6-methyl-2-(phenylethynyl)pyridine and 3-4-dihydroxyphenylglycol

Cancer-related fatigue (CRF) is a common burden in cancer patients and little is known about its underlying mechanism. The primary aim of this study was to identify gene signatures predictive of post-radiotherapy fatigue in prostate cancer patients. We employed Fisher Linear Discriminant Analysis (LDA) to identify predictive genes using whole genome microarray data from 36 men with prostate cancer. Ingenuity Pathway Analysis was used to determine functional networks of the predictive genes. Functional validation was performed using a T lymphocyte cell line, Jurkat E6.1. Cells were pretreated with metabotropic glutamate receptor 5 (mGluR5) agonist (DHPG), antagonist (MPEP), or control (PBS) for 20 min before irradiation at 8 Gy in a Mark-1 γ-irradiator. NF-κB activation was assessed using a NF-κB/Jurkat/GFP Transcriptional Reporter Cell Line. LDA achieved 83.3% accuracy in predicting post-radiotherapy fatigue. "Glutamate receptor signaling" was the most significant (p = 0.0002) pathway among the predictive genes. Functional validation using Jurkat cells revealed clustering of mGluR5 receptors as well as increased regulated on activation, normal T cell expressed and secreted (RANTES) production post irradiation in cells pretreated with DHPG, whereas inhibition of mGluR5 activity with MPEP decreased RANTES concentration after irradiation. DHPG pretreatment amplified irradiation-induced NF-κB activation suggesting a role of mGluR5 in modulating T cell activation after irradiation. These results suggest that mGluR5 signaling in T cells may play a key role in the development of chronic inflammation resulting in fatigue and contribute to individual differences in immune responses to radiation. Moreover, modulating mGluR5 provides a novel therapeutic option to treat CRF.

Topics: Aged; Fatigue; Genome-Wide Association Study; Humans; Jurkat Cells; Machine Learning; Male; Methoxyhydroxyphenylglycol; Middle Aged; NF-kappa B; Prostatic Neoplasms; Pyridines; Radiotherapy; Radiotherapy Dosage; Receptor, Metabotropic Glutamate 5; T-Lymphocytes; Transcriptome

Anhedonia is a core symptom of social defeat stress (SDS)-induced depression associated with the reward system. We previously reported that decreased membrane-bound AMPA-GluR1 in the reward system is associated with lipopolysaccharide-induced anhedonia-like symptoms. Since group I metabotropic glutamate receptor (mGluR) activation reduces the surface density of GluR1, we examined whether group I mGluR-dependent decrease in membrane-bound GluR1 in the reward system is involved in SDS-induced anhedonia-like symptoms. Mice exposed to SDS for 4 consecutive days had markedly decreased membrane-bound GluR1 and GluR2 in the prefrontal cortex (PFC) and membrane-bound GluR1 in the ventral midbrain (VM) along with lower sucrose preference (SP). Intra-PFC injection of the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG; 100 μmol) demonstrated decrease in membrane-bound GluR1 and GluR2 in the PFC 2 and 24 h and membrane-bound GluR1 in the VM 24 h after injection. Moreover, intra-PFC injection of DHPG decreased SP only in the second 24-h (24-48 h) period. Conversely, intra-VM injection of DHPG decreased SP in both the first and second 24-h period and decreased membrane-bound GluR1 in the VM 2 and 24 h after injection. Pre-treatment with the mGluR1 antagonist JNJ16259685 (30 mg/kg, subcutaneous) prevented SDS-decreased SP and membrane-bound GluR1 in the VM. The mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP; 10 mg/kg, subcutaneous) prevented SDS-induced decrease in membrane-bound GluR1 and GluR2 in the PFC, whereas MPEP did not affect SDS-induced decrease in SP and membrane-bound GluR1 in the VM. These results suggest that mGluR1-mediated decrease in membrane-bound GluR1 in VM is involved in SDS-induced anhedonia-like symptoms.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Anhedonia; Animals; Glycine; Male; Mesencephalon; Methoxyhydroxyphenylglycol; Mice; Prefrontal Cortex; Pyridines; Receptors, AMPA; Receptors, Metabotropic Glutamate; Resorcinols; Social Dominance; Stress, Psychological

Disrupted metabotropic glutamate receptor 5 (mGluR5) signaling is implicated in many neuropsychiatric disorders, including autism spectrum disorder, found in fragile X syndrome (FXS). Here we report that intracellular calcium responses to the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) are augmented, and calcium-dependent mGluR5-mediated mechanisms alter the differentiation of neural progenitors in neurospheres derived from human induced pluripotent FXS stem cells and the brains of mouse model of FXS. Treatment with the mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) prevents an abnormal clustering of DHPG-responsive cells that are responsive to activation of ionotropic receptors in mouse FXS neurospheres. MPEP also corrects morphological defects of differentiated cells and enhanced migration of neuron-like cells in mouse FXS neurospheres. Unlike in mouse neurospheres, MPEP increases the differentiation of DHPG-responsive radial glial cells as well as the subpopulation of cells responsive to both DHPG and activation of ionotropic receptors in human neurospheres. However, MPEP normalizes the FXS-specific increase in the differentiation of cells responsive only to N-methyl-d-aspartate (NMDA) present in human neurospheres. Exposure to MPEP prevents the accumulation of intermediate basal progenitors in embryonic FXS mouse brain suggesting that rescue effects of GluR5 antagonist are progenitor type-dependent and species-specific differences of basal progenitors may modify effects of MPEP on the cortical development. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419-437, 2017.

Topics: Animals; Cell Differentiation; Cerebral Cortex; Disease Models, Animal; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; Fragile X Syndrome; Humans; Male; Methoxyhydroxyphenylglycol; Mice; Mice, Inbred C57BL; Mice, Knockout; N-Methylaspartate; Neural Stem Cells; Pyridines; Receptor, Metabotropic Glutamate 5

Angelman syndrome (AS) is caused by the loss of Ube3A, an ubiquitin ligase that commits specific proteins to proteasomal degradation. How this defect causes autism and other pathological phenotypes associated with AS is unknown. Long-term depression (LTD) of excitatory synaptic transmission mediated by type 5 metabotropic glutamate (mGlu5) receptors was enhanced in hippocampal slices of Ube3A(m-/p+) mice, which model AS. No changes were found in NMDA-dependent LTD induced by low-frequency stimulation. mGlu5 receptor-dependent LTD in AS mice was sensitive to the protein synthesis inhibitor anisomycin, and relied on the same signaling pathways as in wild-type mice, e.g., the mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycine pathway, and protein tyrosine phosphatase. Neither the stimulation of MAPK and PI3K nor the increase in Arc (activity-regulated cytoskeleton-associated protein) levels in response to mGlu5 receptor activation were abnormal in hippocampal slices from AS mice compared with wild-type mice. mGlu5 receptor expression and mGlu1/5 receptor-mediated polyphosphoinositide hydrolysis were also unchanged in the hippocampus of AS mice. In contrast, AS mice showed a reduced expression of the short Homer protein isoform Homer 1a, and an increased coupling of mGlu5 receptors to Homer 1b/c proteins in the hippocampus. These findings support the link between Homer proteins and monogenic autism, and lay the groundwork for the use of mGlu5 receptor antagonists in AS.

Topics: Angelman Syndrome; Animals; Carrier Proteins; Disease Models, Animal; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Hemizygote; Hippocampus; Homer Scaffolding Proteins; Immunosuppressive Agents; In Vitro Techniques; Long-Term Synaptic Depression; Methoxyhydroxyphenylglycol; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitogen-Activated Protein Kinase Kinases; Pyridines; Receptor, Metabotropic Glutamate 5; Signal Transduction; Sirolimus; Ubiquitin-Protein Ligases

Alterations of the glutamatergic system have been implicated in the pathophysiology and treatment of major depression. In order to investigate the expression and function of mGlu5 receptors in an animal model for treatment-resistant depression we used rats bred for congenital learned helplessness (cLH) and the control strain, bred for resistance against inescapable stress, congenitally. not learned helpless rats (cNLH). Western blot analysis showed an increased expression of mGlu5 (but not mGlu1a) receptors in the hippocampus of cLH rats, as compared with control cNLH rats. We also examined mGlu1/5 receptor signaling by in vivo measurement of DHPG-stimulated polyphosphoinositides hydrolysis. Stimulation of (3)H-inositolmonophosphate formation induced by i.c.v. injection of DHPG was enhanced by about 50% in the hippocampus of cLH rats. Correspondingly, DHPG-induced long-term depression (LTD) at Schaffer collateral/CA1 pyramidal cell synapses was amplified in hippocampal slices of cLH rats, whereas LTD induced by low frequency stimulation of the Schaffer collaterals did not change. Moreover, these effects were associated with decreased basal dendritic spine density of CA1 pyramidal cell in cLH rats. These data raise the attractive possibility that changes in the expression and function of mGlu5 receptors in the hippocampus might underlie the changes in synaptic plasticity associated with the depressive-like phenotype of cLH rats. However, chronic treatment of cLH rats with MPEP did not reverse learned helplessness, indicating that the enhanced mGlu5 receptor function is not the only player in the behavioral phenotype of this genetic model of depression. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Topics: Animals; CA1 Region, Hippocampal; CA3 Region, Hippocampal; Dendritic Spines; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; Helplessness, Learned; Hydrolysis; Long-Term Synaptic Depression; Male; Methoxyhydroxyphenylglycol; Phosphatidylinositol Phosphates; Pyramidal Cells; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Synapses

Metabotropic glutamate (mGlu) receptors modulate pain from within the midbrain periaqueductal grey (PAG). In the present study, the postsynaptic mGlu receptor mediated effects on rat PAG neurons were examined using whole-cell patch-clamp recordings in brain slices. The selective group I agonist DHPG (10 μM) produced an inward current in all PAG neurons tested which was associated with a near parallel shift in the current-voltage relationship. By contrast, the group II and III mGlu receptor agonists DCG-IV (1 μM) and l-AP4 (3 μM) produced an outward current in only 10-20% of PAG neurons tested. The DHPG induced current was concentration dependent (EC(50) = 1.4 μM), was reduced by the mGlu1 antagonist CPCCOEt (100 μM), and was further reduced by CPCCOEt in combination with the mGlu5 antagonist MPEP (10 μM). The glutamate transport blocker TBOA (30 μM) also produced an inward current, however, this was largely abolished by CNQX (10 μM) plus AP5 (25 μM). Slow EPSCs were evoked following train, but not single shock stimulation, which were enhanced by TBOA (30 μM). The TBOA enhancement of slow EPSCs was abolished by MPEP plus CPCCOEt. These findings indicate that endogenously released glutamate, under conditions in which neurotransmitter spill-over is enhanced, activates group I mGlu receptors to produce excitatory currents within PAG. Thus, postsynaptic group I mGlu receptors have the potential to directly modulate the analgesic, behavioural and autonomic functions of the PAG. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Aminobutyrates; Animals; Aspartic Acid; Chromones; Cyclopropanes; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; Glycine; Male; Membrane Potentials; Methoxyhydroxyphenylglycol; Neurons; Periaqueductal Gray; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate

Anomalies in prefrontal cortex (PFC) function are posited to underpin difficulties in learning to suppress drug-seeking behavior during abstinence. Because group 1 metabotropic glutamate receptors (mGluRs) regulate drug-related learning, we assayed the consequences of extended access to intravenous cocaine (6 h/d; 0.25 mg/infusion for 10 d) on the PFC expression of group 1 mGluRs and the relevance of observed changes for cocaine seeking. After protracted withdrawal, cocaine-experienced animals exhibited a time-dependent intensification of cue-induced cocaine-seeking behavior and an impaired extinction of this behavior. These behavioral phenomena were associated with a time-dependent reduction in mGluR1/5 expression within ventromedial PFC (vmPFC) of cocaine-experienced animals exposed to extinction testing but not in untested ones. Interestingly, pharmacological manipulations of vmPFC mGluR1/5 produced no immediate effects on cue-induced cocaine-seeking behavior but produced residual effects on a subsequent test for cocaine seeking. At 3 d withdrawal, cocaine-experienced rats infused intra-vmPFC with mGluR1/5 antagonists, either before or after an initial test for cocaine seeking, persisted in their cocaine seeking akin to cocaine-experienced rats in protracted withdrawal. Conversely, cocaine-experienced rats infused with an mGluR1/5 agonist before the initial test for cocaine-seeking at 30 d withdrawal exhibited a facilitation of extinction learning. These data indicate that cue-elicited deficits in vmPFC group 1 mGluR function mediate resistance to extinction during protracted withdrawal from a history of extensive cocaine self-administration and pose pharmacological stimulation of these receptors as a potential approach to facilitate learned suppression of drug-seeking behavior that may aid drug abstinence.

Topics: Animals; Blotting, Western; Cocaine-Related Disorders; Conditioning, Operant; Cues; Excitatory Amino Acid Antagonists; Extinction, Psychological; Injections; Male; Methoxyhydroxyphenylglycol; Prefrontal Cortex; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Recurrence; Self Administration; Substance Withdrawal Syndrome

Acute stress reduces pain sensitivity by engaging an endocannabinoid signaling circuit in the midbrain. The neural mechanisms governing this process and molecular identity of the endocannabinoid substance(s) involved are unknown. We combined behavior, pharmacology, immunohistochemistry, RNA interference, quantitative RT-PCR, enzyme assays, and lipidomic analyses of endocannabinoid content to uncover the role of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) in controlling pain sensitivity in vivo. Here, we show that footshock stress produces antinociception in rats by activating type 5 metabotropic glutamate receptors (mGlu(5)) in the dorsolateral periaqueductal gray (dlPAG) and mobilizing 2-AG. Stimulation of mGlu(5) in the dlPAG with DHPG [(S)-3,5-dihydroxyphenylglycine] triggered 2-AG formation and enhanced stress-dependent antinociception through a mechanism dependent upon both postsynaptic diacylglycerol lipase (DGL) activity, which releases 2-AG, and presynaptic CB(1) cannabinoid receptors. Pharmacological blockade of DGL activity in the dlPAG with RHC80267 [1,6-bis(cyclohexyloximinocarbonylamino)hexane] and (-)-tetrahydrolipstatin (THL), which inhibit activity of DGL-α and DGL-β isoforms, suppressed stress-induced antinociception. Inhibition of DGL activity in the dlPAG with THL selectively decreased accumulation of 2-AG without altering levels of anandamide. The putative 2-AG-synthesizing enzyme DGL-α colocalized with mGlu(5) at postsynaptic sites of the dlPAG, whereas CB(1) was confined to presynaptic terminals, consistent with a role for 2-AG as a retrograde signaling messenger. Finally, virally mediated silencing of DGL-α, but not DGL-β, transcription in the dlPAG mimicked effects of DGL inhibition in suppressing both endocannabinoid-mediated stress antinociception and 2-AG formation. The results indicate that activation of the postsynaptic mGlu(5)-DGL-α cascade triggers retrograde 2-AG signaling in vivo. This pathway is required for endocannabinoid-mediated stress-induced analgesia.

Topics: Analgesia; Analysis of Variance; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cyclohexanones; Dose-Response Relationship, Drug; Electroconvulsive Therapy; Endocannabinoids; Excitatory Amino Acid Antagonists; Glycerides; Lipoprotein Lipase; Male; Methoxyhydroxyphenylglycol; Mice; Microscopy, Immunoelectron; Pain; Periaqueductal Gray; Piperidines; Protease Inhibitors; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Rimonabant; RNA, Messenger; RNA, Small Interfering; Synapses; Tandem Mass Spectrometry

The globus pallidus plays a critical role in movement regulation. Morphological studies have shown that group I mGluRs including mGluR1 and mGluR5 are expressed in the globus pallidus. Up to now, little is known about the in vivo electrophysiological effects of group I mGluRs on the pallidal neurons. The present study investigated the electrophysiological effects of group I mGluRs on the firing rate of pallidal neurons in anesthetized rats. Single unit in vivo extracellular recordings showed that micropressure ejection of group I mGluRs agonist, 3,5-dihydroxyphenylglycine (DHPG), increased the spontaneous firing rate of pallidal neurons. DHPG-induced excitation could be blocked by mGluR1 antagonist, (S)-(+)-α-amino-4-carboxy-2-methylb-enzeneacetic acid (LY367385), but not mGluR5 antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP). LY367385 alone had no effect but MPEP alone increased the excitability of pallidal neurons. Unilateral microinjection of DHPG into the globus pallidus induced a contralateral dystonic posturing in the presence of systemic haloperidol administration and this effect could be blocked by LY367385 but not MPEP. The present in vivo electrophysiological and behavioral studies indicate that group I mGluRs could produce excitatory effect on pallidal neurons via mGluR1, and blockade of mGluR5 also has an excitatory effect on pallidal neurons. Our findings suggest that the effects of globus pallidus in movement regulation is partly mediated by group I mGluRs.

Topics: Action Potentials; Animals; Benzoates; Dopamine Antagonists; Drug Interactions; Excitatory Amino Acid Antagonists; Globus Pallidus; Glycine; Haloperidol; Male; Methoxyhydroxyphenylglycol; Movement; Neurons; Pyridines; Rats; Rats, Wistar; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate

Abnormal dendritic spine morphology is a significant neuroanatomical defect in fragile X mental retardation. It has been suggested that overactive group 1 metabotropic glutamate receptor (mGlu) signaling is associated with the spine dysmorphology occurring in fragile X syndrome (FXS). Thus, group 1 mGlu became a new therapeutic target for the treatment of FXS.. The purpose of this study was to identify the effect of inhibition of mGlu signaling in FXS.. We observed the changes in dendritic spines after pharmacological modulation of mGlu signaling in an Fmr1 knockout (KO) mouse model.. The activation of group 1 mGlu resulted in elongation of dendritic spines in the cultured neurons derived from Fmr1 KO mice and wild-type (WT) mice. Antagonism of group 1 mGlu reduced the average spine length of Fmr1 KO neurons. Furthermore, systemic administration of the selective group 1 mGlu5 antagonist 2-methyl-6-phenylethynyl pyridine (MPEP) reduced the average spine length and density in the cortical neurons of Fmr1 KO mice at developmental age. For the adult mice, MPEP administration was less effective for the restoration of spine length. The percentage of immature spines showed a similar reduction in parallel to the changes of spine length. Temporary MPEP intervention with single-dose treatment did not show any effect.. These results show that MPEP administration could partially rescue the morphological deficits of dendritic spines in Fmr1 KO mice at developmental age.

Topics: Age Factors; Analysis of Variance; Animals; Animals, Newborn; Benzopyrans; Cells, Cultured; Dendritic Spines; Disease Models, Animal; Fragile X Mental Retardation Protein; Fragile X Syndrome; Hippocampus; Methoxyhydroxyphenylglycol; Mice; Mice, Inbred C57BL; Mice, Knockout; Neural Inhibition; Neurons; Pyridines; Receptors, Metabotropic Glutamate; Signal Transduction; Silver Staining

Recently, we demonstrated that mRNA for the neuronal glutamate transporter, excitatory amino acid carrier 1 (EAAC1), is found in dendrites of hippocampal neurons in culture and in dendrites of hippocampal pyramidal cells after pilocarpine-induced status epilepticus (SE). We also showed that SE increased the levels of EAAC1 mRNA ~15-fold in synaptoneurosomes. In this study, the effects of SE on the distribution EAAC1 protein in hippocampus were examined. In addition, the effects of Group 1 mGluR receptor activation on the levels of EAAC1 protein were examined in synaptoneurosomes prepared from sham control animals and from animals that experience pilocarpine-induced SE. We find that EAAC1 immunoreactivity increases in pyramidal cells of the hippocampus after 3 h of SE. In addition, the group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine (DHPG), caused an increase in EAAC1 protein levels in hippocampal synaptoneurosomes; this effect of DHPG was much larger (~3- to 5-fold) after 3 h of SE. The DHPG-induced increases in EAAC1 protein were blocked by two different inhibitors of translation but not by inhibitors of transcription. mGluR1 or mGluR5 antagonists completely blocked the DHPG-induced increases in EAAC1 protein. DHPG also increased the levels of glutamate receptor 2/3 protein, but this effect was not altered by SE. The DHPG-induced increase in EAAC1 protein was blocked by an inhibitor of the mammalian target of rapamycin or an inhibitor of extracellular signal-regulated kinase. These studies provide the first evidence EAAC1 translation can be regulated, and they show that regulated translation of EAAC1 is up-regulated after SE.

Topics: Animals; Blotting, Western; Convulsants; Dendrites; Dose-Response Relationship, Drug; Excitatory Amino Acid Transporter 3; Extracellular Signal-Regulated MAP Kinases; Fluorescent Antibody Technique; Hippocampus; Male; Methoxyhydroxyphenylglycol; Neurons; Pilocarpine; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; RNA, Messenger; Status Epilepticus; Synaptosomes; TOR Serine-Threonine Kinases

Astrocytes show a complex structural and physiological interplay with neurons and respond to neuronal activation in vitro and in vivo with intracellular calcium elevations. These calcium changes enable astrocytes to modulate synaptic transmission and plasticity through various mechanisms. However, the response pattern of astrocytes to single neuronal depolarization events still remains unresolved. This information is critical for fully understanding the coordinated network of neuron-glial signaling in the brain. To address this, we developed a system to map astrocyte calcium responses along apical dendrites of CA1 pyramidal neurons in hippocampal slices using single-neuron stimulation with channelrhodopsin-2. This technique allowed selective neuronal depolarization without invasive manipulations known to alter calcium levels in astrocytes. Light-evoked neuronal depolarization was elicited and calcium events in surrounding astrocytes were monitored using the calcium-sensitive dye Calcium Orange. Stimulation of single neurons caused calcium responses in populations of astrocytes along the apical axis of CA1 cell dendrites. Calcium responses included single events that were synchronized with neuronal stimulation and poststimulus changes in calcium event frequency, both of which were modulated by glutamatergic and purinergic signaling. Individual astrocytes near CA1 cells showed low ability to respond to repeated neuronal depolarization events. However, the response of the surrounding astrocyte population was remarkably accurate. Interestingly, the reliability of responses was graded with respect to astrocyte location along the CA1 cell dendrite, with astrocytes residing in the primary dendrite subregion being most responsive. This study provides a new perspective on the dynamic response property of astrocyte ensembles to neuronal activity.

Topics: Action Potentials; Analysis of Variance; Animals; Animals, Newborn; Aspartic Acid; Astrocytes; Bacterial Proteins; Benzoates; Benzoxazines; Calcium; Calcium Channel Blockers; Carbenoxolone; Channelrhodopsins; Electric Stimulation; Excitatory Amino Acid Antagonists; Female; Glial Fibrillary Acidic Protein; Glycine; Green Fluorescent Proteins; Hippocampus; In Vitro Techniques; Luminescent Proteins; Male; Methoxyhydroxyphenylglycol; Mice; Mice, Inbred C57BL; Models, Biological; Morpholines; Naphthalenes; Neurons; Patch-Clamp Techniques; Peptide Fragments; Phosphopyruvate Hydratase; Photic Stimulation; Piperidines; Pyrazoles; Pyridines; Sodium Channel Blockers; Statistics, Nonparametric; Tetanus Toxin; Tetrodotoxin; Transduction, Genetic

The NMDA (N-methyl-D-aspartate)-receptor is fundamentally involved in cognitive functions. Recent studies demonstrated a functional interaction between the metabotropic glutamate receptor 5 (mGlu(5) receptor) and the NMDA-receptor in neurons. In rat hippocampal slices, it was shown that activation of mGlu(5) receptor by a positive modulator in the presence of a subthreshold agonist concentration potentiated NMDA-receptor mediated currents and phosphorylation of intracellular signalling proteins. In the present study, we investigated the functional interaction of mGlu(5) receptor and NMDA-receptor by the selective mGlu(5) receptor positive modulator ADX-47273 in-vitro and in-vivo. In rat primary neurons, this compound potentiated Ca(2+) mobilization in the presence of a subthreshold concentration of the mGluR(1/5) agonist DHPG (0.3 microM) with an EC(50) of 0.28+/-0.05 microM. NMDA-induced Ca(2+)-mobilization in primary neurons could be potentiated when neurons were pre-stimulated with 1 microM ADX-47273 in the presence of 0.3 microM DHPG. The specific mGlu(5) receptor antagonist MPEP and the Src-family kinase inhibitor PP2 blocked this potentiation demonstrating the functional interaction of the NMDA-receptor and mGlu(5) receptor in neurons. Furthermore, ADX-47273 elicited an enhancement of NMDA-receptor dependent long-term potentiation in rat hippocampal slices that could be reversed by MPEP. After intraperitoneal administration to rats, ADX-47273 showed a dose-dependent reduction of NMDA-receptor antagonist (ketamine) induced hyperlocomotion, supporting the mechanistic interaction of the NMDA-receptor and mGlu(5) receptor in-vivo. In conclusion, these findings further support the idea of a functional interaction between the mGlu(5) receptor and NMDA-receptor, which may provide a pharmacological strategy for addressing CNS diseases with cognitive impairments linked to NMDA-receptor hypofunction.

Topics: Allosteric Regulation; Animals; Calcium; Cognition; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Hippocampus; Injections, Intraperitoneal; Male; Methoxyhydroxyphenylglycol; Neurons; Oxadiazoles; Phosphorylation; Piperidines; Pyridines; Rats; Rats, Wistar; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; src-Family Kinases

The central nucleus of the amygdala (CeA) has been identified as a site of nociceptive processing important for sensitization induced by peripheral injury. However, the cellular signaling components underlying this function remain unknown. Here, we identify metabotropic glutamate receptor 5 (mGluR5) as an integral component of nociceptive processing in the CeA. Pharmacological activation of mGluRs with (R,S)-3,5-dihydroxyphenylglycine (DHPG) in the CeA of mice is sufficient to induce peripheral hypersensitivity in the absence of injury. DHPG-induced peripheral hypersensitivity is reduced via pharmacological blockade of mGluR5 or genetic disruption of mGluR5. Furthermore, pharmacological blockade or conditional deletion of mGluR5 in the CeA abrogates inflammation-induced hypersensitivity, demonstrating the necessity of mGluR5 in CeA-mediated pain modulation. Moreover, we demonstrate that phosphorylation of extracellular-signal regulated kinase 1/2 (ERK1/2) is downstream of mGluR5 activation in the CeA and is necessary for the full expression of peripheral inflammation-induced behavioral sensitization. Finally, we present evidence of right hemispheric lateralization of mGluR5 modulation of amygdalar nociceptive processing. We demonstrate that unilateral pharmacological activation of mGluR5 in the CeA produces distinct behavioral responses depending on whether the right or left amygdala is injected. We also demonstrate significantly higher levels of mGluR5 expression in the right amygdala compared with the left under baseline conditions, suggesting a potential mechanism for right hemispheric lateralization of amygdala function in pain processing. Together, these results establish an integral role for mGluR5 and ERK1/2 in nociceptive processing in the CeA.

Topics: Amygdala; Analysis of Variance; Animals; Butadienes; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Formaldehyde; Functional Laterality; Gene Expression Regulation; Green Fluorescent Proteins; Hyperalgesia; Methoxyhydroxyphenylglycol; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogen-Activated Protein Kinase 3; Nitriles; Pain; Pain Measurement; Pyridines; Receptors, Glucocorticoid; Receptors, Kainic Acid

The expression of Arc is tightly coupled to synaptic activities. Recent studies suggested the functional relevance of Arc translation in group I metabotropic glutamate receptor (mGluR)-mediated long-term depression. The present study investigated the transcription-dependent changes of Arc in response to the activation of group I mGluR by (R,S)-3,5-dihydroxyphenylglycine (DHPG) in cultured cortical neurons. The increase in Arc mRNA did not require de novo protein synthesis, indicating that Arc is an immediate early gene upon DHPG stimulation. We further examined the major pathways involved in group I mGluR signaling, and found that DHPG-induced Arc up-regulation depended on CaMK, PLC, and ERK1/2 activity. Moreover, the activity of NMDA receptors, but not l-type voltage gated calcium channels (l-VGCC), was required for Arc transcription. Interestingly, blocking CaMK, PLC, and NMDAR, but not l-VGCC, suppressed DHPG-stimulated ERK1/2 activation. These data suggest the central role of ERK1/2 in group I mGluR-mediated Arc transcription.

Topics: Animals; Cells, Cultured; Cerebral Cortex; CREB-Binding Protein; Cytoskeletal Proteins; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Extracellular Signal-Regulated MAP Kinases; Methoxyhydroxyphenylglycol; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Neurons; Proto-Oncogene Proteins c-fos; Pyridines; Receptors, Metabotropic Glutamate; RNA, Messenger; Signal Transduction; Time Factors; Up-Regulation

Serotonin and norepinephrine, in addition to direct postsynaptic excitatory effects, also enhances glutamate release onto layer V pyramidal cells of the prefrontal cortex/neocortex via G(q/11)-coupled 5-hydroxytryptamine(2A) (5-HT(2A)) and alpha(1)-adrenergic receptors, respectively. Therefore, the present study was designed to test whether a metabotropic glutamate (mGlu) receptor subtype also coupled to G(q/11)-proteins, the mGlu5 receptor, also induces EPSCs when recording from layer V cortical pyramidal cells of the rat medial prefrontal cortex (mPFC). The mGlu1/5 receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG) induces EPSCs at a similar frequency as a near-maximally effective 5-HT concentration. The mGlu5 receptor negative allosteric modulator 2-methyl-6-(phenylethynyl)pyridine (MPEP, 300 nM) potently blocked DHPG-induced EPSCs. Previous work has suggested that activation of 5-HT(2A) and OX2 receptors induces glutamate release, while mGlu2, mGlu4, and mGlu8 receptor activation suppress transmitter release from thalamocortical terminals. Taken together with past results, these findings suggest that mGlu2, mGlu4, mGlu5 and mGlu8 may all act to modulate glutamate release from afferents impinging on layer V pyramidal cells of the mPFC. These findings further suggest that monoamines, neuropeptides and glutamate itself all enhance the excitability of prefrontal cortical output cells indirectly via modulation of glutamate release.

Topics: Animals; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Male; Methoxyhydroxyphenylglycol; Organ Culture Techniques; Patch-Clamp Techniques; Prefrontal Cortex; Pyramidal Cells; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Synapses; Synaptic Transmission

Synaptic plasticity in inhibitory interneurons is essential to maintain a proper equilibrium between excitation and inhibition in hippocampal network. Recent studies showed that theta-burst-induced long-term potentiation (LTP) at excitatory synapses of oriens/alveus (O/A) interneurons in CA1 hippocampal region required the activation of metabotropic glutamate receptor (mGluR) 1. However these interneurons also express mGluR5 and the contribution of this receptor subtype in interneuron synaptic plasticity remains unexplored. We combined pharmacological and transgenic approaches to examine the relative contribution of mGluR1/5 in LTP at excitatory synapses on O/A interneurons. Bath-application of the selective mGluR1/5 agonist (s)-3,5-dihydroxyphenylglycine (DHPG) induced LTP of compound excitatory postsynaptic potentials. DHPG-induced LTP was not prevented by application of either mGluR1 or mGluR5 antagonists, was still present in mGluR1 knockout mice, but was blocked by co-application of both antagonists. These results indicate that LTP can be induced at O/A interneuron synapses by either mGluR1 or mGluR5 activation. As previously reported for mGluR1-dependent LTP, the mGluR5-dependent LTP was independent of N-methyl-d-aspartate receptors. Pairing DHPG application with postsynaptic depolarization induced mGluR1- and mGluR5-dependent LTP of minimally-evoked excitatory postsynaptic currents, which were composed of calcium-permeable AMPA receptor and presynaptically modulated by group II mGluRs, hence confirming that both forms of LTP occurred directly at interneuron excitatory synapses. These findings uncover a new mGluR5-dependent form of LTP at O/A interneuron synapses and indicate that activation of mGluR1 or mGluR5 is sufficient to induce LTP at these synapses. Thus, a rich repertoire of adaptive changes may take place at these interneuron synapses to regulate hippocampal feedback inhibition.

Topics: Animals; Benzoates; Electrophysiology; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glycine; Hippocampus; Interneurons; Long-Term Potentiation; Methoxyhydroxyphenylglycol; Mice; Mice, Knockout; Patch-Clamp Techniques; Pyridines; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Synapses

Endocannabinoids released from the postsynaptic neuronal membrane can activate presynaptic CB1 receptors and inhibit neurotransmitter release. In hippocampal slices, depolarization of the CA1 pyramidal neurons elicits an endocannabinoid-mediated inhibition of gamma-aminobutyric acid release known as depolarization-induced suppression of inhibition (DSI). Using the highly reduced neuron/synaptic bouton preparation from the CA1 region of hippocampus, we have begun to examine endocannabinoid-dependent short-term depression (STD) of inhibitory synaptic transmission under well-controlled physiological and pharmacological conditions in an environment free of other cells. Application of the CB1 synthetic agonist WIN55212-2 and endogenous cannabinoids 2-AG and anandamide produced a decrease in spontaneous inhibitory postsynaptic current (sIPSC) frequency and amplitude, indicating the presence of CB1 receptors at synapses in this preparation. Endocannabinoid-dependent STD is different from DSI found in hippocampal slices and the neuron/bouton preparation from basolateral amygdala (BLA) since depolarization alone was not sufficient to induce suppression of sIPSCs. However, concurrent application of the metabotropic glutamate receptor (mGluR) agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) and postsynaptic depolarization resulted in a transient (30-50 s) decrease in sIPSC frequency and amplitude. Application of DHPG alone had no effect on sIPSCs. The depolarization/DHPG-induced STD was blocked by the CB1 antagonist SR141716A and the mGluR5 antagonist MPEP and was sensitive to intracellular calcium concentration. Comparing the present findings with earlier work in hippocampal slices and BLA, it appears that endocannabinoid release is less robust in isolated hippocampal neurons.

Topics: Animals; Animals, Newborn; Arachidonic Acids; Benzoxazines; Cannabinoid Receptor Modulators; Drug Interactions; Endocannabinoids; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Glycerides; Hippocampus; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Methoxyhydroxyphenylglycol; Morpholines; Naphthalenes; Neurons; Patch-Clamp Techniques; Piperidines; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Rimonabant; Synapses; Synapsins

Group I mGlu receptors have been implicated in the control of brain dopamine release. However, the receptor subtype involved and the precise site of action have not been determined. In this study we show that (R,S)3,5-dihydroxyphenylglycine (DHPG; 6 and 60 nmol ICV), a selective group I mGlu receptor agonist, raised extracellular dopamine respectively by 176% and 243% of basal values in the medial prefrontal cortex as assessed by in vivo microdialysis in conscious rats. (R,S)2-chloro-5-hydroxyphenylglycine (60 nmol ICV), a selective mGlu5 receptor agonist, raised extracellular dopamine by 396% of basal values. Intra-VTA DHPG (0.6-6 nmol) mimicked ICV injection whereas intracortical infusion (1-1000 micromol/L) had no effect. DHPG-induced rise of extracellular dopamine was reversed by tetrodotoxin and by the selective mGlu1 and mGlu5 receptor antagonists 7(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate (CPCCOEt) and 2-methyl-6-(phenylethynyl)pyridine (MPEP) either ICV or into the ventrotegmental area (VTA), suggesting that neuronal release and both mGlu1 and mGlu5 receptors were involved. These results support the existence of functional mGlu1 and mGlu5 receptors in the VTA regulating the release of dopamine in the medial prefrontal cortex.

Topics: Animals; Chromatography, High Pressure Liquid; Chromones; Dopamine; Drug Interactions; Electrochemistry; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Extracellular Fluid; Male; Methoxyhydroxyphenylglycol; Microdialysis; Prefrontal Cortex; Pyridines; Rats; Receptors, Metabotropic Glutamate; Ventral Tegmental Area

We previously demonstrated that peripherally located N-methyl-D-aspartic acid (NMDA) receptors contribute to acute muscle nociception and the development of chronic muscular hyperalgesia. In the present study, we investigated the potential role of peripheral group I metabotropic glutamate receptors (mGluRs 1/5) in the development of muscular hypersensitivity to mechanical stimulation, and attempted to elucidate intracellular signaling mechanisms associated with the mGluR activation in male Sprague-Dawley rats. First, our Western blot analyses revealed that mGluR 5 protein, but not mGluR 1 protein, is reliably detected in trigeminal ganglia and the masseter nerve. Subsequent behavioral studies demonstrated that the group I mGluR agonist, R,S-3,5-dihydroxyphenylglycol (DHPG), significantly decreased the mechanical threshold to noxious stimulation of the masseter, and that the DHPG-induced mechanical hypersensitivity can be effectively prevented by pretreatment of the masseter with 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), a selective mGluR 5 antagonist, but not by 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), a selective mGluR 1 antagonist. Moreover, the DHPG-induced mechanical hypersensitivity was significantly blocked by inhibiting either the alpha or epsilon isoform of protein kinase C (PKC). Collectively, these data provide evidence that peripherally located mGluR 5 may play an important role in the development of masseter hypersensitivity, and that PKC activation is required for the modulatory effect of peripheral mGluR 5 in the craniofacial muscle tissue. Thus, selective targeting of peripheral mGluR 5 and PKCalpha, as well as PKCepsilon, might serve as an effective therapeutic strategy in the management of chronic muscle pain conditions, such as temporomandibular disorders.

Topics: Analysis of Variance; Animals; Area Under Curve; Behavior, Animal; Chromones; Dose-Response Relationship, Drug; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Hyperalgesia; Male; Masseter Muscle; Methoxyhydroxyphenylglycol; Pain Threshold; Physical Stimulation; Protein Kinase C; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate

Fragile X syndrome is a common form of inherited mental retardation and is caused by loss of fragile X mental retardation protein (FMRP), a selective RNA-binding protein that influences the translation of target messages. Here, we identify protein phosphatase 2A (PP2A) as an FMRP phosphatase and report rapid FMRP dephosphorylation after immediate group I metabotropic glutamate receptor (mGluR) stimulation (<1 min) in neurons caused by enhanced PP2A enzymatic activity. In contrast, extended mGluR activation (1-5 min) resulted in mammalian target of rapamycin (mTOR)-mediated PP2A suppression and FMRP rephosphorylation. These activity-dependent changes in FMRP phosphorylation were also observed in dendrites and showed a temporal correlation with the translational profile of select FMRP target transcripts. Collectively, these data reveal an immediate-early signaling pathway linking group I mGluR activity to rapid FMRP phosphorylation dynamics mediated by mTOR and PP2A.

Topics: Animals; Cells, Cultured; Embryo, Mammalian; Enzyme Activation; Excitatory Amino Acid Agents; Fragile X Mental Retardation Protein; Hippocampus; Immunoprecipitation; Methoxyhydroxyphenylglycol; Mutation; Neurons; Phosphorylation; Protein Phosphatase 2; Pyridines; Rats; Receptors, Metabotropic Glutamate; Signal Transduction; Time Factors; Transfection

Group I metabotropic glutamate receptors (mGluRs) are Galphaq-protein-coupled receptors and are densely expressed in medium-sized spiny projection neurons of the neostriatum. Among different subtypes of glutamate receptors, group I mGluRs have been demonstrated to actively interact with the ionotropic glutamate receptor N-methyl-d-aspartate (NMDA) subtypes for regulating various forms of cellular activities and synaptic plasticity. In this study, the possible role of group I mGluRs in regulating serine phosphorylation of NMDA receptor NR1 subunits in the neostriatum was investigated in vivo. We found in chronically cannulated rats that injection of the group I mGluR agonist 3,5-dihydroxyphenylglycine (DHPG) into the dorsal striatum (caudate putamen) significantly increased phosphorylation of the two serine residues (serine 896 and serine 897) on the intracellular C-terminus of the NR1. The increase in NR1 phosphorylation was dose-dependent and DHPG had no effect on basal levels of NR1 proteins. Intrastriatal infusion of the group I mGluR antagonist N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) significantly attenuated the DHPG-stimulated NR1 phosphorylation. Pretreatment with the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) also produced the same effect. These data suggest that group I mGluRs, likely mGluR5 subtypes, possess the ability to upregulate protein phosphorylation of NMDA receptor NR1 subunits in striatal neurons in vivo.

Topics: Animals; Benzopyrans; Blotting, Western; Electrophoresis, Polyacrylamide Gel; Immunohistochemistry; Male; Methoxyhydroxyphenylglycol; Neostriatum; Phosphorylation; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; Serine

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurones accompanied by intense gliosis in lesioned areas of the brain and spinal cord. Glutamate-mediated excitotoxicity resulting from impaired astroglial uptake constitutes one of the current pathophysiological hypotheses explaining the progression of the disease. In this study, we examined the regulation of glutamate transporters by type 5 metabotropic glutamate receptor (mGluR5) in activated astrocytes derived from transgenic rats carrying an ALS-related mutated human superoxide dismutase 1 (hSOD1(G93A)) transgene. Cells from transgenic animals and wild-type littermates showed similar expression of glutamate-aspartate transporter and glutamate transporter 1 (GLT-1) after in vitro activation, whereas cells carrying the hSOD1 mutation showed a three-fold higher expression of functional mGluR5, as observed in the spinal cord of end-stage animals. In cells from wild-type animals, (S)-3,5-dihydroxyphenylglycine (DHPG) caused an immediate protein kinase C (PKC)-dependent up-regulation of aspartate uptake that reflected the activation of GLT-1. Although this effect was mimicked in both cultures by direct activation of PKC using phorbol myristate acetate, DHPG failed to up-regulate aspartate uptake in cells derived from the transgenic rats. The failure of activated mGluR5 to increase glutamate uptake in astrocytes derived from this animal model of ALS supports the theory of glutamate excitotoxicity in the pathogenesis of the disease.

Topics: Amyotrophic Lateral Sclerosis; Animals; Animals, Genetically Modified; Aspartic Acid; Astrocytes; Blotting, Northern; Calcium; Carbachol; Cholinergic Agonists; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Antagonists; Excitatory Amino Acid Transporter 2; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Glutamic Acid; Humans; Immunohistochemistry; Male; Methoxyhydroxyphenylglycol; Protein Kinase C; Pyridines; Rats; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium; Superoxide Dismutase; Tritium

Although metabotropic glutamate receptors (mGluRs) mGluR1 and mGluR5 are often found to have similar functions, there is considerable evidence that the two receptors also serve distinct functions in neurons. In hippocampal area CA1, mGluR5 has been most strongly implicated in long-term synaptic depression (LTD), whereas mGluR1 has been thought to have little or no role. Here we show that simultaneous pharmacological blockade of mGluR1 and mGluR5 is required to block induction of LTD by the group 1 mGluR agonist, (RS)-3,5-dihydroxyphenylglycine (DHPG). Blockade of mGluR1 or mGluR5 alone has no effect on LTD induction, suggesting that activation of either receptor can fully induce LTD. Consistent with this conclusion, mGluR1 and mGluR5 both contribute to activation of extracellular signal-regulated kinase (ERK), which has previously been shown to be required for LTD induction. In contrast, selective blockade of mGluR1, but not mGluR5, reduces the expression of LTD and the associated decreases in AMPA surface expression. LTD is also reduced in mGluR1 knockout mice confirming the involvement of mGluR1. This shows a novel role for mGluR1 in long-term synaptic plasticity in CA1 pyramidal neurons. In contrast to DHPG-induced LTD, synaptically induced LTD with paired-pulse low-frequency stimulation persists in the pharmacological blockade of group 1 mGluRs and in mGluR1 or mGluR5 knockout mice. This suggests different receptors and/or upstream mechanisms for chemically and synaptically induced LTD.

Topics: Action Potentials; Amino Acids; Animals; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Extracellular Signal-Regulated MAP Kinases; Hippocampus; Long-Term Synaptic Depression; Methoxyhydroxyphenylglycol; Mice; Mice, Knockout; Neuronal Plasticity; Neurons; Neurotransmitter Agents; Phosphorylation; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Metabotropic Glutamate 5; Receptors, AMPA; Receptors, Metabotropic Glutamate; Synapses; Xanthenes

Group I metabotropic glutamate receptors (mGluRs) have been reported to regulate N-methyl-d-aspartate (NMDA) receptor function in various brain regions. The selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) can potentiate NMDA antagonists such as PCP and MK-801-induced behavioural responses. In the present study, the role of group I mGluRs on ketamine- and propofol-induced general anaesthesia was examined.. Mice were pretreated with various doses of the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG), selective mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), mGluR1 antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) and mGluR5 antagonist MPEP followed by administration of ketamine (120 mg kg(-1)) or propofol (140 mg kg(-1)) to induce anaesthesia. The duration of loss of righting reflex was recorded.. DHPG and CHPG antagonized and MPEP potentiated ketamine-induced anaesthesia in a dose-dependent manner. CPCCOEt was ineffective. However, propofol-induced anaesthesia was not affected after manipulating mGluR1 and mGluR5 receptors.. mGluR5 receptors play an important role in modulation of anaesthesia induced by ketamine, but not propofol.

Topics: Anesthetics, Dissociative; Anesthetics, Intravenous; Animals; Chromones; Dose-Response Relationship, Drug; Glycine; Ketamine; Male; Methoxyhydroxyphenylglycol; Mice; Phenylacetates; Propofol; Pyridines; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Reflex

An increasing amount of evidence suggests that the family of nuclear factor kappaB (NF-kappaB) transcription factors plays an important role in synaptic plasticity and long-term memory formation. The present study investigated the regulation of NF-kappaB family members p50, p65/RelA, and c-Rel in the hippocampus in response to metabotropic glutamate receptor (mGluR) signaling. Activation of group I metabotropic glutamate receptors (GpI-mGluRs) with the agonist (S)-3,5-dihydroxyphenylglycine (DHPG) resulted in a time-dependent increase in DNA binding activity of p50, p65, and c-Rel in area CA1 of the hippocampus. An antagonist of mGluR5, 2-Methyl-6-(phenylethynyl)pyridine, inhibited the DHPG-induced activation of NF-kappaB, whereas an antagonist of mGluR1, (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid, did not. Using a series of inhibitors, we investigated the signaling pathways necessary for DHPG-induced activation of NF-kappaB and found that they included the phosphatidyl inositol 3-kinase, protein kinase C, mitogen-activated protein kinase kinase, and p38-mitogen-activated protein kinase pathways. To determine the functional significance of mGluR-induced regulation of NF-kappaB, we measured long-term depression (LTD) of Schaffer-collateral synapses in the hippocampus of c-Rel knock-out mice. Early phase LTD was normal in c-rel(-/-) mice. However, late-phase LTD (>90 min) was impaired in c-rel(-/-) mice. The observations of this deficit in hippocampal synaptic plasticity prompted us to further investigate long-term memory formation in c-rel(-/-) mice. c-rel(-/-) mice exhibited impaired performance in a long-term passive avoidance task, providing additional evidence for c-Rel in long-term memory formation. These results demonstrate that the NF-kappaB transcription factor family is regulated by GpI-mGluRs in the hippocampus and that the c-Rel transcription factor is necessary for long-term maintenance of LTD and formation of long-term memory.

Topics: Animals; Animals, Newborn; Avoidance Learning; Behavior, Animal; Blotting, Western; Dose-Response Relationship, Radiation; Electric Stimulation; Enzyme Activation; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Excitatory Postsynaptic Potentials; Gene Expression; Glycine; Hippocampus; In Vitro Techniques; Long-Term Synaptic Depression; Methoxyhydroxyphenylglycol; Mice; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B; Patch-Clamp Techniques; Phenylacetates; Protein Binding; Protein Subunits; Proto-Oncogene Proteins c-rel; Pyridines; Receptors, Metabotropic Glutamate; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Synaptic Transmission; Time Factors

Genetic deletion of fragile X mental retardation protein (FMRP) has been shown to enhance mGluR-dependent long-term depression (LTD). Herein, we demonstrate that mGluR-LTD induces a transient, translation-dependent increase in FMRP that is rapidly degraded by the ubiquitin-proteasome pathway. Moreover, proteasome inhibitors abolished mGluR-LTD, and LTD was absent in mice that overexpress human FMRP. Neither translation nor proteasome inhibitors blocked the augmentation of mGluR-LTD in FMRP-deficient mice. In addition, mGluR-LTD is associated with rapid increases in the protein levels of FMRP target mRNAs in wild-type mice. Interestingly, the basal levels of these proteins were elevated and their synthesis was improperly regulated during mGluR-LTD in FMRP-deficient mice. Our findings indicate that hippocampal mGluR-LTD requires the rapid synthesis and degradation of FMRP and that mGluR-LTD triggers the synthesis of FMRP binding mRNAs. These findings indicate that the translation, ubiquitination, and proteolysis of FMRP functions as a dynamic regulatory system for controlling synaptic plasticity.

Topics: Animals; Animals, Newborn; Anisomycin; Benzoates; Blotting, Western; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Antagonists; Fluorescent Antibody Technique; Fragile X Mental Retardation Protein; Glycine; In Vitro Techniques; Leupeptins; Long-Term Synaptic Depression; Male; Methoxyhydroxyphenylglycol; Mice; Mice, Knockout; Microtubule-Associated Proteins; Models, Biological; Proteasome Endopeptidase Complex; Protein Biosynthesis; Protein Synthesis Inhibitors; Pyridines; Receptors, Metabotropic Glutamate; RNA, Messenger; Signal Transduction

Astrocytes express mainly metabotropic glutamate receptor 3 and metabotropic glutamate receptor 5 receptor subtypes, which show opposing effects on cellular proliferation upon activation. In this study, we investigated the mechanisms by which activation of these receptors modulates astrocyte proliferation. Activation of metabotropic glutamate receptor 5 with (S)-3,5-dihydroxyphenylglycine increased phospholipase D activity in astrocytes as well as astrocyte proliferation. The 3,5-dihydroxyphenylglycine-induced proliferation was inhibited in the presence of the metabotropic glutamate receptor 5 antagonist (2-methyl-6-(phenylethynyl)pyridine), the protein kinase C inhibitor GF109203X, brefeldin A and 1-butanol. Activation of metabotropic glutamate receptor 3 with (2'S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine-IV (DCG-IV) inhibited astrocyte proliferation without affecting metabotropic glutamate receptor 5-mediated phospholipase D activity. Metabotropic glutamate receptor 3 activation, however, only partially inhibited metabotropic glutamate receptor 5-mediated proliferation. In conclusion, metabotropic glutamate receptor 5 stimulates astrocyte proliferation via a protein kinase C-phospholipase D-phosphatidic acid-dependent pathway, whereas metabotropic glutamate receptor 3-mediated inhibition of astrocyte proliferation does not involve phospholipase D, and is independent of metabotropic glutamate receptor 5-mediated effects.

Topics: Animals; Animals, Newborn; Astrocytes; Cell Count; Cell Proliferation; Cells, Cultured; Cerebral Cortex; Cycloleucine; Cyclopropanes; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glycerophospholipids; Glycine; Indoles; Maleimides; Methoxyhydroxyphenylglycol; Phospholipase D; Pyridines; Rats; Rats, Wistar; Receptors, Glutamate; Tritium

Hyperammonemia impairs signal transduction associated to glutamate receptors and phosphorylation of some neuronal proteins including microtubule-associated protein 2 (MAP-2). The aim of this work was to analyze the effects of hyperammonemia on modulation of MAP-2 phosphorylation by metabotropic glutamate receptors (mGluRs) in rat cerebellar neurons in culture. Hyperammonemia increased basal phosphorylation of MAP-2 (180%). Activation of mGluRs 1 and 5 with (S)-3,5-dihydroxyphenylglycine (DHPG) increased MAP-2 phosphorylation (170%) in control neurons but not in neurons exposed to ammonia. Activation of mGluRs 2 and 3 with (2S,3S,4S)-CCG/(2S, 1'S,2'S)-2-(carboxycyclopropyl)glycine increased slightly (25%) MAP-2 phosphorylation in neurons exposed to ammonia or not. Activation of mGluR5 with (+/-)-trans-azetidine-2,4-dicarboxylic acid increased MAP-2 phosphorylation (24%) in control neurons but decreased it by 56% in neurons exposed to ammonia. Activation of mGluR1 using 2-methyl-6-(phenylethynyl)pyridine and DHPG increased MAP-2 phosphorylation 183% in control neurons but only 89% in neurons exposed to ammonia. In control neurons mGluR1 activation greatly increases phosphorylation of MAP-2, while activation of mGluRs 5, 2 or 3 increased it slightly. Taken together, hyperammonemia reduces the increase in MAP-2 phosphorylation induced by mGluR1activation. Moreover, in neurons exposed to ammonia activation of mGluR5 reduces MAP-2 phosphorylation. These effects reflect significant alterations in signal transduction associated to mGluR1 and mGluR5 in hyperammonemia that may contribute to altered glutamatergic neurotransmission and to the neurological alterations in hyperammonemia and hepatic encephalopathy.

Topics: Amino Acids, Dicarboxylic; Ammonia; Animals; Azetidinecarboxylic Acid; Cells, Cultured; Cerebellum; Excitatory Amino Acid Agonists; Immunoprecipitation; Methoxyhydroxyphenylglycol; Microtubule-Associated Proteins; Neurons; Phosphorylation; Pyridines; Rats; Rats, Wistar; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate

This study examined whether activation of group I metabotropic glutamate receptors (mGluRs) could modulate synaptic inhibition of spinal motoneurons in the neonatal rat isolated spinal cord. Recurrent inhibitory postsynaptic potentials (IPSPs) generated by Renshaw cells were evoked via antidromic stimulation of motor axon collaterals and recorded intracellularly from lumbar motoneurons. The selective agonist of group I mGluRs DHPG (5 micromol L-1) depressed the recurrent IPSP, an effect prevented by the selective antagonist AIDA (500 micromol L-1). The depression by DHPG was use-independent and could be partly counteracted by increasing stimulus strength. Paired pulse depression observed at

Topics: Analysis of Variance; Animals; Animals, Newborn; Dicarboxylic Acids; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamates; In Vitro Techniques; Membrane Potentials; Methoxyhydroxyphenylglycol; Motor Neurons; Neural Inhibition; Patch-Clamp Techniques; Pyridines; Rats; Rats, Wistar; Receptors, Metabotropic Glutamate; Spinal Cord; Synaptic Transmission

It is well known that prolonged exposure to morphine results in tolerance to morphine-induced antinociception. In the present study, we found that either intrathecal (i.t.) or subcutaneous (s.c.) injection of the selective metabotropic glutamate receptor 5 (mGluR5) antagonist, methyl-6-(phenylethynyl)-pyridine hydrochloride (MPEP), attenuated the development of tolerance to morphine-induced antinociception. Using the receptor binding assay, we found here that the number of mGluR5 in the mouse spinal cord was significantly increased by repeated treatment with morphine. Furthermore, repeated treatment with morphine produced a significant increase in the level of mGluR5 immunoreactivity in the dorsal horn of the mouse spinal cord. Double-labeling experiments showed that the increased mGluR5 was predominantly expressed in the neurons and sparsely expressed in the processes of astrocytes following repeated treatment with morphine. Consistent with these results, the response of Ca2+ to the selective group I mGluR agonist, 3,5-dihydroxyphenylglycine (DHPG), in cultured spinal cord neurons was potently enhanced by 3 days of in vitro treatment with morphine. These findings support the idea that the increased mGluR5 following repeated treatment with morphine leads to enhanced neuronal excitability and synaptic transmission in the dorsal horn of the spinal cord and, in turn, suppresses the morphine-induced antinociception in mice.

Topics: Analgesics, Opioid; Animals; Calcium; Coculture Techniques; Drug Tolerance; Excitatory Amino Acid Antagonists; Intracellular Membranes; Male; Methoxyhydroxyphenylglycol; Mice; Mice, Inbred ICR; Morphine; Neuroglia; Neurons; Nociceptors; Osmolar Concentration; Pyridines; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Spinal Cord

Group I metabotropic glutamate (mGlu) receptors (i.e. mGlu1 and mGlu5) coupled to phospholipase C have been widely investigated for their possible role in excitotoxic and post-ischemic neuronal death. Recently, phospholipase C has been shown to directly stimulate the activity of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair that has been proposed to play a key role in necrotic cell death. In this study, we investigated whether the stimulation of group I mGlu receptors leads to an increase in PARP activity, as detected by flow cytometry, immunodot blot and immunocytochemistry, both in baby hamster kidney cells transfected with mGlu1a or mGlu5a receptors and in cultured cortical cells. Our results show that the group I mGlu receptor agonist DHPG elicited a significant increase in PARP activity that was completely abolished by the administration of the mGlu1 antagonist 3-MATIDA and partially prevented, in cortical neurons, by the mGlu5 antagonist MPEP. To evaluate whether this pathway is involved in post-ischemic neuronal death, we used a sublethal model of oxygen-glucose deprivation in mixed cortical cell cultures. DHPG exacerbated neuronal death, and this effect was significantly prevented by the application of the PARP inhibitor DPQ. This novel pathway may contribute to the effects of mGlu1 receptors in the mechanisms leading to post-ischemic neuronal death.

Topics: Animals; Animals, Newborn; Blotting, Western; Cells, Cultured; Cerebral Cortex; Cricetinae; Drug Interactions; Enzyme Activation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Fluorescent Antibody Technique; Gene Expression Regulation, Enzymologic; Glial Fibrillary Acidic Protein; Glucose; Hydrogen Peroxide; Hypoxia; Isoquinolines; Methoxyhydroxyphenylglycol; Mice; Neuroglia; Neurons; Piperidines; Poly(ADP-ribose) Polymerases; Pyridines; Receptors, Metabotropic Glutamate; Thiophenes; Time Factors; Transfection

Activation of glycogen synthase kinase 3beta (Gsk3beta) has been shown to be a key component in signaling pathways that underlie neurodegeneration and neurodegenerative disease. Conversely, inactivation of Gsk3beta by phosphoinositide 3-kinase (PI3K)/Akt is an important neuroprotective mechanism. Previous studies have shown that agonist activation of group I metabotropic glutamate receptors (mGluRs) can increase neuronal survival and prevent apoptosis. However, little is known about the signaling pathways that couple mGluR5 to neuroprotection. In this report, we investigated whether activation of the PI3K/Akt/Gsk3beta pathway, which has been shown to have an important neuroprotective mechanism, is required for mGluR5 activation mediated neuroprotection against beta-amyloid. We found that brief incubations of mouse hippocampal slices with (R,S)-3,5-dihydroxyphenylglycine (DHPG) resulted in increased phosphorylation of Akt and Gsk3beta. The PI3K inhibitors, LY294002 and wortmannin, blocked the DHPG-induced increased phosphorylation of Akt and Gsk3beta. Similar results were observed in rat primary hippocampal cultures. Finally, we found that the PI3K inhibitor LY294002 can block (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) mediated neuroprotection against beta-amyloid. Thus, these findings suggest that mGluR5 can modulate the PI3K/Akt/Gsk3beta pathway in the hippocampus, and that modulation of this signaling pathway can reverse beta-amyloid-induced neuronal toxicity.

Topics: Amyloid beta-Peptides; Animals; Benzoates; Cells, Cultured; Chromones; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glycine; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hippocampus; In Vitro Techniques; Indoles; Methoxyhydroxyphenylglycol; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Morpholines; Neurons; Oncogene Protein v-akt; Peptide Fragments; Phosphorylation; Pyridines; Rats; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Resorcinols; Serine; Signal Transduction

The laterocapsular division of the central nucleus of the amygdala (CeA) is now defined as the "nociceptive amygdala" because of its high content of neurons that respond to painful stimuli. The majority of these neurons become sensitized in a model of arthritis pain. Here we address the role of G protein-coupled group I metabotropic glutamate receptor subtypes mGluR1 and mGluR5 in nociceptive processing under normal conditions and in pain-related sensitization. Extracellular single-unit recordings were made from 65 CeA neurons in anesthetized rats. Each neuron's responses to brief mechanical stimuli, background activity, receptive field size, and threshold were measured before and after induction of the kaolin/carrageenan mono-arthritis in one knee and before and during applications of agonists and antagonists into the CeA by microdialysis. All neurons received excitatory input from the knee(s) and responded most strongly to noxious stimuli. Before arthritis, a group I mGluR1 and mGluR5 agonist (DHPG, n = 10) potentiated the responses to innocuous and noxious stimuli. This effect was mimicked by an mGluR5 agonist (CHPG, n = 15). In the arthritis pain state (>6 h after induction), the facilitatory effects of DHPG (n = 9), but not CHPG (n = 7), increased. An mGluR1 antagonist (CPCCOEt) had no effect before arthritis (n = 12) but inhibited the responses of sensitized neurons in the arthritis pain state (n = 8). An mGluR5 antagonist (MPEP) inhibited brief nociceptive responses under normal conditions (n = 19) and prolonged nociception in arthritis (n = 8). These data suggest a change of mGluR1 function and activation in the amygdala in pain-related sensitization, whereas mGluR5 is involved in brief as well as prolonged nociception.

Topics: Action Potentials; Amygdala; Animals; Arthritis; Chromones; Dose-Response Relationship, Drug; Glycine; Kaolin; Male; Methoxyhydroxyphenylglycol; Neurons; Phenylacetates; Physical Stimulation; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate

Activation of spinal cord dorsal horn ionotropic glutamate receptors leads to pain-related behaviors. However, the role of spinal metabotropic glutamate receptors (mGlu), particularly the mGlu5 receptor subtype, in nociception has not been well characterized. A recently described subtype selective and potent mGluR5 antagonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP) was used to evaluate the role of the mGlu5 receptor in cold sensitivity. Intrathecal (i.t.) injection of group I (mGlu1 and mGlu5 receptors) mGlu receptor-selective agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) increased the hind paw frequency and duration of lifting of rats placed on a cold (4 degrees C) surface, a behavior similarly observed in rats with a chronic constriction injury (CCI) of the sciatic nerve. In contrast, rats i.t. injected with DHPG did not display increased lifting when placed on a room temperature surface. I.t. injection of MPEP before i.t. injection of DHPG blocked DHPG-evoked cold hypersensitivity, suggesting that activation of spinal mGlu5 receptors induces this behavioral response. In contrast, i.t. injection of MPEP after i.t. injection of DHPG had no effect. In addition, i.t. injection of MPEP did not affect cold hypersensitivity in rats with a CCI. These data suggest that acute activation of spinal cord mGlu5 receptors results in increased sensitivity to cold, but ongoing cold hypersensitivity does not involve activation of the mGlu5 receptor.

Topics: Animals; Behavior, Animal; Cold Temperature; Constriction, Pathologic; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Injections, Spinal; Male; Methoxyhydroxyphenylglycol; Pain; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Sciatic Nerve; Spinal Cord

The idea that excitatory amino acid transporters (EAATs) can control the activation of specific metabotropic glutamate receptors (mGluRs) was investigated in rat hippocampal slices. Using the accumulation of inositol phosphates as a measure of group I mGluR activity, we have shown that the broad spectrum, non-transportable EAAT blocker, TBOA, produces a significant shift to the left of agonist concentration-response curves. Moreover, this increase in potency did not occur if endogenous glutamate was enzymatically removed, suggesting a glutamate-dependent mechanism. This shift in potency was shown to be NMDA and group II mGlu receptor independent. Additionally, experiments with selective antagonists indicated that the group I receptor responsible for the stimulation of inositol phosphate production in this preparation is likely to be mGluR5. Inhibition of forskolin-stimulated cyclic AMP (cAMP) production was used as an index of group II/III mGluR activity. TBOA produced a rightward shift of the forskolin concentration-response curve. A group III, but not a group II, mGluR agonist also produced this effect, suggesting that the TBOA-mediated increase in glutamate activates a receptor, which appears to be a member of the group III mGluR subset. This was confirmed by the observation that an antagonist of group III mGluRs, prevented the TBOA-induced rightward shift in forskolin potency. These results provide evidence of a role for EAATs in the regulation of mGluR5 and group III mGluRs in the rat hippocampus, which may have therapeutic implications.

Topics: Animals; Aspartic Acid; Benzoates; Colforsin; Cyclic AMP; Excitatory Amino Acid Antagonists; Excitatory Amino Acid Transporter 1; Excitatory Amino Acid Transporter 2; Glycine; Hippocampus; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Male; Methoxyhydroxyphenylglycol; Pyridines; Quisqualic Acid; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate

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.

Topics: Animals; Animals, Newborn; Brain; Cell Line; Cyclic AMP; Excitatory Amino Acid Antagonists; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Lithium Chloride; Male; Methoxyhydroxyphenylglycol; Oocytes; Phosphatidylinositols; Pyridines; Quisqualic Acid; Radioligand Assay; Rats; Rats, Inbred Strains; Rats, Sprague-Dawley; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Recombinant Proteins; Sulfur Radioisotopes; Transfection; Xenopus laevis