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Trafficking of kainate receptors.
Membranes (Basel)
PUBLISHED: 08-20-2014
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Ionotropic glutamate receptors (iGluRs) mediate the vast majority of excitatory neurotransmission in the central nervous system of vertebrates. In the protein family of iGluRs, kainate receptors (KARs) comprise the probably least well understood receptor class. Although KARs act as key players in the regulation of synaptic network activity, many properties and functions of these proteins remain elusive until now. Especially the precise pre-, extra-, and postsynaptic localization of KARs plays a critical role for neuronal function, as an unbalanced localization of KARs would ultimately lead to dysregulated neuronal excitability. Recently, important advances in the understanding of the regulation of surface expression, function, and agonist-dependent endocytosis of KARs have been achieved. Post-translational modifications like PKC-mediated phosphorylation and SUMOylation have been reported to critically influence surface expression and endocytosis, while newly discovered auxiliary proteins were shown to shape the functional properties of KARs.
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Auxiliary subunits: shepherding AMPA receptors to the plasma membrane.
Membranes (Basel)
PUBLISHED: 08-08-2014
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Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated cation channels that mediate excitatory signal transmission in the central nervous system (CNS) of vertebrates. The members of the iGluR subfamily of ?-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPARs) mediate most of the fast excitatory signal transmission, and their abundance in the postsynaptic membrane is a major determinant of the strength of excitatory synapses. Therefore, regulation of AMPAR trafficking to the postsynaptic membrane is an important constituent of mechanisms involved in learning and memory formation, such as long-term potentiation (LTP) and long-term depression (LTD). Auxiliary subunits play a critical role in the facilitation and regulation of AMPAR trafficking and function. The currently identified auxiliary subunits of AMPARs are transmembrane AMPA receptor regulatory proteins (TARPs), suppressor of lurcher (SOL), cornichon homologues (CNIHs), synapse differentiation-induced gene I (SynDIG I), cysteine-knot AMPAR modulating proteins 44 (CKAMP44), and germ cell-specific gene 1-like (GSG1L) protein. In this review we summarize our current knowledge of the modulatory influence exerted by these important but still underappreciated proteins.
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Type I TARPs promote dendritic growth of early postnatal neocortical pyramidal cells in organotypic cultures.
Development
PUBLISHED: 03-25-2014
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The ionotropic ?-amino-3-hydroxy-5-methyl-4-isoxazole propionate glutamate receptors (AMPARs) have been implicated in the establishment of dendritic architecture. The transmembrane AMPA receptor regulatory proteins (TARPs) regulate AMPAR function and trafficking into synaptic membranes. In the current study, we employ type I and type II TARPs to modulate expression levels and function of endogenous AMPARs and investigate in organotypic cultures (OTCs) of rat occipital cortex whether this influences neuronal differentiation. Our results show that in early development [5-10 days in vitro (DIV)] only the type I TARP ?-8 promotes pyramidal cell dendritic growth by increasing spontaneous calcium amplitude and GluA2/3 expression in soma and dendrites. Later in development (10-15 DIV), the type I TARPs ?-2, ?-3 and ?-8 promote dendritic growth, whereas ?-4 reduced dendritic growth. The type II TARPs failed to alter dendritic morphology. The TARP-induced dendritic growth was restricted to the apical dendrites of pyramidal cells and it did not affect interneurons. Moreover, we studied the effects of short hairpin RNA-induced knockdown of endogenous ?-8 and showed a reduction of dendritic complexity and amplitudes of spontaneous calcium transients. In addition, the cytoplasmic tail (CT) of ?-8 was required for dendritic growth. Single-cell calcium imaging showed that the ?-8 CT domain increases amplitude but not frequency of calcium transients, suggesting a regulatory mechanism involving the ?-8 CT domain in the postsynaptic compartment. Indeed, the effect of ?-8 overexpression was reversed by APV, indicating a contribution of NMDA receptors. Our results suggest that selected type I TARPs influence activity-dependent dendritogenesis of immature pyramidal neurons.
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Autoantibodies to glutamate receptor antigens in multiple sclerosis and Rasmussen's encephalitis.
Neuroimmunomodulation
PUBLISHED: 02-06-2014
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Glutamate and its specific ionotropic receptors, including N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, are supposed to play an important role in neurodegeneration as well as neuronal regeneration. Although autoantibodies (aab) to glutamate receptors (GluR) have been identified in several neurologic diseases, including paraneoplastic encephalitis and Rasmussen's encephalitis (RE) with an increasing prevalence, the presence and role of anti-GluR aab in multiple sclerosis (MS) have not been studied yet.
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Structural basis of PI(4,5)P2-dependent regulation of GluA1 by phosphatidylinositol-5-phosphate 4-kinase, type II, alpha (PIP5K2A).
Pflugers Arch.
PUBLISHED: 01-05-2014
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Ionotropic glutamate receptors are the most important excitatory receptors in the central nervous system, and their impairment can lead to multiple neuronal diseases. Here, we show that glutamate-induced currents in oocytes expressing GluA1 are increased by coexpression of the schizophrenia-associated phosphoinositide kinase PIP5K2A. This effect was due to enhanced membrane abundance and was blunted by a point mutation (N251S) in PIP5K2A. An increase in GluA1 currents was also observed upon acute injection of PI(4,5)P2, the main product of PIP5K2A. By expression of wild-type and mutant PIP5K2A in human embryonic kidney cells, we were able to provide evidence of impaired kinase activity of the mutant PIP5K2A. We defined the region K813-K823 of GluA1 as critical for the PI(4,5)P2 effect by performing an alanine scan that suggested PI(4,5)P2 binding to this area. A PIP strip assay revealed PI(4,5)P2 binding to the C-terminal GluA1 peptide. The present observations disclose a novel mechanism in the regulation of GluA1.
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A plant homolog of animal glutamate receptors is an ion channel gated by multiple hydrophobic amino acids.
Sci Signal
PUBLISHED: 06-13-2013
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Ionotropic glutamate receptors (iGluRs) are ligand-gated cation channels that mediate neurotransmission in animal nervous systems. Homologous proteins in plants have been implicated in root development, ion transport, and several metabolic and signaling pathways. AtGLR3.4, a plant iGluR homolog from Arabidopsis thaliana, has ion channel activity and is gated by asparagine, serine, and glycine. Using heterologous expression in Xenopus oocytes, we found that another Arabidopsis iGluR homolog, AtGLR1.4, functioned as a ligand-gated, nonselective, Ca(2+)-permeable cation channel that responded to an even broader range of amino acids, none of which are agonists of animal iGluRs. Seven of the 20 standard amino acids--mainly hydrophobic ones--acted as agonists, with methionine being most effective and most potent. Nine amino acids were antagonists, and four, including glutamate and glycine, had no effect on channel activity. We constructed a model of this previously uncharacterized ligand specificity and used knockout mutants to show that AtGLR1.4 accounts for methionine-induced membrane depolarization in Arabidopsis leaves.
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The delta subfamily of glutamate receptors: characterization of receptor chimeras and mutants.
Eur. J. Neurosci.
PUBLISHED: 02-08-2013
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The delta receptors, GluD1 and GluD2, are regarded as a subfamily of the ionotropic glutamate receptors solely because of sequence homology. While they play important roles in cerebellar function and high-frequency hearing and appear to serve structural functions at synapses, ligand-gated ion channel function has not been observed. However, we have previously shown that GluD2 can form functional ion channels when grafted with the ligand binding domain of a kainate receptor. In this study, we characterized this chimera as well as additional rat delta receptor chimeras and point mutants in more detail. We found that the kainate receptor ligand binding domain renders GluD1 functional as well, and GluD2 becomes a functional ion channel also when provided with an AMPA receptor ligand binding domain. Point mutations indicate that the GluD2 ion pore operates similarly but not identically to that of AMPA (?-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) and kainate receptors. GluD2 mutated at a conserved arginine within the linker region connecting the ligand binding domain to the ion pore domain displays spontaneous currents that occur in the absence of agonists and are inhibited by agonist application - a behavior reminiscent of that of the previously characterized lurcher mutant. Using our chimeric approach, we provide evidence that this inhibition of spontaneous currents by agonists may be caused by desensitization. Our results show that delta receptors have functional gating machineries and ion permeation pathways similar but not identical to those of AMPA and kainate receptors, while the key differences seem to be located within the ligand binding domain.
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Undifferentiated embryonic stem cells express ionotropic glutamate receptor mRNAs.
Front Cell Neurosci
PUBLISHED: 01-01-2013
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Ionotropic glutamate receptors (iGluRs) do not only mediate the majority of excitatory neurotransmission in the vertebrate CNS, but also modulate pre- and postnatal neurogenesis. Most of the studies on the developmental role of iGluRs are performed on neural progenitors and neural stem cells (NSCs). We took a step back in our study by examining the role of iGluRs in the earliest possible cell type, embryonic stem cells (ESCs), by looking at the mRNA expression of the major iGluR subfamilies in undifferentiated mouse ESCs. For that, we used two distinct murine ES cell lines, 46C ESCs and J1 ESCs. Regarding 46C ESCs, we found transcripts of kainate receptors (KARs) (GluK2 to GluK5), AMPA receptors (AMPARs) (GluA1, GluA3, and GluA4), and NMDA receptors (NMDARs) (GluN1, and GluN2A to GluN2D). Analysis of 46C-derived cells of later developmental stages, namely neuroepithelial precursor cells (NEPs) and NSCs, revealed that the mRNA expression of KARs is significantly upregulated in NEPs and, subsequently, downregulated in NSCs. However, we could not detect any protein expression of any of the KAR subunits present on the mRNA level either in ESCs, NEPs, or NSCs. Regarding AMPARs and NMDARs, GluN2A is weakly expressed at the protein level only in NSCs. Matching our findings for iGluRs, all three cell types were found to weakly express pre- and postsynaptic markers of glutamatergic synapses only at the mRNA level. Finally, we performed patch-clamp recordings of 46C ESCs and could not detect any current upon iGluR agonist application. Similar to 46C ESCs, J1 ESCs express KARs (GluK2 to GluK5), AMPARs (GluA3), and NMDARs (GluN1, and GluN2A to GluN2D) at the mRNA level, but these transcripts are not translated into receptor proteins either. Thus, we conclude that ESCs do not contain functional iGluRs, although they do express an almost complete set of iGluR subunit mRNAs.
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Cell class-specific regulation of neocortical dendrite and spine growth by AMPA receptor splice and editing variants.
Development
PUBLISHED: 08-24-2011
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Glutamatergic transmission converging on calcium signaling plays a key role in dendritic differentiation. In early development, AMPA receptor (AMPAR) transcripts are extensively spliced and edited to generate subunits that differ in their biophysical properties. Whether these subunits have specific roles in the context of structural differentiation is unclear. We have investigated the role of nine GluA variants and revealed a correlation between the expression of flip variants and the period of major dendritic growth. In interneurons, only GluA1(Q)-flip increased dendritic length and branching. In pyramidal cells, GluA2(Q)-flop, GluA2(Q)-flip, GluA3(Q)-flip and calcium-impermeable GluA2(R)-flip promoted dendritic growth, suggesting that flip variants with slower desensitization kinetics are more important than receptors with elevated calcium permeability. Imaging revealed significantly higher calcium signals in pyramidal cells transfected with GluA2(R)-flip as compared with GluA2(R)-flop, suggesting a contribution of voltage-activated calcium channels. Indeed, dendritic growth induced by GluA2(R)-flip in pyramidal cells was prevented by blocking NMDA receptors (NMDARs) or voltage-gated calcium channels (VGCCs), suggesting that they act downstream of AMPARs. Intriguingly, the action of GluA1(Q)-flip in interneurons was also dependent on NMDARs and VGCCs. Cell class-specific effects were not observed for spine formation, as GluA2(Q)-flip and GluA2(Q)-flop increased spine density in pyramidal cells as well as in interneurons. The results suggest that AMPAR variants expressed early in development are important determinants for activity-dependent dendritic growth in a cell type-specific and cell compartment-specific manner.
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Brain evolution triggers increased diversification of electric fishes.
Science
PUBLISHED: 04-30-2011
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Communication can contribute to the evolution of biodiversity by promoting speciation and reinforcing reproductive isolation between existing species. The evolution of species-specific signals depends on the ability of individuals to detect signal variation, which in turn relies on the capability of the brain to process signal information. Here, we show that evolutionary change in a region of the brain devoted to the analysis of communication signals in mormyrid electric fishes improved detection of subtle signal variation and resulted in enhanced rates of signal evolution and species diversification. These results show that neural innovations can drive the diversification of signals and promote speciation.
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Expression of NMDA receptors and Ca2+-impermeable AMPA receptors requires neuronal differentiation and allows discrimination between two different types of neural stem cells.
Cell. Physiol. Biochem.
PUBLISHED: 11-10-2010
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Glutamate and its receptors are ascribed a pivotal role during acitivity-dependent neurogenesis. Nevertheless, their precise expression patterns during embryonic and adult differentiation remain elusive. An in vitro-approach that includes cells representing embryonic as well as adult neural stem cells that are both amenable to retinoic acid treatment is well-suited for assessing the developmental regulation of ionotropic glutamate receptors (iGluRs). The chosen system provides a continuous time line from embryonic to adult neurogenesis via two distinguishable cell populations, namely neuroepithelial precursors (NEPs) and radial glia-like neural stem cells (NSCs). We investigated the expression of cell type-specific differentiation markers and iGluR subunits before and after neuronal induction. A quantitative PCR assay was established for the determination of a hypothetical correlation of neuronal differentiation and iGluR expression. The NMDAR subunits NR1 and NR2B as well as the AMPAR subunit GluR2 present in Ca(2+)-impermeable AMPARs were found to be upregulated at the mRNA level in differentiated neuroepithelial precursors, indicating their likely contribution to neurotransmission after the first establishment of neuronal networks. Furthermore, with this approach, discrimination between NEPs and NSCs regarding their iGluR subunit expression patterns before and after the induction of neuronal differentiation was possible and pointed to diverse functions in these two cell types carried out by differentially assembled iGluRs.
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Residues at the tip of the pore loop of NR3B-containing NMDA receptors determine Ca2+ permeability and Mg2+ block.
BMC Neurosci
PUBLISHED: 10-19-2010
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Members of the complex N-methyl-D-aspartate receptor (NMDAR) subfamily of ionotropic glutamate receptors (iGluRs) conventionally assemble from NR1 and NR2 subunits, the composition of which determines receptor properties. Hallmark features of conventional NMDARs include the requirement for a coagonist, voltage-dependent block by Mg2+, and high permeability for Ca2+. Both Mg2+ sensitivity and Ca2+ permeability are critically dependent on the amino acids at the N and N+1 positions of NR1 and NR2. The recently discovered NR3 subunits feature an unprecedented glycine-arginine combination at those critical sites within the pore. Diheteromers assembled from NR1 and NR3 are not blocked by Mg2+ and are not permeable for Ca2+.
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Bridging the synaptic cleft: lessons from orphan glutamate receptors.
Sci Signal
PUBLISHED: 08-17-2010
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For neurons to communicate, signals must cross the cell-to-cell distance at their points of contact. At the predominant cell-cell contact in the central nervous system, the chemical synapse, the synaptic cleft spans roughly 20 nanometers. To signal across this distance, the presynaptic neuron secretes a diffusible neurotransmitter, which is detected by receptors on the postsynaptic neuron. Although this signaling mechanism has become common knowledge, it remains unclear how synapses are maintained when they are not in immediate use. New evidence reveals how Nature solved this problem at a particular type of synapse in the cerebellum: Three old acquaintances bridge the cleft. The ionotropic glutamate receptor GluD2 constitutes the postsynaptic anchor that indirectly interacts with the presynaptic anchor neurexin through a presynaptically secreted soluble factor, a member of the C1q protein family named Cbln1. This trio collaborates to align pre- and postsynaptic sides.
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The Transmembrane Domain C of AMPA Receptors is Critically Involved in Receptor Function and Modulation.
Front Mol Neurosci
PUBLISHED: 05-21-2010
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Ionotropic glutamate receptors are major players in synaptic transmission and are critically involved in many cognitive events. Although receptors of different subfamilies serve different functions, they all show a conserved domain topology. For most of these domains, structure-function relationships have been established and are well understood. However, up to date the role of the transmembrane domain C in receptor function has been investigated only poorly. We have constructed a series of receptor chimeras and point mutants designed to shed light on the structural and/or functional importance of this domain. We here present evidence that the role of transmembrane domain C exceeds that of a mere scaffolding domain and that several amino acid residues located within the domain are crucial for receptor gating and desensitization. Furthermore, our data suggest that the domain may be involved in receptor interaction with transmembrane AMPA receptor regulatory proteins.
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The C-terminal domains of TARPs: unexpectedly versatile domains.
Channels (Austin)
PUBLISHED: 03-13-2010
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AMPA receptors mediate the majority of fast synaptic transmission in the central nervous system and are therefore among the most intensively studied ligand-gated ion channels over the last decades. However, the recent discovery that native AMPA receptor complexes contain auxiliary subunits classified as transmembrane AMPA receptor regulatory proteins (TARPs) was quite a surprise and dramatically changed the field of AMPA receptor research. TARPs regulate trafficking as well as synaptic localization of AMPA receptors, and alter their pharmacological and biophysical properties, generally resulting in strongly elevated receptor-mediated currents. Thus, the association of AMPA receptors with TARPs increases receptor heterogeneity and diversity of postsynaptic currents. In this regard, unravelling the mechanisms by which TARPs modulate AMPA receptor function is an intriguing challenge. Studying the functional importance of the carboxy-terminal domain (CTD) of TARPs for receptor modulation, we found that the increased trafficking mediated by the two TARPs ?2 and ?3 is attributable to their CTDs. Furthermore, we demonstrated that the CTD additionally determines the differences between TARPs regarding their modulation of AMPA receptor function. As a case in point, we showed a unique role of the CTD of ?4, suggesting that TARPs modulate AMPA receptor function via individual mechanisms.
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Cave Canalem: how endogenous ion channels may interfere with heterologous expression in Xenopus oocytes.
Methods
PUBLISHED: 01-26-2010
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Xenopus laevis oocytes are an outstanding heterologous expression system for the investigation of ion channels. However, oocytes express an amazing variety of endogenous ion channels that can severely interfere with electrophysiological measurements. It is therefore necessary to be aware of the channels present in the oocyte and to be able to exclude artifacts they might cause during the analysis of heterologously expressed ion channels. Research on Xenopus endogenous ion channels has started over 30 years ago, and many channels have been described since then. This does not only include voltage-gated channels conducting Na(+), K(+), Ca(2+), and Cl(-), but also ion channels activated by ligand binding such as ionotropic neurotransmitter receptors. Furthermore, there are other channels such as those triggered by changes in osmolarity or mechanical stress, as well as conductances caused by yet uncharacterized molecules. Here, we present an overview of ion channels endogenous to the oocyte described in the literature so far, and provide procedures and methods to abolish or minimize their impact on electrophysiological recordings of exogenous channels.
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Oligomerization in the endoplasmic reticulum and intracellular trafficking of kainate receptors are subunit-dependent but not editing-dependent.
J. Neurochem.
PUBLISHED: 12-26-2009
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Investigating subunit assembly of ionotropic glutamate receptor complexes and their trafficking to the plasma membrane under physiological conditions in live cells has been challenging. By confocal imaging of fluorescently labeled kainate receptor (KAR) subunits combined with digital co-localization and fluorescence resonance energy (FRET) transfer analyses, we investigated the assembly of homomeric and heteromeric receptor complexes and identified the subcellular location of subunit interactions. Our data provide direct evidence for oligomerization of KAR subunits as early as following their biosynthesis in the endoplasmic reticulum (ER). These oligomeric assemblies pass through the Golgi apparatus en route to the plasma membrane. We show that the amino acid at the Q/R editing site of the KAR subunit GluR6 neither determines subunit oligomerization in the ER nor ER exit or plasma membrane expression, and that it does not alter GluR6 interaction with KA2. This finding sets KARs apart from alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors, where in the absence of auxiliary proteins Q isoforms exit the ER much more efficiently than R isoforms. Furthermore, although KA2 subunits do not form functional homotetrameric complexes, we visualized their oligomerization (at least dimerization) in the ER. Finally, we demonstrate that plasma membrane expression of GluR6/KA2 heteromeric complexes is modulated not only by GluR6 but also KA2.
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Functional excitatory GABAA receptors precede ionotropic glutamate receptors in radial glia-like neural stem cells.
Mol. Cell. Neurosci.
PUBLISHED: 11-10-2009
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The involvement of neurotransmission in neuronal development is a generally accepted concept. Nevertheless, the precise regulation of neurotransmitter receptor expression is still unclear. To investigate the expression profiles of the most important ionotropic neurotransmitter receptors, namely GABA(A) receptors (GABA(A)Rs), NMDA receptors (NMDARs), and AMPA receptors (AMPARs), quantitative RT-PCR, immunoblot analysis and patch clamp studies were performed in in vitro-generated neural stem cells (NSCs). This clearly defined cell line is closely related to radial glia cells, the stem cells in the neonate brain. We found functional GABA(A)Rs of the subunit composition alpha2, beta3, and gamma1 to be expressed. Unexpectedly, functional ionotropic glutamate receptors were absent. However, NSCs expressed the NMDAR subunits NR2A and NR3A, and the AMPAR subunit GluR4 at the protein level, and GluR3 at the mRNA level. The overexpression of functional NMDARs in NSCs led to an increased mRNA level of AMPAR subunits, indicating a role in synaptogenesis. Early neuronal markers remained unchanged. These data extend our knowledge about ionotropic neurotransmitter receptor expression during neuronal development and will aid further investigations on activity-dependent neurogenesis.
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C-terminal domains of transmembrane alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor regulatory proteins not only facilitate trafficking but are major modulators of AMPA receptor function.
J. Biol. Chem.
PUBLISHED: 09-22-2009
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Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors are essential players in fast synaptic transmission in the vertebrate central nervous system. Their synaptic delivery and localization as well as their electrophysiological properties are regulated by transmembrane AMPA receptor regulatory proteins (TARPs). However, the exact mechanisms of how the four originally designated TARPs (gamma2, gamma3, gamma4, and gamma8) modulate AMPA receptor function remain largely unknown. Previous studies suggested the C-terminal domain (CTD) of gamma2 to mediate increased trafficking and reduced desensitization of AMPA receptors. As it remained unclear whether these findings extend to other TARPs, we set out to investigate and compare the role of the CTDs of the four original TARPs in AMPA receptor modulation. To address this issue, we replaced the TARP CTDs with the CTD of the homologous subunit gamma1, a voltage-dependent calcium channel subunit expressed in skeletal muscle that lacks TARP properties. We analyzed the impact of the resulting chimeras on GluR1 functional properties in Xenopus oocytes and HEK293 cells. Interestingly, the CTDs of all TARPs not only modulate the extent and kinetics of desensitization but also modulate agonist potencies of AMPA receptors. Furthermore, the CTDs are required for TARP-induced modulation of AMPA receptor gating, including conversion of antagonists to partial agonists and constitutive channel openings. Strikingly, we found a special role of the cytoplasmic tail of gamma4, suggesting that the underlying mechanisms of modulation of AMPA receptor function are different among the TARPs. We propose that the intracellularly located CTD is the origin of TARP-specific functional modulation and not merely a facilitator of trafficking.
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The glutamate receptor subunit delta2 is capable of gating its intrinsic ion channel as revealed by ligand binding domain transplantation.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 06-08-2009
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The family of ionotropic glutamate receptors includes 2 subunits, delta1 and delta2, the physiological relevance of which remains poorly understood. Both are nonfunctional in heterologous expression systems, although the isolated, crystallized ligand binding domain (LBD) of delta2 is capable of binding D-serine. To investigate these seemingly contradictory observations we tested whether delta receptors can be ligand gated at all. We used a strategy that replaced the native LBD of delta2 by a proven glutamate-binding LBD. Test transplantations between alpha-amino-3-hydroxy-5-methylisoxazole propionate (AMPA) and kainate receptors (GluR1 and GluR6, respectively) showed that this approach can produce functional chimeras even if only one part of the bipartite LBD is swapped. Upon outfitting delta2 with the LBD of GluR6, the chimera formed glutamate-gated ion channels with low Ca(2+) permeability and unique rectification properties. Ligand-induced conformational changes can thus gate delta2, suggesting that the LBD of this receptor works fundamentally differently from that of other ionotropic glutamate receptors.
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Effects of NR1 splicing on NR1/NR3B-type excitatory glycine receptors.
BMC Neurosci
PUBLISHED: 04-06-2009
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N-methyl-D-aspartate receptors (NMDARs) are the most complex of ionotropic glutamate receptors (iGluRs). Subunits of this subfamily assemble into heteromers, which - depending on the subunit combination - may display very different pharmacological and electrophysiological properties. The least studied members of the NMDAR family, the NR3 subunits, have been reported to assemble with NR1 to form excitatory glycine receptors in heterologous expression systems. The heterogeneity of NMDARs in vivo is in part conferred to the receptors by splicing of the NR1 subunit, especially with regard to proton sensitivity.
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Xenopus laevis oocytes endogenously express all subunits of the ionotropic glutamate receptor family.
J. Mol. Biol.
PUBLISHED: 03-09-2009
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Xenopus laevis oocytes are commonly used as a heterologous expression system for the electrophysiological characterization of ionotropic glutamate receptors (iGluRs). Recently, however, several glutamate receptor subunits of the N-methyl-d-aspartate receptor subfamily have been found to be expressed endogenously in Xenopus oocytes, thus limiting the use of this expression system for such receptors. We therefore screened oocytes for the Xenopus homologs of all iGluR subunits known to be expressed in mammals to investigate which additional subunits may be present endogenously in oocytes and what, if any, influence such proteins might have on the functional analysis of heterologously expressed receptors. We found Xenopus homologs of every mammalian iGluR subunit to be expressed at the mRNA level. We then cloned, from oocytes, full-length copies of the four Xenopus alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunits XenGluR1 through XenGluR4 and, additionally, XenGluR6 as a representative subunit for the kainate receptor subfamily and electrophysiologically characterized them. Upon analysis, we found only minor functional differences between homologous subunits from Xenopus and rat. Next, we investigated whether endogenous iGluR subunits can be detected electrophysiologically in oocytes. We found no indication for any functional glutamate receptors in native oocytes; however, after heterologous expression of the auxiliary subunit stargazin, endogenous alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors were detected. These data demonstrate that Xenopus oocytes express glutamate receptor subunits endogenously, albeit at very low levels. Such endogenous receptor proteins can, under certain circumstances, become electrophysiologically detectable and then might influence electrophysiological recordings performed on recombinant receptors in oocytes.
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Functional complementation of Glra1(spd-ot), a glycine receptor subunit mutant, by independently expressed C-terminal domains.
J. Neurosci.
PUBLISHED: 02-27-2009
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The oscillator mouse (Glra1(spd-ot)) carries a 9 bp microdeletion plus a 2 bp microinsertion in the glycine receptor alpha1 subunit gene, resulting in the absence of functional alpha1 polypeptides from the CNS and lethality 3 weeks after birth. Depending on differential use of two splice acceptor sites in exon 9 of the Glra1 gene, the mutant allele encodes either a truncated alpha1 subunit (spd(ot)-trc) or a polypeptide with a C-terminal missense sequence (spd(ot)-elg). During recombinant expression, both splice variants fail to form ion channels. In complementation studies, a tail construct, encoding the deleted C-terminal sequence, was coexpressed with both mutants. Coexpression with spd(ot)-trc produced glycine-gated ion channels. Rescue efficiency was increased by inclusion of the wild-type motif RRKRRH. In cultured spinal cord neurons from oscillator homozygotes, viral infection with recombinant C-terminal tail constructs resulted in appearance of endogenous alpha1 antigen. The functional rescue of alpha1 mutants by the C-terminal tail polypeptides argues for a modular subunit architecture of members of the Cys-loop receptor family.
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Molecular and functional characterization of Xenopus laevis N-methyl-d-aspartate receptors.
Mol. Cell. Neurosci.
PUBLISHED: 01-15-2009
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N-methyl-d-aspartate (NMDA) receptors of many different vertebrates have been characterized in the past. However, little information is available about amphibian NMDA receptors. Here, we investigated the South African clawed frog Xenopus laevis NR1 subunit at the molecular and functional level. In this subunit, which is obligatory for functional NMDA receptor complexes, we found three exons, the N1, C1, and C3 cassettes, being alternatively spliced. Combinations of these cassettes generated six different splice variants, which were functionally characterized in oocytes. The Xenopus NR1 isoforms generally showed the same functional properties as their mammalian homologs when coexpressed with rat NR2B. In coexpression with Xenopus NR2B, however, some properties changed significantly. This included a Zn(2+)-mediated potentiation of current amplitudes for some subunit combinations which lasted for several minutes. This mechanism presents a novel form of Xenopus NMDA receptor modulation, possibly mediating a form of short-term potentiation in the Xenopus central nervous system.
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Pressure-selective modulation of NMDA receptor subtypes may reflect 3D structural differences.
Front Cell Neurosci
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Professional deep-water divers exposed to high pressure (HP) above 1.1 MPa suffer from High Pressure Neurological Syndrome (HPNS), which is associated with CNS hyperexcitability. We have previously reported that HP augments N-methyl-D-aspartate receptor (NMDAR) synaptic responses, increases neuronal excitability, and potentially causes irreversible neuronal damage. We now report that HP (10.1 MPa) differentially affects eight specific NMDAR subtypes. GluN1(1a or 1b) was co-expressed with one of the four GluN2(A-D) subunits in Xenopus laevis oocytes. HP increased ionic currents (measured by two electrode voltage clamps) of one subtype, reduced the current in four others, and did not affect the current in the remaining three. 3D theoretical modeling was aimed at revealing specific receptor domains involved with HP selectivity. In light of the information on the CNS spatial distribution of the different NMDAR subtypes, we conclude that the NMDARs diverse responses to HP may lead to selective HP effects on different brain regions. These discoveries call for further and more specific investigation of deleterious HP effects and suggest the need for a re-evaluation of deep-diving safety guidelines.
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Sensory receptor diversity establishes a peripheral population code for stimulus duration at low intensities.
J. Exp. Biol.
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Peripheral filtering is a fundamental mechanism for establishing frequency tuning in sensory systems. By contrast, detection of temporal features, such as duration, is generally thought to result from temporal coding in the periphery, followed by an analysis of peripheral response times within the central nervous system. We investigated how peripheral filtering properties affect the coding of stimulus duration in the electrosensory system of mormyrid fishes using behavioral and electrophysiological measures of duration tuning. We recorded from individual knollenorgans, the electrosensory receptors that mediate communication, and found correlated variation in frequency tuning and duration tuning, as predicted by a simple circuit model. In response to relatively high intensity stimuli, knollenorgans responded reliably with fixed latency spikes, consistent with a temporal code for stimulus duration. At near-threshold intensities, however, both the reliability and the temporal precision of responses decreased. Evoked potential recordings from the midbrain, as well as behavioral responses to electrosensory stimulation, revealed changes in sensitivity across the range of durations associated with the greatest variability in receptor sensitivity. Further, this range overlapped with the natural range of variation in species-specific communication signals, suggesting that peripheral duration tuning affects the coding of behaviorally relevant stimuli. We measured knollenorgan, midbrain and behavioral responses to natural communication signals and found that each of them were duration dependent. We conclude that at relatively low intensities for which temporal coding is ineffective, diversity among sensory receptors establishes a population code, in which duration is reflected in the population of responding knollenorgans.
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Plasticity in D1-like receptor expression is associated with different components of cognitive processes.
PLoS ONE
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Dopamine D1-like receptors consist of D1 (D1A) and D5 (D1B) receptors and play a key role in working memory. However, their possibly differential contribution to working memory is unclear. We combined a working memory training protocol with a stepwise increase of cognitive subcomponents and real-time RT-PCR analysis of dopamine receptor expression in pigeons to identify molecular changes that accompany training of isolated cognitive subfunctions. In birds, the D1-like receptor family is extended and consists of the D1A, D1B, and D1D receptors. Our data show that D1B receptor plasticity follows a training that includes active mental maintenance of information, whereas D1A and D1D receptor plasticity in addition accompanies learning of stimulus-response associations. Plasticity of D1-like receptors plays no role for processes like response selection and stimulus discrimination. None of the tasks altered D2 receptor expression. Our study shows that different cognitive components of working memory training have distinguishable effects on D1-like receptor expression.
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Identification of a novel signaling pathway and its relevance for GluA1 recycling.
PLoS ONE
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We previously showed that the serum- and glucocorticoid-inducible kinase 3 (SGK3) increases the AMPA-type glutamate receptor GluA1 protein in the plasma membrane. The activation of AMPA receptors by NMDA-type glutamate receptors eventually leads to postsynaptic neuronal plasticity. Here, we show that SGK3 mRNA is upregulated in the hippocampus of new-born wild type Wistar rats after NMDA receptor activation. We further demonstrate in the Xenopus oocyte expression system that delivery of GluA1 protein to the plasma membrane depends on the small GTPase RAB11. This RAB-dependent GluA1 trafficking requires phosphorylation and activation of phosphoinositol-3-phosphate-5-kinase (PIKfyve) and the generation of PI(3,5)P(2). In line with this mechanism we could show PIKfyve mRNA expression in the hippocampus of wild type C57/BL6 mice and phosphorylation of PIKfyve by SGK3. Incubation of hippocampal slices with the PIKfyve inhibitor YM201636 revealed reduced CA1 basal synaptic activity. Furthermore, treatment of primary hippocampal neurons with YM201636 altered the GluA1 expression pattern towards reduced synaptic expression of GluA1. Our findings demonstrate for the first time an involvement of PIKfyve and PI(3,5)P(2) in NMDA receptor-triggered synaptic GluA1 trafficking. This new regulatory pathway of GluA1 may contribute to synaptic plasticity and memory.
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GluN3 subunit-containing NMDA receptors: not just one-trick ponies.
Trends Neurosci.
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The two GluN3 subunits were the last NMDA receptor subunits to be cloned some 15 years ago. Strikingly, despite the steadily growing interest in their function, their physiological role remains elusive. The original billing as dominant-negative modulators of classical NMDA receptors composed of GluN1 and GluN2 subunits has given way to proposals of much more complex functions, including roles in synaptogenesis and synaptic plasticity. In addition, GluN3 subunits in the absence of GluN2 surprisingly assemble with GluN1 into excitatory glycine receptors. This review provides an overview of the unique spatial and temporal expression patterns of the GluN3 subunits, discusses proposed functions and physiological roles for receptors comprising these subunits, and briefly summarizes their putative involvement in several neural diseases.
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From the Schnauzenorgan to the back: morphological comparison of mormyromast electroreceptor organs at different skin regions of Gnathonemus petersii.
J. Morphol.
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The nocturnally active weakly electric fish Gnathonemus petersii is known to employ active electrolocation for the detection of objects and for orientation in its environment. The fish emits pulse-type electric signals with an electric organ and perceives these signals with more than 3,000 epidermal electroreceptor organs, the mormyromasts, which are distributed over the animals skin surface. In this study, we measured the metric dimensions of the mormyromasts from different body regions to find structural and functional specialization of the various body parts. We focused on the two foveal regions of G. petersii, which are located at the elongated and movable chin (the Schnauzenorgan; SO) and at the nasal region (NR), the skin region between the mouth and the nares. These two foveal regions were compared to the dorsal part (back) of the fish, which contains typical nonfoveal mormyromasts. While the gross anatomy of the mormyromasts from all skin regions is similar, the metric dimensions of the main substructures differed. The mormyromasts at the SO are the smallest and contain the smallest receptor cells. In addition, the number of receptor cells per organ is lowest at the SO. In contrast, at the back the biggest receptor organs with the highest amount of receptor cells per organ occur. The mormyromasts at the NR are in several respects intermediate between those from the back and the SO. However, mormyromasts at the NR are longer than those at all other skin regions, the canal leading from the receptor pore to the inner chambers were the longest and the overlaying epidermal layers are the thickest. These results show that mormyromasts and the epidermis they are embedded in at both foveal regions differ specifically from those found on the rest of the body. The morphological specializations lead to functional specialization of the two foveae.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.