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Metabotropic glutamate receptor subtype 5 (mGlu5) is implicated in the pathophysiology of Alzheimer´s disease (AD). However, its alteration at the subcellular level in neurons is still unexplored. Here, we provide a quantitative description on the expression and localisation patterns of mGlu5 in the APP/PS1 model of AD at 12 months of age, combining immunoblots, histoblots and high-resolution immunoelectron microscopic approaches. Immunoblots revealed that the total amount of mGlu5 protein in the hippocampus, in addition to downstream molecules, i.e., Gq/11 and PLCβ1, was similar in both APP/PS1 mice and age-matched wild type mice. Histoblots revealed that mGlu5 expression in the brain and its laminar expression in the hippocampus was also unaltered. However, the ultrastructural techniques of SDS-FRL and pre-embedding immunogold demonstrated that the subcellular localisation of mGlu5 was significantly reduced along the neuronal surface of hippocampal principal cells, including CA1 pyramidal cells and DG granule cells, in APP/PS1 mice at 12 months of age. The decrease in the surface localisation of mGlu5 was accompanied by an increase in its frequency at intracellular sites in the two neuronal populations. Together, these data demonstrate, for the first time, a loss of mGlu5 at the plasma membrane and accumulation at intracellular sites in different principal cells of the hippocampus in APP/PS1 mice, suggesting an alteration of the excitability and synaptic transmission that could contribute to the cognitive dysfunctions in this AD animal model. Further studies are required to elucidate the specificity of mGlu5-associated molecules and downstream signalling pathways in the progression of the pathology.

Perinatal hypoxia causes long-term neurobiological consequences, including alterations in mechanisms of activity-dependent synaptic plasticity and cognitive dysfunction. Changes in neurotransmitter receptors have been associated with these alterations, but little is known on how early hypoxia influences the expression and function of metabotropic glutamate (mGlu) receptors in adult life. This is an important issue because mGlu receptors are implicated in mechanisms of synaptic plasticity. Here, we examined the expression of mGlu1, mGlu5, and mGlu2/3 receptor subtypes in the hippocampus, nucleus accumbens, prefrontal cortex, and dorsal striatum in 6-month old Wistar rats (a) born by vaginal delivery; (b) born by caesarean section; and (c) born by caesarean section followed by 20 min of asphyxia. Unexpectedly, we found a large reduction of mGlu1α protein levels in the hippocampus of rats born by caesarean section regardless of the presence of asphyxia. No changes in mGlu1α receptor protein levels were found in the other brain regions. Levels of mGlu5 and mGlu2/3 receptors and levels of GluA2/3 and GluN1 subunits of AMPA and NMDA receptors did not differ among the three groups of rats in any brain region. These results are consistent with previous findings showing that changes in mGlu1 receptors occur within the epigenetic programming caused by early-life events.

Fabry disease (FD) is a X-linked lysosomal storage disorder caused by deficient function of the alpha-galactosidase A (α-GalA) enzyme. α-GalA deficiency leads to multisystemic clinical manifestations caused by the preferential accumulation of globotriaosylceramide (Gb3). A hallmark symptom of FD patients is neuropathic pain that appears in the early stage of the disease as a result of peripheral small fiber damage. Previous studies have shown that Acetyl-L-carnitine (ALC) has neuroprotective, neurotrophic, and analgesic activity in animal models of neuropathic pain. To study the action of ALC on neuropathic pain associated with FD, we treated α-GalA gene null mice (α-GalA(-/0)) with ALC for 30 days. In α-Gal KO mice, ALC treatment induced acute and long-lasting analgesia, which persisted 1 month after drug withdrawal. This effect was antagonized by single administration of LY341495, an orthosteric antagonist of mGlu2/3 metabotropic glutamate receptors. We also found an up-regulation of mGlu2 receptors in cultured DRG neurons isolated from 30-day ALC-treated α-GalA KO mice. However, the up-regulation of mGlu2 receptors was no longer present in DRG neurons isolated 30 days after the end of treatment. Taken together, these findings suggest that ALC induces analgesia in an animal model of FD by up-regulating mGlu2 receptors, and that analgesia is maintained by additional mechanisms after ALC withdrawal. ALC might represent a valuable pharmacological strategy to reduce pain in FD patients.

Acid-sensing ion channels (ASICs), members of the degenerin/epithelial Na+ channel superfamily, are widely distributed in the mammalian nervous system. ASIC1a are highly permeable to Ca2+ and are thought to be important in a variety of physiological processes, including synaptic plasticity, learning and memory. To further understand the role of ASIC1a in synaptic transmission and plasticity, we investigated metabotropic glutamate (mGlu) receptor-dependent long-term depression (LTD) in the hippocampus. We found that ASIC1a channels mediate a component of LTD in P30-40 animals, since the ASIC1a selective blocker psalmotoxin-1 (PcTx1) reduced the magnitude of LTD induced by application of the group I mGlu receptor agonist (S)-3,5-Dihydroxyphenylglycine (DHPG) or induced by paired-pulse low frequency stimulation (PP-LFS). Conversely, PcTx1 did not affect LTD in P13-18 animals. We also provide evidence that ASIC1a is involved in group I mGlu receptor-induced increase in action potential firing. However, blockade of ASIC1a did not affect DHPG-induced polyphosphoinositide hydrolysis, suggesting the involvement of some other molecular partners in the functional crosstalk between ASIC1a and group I mGlu receptors. Notably, PcTx1 was able to prevent the increase in GluA1 S845 phosphorylation at the post-synaptic membrane induced by group I mGlu receptor activation. These findings suggest a novel function of ASIC1a channels in the regulation of group I mGlu receptor synaptic plasticity and intrinsic excitability.

Chronic stress-related psychiatric conditions such as anxiety, depression, and alcohol abuse are an enormous public health concern. The etiology of these pathologies is complex, with psychosocial stressors being among the most frequently discussed risk factors. The brain glutamatergic neurotransmitter system has often been found involved in behaviors and pathophysiologies resulting from acute stress and fear. Despite this, relatively little is known about the role of glutamatergic system components in chronic psychosocial stress, neither in rodents nor in humans. Recently, drug discovery efforts at the metabotropic receptor subtypes of the glutamatergic system (mGlu1-8 receptors) led to the identification of pharmacological tools with emerging potential in psychiatric conditions. But again, the contribution of individual mGlu subtypes to the manifestation of physiological, molecular, and behavioral consequences of chronic psychosocial stress remains still largely unaddressed. The current review will describe animal models typically used to analyze acute and particularly chronic stress conditions, including models of psychosocial stress, and there we will discuss the emerging roles for mGlu receptor subtypes. Indeed, accumulating evidence indicates relevance and potential therapeutic usefulness of mGlu2/3 ligands and mGlu5 receptor antagonists in chronic stress-related disorders. In addition, a role for further mechanisms, e.g. mGlu7-selective compounds, is beginning to emerge. These mechanisms are important to be analyzed in chronic psychosocial stress paradigms, e.g. in the chronic subordinate colony housing (CSC) model. We summarize the early results and discuss necessary future investigations, especially for mGlu5 and mGlu7 receptor blockers, which might serve to suggest improved therapeutic strategies to treat stress-related disorders.

We examined the interaction between ephrins and metabotropic glutamate (mGlu) receptors in the developing brain and cultured neurons. EphrinB2 coimmunoprecipitated with mGlu1a receptors, in all of the brain regions examined, and with mGlu5 receptors in the corpus striatum. In striatal slices, activation of ephrinB2 by a clustered form of its target receptor, EphB1, amplified the mGlu receptor-mediated stimulation of polyphosphoinositide (PI) hydrolysis. This effect was abolished in slices treated with mGlu1 or NMDA receptor antagonists but was not affected by pharmacological blockade of mGlu5 receptors. An interaction among ephrinB2, mGlu1 receptor, and NMDA was supported by the following observations: (1) the NR1 subunit of NMDA receptors coimmunoprecipitated with mGlu1a receptors and ephrinB2 in striatal lysates; (2) clustered EphB1 amplified excitatory amino acid-stimulated PI hydrolysis in cultured granule cells grown under conditions that favored the expression of mGlu1a receptors; and (3) clustered EphB1 amplified the enhancing effect of mGlu receptor agonists on NMDA toxicity in cortical cultures, and its action was sensitive to mGlu1 receptor antagonists. Finally, fluorescence resonance energy transfer and coclustering analysis in human embryonic kidney 293 cells excluded a physical interaction between ephrinB2 and mGlu1a (or mGlu5 receptors). A functional interaction between ephrinB and mGlu1 receptors, which likely involves adaptor or scaffolding proteins, might have an important role in the regulation of developmental plasticity.

Group-II metabotropic glutamate (mGlu) receptors (mGlu2/3 receptors) were highly expressed in various regions (telencephalon, optic tectum, and cerebellum, but not vagal lobe) of the goldfish brain. In the goldfish telencephalon, expression of mGlu2/3 receptors was even higher than in the rat cerebral cortex. In contrast, mGlu5 receptors showed low levels of expression in all goldfish brain regions, whereas mGlu1a receptors were only expressed in the goldfish cerebellum. Pharmacological activation of group-II mGlu receptors with the selective agonists, 2 R ,4 R -4-aminopyrrolidine-2,4-dicarboxylic acid and (2 S ,2′ R ,3′ R )-2-(2,3-dicarboxycyclopropyl) glycine, reduced the evoked release of glutamate from goldfish brain synaptosomes, whereas agonists of group-I and -III mGlu receptors (3,5-dihydroxyphenylglycine and l -2-amino-4-phosphonobutanoate) were inactive. The predominance of group-II over group-I mGlu receptors in the goldfish brain may provide a natural defense against excitotoxic neuronal death and contribute to the unusually high resistance of goldfishes against hypoxic brain damage.

Painful bladder syndrome/Interstitial cystitis (PBS/IC) is a chronic disorder characterized clinically by recurring episodes of pelvic pain and increased urination frequency, significantly impairing patients' quality of life. Despite this, there is an unmet medical need in terms of effective diagnostics and treatment. Animal models are crucial in this endeavor. Systemic chronic administration of cyclophosphamide (CYP) in mice has been proposed as a relevant preclinical model of chronic bladder pain. However, molecular mechanisms underlying the pathogenesis of this model are lacking. Here, we show that mice, subjected to repetitive systemic injections of CYP, developed mild inflammatory response in bladder tissue characterized by submucosal edema, moderate increase in proinflammatory cytokine gene expression, and mastocytosis. No signs of massive inflammatory infiltrate, tissue hemorrhages, mucosal ulcerations and urothelium loss were observed. Instead, CYP treatment induced urothelium hyperplasia, accompanied by activation of proliferative signaling cascades, and a decrease in the expression of urothelium‐specific markers. Metabotropic glutamate (mGlu) receptors have been implicated in chronic pain disorders. CYP administration induced differential changes in mGlu receptors mRNA levels in bladder tissue, without affecting gene expression at spinal cord level, pointing to the potential link between peripheral mGlu receptors and inflammation‐induced bladder malfunction and hyperalgesia. Taken together, these data indicate that chronic CYP treatment in mice is a model of PBS mostly relevant to the major, nonulcerative subtype of the syndrome, characterized by a relatively unaltered mucosa and a sparse inflammatory response. This model can help to elucidate the pathogenetic mechanisms of the disease., We show that chronic cyclophosphamide (CYP)‐induced cystitis in mice is characterized by a mild inflammatory infiltration and a moderate increase in proinflammatory cytokine gene expression in bladder tissue, accompanied by activated proliferation of morphologically intact urothelium. These findings set the mouse model apart from the majority of rodent cystitis models, which exhibit severe bladder inflammation and urothelial damage, and suggest that this model is more relevant to the predominant, nonulcerative subtype of painful bladder syndrome patients. Also, we demonstrate that CYP administration induces differential changes in mGlu receptor mRNA levels locally in bladder tissue without affecting gene expression at spinal cord level, thus pointing to a potential link between peripheral mGlu receptors and inflammation‐induced bladder malfunction.

Metabotropic glutamate (mGlu) receptors are positioned at synapses of the thalamocortical network that underlie the development of spike-and-wave discharges (SWDs) associated with absence epilepsy. The modulatory role of individual mGlu receptor subtypes on excitatory and inhibitory synaptic transmission in the cortico-thalamo-cortical circuitry makes subtype-selective mGlu receptor ligands potential candidates as novel antiabsence drugs. Some of these compounds are under clinical development for the treatment of numerous neurologic and psychiatric disorders, and might be soon available for clinical studies in patients with absence seizures refractory to conventional medications. Herein we review the growing evidence that links mGlu receptors to the pathophysiology of pathologic SWDs moving from the anatomic localization and function of distinct mGlu receptor subtypes in the cortico-thalamo-cortical network to in vivo studies in mouse and rat models of absence epilepsy.

Activation of metabotropic glutamate 2 (mGlu2) receptors inhibits pain transmission at the synapses between primary afferent fibers and neurons in the dorsal horn of the spinal cord. In addition, mGlu2 receptors are found in peripheral nociceptors, and in pain-regulatory centers of the brain stem and forebrain. mGlu2 receptor agonists produce analgesia in models of inflammatory and neuropathic pain, but their use is limited by the development of tolerance. A new therapeutic strategy could be based on the transcriptional regulation of mGlu2 receptors via the acetylation-promoted activation of the p65/RelA transcription factor. "Epigenetic" drugs that increase mGlu2 receptor expression, including l-acetylcarnitine and inhibitors of histone deacetylases, have a different analgesic profile with no tolerance to the therapeutic effect after repeated dosing.

The motivation to maintain nicotine self-administration and dependence may involve alterations in glutamatergic neurotransmission. Metabotropic glutamate (mGlu) 2/3 receptors regulate glutamate and dopamine release in the ventral tegmental area (VTA) and the nucleus accumbens (NAc) shell, two brain areas critically involved in reward and motivational processes. We found that acute systemic, as well as intra-VTA or intra-NAc, administration of the mGlu2/3 receptor agonist LY379268 [(−)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate] decreased nicotine, but not food, self-administration in rats. In addition, nicotine self-administration downregulated mGlu2/3 receptor function in corticolimbic rat brain sites including the VTA and the NAc, demonstrated by decreased coupling of mGlu2/3 receptors to G-proteins in the [35S]GTPγS binding assay. Furthermore, repeated treatment with LY379268 reduced nicotine self-administration at the beginning of a 14 d treatment period; however, the number of nicotine infusions earned gradually returned to baseline levels, indicating tolerance to the effects of repeated LY379268 treatment. Finally, LY379268 administration decreased both cue-induced reinstatement of nicotine- and food-seeking behavior. Together, these findings indicate an important role for mGlu2/3 receptors in the posterior VTA and the NAc shell in the mediation of the rewarding effects of nicotine and potentially in cue-induced nicotine-seeking behavior.

Medicinal therapies for mood disorders neither fully serve the efficacy needs of patients nor are they free of side-effect issues. Although monoamine-based therapies are the primary current treatment approaches, both preclinical and clinical findings have implicated the excitatory neurotransmitter glutamate in the pathogenesis of major depressive disorders. The present commentary focuses on the metabotropic glutamate receptors and their relationship to mood disorders. Metabotropic glutamate (mGlu) receptors regulate glutamate transmission by altering the release of neurotransmitter and/or modulating the post-synaptic responses to glutamate. Convergent biochemical, pharmacological, behavioral, and clinical data will be reviewed that establish glutamatergic neurotransmission via mGlu receptors as a biologically relevant process in the regulation of mood and that these receptors may serve as novel targets for the discovery of small molecule modulators with unique antidepressant properties. Specifically, compounds that antagonize mGlu2, mGlu3, and/or mGlu5 receptors (e.g. LY341495, MGS0039, MPEP, MTEP) exhibit biochemical effects indicative of antidepressant effects as well as in vivo activity in animal models predictive of antidepressant efficacy. Both preclinical and clinical data have previously been presented to define NMDA and AMPA receptors as important targets for the modulation of major depression. In the present review, we present a model suggesting how the interplay of glutamate at the mGlu and at the ionotropic AMPA and NMDA receptors might account for the antidepressant-like effects of glutamatergic- and monoaminergic-based drugs affecting mood in patients. The current data lead to the hypothesis that mGlu-based compounds and conventional antidepressants impact a network of interactive effects that converge upon a down regulation of NMDA receptor function and an enhancement in AMPA receptor signaling.

Cultured mouse embryonic stem (ES) cells maintained under undifferentiated conditions (i.e. grown in medium containing 15% FCS and leukemia inhibitory factor--LIF) expressed mGlu5 metabotropic glutamate receptors. Activation of these receptors with quisqualate increased [Ca2+]i but only when cultures were deprived of extracellular glutamate, indicating that the receptor was saturated by the endogenous glutamate. Pharmacological blockade of mGlu5 receptors with 2-methyl-6-(phenylethynyl)pyridine (MPEP) or antisense-induced knock-down of mGlu5 receptors decreased the expression of the two main transcription factors that sustain ES cell self-renewal, i.e. Oct-4 and Nanog, as assessed by real-time PCR and immunoblotting. Exposure of ES cell cultures to MPEP also reduced alkaline phosphatase activity, a marker of undifferentiated ES cells. These data support a critical role for mGlu receptors in early development showing that mGlu5 receptors are expressed by ES cells and their activation sustains ES cell self-renewal in culture.

Cultured cerebellar granule cells grown in medium containing 10 mM K+ undergo apoptosis after 4-5 days in vitro (DIV), and, at that time, the activity of metabotropic glutamate (mGlu) receptors coupled to polyphosphoinositide (PI) hydrolysis begins to decline. In granule cells at 4 DIV, the mGlu receptor subtype mGlu5 was expressed at high levels. The expression of another PI-coupled mGlu receptor, the mGlu1a, was low at 4 DIV but increased during the following days. In cultures at 4-5 DIV, the few cells that already showed an apoptotic phenotype were devoid of mGlu5 receptors, but they all expressed mGlu1a receptors. The development of apoptosis was accelerated after treating the cultures with: (i) mGlu5 antisense oligonucleotides; (ii) the mixed mGlu receptor antagonist, (+)-alpha-methyl-4-carboxyphenylglycine; or (iii) the glutamate depleting enzyme, alanine aminotransferase. In contrast, an induced overexpression of mGlu5 receptors protected cultured granule cells against apoptotic death. We suggest that the activity of mGlu5 receptors supports cell survival, and a decline in the expression of mGlu5 receptors gives access to programmed cell death in cerebellar granule cells developing in primary cultures.

We examined the expression of metabotropic glutamate (mGlu) receptors in species of fish that differ for their vulnerability to anoxic brain damage. Although expression of mGlu1a and mGlu5 receptors was similar in the brain of all species examined, expression of mGlu2/3 receptors was substantially higher in the brain of anoxia-tolerant species (i.e., the carpCarassius carassiusand the goldfishCarassius auratus) than in the brain of species that are highly vulnerable to anoxic damage, such as the troutsSalmo truttaandOncorhynchus mykiss. This difference was confirmed by measuring the mGlu2/3 receptor-mediated inhibition of forskolin-stimulated cAMP formation in slices prepared from the telencephalon ofC. auratusandS. trutta. We exposed the goldfishC. auratusto water deprived of oxygen for 4 hr for the induction of hypoxic brain damage. Although the goldfish survived this treatment, the occurrence of apoptotic cell death could be demonstrated by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling staining and by the assessment of caspase-3 activity in different brain region. The extent of cell death was highest in the medulla oblongata, followed by the optic tectum, cerebellum, and hypothalamus. No cell death was found in the telencephalon. This regional pattern of hypoxic damage was inversely related to the expression of mGlu2/3 receptors, which was lowest in the medulla oblongata and highest in the telencephalon. Treatment of the goldfish with the brain permeant mGlu2/3 receptor antagonist LY341495 (1 mg/kg, i.p.) amplified anoxic damage throughout the brain and enabled the induction of cell death by anoxia in the telencephalon. In contrast, treatment of the goldfish with the mGlu2/3 receptor agonist LY379268 (0.5 or 1 mg/kg, i.p.) was highly protective against anoxic brain damage. Finally, exposure to the antagonist LY341495 (0.5 μm) greatly amplified the release of glutamate induced by hypoxia in slices prepared from the medulla oblongata and the telencephalon of the goldfish. We conclude that expression of mGlu2/3 receptors provides a major defensive mechanism against brain damage in anoxia-tolerant species.

Schizophrenia and Bipolar Disorder are chronic psychiatric disorders, both considered as "major psychosis"; they are thought to share some pathogenetic factors involving a dysfunctional gene x environment interaction. Alterations in the glutamatergic transmission have been suggested to be involved in the pathogenesis of psychosis. Our group developed an epigenetic model of schizophrenia originated by Prenatal Restraint Stress (PRS) paradigm in mice. PRS mice developed some behavioral alterations observed in schizophrenic patients and classic animal models of schizophrenia, i.e. deficits in social interaction, locomotor activity and prepulse inhibition. They also showed specific changes in promoter DNA methylation activity of genes related to schizophrenia such as reelin, BDNF and GAD67, and altered expression and function of mGlu2/3 receptors in the frontal cortex. Interestingly, behavioral and molecular alterations were reversed by treatment with mGlu2/3 agonists. Based on these findings, we speculate that pharmacological modulation of these receptors could have a great impact on early phase treatment of psychosis together with the possibility to modulate specific epigenetic key protein involved in the development of psychosis. In this review, we will discuss in more details the specific features of the PRS mice as a suitable epigenetic model for major psychosis. We will then focus on key proteins of chromatin remodeling machinery as potential target for new pharmacological treatment through the activation of metabotropic glutamate receptors.