Activation Of Ionotropic Glutamate Receptors Research Articles

Functional Characterization and In Silico Docking of Full and Partial GluK2 Kainate Receptor Agonists

Two structural models have been developed to explain how agonist binding leads to ionotropic glutamate receptor (iGluR) activation. At alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) iGluRs, full and partial agonists close the agonist-binding domain (ABD) to different degrees whereas agonist-induced domain closure is apparently fixed at N-methyl-D-aspartate receptors. Although kainate (KA) iGluRs are thought to behave like AMPA receptors, the issue has not been formally tested because of the paucity of available receptor agonists. Here we identify a series of structurally related full and partial agonists at GluK2 (formerly GluR6) KARs and predict their docking mode using the in silico ligand-docking program FITTED. As expected, the neurotransmitter L-Glu behaved as a full agonist but modest reduction (e.g., L-serine or L-aspartate) or elongation (e.g., L-alpha-aminoadipate) in chain length generated weak partial agonists. It is noteworthy that in silico ligand-docking predicted that most partial agonists select for the closed and not, as expected, the open or intermediate conformations of the GluK2 ABD. Experiments using concanavalin-A to directly report conformations in the intact GluK2 receptor support this prediction with the full agonist, L-Glu, indistinguishable in this regard from weak partial agonists, D- and L-Asp. Exceptions to this were KA and domoate, which failed to elicit full closure as a result of steric hindrance by a key tyrosine residue. Our data suggest that alternative structural models need to be considered to describe agonist behavior at KARs. Finally, our study identifies the responsiveness to several neurotransmitter candidates establishing the possibility that endogenous amino acids other than L-Glu may regulate native KARs at central synapses.

Effects of glutamate receptor activation on NG2-glia in the rat optic nerve.

NG2-glia are a substantial population of cells in the central nervous system (CNS) that can be identified by their specific expression of the NG2 chondroitin sulphate (CSPG). NG2-glia can generate oligodendrocytes, but it is unlikely this is their only function; indeed, they may be multipotent neural stem cells. Moreover, NG2-glia are a highly reactive cell type and a major function is to help form the axon growth inhibitory glial scar in response to CNS injury. The factors that regulate these diverse behaviours of NG2-glia are not fully resolved, but NG2-glia express receptors to the neurotransmitter glutamate, which has known potent effects on other glia. Here, we have examined the actions of glutamate receptor activation on NG2-glia in the rat optic nerve, a typical CNS white matter tract that does not contain neuronal cell bodies. Glutamate induces an increase in [Ca(2+)](i) in immuno-identified NG2-glia in situ and in vitro. In addition, we examined the effects of glutamate receptor activation in vivo by focal injection of the glutamate receptor agonist kainate into the optic nerve; saline was injected in controls. Changes in glial and axonal function were determined at 7 days post injection (dpi), by immunohistochemistry and electrophysiological measurement of the compound action potential (CAP). Injection of kainate resulted in a highly localized 'injury response' in NG2-glia, marked by dense labelling for NG2 at the lesion site, as compared to astrocytes, which displayed a more extensive reactive astrogliosis. Furthermore, injection of kainate resulted in an axonal conduction block. These glial and axonal changes were not observed following injection of saline vehicle. In addition, we provide evidence that endogenous glutamate induces calcium-dependent phosphorylation of extracellular signal-regulated kinases (ERK1/2), which may provide a potential mechanism by which glutamate-mediated changes in raised intracellular calcium could regulate the observed gliosis. The results provide evidence that activation of AMPA-kainate type ionotropic glutamate receptors evoke raised calcium in NG2-glia and induces an injury response in NG2-glia.

Capsaicin-Induced Glutamate Release Is Implicated in Nociceptive Processing Through Activation of Ionotropic Glutamate Receptors and Group I Metabotropic Glutamate Receptor in Primary Afferent Fibers

Glutamate (Glu) is the major excitatory neurotransmitter in the central nervous system. The role of peripheral Glu and Glu receptors (GluRs) in nociceptive transmission is, however, still unclear. In the present study, we examined Glu levels released in the subcutaneous perfusate of the rat hind instep using a microdialysis catheter and the thermal withdrawal latency using the Plantar Test following injection of drugs associated with GluRs with/without capsaicin into the hindpaw. The injection of capsaicin into the rat hind instep caused an increase of Glu level in the s.c. perfusate. Capsaicin also significantly decreased withdrawal latency to irradiation. These effects of capsaicin were inhibited by pretreatment with capsazepine, a transient receptor potential vanilloid receptor 1 (TRPV1) competitive antagonist. Capsaicin-induced Glu release was also suppressed by combination with each antagonist of ionotropic GluRs (iGluRs: NMDA/AMPA receptors) and group I metabotropic GluR (mGluR), but not group II and group III mGluRs. Furthermore, these GluRs antagonists showed remarkable inhibition against capsaicin-induced thermal hyperalgesia. These results suggest that Glu is released from the peripheral endings of small-diameter afferent fibers by noxious stimulation and then activates peripheral iGluRs and group I mGluR in development and/or maintenance of nociception. Furthermore, the activation of peripheral NMDA/AMPA receptors and group I mGluR may be important in mechanisms whereby capsaicin evokes nociceptive responses.

Stimulation of NMDA and AMPA glutamate receptors elicits distinct concentration dynamics of nitric oxide in rat hippocampal slices

Nitric oxide ((*)NO) is an intercellular messenger implicated in memory formation and neurodegeneration in the hippocampus. Owing to its physical and chemical properties, the concentration dynamics of (*)NO is a critical issue in determining its bioactivity as a signaling molecule. Its production is closely related to glutamate N-methyl-D-aspartate (NMDA) receptors, following a rise in intracellular calcium levels. However, that dependent on alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors remains elusive and controversial, despite reports describing a role for these receptors in other brain regions, largely because of lack of quantitative and dynamic measurements of (*)NO. Using a (*)NO-selective microsensor inserted in the diffusional spread of (*)NO in the CA1 region of rat hippocampal slices, we measured its real-time endogenous production, following activation of ionotropic glutamate receptors and under tissue physiological oxygen tension. Both NMDA and AMPA stimulation resulted in a concentration-dependent (*)NO production but encompassing distinct kinetics for lag phases and slower rates of (*)NO production were observed for AMPA stimulation. Robustness of the results was achieved instrumentally and pharmacologically, by means of nitric oxide synthase (NOS) inhibitors and antagonists of NMDA (D-(-)-2-amino-5-phosphonopentanoic acid, AP5) and AMPA (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide, NBQX) receptors. When using glutamate as a stimulus, (*)NO production was of lower magnitude in the presence of AP5 plus NBQX than with AP5 alone, suggesting that even when NMDA receptors are inhibited Ca(2+) rises to levels to induce a peak of (*)NO from the background. Whereas extracellular Ca(2+) was required for the (*)NO signals, Philanthotoxin-4,3,3 (PhTX-4,3,3) a toxin used to target Ca(2+)-permeable AMPA receptors, attenuated (*)NO production. These observations are interpreted on basis of a distinct coupling between the glutamate receptors and neuronal NOS. A role for Ca(2+)-permeable AMPA receptors in the Ca(2+) activation of neuronal NOS is suggested.

Recovery of network-driven glutamatergic activity in rat hippocampal neurons during chronic glutamate receptor blockade

Previous studies indicated that a long-term decrease in the activity of ionotropic glutamate receptors induces cholinergic activity in rat and mouse hypothalamic neuronal cultures. Here we studied whether a prolonged inactivation of ionotropic glutamate receptors also induces cholinergic activity in hippocampal neurons. Receptor activity was chronically suppressed in rat hippocampal primary neuronal cultures with two proportionally increasing sets of concentrations of NMDA plus non-NMDA receptor antagonists: 100 μM/10 μM AP5/CNQX ( 1X cultures) and 200 μM/20 μM AP5/CNQX ( 2X cultures). Using calcium imaging we demonstrate that cholinergic activity does not develop in these cultures. Instead, network-driven glutamate-dependent activity, that normally is detected in hyper-excitable conditions, reappears in each culture group in the presence of these antagonists and can be reversibly suppressed by higher concentrations of AP5/CNQX. This activity is mediated by non-NMDA receptors and is modulated by NMDA receptors. Further, non-NMDA receptors, the general level of glutamate receptor activity and CaMK-dependent signaling are critical for development of this network-driven glutamatergic activity in the presence of receptor antagonists. Using electrophysiology, western blotting and calcium imaging we show that some neuronal parameters are either reduced or not affected by chronic glutamate receptor blockade. However, other parameters (including neuronal excitability, mEPSC frequency, and expression of GluR1, NR1 and βCaMKII) become up-regulated and, in some cases, proportionally between the non-treated, 1X and 2X cultures. Our data suggest recovery of the network-driven glutamatergic activity after chronic glutamate receptor blockade. This recovery may represent a form of neuronal plasticity that compensates for the prolonged suppression of the activity of glutamate receptors.

Gene Expression Profiles in Zebrafish Brain after Acute Exposure to Domoic Acid at Symptomatic and Asymptomatic Doses

Domoic acid (DA) is a neuroexcitatory amino acid that is naturally produced by some marine diatom species of the genus Pseudo-nitzschia. Ingestion of DA-contaminated seafood by humans results in a severe neurotoxic disease known as amnesic shellfish poisoning (ASP). Clinical signs of ASP include seizures and neuronal damage from activation of ionotropic glutamate receptors. However, the impacts of DA exposure at levels below those known to induce outward signs of neurobehavioral exicitotoxicity have not been well characterized. To further understand the mechanisms of neurotoxic injury associated with DA exposure, we examined the transcriptome of whole brains from zebrafish (Danio rerio) receiving intracoelomic (IC) injection of DA at both symptomatic and asymptomatic doses. A majority of zebrafish exposed to high-dose DA (1.2 microg DA/g) exhibited clinical signs of neuroexcitotoxicity (EC(50) of 0.86 microg DA/g) within 5-20 min of IC injection. All zebrafish receiving low-dose DA (0.47 microg DA/g) or vehicle only maintained normal behavior. Microarray analysis of symptomatic and asymptomatic exposures collectively yielded 306 differentially expressed genes (1.5-fold, p </= 0.05) predominately represented by signal transduction, ion transport, and transcription factor functional categories. Transcriptional profiles were suggestive of neuronal apoptosis following an overwhelming of protective adaptive pathways. Further, potential molecular biomarkers of neuropathic injury, including the zebrafish homolog of human NDRG4, were identified and may be relevant to DA exposure levels below that causing neurobehavioral injury. In general, DA-modulated gene expression was consistent with other model species thereby validating zebrafish as an appropriate vertebrate model to study mechanisms of DA neurotoxicity. These data provide a basis for identifying pathways of DA-induced injury as well as biomarkers of asymptomatic and symptomatic DA exposure levels.

Nanomolar concentrations of pregnenolone sulfate enhance striatal dopamine overflow in vivo.

The balance between GABA-mediated inhibitory and glutamate-mediated excitatory synaptic transmission represents a fundamental mechanism for controlling nervous system function, and modulators that can alter this balance may participate in the pathophysiology of neuropsychiatric disorders. Pregnenolone sulfate (PS) is a neuroactive steroid that can modulate the activity of ionotropic glutamate and GABA(A) receptors either positively or negatively, depending upon the particular receptor subtype, and modulates synaptic transmission in a variety of experimental systems. To evaluate the modulatory effect of PS in vivo, we infused PS into rat striatum for 20 min via a microdialysis probe while monitoring local extracellular dopamine (DA) levels. The results demonstrate that PS at low nanomolar concentrations significantly increases extracellular DA levels. The PS-induced increase in extracellular DA is antagonized by the N-methyl-d-aspartate (NMDA) receptor antagonist, d-AP5 [d-(-)-2-amino-5-phosphonopentanoic acid], but not by the sigma receptor antagonist, BD 1063 [1(-)[2-(3,4-dichlorophenyl)-ethyl]-4-methylpiperazine]. The results demonstrate that exogenous PS, at nanomolar concentrations, is able to increase DA overflow in the striatum through an NMDA receptor-mediated pathway.

Positive association between SAT‐1 −1415T/C polymorphism and anxiety

Limbic glutamatergic neurotransmission plays a pivotal role in the pathogenesis of anxiety disorders. Polyamines modulate the activity of several ionotropic glutamate receptors and have been involved in the regulation of fear-conditioning response. Spermidine/spermine N1-acetyltransferase (SSAT-1) is the main enzyme regulating polyamine catabolism. The aim of the present study was to examine the association between anxiety disorders and the -1415T/C (rs1960264) single nucleotide polymorphism (SNP) of the gene (SAT1) coding for SSAT-1. A case-control design was used in order to compare the genotypes for the -1415T/C (rs1960264) SNP between anxiety patients (n = 218), other non-anxiety psychiatric patients (n = 362), and healthy controls (n = 251). DSM-IV diagnoses were provided using MINI 4.4. Genomic DNA was extracted from peripheral blood samples collected from participants. In males, there was a significant difference in the distribution of the two genotypes (T and C) for the SAT-1 -1415T/C SNP between anxiety patients, non-anxiety psychiatric controls, and healthy controls. The T genotype was significantly more frequent in males suffering from anxiety disorders than in male psychiatric controls and healthy controls. This is the first study linking polymorphic variants of genes involved in polyamine metabolism with anxiety disorders.

Increased expression, but not postsynaptic localisation, of ionotropic glutamate receptors during the late-phase of long-term potentiation in the dentate gyrus in vivo

Long-term potentiation (LTP) is extensively studied as a cellular mechanism of information storage in the brain. The induction and early expression mechanisms of LTP depend on activation and rapid modulation of ionotropic glutamate receptors. However, the mechanisms that underlie maintenance of LTP over the course of days or longer are poorly understood. Here, we have investigated the overall expression of AMPA- and NMDA-type glutamate receptors (AMPARs and NMDARs, respectively), as well as their levels at the synaptic surface membrane and in the postsynaptic density (PSD), in the dentate gyrus at 48h following the induction of LTP at perforant path synapses in awake rats. We found a high-frequency stimulation-dependent increase in the overall levels of AMPAR subunits GluA1 and GluA2, but not GluA3 in the dentate gyrus. The increases in GluA1 and GluA2 levels were partially NMDAR-dependent, but were not found in biochemically isolated synaptic surface membrane or PSD fractions. In contrast, we found that the core NMDAR subunit, GluN1, increased in the synaptic surface-membrane fraction but it also was not targeted to the PSD. The GluA1 and GluA2 expression and the surface localisation of GluN1 returned to baseline levels by 2 weeks post-LTP induction. These data suggest that the late-phase LTP is not mediated by an overt increase in the AMPAR content of perforant path synapses. The increase in surface expression NMDARs may influence thresholds for future plasticity events.

Hypoxia-ischemia and retinal ganglion cell damage

Retinal hypoxia is the potentially blinding mechanism underlying a number of sight-threatening disorders including central retinal artery occlusion, ischemic central retinal vein thrombosis, complications of diabetic eye disease and some types of glaucoma. Hypoxia is implicated in loss of retinal ganglion cells (RGCs) occurring in such conditions. RGC death occurs by apoptosis or necrosis. Hypoxia-ischemia induces the expression of hypoxia inducible factor-1alpha and its target genes such as vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS). Increased production of VEGF results in disruption of the blood retinal barrier leading to retinal edema. Enhanced expression of NOS results in increased production of nitric oxide which may be toxic to the cells resulting in their death. Excess glutamate release in hypoxic-ischemic conditions causes excitotoxic damage to the RGCs through activation of ionotropic and metabotropic glutamate receptors. Activation of glutamate receptors is thought to initiate damage in the retina by a cascade of biochemical effects such as neuronal NOS activation and increase in intracellular Ca(2+) which has been described as a major contributing factor to RGC loss. Excess production of proinflammatory cytokines also mediates cell damage. Besides the above, free-radicals generated in hypoxic-ischemic conditions result in RGC loss because of an imbalance between antioxidant- and oxidant-generating systems. Although many advances have been made in understanding the mediators and mechanisms of injury, strategies to improve the damage are lacking. Measures to prevent neuronal injury have to be developed.

Adrenomedullin acts in the lateral parabrachial nucleus to increase arterial blood pressure through mechanisms mediated by glutamate and nitric oxide

Adrenomedullin (ADM) acts in a site-specific manner within autonomic centers of the brain to modulate mean arterial pressure (MAP). To determine the role of ADM in the pontine autonomic center, the lateral parabrachial nucleus (LPBN), we used urethane-anesthetized adult Sprague-Dawley male rats to test the hypothesis that ADM increases MAP at this site through glutamate- and nitric oxide (NO)-dependent mechanisms. ADM microinjected into the LPBN increased MAP in a dose-dependent manner. The pressor effect of ADM (0.01 pmol) had a peak value of 11.9 +/- 1.9 mmHg at 2 min and lasted for 7 min. We demonstrated that ADM's effect is receptor mediated by blocking the effect with the ADM receptor antagonist, ADM22-52. We showed that glutamate mediates ADM's pressor response, as this response was blocked using coinjections of ADM with dizolcipine hydrogen maleate or 6-cyano-7-nitroquinoxaline-2,3-dione, N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor antagonists, respectively. We tested the roles of NO with coinjections of ADM with either N5-(1-iminoethyl)-L-ornithine or 7-nitroindazole monosodium salt, nonspecific and neuronal NO synthase (NOS) inhibitors, respectively; both inhibitors blocked ADM's pressor effect. Finally, we studied the role of calcium influx in ADM's pressor effect, as intracellular calcium is important in both glutamate and NO neurotransmission. ADM's effect was blocked when nifedipine, an L-type calcium channel blocker, was coinjected with ADM into the LPBN. This study is the first to show that ADM acts in the LPBN to increase MAP through mechanisms dependent on activation of ionotropic glutamate receptors, neuronal and endothelial NOS-mediated NO synthesis, and L-type calcium channel activation.

Estradiol Induces Hypothalamic Dendritic Spines by Enhancing Glutamate Release: A Mechanism for Organizational Sex Differences

The naturally occurring sex difference in dendritic spine number on hypothalamic neurons offers a unique opportunity to investigate mechanisms establishing synaptic patterning during perinatal sensitive periods. A major advantage of the rat as a model of sexual differentiation is that treatment of neonatal females with estradiol will permanently induce the male phenotype. During the development of other systems, exuberant innervation is followed by activity-dependent pruning necessary for elimination of spurious synapses. In contrast, we demonstrate that estradiol-induced organization in the hypothalamus involves the induction of new synapses on dendritic spines. Activation of estrogen receptors by estradiol triggers a nongenomic activation of PI3 kinase that results in enhanced glutamate release from presynaptic neurons. Subsequent activation of ionotropic glutamate receptors activates MAP kinases, thereby inducing dendritic spine formation. These results reveal a transneuronal mechanism by which estradiol acts during a sensitive period to establish a profound and lasting sex difference in hypothalamic synaptic patterning.

Activation of ionotropic excitatory amino acid (EAA) receptors modulates both eupnea and gasping in the arterially‐perfused adult rat preparation

Previous work from our laboratory in an arterially‐perfused adult rat preparation has shown that blockade of ionotropic EAA receptors reduces or abolishes basal phrenic nerve discharge and markedly enhances inspiratory neural network complexity. Further, we have demonstrated that during EAA receptor blockade, gasping can still be elicited. The effect of activation of ionotropic glutamate receptors on phrenic motor discharge requires clarification. We examined the effects of perfusion with the glutamate analog DLH on phrenic burst timing and inspiratory neural network complexity in the arterially‐perfused adult rat preparation. DLH (500 μM, n=8) transiently increased phrenic burst frequency by 41% (P=0.005) mediated by decreasing the duration between bursts by 55% (p&lt;0.001), followed by a 36% increased stabilization of burst frequency (P=0.074). Post‐inspiratory activity was also increased. DLH decreased the duration of the peripheral chemoreflex from 23.8±1.7 to 13.2±1.4 s (P&lt;0.001), the duration of ischemia‐induced gasping from 58.6±5.3 to 33.4±2.3 s (P&lt;0.001), and the onset latency to gasping by 79% (P&lt;0.001). These findings support a role for ionotropic EAA neurotransmission in establishing the temporal dynamics of basal phrenic nerve discharge and suggest that it also has a role in determining the onset and duration of the peripheral chemoreflex and gasping. Supported by NS045321 and NS049310

Neural circuits engaged in ventral hippocampal modulation of dopamine function in medial prefrontal cortex and ventral striatum

Dopamine (DA) transmission in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) is crucial for various cognitive processes. However, our understanding of the regulation of DA efflux by glutamatergic afferents to these areas is incomplete. Using microdialysis in freely moving rats, we provide evidence in the present study that brief stimulation (20 Hz, 10 s) of the ventral hippocampus potently increases DA efflux in the mPFC, NAc, and ventral tegmental area for 30-40 min. Subsequent experiments show that the stimulation-evoked increase in DA efflux in the mPFC depends on local activation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate, but not N-methyl-D-aspartate, receptors in the mPFC. Additionally, neural activity and ionotropic glutamate receptor activation in the ventral tegmental area are necessary for ventral hippocampal stimulation to increase mPFC DA efflux. Blocking neural activity or ionotropic glutamate receptors in the ventral tegmental area also attenuated the stimulation-evoked increase in DA efflux in the NAc. Evidence in support of a role for the mPFC in the stimulation-evoked increase in NAc DA was not obtained. Taken together, these observations highlight the important role of the ventral hippocampus in modulating forebrain DA efflux via separate neural circuits.

Antagonists of AMPA/KA and NMDA (glycine site) glutamate receptors

The present review systematizes studies of recent years on the structure-activity relationship of antagonists of the glycine site of NMDA-type glutamate receptors and antagonists of AMPA-kainate-type glutamate receptors. Extreme activation of ionotropic glutamate receptors is known to promote the development of a number of socially important diseases, such as Alzheimer's disease and Parkinson's disease. Construction of inhibitors of these receptors is therefore one of the major directions in contemporary medicinal chemistry. This review may be useful for specialists engaged in the development of new pharmacologically active substances.

Metabotropic Glutamate Receptors (mGluRs) and Diabetic Neuropathy

Multiple in vivo and in vitro studies show that excessive release of glutamate, and subsequent activation of ionotropic glutamate receptors (iGluRs) and some metabotropic glutamate receptors (mGluRs) cause neuronal cell death through either necrosis or apoptosis. However, recently alternative evidence has shown that mGluRs have modulatory effects on excitotoxicity and neuronal cell death. Metabotropic glutamate receptors form a family of eight subtypes (mGluR1-8), subdivided into three groups (I-III) that initiate their biological effects by G protein-linked intracellular signal transduction. Their expression throughout the mammalian nervous system implicates these receptors as essential mediators of a cell's fate during injury to the nervous system. Activation of group-II (mGluR2 and -3) or group-III metabotropic glutamate receptors (mGluR4, -6, -7 and -8) has been established to be neuroprotective in vitro and in vivo. In contrast, group-I mGluRs (mGluR1 and -5) need to be antagonized in order to evoke protection. The pathological signaling pathways associated with diabetic neuropathy are complex and this influences development of appropriate therapies. The Group II mGluRs target several signaling pathways affected in diabetic neuropathy, prevent cellular injury in the peripheral nervous system, and may provide a novel mechanism for treatment of diabetic neuropathy. Direct or indirect activation of mGluR2/3 in animal models protects against development of diabetic neuropathy. The potential mechanisms and role of mGluRs in protection against diabetic neuropathy will be reviewed.

Activity-dependent Regulation of h Channel Distribution in Hippocampal CA1 Pyramidal Neurons

The hyperpolarization-activated cation current, I(h), plays an important role in regulating intrinsic neuronal excitability in the brain. In hippocampal pyramidal neurons, I(h) is mediated by h channels comprised primarily of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel subunits, HCN1 and HCN2. Pyramidal neuron h channels within hippocampal area CA1 are remarkably enriched in distal apical dendrites, and this unique distribution pattern is critical for regulating dendritic excitability. We utilized biochemical and immunohistochemical approaches in organotypic slice cultures to explore factors that control h channel localization in dendrites. We found that distal dendritic enrichment of HCN1 is first detectable at postnatal day 13, reaching maximal enrichment by the 3rd postnatal week. Interestingly we found that an intact entorhinal cortex, which projects to distal dendrites of CA1 but not area CA3, is critical for the establishment and maintenance of distal dendritic enrichment of HCN1. Moreover blockade of excitatory neurotransmission using tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione, or 2-aminophosphonovalerate redistributed HCN1 evenly throughout the dendrite without significant changes in protein expression levels. Inhibition of calcium/calmodulin-dependent protein kinase II activity, but not p38 MAPK, also redistributed HCN1 in CA1 pyramidal neurons. We conclude that activation of ionotropic glutamate receptors by excitatory temporoammonic pathway projections from the entorhinal cortex establishes and maintains the distribution pattern of HCN1 in CA1 pyramidal neuron dendrites by activating calcium/calmodulin-dependent protein kinase II-mediated downstream signals.

Glial cell‐derived glutamate mediates autocrine cell volume regulation in the retina: activation by VEGF

Astroglial cells are a source for gliotransmitters such as glutamate and ATP. We demonstrate here that gliotransmitters have autocrine functions in the regulation of cellular volume. Hypoosmotic stress in the presence of inflammatory mediators or oxidative stress, and during blockade or down-regulation of potassium channels, induces swelling of retinal glial cells. Vascular endothelial growth factor inhibits the osmotic swelling of glial cells in retinal slices or isolated cells. This effect was mediated by a kinase domain region/flk-1 receptor-evoked calcium dependent release of glutamate from glial cells, and subsequent stimulation of glial group I/II metabotropic glutamate receptors. Activation of kinase domain region/flk-1 or glutamate receptors evoked an autocrine swelling-inhibitory purinergic signaling cascade that was calcium-independent. This cascade involved the release of ATP and adenosine, and the activation of purinergic P2Y(1) and adenosine A1 receptors, resulting in the opening of potassium and chloride channels and inhibition of cellular swelling. The glutamatergic-purinergic regulation of the glial cell volume may be functionally important in the homeostasis of the extracellular space volume during intense neuronal activation which is associated with a swelling of neuronal cell structures in the retina. However, glial cell-derived glutamate may also contribute to the swelling of activated neurons since metabolic poisoning of glial cells by iodoacetate inhibits the neuronal cell swelling mediated by activation of ionotropic glutamate receptors.

Hormesis: A promising strategy to sustain endogenous neuronal survival pathways against neurodegenerative disorders

The brain developed adaptive mechanisms in the face of changing environments and stresses imposed on the nervous system. The addition of glutamate as the major excitatory amino acid neurotransmitter to the brain's complement of amino acids and peptides dictated a coordinated transcriptional and translational program to meet the demands of excitatory neurotransmission. One such program is the ability of neurons to sustain and maintain their survival given the nature of glutamate-mediated receptor activation. The unique development of endogenous neuronal pathways activated by glutamate receptors transformed neurons and allowed them to survive under conditions of high energy demands. These same endogenous survival pathways also mediate plastic responses to meet another demand of the brain, adaptation. An endogenous protein that plays a central role in glutamate receptor-mediated survival pathways is brain-derived neurotrophic factor (BDNF). Intermittent but frequent synaptic ionotropic glutamate receptor activation ensures neuronal survival through a BDNF autocrine loop. In sharp contrast, overactivation of ionotropic glutamate receptors leads to neuronal cell death. Thus, innovative strategies that induce endogenous neuronal survival pathways through low-level activation of ionotropic glutamate receptors or those that bypass receptor activation but upregulate endogenous survival pathways may not only prevent neurodegenerative disorders that involve glutamate as a final common pathway that kills neurons, but may also provide treatment alternatives critical for neurons to survive stressful conditions such as stroke, status epilepticus and hypoglycemic-induced neuronal cell death.

Modulation of interleukin‐6 and matrix metalloproteinase 2 expression in human fibroblast‐like synoviocytes by functional ionotropic glutamate receptors

Patients with rheumatoid arthritis (RA) have increased concentrations of the amino acid glutamate in synovial fluid. This study was undertaken to determine whether glutamate receptors are expressed in the synovial joint, and to determine whether activation of glutamate receptors on human synoviocytes contributes to RA disease pathology. Glutamate receptor expression was examined in tissue samples from rat knee joints and in human fibroblast-like synoviocytes (FLS). FLS from 5 RA patients and 1 normal control were used to determine whether a range of glutamate receptor antagonists influenced expression of the proinflammatory cytokine interleukin-6 (IL-6), enzymes involved in matrix degradation and cytokine processing (matrix metalloproteinase 2 [MMP-2] and MMP-9), and the inhibitors of these enzymes (tissue inhibitor of metalloproteinases 1 [TIMP-1] and TIMP-2). IL-6 concentrations were determined by enzyme-linked immunosorbent assay, MMP activity was measured by gelatin zymography, and TIMP activity was determined by reverse zymography. Fluorescence imaging of intracellular calcium concentrations in live RA FLS stimulated with specific antagonists was used to reveal functional activation of glutamate receptors that modulated IL-6 or MMP-2. Ionotropic and metabotropic glutamate receptor subunit mRNA were expressed in the patella, fat pad, and meniscus of the rat knee and in human articular cartilage. Inhibition of N-methyl-D-aspartate (NMDA) receptors in RA FLS increased proMMP-2 release, whereas non-NMDA ionotropic glutamate receptor antagonists reduced IL-6 production by these cells. Stimulation with glutamate, NMDA, or kainate (KA) increased intracellular calcium concentrations in RA FLS, demonstrating functional activation of specific ionotropic glutamate receptors. Our findings indicate that activation of NMDA and KA glutamate receptors on human synoviocytes may contribute to joint destruction by increasing IL-6 expression.