Involvement of spinal metabotropic glutamate receptor 5 in the development of tolerance to morphine-induced antinociception

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.     

Arabidopsis thaliana glutamate receptor ion channel function demonstrated by ion pore transplantation

Ionotropic glutamate receptors (iGluRs) are ligand-gated cation channels that mediate fast excitatory neurotransmission in the mammalian central nervous system. In the model plant Arabidopsis thaliana, a large family of 20 genes encoding proteins that share similarities with animal iGluRs in sequence and predicted secondary structure has been discovered. Members of this family, termed AtGLRs (A. thaliana glutamate receptors), have been implicated in root development, ion transport, and several metabolic and signalling pathways. However, there is still no direct proof of ligand-gated ion channel function of any AtGLR subunit. We used a domain transplantation technique to directly test whether the putative ion pore domains of AtGLRs can conduct ions. To this end, we transplanted the ion pore domains of 17 AtGLR subunits into rat α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (GluR1) and kainate (GluR6) receptor subunits and tested the resulting chimaeras for ion channel function in the Xenopus oocyte expression system. We show that AtGLR1.1 and AtGLR1.4 have functional Na⁺-, K⁺-, and Ca²⁺-permeable ion pore domains. The properties of currents through the AtGLR1.1 ion pore match those of glutamate-activated currents, depolarisations, and glutamate-triggered Ca²⁺ influxes observed in plant cells. We conclude that some AtGLRs have functional non-selective cation pores.     

Extent of RNA editing of glutamate receptor subunit GluR5 in different brain regions of the rat

Summary 1. The structure and function of glutamate receptor subunits GluR2, GluR5, and GluR6 are changed by RNA editing. This reaction produces a base transition in the second transmembrane spanning region. The triplet CAG (coding for glutamine) is changed to CGG (coding for arginine). This transition has a pronounced effect on calcium fluxes through the respective ion channels, because calcium currents decrease with the rate of editing.2. In the present study the extent of RNA editing of the glutamate receptor subunit GluR5 was studied in different brain regions of control rats using a newly developed analysis system. This system is based on restriction analysis of the polymerase chain reaction (PCR) product, derived from reverse-transcribed mRNA as template, with the enzymeBbv1.Bbv1 recognizes the sequence of the nonedited receptor subunit around the edited base (sequence GCAGC) but not that of the edited subunit (sequence GCGGC; A edited to G).3. Total RNA was isolated from the cerebral cortex, striatum, hippocampus, thalamus, hypothalamus, cerebellum, pons/medulla oblongata, and white matter and reverse transcribed into cDNA. The region across the edited sequence was amplified by PCR using GluR5-specific primers and the cDNA as template. PCR products were cleaned by ethanol precipitation, incubated withBbv1, and electrophoresed on an agarose gel together with standards. Gels were photographed and the extent of GluR5 mRNA editing was quantified using an image analysis system. A calibration curve was obtained using PCR products amplified from plasmids with edited and nonedited GluR5 as inserts.4. In the brain of control rats the extent of RNA editing of the GluR5 subunit amounted to 62±6.0% of total (cortex), 43±5.3% (striatum), 52±5.3% (hippocampus), 91±6.3% (thalamus), 85±10.2% (hypothalamus), 82±6.5% (cerebellum), 88±6.8% (pons/medulla oblongata), and 41±2.7% (white matter).5. The extent of RNA editing varied, thus, considerably in different brain regions, being lowest in the white matter and striatum and highest in the thalamus and pons/medulla oblongate. RNA editing of glutamate receptor subunits may play an important role in the control of calcium fluxes through non-N-methyl-D-aspartate receptor channels in different physiological and/or pathological states of the brain.     

Making sense with TRP channels: store-operated calcium entry and the ion channel Trpm5 in taste receptor cells

The sense of taste plays a critical role in the life and nutritional status of organisms. During the last decade, several molecules involved in taste detection and transduction have been identified, providing a better understanding of the molecular physiology of taste receptor cells. However, a comprehensive catalogue of the taste receptor cell signaling machinery is still unavailable. We have recently described the occurrence of calcium signaling mechanisms in taste receptor cells via apparent store-operated channels and identified Trpm5, a novel candidate taste transduction element belonging to the mammalian family of transient receptor potential channels. Trpm5 is expressed in a tissue-restricted manner, with high levels in gustatory tissue. In taste cells, Trpm5 is co-expressed with taste-signaling molecules such as alpha-gustducin, Ggamma(13), phospholipase C beta(2) and inositol 1,4,5-trisphosphate receptor type III. Biophysical studies of Trpm5 heterologously expressed in Xenopus oocytes and mammalian CHO-K1 cells indicate that it functions as a store-operated channel that mediates capacitative calcium entry. The role of store-operated channels and Trpm5 in capacitative calcium entry in taste receptor cells in response to bitter compounds is discussed.     

Structure of the S1S2 glutamate binding domain of GLuR3

Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system. Determining the structural differences between the binding sites of different subtypes is crucial to our understanding of neuronal circuits and to the development of subtype specific drugs. The structures of the binding domain (S1S2) of the GluR3 (flip) AMPA receptor subunit bound to glutamate and AMPA and the GluR2 (flop) subunit bound to glutamate were determined by X‐ray crystallography to 1.9, 2.1, and 1.55 Å, respectively. Overall, the structure of GluR3 (flip) S1S2 is very similar to GluR2 (flop) S1S2 (backbone RMSD of 0.30 ± 0.05 for glutamate‐bound and 0.26 ± 0.01 for AMPA‐bound). The differences in the flip and flop isoforms are subtle and largely arise from one hydrogen bond across the dimer interface and associated water molecules. Comparison of the binding affinity for various agonists and partial agonists suggest that the S1S2 domains of GluR2 and GluR3 show only small differences in affinity, unlike what is found for the intact receptors (with the exception of one ligand, Cl‐HIBO, which has a 10‐fold difference in affinity for GluR2 vs. GluR3). Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Reduction in glutamate uptake is associated with extrasynaptic NMDA and metabotropic glutamate receptor activation at the hippocampal CA1 synapse of aged rats

This study aims to determine whether the regulation of extracellular glutamate is altered during aging and its possible consequences on synaptic transmission and plasticity. A decrease in the expression of the glial glutamate transporters GLAST and GLT‐1 and reduced glutamate uptake occur in the aged (24–27 months) Sprague–Dawley rat hippocampus. Glutamatergic excitatory postsynaptic potentials recorded extracellularly in ex vivo hippocampal slices from adult (3–5 months) and aged rats are depressed by DL‐TBOA, an inhibitor of glutamate transporter activity, in an N‐Methyl‐d‐ Aspartate (NMDA)‐receptor‐dependent manner. In aged but not in young rats, part of the depressing effect of DL‐TBOA also involves metabotropic glutamate receptor (mGluRs) activation as it is significantly reduced by the specific mGluR antagonist d‐methyl‐4‐carboxy‐phenylglycine (MCPG). The paired‐pulse facilitation ratio, a functional index of glutamate release, is reduced by MCPG in aged slices to a level comparable to that in young rats both under control conditions and after being enhanced by DL‐TBOA. These results suggest that the age‐associated glutamate uptake deficiency favors presynaptic mGluR activation that lowers glutamate release. In parallel, 2 Hz‐induced long‐term depression is significantly decreased in aged animals and is fully restored by MCPG. All these data indicate a facilitated activation of extrasynaptic NMDAR and mGluRs in aged rats, possibly because of an altered distribution of glutamate in the extrasynaptic space. This in turn affects synaptic transmission and plasticity within the aged hippocampal CA1 network.     

Metabotropic glutamate receptor 5 and calcium signaling in retinal amacrine cells

To begin to understand the modulatory role of glutamate in the inner retina, we examined the mechanisms underlying metabotropic glutamate receptor 5 (mGluR5)-dependent Ca(2+) elevations in cultured GABAergic amacrine cells. A partial sequence of chicken retinal mGluR5 encompassing intracellular loops 2 and 3 suggests that it can couple to both G(q) and G(s). Selective activation of mGluR5 stimulated Ca(2+) elevations that varied in waveform from cell to cell. Experiments using high external K(+) revealed that the mGluR5-dependent Ca(2+) elevations are distinctive in amplitude and time course from those engendered by depolarization. Experiments with a Ca(2+) -free external solution demonstrated that the variability in the time course of mGluR5-dependent Ca(2+) elevations is largely due to the influx of extracellular Ca(2+). The sensitivity of the initial phase of the Ca(2+) elevation to thapsigargin indicates that this phase of the response is due to the release of Ca(2+) from the endoplasmic reticulum. Pharmacological evidence indicates that mGluR5-mediated Ca(2+) elevations are dependent upon the activation of phospholipase C. We rule out a role for L-type Ca(2+) channels and cAMP-gated channels as pathways for Ca(2+) entry, but provide evidence of transient receptor potential (TRP) channel-like immunoreactivity, suggesting that Ca(2+) influx may occur through TRP channels. These results indicate that GABAergic amacrine cells express an avian version of mGluR5 that is linked to phospholipase C-dependent Ca(2+) release and Ca(2+) influx, possibly through TRP channels.     

The role of the N-methyl-d-aspartate receptor in the proliferation of adult hippocampal neural stem and precursor cells

New neurons are continuously generated from resident pools of neural stem and precursor cells(NSPCs)in the adult brain.There are multiple pathways through which adult neurogenesis is regulated,and here we review the role of the N-methyl-D-aspartate receptor(NMDAR)in regulating the proliferation of NSPCs in the adult hippocampus.Hippocampal-dependent learning tasks,enriched environments,running,and activity-dependent synaptic plasticity,all potently up-regulate hippocampal NSPC proliferation.We first consider the requirement of the NMDAR in activity-dependent synaptic plasticity,and the role the induction of synaptic plasticity has in regulating NSPCs and newborn neurons.We address how specific NMDAR agonists and antagonists modulate proliferation,both in vivo and in vitro,and then review the evidence supporting the hypothesis that NMDARs are present on NSPCs.We believe it is important to understand the mechanisms underlying the activation of adult neurogenesis,given the potential that endogenous stem cell populations have for repopulating the hippocampus with functional new neurons.In conditions such as age-related memory decline,neurodegeneration and psychiatric disease,mature neurons are lost or become defective;as such,stimulating adult neurogenesis may provide a therapeutic strategy to overcome these conditions.     

PICK1 is required for the control of synaptic transmission by the metabotropic glutamate receptor 7

Both postsynaptic density and presynaptic active zone are structural matrix containing scaffolding proteins that are involved in the organization of the synapse. Little is known about the functional role of these proteins in the signaling of presynaptic receptors. Here we show that the interaction of the presynaptic metabotropic glutamate (mGlu) receptor subtype, mGlu7a, with the postsynaptic density-95 disc-large zona occludens 1 (PDZ) domain-containing protein, PICK1, is required for specific inhibition of P/Q-type Ca(2+) channels, in cultured cerebellar granule neurons. Furthermore, we show that activation of the presynaptic mGlu7a receptor inhibits synaptic transmission and this effect also requires the presence of PICK1. These results indicate that the scaffolding protein, PICK1, plays an essential role in the control of synaptic transmission by the mGlu7a receptor complex.     

NCAM-derived peptides function as agonists for the fibroblast growth factor receptor

The neural cell adhesion molecule (NCAM) directly interacts with the fibroblast growth factor receptor (FGFR). Both fibronectin type III (FN3) modules of NCAM are involved in this interaction. One of the NCAM–FGFR contact sites has been localized recently to the upper N-terminal part of the second NCAM FN3 module encompassing the F and G β-strands and the interconnecting loop region. Here, we investigated whether any of the six putative strand-loop-strand regions in the first NCAM FN3 module are involved in FGFR interactions. Peptide sequences encompassing these regions, termed encamins, were synthesized and tested for their ability to bind and activate FGFR. Encamins localized to the N-terminal part of the first FN3 module did not interact with FGFR, whereas encamins localized to the C-terminal part, termed EncaminA, C and E, bound to and activated FGFR. The encamins induced FGFR-dependent neurite outgrowth, and EncaminC and E promoted neuronal survival and enhanced pre-synaptic function. In conclusion, the interaction between NCAM and FGFR probably involves multiple contact sites at an interface formed by the two NCAM FN3 modules and FGFR, and encamins could constitute important pharmacological tools for the study of specific functional aspects of NCAM, including neuroprotection and modulation of plasticity.     

Crystallization of the first three domains of the human insulin-like growth factor-1 receptor.

The insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor of central importance in cell proliferation. A fragment (residues 1-462) comprising the L1-cysteine rich-L2 domains of the human IGF-1R ectodomain has been overexpressed in glycosylation-deficient Lec8 cells and has been affinity-purified via a c-myc tag followed by gel filtration. The fragment was recognized by two anti-IGF-1R monoclonal antibodies, 24-31 and 24-60, but showed no detectable binding of IGF-1 or IGF-2. Isocratic elution of IGF-1R/462 on anion-exchange chromatography reduced sample heterogeneity, permitting the production of crystals that diffracted to 2.6 A resolution with cell dimensions a = 77.0 A, b = 99.5 A, c = 120.1 A, and space group P2(1)2(1)2(1).     

AMPA and NMDA glutamate receptor trafficking: multiple roads for reaching and leaving the synapse

Glutamate receptor trafficking in and out of synapses is one of the core mechanisms for rapid changes in the number of functional receptors during synaptic plasticity. Recent data have shown that the fast gain and loss of receptors from synaptic sites are accounted for by endocytic/exocytic processes and by their lateral diffusion in the plane of the membrane. These events are interdependent and regulated by neuronal activity and interactions with scaffolding proteins. We review here the main cellular steps for AMPA and NMDA receptor synthesis, traffic within intracellular organelles, membrane exocytosis/endocytosis and surface trafficking. We focus on new findings that shed light on the regulation of receptor cycling events and surface trafficking and the way that this might reshape our thinking about the specific regulation of receptor accumulation at synapses.     

Positive and negative allosteric modulation of GluK2 kainate receptors by BPAM344 and antiepileptic perampanel

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NMDA receptor as a newly identified member of the metabotropic glutamate receptor family: Clinical implications for neurodegenerative diseases

Recent reports have proposed a novel function for the N-methyl-d-aspartate (NMDA) receptor (NMDAR), a well-known excitatory, ionotropic receptor. A series of observations employing pharmacological techniques has proposed that upon ligand binding, this ionotropic receptor can actually function via signaling cascades independent of traditional ionotropic action. Moreover, the “metabotropic” action of NMDARs is suggested to mediate a form of synaptic plasticity, namely long-term synaptic depression (LTD), which shares cellular mechanisms with the synaptic deficits observed in Alzheimer’s disease. Given that a growing body of clinical and preclinical evidence strongly recommends NMDAR antagonists for their therapeutic potentials and advantages in a variety of diseases, further investigation into their molecular and cellular mechanisms is required to better understand the “metabotropic” action of NMDARs.     

Regulation of the intracellular distribution, cell surface expression, and protein levels of AMPA receptor GluR2 subunits by the monocarboxylate transporter MCT2 in neuronal cells

The neuronal monocarboxylate transporter, MCT2, is not only an energy substrate carrier but it is also purported to be a binding partner for the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit. To unravel a putative role of MCT2 in the regulation of GluR2 subcellular distribution, Neuro2A cells and primary cultures of mouse cortical neurons were co-transfected with plasmids containing sequences to express the fluorescent proteins mStrawberry (mStb)-fused MCT2 and Venus-fused GluR2. Subsequently, their subcellular distribution was visualized by fluorescence microscopy. GluR2 was led to form perinuclear and dendritic clusters together with MCT2 when co-transfected in Neuro2A cells or in neurons, following the original distribution of MCT2. MCT2 co-transfection had no effect on the intracellular distribution of several other post-synaptic proteins, although it partially affected the intracellular distribution of GluR1 similarly to GluR2. Both cell surface and total protein expression levels of GluR2 were significantly reduced by co-expression with MCT2. Finally, partial perinuclear and dendritic co-localization between MCT2 and Rab8, a member of the small GTPase family involved in membrane trafficking of AMPA receptors, was also observed in co-transfected neurons. These results suggest that MCT2 could influence AMPA receptor trafficking within neurons by modulating GluR2 sorting between different subcellular compartments.     

D1 and D4 dopaminergic receptor interplay mediates coincident G protein-independent and dependent regulation of glutamate NMDA receptors in the lateral amygdala

Dopamine (DA) receptor and NMDA receptor (NMDAR) activation in the lateral (LA) nucleus of the amygdala plays a critical role in emotional processing. Several distinct mechanisms regulate the molecular cross-talk between DA receptors and NMDARs in different brain regions; however, the cellular mechanism through which DA modulates NMDARs in LA projection neurons has not been studied. Here, we investigated the effect of DA receptor activation on NMDAR currents in LA projection neurons recorded in amygdala slices obtained from young rats. We found that DA reduces NMDAR current amplitudes in an additive manner through the activation of both D1-like and D2-like receptors. The reduction of NMDAR current amplitudes by D1-like receptor activation is mediated by a protein-protein interaction between the D1R and the NMDAR, while the regulation of NMDAR activity by D2-like receptors is elicited through a G protein-dependent pathway controlled by D4R. The results of our investigation show for the first time a functional interplay between D1R and D4R that mediates coincident G protein-independent and dependent regulation of NMDARs.