Activation Of mGlu5 Receptors Research Articles

Presynaptic mGlu1 and mGlu5 autoreceptors facilitate glutamate exocytosis from mouse cortical nerve endings

The effects of mGlu1 and mGlu5 receptor activation on the depolarization-evoked release of [ 3H] d-aspartate ([ 3H] d-ASP) from mouse cortical synaptosomes were investigated. The mGlu1/5 receptor agonist 3,5-DHPG (0.1–100 μM) potentiated the K +(12 mM)-evoked [ 3H] d-ASP overflow. The potentiation occurred in a concentration-dependent manner showing a biphasic pattern. The agonist potentiated [ 3H] d-ASP exocytosis when applied at 0.3 μM; the efficacy of 3,5-DHPG then rapidly declined and reappeared at 30–100 μM. The fall of efficacy of agonist at intermediate concentration may be consistent with 3,5-DHPG-induced receptor desensitization. Facilitation of [ 3H] d-ASP exocytosis caused by 0.3 μM 3,5-DHPG was prevented by the selective mGlu5 receptor antagonist MPEP, but was insensitive to the selective mGlu1 receptor antagonist CPCCOEt. In contrast, CPCCOEt prevented the potentiation by 50 μM 3,5-DHPG, while MPEP had minimal effect. Unexpectedly, LY 367385 antagonized both the 3,5-DHPG-induced effects. A total of 0.3 μM 3,5-DHPG failed to facilitate the K +-evoked [ 3H] d-ASP overflow from mGlu5 receptor knockout (mGlu5 −/−) cortical synaptosomes, but not from nerve terminals prepared from the cortex of animals lacking the mGlu1 receptors, the crv4/crv4 mice. On the contrary, 50 μM 3,5-DHPG failed to affect the [ 3H] d-ASP exocytosis from cortical synaptosomes obtained from crv4/ crv4 and mGlu5 −/−mice. Western blot analyses in subsynaptic fractions support the existence of both mGlu1 and mGlu5 autoreceptors located presynaptically, while immunocytochemistry revealed their presence at glutamatergic terminals. We propose that mGlu1 and mGlu5 autoreceptors exist on mouse glutamatergic cortical terminals; mGlu5 receptors may represent the “high affinity” binding sites for 3,5-DHPG, while mGlu1 autoreceptors represent the “low affinity” binding sites.

Group I metabotropic glutamate receptors control proliferation, survival and differentiation of cultured neural progenitor cells isolated from the subventricular zone of adult mice

Neural progenitor cells (NPCs) are found in the subventricular zone (SVZ) of the adult brain, a specialized neurogenic niche that might provide a substrate for brain repair after injury. The incomplete knowledge of how NPCs in the niche respond to local signals limits the use of cultured NPCs in the development of cell transplantation strategies. We show that neurospheres obtained from the SVZ of the adult mouse expressed functional mGlu1 and mGlu5 metabotropic glutamate receptors. Pharmacological blockade of mGlu5 receptors promoted the apoptotic death of progenitors undergoing differentiation into neurons (PSA/NCAM + cells for the most part), whereas blockade of mGlu1 receptors reduced the proliferation rate of NPCs, and promoted their differentiation towards the neuronal lineage. We conclude that endogenous activation of mGlu5 receptors might support specifically the survival of neuronal-restricted precursors, whereas endogenous activation of mGlu1 receptors might sustain the proliferation of earlier progenitors. Moreover, mGlu1 receptor antagonists increased the survival of NPCs, suggesting that endogenously activated mGlu1 receptors might play a role in the natural cell loss regulating the number or the type of progenitors.

Activated Nuclear Metabotropic Glutamate Receptor mGlu5 Couples to Nuclear Gq/11 Proteins to Generate Inositol 1,4,5-Trisphosphate-mediated Nuclear Ca2+ Release

Recently we have shown that the metabotropic glutamate 5 (mGlu5) receptor can be expressed on nuclear membranes of heterologous cells or endogenously on striatal neurons where it can mediate nuclear Ca2+ changes. Here, pharmacological, optical, and genetic techniques were used to show that upon activation, nuclear mGlu5 receptors generate nuclear inositol 1,4,5-trisphosphate (IP3) in situ. Specifically, expression of an mGlu5 F767S mutant in HEK293 cells that blocks Gq/11 coupling or introduction of a dominant negative Galphaq construct in striatal neurons prevented nuclear Ca2+ changes following receptor activation. These data indicate that nuclear mGlu5 receptors couple to Gq/11 to mobilize nuclear Ca2+. Nuclear mGlu5-mediated Ca2+ responses could also be blocked by the phospholipase C (PLC) inhibitor, U73122, the phosphatidylinositol (PI) PLC inhibitor 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3), or by using small interfering RNA targeted against PLCbeta1 demonstrating that PI-PLC is involved. Direct assessment of inositol phosphate production using a PIP2/IP3 "biosensor" revealed for the first time that IP3 can be generated in the nucleus following activation of nuclear mGlu5 receptors. Finally, both IP3 and ryanodine receptor blockers prevented nuclear mGlu5-mediated increases in intranuclear Ca2+. Collectively, this study shows that like plasma membrane receptors, activated nuclear mGlu5 receptors couple to Gq/11 and PLC to generate IP3-mediated release of Ca2+ from Ca2+-release channels in the nucleus. Thus the nucleus can function as an autonomous organelle independent of signals originating in the cytoplasm, and nuclear mGlu5 receptors play a dynamic role in mobilizing Ca2+ in a specific, localized fashion.

Endocannabinoid Interactions

Noting that stimulation of metabotropic glutamate (mGlu) receptors attenuates release of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) in various brain regions by means of retrograde endocannabinoid signaling through presynaptic CB1 receptors (see Di Marzo and Cristino), Maccarrone et al . investigated endocannabinoid signaling in the striatum. A combination of pharmacological and electrophysiological analysis of striatal slices revealed that activation of mGlu5 receptors with 3,5-dihydroxyphenylglycine (DHPG) led to the CB1-dependent inhibition of GABA release. There are two principal endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG), and DHPG increased 2-AG concentration, but not that of anandamide, by stimulating activity of the 2-AG synthetic enzyme diacylglycerol lipase (DAGL) and inhibiting activity of the 2-AG degrading enzyme monoacylglycerol lipase (MAGL). Although functional interactions between anandamide and 2-AG had not previously been shown, pharmacological inhibition of fatty acid amide hydrolase (FAAH, the enzyme that degrades anandamide) blocked the DHPG-mediated increase in 2-AG and prevented its effects on GABAergic transmission, as did FAAH knockout. The stable anandamide derivative methanandamide also blocked DHPG-mediated increase in 2-AG and decreased basal 2-AG. Anandamide activates transient receptor potential vanilloid 1 (TRPV1) channels, and the TRPV1 agonist capsaicin inhibited the effects of DHPG on 2-AG concentration and GABAergic transmission as well, whereas pharmacological inhibition of FAAH and capsaicin failed to block the effects of DHPG on 2-AG and GABAergic transmission in mice lacking TRPV1. Manipulations that increased anandamide concentration were associated with decreased DAGL activity, and capsaicin blocked a DHPG-dependent increase in striatal glutathione (which stimulates DAGL). Thus, anandamide can inhibit the production and effects of striatal 2-AG through activation of TRPV1, effects that seem to involve a glutathione-dependent pathway. M. Maccarrone, S. Rossi, M. Bari, V. De Chiara, F. Fezza, A. Musella, V. Gasperi, C. Prosperetti, G. Bernardi, A. Finazzi-Agrò, B. F Cravatt, D. Centonze, Anandamide inhibits metabolism and physiological actions of 2-arachidonoylglycerol in the striatum. Nat. Neurosci. 11 , 152-159 (2008). [PubMed] V. Di Marzo, L. Cristino, Why endocannabinoids are not all alike. Nat. Neurosci. 11 , 124-126 (2008). [PubMed]

Modulation by group I mGLU receptor activation and group III mGLU receptor blockade of locomotor responses induced by D1-like and D2-like receptor agonists in the nucleus accumbens

Evidence for functional motor interactions between group I and group III metabotropic glutamatergic (mGlu) receptors and dopamine neurotransmission is now clearly established [David, H.N., Abraini, J.H., 2001a. The group I metabotropic glutamate receptor antagonist S-4-CPG modulates the locomotor response produced by the activation of D1-like, but not D2-like, dopamine receptors in the rat nucleus accumbens. Eur. J. Neurosci. 15, 2157–2164, David, H.N., Abraini, J.H., 2002. Group III metabotropic glutamate receptors and D1-like and D2-like dopamine receptors interact in the rat nucleus accumbens to influence locomotor activity. Eur. J. Neurosci. 15, 869–875]. Nevertheless, whether or not and how, activation of group I and blockade of group III mGlu receptors modulate the motor responses induced by the activation of dopaminergic receptors in the NAcc still remains unknown. Answering this question needs to be assessed since functional interactions between neurotransmitters in the NAcc are well known to depend upon the level of activation of glutamatergic and/or dopaminergic receptors and because the effects of glutamatergic receptor agonists and antagonists on dopaminergic receptor-mediated locomotor responses are not always reciprocal as shown in previous studies. Our results show that activation of group I mGlu receptors by DHPG in the NAcc potentiated the locomotor response induced by intra-NAcc activation of D1-like receptors and blocked those induced by D2-like presynaptic or postsynaptic receptors. Alternatively, blockade of group III mGlu receptors by MPPG in the NAcc potentiated the locomotor responses mediated by D1-like receptors and by D2-like postsynaptic receptors and inhibited that induced by D2-like presynaptic receptors. These results compiled with previous data demonstrate that group I mGlu receptors and group III mGlu receptors can modulate the locomotor responses produced by D 1-like and/or D 2-like receptor agonists in a complex phasic and tonic fashion.

Activation of NF-κB p65/c-Rel dimer is associated with neuroprotection elicited by mGlu5 receptor agonists against MPP+ toxicity in SK-N-SH cells

Nuclear factor-kappaB (NF-kappaB) is a transcriptional regulator of neuron survival eliciting diverse effects according to the specific composition of its active dimer. While p50/p65 mediates neurodegenerative events, c-Rel-containing dimers promote cell survival. Stimulation of metabotropic glutamate receptors type 5 (mGlu5) reduces neuron vulnerability to amyloid-beta through activation of anti-apoptotic, c-Rel-dependent transcription of Bcl-X(L) pathway. We here evaluated the protective activity of mGlu5 agonists in dopaminergic SK-N-SH cells exposed to 1-methyl-4-phenylpyridinium (MPP(+)), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causing parkinsonism in experimental animals. MPP(+) produced a concentration-dependent cell loss. Activation of mGlu5 receptors by CHPG (1 mM) and 3HPG (50 microM) abolished the toxic effect produced by 3 microM MPP(+). The neuroprotection was associated with activation of NF-kappaB p65/c-Rel dimer and reduction of p50/p65. These effects were prevented by the mGlu5 receptor antagonist MPEP (5 microM). It is suggested that mGlu5 receptor agonists through activation of a c-Rel-dependent anti-apoptotic pathway can rescue dopaminergic cell from mitochondrial toxicity.

Development of Metabotropic Glutamate Receptor Ligands for Neuroimaging

Metabotropic glutamate receptors (mGluRs) belong to a superfamily of G protein coupled receptors. In the central nervous system (CNS), mGluRs modulate glutamatergic neurotransmission and are considered to affect also on dopaminergic and adrenergic neurotransmission. MGluRs have various physiological functions in CNS and are suggested to be involved in a number of neurological disorders. Receptor ligands binding to mGluRs are also considered as potential therapeutic agents. Classic competitive ligands, which are amino acid derivatives, bind to the orthosteric binding site as endogenous glutamate. It is believed that developing highly selective competitive agonists and antagonists for specific mGluR subtypes has been exceedingly difficult, because of the high conservation of the glutamate binding site across members of this receptor family and the restricted structural requirements for pharmacophores that occupy the binding pocket. Another shortcoming of this kind of ligands is that the carboxyl and amino groups make them too polar to penetrate the blood brain barrier. Although these ligands have had and still have important roles in elucidating physiological and pathophysiological properties of mGluRs, their poor selectivity within group presupposes some knowledge of complexity. In addition, their poor CNS penetration limits their candidacy as promising radiotracers for in vivo imaging of mGluRs. Over the last decade, a number of non-competitive mGluR ligands, which are structurally diverse and bind to mGluRs in the allosteric binding sites located in the seven strand transmembrane domain, have been identified. These include negative, positive and neutral allosteric modulators. Modulating mGlu receptor activity through an allosteric binding site is a relatively new strategy with the first example appeared in 1996. Based on these modulators, a number of radiotracers useful for imaging specific metabotropic glutamate receptors have been developed and their in vivo biological properties have been characterized. We have developed five ligands for PET imaging of metabotropic glutamate subgroup 5 receptors and investigated mGluR functions in animal models of different neurodegenerative disorders. In this review we evaluate our data in comparison to the theoretical and experimental aspects of developing selective and sensitive imaging ligands for in vivo imaging. Emphasis will be given to those highly potent and subtype selective allosteric modulators which are good candidates for radiolabeling with fluorine-18 and carbon-11. Keywords: Allosteric modulator, glutamate, metabotropic glutamate receptor, mGluR5, positron emission tomography, tracer

Group I Metabotropic Glutamate Receptors: A Role in Neurodevelopmental Disorders?

Group I metabotropic glutamate receptors (mGlu1 and mGlu5) are coupled to polyphosphoinositide hydrolysis and are involved in activity-dependent forms of synaptic plasticity, both during development and in the adult life. Group I mGlu receptors can also regulate proliferation, differentiation, and survival of neural stem/progenitor cells, which further support their role in brain development. An exaggerated response to activation of mGlu5 receptors may underlie synaptic dysfunction in Fragile X syndrome, the most common inherited form of mental retardation. In addition, group I mGlu receptors are overexpressed in dysplastic neurons of focal cortical dysplasia and hemimegaloencephaly, which are disorders of cortical development associated with chronic epilepsy. Drugs that block the activity of group I mGlu receptors (in particular, mGlu5 receptors) are potentially helpful for the treatment of Fragile X syndrome and perhaps other neurodevelopmental disorders.

Evidence for a role for the group I metabotropic glutamate receptor in the inhibitory effect of tumor necrosis factor-α on long-term potentiation

Pro-inflammatory cytokines are known to be elevated in several neuropathological states that are associated with learning and memory. We have previously demonstrated in our laboratory that the inhibition of long-term potentiation (LTP) in the dentate gyrus region of the rat hippocampus, by tumor necrosis factor (TNF)-α, represents a biphasic response, an early phase dependent on p38 mitogen activated protein kinase (MAPK) activation and a later phase, possible dependent on protein synthesis. Many of the factors involved in the early modulation of LTP by TNF-α have yet to be elucidated. This study investigated if metabotropic glutamate receptors (mGluRs) are functionally linked to the inhibitory effect of TNF-α on LTP in the rat dentate gyrus in vitro. We report that the impairment of early-LTP by TNF-α is significantly attenuated by prior application of the group I/II mGluR antagonist MCPG and more specifically the mGluR5 antagonist MPEP. Since TNF-α is now known to cause transient increases in intracellular Ca 2+ levels from ryanodine-sensitive stores, we explored the possibility that disruption of intracellular Ca 2+ homeostasis could be involved. Ryanodine was found to significantly reverse the inhibition of LTP by TNF-α. From these studies we propose that the TNF-α inhibition of LTP is dependent upon the activation of TNFR1 and mGlu5-receptors. Importantly this study provides the first proof of the involvement of ryanodine-sensitive intracellular Ca 2+ stores in TNF-α mediated inhibition of LTP.

Blockade of metabotropic glutamate receptor 5 activation inhibits mechanical hypersensitivity following abdominal surgery

This study used the metabotropic glutamate 5 (mGlu5) receptor subtype-selective antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) to characterise the contribution of mGlu5 receptor activity to pain and hypersensitivity in an animal model of post-surgical pain. Adult female Wistar rats (200–250 g) were anaesthetised with isoflurane (2%) and underwent a midline laparotomy with gentle manipulation of the viscera, and the effects of pre- (30 min) or post- (5 h) operative treatment with MPEP (1, 3 or 10 mg kg −1; i.p.) or drug-vehicle on hindpaw withdrawal latency (in seconds) to thermal stimulation (Hargreave’s Test) and response threshold (in grams) to mechanical stimulation (using a dynamic plantar aesthesiometer) were measured. Animals that underwent surgery displayed significant hypersensitivity to mechanical stimulation of the hindpaws. Hypersensitivity was maximum at 6 h post-surgery (44.5 ± 2.4% decrease; p < 0.01 vs. anaesthesia only controls) and persisted for 48 h. Surgery had no effect on thermal withdrawal latency. Both pre-operative and post-operative administration of 10 mg kg −1MPEP blocked mechanical hypersensitivity induced by surgery ( p < 0.01 vs. vehicle treatment). MPEP had no effect on acute nociceptive thresholds in naïve animals. These data suggest that activity at mGlu5 receptors contributes to development of pain and hypersensitivity following surgery.

Developmental switch from LTD to LTP in low frequency‐induced plasticity

The stimulation of the Schaffer collateral/commissural fibers at low frequency (1 Hz) for 3-5 min can trigger a slow-onset form of low-frequency stimulation (LFS)-long-term potentiation (LTP) (LFS-LTP) in the CA1 area of the adult rat hippocampus. Here we have examined the developmental profile of this plasticity. In 9-15 day-old rats, the application of 1 Hz for 5 min induced long-term depression (LFS-LTD). In 17-21 day-old rats, 1 Hz stimulation had no effect when applied for 5 min but mediated LTD when stimulus duration was increased to 15 min. Over 25 day-old, 1 Hz stimulation mediated LFS-LTP. LFS-LTD was dependent on both N-methyl-D-aspartate (NMDA) and mGlu5 receptor activation. Antagonists of mGlu1alpha and cannabinoid type 1 receptor were ineffective to block LTD induction. LFS-LTD was not associated with a change in paired-pulse facilitation ratio, suggesting a postsynaptic locus of expression of this plasticity. Next, we examined whether LFS-LTD was related to 'chemical' LTDs obtained by the direct stimulation of mGlu5 and NMDA receptors. The saturation of LFS-LTD completely occluded NMDA- and (RS)-2-Chloro-5-hydroxyphenylglycine (CHPG)-induced LTD. CHPG-LTD and NMDA-LTD occluded each other. In addition, we observed that NMDA-LTD was dependent on mGlu5 receptor activation in 9-12 day old rats while it was not in animals older than 15 day-old. Therefore we postulate that during LFS application, NMDA and mGlu5 receptor could interact to trigger LTD. Low-frequency-mediated synaptic plasticity is subject to a developmental switch from NMDA- and mGlu5 receptor-dependent LTD to mGlu5 receptor-dependent LTP with a transient period (17-21 day-old) during which LFS is ineffective.

Endogenously activated mGlu5 metabotropic glutamate receptors sustain the increase in c‐Myc expression induced by leukaemia inhibitory factor in cultured mouse embryonic stem cells

We have shown that endogenous activation of type 5 metabotropic glutamate (mGlu5) receptors supports the maintenance of a pluripotent, undifferentiated state in D3 mouse embryonic stem cells cultured in the presence of leukaemia inhibitory factor (LIF). Here, we examined the interaction between LIF and mGlu5 receptors using as a read-out the immediate early gene, c-Myc. The selective mGlu5 receptor antagonist, 2-methyl-6-(phenylenthynyl)pyridine (MPEP; 1 mum), reduced the increase in c-Myc protein levels induced by LIF by enhancing c-Myc ubiquitination. A reduction in c-Myc levels was also observed following small interfering RNA-mediated mGlu5 receptor gene silencing. MPEP reduced glycogen synthase kinase-3beta phosphorylation on Ser9, but increased phosphorylation of the phosphatidylinositol-3-kinase (PI-3-K) substrate, AKT. In our hands, activated PI-3-K reduced the stability of c-Myc, because (i) the PI-3-K inhibitor, LY294002, prevented the reduction in c-Myc levels induced by MPEP; and (ii) over-expression of AKT promoted c-Myc ubiquitination. All effects of MPEP were mimicked by protein kinase C (PKC) inhibitors and reversed by the PKC activator, tetradecanoylphorbol-13-acetate. We conclude that endogenous activation of mGlu5 receptors sustains the increase in c-Myc induced by LIF in embryonic stem cells by inhibiting both glycogen synthase kinase-3beta and PI-3-K, both effects resulting from the activation of PKC.

Group III metabotropic glutamate receptor-mediated, chemically induced long-term depression differentially affects cell viability in the hippocampus

In vivo, activation of group III metabotropic glutamate (mGlu) receptors leads to a reduction of basal synaptic transmission in the hippocampus, and depending on the experimental conditions in vitro, leads to neuroprotection or neurotoxicity. Here, the cellular response to cerebral application of l(+)-2-amino-4-phosphonobutanoic acid (AP4) was investigated in the CA1 region and dentate gyrus of freely moving rats. Drugs were applied via the lateral ventricle, and electrophysiological measurements were obtained via chronically implanted electrodes. AP4 produced a slowly developing depression of evoked responses in both hippocampal regions which lasted for over 4 h. Effects could be reversed by application of high frequency tetanus. Histological evaluation, 4 h or 7 d, following a single, acute AP4 injection into the lateral cerebral ventricle showed that AP4-mediated synaptic depression either amplified (CA1) or attenuated (dentate gyrus) excitotoxic neuronal death, strongly depending on the sub-region investigated. Effects were long-lasting, being still evident 7 days after AP4 application. In both hippocampal areas, the effects obtained were subtle, however, with the CA1 region being more potently affected. Interestingly, effects in the dentate gyrus comprised a slight enhancement of live cell number coupled with deterioration in cell area, suggesting that cell proliferation triggered by group III mGlu receptor activation may have masked neurotoxic effects mediated by activation of this receptor. These results show that although AP4 induces a slow-onset synaptic depression in both sub-regions, cell viability is differentially influenced by activation of group III mGlu receptors in the CA1 region and dentate gyrus.

Insulin Secretion Is Controlled by mGlu5 Metabotropic Glutamate Receptors

Recent evidence suggests that metabotropic glutamate (mGlu) receptors are involved in the regulation of hormone secretion in the endocrine pancreas. We report here that endogenous activation of mGlu5 receptors is required for an optimal insulin response to glucose both in clonal beta-cells and in mice. In clonal beta-cells, mGlu5 receptors were expressed at the cell surface and were also found in purified insulin-containing granules. These cells did not respond to a battery of mGlu5 receptor agonists that act extracellularly, but instead responded to a cell-permeant analog of glutamate with an increase in [Ca2+]i and insulin secretion. Both effects were largely attenuated by the mGlu5 receptor antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP). MPEP and its structural analog, (E)-2-methyl-6-styryl-pyridine (SIB-1893), reduced the increase in [Ca2+]i and insulin secretion induced by glucose in clonal beta-cells, whereas a mGlu1 receptor antagonist was inactive. mGlu5 knockout mice showed a defective insulin response at all times after a glucose pulse (1.5 g/kg, i.p.), whereas wild-type mice treated with MPEP (10 mg/kg, i.p.) showed a selective impairment in the late phase of insulin secretion in response to glucose challenge. Mice injected with MPEP or lacking mGlu5 receptors also showed a blunted glucagon response to an insulin challenge. We conclude that insulin secretion is under the control of mGlu5 receptors both in clonal beta-cells and in vivo. Drugs that modulate the function of mGlu5 receptors might affect glucose homeostasis by altering the secretion of pancreatic hormones.

Among the twenty classical l-amino acids, only glutamate directly activates metabotropic glutamate receptors

Under pathophysiological conditions, cellular amino acids can be profusely released from cells into the cerebral interstitial space. Because several class-C G protein coupled receptors (GPCRs) display a broad natural ligand spectrum, being sensitive to more than one endogenous ligand, we wondered whether the related metabotropic glutamate (mGlu) receptors could be modulated by various types of l-amino acids, allowing them to sense large increase in extracellular amino acid concentration. Here, the agonist, antagonist and allosteric effects of the twenty classical l-amino acids were evaluated on the eight mGlu receptor subtypes. We show that, in addition to glutamate (Glu), cysteine, aspartate and asparagine also lead to the activation of mGlu3, 4 and 5. Interestingly, our data demonstrate that the effect of these three amino acids did not result from a direct activation of the receptors, but from an indirect action involving Glu-transporters/exchangers. These data first demonstrate that mGlu receptors, unlike other class-C GPCRs, display an extremely high selectivity towards one ligand. Moreover, our results also show that Glu transport systems allow mGlu receptors to sense large increase in the extracellular concentration of some amino acids. Such a system will certainly lead to a large increase in some mGlu receptor activity under pathological conditions, such as seizure, ischemia or other brain injuries.

Group-II metabotropic glutamate receptors negatively modulate NMDA transmission at striatal cholinergic terminals: Role of P/Q-type high voltage activated Ca ++ channels and endogenous dopamine

Striatal cholinergic nerve terminals express functional group-II metabotropic (mGlu) and NMDA glutamate receptors. To investigate whether these receptors interact to regulate ACh release, LY354740 (a group-II mGlu receptor agonist) and NMDA were co-applied in striatal synaptosomes and slices. LY354740 prevented the NMDA-evoked [ 3H]-choline release from synaptosomes and ACh release from slices. In synaptosomes, this modulation was prevented by ω-agatoxin IVA, suggesting that it was mediated by P/Q-type high voltage activated Ca ++ channels. In slices, LY341495 (a group-II mGlu receptor antagonist) enhanced the NMDA-induced ACh release, suggesting that group-II mGlu receptor activation by endogenous glutamate inhibits NMDA transmission. Co-immunoprecipitation studies excluded direct group-II mGlu-NMDA receptor interactions. Finally, group-II mGlu negative modulation of NMDA transmission was abolished in dopamine-depleted synaptosomes and slices, suggesting that it relied on endogenous dopamine. We conclude that group-II mGlu receptors attenuate NMDA inputs at striatal cholinergic terminals via Ca ++ channel modulation and dopamine-sensitive pathways.

Altered Expression of Gq/11α Protein Shapes mGlu1 and mGlu5 Receptor-Mediated Single Cell Inositol 1,4,5-Trisphosphate and Ca2+Signaling

The metabotropic glutamate (mGlu) receptors mGlu1 and mGlu5 mediate distinct inositol 1,4,5-trisphosphate (IP(3)) and Ca(2+) signaling patterns, governed in part by differential mechanisms of feedback regulation after activation. Single cell imaging has shown that mGlu1 receptors initiate sustained elevations in IP(3) and Ca(2+), which are sensitive to agonist concentration. In contrast, mGlu5 receptors are subject to cyclical PKC-dependent uncoupling and consequently mediate coincident IP(3) and Ca(2+) oscillations that are largely independent of agonist concentration. In this study, we investigated the contribution of G(q/11)alpha protein expression levels in shaping mGlu1/5 receptor-mediated IP(3) and Ca(2+) signals, using RNA interference (RNAi). RNAi-mediated knockdown of G(q/11)alpha almost abolished the single-cell increase in IP(3) caused by mGlu1 and mGlu5 receptor activation. For the mGlu1 receptor, this unmasked baseline Ca(2+) oscillations that persisted even at maximal agonist concentrations. mGlu5 receptor-activated Ca(2+) oscillations were still observed but were only initiated at high agonist concentrations. Recombinant overexpression of G(q)alpha enhanced IP(3) signals after mGlu1 and mGlu5 receptor activation. It is noteworthy that although mGlu5 receptor-mediated IP(3) and Ca(2+) oscillations in control cells were largely insensitive to agonist concentration, increasing G(q)alpha expression converted these oscillatory signatures to sustained plateau responses in a high proportion of cells. In addition to modulating temporal Ca(2+) signals, up- or down-regulation of G(q/11)alpha expression alters the threshold for the concentration of glutamate at which a measurable Ca(2+) signal could be detected. These experiments indicate that altering G(q/11)alpha expression levels differentially affects spatiotemporal aspects of IP(3) and Ca(2+) signaling mediated by the mGlu1 and mGlu5 receptors.

Functional Metabotropic Glutamate Receptors on Nuclei from Brain and Primary Cultured Striatal Neurons: ROLE OF TRANSPORTERS IN DELIVERING LIGAND

G-protein-coupled receptors are well known for converting an extracellular signal into an intracellular response. Here we showed that the metabotropic glutamate receptor 5 (mGlu5) plays a dynamic intracellular role in signal transduction. Activation of endogenously expressed mGlu5 on striatal nuclear membranes leads to rapid, sustained calcium (Ca2+) responses within the nucleoplasm that can be blocked by receptor-specific antagonists. Extracellular ligands such as glutamate and quisqualate reach nuclear receptors via both sodium-dependent transporters and cystine glutamate exchangers. Inhibition of either transport system blocks radiolabeled agonist uptake as well as agonist-induced nuclear Ca2+ changes. Impermeable antagonists like LY393053 and LY367366 not only blocked [3H]quisqualate binding but also prevented nontransported agonists such as (RS)-3,5-dihydroxyphenylglycine from inducing intracellular Ca2+ changes in heterologous cells. In contrast, neither LY compound prevented quisqualate or glutamate from activating intracellular receptors leading to Ca2+ responses. Inasmuch as Ca2+ can enter the nucleoplasm via the nuclear pore complex or from the nuclear lumen, the presence of nuclear mGlu5 receptors appeared to amplify the latter process generating a faster nuclear response in heterologous cells. In isolated striatal nuclei, nuclear receptor activation results in the de novo appearance of phosphorylated CREB protein. Thus, activation of nuclear mGlu5 receptors initiates a signaling cascade that is known to alter gene transcription and regulate many paradigms of synaptic plasticity. These studies demonstrated that mGlu5 receptors play a dynamic role in signaling both on and off the plasma membrane.

Stimulation of Endocannabinoid Formation in Brain Slice Cultures through Activation of Group I Metabotropic Glutamate Receptors

Activation of group I metabotropic glutamate (mGlu) receptors drives the endocannabinoid system to cause both short- and long-term changes of synaptic strength in the striatum, hippocampus, and other brain areas. Although there is strong electrophysiological evidence for a role of endocannabinoid release in mGlu receptor-dependent plasticity, the identity of the endocannabinoid transmitter mediating this phenomenon remains undefined. In this study, we show that activation of group I mGlu receptors triggers the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG), but not anandamide, in primary cultures of corticostriatal and hippocampal slices prepared from early postnatal rat brain. Pharmacological studies suggest that 2-AG biosynthesis is initiated by activation of mGlu5 receptors, is catalyzed by phospholipase C (PLC) and 1,2-diacylglycerol lipase (DGL) activities, and is dependent on intracellular Ca2+ ions. Realtime polymerase chain reaction and immunostaining analyses indicate that DGL-beta is the predominant DGL isoform expressed in corticostriatal and hippocampal slices and that this enzyme is highly expressed in striatal neurons, where it is colocalized with PLC-beta1. The results suggest that 2-AG is a primary endocannabinoid mediator of mGlu receptor-dependent neuronal plasticity.

Endogenous activation of metabotropic glutamate receptors supports the proliferation and survival of neural progenitor cells

The use of neural progenitor cells (NPCs) is limited by the incomplete knowledge of the extracellular signals regulating their proliferation and survival. We report that cultured mouse NPCs express functional mGlu3 and mGlu5 metabotropic glutamate receptors. Pharmacological blockade of both receptors reduced NPC proliferation and survival, whereas activation of mGlu5 receptors substantially enhanced cell proliferation. Adult mice lacking mGlu5 receptors or treated with mGlu5 or mGlu3 receptor antagonists showed a dramatic reduction in the number of dividing neuroprogenitors present in the subventricular zone and in the dentate gyrus of the hippocampus. These data disclose a novel function of mGlu receptors and offer new potential strategies for the optimization of cell replacement therapy in neurodegenerative disorders.