Functional interactions between presynaptic NMDA receptors and metabotropic glutamate receptors co-expressed on rat and human noradrenergic terminals.
ABSTRACT: Electrophysiological studies described potentiation of NMDA receptor function by metabotropic glutamate receptors (mGluRs) of group I occurring postsynaptically. Since release-enhancing NMDA receptors exist on noradrenergic terminals and group I mGluRs have recently been identified on these nerve endings, we have investigated if NMDA receptor-mGluR interactions also can occur at the presynaptic level.Rat hippocampus and human neocortex synaptosomes were labelled with [(3)H]noradrenaline and superfused with mGluR agonists and antagonists. NMDA-evoked [(3)H]noradrenaline release was produced by removal of external Mg(2+) or by simultaneous application of NMDA and AMPA in Mg(2+)-containing solutions.The mGluR1/5 agonist 3,5-DHPG, inactive on its own, potentiated both the release of [(3)H]noradrenaline elicited by AMPA/NMDA/glycine and that evoked by NMDA/glycine following Mg(2+) removal. The effect of 3,5-DHPG on the AMPA/NMDA/glycine-induced release was insensitive to the mGluR1 antagonist CPCCOEt, but it was abolished by the mGluR5 antagonist MPEP; moreover, it was potentiated by the mGluR5 positive allosteric modulator DFB. When NMDA receptors were activated by Mg(2+) removal, both mGluR5 and mGluR1 contributed to the evoked release, the mGluR-mediated release being blocked only by CPCCOEt and MPEP in combination. Experiments with human neocortex synaptosomes show NMDA receptor-mGluR interactions qualitatively similar to those observed in rodents.Group I mGluRs, both of the mGluR1 and mGluR5 subtypes, co-localize with NMDA receptors on noradrenergic terminals of rat hippocampus and human neocortex. Depending on the mode of activation, NMDA receptors exert differential permissive roles on the activation of presynaptic mGluR1 and mGluR5.
Project description:Cerebral white matter injury in premature infants, known as periventricular leukomalacia (PVL), is common after hypoxia-ischemia (HI). While ionotropic glutamate receptors (iGluRs) can mediate immature white matter injury, we have previously shown that excitotoxic injury to premyelinating oligodendrocytes (preOLs) in vitro can be attenuated by group I metabotropic glutamate receptor (mGluR) agonists. Thus, we evaluated mGluR expression in developing white matter in rat and human brain, and tested the protective efficacy of a central nervous system (CNS)-penetrating mGluR agonist on injury to developing oligodendrocytes (OLs) in vivo. Group I mGluRs (mGluR1 and mGluR5) were strongly expressed on OLs in neonatal rodent cerebral white matter throughout normal development, with highest expression early in development on preOLs. Specifically at P6, mGluR1 and mGLuR5 were most highly expressed on GalC-positive OLs compared to neurons, axons, astrocytes and microglia. Systemic administration of (1S,3R) 1-aminocyclopentane-trans-1,3,-dicarboxylic acid (ACPD) significantly attenuated the loss of myelin basic protein in the white matter following HI in P6 rats. Assessment of postmortem human tissue showed both mGluR1 and mGluR5 localized on immature OLs in white matter throughout development, with mGluR5 highest in the preterm period. These data indicate group I mGluRs are highly expressed on OLs during the peak period of vulnerability to HI and modulation of mGluRs is protective in a rodent model of PVL. Group I mGluRs may represent important therapeutic targets for protection from HI-mediated white matter injury.
Project description:Group 1 metabotropic glutamate receptors (mGluR1/mGluR5) play an integral role in neurodevelopment and are implicated in psychiatric disorders, such as schizophrenia. mGluR1 and mGluR5 are expressed as homodimers, which is important for their functionality and pharmacology. We examined the protein expression of dimeric and monomeric mGluR1? and mGluR5 in the prefrontal cortex (PFC) and hippocampus throughout development (juvenile/adolescence/adulthood) and in the perinatal phencyclidine (PCP) model of schizophrenia. Under control conditions, mGluR1? dimer expression increased between juvenile and adolescence (209-328%), while monomeric levels remained consistent. Dimeric mGluR5 was steadily expressed across all time points; monomeric mGluR5 was present in juveniles, dramatically declining at adolescence and adulthood (-97-99%). The mGluR regulators, Homer 1b/c and Norbin, significantly increased with age in the PFC and hippocampus. Perinatal PCP treatment significantly increased juvenile dimeric mGluR5 levels in the PFC and hippocampus (37-50%) but decreased hippocampal mGluR1? (-50-56%). Perinatal PCP treatment also reduced mGluR1? dimer levels in the PFC at adulthood (-31%). These results suggest that Group 1 mGluRs have distinct dimeric and monomeric neurodevelopmental patterns, which may impact their pharmacological profiles at specific ages. Perinatal PCP treatment disrupted the early expression of Group 1 mGluRs which may underlie neurodevelopmental alterations observed in this model.
Project description:Activation of cerebellar Purkinje cells by either brief depolarizing steps or bursts of climbing fiber synaptic activation evokes a slow inward current, which we have previously called depolarization-induced slow current or DISC. DISC is triggered by Ca influx via voltage-sensitive Ca channels and is attenuated by inhibitors of vacuolar ATPase or vesicle fusion. This led us to suggest that DISC required vesicular release of glutamate from the somatodendritic region of Purkinje cells. Furthermore, we found that DISC was attenuated by the mGluR1 antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), indicating that DISC required autocrine activation of metabotropic glutamate receptor 1 (mGluR1). Here, we have revisited the role of mGluR1 and found that it is, in fact, not required for DISC. CPCCOEt, but not three other specific mGluR1 antagonists (JNJ16259685, alpha-amino-5-carboxy-3-methyl-2-thiopheneacetic acid (3-MATIDA), Bay 36-7620), attenuated DISC, even though all four of these drugs produced near-complete blockade of current evoked by puffs of the exogenous mGluR1/5 agonist DHPG. Cerebellar slices derived from mGluR1 null mice showed substantial DISC that was still attenuated by CPCCOEt. mGluR5 is functionally similar to mGluR1, but is not expressed at high levels in cerebellar Purkinje cells. 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), an mGluR5 antagonist, did not attenuate DISC, and DISC was still present in Purkinje cells derived from mGluR1/mGluR5 double null mice. Thus, neither mGluR1 nor mGluR5 is required for DISC in cerebellar Purkinje cells.
Project description:Group I metabotropic glutamate receptors (mGluRs), including mGluR1 and mGluR5, are G protein–coupled receptors (GPCRs) that are expressed at excitatory synapses in brain and spinal cord. GPCRs are often negatively regulated by specific G protein–coupled receptor kinases and subsequent binding of arrestin-like molecules. Here we demonstrate an alternative mechanism in which group I mGluRs are negatively regulated by proline-directed kinases that phosphorylate the binding site for the adaptor protein Homer, and thereby enhance mGluR–Homer binding to reduce signaling. This mechanism is dependent on a multidomain scaffolding protein, Preso1, that binds mGluR, Homer and proline-directed kinases and that is required for their phosphorylation of mGluR at the Homer binding site. Genetic ablation of Preso1 prevents dynamic phosphorylation of mGluR5, and Preso1(?/?) mice exhibit sustained, mGluR5-dependent inflammatory pain that is linked to enhanced mGluR signaling. Preso1 creates a microdomain for proline-directed kinases with broad substrate specificity to phosphorylate mGluR and to mediate negative regulation.
Project description:Metabotropic glutamate receptors (mGluRs) function as dimers. Recent work suggests that mGluR1 and mGluR5 may physically interact, but the nature and functional consequences of this relationship have not been addressed. In this study, the functional and pharmacological consequences of this interaction were investigated. Using heterologous expression of mGluR cDNA in rat sympathetic neurons from the superior cervical ganglion and inhibition of the native calcium currents as an assay for receptor activation, a functional interdependence between mGluR1 and mGluR5 was demonstrated. In neurons coexpressing these receptors, combining a selective mGluR1 competitive antagonist with either an mGluR1- or mGluR5-selective negative allosteric modulator (NAM) BAY36-7620 [(3aS,6aS)-hexahydro-5-methylene-6a-(2-naphthalenylmethyl)-1H-cyclopenta[c]furan-1-one] or MPEP [2-methyl-6-(phenylethynyl)pyridine hydrochloride], respectively, strongly occluded signaling by both receptors to an approximately equal degree. By contrast, in cells coexpressing mGluR1 and mGluR2, combining the same mGluR1 competitive inhibitor with an mGluR1 or mGluR2 NAM yielded partial and full inhibition of the response, respectively, as expected for independently acting receptors. In neurons expressing mGluR1 and mGluR5, the selective NAMs each strongly inhibited the response to glutamate, suggesting that these receptors do not interact as heterodimers, which would not be inhibited by selective NAMs. Finally, evidence for a similar mGluR1/mGluR5 functional dependence is shown in medium spiny striatal neurons. Together, these data demonstrate cooperative signaling between mGluR1 and mGluR5 in a manner inconsistent with heterodimerization, and thus suggest an interaction between homodimers.
Project description:The mechanism underlying the upregulation of NMDA receptor function by group I metabotropic glutamate receptors (mGluRs), including mGluR1 and 5, is not known. Here we show that in cortical neurons, brief selective activation of group I mGluRs with (S)-3,5-dihydroxy-phenylglycine (DHPG) induced a Ca(2+)-calmodulin-dependent activation of Pyk2/CAKbeta and the Src-family kinases Src and Fyn that was independent of protein kinase C (PKC). Activation of Pyk2 and Src/Fyn kinases led to increased tyrosine phosphorylation of NMDA receptor subunits 2A and B (NR2A/B) and was blocked by a selective mGluR1 antagonist, 7-(hydroxyamino)cyclopropa[b]chromen-1a-carboxylate ethyl ester, but not an mGluR5 antagonist, 2-methyl-6-(phenylethynyl)pyridine. Functional linkage between mGluR1 activation and NR2A tyrosine phosphorylation through Pyk2 and Src was also demonstrated after expression of these elements in human embryonic kidney 293 cells. Supporting functional consequences, selective activation of mGluR1 by DHPG induced a potentiation of NMDA receptor-mediated currents that was blocked by inhibiting mGluR1 or Src-family kinases. Furthermore, antagonizing calmodulin or mGluR1, but not PKC, reduced the basal tyrosine phosphorylation levels of Pyk2 and Src, suggesting that mGluR1 may control the basal activity of these kinases and thus the tyrosine phosphorylation levels of NMDA receptors.
Project description:Metabotropic glutamate receptors (mGluRs) and Homer proteins play critical roles in neuronal functions including plasticity, nociception, epilepsy, and drug addiction. Furthermore, Homer proteins regulate mGluR1/5 function by acting as adapters and facilitating coupling to effectors such as the inositol triphosphate receptor. However, although Homer proteins and their interaction with mGluRs have been the subject of intense study, direct measurements of Homer-induced changes in postsynaptic mGluR-effector coupling have not been reported. This question was addressed here by examining glutamatergic excitatory postsynaptic currents (EPSCs) in rat autaptic hippocampal cultures. In most neurons, the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine strongly inhibited the EPSC acutely. This modulation occurred postsynaptically, was mediated primarily by mGluR5, and was inositol triphosphate receptor-dependent. Expression of the dominant negative, immediate early form of Homer, Homer 1a, strongly reduced EPSC modulation, but the W24A mutant of Homer 1a, which cannot bind mGluRs, had no effect. (S)-3,5-dihydroxyphenylglycine-mediated intracellular calcium responses in the processes of Homer 1a-expressing neurons were reduced compared with those in Homer 1a W24A-expressing cells. However, neither the distribution of mGluR5 nor the modulation of somatic calcium channels was altered by Homer 1a expression. These data demonstrate that Homer 1a can reduce mGluR5 coupling to postsynaptic effectors without relying on large changes in the subcellular distribution of the receptor. Thus, alteration of mGluR signaling by changes in Homer protein expression may represent a viable mechanism for fine-tuning synaptic strength in neurons.
Project description:Activation of group I metabotropic glutamate receptors (subtypes mGluR1 and mGluR5) regulates neural activity in a variety of ways. In CA1 pyramidal neurons, activation of group I mGluRs eliminates the post-burst afterhyperpolarization (AHP) and produces an afterdepolarization (ADP) in its place. Here we show that upregulation of Ca(v)2.3 R-type calcium channels is responsible for a component of the ADP lasting several hundred milliseconds. This medium-duration ADP is rapidly and reversibly induced by activation of mGluR5 and requires activation of phospholipase C (PLC) and release of calcium from internal stores. Effects of mGluR activation on subthreshold membrane potential changes are negligible but are large following action potential firing. Furthermore, the medium ADP exhibits a biphasic activity dependence consisting of short-term facilitation and longer-term inhibition. These findings suggest that mGluRs may dramatically alter the firing of CA1 pyramidal neurons via a complex, activity-dependent modulation of Ca(v)2.3 R-type channels that are activated during spiking at physiologically relevant rates and patterns.
Project description:Traumatic brain injury (TBI) produces excessive glutamate, leading to excitotoxicity via the activation of glutamate receptors. Postsynaptic density scaffold proteins have crucial roles in mediating signal transduction from glutamate receptors to their downstream mediators. Therefore, studies on the mechanisms underlying regulation of excitotoxicity by scaffold proteins can uncover new treatments for TBI. Here, we demonstrated that the postsynaptic scaffold protein Homer 1a was neuroprotective against TBI in vitro and in vivo, and this neuroprotection was associated with its effects on group I metabotropic glutamate receptors (mGluRs). Upon further study, we found that Homer 1a mainly affected neuronal injury induced by mGluR1 activation after TBI and also influenced mGluR5 function when its activity was restored. The ability of Homer 1a to disrupt mGluR-ERK signaling contributed to its ability to regulate the functions of mGluR1 and mGluR5 after traumatic injury. Intracellular Ca(2+) and PKC were two important factors involved in the mediation of mGluR-ERK signaling by Homer 1a. These results define Homer 1a as a novel endogenous neuroprotective agent against TBI.
Project description:Conventional signalling by the group I metabotropic glutamate receptors, mGluR1 and mGluR5, occurs through G-protein coupling, but evidence suggests they might also utilize other, non-canonical effector pathways. Here we test whether group I mGluRs require ?-arrestin signalling during specific forms of plasticity at hippocampal excitatory synapses. We find that genetic ablation of ?-arrestin2, but not ?-arrestin1, results in deficits in plasticity mediated by mGlu1 receptors in CA3 pyramidal neurons and by mGlu5 receptors in CA1 pyramidal neurons. Pharmacological studies additionally support roles for Src kinases and MAPK/ERK downstream of ?-arrestin2 in CA3 neurons. mGluR1 modulation of intrinsic conductances is otherwise preserved in ?-arrestin2-/- mice with the exception of a rebound depolarization, and non-mGluR-mediated long-term potentiation is unaltered. These results reveal a signalling pathway engaged by group I mGluRs to effect changes in synaptic and cell intrinsic physiology dependent upon ?-arrestin rather than G proteins. Pharmacological manipulation of mGluRs with effector-biased ligands could lead to novel therapies to treat neurological disease.