RESUMO
The early effects of deafferentation on the postsynaptic membrane beneath the end bulb of Held in the anteroventral cochlear nucleus (AVCN) were studied with the freeze-fracture technique. Three distinct responses were seen on the external membrane leaflet after cochlear ablation. Within 12 h the number of nonaggregate particles increased 147% by the addition of new particles to the membrane. The increase in number of nonaggregate particles continued until 4 days after cochlear ablation. The other responses occurred later, after degenerative changes were present in the end bulb. Between 1 and 2 days after cochlear ablation, the number of perisynaptic aggregates surrounding the postsynaptic active zone decreased to 10% of normal numbers. By 4 days, all perisynaptic aggregates had disappeared from the membrane. Coated vesicles may be involved in removing these aggregates. Between 1 and 3 days, the number of junctional aggregates decreased, but the size of the aggregates increased, apparently as a result of coalescence of nearby junctional aggregates. The total number of particles in junctional aggregates in the membrane was not altered during the first 6 days after cochlear ablation. The three separate responses suggest the existence of at least three different types of intramembranous particles on the external leaflet of the principal cell membrane, with each type dependent upon different cues for its maintenance in the membrane.
Assuntos
Nervo Coclear/fisiologia , Sinapses/ultraestrutura , Membranas Sinápticas/ultraestrutura , Vias Aferentes , Animais , Membrana Celular/ultraestrutura , Denervação , Técnica de Fratura por Congelamento , Cobaias , Fatores de TempoRESUMO
When kainic acid, a putative neurotoxin for neurons with glutamatergic input, is injected into the brainstem, it produces a selective pattern of degeneration in the cochlear nucleus. The rate and extent of degeneration is correlated with the distribution of the primary auditory fibers. This evidence supports the hypothesis that glutamate is the neurotransmitter for primary auditory fibers.
Assuntos
Tronco Encefálico/efeitos dos fármacos , Ácido Caínico/farmacologia , Pirrolidinas/farmacologia , Receptores de Neurotransmissores/efeitos dos fármacos , Nervo Vestibulococlear/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Glutamatos/fisiologia , Cobaias , Masculino , Degeneração Neural/efeitos dos fármacos , Neurotransmissores/fisiologia , Nervo Vestibulococlear/fisiologiaRESUMO
To monitor changes in alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor distribution in living neurons, the AMPA receptor subunit GluR1 was tagged with green fluorescent protein (GFP). This protein (GluR1-GFP) was functional and was transiently expressed in hippocampal CA1 neurons. In dendrites visualized with two-photon laser scanning microscopy or electron microscopy, most of the GluR1-GFP was intracellular, mimicking endogenous GluR1 distribution. Tetanic synaptic stimulation induced a rapid delivery of tagged receptors into dendritic spines as well as clusters in dendrites. These postsynaptic trafficking events required synaptic N-methyl-D-aspartate (NMDA) receptor activation and may contribute to the enhanced AMPA receptor-mediatedtransmission observed during long-term potentiation and activity-dependent synaptic maturation.
Assuntos
Dendritos/metabolismo , Plasticidade Neuronal , Neurônios/fisiologia , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Sinapses/fisiologia , Animais , Células Cultivadas , Dendritos/ultraestrutura , Estimulação Elétrica , Hipocampo/citologia , Hipocampo/fisiologia , Humanos , Potenciação de Longa Duração , Técnicas de Cultura de Órgãos , Ratos , Agregação de Receptores , Proteínas Recombinantes de Fusão/metabolismo , Sinapses/metabolismo , Transmissão Sináptica , TetaniaRESUMO
The objective of the present study was to determine if a neuron that expresses multiple glutamate receptors targets the same receptors to all glutamatergic postsynaptic populations, or if the receptors are differentially targeted to specific postsynaptic populations. As a model for this study, we chose the fusiform cell of the dorsal cochlear nucleus. This neuron expresses multiple glutamate receptors and receives two distinct glutamatergic inputs: parallel fibers synapse on apical dendrites, and auditory nerve fibers synapse on basal dendrites. Pre- and postembedding immunocytochemistry were combined with retrograde tracing to identify the receptors expressed on postsynaptic membranes of parallel fiber and auditory nerve synapses. Most receptors were found at both populations of synapses, but the AMPA receptor subunit, GluR4, and the metabotropic receptor, mGluR1 alpha, were found only at the auditory nerve synapse. These results demonstrate that glutamate receptors are targeted to specific postsynaptic populations of glutamatergic synapses.
Assuntos
Núcleo Coclear/metabolismo , Neurônios/metabolismo , Receptores de Glutamato/metabolismo , Membranas Sinápticas/metabolismo , Animais , Mapeamento Encefálico , Compartimento Celular , Núcleo Coclear/citologia , Imuno-Histoquímica , Ratos , Ratos Sprague-DawleyRESUMO
The NMDA receptor NR1 subunit has four splice variants that differ in their C-terminal, cytoplasmic domain. We investigated the contribution of the C-terminal cassettes, C0, C1, C2, and C2', to trafficking of NR1 in heterologous cells and neurons. We identified an ER retention signal (RRR) in the C1 cassette of NR1, which is similar to the RXR motif in ATP-sensitive K(+) channels (Zerangue et al., 1999). We found that surface expression of NR1-3, which contains C1, is due to a site on the C2' cassette, which includes the terminal 4 amino acid PDZ-interacting domain. This site suppresses ER retention of the C1 cassette and leads to surface expression. These findings suggest a role for PDZ proteins in facilitating the transition of receptors from an intracellular pool to the surface of the neuron.
Assuntos
Processamento Alternativo/genética , Retículo Endoplasmático/metabolismo , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Motivos de Aminoácidos/genética , Animais , Membrana Celular/metabolismo , Células Cultivadas , Células HeLa , Humanos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologiaRESUMO
RNA editing and subunit assembly of ionotropic glutamate receptors (GluRs) were examined in an oligodendrocyte progenitor cell line, CG4, which expresses GluR2-GluR4, GluR6, GluR7, KA1, and KA2. AMPA-evoked currents rapidly desensitize, whereas kainate-evoked currents contain a steady-state component with a nearly linear current-voltage relation and a fast desensitizing component that is inwardly rectifying. The Q/R site is edited > 95% to the arginine codon in GluR2(Q607) mRNA, and < 5% in GluR6(Q621) mRNA. Immunoprecipitation experiments demonstrate that GluR6 and/or GluR7 subunits assemble with KA2, but not with GluR2-GluR4. These results indicate that oligodendrocyte progenitor cells selectively edit and assemble glutamate receptors into at least two functionally and structurally distinct heteromeric channels.
Assuntos
Oligodendroglia/fisiologia , Edição de RNA , Receptores de Glutamato/genética , Animais , Arginina , Sequência de Bases , Linhagem Celular , Córtex Cerebral/citologia , Códon , Condutividade Elétrica , Glutamatos/farmacologia , Ácido Glutâmico , Humanos , Técnicas de Imunoadsorção , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Ácido Caínico/farmacologia , Dados de Sequência Molecular , Oligodendroglia/efeitos dos fármacos , Reação em Cadeia da Polimerase , RNA/química , Splicing de RNA , Ratos , Receptores de Glutamato/fisiologia , Células-Tronco/fisiologia , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
Homer is a neuronal immediate early gene (IEG) that is enriched at excitatory synapses and binds group 1 metabotropic glutamate receptors (mGluRs). Here, we characterize a family of Homer-related proteins derived from three distinct genes. Like Homer IEG (now termed Homer 1a), all new members bind group 1 mGluRs. In contrast to Homer 1a, new members are constitutively expressed and encode a C-terminal coiled-coil (CC) domain that mediates self-multimerization. CC-Homers form natural complexes that cross-link mGluRs and are enriched at the postsynaptic density. Homer 1a does not multimerize and blocks the association of mGluRs with CC-Homer complexes. These observations support a model in which the dynamic expression of Homer 1a competes with constitutively expressed CC-Homers to modify synaptic mGluR properties.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/fisiologia , Receptores de Glutamato Metabotrópico/metabolismo , Sinapses/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Arcabouço Homer , Camundongos , Dados de Sequência Molecular , Família Multigênica/genética , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/química , Neuropeptídeos/genética , Ratos , Distribuição TecidualRESUMO
Although synaptic AMPA receptors have been shown to rapidly internalize, synaptic NMDA receptors are reported to be static. It is not certain whether NMDA receptor stability at synaptic sites is an inherent property of the receptor, or is due to stabilization by scaffolding proteins. In this study, we demonstrate that NMDA receptors are internalized in both heterologous cells and neurons, and we define an internalization motif, YEKL, on the distal C-terminus of NR2B. In addition, we show that the synaptic protein PSD-95 inhibits NR2B-mediated internalization, and that deletion of the PDZ-binding domain of NR2B increases internalization in neurons. This suggests an involvement for PSD-95 in NMDA receptor regulation and an explanation for NMDA receptor stability at synaptic sites.
Assuntos
Sistema Nervoso Central/metabolismo , Endocitose/fisiologia , Neurônios/metabolismo , Transporte Proteico/fisiologia , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Membranas Sinápticas/metabolismo , Motivos de Aminoácidos/fisiologia , Animais , Sítios de Ligação/fisiologia , Sistema Nervoso Central/ultraestrutura , Clatrina/metabolismo , Proteína 4 Homóloga a Disks-Large , Feto , Células HeLa/citologia , Células HeLa/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Proteínas do Tecido Nervoso/metabolismo , Neurônios/ultraestrutura , Estrutura Terciária de Proteína/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/ultraestrutura , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Membranas Sinápticas/ultraestruturaRESUMO
Early in postnatal development, glutamatergic synapses transmit primarily through NMDA receptors. As development progresses, synapses acquire AMPA receptor function. The molecular basis of these physiological observations is not known. Here we examined single excitatory synapses with immunogold electron-microscopic analysis of AMPA and NMDA receptors along with electrophysiological measurements. Early in postnatal development, a significant fraction of excitatory synapses had NMDA receptors and lacked AMPA receptors. As development progressed, synapses acquired AMPA receptors with little change in NMDA receptor number. Thus, synapses with NMDA receptors but no AMPA receptors can account for the electrophysiologically observed 'silent synapse'.
Assuntos
Envelhecimento/metabolismo , Animais Recém-Nascidos/metabolismo , Hipocampo/metabolismo , Receptores de AMPA/metabolismo , Sinapses/fisiologia , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Eletrofisiologia , Hipocampo/crescimento & desenvolvimento , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
The regulation of AMPA receptors at the postsynaptic membrane is a fundamental component of synaptic plasticity. In the hippocampus, the induction of long-term potentiation increases the delivery of GluR1, a major AMPA receptor subunit in hippocampal pyramidal neurons, to the synaptic plasma membrane through a mechanism that requires the PDZ binding domain of GluR1. Synapse-associated protein 97 (SAP97), a member of the membrane-associated guanylate kinase family, is believed to associate with AMPA receptors (AMPARs) containing the GluR1 subunit, but the functional significance of these interactions is unclear. We investigated the interaction of GluR1 with SAP97, the only PDZ protein known to interact with GluR1. We find that interactions involving SAP97 and GluR1 occur early in the secretory pathway, while the receptors are in the endoplasmic reticulum or cis-Golgi. In contrast, few synaptic receptors associate with SAP97, suggesting that SAP97 dissociates from the receptor complex at the plasma membrane. We also show that internalization of GluR1, as triggered by NMDAR activation, does not require SAP97. These results implicate GluR1-SAP97 interactions in mechanisms underlying AMPA receptor targeting.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Receptores de AMPA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Biotinilação , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Córtex Cerebral/química , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Hipocampo/química , Hipocampo/citologia , Hipocampo/metabolismo , Imuno-Histoquímica , Proteínas de Membrana , Modelos Neurológicos , Neurônios/metabolismo , Neurônios/ultraestrutura , Testes de Precipitina , Ratos , Ratos Sprague-Dawley , Frações Subcelulares/metabolismo , Sinapses/metabolismo , Sinapses/ultraestruturaRESUMO
The membrane-associated guanylate kinases [Chapsyn-110/postsynaptic density-93 (PSD-93), synapse-associated protein-90 (SAP-90)/PSD-95, and SAP-102] are believed to cluster and anchor NMDA receptors at the synapse and to play a role in signal transduction. We have investigated the developmental changes in expression of these proteins in rat hippocampus using biochemical analyses and quantitative immunogold electron microscopy. At postnatal day 2 (P2), SAP-102 was highly expressed, whereas PSD-93 and PSD-95 were low. SAP-102 expression increased during the first week, stayed stable through P35, and showed a reduced expression at 6 months. From P2 through 6 months, PSD-93 and PSD-95 increased. For PSD-95, the percent of labeled synapses increased almost threefold with age, whereas the number of gold particles per labeled synapse did not change significantly, suggesting that the increase in PSD-95 is attributable primarily to an increase in the number of synapses containing PSD-95. In contrast, for SAP-102, both percent labeled synapses and the number of gold particles per labeled synapse decreased during this time. From Western blots of hippocampus and immunogold analysis of CA1 synapses, the high expression of NR2B at P2 coincides with the high level of SAP-102 at synapses, whereas the later expression of NR2A coincides with that of PSD-93 and PSD-95. To determine whether the changes in PSD-93/95 and SAP-102 reflect preferred associations with NR2A and NR2B, respectively, we measured co-immunoprecipitation in the adult hippocampus. These studies suggest that there is a preference for complexes of NR2A/PSD-93/95 and NR2B/SAP-102. These results indicate that individual receptor-associated proteins may have specific functions that are critical to synapse development.
Assuntos
Envelhecimento/metabolismo , Hipocampo/metabolismo , Proteínas Nucleares , Núcleosídeo-Fosfato Quinase/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Fatores de Transcrição , Animais , Western Blotting , Linhagem Celular , Proteína 4 Homóloga a Disks-Large , Guanilato Quinases , Hipocampo/crescimento & desenvolvimento , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/metabolismo , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/metabolismoRESUMO
The glutamate receptor subunit delta2 has a unique distribution at the parallel fiber-Purkinje cell synapse of the cerebellum, which is developmentally regulated such that delta2 occurs at both parallel fiber synapses and climbing fiber synapses early in development but is restricted to parallel fiber synapses in adult animals. To identify proteins that might be involved in the trafficking or docking of delta2 receptors, we screened a yeast two-hybrid library with the cytosolic C terminus of delta2 and isolated a member of the postsynaptic density (PSD)-95 family of proteins, which are known to interact with the extreme C termini of NMDA receptors. We find that delta2 binds specifically to PSD-93, which is enriched in Purkinje cells. In addition, PSD-93 clusters delta2 when they are coexpressed in heterologous cells, and clustering is disrupted by point mutations of delta2 that disrupt the delta2-PSD-93 interaction. Ultrastructural localization of PSD-93 and delta2 shows they are colocalized at parallel fiber synapses; however, PSD-93 also is present at climbing fiber synapses of the adult rat, where delta2 is not found, indicating that the presence of PSD-93 alone is not sufficient for determining the synaptic expression of delta2.
Assuntos
Fibras Nervosas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Glutamato/metabolismo , Sinapses/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Linhagem Celular , Proteína 4 Homóloga a Disks-Large , Biblioteca Gênica , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , RatosRESUMO
Membrane-associated guanylate kinases (MAGUKs) are abundant postsynaptic density (PSD)-95/discs large/zona occludens-1 (PDZ)-containing proteins that can assemble receptors and associated signaling enzymes at sites of cell-cell contact, including synapses. PSD-93, a postsynaptic neuronal MAGUK, has three PDZ domains that can bind to specific ion channels, including NMDA delta2 type glutamate receptors, as well as Shaker and inward rectifier type K(+) channels, and can mediate clustering of these channels in heterologous cells. Genetic analyses of Drosophila show that MAGUKs play critical roles in synaptic development because mutations of discs large disrupt the subsynaptic reticulum and block postsynaptic clustering of Shaker K(+) channels. It is uncertain whether MAGUKs play an essential role in the development of central synapses. There are four neuronal MAGUKs with overlapping expression patterns in the mammalian brain; however, we find PSD-93 is the only MAGUK expressed in cerebellar Purkinje neurons. Therefore, we targeted disruption of PSD-93 in mouse. Despite the absence of MAGUK immunoreactivity in Purkinje neurons from the knock-outs, these mice have no structural or functional abnormality in cerebellum. Both the dendritic architecture and the postsynaptic localization of PSD-93 interacting proteins remain intact at light and electron microscopic levels in the knock-outs. Postsynaptic Purkinje cell responses, monosynaptic climbing fiber innervation, and cerebellar-dependent behaviors are also normal. Our data demonstrate that MAGUK proteins of the PSD-93/95 family are not essential for development of certain central synapses but may instead participate in specialized aspects of synaptic signaling and plasticity.
Assuntos
Cerebelo/metabolismo , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Núcleosídeo-Fosfato Quinase/metabolismo , Sinapses/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Comportamento Animal , Cerebelo/citologia , Cerebelo/embriologia , Proteína 1 Homóloga a Discs-Large , Proteína 4 Homóloga a Disks-Large , Expressão Gênica , Marcação de Genes , Guanilato Quinases , Imuno-Histoquímica , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neuropeptídeos/metabolismo , Técnicas de Patch-Clamp , Células de Purkinje/citologia , Células de Purkinje/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recombinação Genética , Sinapses/genética , Sinapses/ultraestruturaRESUMO
The electrophysiological effects of N-acetylaspartylglutamate (NAAG), an endogenous peptide restrictively distributed in the central nervous system, were studied using Xenopus oocytes injected with RNAs transcribed from cloned glutamate receptor cDNAs. NAAG induced an inward current, dose dependently, in oocytes injected with RNA for an N-methyl-D-aspartate receptor subunit (NMDAR1). In contrast, the oocytes injected with RNAs for AMPA-selective glutamate receptors (GluR1, GluR3, GluR1+GluR2 and GluR2+GluR3) scarcely responded to NAAG, and the oocytes injected with RNA for kainate receptor (GluR6) did not respond to NAAG. The half-maximal response (ED50) value of NAAG on expressed NMDAR1 was 185 microM, which shows that NAAG is about 115-times less potent than L-glutamate (Glu), the ED50 of which value was 1.6 microM. The maximal current amplitude induced by NAAG was about 70% of that by Glu. NAAG-induced current in NMDAR1-injected oocytes was potentiated by glycine, dose-dependently antagonized by DL-2-amino-5-phosphonovaleric acid, and blocked by magnesium ions in a voltage-dependent fashion. These results suggest that NAAG is one of the endogenous agonists selective for NMDAR1.
Assuntos
Dipeptídeos/farmacologia , Oócitos/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , 2-Amino-5-fosfonovalerato/farmacologia , Animais , Clonagem Molecular , Relação Dose-Resposta a Droga , Feminino , Glutamatos/farmacologia , Ácido Glutâmico , Ácido Caínico/farmacologia , Cinética , Potenciais da Membrana/efeitos dos fármacos , N-Metilaspartato/farmacologia , Oócitos/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Transcrição Gênica , Xenopus laevisRESUMO
Antibodies were made to a thirteen amino acid synthetic peptide corresponding to the C-terminal portion of the glutamate (glu) receptor, GluR-A. The immunoprecipitation of kainic acid (KA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) binding sites by the anti-peptide antibodies was studied using a detergent-solubilized preparation of rat brain membranes. Under these conditions a subpopulation of AMPA binding sites was recognized by the antibodies, but no KA binding sites were recognized. Scatchard analysis of this subpopulation of AMPA binding sites yields a curvilinear plot which fits a two-site model with dissociation constants of 4.6 and 323 nM. These studies show that the glu receptor complex, GluR-A, binds AMPA but not KA and suggest that (i) the binding sites for these two ligands reside on different proteins, and (ii) the KA receptor identified physiologically is not equivalent to the KA binding sites identified with 3H-labelled KA.
Assuntos
Ácido Ibotênico/análogos & derivados , Ácido Caínico/metabolismo , Receptores de Neurotransmissores/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos , Sítios de Ligação , Ligação Competitiva , Encéfalo/metabolismo , Membrana Celular/metabolismo , Glutamatos/metabolismo , Ácido Ibotênico/metabolismo , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Peptídeos/síntese química , Ratos , Receptores de Glutamato , Receptores de Neurotransmissores/imunologia , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol PropiônicoRESUMO
The gene encoding chick kainate-binding protein (c-KBP), a member of the non-NMDA ionotropic glutamate receptor family has been isolated and characterized. The c-KBP gene is at least 13 kilobases long and contains 11 exons interrupted by 10 introns. Primer extension and RNase protection studies identified a major transcription initiation site located 117 bases upstream from the initiation methionine codon ATG. Consensus TATA and CCAAT sequences were detected in the putative promoter region. The structure of the c-KBP gene is strikingly different from that of other members of neurotransmitter-gated ion-channels (cloned at present) although the topology of c-KBP consists of four membrane-spanning domains, a structural characteristic of ionotropic receptor subunits. The c-KBP gene was found to be expressed at high levels in chick cerebellar Bergmann glia and at extremely low levels in the forebrain. The limited expression of the c-KBP gene raises important questions concerning the mechanisms governing the regulation of c-KBP gene transcription.
Assuntos
Encéfalo/metabolismo , Ácido Caínico/metabolismo , Receptores de Neurotransmissores/genética , Animais , Sequência de Bases , Cerebelo/metabolismo , Galinhas , DNA , Éxons , Regulação da Expressão Gênica , Íntrons , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Especificidade de Órgãos/genética , Receptores de Ácido Caínico , Receptores de Neurotransmissores/metabolismo , Transcrição GênicaRESUMO
Since four AMPA-type excitatory amino acid receptor subunits have been cloned recently, it is now possible to localize these important molecules in the nervous system. A comprehensive study of AMPA receptor immunocytochemistry was carried out on vibratome sections of rat brain, which were immunolabeled with antibodies made against peptides corresponding to the C-terminal portions of AMPA-receptor subunits: GluR1, GluR2/3, and GluR4. Labeling was most prominent in forebrain structures such as the olfactory bulb and tubercle, septal nuclei, amygdaloid complex, hippocampus, induseum griseum, habenula, and interpeduncular nucleus, and in the cerebellum. Different patterns of immunolabeling were evident with the antibodies to the four subunits, with marked contrast between densely and lightly stained structures with antibody to GluR1, widespread dense staining with antibody to GluR2/3, and moderate staining with antibody to GluR4. In the parietal cortex, some non-pyramidal neurons were more densely stained than pyramidal cells with antibodies to GluR1. Neurons of the main olfactory bulb, other than granule cells, were most densely stained with antibody to GluR1. In the cerebellum, Bergmann glia were densely stained with antibodies to GluR1 and 4, while neurons, other than granule cells, were most densely stained with antibody to GluR2/3. Immunolabeling patterns of all antibodies were consistent with that of previous in situ hybridization histochemistry studies and with the overall pattern of 3H-AMPA binding. Electron microscopy of thin sections taken from immunolabeled vibratome sections of hippocampus and cerebral cortex showed staining which was restricted mainly to postsynaptic densities and adjacent dendritoplasm, and to neuron cell body cytoplasm. We saw no convincing examples of stained presynaptic terminals, and only limited evidence of glial staining, excepting Bergmann glia.
Assuntos
Encéfalo/anatomia & histologia , Ácido Ibotênico/análogos & derivados , Receptores de Neurotransmissores/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Northern Blotting , Encéfalo/ultraestrutura , Química Encefálica/efeitos dos fármacos , Química Encefálica/fisiologia , Eletroforese em Gel de Poliacrilamida , Ácido Ibotênico/farmacologia , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Ratos , Ratos Endogâmicos , Receptores de AMPA , Receptores de Glutamato , Receptores de Neurotransmissores/ultraestrutura , Inclusão do Tecido , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol PropiônicoRESUMO
The present study provides a survey of the immunolocalization of ionotropic glutamate receptor subunits throughout the rat and cat cerebellar cortex, with emphasis on the unipolar brush cell (UBC), a hitherto neglected cerebellar cell that is densely concentrated in the granular layer of the vestibulocerebellum and that forms giant synapses with mossy fibers. An array of nine previously characterized antibodies has been used, each of which stained a characteristic profile of cerebellar cells. The UBCs of both rat and cat were strongly immunostained by an antibody against the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptor subunits, GluR2 and GluR3; were moderately immunostained by a monoclonal antibody to kainate receptor subunits, GluR5/6/7; were weakly immunostained by antibodies to NR1 subunits; and were not stained by antibodies to GluR1, GluR4, GluR6/7, KA-2, and NR2A/B. Postsynaptic densities of the giant mossy fiber-UBC synapses were GluR2/3, GluR5/6/7, and NR1 immunoreactive. The other cerebellar neurons were all immunolabeled to some extent with the GluR2/3 and NR1 antibodies. In addition, Purkinje cells were immunopositive for GluR1 and GluR5/6/7; granule cells were immunopositive for GluR5/6/7, GluR6/7, KA-2, and NR2A/B. The Golgi-Bergmann glia was densely stained by GluR1 and GluR4 antibodies, whereas astrocytes of the granular layer were stained by the GluR4 antiserum. Data provided herein may guide further electrophysiological and pharmacological studies of cerebellar cells in general and the UBCs in particular.
Assuntos
Gatos/metabolismo , Córtex Cerebelar/química , Fibras Nervosas/química , Ratos Sprague-Dawley/metabolismo , Receptores de Glutamato/análise , Sinapses/química , Vias Aferentes , Animais , Córtex Cerebelar/citologia , Feminino , Imuno-Histoquímica , Masculino , Microscopia , Microscopia Eletrônica , Ratos , Receptores de AMPA/análise , Receptores de Ácido Caínico/análise , Receptores de N-Metil-D-Aspartato/análiseRESUMO
alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors are probably the most widespread excitatory neurotransmitter receptors of the central nervous system, and they play a role in most normal and pathological neural activities. However, previous detailed studies of AMPA subunit distribution have been limited mainly to the brain. Thus, a comprehensive study of AMPA receptor subunit distribution was carried out on sections of rat spinal cord and dorsal root ganglia, which were immunolabeled with antibodies made against peptides corresponding to C-terminal portions of the AMPA receptor subunits: GluR1, GluR2/3, and GluR4. In the spinal cord, labeling was most prominent in the superficial dorsal horn, motoneurons, and nuclei containing preganglionic autonomic neurons. Immunostaining also was observed in neurons in other regions including those known to contain Renshaw cells and Ia inhibitory cells. Although overall immunostaining was lighter with antibody to GluR1 than with GluR2/3 and 4, there were neurons that preferentially stained with antibody to GluR1. These "GluR1 intense" neurons were usually fusiform and most concentrated in lamina X. In dorsal root ganglia, immunostaining of ganglion cell bodies was moderate to dense with antibody to GluR2/3 and light to moderate with antibody to GluR4. Possible neuroglia in the spinal cord (mainly GluR2/3 and 4) and satellite cells in dorsal root ganglia (GluR4) were immunostained. Electron microscopic studies of the superficial dorsal horn and lateral motor column showed staining that was restricted mainly to postsynaptic densities and associated dendritic and cell body cytoplasm. In dorsal horn, colocalization of dense-cored vesicles with clear, round synaptic vesicles was observed in unstained presynaptic terminals apposed to stained postsynaptic densities. Subsynaptic dense bodies (Taxi-bodies) were associated with some stained postsynaptic densities in both the superficial dorsal horn and lateral motor column. Based on several morphological features including vesicle structure and presence of Taxi-bodies, it is likely that at least some of the postsynaptic staining seen in this study is apposed to glutamatergic input from primary sensory afferent terminals.
Assuntos
Receptores de AMPA/metabolismo , Medula Espinal/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/isolamento & purificação , Eletroforese , Gânglios Espinais/metabolismo , Gânglios Espinais/ultraestrutura , Immunoblotting , Imuno-Histoquímica , Masculino , Camundongos , Microscopia Imunoeletrônica , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/imunologia , Receptores de Glutamato/imunologia , Receptores de Glutamato/metabolismo , Medula Espinal/ultraestruturaRESUMO
Kainate receptors are found throughout many regions of the brain and presumably contribute to responses of neurons to glutamate and other excitatory amino acids. Two affinity-purified polyclonal antibodies that recognize the kainate binding subunits, KA2 and GluR6, were made using C-terminus peptides. A previous study demonstrated that each antibody is specific for its subunit, although antibody to GluR6 recognizes GluR7 to some extent (hence the designation GluR6/7). Vibratome sections immunostained with either antibody showed light to moderate staining in many structures in the brain as well as in cervical spinal cord, dorsal root and vestibular ganglia, and pineal and pituitary glands. Moderate levels were seen in the olfactory bulb, cerebral cortex, caudate/putamen, and hypothalamus, whereas much of the thalamus was stained lightly. In the hippocampus, CA3 pyramidal cells were stained more densely than CA1 pyramidal cells--the difference more evident with antibody to GluR6/7. In addition, neuropilar staining was densest in the stratum lucidum of the CA3 region. In the brainstem, staining was moderate to moderately dense in a number of sensory, motor, and reticular nuclei. The moderately dense staining in the reticulothalamic nucleus and pontine nuclei with antibody to GluR6/7 may represent its recognition of GluR7. In the cerebellum, staining was moderate in granular and molecular layers with antibody to KA2 and in the molecular layer with antibody to GluR6/7, whereas it was moderately dense to dense in the granular layer with the GluR6/7 antibody. Outside of the brain, densest staining was seen with antibody to KA2 in the intermediate lobe of the pituitary gland. Ultrastructural localization of immunostaining was examined in the hippocampus, cerebral cortex, and cerebellar cortex. Typically, major staining was in postsynaptic densities apposed by unstained presynaptic terminals with round or mainly round vesicles and in associated dendrites. The light microscope pattern of staining was fairly similar to that of previous [3H]kainate binding and in situ hybridization studies. In addition, comparison with previous studies on distribution of other types of glutamate receptors indicates that KA2 and GluR6/7 are found with various other subunits in many of the same cell populations throughout the nervous system.