NMDA receptors (NMDARs) play an important role in neural plasticity including long-term potentiat... more NMDA receptors (NMDARs) play an important role in neural plasticity including long-term potentiation and long-term depression, which are likely to explain their importance for learning and memory. Cognitive decline is a major problem facing an ageing human population, so much so that its reversal has become an important goal for scientific research and pharmaceutical development. Enhancement of NMDAR function is a core strategy toward this goal. In this review we indicate some of the major ways of potentiating NMDAR function by both direct and indirect modulation. There is good evidence that both positive and negative modulation can enhance function suggesting that a subtle approach correcting imbalances in particular clinical situations will be required. Excessive activation and the resultant deleterious effects will need to be carefully avoided. Finally we describe some novel positive allosteric modulators of NMDARs, with some subunit selectivity, and show initial evidence of their ability to affect NMDAR mediated events. This article is part of a Special Issue entitled 'Cognitive Enhancers'.
Lignans are biologically active phenolic compounds related to lignin, produced in different plant... more Lignans are biologically active phenolic compounds related to lignin, produced in different plants. Arctigenin, a dibenzylbutyrolactone-type lignan, has been used as a neuroprotective agent for the treatment of encephalitis. Previous studies of cultured rat cerebral cortical neurones raised the possibility that arctigenin inhibits kainate-induced excitotoxicity. The aims of the present study were: 1) to analyse the effect of arctigenin on normal synaptic activity in ex vivo brain slices, 2) to determine its receptor binding properties and test the effect of arctigenin on AMPA/kainate receptor activation and 3) to establish its effects on neuronal activity in vivo. Arctigenin inhibited glutamatergic transmission and reduced the evoked field responses. The inhibitory effect of arctigenin on the evoked field responses proved to be substantially dose dependent. Our results indicate that arctigenin exerts its effects under physiological conditions and not only on hyper-excited neurons. F...
The postsynaptic apparatus is associated with binding studies have demonstrated that the oligosac... more The postsynaptic apparatus is associated with binding studies have demonstrated that the oligosaca number of glycoproteins with apparent molecular charide moieties associated with synaptic glycopromasses of 180, 116, and 110 kDa, which are highly con-teins are located within the synaptic cleft (e.g., Matus centrated in and may be uniquely associated with this et al., 1973) where they contribute to the molecular structure. These glycoproteins, purified by concanavalin environment and thereby, potentially, to synaptic func-A lectin-affinity chromatography, showed immunoreactivity in the present study with subunit-specific antibodies to tioning. A knowledge of the glycosylation of individglutamate receptors as follows: GP 180, NMDA receptor ual glycoproteins, therefore, is necessary for a full unsubunits NR2A/NR2B; GP 116, NMDA receptor NA1 derstanding of their molecular function. (1 a); and GP 110, pan-a-amino-3-hydroxy-5-methylisox-Synaptic junctional complexes (SJs) and postsynapazole-4-propionate (pan-AMPA) receptors. Sensitivities tic densities (PSDs) -electron-dense, protein-rich to the glycosidases peptide N-glycosidase F and endostructures underlying the postsynaptic membrane-are /3-N-acetylglucosaminidase H on both western blots and known to be associated with several high-molecularsilver-stained gels suggested that the glutamate recep-weight, concanavalin A (Con A)-binding glycoprotors were at least major constituents of the glycoprotein teins with molecular masses of 110, 116, and 180 kDa bands. Similar detailed glycosylation was observed for all (termed GP 110, GP 116, and GP 180). These are three glycoproteins, with neutral oligosaccharides being highly concentrated in and may be uniquely associated dominant. Oligomannosidic glycans (with from five to nine mannoses) accounted for -~50%of the neutral sug-with the postsynaptic apparatus . Howars, with Man 5 (at almost 20% of the neutral sugars) ever, their complete characterization and function on always the major glycan. Other abundant neutral oligo-the postsynaptic membrane remain to be elucidated. saccharides were of the complex type. Similar sensitivi-A glycoprotein with an apparent molecular mass ties to peptide N-glycosidase F and endo-~3-N-acetylglu-of 116 kDa has been found in the synaptic plasma cosaminidase H were observed for cell line-expressed NMDA receptor subunits, suggesting that irrespective of the glycosylation processing available, the least highly Resubmitted manuscript
The rocker mice are hereditary ataxic mutants that carry a point mutation in the gene encoding th... more The rocker mice are hereditary ataxic mutants that carry a point mutation in the gene encoding the Ca V 2.1 (P ⁄ Q-type) Ca 2+ channel a 1 subunit, and show the mildest symptoms among the reported Ca V 2.1 mutant mice. We studied the basic characteristics of the rocker mutant Ca 2+ channel and their impacts on excitatory synaptic transmission in cerebellar Purkinje cells (PCs). In acutely dissociated PC somas, the rocker mutant channel showed a moderate reduction in Ca 2+ channel current density, whereas its kinetics and voltage dependency of gating remained nearly normal. Despite the small changes in channel function, synaptic transmission in the parallel fiber (PF)-PC synapses was severely impaired. The climbing fiber inputs onto PCs showed a moderate impairment but could elicit normal complex spikes. Presynaptic function of the PF-PC synapses, however, was unexpectedly almost normal in terms of paired-pulse facilitation, sensitivity to extracellular Ca 2+ concentration and glutamate concentration in synaptic clefts. Electron microscopic analyses including freeze-fracture replica labeling revealed that both the number and density of postsynaptic a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors substantially decreased without gross structural changes of the PF-PC synapses. We also observed an abnormal arborization of PC dendrites in young adult rocker mice ( 1 month old). These lines of evidence suggest that even a moderate dysfunction of Ca V 2.1 Ca 2+ channel can cause substantial changes in postsynaptic molecular composition of the PF-PC synapses and dendritic structure of PCs.
In order to define the membrane topology of the GluRl glutamate receptor subunit, we have examine... more In order to define the membrane topology of the GluRl glutamate receptor subunit, we have examined the location of epitopes . Antibodies were produced against peptides corresponding to putative extracellular and intracellular segments of the rat brain GluRl glutamate receptor subunit . Immunocytochemistry at the electron microscopic level in the dentate gyrus of the hippocampal formation showed that epitopes for the antiserum to the N-terminal part of the subunit are located at the extracellular face of the plasma membrane, whereas the antigenic determinants for the antiserum to the C-terminal part are found at the intracellular face of the postsynaptic membrane . Furthermore, antibodies to the N-terminal residues 253-267 reacted similarly with both intact and permeabilized synaptosomes, whereas the binding of antibodies to the C-terminal residues 877-889 increased about 1 .6-fold following permeabilization . Our data suggest that the N-and C-terminal regions are located on the opposite side of the membrane and, therefore, the GluRl subunit probably has an odd number of membrane spanning segments . The antibody cross-reactivities in different species and their effect on ligand binding activity were also established . Key Words : a-Amino-3-hydroxy-5-methyl-4-isoxazole propionate-Antipeptide antibodies-Topology-Hippocampus-Dentategyrus-Immunocytochemistry .
Glutamatergic synapses are the primary source of excitatory transmission in the central nervous s... more Glutamatergic synapses are the primary source of excitatory transmission in the central nervous system (CNS), and their formation is critical in the establishment of neuronal connections. The refinement of these connections occurs during development and also it is postulated during learning and memory. Recent progress in understanding the molecular components of synaptic junctions, together with advances in imaging techniques, has started to offer new insights into the development of excitatory synapses. Studies performed on low-density primary neuronal cultures have enabled dissection of the temporal sequence of events, which have lead to the differentiation of pre- and postsynaptic components. A central feature of the development of excitatory synapses is the accumulation of glutamatergic receptors (GluRs) at the postsynaptic site. These receptors need to be localized and fixed opposite nerve terminals that release glutamate. But for this to occur, neurons require intracellular anchoring molecules, as well as mechanisms that ensure the efficient turnover and transport of receptor proteins. This review focuses on some of the developmental changes observed in the subcellular distribution and molecular organization of AMPA and NMDA type ionotropic GluRs (iGluRs), which mediate the majority of fast excitatory neurotransmission in the CNS.
This Editorial highlights a study by Hunsberger et al. (2015) in the current issue of Journal of ... more This Editorial highlights a study by Hunsberger et al. (2015) in the current issue of Journal of Neurochemistry, in which the authors explore the effects of riluzole (R) treatment on tau-P301L transgenic mice. The authors employed a comprehensive analysis of possible restorative effects of the drug by examining glutamate levels in subregions of the hippocampus, expression of tau and its hyper-phosphorylated forms, and memory function using behavioral tests. The authors report a simultaneous reduction in glutamate reuptake and an increase in glutamate release in the tau-P301L model, both of which are ameliorated with riluzole treatment. The authors' findings have implications for our understanding of synaptic transmission mechanisms also associated with Alzheimer's disease pathology.
We investigated the expression of metabotropic glutamate receptor (mGluR) isoforms in CG-4 rodent... more We investigated the expression of metabotropic glutamate receptor (mGluR) isoforms in CG-4 rodent oligodendroglial progenitor cells (OPC) and rat brain oligodendrocytes. Our RT-PCR analysis detected mRNAs for mGluR3 and mGluR5 isoforms in OPCs. Although neurons express both mGluR5a and mGluR5b splice variants, only mGluR5a was identified in OPCs. Antibodies to mGluR2/3 and mGluR5 detected the corresponding receptor proteins in immunoblots of OPC membrane fractions. Furthermore, immunocytochemical analysis identified mGluR5 in oligodendrocyte marker O4-positive OPCs. The expression of mGluR5 was also demonstrated in oligodendrocyte marker (O4 and O1) positive cells in white matter of postnatal 4- and 7-day-old rat brain sections using immunofluorescent double labelling and confocal microscopy. The mGluR5 receptor function was assessed in CG-4 OPCs with fura-2 microfluorometry. Application of the mGluR1/5 specific agonist (S)-3,5-dihydroxyphenylglycine (DHPG) induced calcium oscillati...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2000
AMPA and NMDA receptors mediate most excitatory synaptic transmission in the CNS. We have develop... more AMPA and NMDA receptors mediate most excitatory synaptic transmission in the CNS. We have developed antibodies that recognize all AMPA or all NMDA receptor variants on the surface of living neurons. AMPA receptor variants were identified with a polyclonal antibody recognizing the conserved extracellular loop region of all four AMPA receptor subunits (GluR1-4, both flip and flop), whereas NMDA receptors were immunolabeled with a polyclonal antibody that binds to an extracellular N-terminal epitope of the NR1 subunit, common to all splice variants. In non-fixed brain sections these antibodies gave labeling patterns similar to autoradiographic distributions with particularly high levels in the hippocampus. Using these antibodies, in conjunction with GluR2-specific and synaptophysin antibodies, we have directly localized and quantified surface-expressed native AMPA and NMDA receptors on cultured living hippocampal neurons during development. Using a quantitative cell ELISA, a dramatic i...
The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry, 2002
Glutamatergic synapses are the primary source of excitatory transmission in the central nervous s... more Glutamatergic synapses are the primary source of excitatory transmission in the central nervous system (CNS), and their formation is critical in the establishment of neuronal connections. The refinement of these connections occurs during development and also it is postulated during learning and memory. Recent progress in understanding the molecular components of synaptic junctions, together with advances in imaging techniques, has started to offer new insights into the development of excitatory synapses. Studies performed on low-density primary neuronal cultures have enabled dissection of the temporal sequence of events, which have lead to the differentiation of pre- and postsynaptic components. A central feature of the development of excitatory synapses is the accumulation of glutamatergic receptors (GluRs) at the postsynaptic site. These receptors need to be localized and fixed opposite nerve terminals that release glutamate. But for this to occur, neurons require intracellular an...
The inhibitory neurotransmitter gamma-aminobutyric acid (GABA), acts at ionotropic (GABA(A) and G... more The inhibitory neurotransmitter gamma-aminobutyric acid (GABA), acts at ionotropic (GABA(A) and GABA(C)) and metabotropic (GABA(B)) receptors. Functional GABA(B) receptors are heterodimers of GABA(B(1)) and GABA(B(2)) subunits. Here we show a robust, direct, and specific interaction between the coiled-coil domain present in the C-terminus of the GABA(B(1)) subunit and the transcription factor ATF4 (also known as CREB2). ATF4 and GABA(B(2)) binding to the GABA(B(1)) subunit were mutually exclusive. In rat hippocampal neurons native GABA(B(1)) showed surprisingly little similarity to GABA(B(2)) in its subcellular distribution. GABA(B(1)) and ATF4, however, were highly colocalized throughout the cell and displayed a punctate distribution within the dendrites. Activation of GABA(B) receptors in hippocampal neurons caused a dramatic translocation of ATF4 out of the nucleus into the cytoplasm. These data suggest a novel neuronal signaling pathway that could regulate the functional express...
The cellular and subcellular localization of the GluRA, GluRB/C and GluRD subunits of the alpha-a... more The cellular and subcellular localization of the GluRA, GluRB/C and GluRD subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate receptor was determined in the rat hippocampus using polyclonal antipeptide antibodies in immunoperoxidase and immunogold procedures. For the localization of the GluRD subunit a new polyclonal antiserum was developed using the C-terminal sequence of the protein (residues 869-881), conjugated to carrier protein and absorbed to colloidal gold for immunization. The purified antibodies immunoprecipitated about 25% of 3[H]AMPA binding activity from the hippocampus, cerebellum or whole brain, but very little from neocortex. These antibodies did not precipitate a significant amount of 3[H]kainate binding activity. The antibodies also recognize the GluRD subunit, but not the other AMPA receptor subunits, when expressed in transfected COS-7 cells and only when permeabilized with detergent, indicating an intracellular epitope. All...
To identify the location of the first transmembrane segment of the GluR1 glutamate receptor subun... more To identify the location of the first transmembrane segment of the GluR1 glutamate receptor subunit artificial stop codons have been introduced into the N-terminal domain at amino acid positions 442, 510, and 563, namely just before and spanning the proposed first two transmembrane regions. The resultant truncated N-terminal fragments of GluR1, termed NT1, NT2, and NT3 respectively were expressed in Cos-7 cells and their cellular distribution and cell-surface expression analysed using an N-terminal antibody to GluR1. All of the fragments were fully glycosylated and were found to be associated with cell membranes but none was secreted. Differential extraction of the cell membranes indicated that both NT1 and NT2 behave as peripheral membrane proteins. In contrast NT3, like the full subunit, has integral membrane protein properties. Furthermore only NT3 is expressed at the cell surface as determined by immunofluorescence and cell-surface biotinylation. Protease protection assays indic...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1994
The cellular and subcellular distribution of the GluR1 subunit of the AMPA-type excitatory amino ... more The cellular and subcellular distribution of the GluR1 subunit of the AMPA-type excitatory amino acid receptor was determined in the cerebellar cortex of rat using immunocytochemistry. Two polyclonal antibodies were raised against the N- and C-terminal regions of the subunit. They both labeled a band in immunoblots of rat cerebellar membranes with a molecular weight corresponding to that predicted for this subunit of 105 kDa molecular mass. In light microscopy the distribution of immunoreactivity for the two antibodies was very similar. The molecular layer was strongly immunoreactive whereas no labeling was observed in the granular layer. Electron microscopy revealed that the antibody raised against the N-terminal part of the subunit recognizes an extracellular epitope(s), whereas the antibody against the C-terminal part recognizes an intracellular epitope(s) along the plasma membrane. In Bergmann glial cells the endoplasmic reticulum, Golgi apparatus, and multivesicular bodies were...
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1992
l. Carp and rabbit sarcoplasmatic reticulum Ca2+-ATPase enzymes were compared with respect to the... more l. Carp and rabbit sarcoplasmatic reticulum Ca2+-ATPase enzymes were compared with respect to their sensitivity to FITC labelling.
Read the full article 'Cdk5/p35 is required for motor coordination and cerebellar plasticity' on ... more Read the full article 'Cdk5/p35 is required for motor coordination and cerebellar plasticity' on page 53. Abbreviations used: AMPAR, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor; CaMKII, Ca 2+ /calmodulin-dependent protein kinase; Cdk5, cyclin-dependent kinase 5; CF, climbing fibre; EPSC, excitatory post-synaptic current; GC, granule cell; GluN2, NMDA receptor subunit; IP3R1, inositol 1,4,5-trisphosphate receptor type 1; LTD, long-term depression; LTP, long-term potentiation; MF, mossy fibre; NMDAR, N-methyl-D-aspartate-type ionotropic glutamate receptor; p35, neuron-specific activator subunit of Cdk5; PC, Purkinje cell; PF, parallel fibre; PSD-95, post-synaptic density protein 95; STEP, striatal-enriched tyrosine phosphatase; VDCC, voltage-dependent Ca 2+ channel.
NMDA receptors (NMDARs) play an important role in neural plasticity including long-term potentiat... more NMDA receptors (NMDARs) play an important role in neural plasticity including long-term potentiation and long-term depression, which are likely to explain their importance for learning and memory. Cognitive decline is a major problem facing an ageing human population, so much so that its reversal has become an important goal for scientific research and pharmaceutical development. Enhancement of NMDAR function is a core strategy toward this goal. In this review we indicate some of the major ways of potentiating NMDAR function by both direct and indirect modulation. There is good evidence that both positive and negative modulation can enhance function suggesting that a subtle approach correcting imbalances in particular clinical situations will be required. Excessive activation and the resultant deleterious effects will need to be carefully avoided. Finally we describe some novel positive allosteric modulators of NMDARs, with some subunit selectivity, and show initial evidence of their ability to affect NMDAR mediated events. This article is part of a Special Issue entitled 'Cognitive Enhancers'.
Lignans are biologically active phenolic compounds related to lignin, produced in different plant... more Lignans are biologically active phenolic compounds related to lignin, produced in different plants. Arctigenin, a dibenzylbutyrolactone-type lignan, has been used as a neuroprotective agent for the treatment of encephalitis. Previous studies of cultured rat cerebral cortical neurones raised the possibility that arctigenin inhibits kainate-induced excitotoxicity. The aims of the present study were: 1) to analyse the effect of arctigenin on normal synaptic activity in ex vivo brain slices, 2) to determine its receptor binding properties and test the effect of arctigenin on AMPA/kainate receptor activation and 3) to establish its effects on neuronal activity in vivo. Arctigenin inhibited glutamatergic transmission and reduced the evoked field responses. The inhibitory effect of arctigenin on the evoked field responses proved to be substantially dose dependent. Our results indicate that arctigenin exerts its effects under physiological conditions and not only on hyper-excited neurons. F...
The postsynaptic apparatus is associated with binding studies have demonstrated that the oligosac... more The postsynaptic apparatus is associated with binding studies have demonstrated that the oligosaca number of glycoproteins with apparent molecular charide moieties associated with synaptic glycopromasses of 180, 116, and 110 kDa, which are highly con-teins are located within the synaptic cleft (e.g., Matus centrated in and may be uniquely associated with this et al., 1973) where they contribute to the molecular structure. These glycoproteins, purified by concanavalin environment and thereby, potentially, to synaptic func-A lectin-affinity chromatography, showed immunoreactivity in the present study with subunit-specific antibodies to tioning. A knowledge of the glycosylation of individglutamate receptors as follows: GP 180, NMDA receptor ual glycoproteins, therefore, is necessary for a full unsubunits NR2A/NR2B; GP 116, NMDA receptor NA1 derstanding of their molecular function. (1 a); and GP 110, pan-a-amino-3-hydroxy-5-methylisox-Synaptic junctional complexes (SJs) and postsynapazole-4-propionate (pan-AMPA) receptors. Sensitivities tic densities (PSDs) -electron-dense, protein-rich to the glycosidases peptide N-glycosidase F and endostructures underlying the postsynaptic membrane-are /3-N-acetylglucosaminidase H on both western blots and known to be associated with several high-molecularsilver-stained gels suggested that the glutamate recep-weight, concanavalin A (Con A)-binding glycoprotors were at least major constituents of the glycoprotein teins with molecular masses of 110, 116, and 180 kDa bands. Similar detailed glycosylation was observed for all (termed GP 110, GP 116, and GP 180). These are three glycoproteins, with neutral oligosaccharides being highly concentrated in and may be uniquely associated dominant. Oligomannosidic glycans (with from five to nine mannoses) accounted for -~50%of the neutral sug-with the postsynaptic apparatus . Howars, with Man 5 (at almost 20% of the neutral sugars) ever, their complete characterization and function on always the major glycan. Other abundant neutral oligo-the postsynaptic membrane remain to be elucidated. saccharides were of the complex type. Similar sensitivi-A glycoprotein with an apparent molecular mass ties to peptide N-glycosidase F and endo-~3-N-acetylglu-of 116 kDa has been found in the synaptic plasma cosaminidase H were observed for cell line-expressed NMDA receptor subunits, suggesting that irrespective of the glycosylation processing available, the least highly Resubmitted manuscript
The rocker mice are hereditary ataxic mutants that carry a point mutation in the gene encoding th... more The rocker mice are hereditary ataxic mutants that carry a point mutation in the gene encoding the Ca V 2.1 (P ⁄ Q-type) Ca 2+ channel a 1 subunit, and show the mildest symptoms among the reported Ca V 2.1 mutant mice. We studied the basic characteristics of the rocker mutant Ca 2+ channel and their impacts on excitatory synaptic transmission in cerebellar Purkinje cells (PCs). In acutely dissociated PC somas, the rocker mutant channel showed a moderate reduction in Ca 2+ channel current density, whereas its kinetics and voltage dependency of gating remained nearly normal. Despite the small changes in channel function, synaptic transmission in the parallel fiber (PF)-PC synapses was severely impaired. The climbing fiber inputs onto PCs showed a moderate impairment but could elicit normal complex spikes. Presynaptic function of the PF-PC synapses, however, was unexpectedly almost normal in terms of paired-pulse facilitation, sensitivity to extracellular Ca 2+ concentration and glutamate concentration in synaptic clefts. Electron microscopic analyses including freeze-fracture replica labeling revealed that both the number and density of postsynaptic a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors substantially decreased without gross structural changes of the PF-PC synapses. We also observed an abnormal arborization of PC dendrites in young adult rocker mice ( 1 month old). These lines of evidence suggest that even a moderate dysfunction of Ca V 2.1 Ca 2+ channel can cause substantial changes in postsynaptic molecular composition of the PF-PC synapses and dendritic structure of PCs.
In order to define the membrane topology of the GluRl glutamate receptor subunit, we have examine... more In order to define the membrane topology of the GluRl glutamate receptor subunit, we have examined the location of epitopes . Antibodies were produced against peptides corresponding to putative extracellular and intracellular segments of the rat brain GluRl glutamate receptor subunit . Immunocytochemistry at the electron microscopic level in the dentate gyrus of the hippocampal formation showed that epitopes for the antiserum to the N-terminal part of the subunit are located at the extracellular face of the plasma membrane, whereas the antigenic determinants for the antiserum to the C-terminal part are found at the intracellular face of the postsynaptic membrane . Furthermore, antibodies to the N-terminal residues 253-267 reacted similarly with both intact and permeabilized synaptosomes, whereas the binding of antibodies to the C-terminal residues 877-889 increased about 1 .6-fold following permeabilization . Our data suggest that the N-and C-terminal regions are located on the opposite side of the membrane and, therefore, the GluRl subunit probably has an odd number of membrane spanning segments . The antibody cross-reactivities in different species and their effect on ligand binding activity were also established . Key Words : a-Amino-3-hydroxy-5-methyl-4-isoxazole propionate-Antipeptide antibodies-Topology-Hippocampus-Dentategyrus-Immunocytochemistry .
Glutamatergic synapses are the primary source of excitatory transmission in the central nervous s... more Glutamatergic synapses are the primary source of excitatory transmission in the central nervous system (CNS), and their formation is critical in the establishment of neuronal connections. The refinement of these connections occurs during development and also it is postulated during learning and memory. Recent progress in understanding the molecular components of synaptic junctions, together with advances in imaging techniques, has started to offer new insights into the development of excitatory synapses. Studies performed on low-density primary neuronal cultures have enabled dissection of the temporal sequence of events, which have lead to the differentiation of pre- and postsynaptic components. A central feature of the development of excitatory synapses is the accumulation of glutamatergic receptors (GluRs) at the postsynaptic site. These receptors need to be localized and fixed opposite nerve terminals that release glutamate. But for this to occur, neurons require intracellular anchoring molecules, as well as mechanisms that ensure the efficient turnover and transport of receptor proteins. This review focuses on some of the developmental changes observed in the subcellular distribution and molecular organization of AMPA and NMDA type ionotropic GluRs (iGluRs), which mediate the majority of fast excitatory neurotransmission in the CNS.
This Editorial highlights a study by Hunsberger et al. (2015) in the current issue of Journal of ... more This Editorial highlights a study by Hunsberger et al. (2015) in the current issue of Journal of Neurochemistry, in which the authors explore the effects of riluzole (R) treatment on tau-P301L transgenic mice. The authors employed a comprehensive analysis of possible restorative effects of the drug by examining glutamate levels in subregions of the hippocampus, expression of tau and its hyper-phosphorylated forms, and memory function using behavioral tests. The authors report a simultaneous reduction in glutamate reuptake and an increase in glutamate release in the tau-P301L model, both of which are ameliorated with riluzole treatment. The authors' findings have implications for our understanding of synaptic transmission mechanisms also associated with Alzheimer's disease pathology.
We investigated the expression of metabotropic glutamate receptor (mGluR) isoforms in CG-4 rodent... more We investigated the expression of metabotropic glutamate receptor (mGluR) isoforms in CG-4 rodent oligodendroglial progenitor cells (OPC) and rat brain oligodendrocytes. Our RT-PCR analysis detected mRNAs for mGluR3 and mGluR5 isoforms in OPCs. Although neurons express both mGluR5a and mGluR5b splice variants, only mGluR5a was identified in OPCs. Antibodies to mGluR2/3 and mGluR5 detected the corresponding receptor proteins in immunoblots of OPC membrane fractions. Furthermore, immunocytochemical analysis identified mGluR5 in oligodendrocyte marker O4-positive OPCs. The expression of mGluR5 was also demonstrated in oligodendrocyte marker (O4 and O1) positive cells in white matter of postnatal 4- and 7-day-old rat brain sections using immunofluorescent double labelling and confocal microscopy. The mGluR5 receptor function was assessed in CG-4 OPCs with fura-2 microfluorometry. Application of the mGluR1/5 specific agonist (S)-3,5-dihydroxyphenylglycine (DHPG) induced calcium oscillati...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2000
AMPA and NMDA receptors mediate most excitatory synaptic transmission in the CNS. We have develop... more AMPA and NMDA receptors mediate most excitatory synaptic transmission in the CNS. We have developed antibodies that recognize all AMPA or all NMDA receptor variants on the surface of living neurons. AMPA receptor variants were identified with a polyclonal antibody recognizing the conserved extracellular loop region of all four AMPA receptor subunits (GluR1-4, both flip and flop), whereas NMDA receptors were immunolabeled with a polyclonal antibody that binds to an extracellular N-terminal epitope of the NR1 subunit, common to all splice variants. In non-fixed brain sections these antibodies gave labeling patterns similar to autoradiographic distributions with particularly high levels in the hippocampus. Using these antibodies, in conjunction with GluR2-specific and synaptophysin antibodies, we have directly localized and quantified surface-expressed native AMPA and NMDA receptors on cultured living hippocampal neurons during development. Using a quantitative cell ELISA, a dramatic i...
The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry, 2002
Glutamatergic synapses are the primary source of excitatory transmission in the central nervous s... more Glutamatergic synapses are the primary source of excitatory transmission in the central nervous system (CNS), and their formation is critical in the establishment of neuronal connections. The refinement of these connections occurs during development and also it is postulated during learning and memory. Recent progress in understanding the molecular components of synaptic junctions, together with advances in imaging techniques, has started to offer new insights into the development of excitatory synapses. Studies performed on low-density primary neuronal cultures have enabled dissection of the temporal sequence of events, which have lead to the differentiation of pre- and postsynaptic components. A central feature of the development of excitatory synapses is the accumulation of glutamatergic receptors (GluRs) at the postsynaptic site. These receptors need to be localized and fixed opposite nerve terminals that release glutamate. But for this to occur, neurons require intracellular an...
The inhibitory neurotransmitter gamma-aminobutyric acid (GABA), acts at ionotropic (GABA(A) and G... more The inhibitory neurotransmitter gamma-aminobutyric acid (GABA), acts at ionotropic (GABA(A) and GABA(C)) and metabotropic (GABA(B)) receptors. Functional GABA(B) receptors are heterodimers of GABA(B(1)) and GABA(B(2)) subunits. Here we show a robust, direct, and specific interaction between the coiled-coil domain present in the C-terminus of the GABA(B(1)) subunit and the transcription factor ATF4 (also known as CREB2). ATF4 and GABA(B(2)) binding to the GABA(B(1)) subunit were mutually exclusive. In rat hippocampal neurons native GABA(B(1)) showed surprisingly little similarity to GABA(B(2)) in its subcellular distribution. GABA(B(1)) and ATF4, however, were highly colocalized throughout the cell and displayed a punctate distribution within the dendrites. Activation of GABA(B) receptors in hippocampal neurons caused a dramatic translocation of ATF4 out of the nucleus into the cytoplasm. These data suggest a novel neuronal signaling pathway that could regulate the functional express...
The cellular and subcellular localization of the GluRA, GluRB/C and GluRD subunits of the alpha-a... more The cellular and subcellular localization of the GluRA, GluRB/C and GluRD subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate receptor was determined in the rat hippocampus using polyclonal antipeptide antibodies in immunoperoxidase and immunogold procedures. For the localization of the GluRD subunit a new polyclonal antiserum was developed using the C-terminal sequence of the protein (residues 869-881), conjugated to carrier protein and absorbed to colloidal gold for immunization. The purified antibodies immunoprecipitated about 25% of 3[H]AMPA binding activity from the hippocampus, cerebellum or whole brain, but very little from neocortex. These antibodies did not precipitate a significant amount of 3[H]kainate binding activity. The antibodies also recognize the GluRD subunit, but not the other AMPA receptor subunits, when expressed in transfected COS-7 cells and only when permeabilized with detergent, indicating an intracellular epitope. All...
To identify the location of the first transmembrane segment of the GluR1 glutamate receptor subun... more To identify the location of the first transmembrane segment of the GluR1 glutamate receptor subunit artificial stop codons have been introduced into the N-terminal domain at amino acid positions 442, 510, and 563, namely just before and spanning the proposed first two transmembrane regions. The resultant truncated N-terminal fragments of GluR1, termed NT1, NT2, and NT3 respectively were expressed in Cos-7 cells and their cellular distribution and cell-surface expression analysed using an N-terminal antibody to GluR1. All of the fragments were fully glycosylated and were found to be associated with cell membranes but none was secreted. Differential extraction of the cell membranes indicated that both NT1 and NT2 behave as peripheral membrane proteins. In contrast NT3, like the full subunit, has integral membrane protein properties. Furthermore only NT3 is expressed at the cell surface as determined by immunofluorescence and cell-surface biotinylation. Protease protection assays indic...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1994
The cellular and subcellular distribution of the GluR1 subunit of the AMPA-type excitatory amino ... more The cellular and subcellular distribution of the GluR1 subunit of the AMPA-type excitatory amino acid receptor was determined in the cerebellar cortex of rat using immunocytochemistry. Two polyclonal antibodies were raised against the N- and C-terminal regions of the subunit. They both labeled a band in immunoblots of rat cerebellar membranes with a molecular weight corresponding to that predicted for this subunit of 105 kDa molecular mass. In light microscopy the distribution of immunoreactivity for the two antibodies was very similar. The molecular layer was strongly immunoreactive whereas no labeling was observed in the granular layer. Electron microscopy revealed that the antibody raised against the N-terminal part of the subunit recognizes an extracellular epitope(s), whereas the antibody against the C-terminal part recognizes an intracellular epitope(s) along the plasma membrane. In Bergmann glial cells the endoplasmic reticulum, Golgi apparatus, and multivesicular bodies were...
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1992
l. Carp and rabbit sarcoplasmatic reticulum Ca2+-ATPase enzymes were compared with respect to the... more l. Carp and rabbit sarcoplasmatic reticulum Ca2+-ATPase enzymes were compared with respect to their sensitivity to FITC labelling.
Read the full article 'Cdk5/p35 is required for motor coordination and cerebellar plasticity' on ... more Read the full article 'Cdk5/p35 is required for motor coordination and cerebellar plasticity' on page 53. Abbreviations used: AMPAR, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor; CaMKII, Ca 2+ /calmodulin-dependent protein kinase; Cdk5, cyclin-dependent kinase 5; CF, climbing fibre; EPSC, excitatory post-synaptic current; GC, granule cell; GluN2, NMDA receptor subunit; IP3R1, inositol 1,4,5-trisphosphate receptor type 1; LTD, long-term depression; LTP, long-term potentiation; MF, mossy fibre; NMDAR, N-methyl-D-aspartate-type ionotropic glutamate receptor; p35, neuron-specific activator subunit of Cdk5; PC, Purkinje cell; PF, parallel fibre; PSD-95, post-synaptic density protein 95; STEP, striatal-enriched tyrosine phosphatase; VDCC, voltage-dependent Ca 2+ channel.
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