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Kainate receptors play fundamental roles in regulating synaptic transmission and plasticity in central nervous system and are regulated by their cognate auxiliary subunits Neuropilin and tolloid-like proteins 1 and 2 (Neto). While electrophysiology-based insights into functions of Neto proteins are known, biophysical and biochemical studies into Neto proteins have been largely missing till-date. Our biochemical, biophysical, and functional characterization of the purified extracellular domain (ECD) of Neto1 shows that Neto1-ECD exists as monomers in solution and has a micromolar affinity for GluK2 receptors in apo state or closed state. Remarkably, the affinity was ~2.8 fold lower for receptors trapped in the desensitized state, highlighting the conformation-dependent interaction of Neto proteins with kainate receptors. SAXS analysis of Neto1-ECD reveals that their dimensions are long enough to span the entire extracellular domain of kainate receptors. The shape and conformation of Neto1-ECD seems to be altered by calcium ions pointing towards its possible role in modulating Neto1 functions. Functional assays using GluK2 receptors and GluK2/GluA2 chimeric receptors reveal a differential role of Neto1 domains in modulating receptor functions. Although the desensitization rate was not affected by the Neto1-ECD, the recovery rates from the desensitized state are altered., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Hippocampal GABAergic neurons are subdivided into more than 20 subtypes that are distinguished by features and functions. We have previously described the subpopulation of GABAergic neurons, which can be identified in hippocampal cell culture by the calcium response to the application of domoic acid (DoA), an agonist of kainate receptors (KARs). Here, we investigate the features of DoA-sensitive neurons and their GABA release mechanism in response to KARs activation. We demonstrate that DoA-sensitive GABAergic neurons express GluK1-containing KARs because ATPA, a selective agonist of GluK1-containing receptors, induces the calcium response exclusively in these GABAergic neurons. Our experiments also show that NASPM, previously considered a selective antagonist of calcium-permeable AMPARs, blocks calcium-permeable KARs. We established using NASPM that GluK1-containing receptors of the studied population of GABAergic neurons are calcium-permeable, and their activation is required for GABA release, at least in particular synapses. Notably, GABA release occurs even in the presence of tetrodotoxin, indicating that propagation of the depolarizing stimulus is not required for GABA release in this case. Thus, our data demonstrate that the activation of GluK1-containing calcium-permeable KARs mediates the GABA release by the studied subpopulation of GABAergic neurons., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.)

Kainate receptors play an important role in the brain. They contribute to postsynaptic depolarization, modulate the release of neurotransmitters such as GABA and glutamate, affect the development of the neuronal network. At the same time, their functions depend not only on the type of neuron expressing them but also on their localization (pre- or postsynaptic). It has been shown in present work that activation of kainate receptors by domoic acid stimulates the secretion of both glutamate and GABA. This effect is observed at a concentration of 100 nM. At higher levels (200-500 nM), domoic acid selectively activates a specific population of GABAergic neurons. The peculiarity of these neurons is increased excitability in the network. This phenomenon can be explained by the weak GABA(A)R-mediated inhibition, as well as by the lower activation threshold of voltage-gated channels. Moreover, activation of these GABAergic neurons by domoic acid leads to the suppression of activity in the network under ammonium-induced hyperexcitation. As shown by inhibitory analysis, this effect is mediated by GABA(A) receptors. The obtained data may be of interest since the suppression of hyperexcitation via the selective activation of GABAergic neurons can be considered as a new potential approach to the treatment of diseases accompanied by increased neuronal activity such as epilepsy, ischemia and hepatic encephalopathy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Classically, endoplasmic reticulum (ER) retention signals in secreted integral membrane proteins impose the requirement to assemble with other cognate subunits to form functional assemblies before they can exit the ER. We report that GluK5 has two ER retention signals in its cytoplasmic C-terminus: an arginine-based signal and a di-leucine motif previously thought to be an endocytic motif. GluK5 assembles with GluK2, but surprisingly GluK2 association does little to block the ER retention signals. We find instead that the ER retention signals are blocked by two proteins involved in intracellular trafficking, SAP97 and CASK. We show that SAP97, in the presence of CASK and the receptor complex, assumes an extended conformation. In the extended conformation, SAP97 makes its SH3 and GuK domains available to bind and sterically mask the ER retention signals in the GluK5 C-terminus. SAP97 and CASK are also necessary for sorting receptor cargoes into the local dendritic secretory pathway in neurons. We show that the ER retention signals of GluK5 play a vital role in sorting the receptor complex in the local dendritic secretory pathway in neurons. These data suggest a new role for ER retention signals in trafficking integral membrane proteins in neurons. SIGNIFICANCE: We present evidence that the ER retention signals in the kainate receptors containing GluK5 impose a requirement for sorting into local dendritic secretory pathways in neurons, as opposed to traversing the somatic Golgi apparatus. There are two ER retention signals in the C-terminus of GluK5. We show that both are blocked by physical association with SAP97 and CASK. The SH3 and GuK domains of SAP97, in the presence of CASK, bind directly to each ER retention signal and form a complex. These results support an entirely new function for ER retention signals in the C-termini of neuronal receptors, such as NMDA and kainate receptors, and define a mechanism for selective entry of receptors into local secretory pathways., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: In addition to general anaesthetic effects, sevoflurane can also induce hyperactive behaviours during induction and recovery, which may contribute to neurotoxicity; however, the mechanism of such effects is unclear. Volatile anaesthetics including isoflurane have been found to activate the kainate (GluK2) receptor. We developed a novel mouse model and further explored the involvement of kainate (GluK2) receptors in sevoflurane-induced hyperactivity., Methods: Maximal speed, mean speed, total movement distance and resting percentage of C57BL/6 mice were quantitatively measured using behavioural tracking software before and after sevoflurane anaesthesia. Age dependence of this model was also analysed and sevoflurane-induced hyperactivity was evaluated after intracerebral injection of the GluK2 receptor blocker NS-102. Neurones from the hippocampal CA3 region were used to undertake in vitro electrophysiological measurement of kainate currents and miniature excitatory postsynaptic potential (mEPSP)., Results: Sevoflurane induced significant hyperactivities in mice under sevoflurane 1% anaesthesia and during the recovery period, characterized as increased movement speed and total distance. The hyperactivity was significantly increased in young mice compared with adults (P<0.01) and pre-injection of NS-102 significantly prevented this sevoflurane-induced hyperactivity. In electrophysiological experiments, sevoflurane significantly increased the frequency of mEPSP at low concentrations and evoked kainate currents at high concentrations., Conclusions: We developed a behavioural model in mice that enabled characterization of sevoflurane-induced hyperactivity. The kainate (GluK2) receptor antagonist attenuated these sevoflurane-induced hyperactivities in vivo, suggesting that kainate receptors might be the underlying therapeutic targets for sevoflurane-induced hyperactivities in general anaesthesia., (© The Author 2017. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Several C1q family members, related to the C1q complement component are extensively expressed in the central nervous system. Cbln1, which belongs to the Cbln subfamily of C1q proteins and released from cerebellar granule cells, plays an indispensable role in the synapse formation and function at parallel fiber-Purkinje cell synapses. This is achieved by formation of a trans-synaptic tripartite complex which is composed of one unit of the Cbln1 hexamer, monomeric neurexin (NRX) containing a splice site 4 insertion at presynaptic terminals and the postsynaptic GluD2 dimers. Recently an increasing number of soluble or transmembrane proteins have been identified to bind directly to the amino-terminal domains of iGluR and regulate the recruitment and function of iGluRs at synapses. Especially at mossy fiber (MF)-CA3 synapses in the hippocampus, postsynaptic kainate-type glutamate receptors (KARs) are involved in synaptic network activity through their characteristic channel kinetics. C1ql2 and C1ql3, which belong to the C1q-like subfamily of C1q proteins, are produced by MFs and serve as extracellular organizers to recruit functional postsynaptic KAR complexes at MF-CA3 synapses via binding to the amino-terminal domains of GluK2 and GluK4 KAR subunits. In addition, C1ql2 and C1ql3 directly bind to NRX3 containing sequences encoded by exon 25b insertion at splice site 5. In the present review, we highlighted the generality of the strategy by tripartite complex formation of the specific type of NRX and iGluR via C1q family members., (Copyright © 2016 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.)

Kainate-type glutamate receptors (KARs) are the third class of ionotropic glutamate receptors whose activation leads to the unique roles in regulating synaptic transmission and circuit functions. In contrast to AMPA receptors (AMPARs), little is known about the mechanism of synaptic localization of KARs. PSD-95, a major scaffold protein of the postsynaptic density, is a candidate molecule that regulates the synaptic KARs. Although PSD-95 was shown to bind directly to KARs subunits, it has not been tested whether PSD-95 regulates synaptic KARs in intact synapses. Using PSD-95 knockout mice, we directly investigated the role of PSD-95 in the KARs-mediated components of synaptic transmission at hippocampal mossy fiber-CA3 synapse, one of the synapses with the highest density of KARs. Mossy fiber EPSCs consist of AMPA receptor (AMPAR)-mediated fast component and KAR-mediated slower component, and the ratio was significantly reduced in PSD-95 knockout mice. The size of KARs-mediated field EPSP reduced in comparison with the size of the fiber volley. Analysis of KARs-mediated miniature EPSCs also suggested reduced synaptic KARs. All the evidence supports critical roles of PSD-95 in regulating synaptic KARs., (Copyright © 2015 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.)

Ligand-gated ion channels (LGICs) are cell surface integral proteins that mediate the fast neurotransmission in the nervous system. LGICs require auxiliary subunits for their trafficking, assembly and pharmacological modulation. Auxiliary subunits do not form functional homomeric receptors, but are reported to assemble with the principal subunits in order to modulate their pharmacological profiles. For example, nACh receptors are built at least by co-assemble of α and β subunits, and the neuronal auxiliary subunits β3 and α5 and muscle type β, δ, γ, and ϵ determine the agonist affinity of these receptors. Serotonergic 5-HT3B, 5-HT3C, 5-HT3D and 5-HT3E are reported to assemble with the 5-HT3A subunit to modulate its pharmacological profile. Functional studies evaluating the role of γ2 and δ auxiliary subunits of GABAA receptors have made important advances in the understanding of the action of benzodiazepines, ethanol and neurosteroids. Glycine receptors are composed principally by α1-3 subunits and the auxiliary subunit β determines their synaptic location and their pharmacological response to propofol and ethanol. NMDA receptors appear to be functional as heterotetrameric channels. So far, the existence of NMDA auxiliary subunits is controversial. On the other hand, Kainate receptors are modulated by NETO 1 and 2. AMPA receptors are modulated by TARPs, Shisa 9, CKAMP44, CNIH2-3 auxiliary proteins reported that controls their trafficking, conductance and gating of channels. P2X receptors are able to associate with auxiliary Pannexin-1 protein to modulate P2X7 receptors. Considering the pharmacological relevance of different LGICs auxiliary subunits in the present work we will highlight the therapeutic potential of these modulator proteins., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Medial prefrontal cortex (mPFC) and orbitofrontal cortex (OFC) play critical roles in cognition and behavioural control. Glutamatergic, GABAergic, and monoaminergic dysfunction in the prefrontal cortex has been hypothesised to underlie symptoms in neuropsychiatric disorders. Here we characterised electrically-evoked field potentials in the mPFC and OFC. Electrical stimulation evoked field potentials in layer V/VI of the mPFC and layer V of the OFC. The earliest component (approximately 2 ms latency) was insensitive to glutamate receptor blockade and was presumed to be presynaptic. Later components were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX (20 µM)) and were assumed to reflect monosynaptic (latency 4-6 ms) and polysynaptic activity (latency 6-40 ms) mediated by glutamate via AMPA/kainate receptor. In the mPFC, but not the OFC, the monosynaptic component was also partly blocked by 2-amino-5-phosphonopentanoic acid (AP-5 (50-100µM)) indicating the involvement of NMDA receptors. Bicuculline (3-10 µM) enhanced the monosynaptic component suggesting electrically-evoked and/or glutamate induced GABA release inhibits the monosynaptic component via GABAA receptor activation. There were complex effects of bicuculline on polysynaptic components. In the mPFC both the mono- and polysynaptic components were attenuated by 5-HT (10-100 µM) and NA (30 and 60 µM) and the monosynaptic component was attenuated by DA (100 µM). In the OFC the mono- and polysynaptic components were also attenuated by 5-HT (100 µM), NA (10-100 µM) but DA (10-100 µM) had no effect. We propose that these pharmacologically characterised electrically-evoked field potentials in the mPFC and OFC are useful models for the study of prefrontal cortical physiology and pathophysiology., (Copyright © 2013 Elsevier B.V. and ECNP. All rights reserved.)

We previously investigated a sample of psychotic patients acutely ill and acutely treated with haloperidol in the search for genetic predictors of response at PANSS scores during the first month of treatment. In the present work we extend the analysis to a wider panel of genetic variations including SNPs harbored by genes whose products are involved in molecular pathways consistent with the latest results of genome-wide association studies (GWAS) of antipsychotic efficacy. 96 Patients were investigated. The results were replicated in an independent sample of bipolar manic patients treated with antipsychotics (n tot=470, the sample was retrieved from the STEP-BD). Outcomes were the PANSS variation through time in the first sample, and changes of mania symptomatology at any two consecutive observations in the public available STEP-BD replication sample. A list of variations harbored by AKAP13, CACNA1, GRIK4 and GRIA1 were found to be significantly associated with outcome in both samples (different set of variations for each sample). Results did not survived multiple testing in the original sample but were replicated in both samples. This finding stresses the relevance of the glutamatergic system and regulatory molecular cascades in antipsychotic response. Nonetheless, the level of significance and the indirect and incomplete replication mandate cautiousness and further replication., (Copyright © 2012 Elsevier B.V. and ECNP. All rights reserved.)

GluR5-containing kainite receptor (GluR5-KAR) plays an important role in the pathophysiology of nervous system diseases, while S-nitrosylation exerts a variety of effects on biological systems. However, the mechanism of GluR5-KAR S-nitrosylation is still unclear up to now. Here our researches found that GluR5-KAR selective agonist ATPA stimulation activated the nonclassical GluR5-KAR-Gq-PLC-IP(3)R pathway and the signalling module GluR5·PSD-95·nNOS (the former is more important), led to Ca(2+) release from intracellular calcium stores endoplasmic reticulum (ER) to cytoplasm and extracellular calcium indrawal, respectively, which further resulted in nNOS activation and GluR5-KAR S-nitrosylation, and then inhibited GluR5-mediated whole-cell current attenuation and induced apoptosis in primary cultured hippocampal neurons. Clarification of the primary mechanisms of GluR5-KAR S-nitrosylation induced by ATPA and identification of critical cysteine for GluR5-2a S-nitrosylation (Cys231 and Cys804) open up a brand-new field for revealing downstream signalling pathway of GluR5-KAR and its molecular characteristics, exploring the pathogenesis of neurological diseases and searching for promising therapies., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Glutamate receptor 6 (GluR6) is well documented to play a pivotal role in ischemic brain injury, which is mediated by the GluR6·PSD95·MLK3 signaling module and subsequent c-Jun N-terminal kinase (JNK) activation. Our recent studies show that GluR6 is S-nitrosylated in the early stages of ischemia-reperfusion. NO (Nitric Oxide) is mainly generated from neuronal nitric oxide synthase (nNOS) in cerebral neurons during the early stages of reperfusion. Here, the effect of nNOS downregulation on GluR6 S-nitrosylation and GluR6-mediated signaling was investigated in cerebral ischemia and reperfusion. Administration of nNOS oligonucleotides confirmed that GluR6 nitrosylation is induced by nNOS-derived endogenous NO and further activates the GluR6·PSD95·MLK3 signaling module and JNK signaling pathway. Moreover, this study revealed for the first time that nNOS can bind with GluR6 during ischemic reperfusion, and PSD95 is involved in this interaction. In summary, our results suggest that nNOS binds with GluR6 via PSD95 and then produces endogenous NO to S-nitrosylate GluR6 in cerebral ischemia-reperfusion, which provides a new approach for stroke therapy., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Dysiherbaine (DH) and neodysiherbaine A (NDH) selectively bind and activate two kainate-type ionotropic glutamate receptors, GluK1 and GluK2. The ligand-binding domains of human GluK1 and GluK2 were crystallized as bound forms with a series of DH analogues including DH, NDH, 8-deoxy-NDH, 9-deoxy-NDH and 8,9-dideoxy-NDH (MSVIII-19), isolated from natural sources or prepared by total synthesis. Since the DH analogues exhibit a wide range of binding affinities and agonist efficacies, it follows that the detailed analysis of crystal structure would provide us with a significant opportunity to elucidate structural factors responsible for selective binding and some aspects of gating efficacy. We found that differences in three amino acids (Thr503, Ser706 and Ser726 in GluK1 and Ala487, Asn690 and Thr710 in GluK2) in the ligand-binding pocket generate differences in the binding modes of NDH to GluK1 and GluK2. Furthermore, deletion of the C(9) hydroxy group in NDH alters the ligand conformation such that it is no longer suited for binding to the GluK1 ligand-binding pocket. In GluK2, NDH pushes and rotates the side chain of Asn690 (substituted for Ser706 in GluK1) and disrupts an interdomain hydrogen bond with Glu409. The present data support the idea that receptor selectivities of DH analogues resulted from the differences in the binding modes of the ligands in GluK1/GluK2 and the steric repulsion of Asn690 in GluK2. All ligands, regardless of agonist efficacy, induced full domain closure. Consequently, ligand efficacy and domain closure did not directly coincide with DH analogues and the kainate receptors., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Ionotropic glutamate receptors (iGluRs) are ligand-gated cation channels that mediate fast excitatory neurotransmission in the mammalian central nervous system. In the model plant Arabidopsis thaliana, a large family of 20 genes encoding proteins that share similarities with animal iGluRs in sequence and predicted secondary structure has been discovered. Members of this family, termed AtGLRs (A. thaliana glutamate receptors), have been implicated in root development, ion transport, and several metabolic and signalling pathways. However, there is still no direct proof of ligand-gated ion channel function of any AtGLR subunit. We used a domain transplantation technique to directly test whether the putative ion pore domains of AtGLRs can conduct ions. To this end, we transplanted the ion pore domains of 17 AtGLR subunits into rat alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (GluR1) and kainate (GluR6) receptor subunits and tested the resulting chimaeras for ion channel function in the Xenopus oocyte expression system. We show that AtGLR1.1 and AtGLR1.4 have functional Na(+)-, K(+)-, and Ca(2+)-permeable ion pore domains. The properties of currents through the AtGLR1.1 ion pore match those of glutamate-activated currents, depolarisations, and glutamate-triggered Ca(2+) influxes observed in plant cells. We conclude that some AtGLRs have functional non-selective cation pores.

This study investigated the effects of the AMPA/Kainate receptor antagonist, NBQX, on cue-elicited cocaine-seeking behavior and concomitant changes in Fos protein expression. After cocaine self-administration training, rats underwent 24 days of abstinence during which they were exposed daily either to the self-administration environment with response-contingent cues previously paired with cocaine infusions available (Extinction group) or to an alternate environment (No Extinction group). Subsequently, rats were tested for cocaine-seeking behavior (i.e., operant responses without cocaine reinforcement) elicited by the cocaine-associated cues after pretreatment with either vehicle or NBQX (10 mg/kg, IP). NBQX markedly attenuated cue-elicited cocaine-seeking behavior relative to vehicle pretreatment in the No Extinction group and also decreased cue-elicited Fos protein expression in a region-specific manner in the anterior cingulate and orbitofrontal cortices, basolateral amygdala, nucleus accumbens core, and dorsal caudate-putamen, suggesting involvement of AMPA glutamate systems in specific subregions of the neuronal circuitry activated by cocaine cues.

AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis. Kainic acid (KA), a prototype excitotoxin is known to induce brain-derived neurotrophic factor (BDNF) in brain. In this study, we examined the role of AMPK in KA-induced BDNF expression in C6 glioma cells. We showed that KA and KA receptor agonist induced activation of AMPK and KA-induced AMPK activation was blocked by inhibition of Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) beta. We then showed that inhibition of AMPK by compound C, a selective inhibitor of AMPK, or small interfering RNA of AMPKalpha1 blocked KA-induced BDNF mRNA and protein expression. Inhibition of AMPK blocked KA-induced phosphorylation of CaMKII and I kappaB kinase (IKK) in C6 cells. Finally, we showed that inhibition of AMPK reduced DNA binding and transcriptional activation of nuclear factor-kappaB (NF-kappaB) in KA-treated cells. These results suggest that AMPK mediates KA-induced BDNF expression by regulating NF-kappaB activation.

Background: N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxazolone-4-propionic acid (AMPA), and kainate (KA) receptors are members of the ionotropic glutamate receptor (iGluR) family and are increased in inflamed rat skin. These receptors contribute to inflammatory pain. In this study, we have examined whether there is a similar increase in iGluRs in inflamed human skin in the presence of inflammatory pain., Methods: Normal and inflamed-skin biopsies were obtained from eight patients undergoing elective wound-debridement surgery. Real-time-polymerase chain reaction (PCR) and western blot analysis were used for quantitation of iGluR mRNA and protein in normal and inflamed human skin., Results: A significant increase in mRNA and protein for NMDA, AMPA, and KA receptor subunits was detected in inflamed compared with normal skin. The amounts of NMDA (NR1 subunit), AMPA (GluR2 subunit), and KA (GluR6 subunit) mRNA in inflamed skin were mean 6 (SD 1.6-fold), 2.5 (0.6-fold), and 3.8 (0.9-fold) (P<0.05), respectively, greater than that measured in normal skin. The ratio of NR1, GluR2, and GluR6 protein in inflamed compared with normal skin was 5.7 (1.2), 2.4 (0.5), and 3.6 (0.9) (P<0.05), respectively., Conclusions: These results, in human tissue, demonstrate that iGluR mRNA and protein expression are increased during persistent inflammation and that this increased activity may be involved in mediating clinical inflammatory pain in human skin.

Glutamate receptor (GluR) subunit composition of inferior salivatory nucleus (ISN) neurons was studied by immunohistochemical staining of retrogradely labeled neurons. Preganglionic ISN neurons innervating the von Ebner or parotid salivary glands were labeled by application of a fluorescent tracer to the lingual-tonsilar branch of the glossopharyngeal nerve or the otic ganglion respectively. We used polyclonal antibodies to glutamate receptor subunits NR1, NR2A, NR2B, (NMDA receptor subunits) GluR1, GluR2, GluR3, GluR4 (AMPA receptor subunits), and GluR5-7, KA2 (kainate receptor subunits) to determine their expression in ISN neurons. The distribution of the NMDA, AMPA and kainate receptor subunits in retrogradely labeled ISN neurons innervating the von Ebner and parotid glands was qualitatively similar. The percentage of retrogradley labeled ISN neurons innervating the parotid gland expressing the GluR subunits was always greater than those innervating the von Ebner gland. For both von Ebner and parotid ISN neurons, NR2A subunit staining had the highest expression and the lowest expression of GluR subunit staining was NR2B for von Ebner ISN neurons and GluR1 for parotid ISN neurons. The percentage of NR2B and GluR4 expressing ISN neurons was significantly different between the two glands. The percentage of ISN neurons that expressed GluR receptor subunits ranged widely indicating that the distribution of GluR subunit expression differs amongst the ISN neurons. While ISN preganglionic neurons express all the GluR subunits, differences in the percentage of ISN neurons expression between neurons innervating the von Ebner and parotid glands may relate to the different functional roles of these glands.

In this article we investigate the distributions of kainate glutamate receptor subtypes GluR5-7 and KA1, 2 on the dendritic arbors of direction-selective (DS) retinal ganglion cells (RGCs) of the rabbit retina to search for anisotropies, which might contribute to a directional preference of DS RGCs. The distribution of the kainate receptor subunits on the DS RGCs was determined using antibody immunocytochemistry. DS RGCs were injected with Lucifer yellow and the cells were identified by their characteristic morphology. The double-labeled images of dendrites and receptors were visualized using confocal microscopy and were reconstructed from high-resolution confocal images. We found no evidence of asymmetry in any of the kainate receptor subunits examined on the dendritic arbors of both On and Off layers of DS RGCs. Our results indicate that direction selectivity appears to lie in the neuronal circuitry afferent to the ganglion cell.

Microglia are reported to have alpha-amino-hydroxy-5-methyl-isoxazole-4-propionate/kainate (KA) types. However, only small population of primary cultured rat microglia (approximately 20%) responded to KA. In the present study, we have attempted to elucidate the regulatory mechanism of responsiveness to KA in GMIR1 rat microglial cell line. When the GMIR1 cells were plated at a low density in the presence of granulocyte macrophage colony-stimulating factor, the proliferation rate increased and reached the peak after 2 days in culture and then gradually decreased because of density-dependent inhibition. At cell proliferation stage, approximately 80% of the GMIR1 cells exhibited glutamate (Glu)- and KA-induced inward currents at cell proliferation stage, whereas only 22.5% of the cells showed responsiveness to Glu and KA at cell quiescent stage. Furthermore, the mean amplitudes of inward currents induced by Glu and KA at cell proliferation stage (13.8+/-3.0 and 8.4+/-0.6 pA) were significantly larger than those obtained at cell quiescent stage (4.7+/-0.8 and 6.2+/-1.2 pA). In the GMIR1 cells, KA-induced inward currents were markedly inhibited by (RS)-3-(2-carboxybenzyl) willardiine (UBP296), a selective antagonist for KA receptors. The KA-responsive cells also responded to (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), a selective agonist for GluR5, in both GMIR1 cells and primary cultured rat microglia. Furthermore, mRNA levels of the KA receptor subunits, GluR5 and GluR6, at the cell proliferation stage were significantly higher than those at the cell quiescent stage. Furthermore, the immunoreactivity for GluR6/7 was found to increase in activated microglia in the post-ischemic hippocampus. These results strongly suggest that microglia have functional KA receptors mainly consisting of GluR5 and GluR6, and the expression levels of these subunits are closely regulated by the cell cycle mechanism.

Allodynia or hyperalgesia induced by peripheral nerve injury may be involved in changes in the sensitivity of neurotransmitters at the spinal cord level. In order to clarify the functional role of neurotransmitters in peripheral nerve injury, we used rats with nerve injury induced by chronic constriction of the sciatic nerve (CCI rat model) and estimated the effects of the intrathecal injection of drugs known to affect glutamate and tachykinin receptors. In sham-operated rats, the NMDA receptor agonist NMDA and AMPA-kinate receptor agonist RS-(5)-bromowillardin reduced withdrawal latency. The non-competitive NMDA receptor antagonist MK-801, competitive NMDA receptor antagonist AP-5 and AMPA-kinate receptor antagonist NBQX increased withdrawal latency. Substance P (SP) increased the withdrawal latency but only transitorily. The NK1 receptor antagonist RP67580 increased withdrawal latency, but the NK2 receptor antagonist SR48968 did not show an effect. In CCI rats, RS-(5)-bromowillardin reduced withdrawal latency, but NMDA did not show an effect. NBQX increased withdrawal latency, while MK-801 and AP-5 showed little or no effect. SP reduced withdrawal latency, and both RP67580 and SR48968 increased it. These results indicate that the alteration in sensitivity of ionotropic glutamate receptors and tachykinin receptors in the spinal cord contribute to development and maintenance of nerve injury-evoked neuropathic pain.

Background: Several reports have shown that the glutamatergic system is involved in both the pathogenesis of affective and stress-related disorders and in the action of antidepressant drugs. In particular, antidepressant treatment was shown to modulate expression and function of ionotropic glutamate receptors, to inhibit glutamate release and to restore synaptic plasticity impaired by stress., Methods: We analyzed the mRNA expression and RNA editing of alpha-amino-propionic-acid (AMPA) and kainate (KA) receptor subunits, in the pre-frontal/frontal cortex (P/FC) and hippocampus (HI) of rats chronically treated with three different drugs: the selective serotonin (5-HT) reuptake inhibitor fluoxetine, the selective noradrenaline (NA) reuptake inhibitor reboxetine and the tricyclic antidepressant desipramine., Results: Our data showed that fluoxetine and desipramine exerted moderate but selective effects on glutamate receptor expression and editing, while reboxetine appeared to be the drug that affects glutamate receptors (GluR) most. The most consistent effect, observed with pronoradrenergic drugs (desipramine and reboxetine), was a decrease of GluR3 expression both in P/FC and HI. Interestingly, in HI, the same drugs also decreased the editing levels of either the flip (desipramine) or flop (reboxetine) form of GluR3., Conclusions: Overall, these results point to specific and regionally discrete changes in the expression and editing level of glutamate receptors and, in particular, to a selective reduction of conductance for GluR3-containing receptors following treatment with antidepressant drugs. These data support the hypothesis that changes in glutamate neurotransmission are involved in the therapeutic effects induced by these drugs.

Glial precursor cells (GPCs) are present in the adult human central nervous system (CNS) and they can be isolated and maintained in culture for in vitro studies. This study analysed expression of mGluR3 and mGluR5 metabotropic glutamate receptor (mGluR) mRNAs in GPCs. A2B5 surface antigen positive GPCs were isolated using immunomagnetic selection from dissociated temporal lobe subcortical white matter cells. The separated GPCs were maintained in cultures and characterised by immunoreactivity for the differentiation markers A2B5 and human platelet-derived growth factor-alpha receptor (PDGFalphaR). Reverse transcription followed by multiplex PCR analysis showed that the GPCs expressed both mGluR3 and mGluR5a mRNAs. Double immunostaining for glial progenitor markers and mGluR5 proteins demonstrated that all A2B5 and PDGFalphaR-positive cells were also positive for mGluR5. The results indicate that GPCs present in the adult human CNS express mGluR3 and mGluR5a. These neurotransmitter receptors may be involved in the proliferation and differentiation of glial cells.

We measured the MLK3 expression, activity and backphosphorylation following cerebral ischemia. Our data showed that MLK3 protein levels were unalterable during ischemia and reperfusion. However, during ischemia MLK3 activity gradually increased and reached its peak at 30 min of ischemia. While its backphosphorylation reduced from 5 min of ischemia to 30 min of ischemia. In addition, we also detected MLK3 alteration at various time points of reperfusion after 15 min of ischemia, which showed that MLK3 activity increased twice, whereas MLK3 backphosphorylation was similarly consistent with its activity during reperfusion. To further analyze the reason of MLK3 activation, antioxidant N-acetylcysteine (NAC) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA)/kainate (KA) receptor antagonist 6,7-dinitroquinoxaline-2,3(1H, 4H)-dione (DNQX) were given to the rats 20 min prior to ischemia. The results illustrated that NAC preferably inhibited the MLK3 activation during the ischemia and the early reperfusion, whereas DNQX effectively attenuated the MLK3 activation of the late reperfusion. We think that MLK3 activation is certainly associated with reactive oxygen species (ROS) and AMPA/KA receptor in response to ischemic insult.

The glutamate receptor subtypes AMPA and kainate are involved in synaptic transmission and synaptic plasticity in the CNS. Recently there has been considerable interest in understanding the molecular regulation of these receptors by proteins that directly bind to AMPA and kainate receptor subunits. Amongst the first interaction partners to be discovered were NSF, ABP, GRIP and PICK1, which bind the AMPA receptor subunit GLUA2. We have studied the functional roles of the interactions of these proteins in regulating AMPA receptor-mediated synaptic transmission and synaptic plasticity in the hippocampus. We have also started to investigate the functions of PICK1 and GRIP on kainate receptor-mediated synaptic transmission in this region. In this article we reflect upon this work, which has led to some new ideas about how AMPA and kainate receptors are regulated at synapses.

The ionotropic glutamate receptor subunits expressed by vagal preganglionic neurones in the rat medulla oblongata were examined by using fluorescence immunolabelling combined with retrograde neuronal tracing. The general population of these neurones in the medulla was identified by intraperitoneal injections of Fluorogold and also with choline acetyltransferase antibodies. Cardiac projecting neurones were specifically identified by applying the fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine (DiI) to the heart or by injecting cholera toxin B-subunit into the pericardium. Both tracers labelled populations of neurones lying in the dorsal vagal nucleus, intermediate reticular formation and nucleus ambiguus, and when both tracers were applied simultaneously, approximately 50% of cells were dual-labelled. Control experiments established that the labelling was specific for neurones projecting to the heart. Most vagal preganglionic neurones, including those projecting to the heart, irrespective of their location in the medulla, had a similar profile of glutamate receptor immunoreactivity. Labelling of somata for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) subunit GluR1 was weak or absent, while labelling with antibodies directed to GluR2, a common sequence of GluR2 and GluR3, and GluR4 was moderate or intense. All neurones studied appeared to express the N-methyl-D-aspartate (NMDA) receptor subunit NR1, and while antibodies recognising the NR2A and NR2B splice variants gave strong labelling, immunoreactivity with a NR2B specific antibody was weaker. Weak to moderate labelling was seen in some neurones using antibodies to the kainate receptor subunits KA2 and GluR5-7. These results are consistent with neurophysiological data indicating the presence of AMPA, NMDA and kainate responses in cardiac vagal preganglionic neurones, and suggest that these neurones are similar to other vagal parasympathetic preganglionic neurones in expressing mainly AMPA receptor subunits GluR2-4.

Kainate-type ionotropic glutamate receptors (KARs) distribute widely and heterogenously throughout the central nervous system (CNS). There is now increasing evidence showing that, in addition to conventional action to mediate postsynaptic excitation, KAR also exerts presynaptic action modulating the amount of transmitter release at certain synapses in the CNS. The mechanism and physiological function of presynaptic KARs have been studied most extensively at the hippocampal mossy fiber (MF)-CA3 synapse, one of the CNS regions where the highest density of KAR subunits is expressed. One unique feature of presynaptic KARs is that their activation modulates transmitter release bi-directionally; weak activation enhances glutamate release, while strong activation leads to inhibition. These findings may be explained by their possible ionotropic action leading to axonal depolarization, which in turn regulates several voltage-dependent channels involved in action potential-dependent Ca2+ entry processes. Furthermore, physiological activation of presynaptic KAR involves an activity-dependent process. Large frequency facilitation, a form of short-term plasticity characteristic of the MF-CA3 synapse, is mediated, at least partly, by presynaptic KAR. Bi-directional and activity-dependent regulation of transmitter release by kainate autoreceptors might have physiological significance in information processing in the hippocampus and other CNS regions, as well as its well-known pathological action contributing to epileptogenesis.

Objective: Our purpose was to evaluate the effect of seizures on kainate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor binding in maternal rat brain and whether maternal peripheral administration of magnesium sulfate can decrease this effect., Study Design: Rats were implanted with a bipolar electrode into the hippocampus. One week of recovery was allowed before breeding. Pregnant rats were randomly assigned to 1 of 4 groups, as follows: group 1, sodium chloride and no seizures (n = 5); group 2, magnesium sulfate and no seizures (n = 4); group 3, sodium chloride and seizures (n = 8); and group 4, magnesium sulfate and seizures (n = 9). Doses of sodium chloride or magnesium sulfate were administered every 20 minutes for 4 hours to all rats on gestational days 9, 11, 13, 15, 17, and 19, followed by seizure induction (groups 3 and 4). On gestational day 20, rats were perfused, brains were dissected, and cryostat sections were taken, labeled in vitro, and placed on Hyperfilm for 4 weeks. The ligands used included kainate receptor agonist and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor agonist and antagonist. Optical density measurements of binding in 15 brain regions on each section were evaluated by 1- and 2-way analysis of variance., Results: Seizure activity was associated with decreased alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor binding of its agonist in pregnant rat brains (seizure effect, 25.9 +/- 3.2 and 92.6 +/- 3.4 fmol/mg tissue in hindbrain and forebrain, respectively; no seizure effect, 44.5 +/- 4.7 and 110. 7 +/- 5.0 fmol/mg tissue in hindbrain and forebrain, respectively; P <.01). Magnesium administration was associated with increased binding of tritiated alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (magnesium effect, 44.9 +/- 4.2 and 110.4 +/- 4.5 fmol/mg tissue in hindbrain and forebrain, respectively; sodium chloride effect, 25.5 +/- 3.7 and 92.9 +/- 4.0 fmol/mg tissue in hindbrain and forebrain, respectively; P <.01). The same trend was seen with the kainate receptor in the hippocampus and hypothalamus, with a significant interaction effect between seizure and magnesium (P <.05)., Conclusions: The mechanism for maternal rat brain injury resulting from seizure activity may be, at least in part, associated with alteration in the function of excitatory amino acid receptors. Administration of magnesium sulfate can counteract this effect and may reduce resultant maternal brain damage.

Thus far, five members including Dlg, SAP97/hDlg, SAP90/PSD-95, SAP102, and PSD-93/chapsyn110 which belong to SAP family have been identified. Recent studies have revealed that these proteins play important roles in the localization and function of glutamate receptors and K(+) channels. Although most of them have been reported to be localized to the synapse, only one member, SAP97, is expressed also in the epithelial cells. In this review, we have summarized structural characters of SAP family proteins and discuss their functions in neurons and epithelial cells., (Copyright 2000 Academic Press.)

Glutamate receptors have been studied extensively in mammals but less explored in lower vertebrates. These receptors are present in amphibians. Using a recent method based upon agonist-induced cobalt uptake, we were able to detect the presence of functional alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors permeable to divalent cations in tadpoles and in adults. The uptake specificity was checked by co-application of an antagonist. We studied the distribution of receptor-bearing cells in the principal brain regions. The distribution was similar in the two species studied: Rana esculenta (green frog) and Bufo bufo (common toad). The high number of cobalt-positive cells suggests that the AMPA/kainate receptors permeable to divalent cations play an important role in the anuran nervous system.

An assessment of glutamate receptor subunit profiles was made in hippocampus and temporal lobe cortex of patients with refractory epilepsy. Molecular biological analyses using reverse transcription reaction (RT) followed by polymerase chain reaction (PCR) revealed changes in the distribution profile of the transcripts of AMPA/KA glutamate receptor subunits in hippocampal and cortical tissue from patients with refractory epilepsy when compared to similar tissue from six human and four non-human primate samples with no history of seizures or seizure medication. A severe mean decrease (38% of control) in mRNA for the GluR1 subunit was found in 400 mm cross-sections of hippocampus from patients with epilepsy. Less severe but significant reductions in that GluR1 subunit expression (54% of control) were exhibited in samples of excised temporal pole cortex from the same subjects. Message for the GluR4 subunit was also significantly decreased in hippocampus (68% of control), but in contrast to GluR1, GluR4 mRNA level was not decreased in temporal cortex. Levels of GluR2 mRNA were not significantly changed in epileptic hippocampal and cortical tissue relative to control samples. Protein levels of the GluR1 and GluR4 subunits quantified by Western blot analysis were also reduced in hippocampal and cortical tissue from epilepsy patients. Two other kainate subunit transcripts, GluR6 and KA1 also showed significant changes compared to non-epileptic tissue (136% and 71% of control, respectively). Results are discussed in terms of possible mechanisms by which protracted seizures could produce selective loss of certain AMPA/KA subunits.

The aim of the present work was to evaluate the neurochemical effects of early unilateral visual deprivation as a model of impaired visual maturation. For this purpose, binding to the different ionotropic glutamate receptor subtypes was quantified in vision-related and vision-unrelated brain structures of control and unilaterally deprived newborn rats. At post-natal (PN) day 10, male Sprague-Dawley rats underwent either unilateral eyeball enucleation (enucleation group, n = 12) or sham operation (control group, n = 12). In each group, brains were obtained either at post-natal day 20 (n = 6) or post-natal day 30 (n = 6) and processed for quantitative in vitro autoradiography selective for NMDA, kainate, and AMPA glutamate-binding sites, as well as for the presynaptic adenosine A1 receptor as a control of the deafferentation efficacy. In control animals, quantitative autoradiography revealed an increase in NMDA (e.g. +45% in superior colliculus) and kainate receptor binding (e.g. +55% in visual cortex, layer IV) from post-natal day 20 to post-natal day 30, associated with stable levels of AMPA receptor binding, in the vision-related structures. In the deafferented visual structures, monocular enucleation induced a marked decrease in A1 site density (e.g. -38 to -52%, in the superficial layer of superior colliculi, at PN day 20 and PN day 30, respectively) in parallel with a mild increase in both NMDA (e.g. +8 to 9%, in superior colliculi and visual cortex, layer IV at PN day 30, respectively) and AMPA (e.g. +16%, in layer IV of the visual cortex at PN day 30). Superimposed on marked bilateral decreases at PN day 30 in the enucleated rats, kainate receptor binding also revealed a slight but significant decrease (-5%) in the deafferented superior colliculus as compared to the non-deafferented side. The present findings (different time-courses of, and differential effects of deafferentation on, the NMDA, kainate and AMPA glutamate receptor subtypes throughout the visual brain structures) further support the involvement of these receptors in distinctive roles during maturation of the visual system.

Objective: Our purpose was to determine the effect of peripherally administered magnesium sulfate on kainate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors in the rat brain., Study Design: Six rats were injected intraperitoneally with 270 mg/kg magnesium sulfate, followed by 27 mg/kg every 20 minutes for 4 hours. Controls (n = 6) received saline solution. Six rats received intraperitoneal injections of magnesium sulfate (270 mg/kg) every 4 hours for 24 hours and six received saline solution. Then 6 rats received intraperitoneal magnesium sulfate (270 mg/kg) every 12 hours for 2 weeks and six received saline solution. Rats were subsequently perfused and killed; their brains were dissected and frozen. Cryostat sections were labeled in vitro for autoradiography assay. The ligands used were tritiated kainate agonist, kainate binding site; tritiated alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid agonist, and tritiated 6-cyano-7-nitroquinoxaline-2,3-dione antagonist, both at the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding site., Results: Magnesium sulfate caused decreased binding of the agonist to the kainate receptor recognition site after both short-term and intermediate-term systemic administration, whereas long-term treatment resulted in increased binding. No significant consistent effect on the binding to the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor agonist site was recorded after magnesium administration. The receptor antagonist showed an increased binding after short-term treatment. Long-term administration also resulted in increased binding of the antagonist, an effect that was limited to the hippocampus., Conclusions: These data suggest down-regulation of the kainate receptor population during short- and intermediate-term magnesium sulfate treatment. However, long-term inhibition by magnesium resulted in up-regulation of the receptor population. The results may also reflect an increased inhibitory effect of magnesium sulfate on the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor.

On the pattern of well known dialkylaminoacyl anilides, a set of N-homolupinanoyl anilides was prepared and subjected to a broad pharmacological screening with in vivo and in vitro assays. As expected most compounds exhibited a strong antiarrhythmic activity, often comparable or superior to that of lidocaine and quinidine. Compound 1 exhibited an unusual profile as antiarrhythmic, being devoid of local anesthetic activity, calcium channel and beta adrenoceptor antagonism. Calcium channel blocking activity was seen in all aminobenzophenone derivatives, but not in the simpler anilides. Noteworthy are also the capacity of compound 7 to protect mice from a lethal dose of KCN, the moderate antihypertensive activity of 10 and, above all, the antagonism to guinea pig ileum contractile responses induced by several agents exhibited by compound 11, which deserves further investigation for a potential use in irritable bowel syndrome. Compound 11 showed also good relaxant activity on tracheal strips and inhibitory activity against arachidonate induced platelet aggregation. Finally, compound 11 displaced several radioligands from their respective binding sites. Most potent was displacement of [3H]pirenzepine (IC50 < or = 0.01 microM) from M1 binding sites of rat brain, while displacement of [3H] methylscopolamine from rat heart (M2) and submaxillary salivary glands (M3) preparations was much weaker (IC50 approximately equal to 2.4 and 1.3 microM, respectively).