Deletion of the type 2 metabotropic glutamate receptor increases heroin abuse vulnerability in transgenic rats.

Opioid abuse is a rapidly growing public health crisis in the USA. Despite extensive research in the past decades, little is known about the etiology of opioid addiction or the neurobiological risk factors that increase vulnerability to opioid use and abuse. Recent studies suggest that the type 2 metabotropic glutamate receptor (mGluR2) is critically involved in substance abuse and addiction. In the present study, we evaluated whether low-mGluR2 expression may represent a risk factor for the development of opioid abuse and addiction using transgenic mGluR2-knockout (mGluR2-KO) rats. Compared to wild-type controls, mGluR2-KO rats exhibited higher nucleus accumbens (NAc) dopamine (DA) and locomotor responses to heroin, higher heroin self-administration and heroin intake, more potent morphine-induced analgesia and more severe naloxone-precipitated withdrawal symptoms. In contrast, mGluR2-KO rats displayed lower motivation for heroin self-administration under high price progressive-ratio (PR) reinforcement conditions. Taken together, these findings suggest that mGluR2 may play an inhibitory role in opioid action, such that deletion of this receptor results in an increase in brain DA responses to heroin and in acute opioid reward and analgesia. Low-mGluR2 expression in the brain may therefore be a risk factor for the initial development of opioid abuse and addiction.

Cardiac mGluR1 metabotropic receptors in cardioprotection.

AIMS: In a previous study using a genome-wide microarray strategy, we identified metabotropic glutamate receptor 1 (mGluR1) as a putative cardioprotective candidate in ischaemic postconditioning (PostC). In the present study, we investigated the role of cardiac mGluR1 receptors during cardioprotection against myocardial ischaemia-reperfusion injury in the mouse myocardium. METHODS AND RESULTS: mGluR1 activation by glutamate administered 5 min before reperfusion in C57Bl/6 mice subjected to a myocardial ischaemia protocol strongly decreased both infarct size and DNA fragmentation measured at 24 h reperfusion. This cardioprotective effect was mimicked by the mGluR1 agonist, DHPG (10 µM), and abolished when glutamate was coinjected with the mGluR1 antagonist YM298198 (100 nM). Wortmannin (100 nM), an inhibitor of PI3-kinase, was able to prevent glutamate-induced cardioprotection. A glutamate bolus at the onset of reperfusion failed to protect the heart of mGluR1 knockout mice subjected to a myocardial ischaemia-reperfusion protocol, although PostC still protected the mGluR1 KO mice. Glutamate-treatment improved post-infarction functional recovery as evidenced by an echocardiographic study performed 15 days after treatment and by a histological evaluation of fibrosis 21 days post-treatment. Interestingly, restoration of functional mGluR1s by a PostC stimulus was evidenced at the transcriptional level. Since mGluR1s were localized at the surface membrane of cardiomyocytes, they might contribute to the cardioprotective effect of ischaemic PostC as other Gq-coupled receptors. CONCLUSION: This study provides the first demonstration that mGluR1 activation at the onset of reperfusion induces cardioprotection and might represent a putative strategy to prevent ischaemia-reperfusion injury.

Prevalence and influence of cys407* Grm2 mutation in Hannover-derived Wistar rats: mGlu2 receptor loss links to alcohol intake, risk taking and emotional behaviour.

Modulation of metabotropic glutamate 2 (mGlu2) receptor function has huge potential for treating psychiatric and neurological diseases. Development of drugs acting on mGlu2 receptors depends on the development and use of translatable animal models of disease. We report here a stop codon mutation at cysteine 407 in Grm2 (cys407*) that is common in some Wistar rats. Therefore, researchers in this field need to be aware of strains with this mutation. Our genotypic survey found widespread prevalence of the mutation in commercial Wistar strains, particularly those known as Han Wistar. Such Han Wistar rats are ideal for research into the separate roles of mGlu2 and mGlu3 receptors in CNS function. Previous investigations, unknowingly using such mGlu2 receptor-lacking rats, provide insights into the role of mGlu2 receptors in behaviour. The Grm2 mutant rats, which dominate some selectively bred lines, display characteristics of altered emotionality, impulsivity and risk-related behaviours and increased voluntary alcohol intake compared with their mGlu2 receptor-competent counterparts. In addition, the data further emphasize the potential therapeutic role of mGlu2 receptors in psychiatric and neurological disease, and indicate novel methods of studying the role of mGlu2 and mGlu3 receptors. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.

Effects of alcohol on c-Myc protein in the brain.

Alcoholism is a disorder categorized by significant impairment that is directly related to persistent and extreme use of alcohol. The effects of alcoholism on c-Myc protein expression in the brain have been scarcely studied. This is the first study to investigate the role different characteristics of alcoholism have on c-Myc protein in the brain. We analyzed c-Myc protein in the hypothalamus and amygdala from five different animal models of alcohol abuse. c-Myc protein was increased following acute ethanol exposure in a mouse knockout model and following chronic ethanol consumption in vervet monkeys. We also observed increases in c-Myc protein exposure in animals that are genetically predisposed to alcohol and methamphetamine abuse. Lastly, c-Myc protein was increased in animals that were acutely exposed to methamphetamine when compared to control treated animals. These results suggest that in substance abuse c-Myc plays an important role in the brain's response.

Functional effects of GRM1 suppression in human melanoma cells.

Ectopic expression of a neuronal receptor, metabotropic glutamate receptor 1 (Grm1), in melanocytes has been implicated in melanoma development in mouse models. The human relevance of this receptor's involvement in melanoma pathogenesis was shown by detecting GRM1 expression in subsets of human melanomas, an observation lacking in benign nevi or normal melanocytes. Grm1-transformed mouse melanocytes and a conditional Grm1 transgenic mouse model confirmed a requirement for sustained expression of Grm1 for the maintenance of transformed phenotypes in vitro and tumorigenicity in vivo. Here, we investigate if continued GRM1 expression is also required in human melanoma cell lines by using two inducible, silencing RNA systems: the ecdysone/Ponasterone A and tetracycline on/off approaches to regulate GRM1 expression in the presence of each inducer. Various in vitro assays were conducted to assess the consequences of a reduction in GRM1 expression on cell proliferation, apoptosis, downstream targeted signaling pathways, and in vivo tumorigenesis. We showed that suppression of GRM1 expression in several human melanoma cell lines resulted in a reduction in the number of viable cells and a decrease in stimulated mitogen-activated protein kinase (MAPK) and PI3K/AKT and suppressed tumor progression in vivo. These results reinforce earlier observations where a reduction in cell growth in vitro and tumorigenesis in vivo were correlated with decreased GRM1 activities by pharmacologic inhibitors of the receptor, supporting the notion that GRM1 plays a role in the maintenance of transformed phenotypes in human melanoma cells in vitro and in vivo and could be a potential therapeutic target for the treatment of melanoma.

The glutamatergic compounds sarcosine and N-acetylcysteine ameliorate prepulse inhibition deficits in metabotropic glutamate 5 receptor knockout mice.

RATIONALE: Mice lacking metabotropic glutamate receptors 5 (mGluR5) exhibit reduced glutamatergic function and behavioral abnormalities, including deficits in prepulse inhibition (PPI) of the startle response that may be relevant to schizophrenia. Thus, these mice are an animal model that may be used for preclinical evaluation of potentially new classes of antipsychotic compounds. Recent clinical studies have suggested several compounds that modulate glutamatergic transmission through distinct mechanisms, such as potentiation of the N-methyl-D: -aspartate (NMDA) receptor glycine site, activation of group II mGluR, and activation of glutamate-cysteine antiporters, as being efficacious in the treatment of schizophrenia. OBJECTIVES: The aim of this work is to evaluate the effects of sarcosine (a selective inhibitor of the glycine transporter 1 [GlyT1]), LY379268 (a group II mGluR agonist), and N-acetylcysteine (a cysteine prodrug that indirectly activates cystine-glutamate antiporters to increase glutamate levels in the extrasynaptic space) on PPI deficits in mGluR5 knockout mice. RESULTS: Sarcosine and N-acetylcysteine, but not LY379268, ameliorated PPI deficits in mGluR5 knockout mice. The ability of N-acetylcysteine to restore PPI deficits was not blocked by the group II mGluR antagonist LY341495, indicating that the effects of N-acetylcysteine were not attributable to activation of group II mGluRs by glutamate. CONCLUSIONS: These findings provide evidence that the interactions between mGluR5 and NMDA receptors are involved in the regulation of PPI and suggest that activation of glutamate receptors, other than group II receptors, by increased endogenous glutamate transmission, may ameliorate the behavioral abnormalities associated with mGluR5 deficiency.

Individual contribution of metabotropic glutamate receptor (mGlu) 2 and 3 to c-Fos expression pattern evoked by mGlu2/3 antagonism.

UNLABELLED: OBJECTIVES AND MATERIALS AND METHODS: The aims of the present study were (1) to determine the neuronal activation pattern elicited by the group II mGlu antagonist LY341495 and (2) to evaluate the contribution of each group II mGlu subtype by using wild-type (WT) and knockout (KO) mice lacking either mGlu2 or mGlu3. c-Fos expression was used as a marker of neuronal activation. RESULTS AND DISCUSSION: In WT mice, LY341495 induced widespread c-Fos expression in 68 out of 92 brain areas, including limbic areas such as the amygdala, septum, prefrontal cortex, and hippocampus. LY341495-induced c-Fos response was markedly decreased in the medial part of the central amygdala (CeM) and lateral septum (LS) in mGlu3-KO mice, as well as in the lateral parabrachial nucleus (LPB) in both KO strains. In the majority of investigated areas, LY341495-induced c-Fos expression was similar in KO and WT mice. Analysis of the cellular and subcellular distribution of mGlu2 and mGlu3 revealed a prevailing presence of mGlu3-immunoreactivity in the CeM in glial processes and in postsynapstic neuronal elements, whereas only rare presynaptic axon terminals were found immunoreactive for mGlu2. CONCLUSION: In conclusion, our data indicate that group II mGlu blockade increases neuronal activation in a variety of brain areas, including many stress- and anxiety-related areas. The activation of two key brain areas, the CeM and LS, is mediated via mGlu3, while activation in the LPB involves both subtypes. Moreover, in the majority of investigated areas, LY341495-mediated neuronal activation appears to require a complex cross talk between group II mGlu subtypes or the action of LY341495 on additional receptors.

Defective group-II metaboropic glutamate receptors in the hippocampus of spontaneously depressed rats.

Spontaneously depressed flinders sensitive line (FSL) rats showed a reduced expression of mGlu2/3 metabotropic glutamate receptors in the hippocampus, as compared to "non-depressed" flinders resistant line (FRL) rats. No changes in mGlu2/3 receptor protein levels were found in other brain regions, including the amygdala, hypothalamus, and cerebral cortex. Biochemical analysis of receptor signalling supported the reduction of mGlu2/3 receptors in the hippocampus of FSL rats. Accordingly, the selective mGlu2/3 receptor agonist, LY379268 (1microM) reduced forskolin-stimulated cAMP formation by 56% and 32% in hippocampal slices from FRL and FSL rats, respectively. In addition, LY379268 enhanced 3,5-dihydroxyphenylglycine-stimulated inositol phospholipid hydrolysis from 65% to 215% in hippocampal slices from FRL rats, whereas it was inactive in slices from FRL rats. We also examined the behavioural response of FSL rats to systemic injection of LY379268 (0.5mg/kg, i.p., once a day for 1-21 days) by measuring the immobility time in the forced swim test, which is known to be increased in these rats. LY379268 was administered alone or combined with the classical antidepressant, chlorimipramine (10mg/kg, i.p.). LY379268 alone had no effect at any of the selected time-points, whereas chlorimipramine alone reduced the immobility time only after 21 days of treatment. In contrast, when combined with LY379268, chlorimipramine reduced the immobility time during the first 14 days of treatment. These data support the view that mGlu2/3 receptors might be involved in the pathophysiology of depressive disorders, and that pharmacological activation of these receptors may shorten the latency of antidepressant medication.

A novel connection between rods and ON cone bipolar cells revealed by ectopic metabotropic glutamate receptor 7 (mGluR7) in mGluR6-deficient mouse retinas.

Since the discovery of direct chemical synapses between rod photoreceptor and OFF cone bipolar cells in mouse retinas, whether the ON cone bipolar cell also receive direct chemical input from rod has been a pending question. In finding that metabotropic glutamate receptor 7 (mGluR7) was uniquely expressed in dendrites of ON cone bipolar cells in the mGluR6-deficient mouse retina, we used this ectopic mGluR7 immunoreactivity as a specific marker for the ON cone bipolar to search for its rod connection. Here, we show that a certain type of ON cone bipolar cell forms ribbon-associated synapses not only with cones, but also rods. This finding was verified in the wild-type mouse retina by three-dimensional reconstruction of bipolar cells from serial electron micrographs. These ON cone bipolars were further identified as corresponding to type 7 of mouse bipolar cell described by Ghosh et al. (2004) and also to the green fluorescent protein (GFP)-labeled type 7 bipolars in the alpha-gustducin-GFP transgenic mouse. Our findings suggest that, in mice, rod signals bifurcate into a third ON and OFF pathway in addition to the two known routes to cone bipolar cells: (1) via rod chemical synapse --> rod bipolar --> AII amacrine --> ON and OFF cone bipolar cells; (2) via rod-cone gap junction --> cone chemical synapse --> ON and OFF cone bipolar cells; and (3) via rod chemical synapse --> ON and OFF cone bipolar cells. This third novel pathway is thought to transmit fast and moderately light-sensitive rod signals, functioning to smooth out the intensity changes at the scotopic-mesopic interface.

Assessment of NMDA receptor activation in vivo by Fos induction after challenge with the direct NMDA agonist (tetrazol-5-yl)glycine: effects of clozapine and haloperidol.

Induction of Fos protein by the potent and direct NMDA agonist (tetrazol-5-yl)glycine (TZG) was examined in mice. Effects of antipsychotic drugs were assessed on this in vivo index of NMDA receptor activation. TZG induced the expression of Fos in a neuroanatomically selective manner, with the hippocampal formation showing the most robust response. In mice genetically altered to express low levels of the NR1 subunit of the NMDA receptor, TZG-induced Fos was reduced markedly in comparison to the wild type controls. TZG-induced Fos was also blocked by the selective NMDA antagonist MK-801. Pretreatment of mice with clozapine (3 and 10 mg/kg) reduced TZG-induced Fos in the hippocampal formation but not in other brain regions. Haloperidol at a dose of 0.5 mg/kg did not antagonize TZG induced Fos in any region. Haloperidol at a dose of 1.0 mg/kg did attenuate the induction of Fos by TZG in the hippocampus but not in other brain regions. The relatively high dose (1 mg/kg) of haloperidol required to block effects of TZG suggests that this action may not be related to the D(2) dopamine receptor-blocking properties, since maximal D(2) receptor blockade was probably achieved by the 0.5 mg/kg dose of haloperidol. The antidepressant drug imipramine (10 or 20 mg/kg) did not antagonize TZG induced Fos in any brain region. The data suggest that clozapine can reduce excessive activation of NMDA receptors by TZG administration in vivo at doses relevant to the drugs' actions in rodent models of antipsychotic activity. Whether or not this action of clozapine contributes to its therapeutic properties will require further study.

Interactions between metabotropic glutamate 5 and adenosine A2A receptors in normal and parkinsonian mice.

Evidence for heteromeric receptor complexes comprising adenosine A2A and metabotropic glutamate 5 (mGlu5) receptors in striatum has raised the possibility of synergistic interactions between striatal A2A and mGlu5 receptors. We investigated the role of striatal A2A receptors in the locomotor stimulant and antiparkinsonian properties of mGlu5 antagonists using complementary pharmacologic and genetic approaches. Locomotion acutely stimulated by the mGlu5 antagonist [2-methyl-6-(phenylethynyl)-pyridine (MPEP)] was absent in mGlu5 knock-out (KO) mice and was potentiated by an A2A antagonist KW-6002 [(E)-1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methylxanthine], both in normal and in dopamine-depleted (reserpinized) mice. Conversely, the MPEP-induced motor response was markedly attenuated in single and double A2A and D2 receptor KO mice. In contrast, motor stimulation by a D1 dopamine agonist was not attenuated in the KO mice. The A2A receptor dependence of MPEP-induced motor stimulation was investigated further using a postnatal forebrain-specific conditional (Cre/loxP system) KO of the A2A receptor. MPEP loses the ability to stimulate locomotion in conditional KO mice, suggesting that this mGlu5 antagonist effect requires the postdevelopmental action of striatal A2A receptors. The potentiation of mGlu5 antagonist-induced motor stimulation by an A2A antagonist and its dependence on both D2 and forebrain A2A receptors highlight the functional interdependence of these receptors. These data also strengthen a rationale for pursuing a combinational drug strategy for enhancing the antiparkinsonian effects of A2A and mGlu5 antagonists.

Grm5 expression is not required for the oncogenic role of Grm1 in melanocytes.

Melanoma is the aberrant proliferation of melanocytes, the cells in the skin responsible for pigment (melanin) production. In its early stages, melanoma can be surgically removed with great success, however, advanced stages of melanoma have a high mortality rate due to the lack of responsiveness to currently available therapies. We have previously characterized a mouse melanoma model, TG-3, which has implicated the ectopic expression of metabotropic glutamate receptor 1 (Grm1, formerly mGluR1), in melanomagenesis and metastasis [Pollock et al., 2003. Melanoma mouse model implicates metabotropic glutamate signaling in melanocytic neoplasia. Nat Genet. 34, 108-112.]. Here we report the characterization of several in vitro cell lines derived from independent mouse melanoma tumors. These cell lines show characteristic phenotypes of transformed melanocytes, and express Grm1, and Grm5 (another metabotropic glutamate receptor), as well as melanocyte-specific protein markers. To investigate the possible role of Grm5 in vivo during melanoma development in our mice, we have crossed Grm5 null mice with TG-3, generating a new line of transgenic mice, TGM. TGMs, which are homozygote knockouts for Grm5 and carry the TG transgene, develop tumors with onset, progression, and metastasis very similar to that described for TG-3. Taken together, these results indicate that Grm1 can act as an oncogene in melanocytes independently of Grm5 expression.

Interactions between ephrin-B and metabotropic glutamate 1 receptors in brain tissue and cultured neurons.

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.

Systemic administration of the potent mGlu8 receptor agonist (S)-3,4-DCPG induces c-Fos in stress-related brain regions in wild-type, but not mGlu8 receptor knockout mice.

The effect of a novel and potent metabotropic glutamate 8 (mGlu8) receptor agonist, (S)-3,4-dicarboxyphenylglycine (DCPG), was studied in vivo in mouse brain. c-Fos expression was used as a marker of neuronal activity in specific brain regions 2 h after systemic (S)-3,4-DCPG (3-100 mg/kg, i.p.). The selectivity of (S)-3,4-DCPG on mGlu8 receptors was determined in mGlu8 receptor knockout mice. In wild-type mice, (S)-3,4-DCPG (100 mg/kg) significantly increased c-Fos expression in several stress-related brain regions: paraventricular nucleus of the hypothalamus, central nucleus of the amygdala, lateral parabrachial nucleus and locus coeruleus. In the central nucleus of the amgydala, more than 92% of c-Fos positive neurons were identified as GABAergic inhibitory neurons after (S)-3,4-DCPG. Moreover, (S)-3,4-DCPG significantly induced c-Fos in the superficial gray layer of the superior colliculus, a central visual region. c-Fos expression was unchanged by (S)-3,4-DCPG in mGlu8 receptor knockout mice. Our results indicate that systemic (S)-3,4-DCPG alters neuronal excitability in specific brain regions via mGlu8 receptor. The prominent activation of stress areas suggests a role for mGlu8 receptors in the central integration of stress responses. Furthermore, our results indicate that systemic (S)-3,4-DCPG can be used as a tool to explore behavioral and cellular consequences of mGlu8 receptor activation.

Increased c-Fos expression in the centromedial nucleus of the thalamus in metabotropic glutamate 8 receptor knockout mice following the elevated plus maze test.

Ligands for metabotropic glutamate 8 (mGlu8) receptors, such as (S)-2-amino-4-phosphonobutanoic acid and (S)-3,4-dicarboxyphenylglycine suppress CNS excitability via presynaptic regulation of glutamate release and are anticonvulsant in mice. These observations suggest that mGlu8 receptors play a role in the regulation of neuronal excitability. To further characterize the role of mGlu8 receptors in vivo, the mGlu8 receptor knockout mouse was generated. Recently, we reported that mGlu8 receptor knockout mice showed increased anxiety in the elevated plus maze (EPM). Here, the pattern of c-Fos expression was studied in mGlu8 receptor knockout and wild-type mice after exposure to the EPM test for 5 min. The present study shows that the increased anxiety-related behavior of mGlu8 receptor knockout mice in the EPM was associated with a 2.3-fold higher (P<0.05) number of c-Fos positive cells in the centromedial nucleus of the thalamus compared with wild-type mice (when prehandled mice were used). The increased neuronal activity in the centromedial nucleus of the thalamus in the mGlu8 receptor knockout mouse was also observed in a separate experiment with naive mice (no prehandling). In these naive mGlu8 receptor knockouts, c-Fos expression was significantly induced by the EPM in the centrolateral nucleus of the thalamus, paraventricular nucleus of the hypothalamus, and granular cell layer of the dentate gyrus, but in naive wild-type mice c-Fos was significantly increased only in the piriform cortex. Basal c-Fos expression in the absence of EPM exposure did not differ between wild-type and mGlu8 receptor knockout mice in any brain region we examined. As the centromedial nucleus of the thalamus is important in regulating sensory information to higher brain regions, these results support the hypothesis that mGlu8 receptors are involved in the response to certain novel, aversive environments. In particular, the deletion of the mGlu8 receptor reduced the threshold of neuronal activation in stress-related brain regions such as the centromedial nucleus of the thalamus.

The metabotropic glutamate receptor 1 is not involved in the facilitation of glutamate release in cerebrocortical nerve terminals.

In this study we have addressed the identification of the metabotropic glutamate receptor (mGluR) involved in the facilitation of glutamate release in nerve terminals from the cerebral cortex. mGluR1 and 5 are coupled to phosphoinositide hydrolysis and the activation of these receptors with the specific agonist 3,5-dihydroxyphenylglycine (DHPG) enhances the release of glutamate. We have examined whether mGluR1 is responsible for this modulatory effect by preparing nerve terminals from mGluR 1 deficient mice. The Ca2+-dependent glutamate release evoked by a submaximal depolarization is enhanced by the agonist DHPG in nerve terminals from both wild and mutant mice. This result is consistent with the finding that the mGluR agonist also induces a similar increase in the levels of diacylglycerol (DAG) in the nerve terminals from wild and mutant mice. Moreover, the activity-dependent switch from facilitation to inhibition of release, observed when a second stimulation of the receptor is applied shortly after (5 min) the first pulse, was also observed in the mutant mice. These results indicate therefore, that the facilitation of glutamate release is unlikely to be due to the activation of mGluR1 but related to another phosphoinositide coupled mGluR.