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In cerebellar Purkinje cells (PCs) type-1 metabotropic glutamate (mGlu1) receptors play a key role in motor learning and drive the refinement of synaptic innervation during postnatal development. The cognate mGlu5 receptor is absent in mature PCs and shows low expression levels in the adult cerebellar cortex. Here we found that mGlu5 receptors were heavily expressed by PCs in the early postnatal life, when mGlu1α receptors were barely detectable. The developmental decline of mGlu5 receptors coincided with the appearance of mGlu1α receptors in PCs, and both processes were associated with specular changes in CpG methylation in the corresponding gene promoters. It was the mGlu1 receptor that drove the elimination of mGlu5 receptors from PCs, as shown by data obtained with conditional mGlu1α receptor knockout mice and with targeted pharmacological treatments during critical developmental time windows. The suppressing activity of mGlu1 receptors on mGlu5 receptor was maintained in mature PCs, suggesting that expression of mGlu1α and mGlu5 receptors is mutually exclusive in PCs. These findings add complexity to the the finely tuned mechanisms that regulate PC biology during development and in the adult life and lay the groundwork for an in-depth analysis of the role played by mGlu5 receptors in PC maturation.

Presynaptic kainate-type glutamate receptors (KARs) regulate glutamate release probability and short-term plasticity in various areas of the brain. Here we show that long-term depression (LTD) in the area CA1 of neonatal rodent hippocampus is associated with an upregulation of tonic inhibitory KAR activity, which contributes to synaptic depression and causes a pronounced increase in short-term facilitation of transmission. This increased KAR function was mediated by high-affinity receptors and required activation of NMDA receptors, nitric oxide (NO) synthetase, and postsynaptic calcium signaling. In contrast, KAR activity was irreversibly downregulated in response to induction of long-term potentiation in a manner that depended on activation of the TrkB-receptor of BDNF. Both tonic KAR activity and its plasticity were restricted to early stages of synapse development and were lost in parallel with maturation of the network due to ongoing BDNF-TrkB signaling. These data show that presynaptic KARs are targets for activity-dependent modulation via diffusible messengers NO and BDNF, which enhance and depress tonic KAR activity at immature synapses, respectively. The plasticity of presynaptic KARs in the developing network allows nascent synapses to shape their response to incoming activity. In particular, upregulation of KAR function after LTD allows the synapse to preferentially pass high-frequency afferent activity. This can provide a potential rescue from synapse elimination by uncorrelated activity and also increase the computational dynamics of the developing CA3-CA1 circuitry., (Copyright © 2014 the authors 0270-6474/14/3416902-15$15.00/0.)

Glutamate receptors are fundamental for control synaptic transmission, synaptic plasticity, and neuronal excitability. However, many of the molecular mechanisms underlying their trafficking remain elusive. We previously demonstrated that the small GTPase Rab17 regulates dendritic trafficking in hippocampal neurons. Here, we investigated the role(s) of Rab17 in AMPA receptor (AMPAR) and kainate receptor (KAR) trafficking. Although Rab17 knockdown did not affect surface expression of the AMPAR subunit GluA1 under basal or chemically induced long term potentiation conditions, it significantly reduced surface expression of the KAR subunit GluK2. Rab17 co-localizes with Syntaxin-4 in the soma, dendritic shaft, the tips of developing hippocampal neurons, and in spines. Rab17 knockdown caused Syntaxin-4 redistribution away from dendrites and into axons in developing hippocampal neurons. Syntaxin-4 knockdown reduced GluK2 but had no effect on GluA1 surface expression. Moreover, overexpression of constitutively active Rab17 promoted dendritic surface expression of GluK2 by enhancing Syntaxin-4 translocation to dendrites. These data suggest that Rab17 mediates the dendritic trafficking of Syntaxin-4 to selectively regulate dendritic surface insertion of GluK2-containing KARs in rat hippocampal neurons.

While group 1 metabotropic glutamate receptors (mGluRs) and ionotropic N-methyl-d-aspartate (NMDA) receptors regulate nociception, the precise molecular mechanism(s) contributing to glutamate signaling in chronic pain remain unclear. Here we not only confirmed the key involvement of Homer proteins in neuropathic pain, but also distinguished between the functional roles for different Homer family members and isoforms. Chronic constriction injury (CCI) of the sciatic nerve induced long-lasting, time-dependent increases in the postsynaptic density expression of the constitutively expressed (CC) isoforms Homer1b/c and/or Homer2a/b in the spinal dorsal horn and supraspinal structures involved in nociception (prefrontal cortex, thalamus), that co-occurred with increases in their associated mGluRs, NR2 subunits of the NMDA receptor, and the activation of downstream kinases. Virus-mediated overexpression of Homer1c and Homer2b after spinal (intrathecal) virus injection exacerbated CCI-induced mechanical and cold hypersensitivity, however, Homer1 and Homer2 gene knockout (KO) mice displayed no changes in their neuropathic phenotype. In contrast, overexpression of the immediate early gene (IEG) Homer1a isoform reduced, while KO of Homer1a gene potentiated neuropathic pain hypersensitivity. Thus, nerve injury-induced increases in CC-Homers expression promote pain in pathological states, but IEG-Homer induction protects against both the development and maintenance of neuropathy. Additionally, exacerbated pain hypersensitivity in transgenic mice with reduced Homer binding to mGluR5 supports also an inhibitory role for Homer interactions with mGluR5 in mediating neuropathy. Such data indicate that nerve injury-induced changes in glutamate receptor/Homer signaling contribute in dynamic but distinct ways to neuropathic pain processing, which has relevance for the etiology of chronic pain symptoms and its treatment., (Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Parallel representations of the visual world are already established at the very first synapse of the visual system. Cone photoreceptors, which hyperpolarize in response to light, forward the visual signal onto distinct types of ON and OFF cone bipolar cells (BCs). In the case of OFF BCs, the glutamatergic cone input is integrated by ionotropic glutamate receptors, giving rise to a sign-preserving mode of synaptic transmission. The combination of glutamate receptor (GluR) subunits, i.e. AMPA or kainate subunits, importantly contributes to shaping the OFF bipolar cells' distinct response properties. The mouse is one of the few mammals in which the (most likely) complete set of (five) retinal OFF BC types is identified. However, it is not clear which GluR subtypes are expressed by the different mouse OFF BC types. We addressed this question by combining immunolabeling, electrical whole-cell recordings and pharmacology, and present evidence that the different types of OFF BCs express distinct types of glutamate receptors: Type 1 BCs exclusively expressed AMPA receptors, whereas type 2 and type 3a BCs expressed kainate receptors of different subunit compositions. Additionally, we found that two OFF BC types (3b and 4) very likely express both AMPA and kainate receptors but, interestingly, the two receptor subunits were not co-localized at the same dendritic site. The complex, BC type-specific expression pattern of GluRs we describe here supports their essential role in establishing parallel pathways at the first synapse of the mouse visual system., (Copyright © 2013 IBRO. All rights reserved.)

Kainate receptors, a subtype of ionotropic glutamate receptors, perform important functions in the spinal cord. This study aimed to examine the expression pattern of various kainate receptor subunits in the spinal cord over different stages of development. The regional distribution and levels of Grik1-5 mRNAs, which encode kainate receptor subunits, were examined in the spinal cord of embryonic, perinatal, and adult mice using in-situ hybridization and real-time PCR. At different developmental stages, the expression of Grik1-5 genes showed different regional distributions in the spinal cord. At E16.5, Grik2 and Grik3 were mainly expressed in the dorsal horns whereas Grik5 was expressed in the entire spinal cord. At P0 and P7, Grik2 expression accumulated at laminae II-IV, whereas Grik1 accumulated at the superficial laminae of the dorsal horns. At P30 and P60, the expression of Grik1-5 was concentrated in the superficial laminae of the dorsal horns. Development-related changes were observed in the expression pattern of Grik1-5. Grik5 was expressed in the entire spinal cord up to the perinatal period, whereas from P7 to adult stages, Grik5 expression was almost exclusively restricted to the dorsal horns. Similar observations were present with Grik1, Grik2, and Grik3. Consistently, quantitative determination of the expression levels of Grik1-5 was in accordance with the in-situ hybridization results. This age-related dynamic expression of kainate receptors may act as one driving force for the development of the anatomofunctional pattern and the maturation of the somatosensory circuitry in the spinal cord.

In the current study, we investigated changes in N-methyl D-aspartate (NMDA) and kainate receptor expression, long-term potentiation (LTP), and neurogenesis in response to neurotoxic stress in long-living Ames dwarf mice. We hypothesized that Ames dwarf mice have enhanced neurogenesis that enables retention of spatial learning and memory with age and promotes neurogenesis in response to injury. Levels of the NMDA receptors (NR)1, NR2A, NR2B, and the kainate receptor (KAR)2 were increased in Ames dwarf mice, relative to wild-type littermates. Quantitative assessment of the excitatory postsynaptic potential in Schaffer collaterals in hippocampal slices from Ames dwarf mice showed an increased response in high-frequency induced LTP over time compared with wild type. Kainic acid (KA) injection was used to promote neurotoxic stress-induced neurogenesis. KA mildly increased the number of doublecortin-positive neurons in wild-type mice, but the response was significantly enhanced in the Ames dwarf mice. Collectively, these data support our hypothesis that the enhanced learning and memory associated with the Ames dwarf mouse may be due to elevated levels of NMDA and KA receptors in hippocampus and their ability to continue producing new neurons in response to neuronal damage.

The present study was performed to investigate the effects of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) antisense oligodeoxynucleotides (ODNs) on the assembly of the CaMKII·GluR6·PSD-95 signaling module, GluR6 serine phosphorylation and c-Jun N-terminal kinase 3 (JNK3) activation. A further aim was to determine the neuroprotective mechanism of CaMKII antisense ODNs against ischemia-reperfusion (I/R)-induced neuronal death in the rat hippocampus. CaMKII antisense ODNs were intracerebroventricularly infused to inhibit CaMKII expression once daily for 3 days prior to the induction of ischemia. Transient cerebral ischemia (15 min) and reperfusion were induced by four-vessel occlusion in Sprague-Dawley rats as an animal model for transient cerebral I/R. The expression of related proteins was examined by immunoprecipitation and immunoblotting. Neuronal death in the rat hippocampus was detected by histology and histochemistry. The results indicate that CaMKII antisense ODNs inhibit several of the processes that are normally induced by cerebral I/R, including CaMKII expression, increased CaMKII·GluR6·PSD-95 signaling module assembly, GluR6 serine phosphorylation and JNK3 activation. Alternatively, CaMKII antisense ODNs also exhibit a significant neuroprotective role against cerebral I/R-induced cell death. These results provide the first evidence that CaMKII antisense ODNs can exert neuroprotective effects on cerebral I/R-induced cell death. The possible molecular mechanisms underlying this effect include 1) an inhibition of CaMKII expression and subsequent suppression of the assembly of the CaMKII·GluR6·PSD-95 signaling module, 2) GluR6 serine phosphorylation, and 3) reduced JNK3 activation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Glutamate is thought to be involved in the pathophysiology of major depressive disorder and bipolar disorder; however, the molecular changes underlying abnormal glutamatergic signalling remain poorly understood. Whilst previous studies have suggested that the NMDA receptor may be involved in the pathophysiology of mood disorders, it is unclear whether the non-NMDA receptors are also involved. Therefore, we sought to examine whether the expression of the non-NMDA, ionotropic glutamate receptors, AMPA receptor and kainate receptor, is altered in mood disorders., Methods: We used [3H]AMPA and [3H]kainate to measure the levels of AMPA and kainate receptor, respectively, in the anterior cingulate (BA 24) and dorsolateral prefrontal cortex (BA 46) from post-mortem CNS in 10 subjects with major depressive disorder, 10 subjects with bipolar disorder and 10 control subjects., Results: A 20.7% to 27.7% increase in [3H]AMPA binding density was seen in BA 24 (p<0.05) but not BA 46 (p>0.05) in major depressive disorder compared to control levels. [3H]AMPA binding density was not changed in bipolar disorder in either BA 24 or BA 46 (p>0.05) compared to controls. [3H]Kainate binding was not changed in either BA 24 or BA 46 in either disorder compared to controls (p>0.05)., Limitations: Small sample sizes (n=10) were used in this study. The subjects were not drug naïve., Conclusions: Our data suggests increased in AMPA receptor levels in the anterior cingulate are involved in the pathophysiology of major depressive disorder. This data has relevance for the development of new anti-depressant drugs targeted towards the AMPA receptors., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Kainate receptors (KARs) have been implicated in a number of neurological disorders, including epilepsy. KARs are tetrameric, composed of a combination of GluK1-GluK5 subunits. We examined the contribution of GluK2 and GluK5 subunits to activation and desensitization of the heteromeric receptor. Heteromeric GluK2/K5 receptors expressed in HEK-293T cells showed markedly higher glutamate sensitivity than GluK2 homomers and did not desensitize at low glutamate concentrations. Mutation of residue E738 in GluK2 substantially lowered its glutamate sensitivity. However, heteromeric KARs containing this mutant GluK2 [GluK2(E738D)] assembled with wild-type GluK5 showed no change in glutamate EC(50) compared with wild-type heteromeric KARs. Instead, higher concentrations of glutamate were required to produce desensitization. This suggested that, within the heteromeric receptor, glutamate binding to the high-affinity GluK5 subunit alone was sufficient for channel activation but not desensitization, whereas agonist binding to the low-affinity GluK2 subunit was not necessary to open the channel but instead caused the channel to enter a closed, desensitized state. To test this hypothesis in wild-type receptors, we used the competitive antagonist kynurenate, which has higher affinity for the GluK2 than the GluK5 subunit. Coapplication of kynurenate with glutamate to heteromeric receptors reduced the onset of desensitization without affecting the peak current response, consistent with our hypothesis. Our results suggest that GluK2 and GluK5 subunits can be individually activated within the heteromeric receptor and that these subunits serve dramatically different functional roles.

Background: We sought to study the effects of chronic exposure to fluoxetine - a selective serotonin reuptake inhibitor (SSRI) and specific 5-HT(2B) receptor agonist in astrocytes - on the expression of kainate receptors (GluK1-5) in cultured astrocytes and in intact brains in mice and on GluK2 editing by adenosine deaminase acting on RNA (ADAR), as well as the ensuing effects of fluoxetine on glutamate-mediated Ca(2+) influx and extracellular signal-regulated kinase (ERK)(1/2) phosphorylation in astrocytes., Methods: We performed reverse transcription-polymerase chain reaction (PCR) to assess mRNA expression. We analyzed RNA editing with amplification refractory mutation system PCR and complementary DNA sequencing. Protein expression and ERK phosphorylation were assessed using Western blots. We studied gene silencing with specific small interfering RNAs (siRNA), and we studied intracellular Ca(2+) using fluorometry., Results: All GluK subunits were present in the brain in vivo, and GluK2-5 subunits were present in cultured astrocytes. Fluoxetine upregulated GluK2 and ADAR2. Enhanced GluK2 editing by fluoxetine abolished glutamate-mediated increases in intra cellular Ca(2+) and ERK(1/2) phosphorylation. Enhanced editing of GluK2 was prevented by siRNA against the 5-HT(2B) receptor or ADAR2., Limitations: Limitations of our study include the use of an in vitro system, but our cultured cells in many respects behave like in vivo astrocytes., Conclusion: Fluoxetine alters astrocytic glutamatergic function.

Presynaptic kainate receptors play an important role in synaptic transmission and short-term plasticity to profoundly regulate network activity in many parts of the mammalian brain. In primary sensory neocortex, where short-term synaptic plasticity is important for receptive field structure and information processing, kainate receptors are highly expressed and regulate thalamocortical inputs, particularly during development. However, the mechanisms of the kainate receptor-dependent presynaptic regulation of thalamocortical transmission are unclear. We therefore investigated this issue using electrophysiology in neonatal thalamocortical slices of barrel cortex combined with pharmacology and biochemical analyses. We show that presynaptic kainate receptors can both facilitate or depress synaptic transmission depending on the extent of their activation. This bi-directional regulation is mediated in part by kainate receptors that directly influence thalamocortical axonal excitability, but also likely involves receptors acting at thalamocortical terminals to regulate transmitter release. The efficacy of kainate in regulating thalamocortical transmission is low compared to that reported for other inputs. Consistent with this low efficacy, our biochemical analyses indicate that the presynaptic kainate receptors regulating neonatal thalamocortical inputs likely lack the high kainate affinity GluK4 and 5 subunits. Thus thalamocortical transmission can be bi-directionally regulated by low affinity kainate receptors through two mechanisms. Such presynaptic regulation provides a potentially powerful mechanism to influence sensory processing during development of barrel cortex., (Copyright © 2010. Published by Elsevier Ltd.)

Background: There is no consistent evidence of specific gene(s) or molecular pathways that contribute to the pathogenesis, therapeutic intervention or diagnosis of chronic fatigue syndrome (CFS). While multiple studies support a role for genetic variation in CFS, genome-wide efforts to identify associated loci remain unexplored. We employed a novel convergent functional genomics approach that incorporates the findings from single-nucleotide polymorphism (SNP) and mRNA expression studies to identify associations between CFS and novel candidate genes for further investigation., Methods: We evaluated 116,204 SNPs in 40 CFS and 40 nonfatigued control subjects along with mRNA expression of 20,160 genes in a subset of these subjects (35 CFS subjects and 27 controls) derived from a population-based study., Results: Sixty-five SNPs were nominally associated with CFS (p<0.001), and 165 genes were differentially expressed (≥4-fold; p≤0.05) in peripheral blood mononuclear cells of CFS subjects. Two genes, glutamate receptor, ionotropic, kinase 2 (GRIK2) and neuronal PAS domain protein 2 (NPAS2), were identified by both SNP and gene expression analyses. Subjects with the G allele of rs2247215 (GRIK2) were more likely to have CFS (p=0.0005), and CFS subjects showed decreased GRIK2 expression (10-fold; p=0.015). Subjects with the T allele of rs356653 (NPAS2) were more likely to have CFS (p=0.0007), and NPAS2 expression was increased (10-fold; p=0.027) in those with CFS., Conclusion: Using an integrated genomic strategy, this study suggests a possible role for genes involved in glutamatergic neurotransmission and circadian rhythm in CFS and supports further study of novel candidate genes in independent populations of CFS subjects., (Copyright © 2011 S. Karger AG, Basel.)

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) has been implicated in the processing of nociceptive information from the orofacial region. Kainate receptors (KARs) play an important role in sensory transmission. Five different KAR subunits have been cloned and the expression of the KAR subunits showed developmental changes. In this study, RT-PCR, western blotting, immunohistochemistry and a patch clamp technique were used examine the functional expression of the GluR5 subunit in the SG of the Vc in juvenile, peripubertal and/or adult mice. The levels of mRNA and protein expression of the GluR5 subunit in the SG of the Vc were higher in the juvenile mice than in the peripubertal or adult mice. In addition, the KA and ATPA, a GluR5 KAR agonist, induced membrane depolarization on the SG neurons in both juvenile and adult mice in a concentration-dependent manner. However, the juvenile SG neurons showed a stronger response to KA and ATPA than those of adults. The membrane depolarization by KA was suppressed slightly in the presence of the AMPA receptor antagonist, GYKI 52466. These results show that the GluR5 KAR subunits are expressed functionally on the SG neurons of the Vc in mice, and the expression levels of the GluR5 subunits decrease with postnatal development. These postnatal changes in the GluR5 KAR subunit may be a possible mechanism for age-dependent pain processing., (Copyright 2010 Elsevier B.V. All rights reserved.)

GABAergic feedback inhibition from amacrine cells shapes visual signaling in the inner retina. Rod bipolar cells (RBCs), ON-sensitive cells that depolarize in response to light increments, receive reciprocal GABAergic feedback from A17 amacrine cells and additional GABAergic inputs from other amacrine cells located laterally in the inner plexiform layer. The circuitry and synaptic mechanisms underlying lateral GABAergic inhibition of RBCs are poorly understood. A-type and rho-subunit-containing (C-type) GABA receptors (GABA(A)Rs and GABA(C)Rs) mediate both forms of inhibition, but their relative activation during synaptic transmission is unclear, and potential interactions between adjacent reciprocal and lateral synapses have not been explored. Here, we recorded from RBCs in acute slices of rat retina and isolated lateral GABAergic inhibition by pharmacologically ablating A17 amacrine cells. We found that amacrine cells providing lateral GABAergic inhibition to RBCs receive excitatory synaptic input mostly from ON bipolar cells via activation of both Ca(2+)-impermeable and Ca(2+)-permeable AMPA receptors (CP-AMPARs) but not NMDA receptors (NMDARs). Voltage-gated Ca(2+) (Ca(v)) channels mediate the majority of Ca(2+) influx that triggers GABA release, although CP-AMPARs contribute a small component. The intracellular Ca(2+) signal contributing to transmitter release is amplified by Ca(2+)-induced Ca(2+) release from intracellular stores via activation of ryanodine receptors. Furthermore, lateral nonreciprocal feedback is mediated primarily by GABA(C)Rs that are activated independently from receptors mediating reciprocal feedback inhibition. These results illustrate numerous physiological differences that distinguish GABA release at reciprocal and lateral synapses, indicating complex, pathway-specific modulation of RBC signaling.

Kainate receptors (KARs) are crucial for the regulation of both excitatory and inhibitory neurotransmission, but little is known regarding the mechanisms controlling KAR surface expression. We used super ecliptic pHluorin (SEP)-tagged KAR subunit GluR6a to investigate real-time changes in KAR surface expression in hippocampal neurons. Sindbis virus-expressed SEP-GluR6 subunits efficiently co-assembled with native KAR subunits to form heteromeric receptors. Diffuse surface-expressed dendritic SEP-GluR6 is rapidly internalized following either N-methyl-d-aspartate or kainate application. Sustained kainate or transient N-methyl-d-aspartate application resulted in a slow decrease of base-line surface KAR levels. Surprisingly, however, following the initial loss of surface receptors, a short kainate application caused a long lasting increase in surface-expressed KARs to levels significantly greater than those prior to the agonist challenge. These data suggest that after initial endocytosis, transient agonist activation evokes increased KAR exocytosis and reveal that KAR surface expression is bidirectionally regulated. This process may provide a mechanism for hippocampal neurons to differentially adapt their physiological responses to changes in synaptic activation and extrasynaptic glutamate concentration.

Kainate receptors (KARs) are neuronal proteins that exhibit a highly polarized distribution in the mammalian CNS. Assembly, intracellular trafficking, and synaptic targeting of KARs and other ionotropic glutamate receptors are processes controlled, in part, by various determinants within the constituent subunit proteins themselves. Here, we demonstrate that the linker region between the M3 and S2 domains, which in current structural models is thought to transduce ligand-binding energy into channel opening, additionally has an essential role in receptor biogenesis. Our results show that this gating-associated domain is engaged at two distinct critical stages of KAR biogenesis: first, during the transition from dimeric to tetrameric assembly states and, second, at a postassembly trafficking checkpoint within the endoplasmic reticulum. Alteration of a basic residue, arginine 663, altered the desensitization properties of the GluR6 kainate receptor in response to glutamate application, and these changes were weakly correlated with intracellular retention of the mutant receptors. Elimination of the positive charge also significantly attenuated oligomerization and stability of the intracellular subunit protein. Furthermore, charge swapping with an adjacent residue, glutamate 662, normalized the receptor physiological behavior and reversed the deficits in assembly and degradation, but only partially restored plasma membrane expression of the receptors. These results reveal a new role for this linker domain in glutamate receptor biogenesis and contribute to understanding the cellular controls of receptor assembly and trafficking, which will be important for relating receptor stoichiometry to their neuronal targeting and function.

Glutamate receptors are the major excitatory receptors in the vertebrate CNS and have been implicated in a number of physiological and pathological processes. Previous work has shown that glutamate receptor function may be modulated by protein kinase A (PKA)-mediated phosphorylation, although the molecular mechanism of this potentiation has remained unclear. We have investigated the phosphorylation of specific amino acid residues in the C-terminal cytoplasmic domain of the rat kainate receptor subtype 6 (GluR6) as a possible mechanism for regulation of receptor function. The C-terminal tail of rat GluR6 can be phosphorylated by PKA on serine residues as demonstrated using [gamma-32P]ATP kinase assays. Whole cell recordings of transiently transfected human embryonic kidney (HEK) 293 cells showed that phosphorylation by PKA potentiates whole cell currents in wildtype GluR6 and that removal of the cytoplasmic C-terminal domain abolishes this potentiation. This suggested that the C-terminal domain may contain residue(s) involved in the PKA-mediated potentiation. Single mutations of each serine residue in the C-terminal domain (S815A, S825A, S828A, and S837A) and a truncation after position 855, which removes all threonines (T856, T864, and T875) from the domain, do not abolish PKA potentiation. However, the S825A/S837A mutation, but no other double mutation, abolishes potentiation. These results demonstrate that phosphorylation of the C-terminal tail of GluR6 by PKA leads to potentiation of whole cell response, and the combination of S825 and S837 in the C-terminal domain is a vital component of the mechanism of GluR6 potentiation by PKA.

We examined the identities of horizontal cell (HC) lateral components in cone terminals and the expression of glutamate receptors on the tips of HC dendrites. We injected A-type horizontal cells (AHCs) with neurobiotin and demonstrated that neurobiotin labeled completely all AHCs within a patch of retina. We converted neurobiotin by using diaminobenzidine and considered labeled processes to be from AHCs and unlabeled processes to be from B-type horizontal cells (BHCs). Three possible combinations of HC dendrites could exist in cone pedicles: both lateral components originating from AHCs, both from BHCs, or one from an AHC and the other from a BHC. EM observations revealed that a majority of cone terminals contained about equal numbers of lateral components originating from each of the two types of HCs and that each of the three possible combinations was present in equal numbers. Localization of different types of glutamate receptors on HC dendritic tips showed that 55% of AHC dendritic tips expressed AMPA receptors and 30% expressed kainate receptors, whereas, in the case of BHCs, 22% of dendritic tips expressed AMPA receptors and 33% expressed kainate receptors. This study suggests that cone photoreceptors feed the light signal equally into networks of AHCs and BHCs and that differential expression of AMPA/kainate receptors by different HCs could account for different functions., (Copyright 2006 Wiley-Liss, Inc.)

Several findings suggest that glucocorticoid hormones influence the propensity of an individual to develop cocaine abuse. These hormones activate two related transcription factors, the glucocorticoid receptor and the mineralocorticoid receptor. We have shown previously that mice carrying a mutation of the glucocorticoid receptor gene specifically in neural cells, glucocorticoid receptor knock-out in the brain, show a dramatic decrease in cocaine-induced self-administration and no behavioral sensitization to this drug, two experimental procedures considered relevant models of addiction. Here, we investigated in glucocorticoid receptor knock-out in the brain mice the consequences of this mutation at the level of the expression of neuropeptide, dopamine receptor and glutamate receptor subunit mRNAs. We quantified mRNA levels in the cortex, striatum and accumbens under basal conditions and following acute or repeated cocaine treatments. Our results show that, under basal conditions, neuropeptide (substance P, dynorphin) and dopamine receptor (D1, D2) mRNAs were decreased in glucocorticoid receptor knock-out in the brain mice in the dorsal striatum but not in the accumbens. However, cocaine-induced changes in the levels of these mRNAs were not modified in glucocorticoid receptor knock-out in the brain mice. In contrast, mutant mice showed altered response in mRNA levels of N-methyl-D-aspartate, GLUR5 and GLUR6 glutamate receptor subunits as well as of enkephalin following cocaine administration. These modifications may be associated to decrease of behavioral effects of cocaine observed in glucocorticoid receptor knock-out in the brain mice.

Chronic cocaine and withdrawal induce significant alterations in nucleus accumbens (NAc) glutamatergic function in humans and rodent models of cocaine addiction. Dysregulation of glutamatergic function of the prefrontal cortical-NAc pathway has been proposed as a critical substrate for unmanageable drug seeking. Previously, we demonstrated significant up-regulation of NMDA, (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptor subunit mRNAs and protein levels in the ventral tegmental area (VTA), but not the substantia nigra, of cocaine overdose victims (COD). The present study was undertaken to examine the extent of altered ionotropic glutamate receptor (iGluR) subunit expression in the NAc and the putamen in cocaine overdose victims. Results revealed statistically significant increases in the NAc, but not in the putamen, of NMDA receptor subunit (NR)1 and glutamate receptor subunit (GluR)2/3 wit trends in GluR1 and GluR5 in COD. These results extend our previous finding and indicate pathway-specific alterations in iGluRs in COD. In order to determine that changes were related to cocaine intake and not to other factors in the COD victims, we examined the effects of cocaine intravenous self-administration in rhesus monkeys for 18 months (unit dose of 0.1 mg/kg/injection and daily drug intake of 0.5 mg/kg/session). Total drug intake for the group of four monkeys was 37.9 +/- 4.6 mg/kg. Statistically significant elevations were observed for NR1, GluR1, GluR2/3 and GluR5 (p < 0.05) and a trend towards increased NR1 phosphorylated at serine 896 (p = 0.07) in the NAc but not putamen of monkeys self-administering cocaine compared with controls. These results extend previous results by demonstrating an up-regulation of NR1, GluR2/3 and GluR5 in the NAc and suggest these alterations are pathway specific. Furthermore, these changes may mediate persistent drug intake and craving in the human cocaine abuser.

Purpose: Glutamate receptor 6 is strongly implicated in human refractory epilepsy and in kainic acid (KA)-induced status epilepticus (SE). In vitro pharmacologic studies with newer antiepileptic drugs (AEDs) are increasingly indicating the role of glutamate receptor 5 (GluR5) in epilepsy. Glutamate receptor 7 (GluR7) has been the least investigated in the context of epilepsy. We studied the messenger ribonucleic acid (mRNA) and protein expression of GluR5, GluR6, and GluR7 in rat hippocampus 72 h, 90 days, and 180 days after KA-induced SE., Methods: SE was induced by injecting KA intraperitoneally (i.p.) into adult rats. The hippocampi were isolated 72 h, 90 days, and 180 days after SE. Reverse transcription-polymerase chain reaction (RT-PCR) was performed for mRNA expression. Western blots determined the protein expression., Results: A significant increase was noted in GluR5 expression in KA-treated animals compared with controls at 72 h and 180 days, with no significant difference at the intervening 90-day point. Protein levels for GluR5 increased at 72 h and remained elevated until 180 days. GluR7 mRNA showed a significant decrease at 90 days after seizures. Neither the mRNA expression nor the protein levels of GluR6 differed from controls at any of the times after SE., Conclusions: KA-induced SE leads to an upregulation of GluR5 mRNA and protein and a downregulation of GluR7 mRNA in rat hippocampus, with no change in GluR6 mRNA or protein expression.

Kainate receptors (KARs) are mediators of excitatory neurotransmission in the mammalian central nervous system, and their efficient targeting and trafficking is critical for normal synaptic function. A key step in the delivery of KARs to the neuronal plasma membrane is the exit of newly assembled receptors from the endoplasmic reticulum (ER). Here we report the identification of a novel ER retention signal in the alternatively spliced C-terminal domain of the GluR5-2b subunit, which controls receptor trafficking in both heterologous cells and neurons. The ER retention motif consists of a critical arginine (Arg-896) and surrounding amino acids, disruption of which promotes ER exit and surface expression of the receptors, as well as altering their physiological properties. The Arg-896-mediated ER retention of GluR5 is regulated by a mutation that mimics phosphorylation of Thr-898, but not by PDZ interactions. Furthermore, two positively charged residues (Arg-900 and Lys-901) in the C terminus were also found to regulate ER export of the receptors. Taken together, our results identify novel trafficking signals in the C-terminal domain of GluR5-2b and demonstrate that alternative splicing is an important mechanism regulating KAR function.

The presence of Ca2+-permeable AMPA/kainate (Ca-A/K) channels on hippocampal pyramidal neurons (HPNs) has been controversial, although they are present on many forebrain GABAergic neurons. We combined high-resolution fluorescence Ca2+ imaging with surface AMPA receptor (AMPAR) subunit immunocytochemistry to examine the expression of functional Ca-A/K channels in dissociated hippocampal neurons at the subcellular level. In GABAergic neurons [identified by glutamate decarboxylase (GAD) immunocytochemistry], focal application of AMPA induced large dendrosomatic intracellular [Ca2+] ([Ca2+]i) rises, consistent with their known strong Ca-A/K channel expression. Surface immunostaining for the AMPAR subunits GluR1 and GluR2 revealed abundant dendritic GluR1 puncta containing little or no GluR2, which, when present, was limited to diffuse staining in the soma and proximal dendrites. In contrast, the majority of HPNs (putatively identified by morphological criteria and lack of GAD labeling) showed little or no AMPA-induced [Ca2+]i rise. Correspondingly, most HPNs showed strong dendritic labeling for both GluR1 and GluR2 that colocalized extensively. A subpopulation of HPNs, however, displayed noticeable [Ca2+]i rises that began and often reached their highest levels in discrete dendritic regions. In these HPNs, levels of GluR1 relative to GluR2 were higher, and GluR1 was often present without overlying GluR2. The present studies, which are the first to directly examine the relationship between the local complement of cell surface AMPAR and the presence of dendritic Ca-A/K channels, clearly indicate that considerable cell surface GluR2 does not preclude the presence of Ca-A/K channels and further show that HPNs display considerable heterogeneity in terms of apparent Ca-A/K channel expression.

The wide-ranging expression of glutamate receptors in peripheral tissues suggests an unexpectedly wider role(s) of l-glutamate as an intercellular signaling molecule. However, the peripheral glutamatergic system is poorly understood, partly because the sites of l-glutamate signal appearance are less well characterized. Vesicular glutamate transporters (VGLUTs) are potential probes for the sites of vesicular storage and subsequent secretion of l-glutamate. In this study we raised specific polyclonal antibodies against two VGLUT isoforms, VGLUT1 and VGLUT2, and investigated their localization in peripheral tissues of rat. We detected the expression of either VGLUT1 or VGLUT2, or both, in pancreas, stomach, intestine, and testis. In pancreas, VGLUT1 and VGLUT2 are present in pancreatic polypeptide-containing secretory granules in F-cells in the islets of Langerhans. In stomach, VGLUT2 is abundant in the antrum and pylorus and is present in a subset of pancreatic polypeptide-containing cells. In intestine, VGLUT2 is abundant in the ileum and is co-localized with glucagon-like immunoreactive peptide and polypeptide YY (PYY). In testis, VGLUT2 is expressed and localized in the outer acrosomal membrane of spermatids, where KA1 and GluR5, kainate receptor subunits, are almost always localized. Taken together, these results strongly suggest the occurrence of a peripheral glutamatergic system in the gastroenteropancreatic system and testis.

Kainate receptors (KARs) modulate synaptic transmission at both pre-synaptic and post-synaptic sites. The overlap in the distribution of KA-2 and GluR6/7 subunits in several brain regions suggests the co-assembly of these subunits in native KARs. The molecular mechanisms that control the assembly and surface expression of KARs are unknown. Unlike GluR5-7, the KA-2 subunit is unable to form functional homomeric KAR channels. We expressed the KA-2 subunit alone or in combination with other KAR subunits in HEK-293 cells. The cell surface expression of the KAR subunit homo- and heteromers were analysed using biotinylation and agonist-stimulated cobalt uptake. While GluR6 or GluR7 homomers were expressed on the cell surface, KA-2 alone was retained within the endoplasmic reticulum. We found that the cell surface expression of KA-2 was dramatically increased by co-expression with either of the low-affinity KAR subunits GluR5-7. However, co-expression with other related ionotropic glutamate receptor subunits (GluR1 and NR1) does not facilitate the cell surface expression of KA-2. The analysis of subcellular fractions of neocortex revealed that synaptic KARs have a relatively high KA-2 content compared to microsomal ones. Thus, KA-2 is likely to contain an endoplasmic reticulum retention signal that is shielded on assembly with other KAR subunits.

The ionotropic glutamate receptor (GluR) subunits GluR2, GluR5 and GluR6 are subject to RNA editing at their Q/R sites, resulting in significant alterations in the channel properties of the receptors. RNA editing at the Q/R site of GluRs is both developmentally and regionally regulated. Here we provide the first quantitative measurements of both mRNAs of the GluR subunits and mRNAs of the RNA editing enzymes ADAR1-ADAR3 in a comparison of the efficiency of editing at the Q/R site with the expression levels of ADAR mRNA in human brain. We demonstrate that the Q/R site of GluRs in white matter is edited significantly less than in grey matter. In addition, by means of quantitative reverse transcription-polymerase chain reaction methods, we demonstrate that the relative abundance of ADAR2 mRNA to GluR2 mRNA is significantly lower in white matter than in grey matter and that the GluR2 Q/R site editing decreased only when the ratio of ADAR2 mRNA (not that of ADAR1 mRNA) to GluR2 mRNA dropped below a threshold (20 x 10(-3)). These results suggest that Q/R site of GluRs editing is regulated in a regional, and hence presumably cell-specific, manner and that the GluR2 Q/R site editing is critically regulated by ADAR2 in human brain.

Spinal cord white matter is susceptible to AMPA/kainate (KA)-type glutamate receptor-mediated excitotoxicity. To understand this vulnerability, it is important to characterize the distribution of AMPA/KA receptor subunits in this tissue. Using immunohistochemistry and laser confocal microscopy, we studied the expression sites of AMPA/KA receptor subunits in mouse spinal cord. The white matter showed consistent immunoreactivity for AMPA receptor subunit GluR2/3 and KA receptor subunits GluR6/7 and KA2. In contrast, antibodies against GluR1, GluR2, GluR4 (AMPA), and GluR5 (KA) subunits showed only weak and occasional labeling of white matter. However, gray matter neurons did express GluR1 and GluR2, as well as GluR2/3. The white matter astrocytes were GluR2/3 and GluR6/7 immunopositive, while the gray matter astrocytes displayed primarily GluR6/7. Both exclusively and abundantly, KA2 labeled oligodendrocytes and myelin, identified by CNPase expression. Interestingly, myelin basic protein, another myelin marker, showed less correlation with KA2 expression, placing KA2 at specific CNPase-containing subdomains. Focal points of dense KA2 labeling showed colocalization with limited, but distinct, axonal regions. These regions were identified as nodes of Ranvier by coexpressing the nodal marker, ankyrin G. Overall, axonal tracts showed little, if any, AMPA/KA receptor expression. The proximity of oligodendrocytic KA2 to the axonal node and the paucity of axonal AMPA/kainate receptor expression suggest that excitotoxic axonal damage may be secondary and, possibly, mediated by oligodendrocytes. Our data demonstrate differential expression of glutamate AMPA and KA receptor subunits in mouse spinal cord white matter and point to astrocytes and oligodendrocytes as potential targets for pharmacological intervention in white matter glutamate excitotoxicity., (Copyright 2003 Wiley-Liss, Inc.)

Potential neuroprotective effects of the antiepileptic drug (AED) topiramate (TPM) were evaluated using primary neuronal-astroglial cultures or astroglial-enriched cultures from newborn rats exposed to excitotoxic concentrations of glutamate (Glu) or kainate. Neurons expressed functional Glu receptors of the NMDA and AMPA/kainate types as evaluated by immunocytochemistry and Ca(2+) imaging. When Glu (10 mM) was added to 9-10-day cultures incubated with the fluorescent dye calcein/AM for 5h, there was a marked cell loss in both culture types, but was more pronounced in the neuronal-astroglial cultures. When TPM (5-10 microM) was included in the medium together with Glu, the amount of surviving cells was significantly higher in the neuronal-astroglial cultures, but not in the astroglial-enriched cultures. Immuno-labeling of the cultures revealed an enhanced survival of MAP positive neuronal cells when TPM was included in the Glu containing medium. As TPM has a proven negative modulatory effect on kainate activated receptors, neuronal-astroglial cultures were further exposed to excitotoxic concentrations of kainate (100 microM) and analyzed immunohistochemically. Significantly more MAP positive neurons survived in the TPM containing medium and showed a morphology similar to untreated cells. Valproate and phenytoin were used as reference AEDs. In conclusion, our results demonstrate a protective effect of TPM upon neuronal cells in primary culture, exposed to excitotoxic levels of Glu or kainate.

With the ever-growing number of transgenic mice being used in vision research, a precise knowledge of the cellular organization of the mouse retina is required. As with the cat, rabbit, rat, and primate retinae, as many as 10 cone bipolar types and one rod bipolar type can be expected to exist in the mouse retina; however, they still have to be defined. In the current study, several immunocytochemical markers were applied to sections of mouse retina, and the labeling of bipolar cells was studied using confocal microscopy and electron microscopy. By using antibodies against the neurokinin-3 receptor NK3R; the plasma membrane calcium ATPase1 (PMCA1); and the calcium (Ca)-binding proteins CaB1, CaB5, caldendrin, and recoverin, three different OFF-cone bipolar cells could be identified. One type of ON-cone bipolar cell was identified through its immunoreactivity for CaB5 and PMCA1. Rod bipolar cells, comparable in morphology to those of other mammalian retinae, expressed protein kinase Calpha and CaB5. It was also shown that putative OFF-cone bipolar cells receive light signals through flat contacts at the cone pedicle base, whereas ON-cone bipolar signaling involves invaginating contacts. The distribution of the kainate receptor subunit GluR5 was studied by confocal and electron microscopy. GluR5 was expressed at flat bipolar cell contacts; however, it appears to be involved with only certain types of OFF-cone bipolar cells. This suggests that different bipolar cell types receive their light signals through different sets of glutamate receptors., (Copyright 2002 Wiley-Liss, Inc.)

Fast-acting excitatory neurotransmission in the retina is mediated primarily by glutamate, acting at alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) -selective and kainate-selective receptors. To localize these sites of action, cat retinas were stimulated with either AMPA or kainate and processed for histochemical visualization of cobalt uptake through calcium-permeable channels. Treatment with both agonists resulted in staining of A- and B-type horizontal cells and several types of OFF cone bipolar cells; there was no evidence for staining of ON cone bipolar cells or rod bipolar cells. The subpopulations of OFF cone bipolar cells differed in their responses with two distinct types that stained heavily with cobalt after exposure to AMPA and three different types that were preferentially labeled after exposure to kainate. Although many amacrine and ganglion cells appeared to respond to both agonists, AII amacrine cells were stained after stimulation by AMPA but not by kainate. The OFF cone bipolar cells that exhibit AMPA-stimulated cobalt uptake were found to have a high level of correspondence with cells that show immunocytochemical staining for the AMPA-selective glutamate receptor subunits GluR1 and GluR2/3. Similarly, the cone bipolar cells exhibiting kainate-stimulated cobalt uptake resemble those that are immunoreactive for the kainate subunit GluR5. The results indicate that, whereas many retinal neurons express both AMPA and kainate receptors, AII amacrine cells and subpopulations of OFF cone bipolar cells are limited to the expression of either AMPA or kainate receptors. This differential expression may contribute to the unique character of transmission by these cell types., (Copyright 2002 Wiley-Liss, Inc.)

We determined whether neural responses to inflammation and hyperalgesia involve activation of kainate receptors, a subgroup of glutamate receptors. Inflammation was introduced into the hind paw by intraplantar injection of complete Freund's adjuvant. The inflammation-induced thermal hyperalgesia was attenuated by intrathecal administration of a non-selective alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonist, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX), as well as by selective kainate receptor antagonists, 6,7,8,9-tetrohydro-5-nitro-1H-benz[g]indole-2,3-dione 3-oxime (NS-102) and 3S,4aR,6S,8aR-6-(4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-deca-hydroisoquinoline-3-carboxylic acid (LY382884). Reverse transcription-polymerase chain reaction (RT-PCR) indicated that the GluR5 and GluR6, but not the GluR7, KA1 and KA2 subunits, exhibited increased mRNA expression at 2 h to 3 days following inflammation (P<0.05). Western blot showed an increase in GluR6 protein levels (P<0.01) with a time course consistent with the changes in its mRNA levels. cDNA sequence and BbvI endonuclease digestion of the GluR6 PCR product revealed that the upregulated GluR6 mRNAs were predominantly the unedited form (Q). These results suggest that a selective upregulation of kainate receptor subunit expression contributes to inflammatory hyperalgesia.

Reverse transcription polymerase chain reaction revealed expression of mRNA for particular subunits of ionotropic glutamate receptors (iGluR) in primary cultures of rat calvarial osteoblastic cells under immature to mature states. These included GluR3, KA1 and KA2 subunits, in addition to NR1 and NR2D subunits. These results suggest that glutamate may play an unidentified role in mechanisms associated with cellular development through particular subunits of iGluR in rat calvarial osteoblasts.

Background: Although chronic alcohol consumption impairs endothelial nitric oxide synthase-dependent reactivity of cerebral arterioles, the effect of alcohol consumption on vasodilation in response to activation of neuronal nitric oxide synthase (nNOS) has not been examined. Thus, our first goal was to determine whether chronic alcohol consumption impairs nNOS-dependent reactivity of pial arterioles. Our second goal was to examine potential mechanisms for impaired responses of pial arterioles during chronic alcohol consumption., Methods: Sprague Dawley rats were fed liquid diets with or without alcohol for 8 to 12 weeks. By using intravital microscopy, we measured the diameter of pial arterioles in response to nNOS-dependent agonists--NMDA and kainate (KA)--in the absence and presence of N(G)-monomethyl-L-arginine (L-NMMA) or 7-nitroindazole (7-NI). We also measured responses of pial arterioles to nitroglycerin. Next, using Western blot analysis, we measured protein levels of the NMDA receptor subunit, KA receptor subunit, and nNOS protein in cerebral microvessels, parietal cortex, cerebellum, and brainstem of non-alcohol-fed and alcohol-fed rats., Results: Topical application of NMDA (100 and 300 microM) and KA (100 and 300 microM) produced dose-related dilation of pial arterioles in non-alcohol-fed and alcohol-fed rats. However, the magnitude of vasodilation in response to NMDA and KA, but not nitroglycerin, was significantly less in alcohol-fed compared with non-alcohol-fed rats. Topical application of L-NMMA (10 microM) or 7-NI (10 microM) significantly inhibited dilation of pial arterioles in response to NMDA and KA in non-alcohol-fed rats. In alcohol-fed rats, only NMDA-induced vasodilation was inhibited by L-NMMA. In addition, we found that NMDA receptor subunit and KA receptor subunit protein levels increased in the parietal cortex and cerebellum of alcohol-fed compared with non-alcohol-fed rats. However, no significant difference in protein level of nNOS was observed between non-alcohol-fed and alcohol-fed rats., Conclusions: Our findings suggest that chronic alcohol consumption impairs nNOS-dependent dilation of pial arterioles via a mechanism that appears to be unrelated to quantitative changes in NMDA receptors, KA receptors, or nNOS. Because the regulation of cerebral blood flow is influenced by neuronal activation, impaired reactivity of cerebral blood vessels to neuronal activation may contribute to the pathogenesis of cerebrovascular disorders observed during chronic alcohol consumption.

Excitotoxicity is a process in which glutamate or other excitatory amino acids induce neuronal cell death. Accumulating evidence suggests that excitotoxicity may contribute to human neuronal cell loss caused by acute insults and chronic degeneration in the central nervous system. The immediate early gene (IEG) c-fos encodes a transcription factor. The c-Fos proteins form heterodimers with Jun family proteins, and the resulting AP-1 complexes regulate transcription by binding to the AP-1 sequence found in many cellular genes. Emerging evidence suggests that c-fos is essential in regulating neuronal cell survival versus death. Although c-fos is induced by neuronal activity, including kainic acid-induced seizures, whether and how c-fos is involved in excitotoxicity is still unknown. To address this issue, we generated a mouse in which c-fos expression is largely eliminated in the hippocampus. We found that these mutant mice have more severe kainic acid-induced seizures, increased neuronal excitability and neuronal cell death, compared with control mice. Moreover, c-Fos regulates the expression of the kainic acid receptor GluR6 and brain-derived neurotrophic factor (BDNF), both in vivo and in vitro. Our results suggest that c-fos is a genetic regulator for cellular mechanisms mediating neuronal excitability and survival.

The localization and functions of kainate receptors (KARs) in the CNS are still poorly known. In the striatum, GluR6/7 and KA2 immunoreactivity is expressed presynaptically in a subpopulation of glutamatergic terminals and postsynaptically in dendrites and spines. The goal of this study was to further characterize the subcellular and subsynaptic localization of kainate receptor subunits in the monkey striatum. Immunoperoxidase data reveal that the relative abundance of GluR6/7- and KA2-immunoreactive terminals is homogeneous throughout the striatum irrespective of the differential degree of striatal degeneration in Huntington's disease. Pre-embedding and post-embedding immunogold data indicate that >70% of the presynaptic or postsynaptic GluR6/7 and KA2 labeling is expressed intracellularly. In material stained with the post-embedding immunogold method, approximately one-third of plasma membrane-bound gold particles labeling in axon terminals and spines is associated with asymmetric synapses, thereby representing synaptic kainate receptor subunits. On the other hand, >60% of the plasma-membrane bound labeling is extrasynaptic. Both GluR6/7 and KA2 labeling in glutamatergic terminals often occurs in clusters of gold particles along the membrane of large vesicular organelles located at various distances from the presynaptic grid. Anterograde labeling from the primary motor cortex or the centromedian thalamic nucleus indicate that both corticostriatal and thalamostriatal terminals express presynaptic GluR6/7 and KA2 immunoreactivity in the postcommissural putamen. In conclusion, these data demonstrate that kainate receptors in the striatum display a pattern of subcellular distribution different from other ionotropic glutamate receptor subtypes, but consistent with their metabotropic-like functions recently shown in the hippocampus.

Dorsal root ganglion (DRG) neurons first express kainate receptor subunits, predominantly GluR5, during embryonic development. In the DRG and throughout the nervous system, substantial editing of GluR5 mRNA occurs with developmental maturation (Bernard et al., 1999). The accompanying change in Ca(2+) permeability of functional kainate receptors that is the predicted outcome of this developmental regulation of mRNA editing has not been investigated. Here we report that kainate receptors on DRG neurons from late embryonic and newborn rats are predominantly Ca(2+) permeable but then become fully Ca(2+) impermeable later in the first postnatal week. Using multiple markers for nociceptor subpopulations, we show that this switch in Ca(2+) permeability is not caused by the appearance of a new subpopulation of nociceptors with different receptor properties. Instead, the change in Ca(2+) permeability matches the time course of post-transcriptional RNA editing of GluR5 at the Q/R site within the pore of the channel, indicating that the change is probably caused by developmentally regulated RNA editing. We also report that, on the basis of the strong correlation of receptor expression with expression of the surface markers LA4, isolectin B4, and LD2, kainate receptors are present on C-fiber-type neurons projecting to lamina II of spinal cord dorsal horn. These results raise the possibility that kainate receptors in their Ca(2+)-permeable form serve a developmental role in synapse formation between this population of C-fibers and their targets in the spinal cord dorsal horn. Thereafter, the receptors may serve a new function that does not require Ca(2+) permeability.

We describe here the first example of an exonic polymorphism that affects the primary structure of a human ionotropic glutamate receptor. The human kainate receptor GluR7 gene contains a thymine (T)/guanine (G) nucleotide variation that determines a serine or alanine at position 310 in the extracellular region of GluR7 receptor subunits. Our finding contrasts with a previous report that suggested that GluR7 transcripts were RNA-edited at this site. Whole-cell patch-clamp recordings did not detect differences in receptor activation and desensitization between the human GluR7 receptor isoforms expressed in HEK-293 cells. Analysis of 41 tissue samples obtained from 30 human brains revealed expression level differences between GluR7 alleles expressed in the same brain. The expression level of the allelic GluR7 mRNAs differed in 27 samples from 1.2- to 12.7-fold. Unequal expression level of allelic mRNAs is characteristic for genes that are affected by genomic imprinting or that contain mutations. Genomic imprinting in most cases is conserved between human and mice. However, we did not detect unequal expression of allelic GluR7 mRNAs in mice. Our results are important for future studies that explore a potential role or roles for GluR7 receptors in the brain and for neurological disorders.

The expression of the chick kainate-binding protein, a member of the ionotropic glutamate receptor family, is restricted to the cerebellum, specifically to Bergmann glia. Glutamate induces a membrane to nuclei signaling involved in gene expression regulation. Exposure of cultured chick Bergmann glia cells to glutamate leads to an increase in kainate binding protein and mRNA levels, suggesting a transcriptional level of regulation. The 5' proximal region of the chick kainate binding gene was cloned and transfected 4into Bergmann glia cells. Three main regulatory regions could be defined, a minimal promoter region, a negative regulatory region, and interestingly, a glutamate-responsive element. Deletion of this element abolishes the agonist effect. Moreover, electrophoretic mobility shift assays, cotransfection experiments, and site-directed mutagenesis clearly suggest that the glutamate effect is mediated through an AP-1 site by a Fos/Jun heterodimer. The present results favor the notion of a functional role of kainate-binding protein in glutamatergic cerebellar neurotransmission.

Quantitative receptor autoradiography and immunoblotting were used to study the expression and distribution of AMPA, kainate and NMDA receptors in first trimester human spinal cord obtained from elective abortions ranging from 4 to 11.5 weeks of gestational age. Spinal cord tissue sections were processed for receptor autoradiography with the ligands [3H]AMPA, [3H]kainate and [3H]MK-801 and the optical density was measured separately in a dorsal region (alar plate) and ventral region (basal plate) of the autoradiographs. Binding sites for all three ligands were demonstrated already at 4-5.5 weeks of gestation and increased continuously during the first trimester both in the dorsal and ventral regions. [3H]AMPA binding to both high- and low-affinity sites increased from undetectable levels to about 35 and 400 fmol/mg tissue, respectively, during this period. A temporal difference in the distribution of [3H]AMPA binding sites was observed. The early homogeneous pattern of [3H]AMPA binding in both alar and basal plates had changed to a heterogeneous pattern at 11 weeks of gestation with the highest density of [3H]AMPA binding sites in the superficial layers of the immature dorsal horn. [3H]kainate and [3H]MK-801 binding sites were densely and homogeneously distributed already at 4 weeks, and steadily increased six- and two-fold, respectively, to about 100 fmol/mg tissue at 11.5 weeks of gestation. Immunoreactive bands corresponding to the NMDA receptor subunits NR1, NR2A, NR2B, NR2C and NR2D were demonstrated by immunoblotting at the earliest between 4.5 and 7 weeks and increasing concentrations were seen up to 11 weeks of gestation. These results suggest that AMPA, kainate and NMDA receptors are expressed in the human spinal cord early in embryogenesis.

Previous studies showed that recombinant homomeric GluR6 receptors are acutely inhibited by ethanol. This study examined the acute actions of ethanol on recombinant homomeric and heteromeric kainate (KA) receptors with different subunit configurations. Application of 25 to 100 mM ethanol produced inhibition of a similar magnitude of both GluR5-Q and GluR6-R KA receptor-dependent currents in Xenopus oocytes. Ethanol decreased the KA Emax without affecting the EC50 and its effect was independent of the membrane holding potential for both of these receptors subtypes. Ethanol also inhibited homomeric and heteromeric receptors transiently expressed in human embryonic kidney (HEK) 293 cells. In these cells, the expression of heteromeric GluR6-R subunit-containing receptors was confirmed by testing their sensitivity to 1 mM alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid. Ethanol inhibited to a similar extent KA-gated currents mediated by receptors composed of either GluR6 or GluR6 + KA1 subunits, and to a slightly lesser extent receptors composed of GluR6 + KA2 subunits. Acute ethanol's effects were tested on GluR5 KA receptors that are expressed as homomers (GluR5-Q) or heteromers (GluR5-R + KA1 and GluR5-R + KA2). Homomeric and heteromeric GluR5 KA receptors were all inhibited to a similar extent by ethanol; however, there was slightly more inhibition of GluR5-R + KA2 receptors. Thus, recombinant KA receptors with different subunit compositions are all acutely inhibited to a similar extent by ethanol. In light of recent reports that KA receptors regulate neurotransmitter release and mediate synaptic currents, we postulate that these receptors may play a role in acute ethanol intoxication.

The present study examined the effects of chronic ethanol exposure on the expression of N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxalone (AMPA) and kainate receptor subunit proteins in rat cortical neuronal cultures grown in media containing 2 mM (high) or 0.1 mM (low) glutamine. Immunoblot analysis of NMDA (NR1, NR2A, NR2B, and NR2D), AMPA (GluR1 and GluR2/3), and kainate (GluR6/7) subunit polypeptides in 3-, 5-, 8-, 10-, and 12 day-old-cultures showed that NMDA receptor subunits NR1, NR2A, and NR2B and AMPA receptor subunits GluR2/3 progressively increased as a function of time, whereas levels of NMDA subunit NR2D were high at day 3 and progressively declined to barely detectable levels by day 12. Levels of AMPA subunit GluR1 and the kainate subunit GluR6/7 remained stable throughout the time course. Replacing the culture media with low glutamine media at culture day 5 did not alter the levels of subunit proteins measured at culture days 9 and 13. However, exposure of low glutamine cultures to 100 mM ethanol for 4 days (starting at culture day 9) significantly increased the levels of NMDA receptor subunits (NR1, NR2A, and NR2B) and AMPA receptor subunits (GluR1 and GluR2/3), but had no effect upon kainate receptor subunits (GluR6/7) or the synapse-associated proteins synapsin I and PSD-95. In contrast, chronic ethanol did not alter the levels of any of these subunit proteins in cells grown in high glutamine. These data demonstrate that under certain experimental conditions, prolonged exposure to ethanol upregulates NMDA and AMPA receptor subunit proteins, but has no effect upon kainate receptor subunit proteins. Because we have previously shown that acute ethanol can inhibit NMDA and AMPA, but not kainate, receptor function in these cultures, the increase in subunit expression likely reflects an adaptive response to the inhibitory effects of ethanol and suggests that both NMDA and AMPA receptors may play an important role in adaptation of the CNS to chronic ethanol.

The neurotoxic actions of kainate and domoate were studied in cultured murine neocortical neurons at various days in culture and found to be developmentally regulated involving three components of neurotoxicity: (1) toxicity via indirect activation of N-methyl-D-aspartate (NMDA) receptors, (2) toxicity mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, and (3) toxicity that can be mediated by kainate receptors when desensitization of the receptors is blocked. The indirect action at NMDA receptors was discovered because (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine (MK-801), an NMDA receptor antagonist, was able to block part of the toxicity. The activation of NMDA receptors is most likely a secondary effect resulting from glutamate release upon kainate or domoate stimulation. 1-(4-Aminophenyl)-3-methylcarbamyl-4-methyl-3,4-dihydro-7,8-ethyle nedioxy-5H-2,3-benzodiazepine (GYKI 53655), a selective AMPA receptor antagonist, abolished the remaining toxicity. These results indicated that kainate- and domoate-mediated toxicity involves both the NMDA and the AMPA receptors. Pretreatment of the cultures with concanavalin A to prevent desensitization of kainate receptors led to an increased neurotoxicity upon stimulation with kainate or domoate. In neurons cultured for 12 days in vitro a small but significant neurotoxic effect was observed when stimulated with agonist in the presence of MK-801 and GYKI 53655. This indicates that the toxicity is produced by kainate receptors in mature cultures. Examining the subunit expression of the kainate receptor subunits GluR6/7 and KA2 did, however, not reveal any major change during development of the cultures.

1. Patch-clamp recordings were made from rat cerebellar granule cells in primary culture. In cells pre-exposed to concanavalin A (ConA) to remove kainate receptor desensitization, concentration-response data for kainate showed two components. The EC50 value for the high-affinity component (4 microM) was consistent with activation of kainate-type channels. ConA enhanced the apparent potency of the kainate receptor ligand SYM 2081 by 100-fold. 2. In ConA-treated granule cells, currents evoked by 10 microM kainate were not significantly reduced by the AMPA receptor antagonist GYKI 53655, nor were these currents significantly reduced by the co-application of 100 microM AMPA. Currents activated by low concentrations of kainate in the presence of AMPA were completely inhibited by 10 microM La3+. 3. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that granule cells express both unedited (Q) and edited (R) versions of GluR5, with the majority of the GluR5 transcripts being unedited. In contrast, BluR6(R) was detected in seven cells and GluR6(Q) was detected in one granule cell. 4. Whole-cell current-voltage curves for kainate-type currents in granule cells were measured and the ratio of the slope conductances at +40 MV and -40 mV was used as an index of rectification. The mean +40 mV/-40 mV ratio determined from thirty-six granule cells was 1.3 +/- 0.1. Spectral density analysis of kainate-evoked whole-cell current noise gave values for the apparent single-channel conductance, gamma(noise), that were on average about 1 pS. 5. To compare further the properties of recombinant kainate channels with the native kainate-type channels in granule cells, we determined EC50 and gamma(noise) values for SYM 2081 in stable cell lines expressing either (GluR6(R) or GluR6(R) and KA2. Co-expression of KA2 with GluR6(R) shifts the EC50 and gamma(noise) values determined for SYM 2081 closer to the values typically found for native kainate-type channels in granule cells. 6. The results demonstrate that cerebellar granule cells in culture express functional kainate-type channels and that in most cells these channels show properties that are similar to those determined for heteromeric channels formed from GluR6(R) and KA2. However, the results also suggest that different granule cells express different repertoires of kainate-type channels with different, and perhaps variable, subunit composition.

We analyzed the expression and regulation of glutamate receptor subunits in the rat neural tube (10 day embryos) and in cell cultures derived from this tissue. In the cultures, all cells were stained with antibodies against the neural progenitor marker nestin. More than 50% of the cells were also stained by the monoclonal antibodies LB1 or A2B5, which bind to neuronal and glial progenitors. Approximately 6% of the cells were stained with antibodies for the low affinity NGF receptor, a neural crest cell marker. A small percentage of cells differentiated to neurons or astrocytes, as determined by staining with anti-neurofilament and anti-GFAP antibodies, respectively. RT-PCR analysis of neural tube tissue and culture mRNAs demonstrated that the AMPA receptor subunits GluR3 and 4 and the kainate receptor subunits GluR6, 7, KA1 and KA2 were detectable at E10. The kainate receptor subunits GluR6 and KA2 were upregulated by culture conditions which stimulated cell differentiation, as determined by concomitant downregulation of nestin mRNA. Both in neural tube tissue and in cultured cells, GluR6 was 100% unedited. Finally, both GluR6 and KA2 proteins could be detected in subpopulations of neural progenitors and differentiated neurons. Our data indicate that kainate receptor genes are expressed in undifferentiated progenitor cells of the neural tube at E10, and are upregulated during neural cell differentiation.

Here, functional AMPA/kainate receptors in human embryonic (5.5-7.5 gestational weeks) and foetal (8-10 gestational weeks) central nervous system tissue, shown by the cobalt labeling method, are reported. Specific agonist-induced cobalt incorporation was detected in brainstem and spinal cord cells, even in the youngest embryo studied. T-AMPA or kainate, but also vegetal toxins such as L-BOAA or acromelate, induced accumulation of cobalt. In contrast, no labeling was observed after exposure to KCl or NMDA. Cobalt labeled cells were particularly prominent in motor regions of brainstem and spinal cord. Co-application of the diuretic agent cyclothiazide, a desensitization blocker at AMPA receptors, dramatically increased the number of stained cells, which was particularly obvious in sensory regions, suggesting different receptor properties in motor versus sensory regions. This is the first study providing evidence for functional AMPA/kainate receptors, permeable to divalent cations, in brainstem and spinal cord at an early stage of human central nervous system development. Since many developmental processes are influenced by the modulation of cytosolic calcium, exposure at critical stages of embryogenesis to food or drug substances modifying the activity of AMPA/kainate receptors may alter brain development.

GluR5 and GluR6 kainate receptors differ in their responses to a variety of agonists, despite their relatively high primary sequence homology. We carried out a structure-function study to identify amino acids underlying these divergent responses. Patch clamp analysis of chimeric GluR5-GluR6 receptors indicated that several functionally dominant sites were localized to the C-terminal side of M1. All nonconserved amino acids in the region between M3 and M4 of GluR6 were then individually mutated to their GluR5 counterparts. We found that a single amino acid (N721 in GluR6) controls both AMPA sensitivity and domoate deactivation rates. Additionally, mutation of A689 in GluR6 slowed kainate desensitization. These functional effects were accompanied by alterations in binding affinities. These results support a critical role for these residues in receptor binding and gating activity.

The ontogeny of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate (KA) glutamate receptors in rat barrel field cortex was characterized by using receptor autoradiography and immunocytochemistry. A somatotopic pattern of AMPA receptors with fewer [3H]AMPA sites in barrel centers than in surrounding cortex did not emerge until postnatal day 10 (P10). After reaching a peak density at P14, the density of [3H]AMPA receptors declined in both barrel centers and surrounding cortex. Compared with AMPA receptors, the density of [3H]KA sites at all ages was low, a somatotopic expression of [3H]KA sites was missing, and the developmental curve for [3H]KA sites was more shallow than that for [3H]AMPA binding sites. A differential ontogeny of AMPA and KA receptors in barrel field cortex was also demonstrated in immunocytochemical studies with antibodies to the AMPA receptor subunits GluR1 and GluR2,3 and the KA receptor subunits GluR6,7. GluR1 and GluR2,3 staining was more dense in barrel septa than in barrel centers; this pattern persisted into adulthood. GluR1 and GluR2,3 receptors were localized to cell bodies and dendrites as well as the neuropil, but different populations of cortical neurons expressed these receptors. At P10, KA receptor subunits GluR6,7 exhibited a contrasting pattern to that of AMPA receptor subunits, with slightly more neuropil staining in barrel centers than in surrounding cortex. After that point, the somatotopic pattern of GluR6,7 subunit expression was lost. The contrasting developmental patterns of expression of the AMPA and KA receptors in the barrel field suggest that they may play different roles in the whisker-to-barrel pathway.

RNA editing determines receptor kinetics and permeability of glutamate receptors. This post-transcriptional modification alters single nucleotides within an RNA transcript changing the codon specified by the genome resulting in the incorporation of a different amino acid, profoundly affecting the properties of the protein subunit. We have studied the three sites subject to RNA editing within the kainate-specific subunit GluR6 in the mammalian hippocampus to determine developmental changes and cell-specific variation in editing. GluR6, when measured in the whole rat hippocampus, is predominantly expressed in the unedited form at E18, with a gradual progression to the edited form during the 1st post-natal week, and remains stable from P8 through 30 months. Individual neurons from P0 through P8 rat hippocampal slices analyzed with single-cell PCR show predominant expression of fully edited GluR6, unlike the population profile. In contrast, single astrocytes from P0 hippocampal cultures show that the most common variant is partially edited. Thus, editing in neurons and glia differs, and this difference accounts for part of the disparity between single-neuron and whole-hippocampus data. Editing in astrocytes is affected by conditions in the external environment, as purified astrocytes fail to edit GluR6, although editing occurs in astrocytes from hippocampal cultures. The homogeneity of GluR6 editing between species was also determined by comparing editing in avians and mammals. Genomic and cDNA analysis of chick glutamate receptors demonstrates avian editing of GluR2 but not GluR6.