Neuronal glutamate transporters control activation of postsynaptic metabotropic glutamate receptors and influence cerebellar long-term depression.

Neuronal and glial isoforms of glutamate transporters show distinct distributions on membranes surrounding excitatory synapses, but specific roles for transporter subtypes remain unidentified. At parallel fiber (PF) synapses in cerebellum, neuronal glutamate transporters and metabotropic glutamate receptors (mGluRs) have overlapping postsynaptic distributions suggesting that postsynaptic transporters selectively regulate mGluR activation. We examined interactions between transporters and mGluRs by evoking mGluR-mediated excitatory postsynaptic currents (mGluR EPSCs) in slices of rat cerebellum. Selective inhibition of postsynaptic transporters enhanced mGluR EPSCs greater than 3-fold. Moreover, impairing glutamate uptake facilitated mGluR-dependent long-term depression at PF synapses. Our results demonstrate that uniquely positioned glutamate transporters strongly influence mGluR activation at cerebellar PF synapses. Postsynaptic glutamate uptake may serve as a general mechanism for regulating mGluR-initiated synaptic depression.     

Depression of excitatory transmission at PF-PC synapse by group III metabotropic glutamate receptors is provided exclusively by mGluR4 in the rodent cerebellar cortex

In the rodent cerebellum, pharmacological activation of group III pre-synaptic metabotropic glutamate receptors (mGluRs) by the broad spectrum agonist l -2-amino-4-phosphonobutyric acid, acutely depresses excitatory synaptic transmission at parallel fiber (PF)-Purkinje cell (PC) synapses. Among the group III mGluR subtypes, cerebellar granule cells express predominantly mGluR4, but also mGluR7 and mGluR8 mRNA. Taking into account that previous functional and pharmacological studies have used group III mGluR broad spectrum agonists that do not differentiate between these various subtypes, their relative contribution to the modulation of glutamatergic transmission at PF-PC synapses remains to be elucidated. In order to clarify this issue, we applied conventional whole-cell patch-clamp recordings and pre-synaptic calcium influx measurements, combined with pharmacological manipulations to rat and mice cerebellar slices. With the use of (1 S ,2 R )-1-amino-2-phosphonomethylcyclopropanecarboxylic acid, a new and selective group III mGluR agonist, N -phenyl-7-(hydroxylimino)cyclopropa[b]-chromen-1a-carboxamide, the specific positive allosteric modulator of mGluR4, ( S )-3,4-dicarboxyphenylglycine, a selective mGluR8 agonist, and mGluR4 knock-out mice, we demonstrate that the inhibitory control of group III mGluRs on excitatory neurotransmission at PF-PC synapses of the rodent cerebellar cortex, is totally because of the activation of pre-synaptic mGluR4 autoreceptors.     

Immunolocalization of the mGluR1b splice variant of the metabotropic glutamate receptor 1 at parallel fiber-Purkinje cell synapses in the rat cerebellar cortex

Several metabotropic glutamate receptor (mGluR) subtypes have been identified in the cerebellar cortex that are targeted to different compartments in cerebellar cells. In this study, preembedding immunocytochemical methods for electron microscopy were used to investigate the subcellular distribution of the mGluR1b splice variant in the rat cerebellar cortex. Dendritic spines of Purkinje cells receiving parallel fiber synaptic terminals were immunoreactive for mGluR1b. With a preembedding immunogold method, approximately 25% of the mGluR1b immunolabeling was observed perisynaptically within 60 nm from the edge of the postsynaptic densities. Values of extrasynaptic gold particles beyond the first 60 nm were maintained at between 10 and 18% along the whole intracellular surface of the dendritic spine membranes of Purkinje cells. For comparison, the distribution of mGluR1a was studied. A predominant (approximately 37%) perisynaptic localization of mGluR1a was seen in dendritic spines of Purkinje cells, dropping the extrasynaptic labeling to 15% in the 60-120-nm bin from the edge of the postsynaptic specialization. Our results reveal that mGluR1b and mGluR1a are localized to the same subcellular compartments in Purkinje cells but that the densities of the perisynaptic and extrasynaptic pools were different for both isoforms. The compartmentalization of mGluR1b and mGluR1a might serve distinct requirements in cerebellar neurotransmission.     

Pharmacological characterization of metabotropic glutamate receptors in cultured cerebellar granule cells

A detailed pharmacological characterization of metabotropic glutamate receptors (mGluR) was performed in primary cultures of cerebellar granule cells at 6 days in vitro (DIV). The rank order of agonists induced polyphosphoinositide (PPI) hydrolysis (after correcting for the ionotropic component in the response) was as follows: in terms of efficiency, Glu>quisqualate (quis)=ibotenate (ibo)>(1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD)>-methyl-amino-l-alanine (BMAA) and in terms of potency, quis>ACPD>Glu>ibo=BMAA. Ionotropic excitatory amino acid (EAA) receptor agonists, such as -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) were relatively inactive (in the presence of Mg²⁺). Quis and ACPD-induced PPI hydrolysis was unaffected by ionotropic Glu receptor antagonists, but was inhibited, in part by L-2-amino-3-phosphonopropionate (AP3). In contrast, Glu-or ibo- induced PPI hydrolysis was reduced, in part, by both AP3 and NMDA receptor antagonists. Characteristic interactions involving different transmitter receptors were noted. PPI hydrolysis evoked by quis and 1_S,3_R-ACPD was not additive. In contrast, PPI hydrolysis stimulated by quis/ACPD and carbamylcholine was additive (indicating different receptors/transduction pathways). In the presence of Mg²⁺, the metabotropic response to quis/AMPA and NMDA was synergistic (this being consistent with AMPA receptor-induced depolarization activating NMDA receptor). On the other hand, in Mg²⁺-free buffer the effects of quis and NMDA, at concentrations causing maximal PPI hydrolysis, were additive (indicating that PPI hydrolysis was effected by two different mechanisms). Thus, in cerebellar granule cells EAAs elicit PPI hydrolysis by acting at two distinct receptor types: (i) metabotropic Glu receptors (mGluR), with pharmacological characteristics suggesting the expression of a unique mGluR receptor that shows certain similarities to those observed for the mGluR1 subtype (Aramori and Nakanishi, 1992) and (ii) NMDA receptors. The physiological agonist, Glu, is able to stimulate both receptor classes.Abbreviations ACPD (1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid - AMPA -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid - AP₃ L-2-amino-3-phosphono-propionate - AP₅ D-2-amino-5-phosphonopentenoate - BMAA -methyl-amino-L-alanine - DIV days in vitro - DNOX 6,7-dinitroouinoxoline-2,3-dione - EAA excitatory amino acids - Glu glutamate - InsP inositol monophosphate - mGluR metabotropic glutamate receptors - MK-801 (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohept-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate - PPI polyphosphoinositide - quis quisqualate     

Localization of the mGlu4a metabotropic glutamate receptor in rat cerebellar cortex

 The subcellular localization of the mGlu4a metabotropic glutamate receptor was investigated in rat cerebellum. At the light microscopical level, strong mGlu4a immunoreactivity was found in the molecular layer. A post-embedding immunogold method for electron microscopy revealed gold particles at the presynaptic sites of synapses made by parallel fiber terminals with dendritic spines of Purkinje cells. These observations support electrophysiological evidence indicating an autoreceptor function of mGlu4 receptors at these synapses. Accepted: 1 July 1997     

Climbing fiber activation of metabotropic glutamate receptors on cerebellar purkinje neurons

In the cerebellum, metabotropic glutamate receptors (mGluRs) are required for distinct forms of synaptic plasticity expressed at parallel fiber (PF) and climbing fiber (CF) synapses. At PF synapses, mGluR activation generates a slow synaptic current and triggers intracellular calcium release; at CF synapses, mGluR activation has not been observed. This has led some investigators to propose that mGluR-dependent changes in CF synaptic strength are induced heterosynaptically. Here we describe an mGluR-mediated response to CF stimulation consisting of two parallel signaling pathways: one leading to a slow synaptic conductance and the other leading to internal calcium release. This additional target for glutamate broadens the signaling capabilities of CF synapses and raises the possibility that changes in CF strength are homosynaptically triggered.     

Development and function of metabotropic glutamate receptors in cat visual cortex.

Metabotropic glutamate receptors have been implicated in plasticity in the hippocampus and cerebellum. Are they also involved in plasticity in the visual cortex? This is a complicated question because of the diversity of metabotropic glutamate receptors and the variations in both receptors and plasticity with layer. Inhibition driven by group II metabotropic glutamate receptors is certainly correlated with ocular dominance segregation in layer IV of the cortex. Of the group I metabotropic glutamate receptors, mGluR5 may be involved in plasticity, but mGluR1 is unlikely to be. Both group I and group II receptors produce increases in cyclic adenosine monophosphate which are clearly related to plasticity. Further conclusions await the development of agonists and antagonists specific for individual metabotropic glutamate receptors, as opposed to groups of the receptors.     

Role of group I metabotropic glutamate receptors and NMDA receptors in homocysteine-evoked acute neurodegeneration of cultured cerebellar granule neurones

Hyperhomocysteinemia is a risk factor in neurodegeneration. It has been suggested that apart from disturbances in methylation processes, the mechanisms of this effect may include excitotoxicity mediated by the N-methyl-D-aspartate (NMDA) receptors. In this study we demonstrate that apart from NMDA receptors, also group I metabotropic glutamate receptors participate in acute homocysteine (Hcy)-induced neurotoxicity in cultured rat cerebellar granule neurones. Primary neuronal cultures were incubated for 30 min in the Mg(2+)-free ionic medium containing homocysteine and other ligands, and neurodegenerative changes were assessed 24h later using propidium iodide staining. D,L-Homocysteine given alone appeared to be a weak neurotoxin, with EC(50) of 17.4mM, whereas EC(50) for L-glutamate was 0.17 mM. Addition of 50 microM glycine enhanced homocysteine neurotoxicity, and only that portion of neurotoxicity was abolished by 0.5 microM MK-801, an uncompetitive NMDA receptor antagonist. The net stimulation of 45Ca uptake by granule cells incubated in the presence of 25 mM D,L-homocysteine with 50 microM glycine was only 3% of the net uptake evoked by 1mM glutamate. Application of an antagonist of group I metabotropic glutamate receptors (mGluRs) LY367385 at 25 and 250 microM concentrations, induced a dose-dependent partial neuroprotection, whereas given together with MK-801 completely prevented neurotoxicity. In the absence of glycine, LY367385 and MK-801 given alone failed to induce neuroprotection, while applied together completely prevented homocysteine neurotoxicity. Agonist of group I mGluRs, 10 trans-azetidine-2,3-dicarboxylic acid (t-ADA) induced significant neurotoxicity. This study shows for the first time that acute homocysteine-induced neurotoxicity is mediated both by group I mGluRs and NMDA receptors, and is not accompanied by massive influx of extracellular Ca(2+) to neurones.     

Extracellular glutamate diffusion determines the occupancy of glutamate receptors at CA1 synapses in the hippocampus

Following exocytosis at excitatory synapses in the brain, glutamate binds to several subtypes of postsynaptic receptors. The degree of occupancy of AMPA and NMDA receptors at hippocampal synapses is, however, not known. One approach to estimate receptor occupancy is to examine quantal amplitude fluctuations of postsynaptic signals in hippocampal neurons studied in vitro. The results of such experiments suggest that NMDA receptors at CA1 synapses are activated not only by glutamate released from the immediately apposed presynaptic terminals, but also by glutamate spillover from neighbouring terminals. Numerical simulations point to the extracellular diffusion coefficient as a critical parameter that determines the extent of activation of receptors positioned at different distances from the release site. We have shown that raising the viscosity of the extracellular medium can modulate the diffusion coefficient, providing an experimental tool to investigate the role of diffusion in activation of synaptic and extrasynaptic receptors. Whether intersynaptic cross-talk mediated by NMDA receptors occurs in vivo remains to be determined. The theoretical and experimental approaches described here also promise to shed light on the roles of metabotropic and kainate receptors, which often occur in an extrasynaptic distribution, and are therefore positioned to sense glutamate escaping from the synaptic cleft.     

Involvement of metabotropic glutamate receptors in excitatory amino acid and GABA release following spinal cord injury in rat

Spinal cord injury (SCI) leads to an increase in extracellular excitatory amino acid (EAA) concentrations resulting in glutamate receptor-mediated excitotoxic events. The glutamate receptors include ionotropic (iGluRs) and metabotropic (mGluR) receptors. Of the three groups of mGluRs, group-I activation can initiate intracellular pathways that lead to further transmitter release. Groups II and III mGluRs function mainly as autoreceptors to regulate neurotransmitter release. In an effort to examine the role of mGluRs in the increase in EAAs following SCI, we administered AIDA, a potent group-I mGluR antagonist immediately after injury. To determine subtype specific roles of the group-I mGluRs, we evaluated EAA release following LY 367385 (mGluR1 antagonist) and MPEP (mGluR5 antagonist) administration. To evaluate group-II and -III mGluRs we administered APDC (group-II agonist) and L-AP4 (group-III agonist) immediately following injury; additionally, we initiated treatment with CPPG (group-II/-III antagonist) and LY 341495 (group-II antagonist) 5 min prior to injury. Subjects were adult male Sprague-Dawley rats (225-250 g), impact injured at T10 with an NYU impactor (12.5 mm drop). Agents were injected into the epicenter of injury, amino acids where collected by microdialysis fibers inserted 0.5 mm caudal from the edge of the impact region and quantified by HPLC. Treatment with AIDA significantly decreased extracellular EAA and GABA concentrations. MPEP reduced EAA concentrations without affecting GABA. Combining LY 367385 and MPEP resulted in a decrease in EAA and GABA concentrations greater than either agent alone. L-AP4 decreased EAA levels, while treatment with LY 341495 increased EAA levels. These results suggest that mGluRs play an important role in EAA toxicity following SCI.     

High-resolution quantitative visualization of glutamate and GABA receptors at central synapses

Glutamate and GABA are the main transmitters in the central nervous system and their effects are mediated by ionotropic and metabotropic receptors. Immunogold electron microscopy has revealed the quantitative localization of these receptors at 20-30nm resolution. SDS-digested freeze-fracture replica labeling (SDS-FRL), a newly developed immunogold method, provides an accurate estimate of molecule numbers. Here, we summarize the recent advances in quantitative receptor localization, including use of SDS-FRL analyses to determine numbers of AMPA-type glutamate receptors in the cerebellum. The two-dimensional view and high sensitivity of SDS-FRL have revealed small, irregularly shaped AMPA receptor clusters within cerebellar synapses.     

Direct interaction enables cross-talk between ionotropic and group I metabotropic glutamate receptors

Functional interplay between ionotropic and metabotropic receptors frequently involves complex intracellular signaling cascades. The group I metabotropic glutamate receptor mGlu5a co-clusters with the ionotropic N-methyl-d-aspartate (NMDA) receptor in hippocampal neurons. In this study, we report that a more direct cross-talk can exist between these types of receptors. Using bioluminescence resonance energy transfer in living HEK293 cells, we demonstrate that mGlu5a and NMDA receptor clustering reflects the existence of direct physical interactions. Consequently, the mGlu5a receptor decreased NMDA receptor current, and reciprocally, the NMDA receptor strongly reduced the ability of the mGlu5a receptor to release intracellular calcium. We show that deletion of the C terminus of the mGlu5a receptor abolished both its interaction with the NMDA receptor and reciprocal inhibition of the receptors. This direct functional interaction implies a higher degree of target-effector specificity, timing, and subcellular localization of signaling than could ever be predicted with complex signaling pathways.     

代谢型谷氨酸受体在突触可塑性中的作用

突触可塑性是近几年神经科学研究的热点之一,因为它对于理解神经系统的学习、学习和记忆、多咱神经疾病等许多过程有着重要的意义。除了离子型谷氨酸受体外,代谢型谷氨酸受体也参与了一些脑区中不同形式的突触可塑性变化。本文就代谢型谷氨酸受体选择性激动剂和拮抗剂对长时程增强和长时程抑制的作用进行了综述,以助于人们进一步理解突触可塑性的细胞和分子机制。     

Group 1 metabotropic glutamate receptors 1 and 5 form a protein complex in mouse hippocampus and cortex

The group 1 metabotropic glutamate receptors 1 and 5 (mGluR1/5) have been implicated in mechanisms of synaptic plasticity and may serve as potential therapeutic targets in autism spectrum disorders. The interactome of group 1 mGluRs has remained largely unresolved. Using a knockout‐controlled interaction proteomics strategy we examined the mGluR5 protein complex in two brain regions, hippocampus and cortex, and identified mGluR1 as its major interactor in addition to the well described Homer proteins. We confirmed the presence of mGluR1/5 complex by (i) reverse immunoprecipitation using an mGluR1 antibody to pulldown mGluR5 from hippocampal tissue, (ii) coexpression in HEK293 cells followed by coimmunoprecipitation to reveal the direct interaction of mGluR1 and 5, and (iii) superresolution microscopy imaging of hippocampal primary neurons to show colocalization of the mGluR1/5 in the synapse.     

A Brownian model of glutamate diffusion in excitatory synapses of hippocampus

We simulated the diffusion of glutamate, following the release of a single vesicle from a pre-synaptic terminal, in the synaptic cleft by using a Brownian diffusion model based on Langevin equations. The synaptic concentration time course and the time course of quantal excitatory post-synaptic current have been analyzed. The results showed that they depend on the number of receptors located at post-synaptic membrane. Their time course are dependent both on the total number of the post-synaptic receptors and on the eccentricity of the pre-synaptic glutamate vesicle.     

Repeated exposure to cocaine alters the modulation of mesocorticolimbic glutamate transmission by medial prefrontal cortex Group II metabotropic glutamate receptors

Repeated cocaine exposure enhances glutamatergic output from the medial prefrontal cortex to subcortical brain regions. Loss of inhibitory control of cortical pyramidal neurons may partly account for this augmented cortical glutamate output. Recent research indicated that repeated cocaine exposure reduced the ability of cortical Group II metabotropic glutamate receptors to modulate behavioral and neurochemical responses to cocaine. Thus, experiments described below examined whether repeated cocaine exposure alters metabotropic glutamate receptor regulation of mesocorticolimbic glutamatergic transmission using in vivo microdialysis. Infusion of the Group II metabotropic glutamate receptor antagonist LY341495 into the medial prefrontal cortex enhanced glutamate release in this region, the nucleus accumbens and the ventral tegmental area in sensitized animals, compared to controls, following short-term withdrawal but not after long-term withdrawal. Additional studies demonstrated that vesicular (K(+)-evoked) and non-vesicular (cystine-evoked) glutamate release in the medial prefrontal cortex was enhanced in sensitized animals, compared to controls, that resulted in part from a reduction in Group II metabotropic glutamate receptor modulation of these pools of glutamate. In summary, these findings indicate that the expression of sensitization to cocaine is correlated with an altered modulation of mesocorticolimbic glutamatergic transmission via reduction of Group II metabotropic glutamate receptor function.     

Heterodimerization of calcium sensing receptors with metabotropic glutamate receptors in neurons

Calcium sensing (CaR) and Group I metabotropic glutamate receptors exhibit overlapping expression patterns in brain, and share common signal transduction pathways. To determine whether CaR and Group I metabotropic glutamate receptors (mGluRs) (mGluR1alpha and mGluR5) can form heterodimers, we immunoprecipitated CaR from bovine brain and observed co-precipitation of mGluR1alpha. CaR and mGluR1alpha co-localize in hippocampal and cerebellar neurons, but are expressed separately in other brain regions. In vitro transfection studies in HEK-293 cells established the specificity and disulfide-linked nature of the CaR:mGluR1alpha (CaR:mGluR5) interactions. CaR:mGluR1alpha (CaR:mGluR5) heterodimers exhibit altered trafficking via Homer 1c when compared with CaR:CaR homodimers. CaR becomes sensitive to glutamate-mediated internalization when present in CaR:mGluR1alpha heterodimers. These results demonstrate cross-family covalent heterodimerization of CaR with Group I mGluRs, and increase the potential role(s) for CaR in modulating neuronal function.     

代谢型谷氨酸受体在突触可塑性中的作用研究进展

突触可塑性是近 30年来神经科学领域的研究热点之一 ,它主要包括长时程增强 (long termpotentiation ,LTP)和长时程抑制 (long termdepression ,LTD)。以往的研究已经证实 ,离子型谷氨酸受体 (iGluRs)中的NMDA受体和AMPA受体 ,在LTP和LTD的诱导和维持中通过阳离子内流 ,引起细胞内的级联反应而起作用。新近的研究发现 ,代谢型谷氨酸受体 (mGluRs)与G蛋白偶联 ,通过细胞内的多种信使系统介导慢突触传递。本文主要就mGluRs在不同脑区LTP和LTD中的作用进行综述