Activation of mGlu 2/3 receptors with the orthosteric agonist LY-404,039 alleviates dyskinesia in experimental parkinsonism.

LY-404,039 is an orthosteric agonist at metabotropic glutamate 2 and 3 (mGlu 2/3 ) receptors, with a possible additional agonist effect at dopamine D 2 receptors. LY-404,039 and its pro-drug, LY-2140023, have previously been tested in clinical trials for psychiatric indications and could therefore be repurposed if they were shown to be efficacious in other conditions. We have recently demonstrated that the mGlu 2/3 orthosteric agonist LY-354,740 alleviated L-3,4-dihydroxyphenylalanine (L-DOPA)-induced abnormal involuntary movements (AIMs) in the 6-hydroxydopamine (6-OHDA)-lesioned rat without hampering the anti-parkinsonian action of L-DOPA. Here, we seek to take advantage of a possible additional D 2 -agonist effect of LY-404,039 and see if an anti-parkinsonian benefit might be achieved in addition to the antidyskinetic effect of mGlu 2/3 activation. To this end, we have administered LY-404,039 (vehicle, 0.1, 1 and 10 mg/kg) to 6-OHDA-lesioned rats, after which the severity of axial, limbs and oro-lingual (ALO) AIMs was assessed. The addition of LY-404,039 10 mg/kg to L-DOPA resulted in a significant reduction of ALO AIMs over 60-100 min (54%, P  < 0.05). In addition, LY-404,039 significantly enhanced the antiparkinsonian effect of L-DOPA, assessed through the cylinder test (76%, P  < 0.01). These results provide further evidence that mGlu 2/3 orthosteric stimulation may alleviate dyskinesia in PD and, in the specific case of LY-404,039, a possible D 2 -agonist effect might also make it attractive to address motor fluctuations. Because LY-404,039 and its pro-drug have been administered to humans, they could possibly be advanced to Phase IIa trials rapidly for the treatment of motor complications in PD.

A nanobody activating metabotropic glutamate receptor 4 discriminates between homo- and heterodimers.

There is growing interest in developing biologics due to their high target selectivity. The G protein-coupled homo- and heterodimeric metabotropic glutamate (mGlu) receptors regulate many synapses and are promising targets for the treatment of numerous brain diseases. Although subtype-selective allosteric small molecules have been reported, their effects on the recently discovered heterodimeric receptors are often not known. Here, we describe a nanobody that specifically and fully activates homodimeric human mGlu4 receptors. Molecular modeling and mutagenesis studies revealed that the nanobody acts by stabilizing the closed active state of the glutamate binding domain by interacting with both lobes. In contrast, this nanobody does not activate the heterodimeric mGlu2-4 but acts as a pure positive allosteric modulator. These data further reveal how an antibody can fully activate a class C receptor and bring further evidence that nanobodies represent an alternative way to specifically control mGlu receptor subtypes.

Conformational rearrangement during activation of a metabotropic glutamate receptor.

G protein-coupled receptors (GPCRs) relay information across cell membranes through conformational coupling between the ligand-binding domain and cytoplasmic signaling domain. In dimeric class C GPCRs, the mechanism of this process, which involves propagation of local ligand-induced conformational changes over 12 nm through three distinct structural domains, is unknown. Here, we used single-molecule FRET and live-cell imaging and found that metabotropic glutamate receptor 2 (mGluR2) interconverts between four conformational states, two of which were previously unknown, and activation proceeds through the conformational selection mechanism. Furthermore, the conformation of the ligand-binding domains and downstream domains are weakly coupled. We show that the intermediate states act as conformational checkpoints for activation and control allosteric modulation of signaling. Our results demonstrate a mechanism for activation of mGluRs where ligand binding controls the proximity of signaling domains, analogous to some receptor kinases. This design principle may be generalizable to other biological allosteric sensors.

Reelin Regulates Neuronal Excitability through Striatal-Enriched Protein Tyrosine Phosphatase (STEP61) and Calcium Permeable AMPARs in an NMDAR-Dependent Manner.

Alzheimer's disease (AD) is a progressive neurodegenerative disease marked by the accumulation of amyloid-ß (Aß) plaques and neurofibrillary tangles. Aß oligomers cause synaptic dysfunction early in AD by enhancing long-term depression (LTD; a paradigm for forgetfulness) via metabotropic glutamate receptor (mGluR)-dependent regulation of striatal-enriched tyrosine phosphatase (STEP61). Reelin is a neuromodulator that signals through ApoE (apolipoprotein E) receptors to protect the synapse against Aß toxicity (Durakoglugil et al., 2009) Reelin signaling is impaired by ApoE4, the most important genetic risk factor for AD, and Aß-oligomers activate metabotropic glutamate receptors (Renner et al., 2010). We therefore asked whether Reelin might also affect mGluR-LTD. To this end, we induced chemical mGluR-LTD using DHPG (Dihydroxyphenylglycine), a selective mGluR5 agonist. We found that exogenous Reelin reduces the DHPG-induced increase in STEP61, prevents the dephosphorylation of GluA2, and concomitantly blocks mGluR-mediated LTD. By contrast, Reelin deficiency increased expression of Ca2+-permeable GluA2-lacking AMPA receptors along with higher STEP61 levels, resulting in occlusion of DHPG-induced LTD in hippocampal CA1 neurons. We propose a model in which Reelin modulates local protein synthesis as well as AMPA receptor subunit composition through modulation of mGluR-mediated signaling with implications for memory consolidation or neurodegeneration.SIGNIFICANCE STATEMENT Reelin is an important neuromodulator, which in the adult brain controls synaptic plasticity and protects against neurodegeneration. Amyloid-ß has been shown to use mGluRs to induce synaptic depression through endocytosis of NMDA and AMPA receptors, a mechanism referred to as LTD, a paradigm of forgetfulness. Our results show that Reelin regulates the phosphatase STEP, which plays an important role in neurodegeneration, as well as the expression of calcium-permeable AMPA receptors, which play a role in memory formation. These data suggest that Reelin uses mGluR LTD pathways to regulate memory formation as well as neurodegeneration.

X-ray multiscale 3D neuroimaging to quantify cellular aging and neurodegeneration postmortem in a model of Alzheimer's disease.

PURPOSE: Modern neuroimaging lacks the tools necessary for whole-brain, anatomically dense neuronal damage screening. An ideal approach would include unbiased histopathologic identification of aging and neurodegenerative disease. METHODS: We report the postmortem application of multiscale X-ray phase-contrast computed tomography (X-PCI-CT) for the label-free and dissection-free organ-level to intracellular-level 3D visualization of distinct single neurons and glia. In deep neuronal populations in the brain of aged wild-type and of 3xTgAD mice (a triply-transgenic model of Alzheimer's disease), we quantified intracellular hyperdensity, a manifestation of aging or neurodegeneration. RESULTS: In 3xTgAD mice, the observed hyperdensity was identified as amyloid-ß and hyper-phosphorylated tau protein deposits with calcium and iron involvement, by correlating the X-PCI-CT data to immunohistochemistry, X-ray fluorescence microscopy, high-field MRI, and TEM. As a proof-of-concept, X-PCI-CT was used to analyze hippocampal and cortical brain regions of 3xTgAD mice treated with LY379268, selective agonist of group II metabotropic glutamate receptors (mGlu2/3 receptors). Chronic pharmacologic activation of mGlu2/3 receptors significantly reduced the hyperdensity particle load in the ventral cortical regions of 3xTgAD mice, suggesting a neuroprotective effect with locoregional efficacy. CONCLUSIONS: This multiscale micro-to-nano 3D imaging method based on X-PCI-CT enabled identification and quantification of cellular and sub-cellular aging and neurodegeneration in deep neuronal and glial cell populations in a transgenic model of Alzheimer's disease. This approach quantified the localized and intracellular neuroprotective effects of pharmacological activation of mGlu2/3 receptors.

Group II Metabotropic Glutamate Receptors Reduce Apoptosis and Regulate BDNF and GDNF Levels in Hypoxic-Ischemic Injury in Neonatal Rats.

Birth asphyxia causes brain injury in neonates, but a fully successful treatment has yet to be developed. This study aimed to investigate the effect of group II mGlu receptors activation after experimental birth asphyxia (hypoxia-ischemia) on the expression of factors involved in apoptosis and neuroprotective neurotrophins. Hypoxia-ischemia (HI) on 7-day-old rats was used as an experimental model. The effects of intraperitoneal application of mGluR2 agonist LY379268 (5 mg/kg) and the specific mGluR3 agonist NAAG (5 mg/kg) (1 h or 6 h after HI) on apoptotic processes and initiation of the neuroprotective mechanism were investigated. LY379268 and NAAG applied shortly after HI prevented brain damage and significantly decreased pro-apoptotic Bax and HtrA2/Omi expression, increasing expression of anti-apoptotic Bcl-2. NAAG or LY379268 applied at both times also decreased HIF-1α formation. HI caused a significant decrease in BDNF concentration, which was restored after LY379268 or NAAG administration. HI-induced increase in GDNF concentration was decreased after administration of LY379268 or NAAG. Our results show that activation of mGluR2/3 receptors shortly after HI prevents brain damage by the inhibition of excessive glutamate release and apoptotic damage decrease. mGluR2 and mGluR3 agonists produced comparable results, indicating that both receptors may be a potential target for early treatment in neonatal HI.

Mechanisms Underlying Enhancement of Spontaneous Glutamate Release by Group I mGluRs at a Central Auditory Synapse.

One emerging concept in neuroscience states that synaptic vesicles and the molecular machinery underlying spontaneous transmitter release are different from those underlying action potential-driven synchronized transmitter release. Differential neuromodulation of these two distinct release modes by metabotropic glutamate receptors (mGluRs) constitutes critical supporting evidence. However, the mechanisms underlying such a differential modulation are not understood. Here, we investigated the mechanisms of the modulation by group I mGluRs (mGluR Is) on spontaneous glutamate release in the medial nucleus of the trapezoid body (MNTB), an auditory brainstem nucleus critically involved in sound localization. Whole-cell patch recordings from brainstem slices of mice of both sexes were performed. Activation of mGluR I by 3,5-dihydroxyphenylglycine (3,5-DHPG; 200 µm) produced an inward current at -60 mV and increased spontaneous glutamate release in MNTB neurons. Pharmacological evidence indicated involvement of both mGluR1 and mGluR5, which was further supported for mGluR5 by immunolabeling results. The modulation was eliminated by blocking NaV channels (tetrodotoxin, 1 µm), persistent Na+ current (INaP; riluzole, 10 µm), or CaV channels (CdCl2, 100 µm). Presynaptic calyx recordings revealed that 3,5-DHPG shifted the activation of INaP to more hyperpolarized voltages and increased INaP at resting membrane potential. Our data indicate that mGluR I enhances spontaneous glutamate release via regulation of INaP and subsequent Ca2+-dependent processes under resting condition.SIGNIFICANCE STATEMENT For brain cells to communicate with each other, neurons release chemical messengers, termed neurotransmitters, in response to action potential invasion (evoked release). Neurons also release neurotransmitters spontaneously. Recent work has revealed different release machineries underlying these two release modes, and their different roles in synaptic development and plasticity. Our recent work discovered differential neuromodulation of these two release modes, but the mechanisms are not well understood. The present study showed that activation of group I metabotropic glutamate receptors enhanced spontaneous glutamate release in an auditory brainstem nucleus, while suppressing evoked release. The modulation is dependent on a persistent Na+ current and involves subsequent Ca2+ signaling, providing insight into the mechanisms underlying the different release modes in auditory processing.

Activation of metabotropic glutamate receptor 4 regulates proliferation and neural differentiation in neural stem/progenitor cells of the rat subventricular zone and increases phosphatase and tensin homolog protein expression.

Neural stem/progenitor cells (NSPCs) persist in the mammalian subventricular zone throughout life, where they can be activated in response to physiological and pathophysiological stimuli. A recent study indicates metabotropic glutamate receptor 4 (mGluR4) is involved in regulating NSPCs behaviors. Therefore, defining mGluR4 function in NSPCs is necessary for determining novel strategies to enhance the intrinsic potential for brain regeneration after injuries. In this study, mGluR4 was functionally expressed in SVZ-derived NSPCs from male Sprague-Dawley rats, in which the cyclic adenosine monophosphate concentration was reduced after treatment with the mGluR4-specific agonist VU0155041. Additionally, lateral ventricle injection of VU0155041 significantly decreased 5-bromo-2'-deoxyuridine (BrdU)+ and Ki67+ cells, while increased Doublecortin (DCX)/BrdU double-positive cells in SVZ. In cultured NSPCs, mGluR4 activation decreased the ratio of BrdU+ cells, G2/M-phase cells, and inhibited Cyclin D1 expression, whereas it increased neuron-specific class III ß-tubulin (Tuj1) expression and the number of Tuj1, DCX, and PSA-NCAM-positive cells. However, pharmacological blocking mGluR4 with the antagonist MSOP or knockdown of mGluR4 abolished the effects of VU0155041 on NSPCs proliferation and neuronal differentiation. Further investigation demonstrated that VU0155041 treatment down-regulated AKT phosphorylation and up-regulated expression of the phosphatase and tensin homolog protein (PTEN) in NSPCs culture. Moreover VU0155041-induced proliferating inhibition and neuronal differentiating amplification in NSPCs were significantly hampered by VO-OHpic, a PTEN inhibitor. We conclude that activation of mGluR4 in SVZ-derived NSPCs suppresses proliferation and enhances their neuronal differentiation, and regulation of PTEN may be involved as a potential intracellular target of mGluR4 signal. Cover Image for this issue: https://doi.org/10.1111/jnc.15052.

Cytohesin-2 mediates group I metabotropic glutamate receptor-dependent mechanical allodynia through the activation of ADP ribosylation factor 6 in the spinal cord.

Group I metabotropic glutamate receptors (mGluRs), mGluR1 and mGluR5, in the spinal cord are implicated in nociceptive transmission and plasticity through G protein-mediated second messenger cascades leading to the activation of various protein kinases such as extracellular signal-regulated kinase (ERK). In this study, we demonstrated that cytohesin-2, a guanine nucleotide exchange factor for ADP ribosylation factors (Arfs), is abundantly expressed in subsets of excitatory interneurons and projection neurons in the superficial dorsal horn. Cytohesin-2 is enriched in the perisynapse on the postsynaptic membrane of dorsal horn neurons and forms a protein complex with mGluR5 in the spinal cord. Central nervous system-specific cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia in inflammatory and neuropathic pain models. Pharmacological blockade of cytohesin catalytic activity with SecinH3 similarly reduced mechanical allodynia and inhibited the spinal activation of Arf6, but not Arf1, in both pain models. Furthermore, cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia and ERK1/2 activation following the pharmacological activation of spinal mGluR1/5 with 3,5-dihydroxylphenylglycine (DHPG). The present study suggests that cytothesin-2 is functionally associated with mGluR5 during the development of mechanical allodynia through the activation of Arf6 in spinal dorsal horn neurons.

Metabotropic Glutamate Receptor 2/3 Activation Improves Motor Performance and Reduces Pathology in Heterozygous zQ175 Huntington Disease Mice.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease that leads to progressive motor impairments with no available disease-modifying treatment. Current evidence indicates that exacerbated postsynaptic glutamate signaling in the striatum plays a key role in the pathophysiology of HD. However, it remains unclear whether reducing glutamate release can be an effective approach to slow the progression of HD. Here, we show that the activation of metabotropic glutamate receptors 2 and 3 (mGluR2/3), which inhibit presynaptic glutamate release, improves HD symptoms and pathology in heterozygous zQ175 knockin mice. Treatment of both male and female zQ175 mice with the potent and selective mGluR2/3 agonist LY379268 for either 4 or 8 weeks improves both limb coordination and locomotor function in all mice. LY379268 also reduces mutant huntingtin aggregate formation, neuronal cell death, and microglial activation in the striatum of both male and female zQ175 mice. The reduction in mutant huntingtin aggregates correlates with the activation of a glycogen synthase kinase 3ß-dependent autophagy pathway in male, but not female, zQ175 mice. Furthermore, LY379268 reduces both Akt and ERK1/2 phosphorylation in male zQ175 mice but increases both Akt and ERK1/2 phosphorylation in female zQ175 mice. Taken together, our results indicate that mGluR2/3 activation mitigates HD neuropathology in both male and female mice but is associated with the differential activation and inactivation of cell signaling pathways in heterozygous male and female zQ175 mice. This further highlights the need to take sex into consideration when developing future HD therapeutics. SIGNIFICANCE STATEMENT: The mGluR2/3 agonist LY379268 improves motor impairments and reduces pathology in male and female zQ175 Huntington's disease mice. The beneficial outcomes of LY379268 treatment in Huntington's disease mice were mediated by divergent cell signaling pathways in both sexes. We provide evidence that mGluR2/3 agonists can be repurposed for the treatment of Huntington's disease, and we emphasize the importance of investigating sex as a biological variable in preclinical disease-modifying studies.

Metabotropic glutamate 2,3 receptor stimulation desensitizes agonist activation of G-protein signaling and alters transcription regulators in mesocorticolimbic brain regions.

Metabotropic glutamate (mGlu) receptors are regulators of glutamate release and targets for development of therapies for hyperactive glutamatergic signaling. However, the effects of long-term stimulation of mGlu receptors on cellular signaling in the brain have not been described. This study investigated the effects of 2-day and 14-day osmotic mini-pump administration of the mGlu2,3 agonist LY379268 (3.0 mg kg-1  day-1 ) to rats on receptor-mediated G-protein activation and signaling in mesocorticolimbic regions in rat brain sections. A significant reduction in LY379268-stimulated [35 S]GTPγS binding was observed in the 14-day group in some cortical regions, prefrontal cortex, nucleus accumbens, and ventral pallidum. The 14-day LY379268 treatment group exhibited mGlu2 mRNA levels significantly lower in hippocampus, nucleus accumbens, caudate, and ventral pallidum. In both 2-day and 14-day treatment groups immunodetectable phosphorylated cAMP Response Element-Binding protein (CREB) was significantly reduced across all brain regions. In the 2-day group, we observed significantly lower immunodetectable CREB protein across all brain regions, which was subsequently increased in the 14-day group but failed to achieve control values. Neither immunodetectable extracellular signal-regulated kinase (ERK) protein nor phosphorylated ERK from 2-day or 14-day treatment groups differed significantly from control across all brain regions. However, the ratio of phosphorylated ERK to total ERK protein was significantly greater in the 14-day treatment group compared with the control. These results identify compensatory changes to mGlu2,3 signal transduction in rat brains after chronic systemic administration of agonist, which could be predictive of the mechanism of action in human pharmacotherapies.

The effect of the mGlu8 receptor agonist, (S)-3,4-DCPG on acquisition and expression of morphine-induced conditioned place preference in male rats.

BACKGROUND: The nucleus accumbens (NAc) plays a principal role in drug reward. It has been reported that metabotropic glutamate receptors (mGlu receptors) play a key role in the rewarding pathway(s). Previous studies have shown the vast allocation of the different types of mGlu receptors, including mGlu8 receptors, in regions that are associated with opioid rewards, such as the NAc. The aim of the present study was to evaluate the role of mGlu8 receptors within the NAc in the acquisition and expression phases of morphine induced conditioned place preference (CPP). Adult male Wistar rats were bilaterally implanted by two cannulas' in the NAc and were evaluated in a CPP paradigm. Selective mGlu8 receptor allosteric agonist (S-3,4-DCPG) was administered at doses of 0.03, 0.3, and 3 µg/0.5 µL saline per side into the NAc on both sides during the 3 days of morphine (5 mg/kg) conditioning (acquisition) phase, or before place preference test, or post-conditioning (expression) phase of morphine-induced CPP. RESULTS: The results revealed that intra-accumbal administration of S-3,4-DCPG (0.3 and 3 µg) markedly decreased the acquisition in a dose-dependent manner but had no effect on expression of morphine-induced CPP. CONCLUSIONS: The findings suggest that activation of mGlu8 receptors in the NAc dose-dependently blocks the establishment of morphine-induced CPP and reduces the rewarding properties of morphine which may be related to the glutamate activity into the NAc and in reward pathway(s). These data suggest that mGlu8 receptor may be involved in conditioned morphine reward.

Positive allosteric modulators (PAMs) of the group II metabotropic glutamate receptors: Design, synthesis, and evaluation as ex-vivo tool compounds.

This letter describes synthesis and evaluation of two series of dual mGlu2/mGlu3 positive allosteric modulators with moderate mGlu3 potency and robust mGlu2 potency in thallium flux assays. These compounds were profiled their ability to modulate mGlu3-mediated signaling in central neurons by co-application of a selective mGlu2 NAM to isolate mGlu3-selective effects. Using acute mouse brain slices from the prefrontal cortex, potentiation of group II mGlu receptor agonist Ca2+ signaling in PFC pyramidal cells with either the dual mGlu2/mGlu3 PAM 16e or 23d demonstrated effects mediated selectively via mGlu3.

Endocannabinoid receptors contribute significantly to multiple forms of long-term depression in the rat dentate gyrus.

Cannabinoid receptors are widely expressed throughout the hippocampal formation, but are particularly dense in the dentate gyrus (DG) subregion. We, and others, have shown in mice that cannabinoid type 1 receptors (CB1Rs) are involved in a long-term depression (LTD) that can be induced by prolonged 10 Hz stimulation of the medial perforant path (MPP)-granule cell synaptic input to the DG. Here, we extend this work to examine the involvement of CB1Rs in other common forms of LTD in the hippocampus of juvenile male and female Sprague-Dawley rats (Rattus norvegicus). We found, as in mice, that prolonged 10 Hz stimulation (6000 pulses) could reliably induce a form of LTD that was dependent upon CB1R activation. In addition, we also discovered a role for both CB1R and mGluR proteins in LTD induced with 1 Hz low-frequency stimulation (1 Hz-LTD; 900 pulses) and in LTD induced by bath application of the group I mGluR agonist (RS)-3,5-Dihydroxyphenylglycine (DHPG; DHPG-LTD). This study elucidates an essential role for endocannabinoid receptors in a number of forms of LTD in the rat DG, and identifies a novel role for CB1Rs as potential therapeutic targets for conditions that involve impaired LTD in the DG.

Elfn1-Induced Constitutive Activation of mGluR7 Determines Frequency-Dependent Recruitment of Somatostatin Interneurons.

Excitatory synapses onto somatostatin (SOM) interneurons show robust short-term facilitation. This hallmark feature of SOM interneurons arises from a low initial release probability that regulates the recruitment of interneurons in response to trains of action potentials. Previous work has shown that Elfn1 (extracellular leucine rich repeat and fibronectin Type III domain containing 1) is necessary to generate facilitating synapses onto SOM neurons by recruitment of two separate presynaptic components: mGluR7 (metabotropic glutamate receptor 7) and GluK2-KARs (kainate receptors containing glutamate receptor, ionotropic, kainate 2). Here, we identify how a transsynaptic interaction between Elfn1 and mGluR7 constitutively reduces initial release probability onto mouse cortical SOM neurons. Elfn1 produces glutamate-independent activation of mGluR7 via presynaptic clustering, resulting in a divergence from the canonical "autoreceptor" role of Type III mGluRs, and substantially altering synaptic pharmacology. This structurally induced determination of initial release probability is present at both layer 2/3 and layer 5 synapses. In layer 2/3 SOM neurons, synaptic facilitation in response to spike trains is also dependent on presynaptic GluK2-KARs. In contrast, layer 5 SOM neurons do not exhibit presynaptic GluK2-KAR activity at baseline and show reduced facilitation. GluK2-KAR engagement at synapses onto layer 5 SOM neurons can be induced by calmodulin activation, suggesting that synaptic function can be dynamically regulated. Thus, synaptic facilitation onto SOM interneurons is mediated both by constitutive mGluR7 recruitment by Elfn1 and regulated GluK2-KAR recruitment, which determines the extent of interneuron recruitment in different cortical layers.SIGNIFICANCE STATEMENT This study identifies a novel mechanism for generating constitutive GPCR activity through a transsynaptic Elfn1/mGluR7 structural interaction. The resulting tonic suppression of synaptic release probability deviates from canonical autoreceptor function. Constitutive suppression delays the activation of somatostatin interneurons in circuits, necessitating high-frequency activity for somatostatin interneuron recruitment. Furthermore, variations in the synaptic proteome generate layer-specific differences in facilitation at pyr → SOM synapses. The presence of GluK2 kainate receptors in L2/3 enhances synaptic transmission during prolonged activity. Thus, layer-specific synaptic properties onto somatostatin interneurons are mediated by both constitutive mGluR7 recruitment and regulated GluK2 kainate receptor recruitment, revealing a mechanism that generates diversity in physiological responses of interneurons.

Distinct anti-dyskinetic effects of amantadine and group II metabotropic glutamate receptor agonist LY354740 in a rodent model: An electrophysiological perspective.

L-DOPA-induced dyskinesia (LID) is a major complication of long-term dopamine replacement therapy in Parkinson's disease. Characteristic neural oscillation and abnormal activity of striatal projection neurons (SPNs) are typical pathological events of LID, which would be reliable biomarkers for assessment of novel anti-dyskinetic approach if fully profiled. Glutamate dysregulation plays a critical role in the development of LID, and the group II metabotropic glutamate receptors (mGluR2/3) is believed to regulate the release of glutamate on the presynaptic terminals and inhibits postsynaptic excitation. However, the anti-dyskinetic effect of modulating mGluR2/3 is still unclear. In this study, rats with unilateral dopaminergic lesion were injected with L-DOPA (12 mg/kg, i.p.) for seven days, while motor behavior was correlated with in vivo electrophysiology analyzing LFP and single-cell activity in both primary motor cortex and dorsolateral striatum. Our study showed that as LID established, high γ oscillation (hγ) predominated during LID, the number of unstable responses of SPN to dopamine increased, and the coherence between these patterns of oscillation and spiking activity also increased. We found that pretreatment of NMDA receptor antagonist, amantadine 60 mg/kg, i.p. (AMAN) significantly reduced abnormal involuntary movements (AIMs), in parallel with the reduction of hγ oscillation, and more markedly with a decrease in unstable responses of SPNs. In contrast, a mGluR2/3 agonist, LY354740 12 mg/kg, i.p. (LY) significantly shortened the duration of LID but merely exhibited a weak effect in diminishing the intensity of LID or reversing SPN responses. Together results indicate that AIMs in the rat model of PD are associated with abnormal corticostriatal signaling, which could be reversed by NMDAR antagonism more efficiently than mGluR2/3 agonism.

Mechanisms and functional impact of Group I metabotropic glutamate receptor modulation of excitability in mouse MNTB neurons.

We examined effects of Group I metabotropic glutamate receptors on the excitability of mouse medial nucleus of the trapezoid body (MNTB) neurons. The selective agonist, S-3,5-dihydroxyphenylglycine (DHPG), evoked a dose-dependent depolarization of the resting potential, increased membrane resistance, increased sag depolarization, and promoted rebound action potential firing. Under voltage-clamp, DHPG evoked an inward current, referred to as IDHPG , which was developmentally stable through postnatal day P56. IDHPG had low temperature dependence in the range 25-34°C, consistent with a channel mechanism. However, the I-V relationship took the form of an inverted U that did not reverse at the calculated Nernst potential for K+ or Cl- . Thus, it is likely that more than one ion type contributes to IDHPG and the mix may be voltage dependent. IDHPG was resistant to the Na+ channel blockers tetrodotoxin and amiloride, and to inhibitors of iGluR (CNQX and MK801). IDHPG was inhibited 21% by Ba2+ (500 µM), 60% by ZD7288 (100 µM) and 73% when the two antagonists were applied together, suggesting that KIR channels and HCN channels contribute to the current. Voltage clamp measurements of IH indicated a small (6%) increase in Gmax by DHPG with no change in the voltage dependence. DHPG reduced action potential rheobase and reduced the number of post-synaptic AP failures during high frequency stimulation of the calyx of Held. Thus, activation of post-synaptic Group I mGlu receptors modifies the excitability of MNTB neurons and contributes to the reliability of high frequency firing in this auditory relay nucleus.

Synthesis and pharmacological evaluation of bivalent tethered ligands to target the mGlu2/4 heterodimeric receptor results in a compound with mGlu2/2 homodimer selectivity.

This Letter details our ongoing efforts to develop selective positive allosteric modulators (PAMs) of the mGlu2/4 heterodimeric receptor that exists in the CNS and may represent a novel drug target to modulate the glutamatergic system. As multiple hit-to-lead campaigns from HTS hits failed to produce selective small molecule mGlu2/4 heterodimer PAMs, we were inspired by the work of Portoghese to synthesize and evaluate a set of nine bivalent tethered ligands (possessing an mGlu2 PAM at one terminus and an mGlu4 PAM at the other). Utilizing G protein-Inwardly Rectifying Potassium (GIRK) channel functional assays, we found that the tethered ligands displayed PAM activity in a cell line co-expressing both mGlu2 and mGlu4 but also in cells expressing mGlu2 or mGlu4 alone. In a CODA-RET assay, one of the tethered ligands potentiated mGlu2/4 heterodimers; however, another compound displayed 75-fold preference for the mGlu2/2 homodimer over heterodimeric mGlu2/4 or homomeric mGlu4/4. This work highlights the development of mGlu receptor PAMs with homodimer/heterodimer preference and expands the potential for PAMs as tethered ligands beyond the more classical antagonists and NAMs.

Biphasic Modulation of NMDA Receptor Function by Metabotropic Glutamate Receptors.

A recently reported rapid potentiation of NMDA receptors by Group I metabotropic glutamate receptors (mGluRIs) via a Homer protein link is distinct from the classical, relatively slow inhibitory G-protein-associated signaling triggered by mGluRI activation. The relationship between these two mechanisms remains unknown. Here, we focused on the mGluRI-dependent modulation of NMDAR response in hippocampal dentate gyrus granule cells and cerebellar granule cells of C57BL6-J mice and found that these two contrasting mechanisms overlap competitively on the time scale from hundreds of milliseconds to seconds, with the net effect depending on the cell type. At a shorter time interval (units of millisecond), the Homer-mediated signal from mGluRIs prevails, causing upregulation of NMDAR function, in both dentate gyrus granule cells and cerebellar granule cells. Our results shed light on the possible mechanisms of anti-schizophrenia drugs that disrupt Homer-containing protein link.SIGNIFICANCE STATEMENT Here we study modulation of NMDA receptors triggered by activation of metabotropic glutamate receptors Group I via two distinct pathways: classical G-protein signaling system and newly discovered high-speed modulatory mechanism associated with Homer-protein-containing direct molecular link. We found that these two contrasting mechanisms overlap competitively on the time scale from hundreds of milliseconds to seconds, with the net effect depending on the cell type. We have also found that both crosstalk mechanisms cause significant changes in synaptic strength and plasticity. Our results resolve an apparent discrepancy between earlier studies that demonstrated contradictive effects of Homer-containing protein link disruption on NMDA receptor signaling. On top of that, our data provide a plausible explanation for unclear action mechanisms of anti-schizophrenia drugs.

Glutamate triggers intracellular Ca2+ oscillations and nitric oxide release by inducing NAADP- and InsP3 -dependent Ca2+ release in mouse brain endothelial cells.

The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca2+ concentration ([Ca2+ ]i ) in the target cells, which activates the Ca2+ /Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca2+ ]i and NO production. The current study assessed whether and how glutamate drives Ca2+ -dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca2+ ]i , which was maximally activated at 200 µM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca2+ oscillations were triggered by rhythmic endogenous Ca2+ mobilization and maintained over time by extracellular Ca2+ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca2+ release was mediated by InsP3 -sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca2+ entry mediated Ca2+ entry during ongoing Ca2+ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca2+ signals. Of note, glutamate induced Ca2+ -dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca2+ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.