HT(2A) receptors. In this study, we tested the ability of the selective mGlu2/3 agonist (1S,2S,5R, 6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate (LY354740) and the selective mGlu2/3 antagonist 2S-2-amino-2-(1S, 2S-2-carboxycycloprop-1-yl)-3(xanthy-9-yl)propanoic acid (LY341495) to modulate serotonin(5-HT)-induced EPSPs and electrically evoked EPSPs by using intracellular recording from layer V pyramidal cells in medial prefrontal cortex. The mGlu2/3 antagonist LY341495 increased the frequency and amplitude of 5-HT-induced EPSCs, suggesting a role for mGlu2/3 receptors in mediating the action of endogenous glutamate on autoreceptors. Conversely, the mGlu2/3 agonist LY354740 was highly effective and potent (EC(50) = 89 nM) in suppressing glutamate release induced by 5-HT(2A) receptor activation in the medial prefrontal cortex, probably via a presynaptic mechanism. The mGlu2/3 antagonist LY341495 potently blocked the suppressant effect of LY354740 on 5-HT-induced EPSCs as well as electrically evoked early EPSPs. Autoradiography with the radioligands [(3)H]LY354740 and [(125)I](+/-)-1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane showsa striking overlap of the laminar distribution of mGlu2/3 and 5-HT(2A) receptors in the medial prefrontal cortex that is not apparent in other cortical regions. These findings suggest a close coupling between mGlu2/3 and 5-HT(2A) receptors in the prefrontal cortex that may be relevant for novel therapeutic approaches in the treatment of neuropsychiatric syndromes such as depression and schizophrenia.

Topics: Amino Acids; Animals; Autoradiography; Bridged Bicyclo Compounds; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Free Radical Scavengers; Glutamic Acid; Humans; In Vitro Techniques; Male; Membrane Potentials; Prefrontal Cortex; Protein Binding; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Receptors, Presynaptic; Serotonin; Xanthenes

Recent electrophysiological studies in our laboratory have demonstrated a physiological interaction between 5-HT(2A) and metabotropic glutamate2/3 (mGlu2/3) receptors in the medial prefrontal cortex. Several behavioral studies have found that phenethylamine hallucinogens with partial agonist activity at 5-HT(2A) receptors induce head shakes when directly administered into the medial prefrontal cortex. The purpose of the present experiments was to examine whether an interaction occurs between mGlu2/3 and 5-HT(2A) receptors on a behavioral level using head shakes induced by phenethylamine hallucinogens as a model of 5-HT(2A) receptor activation. Administration of the mGlu2/3 agonist LY354740 (0.3-10 mg/kg, ip) suppressed head shakes induced by the phenethylamine hallucinogen 1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Conversely, administration of the mGlu2/3 antagonist LY341495 (1 mg/kg, ip) enhanced the frequency of DOI-induced head shakes. Taken together, these results raise the possibility that the psychomimetic properties of hallucinogenic drugs may be mediated in part, via increased glutamate release following activation of 5-HT(2A) receptors.

Topics: Amino Acids; Amphetamines; Animals; Behavior, Animal; Bridged Bicyclo Compounds; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hallucinogens; Male; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Receptor, Serotonin, 5-HT2A; Receptors, Metabotropic Glutamate; Receptors, Serotonin; Xanthenes