RESUMO
Neurodevelopmental disorders are multi-faceted and can lead to intellectual disability, autism spectrum disorder and language impairment. Mutations in the Forkhead box FOXP1 gene have been linked to all these disorders, suggesting that it may play a central role in various cognitive and social processes. To understand the role of Foxp1 in the context of neurodevelopment leading to alterations in cognition and behaviour, we generated mice with a brain-specific Foxp1 deletion (Nestin-Cre(Foxp1-/-)mice). The mutant mice were viable and allowed for the first time the analysis of pre- and postnatal neurodevelopmental phenotypes, which included a pronounced disruption of the developing striatum and more subtle alterations in the hippocampus. More detailed analysis in the CA1 region revealed abnormal neuronal morphogenesis that was associated with reduced excitability and an imbalance of excitatory to inhibitory input in CA1 hippocampal neurons in Nestin-Cre(Foxp1-/-) mice. Foxp1 ablation was also associated with various cognitive and social deficits, providing new insights into its behavioural importance.
Assuntos
Transtorno Autístico/genética , Deficiências do Desenvolvimento/genética , Fatores de Transcrição Forkhead/deficiência , Proteínas Repressoras/deficiência , Estimulação Acústica , Animais , Animais Recém-Nascidos , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Proliferação de Células/genética , Dendritos/patologia , Deficiências do Desenvolvimento/patologia , Fatores de Transcrição Forkhead/genética , Hipocampo/patologia , Técnicas In Vitro , Masculino , Transtornos da Memória/genética , Memória de Curto Prazo/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/patologia , Neurônios/fisiologia , Inibição Pré-Pulso/genética , Proteínas Repressoras/genética , Transtornos do Comportamento Social/genética , Transmissão Sináptica/genéticaRESUMO
Functional glutamate receptor (GluRs) were transiently expressed in cultured mammalian cells from cloned complementary DNAs encoding GluR-A, -B, -C, or -D polypeptides. The steady-state current-voltage (I-V) relations of glutamate- and kainate-induced currents through homomeric channels fell into two classes: channels composed of either the GluR-A, -C, and -D subunits showed doubly rectifying I-V curves, and channels composed of the GluR-B subunits displayed simple outward rectification. The presence of GluR-B subunits in heteromeric GluRs determined the I-V behavior of the resulting channels. Site-directed mutagenesis identified a single amino acid difference (glutamine to arginine) in the putative transmembrane segment TM2 responsible for subunit-specific I-V relationships. The properties of heteromeric wild-type and mutant GluRs revealed that the dominance of GluR-B is due to the arginine residue in the TM2 region.
Assuntos
Canais Iônicos/fisiologia , Receptores de Neurotransmissores/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Análise Mutacional de DNA , Glutamatos/fisiologia , Humanos , Ativação do Canal Iônico , Substâncias Macromoleculares , Glicoproteínas de Membrana/fisiologia , Dados de Sequência Molecular , Oligonucleotídeos/química , Receptores de Glutamato , Proteínas Recombinantes , Relação Estrutura-AtividadeRESUMO
The decay of excitatory postsynaptic currents in central neurons mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors is likely to be shaped either by receptor desensitization or by offset after removal of glutamate from the synaptic cleft. Native AMPA receptors show desensitization time constants of 1 to about 10 milliseconds, but the underlying molecular determinants of these large differences are unknown. Cloned AMPA receptors carrying the "flop" splice variants of glutamate receptor subtype C (GluR-C) and GluR-D are shown to have desensitization time constants of around 1 millisecond, whereas those with the "flip" variants are about four times slower. Cerebellar granule cells switch their expression of GluR-D splice variants from mostly flip forms in early stages to predominantly flop forms in the adult rat brain. These findings suggest that rapid desensitization of AMPA receptors can be regulated by the expression and alternative splicing of GluR-D gene transcripts.
Assuntos
Ácido Glutâmico/farmacologia , Receptores de AMPA/fisiologia , Processamento Alternativo , Animais , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Clonagem Molecular , Hibridização In Situ , Oócitos , Técnicas de Patch-Clamp , Ratos , Receptores de AMPA/efeitos dos fármacos , Receptores de AMPA/genética , Proteínas Recombinantes , Transmissão Sináptica , Xenopus laevisRESUMO
Glutamate-operated ion channels (GluR channels) of the L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-kainate subtype are found in both neurons and glial cells of the central nervous system. These channels are assembled from the GluR-A, -B, -C, and -D subunits; channels containing a GluR-B subunit show an outwardly rectifying current-voltage relation and low calcium permeability, whereas channels lacking the GluR-B subunit are characterized by a doubly rectifying current-voltage relation and high calcium permeability. Most cell types in the central nervous system coexpress several subunits, including GluR-B. However, Bergmann glia in rat cerebellum do not express GluR-B subunit genes. In a subset of cultured cerebellar glial cells, likely derived from Bergmann glial cells. GluR channels exhibit doubly rectifying current-voltage relations and high calcium permeability, whereas GluR channels of cerebellar neurons have low calcium permeability. Thus, differential expression of the GluR-B subunit gene in neurons and glia is one mechanism by which functional properties of native GluR channels are regulated.
Assuntos
Cálcio/metabolismo , Cerebelo/fisiologia , Neuroglia/fisiologia , Receptores de Neurotransmissores/fisiologia , Animais , Permeabilidade da Membrana Celular , Células Cultivadas , Expressão Gênica , Glutamatos/fisiologia , Técnicas In Vitro , Ativação do Canal Iônico , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , Ratos , Receptores de Ácido CaínicoRESUMO
The N-methyl D-aspartate (NMDA) receptor subtype of glutamate-gated ion channels possesses high calcium permeability and unique voltage-dependent sensitivity to magnesium and is modulated by glycine. Molecular cloning identified three complementary DNA species of rat brain, encoding NMDA receptor subunits NMDAR2A (NR2A), NR2B, and NR2C, which are 55 to 70% identical in sequence. These are structurally related, with less than 20% sequence identity, to other excitatory amino acid receptor subunits, including the NMDA receptor subunit NMDAR1 (NR1). Upon expression in cultured cells, the new subunits yielded prominent, typical glutamate- and NMDA-activated currents only when they were in heteromeric configurations with NR1. NR1-NR2A and NR1-NR2C channels differed in gating behavior and magnesium sensitivity. Such heteromeric NMDA receptor subtypes may exist in neurons, since NR1 messenger RNA is synthesized throughout the mature rat brain, while NR2 messenger RNA show a differential distribution.
Assuntos
Encéfalo/fisiologia , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA/genética , Glutamatos/farmacologia , Ácido Glutâmico , Glicina/farmacologia , Substâncias Macromoleculares , Magnésio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Família Multigênica , N-Metilaspartato/farmacologia , Sondas de Oligonucleotídeos , Especificidade de Órgãos , Peptídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Homologia de Sequência do Ácido Nucleico , TransfecçãoRESUMO
The N-methyl-D-aspartate (NMDA) receptor forms a cation-selective channel with a high calcium permeability and sensitivity to channel block by extracellular magnesium. These properties, which are believed to be important for the induction of long-term changes in synaptic strength, are imparted by asparagine residues in a putative channel-forming segment of the protein, transmembrane 2 (TM2). In the NR1 subunit, replacement of this asparagine by a glutamine residue decreases calcium permeability of the channel and slightly reduces magnesium block. The same substitution in NR2 subunits strongly reduces magnesium block and increases the magnesium permeability but barely affects calcium permeability. These asparagines are in a position homologous to the site in the TM2 region (Q/R site) of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that is occupied by either glutamine (Q) or arginine (R) and that controls divalent cation permeability of the AMPA receptor channel. Hence AMPA and NMDA receptor channels contain common structural motifs in their TM2 segments that are responsible for some of their ion selectivity and conductance properties.
Assuntos
Asparagina/química , Cálcio/metabolismo , Canais Iônicos/fisiologia , Magnésio/farmacologia , Receptores de N-Metil-D-Aspartato/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cálcio/farmacologia , Linhagem Celular , Condutividade Elétrica , Glutamatos/farmacologia , Ácido Glutâmico , Canais Iônicos/química , Magnésio/metabolismo , Camundongos , Dados de Sequência Molecular , Mutagênese , Oócitos/metabolismo , Permeabilidade , Ratos , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Relação Estrutura-Atividade , Transfecção , XenopusRESUMO
AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor channels mediate the fast component of excitatory postsynaptic currents in the central nervous system. Site-selective nuclear RNA editing controls the calcium permeability of these channels, and RNA editing at a second site is shown here to affect the kinetic aspects of these channels in rat brain. In three of the four AMPA receptor subunits (GluR-B, -C, and -D), intronic elements determine a codon switch (AGA, arginine, to GGA, glycine) in the primary transcripts in a position termed the R/G site, which immediately precedes the alternatively spliced modules "flip" and "flop." The extent of editing at this site progresses with brain development in a manner specific for subunit and splice form, and edited channels possess faster recovery rates from desensitization.
Assuntos
Encéfalo/metabolismo , Edição de RNA , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/embriologia , Núcleo Celular/metabolismo , Éxons , Ácido Glutâmico/farmacologia , Glicina/genética , Íntrons , Cinética , Potenciais da Membrana , Dados de Sequência Molecular , Oócitos , Células PC12 , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismo , XenopusRESUMO
The expression of two alternative splice variants, Flip and Flop, in mRNAs encoding the four AMPA-selective glutamate receptors (GluR-A, -B, -C, and -D) was studied in the developing brain by in situ hybridization. These receptors are expressed prominently before birth, and patterns of distribution for Flip versions remain largely invariant during postnatal brain development. In contrast, the Flop versions are expressed at low levels prior to postnatal day 8. Around this time, the expression of Flop mRNAs increases throughout the brain, reaching adult levels by postnatal day 14. Thus, receptors carrying the Flop module appear to participate in mature receptor forms.
Assuntos
Encéfalo/embriologia , Neurônios/metabolismo , RNA Mensageiro/genética , Receptores de Neurotransmissores/genética , 6-Ciano-7-nitroquinoxalina-2,3-diona , Animais , Encéfalo/metabolismo , Cerebelo/embriologia , Cerebelo/metabolismo , Expressão Gênica , Hipocampo/embriologia , Hipocampo/metabolismo , Ácido Ibotênico/análogos & derivados , Ácido Ibotênico/metabolismo , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , Quinoxalinas/metabolismo , Splicing de RNA , Ratos , Ratos Endogâmicos , Receptores de AMPA , Receptores de Glutamato , Receptores de Neurotransmissores/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol PropiônicoRESUMO
We studied the protective efficacy of novel 21-aminosteroids against several forms of neuronal injury in murine cortical cell cultures. Concentrations of 200 nM to 20 microM partially attenuated the damage induced by glucose deprivation, combined oxygen-glucose deprivation, or exposure to NMDA; maximal protection was less than that produced by NMDA antagonists, but the combination of a 21-aminosteroid plus an NMDA antagonist produced a greater benefit than either drug alone. 21-Aminosteroid addition did not attenuate NMDA-induced whole-cell current, but did block almost all of the damage induced by exposure to iron, a protective action consistent with inhibition of free radical-mediated lipid peroxidation. Lipid peroxidation may be a downstream event mediating a portion of the injury triggered by excess stimulation of NMDA receptors.
Assuntos
Peróxidos Lipídicos/antagonistas & inibidores , Neurônios/metabolismo , Pregnatrienos/farmacologia , Animais , Ácido Aspártico/análogos & derivados , Ácido Aspártico/antagonistas & inibidores , Ácido Aspártico/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Glucose/metabolismo , Peroxidação de Lipídeos , Potenciais da Membrana , Camundongos , N-Metilaspartato , Degeneração Neural , Neurônios/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato , Receptores de Neurotransmissores/metabolismoRESUMO
Functionally diverse GluR channels of the AMPA subtype are generated by the assembly of GluR-A, -B, -C, and -D subunits into homo- and heteromeric channels. The GluR-B subunit is dominant in determining functional properties of heteromeric AMPA receptors. This subunit exists in developmentally distinct edited and unedited forms, GluR-B(R) and GluR-B(Q), which differ in a single amino acid in transmembrane segment TM2 (Q/R site). Homomeric GluR-B(R) channels expressed in 293 cells display a low divalent permeability, whereas homomeric GluR-B(Q) and GluR-D channels exhibit a high divalent permeability. Mutational analysis revealed that both the positive charge and the size of the amino acid side chain located at the Q/R site control the divalent permeability of homomeric channels. Coexpression of Q/R site arginine- and glutamine-containing subunits generates cells with varying divalent permeabilities depending on the amounts of expression vectors used for cell transfection. Intermediate divalent permeabilities were traced to the presence of both divalent permeant homomeric and impermeant heteromeric channels. It is suggested that the positive charge contributed by the arginine of the edited GluR-B(R) subunit determines low divalent permeability in heteromeric GluR channels and that changes in GluR-B(R) expression regulate the AMPA receptor-dependent divalent permeability of a cell.
Assuntos
Canais Iônicos/fisiologia , Receptores de Neurotransmissores/fisiologia , Sequência de Bases , Cálcio/metabolismo , Cálcio/farmacologia , Cátions Bivalentes , Linhagem Celular , Eletrofisiologia , Expressão Gênica , Glutamatos/farmacologia , Ácido Glutâmico , Humanos , Substâncias Macromoleculares , Magnésio/metabolismo , Magnésio/farmacologia , Potenciais da Membrana , Dados de Sequência Molecular , Permeabilidade , RNA Mensageiro/genética , Receptores de AMPA , Receptores de Neurotransmissores/genética , Proteínas Recombinantes/fisiologia , Sódio/farmacologia , TransfecçãoRESUMO
Fast excitatory synaptic transmission in the CNS is mediated by AMPA-type glutamate receptor (GluR) channels. Heterologous expression suggested that the Ca2+ permeability of these receptors critically depends on the subunit composition. Using patch-clamp techniques in brain slices, we found that the Ca2+ permeability of native AMPA-type GluRs was markedly higher in nonpyramidal (PCa/PK approximately 0.63) than in pyramidal (PCa/PK approximately 0.05) neurons of rat neocortex. Analysis of mRNA in single cells indicated that the relative abundance of GluR-B-specific mRNA was significantly lower in nonpyramidal (GluR-B/GluR-non-B approximately 0.3) than in pyramidal (GluR-B/GluR-non-B approximately 3) cells. This suggests that differences in relative abundance of GluR-B-specific mRNA generate functional diversity of AMPA-type GluRs in neurons with respect to Ca2+ permeability.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Córtex Cerebral/fisiologia , Expressão Gênica , Neurônios/fisiologia , Células Piramidais/fisiologia , Receptores de AMPA/fisiologia , Receptores de Glutamato/biossíntese , Córtex Visual/fisiologia , Animais , Sequência de Bases , Primers do DNA , Técnicas In Vitro , Substâncias Macromoleculares , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Ratos , Ratos WistarRESUMO
Pure traumatic neuronal injury was modeled in dispersed neocortical cell cultures derived from fetal mice. A plastic stylet was used to tear the neuronal and glial cell layer; medium oxygen content, pH, and glucose remained unchanged. Adjacent to this local disruption, many neurons developed acute swelling and went on to degenerate over the next day, but glia were relatively spared. If the same mechanical insult was delivered in the presence of the N-methyl-D-aspartate (NMDA) antagonists dextrorphan or D-2-amino-5-phosphonovalerate, resultant neuronal degeneration was markedly reduced. The protective effect of these NMDA antagonists was concentration-dependent between 1 and 100 microM, with EC50 near 10 microM for both compounds. Present findings suggest that endogenous excitatory amino acids may participate significantly in the propagation of central neuronal cell loss in response to a purely mechanical insult.
Assuntos
Ácido Aspártico/análogos & derivados , Dextrorfano/farmacologia , Morfinanos/farmacologia , Tecido Nervoso/lesões , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , 2-Amino-5-fosfonovalerato/farmacologia , Animais , Ácido Aspártico/antagonistas & inibidores , Ácido Aspártico/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Camundongos , N-Metilaspartato , Neuroglia/patologia , Neurônios/patologia , Estresse MecânicoRESUMO
An in situ study of mRNAs encoding NMDA receptor subunits in the developing rat CNS revealed that, at all stages, the NR1 gene is expressed in virtually all neurons, whereas the four NR2 transcripts display distinct expression patterns. NR2B and NR2D mRNAs occur prenatally, whereas NR2A and NR2C mRNAs are first detected near birth. All transcripts except NR2D peak around P20. NR2D mRNA, present mainly in midbrain structures, peaks around P7 and thereafter decreases to adult levels. Postnatally, NR2B and NR2C transcript levels change in opposite directions in the cerebellar internal granule cell layer. In the adult hippocampus, NR2A and NR2B mRNAs are prominent in CA1 and CA3 pyramidal cells, but NR2C and NR2D mRNAs occur in different subsets of interneurons. Recombinant binary NR1-NR2 channels show comparable Ca2+ permeabilities, but marked differences in voltage-dependent Mg2+ block and in offset decay time constants. Thus, the distinct expression profiles and functional properties of NR2 subunits provide a basis for NMDA channel heterogeneity in the brain.
Assuntos
Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Animais Recém-Nascidos , Sequência de Bases , Cerebelo/citologia , Cerebelo/metabolismo , Granulócitos/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Canais Iônicos/metabolismo , Sondas Moleculares/genética , Dados de Sequência Molecular , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Proteínas RecombinantesRESUMO
Sulfhydryl redox agents affect NMDA receptor activity. We investigated a putative redox site in four recombinant NMDA receptors. In 293 cells expressing NR1-NR2A channels dithiothreitol (DTT) rapidly potentiated L-glutamate-activated whole-cell currents and decreased the time course of desensitization and deactivation. Part of the current potentiation (reversible component) and all kinetic changes reversed upon washout. The remaining potentiation (persistent component) was abolished by an oxidizing agent. The N-terminal 370 residues of NR2A mediate the reversible component in chimeric NR2 subunits. In cells expressing the NR1-NR2B, -NR2C, and -NR2D channels DTT elicited only a slowly developing, persistent potentiation and increased the deactivation time course. In these, but not in NR1-NR2A, the DTT effect was rendered insensitive to reoxidation by alkylation. Reduced glutathione mimicked the DTT effects only in the NR1-NR2A receptor. Hence, molecularly distinct NMDA receptors differ profoundly in their responses to sulfhydryl redox agents.
Assuntos
Ditiotreitol/farmacologia , Glutamatos/farmacologia , Glutationa/farmacologia , Canais Iônicos/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Alanina , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Cisteína , Sinergismo Farmacológico , Etilmaleimida/farmacologia , Ácido Glutâmico , Humanos , Canais Iônicos/biossíntese , Canais Iônicos/efeitos dos fármacos , Rim , Cinética , Substâncias Macromoleculares , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Ratos , Receptores de N-Metil-D-Aspartato/biossíntese , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Serina , TransfecçãoRESUMO
Argiotoxin, a component of the spider venom from Argiope lobata, blocks AMPA receptor channels expressed in homomeric and heteromeric configuration in Xenopus oocytes. Argiotoxin acts as an open channel blocker in a voltage-dependent manner and discriminates between the functionally diverse AMPA receptors. Importantly, a transmembrane region 2 determinant for divalent cation permeability also determines argiotoxin sensitivity. Subunit-specific differences in the time courses of block and recovery demonstrate that heteromeric AMPA receptors can assemble in variable ratios. Thus, argiotoxin can be used as a tool in analyzing the subunit composition of AMPA receptors in native membranes.
Assuntos
Canais Iônicos/fisiologia , Oócitos/fisiologia , Fenilacetatos/farmacologia , Poliaminas/farmacologia , Receptores de Glutamato/fisiologia , Venenos de Aranha/farmacologia , Animais , Sequência de Bases , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Potenciais Evocados/efeitos dos fármacos , Feminino , Ácidos Indolacéticos , Canais Iônicos/efeitos dos fármacos , Ácido Caínico/farmacologia , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutagênese , Oligodesoxirribonucleotídeos , Oócitos/efeitos dos fármacos , Conformação Proteica , Receptores de AMPA , Receptores de Glutamato/efeitos dos fármacos , Receptores de Glutamato/genética , Xenopus laevisRESUMO
We examined glutamate-mediated neurotoxicity in cortical cell cultures pretreated with 1-5 micrograms/ml tetanus toxin to attenuate the Ca(2+)-dependent release of neurotransmitters. Efficacy of the tetanus toxin pretreatment was suggested by blockade of electrical burst activity induced by Mg2+ removal and by reduction of glutamate efflux induced by high K+. Tetanus toxin reduced neuronal injury produced by brief exposure to elevated extracellular K+ or to glutamate, situations in which release of endogenous excitatory neurotransmitter is likely to play a role. Furthermore, although glutamate efflux evoked by anoxic conditions may occur largely via Ca(2+)-independent transport, tetanus toxin attenuated both glutamate efflux and neuronal injury following combined oxygen and glucose deprivation. With prolonged exposure periods, the neuroprotective efficacy of tetanus toxin was comparable to that of NMDA receptor antagonists. Presynaptic inhibition of Ca(2+)-dependent glutamate release may be a valuable approach to attenuating hypoxic-ischemic brain injury.
Assuntos
Córtex Cerebral/citologia , Glucose/administração & dosagem , Neurônios/fisiologia , Oxigênio/administração & dosagem , Toxina Tetânica/farmacologia , 6-Ciano-7-nitroquinoxalina-2,3-diona , Animais , Cálcio/farmacologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiologia , Maleato de Dizocilpina/farmacologia , Eletrofisiologia , Glutamatos/metabolismo , Glutamatos/farmacologia , Ácido Glutâmico , Magnésio/administração & dosagem , Camundongos , Neurônios/efeitos dos fármacos , Neurotransmissores/metabolismo , Potássio/farmacologia , Quinoxalinas/farmacologiaRESUMO
Recording of glutamate-activated currents in membrane patches was combined with RT-PCR-mediated AMPA receptor (AMPAR) subunit mRNA analysis in single identified cells of rat brain slices. Analysis of AMPARs in principal neurons and interneurons of hippocampus and neocortex and in auditory relay neurons and Bergmann glial cells indicates that the GluR-B subunit in its flip version determines formation of receptors with relatively slow gating, whereas the GluR-D subunit promotes assembly of more rapidly gated receptors. The relation between Ca2+ permeability of AMPAR channels and the relative GluR-B mRNA abundance is consistent with the dominance of this subunit in determining the Ca2+ permeability of native receptors. The results suggest that differential expression of GluR-B and GluR-D subunit genes, as well as splicing and editing of their mRNAs, account for the differences in gating and Ca2+ permeability of native AMPAR channels.
Assuntos
Cálcio/metabolismo , Sistema Nervoso Central/citologia , Interneurônios/ultraestrutura , Ativação do Canal Iônico/fisiologia , Neurônios/ultraestrutura , RNA Mensageiro/análise , Receptores de AMPA/genética , Animais , Vias Auditivas/citologia , Sequência de Bases , Permeabilidade da Membrana Celular/genética , Cerebelo/citologia , Cerebelo/fisiologia , Hipocampo/citologia , Hipocampo/fisiologia , Interneurônios/metabolismo , Dados de Sequência Molecular , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Neurônios/metabolismo , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , Células Piramidais/fisiologia , Ratos , Ratos Wistar , Receptores de AMPA/metabolismo , Receptores de AMPA/ultraestruturaRESUMO
Neural processing occurs in parallel in distant cortical areas even for simple perceptual tasks. Associated cognitive binding is believed to occur through the interareal synchronization of rhythmic activity in the gamma (30-80 Hz) range. Such oscillations arise as an emergent property of the neuronal network and require conventional chemical neurotransmission. To test the potential role of gap junction-mediated electrical signaling in this network property, we generated mice lacking connexin 36, the major neuronal connexin. Here we show that the loss of this protein disrupts gamma frequency network oscillations in vitro but leaves high frequency (150 Hz) rhythms, which may involve gap junctions between principal cells (Schmitz et al., 2001), unaffected. Thus, specific connexins differentially deployed throughout cortical networks are likely to regulate different functional aspects of neuronal information processing in the mature brain.
Assuntos
Encéfalo/fisiologia , Conexinas/fisiologia , Hipocampo/fisiologia , Rede Nervosa/fisiologia , Neurônios/fisiologia , Envelhecimento , Animais , Encéfalo/crescimento & desenvolvimento , Carbacol/farmacologia , Córtex Cerebral/fisiologia , Conexinas/deficiência , Conexinas/genética , Eletroencefalografia , Junções Comunicantes/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/efeitos dos fármacos , Técnicas In Vitro , Ácido Caínico/farmacologia , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Oscilometria , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transcrição Gênica , Proteína delta-2 de Junções ComunicantesRESUMO
Classic neurotransmitter phenotypes are generally predetermined and develop as a consequence of target-independent lineage decisions. A unique mode of target-dependent phenotype instruction is the acquisition of the cholinergic phenotype in the peripheral sympathetic nervous system. A body of work suggests that the sweat gland plays an important role to determine the cholinergic phenotype at this target site. A key issue is whether neurons destined to innervate the sweat glands express cholinergic markers before or only after their terminals make target contact. We employed cholinergic-specific over-expression of the vesicular acetylcholine transporter (VAChT) in transgenic mice to overcome sensitivity limits in the detection of initial cholinergic sweat gland innervation. We found that VAChT immunoreactive nerve terminals were present around the sweat gland anlage already from the earliest postnatal stages on, coincident selectively at this sympathetic target with tyrosine hydroxylase-positive fibers. Our results provide a new mechanistic model for sympathetic neuron-target interaction during development, with initial selection by the target of pioneering nerve terminals expressing a cholinergic phenotype, and subsequent stabilization of this phenotype during development.
Assuntos
Acetilcolina/metabolismo , Neurônios/metabolismo , Norepinefrina/metabolismo , Fenótipo , Glândulas Sudoríparas/inervação , Sistema Nervoso Simpático/citologia , Fatores Etários , Animais , Animais Recém-Nascidos , Colina O-Acetiltransferase/genética , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Sistema Nervoso Simpático/crescimento & desenvolvimento , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/genética , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismoRESUMO
The functional characterization of the many complex proteins expressed in the CNS is a daunting task. The development of nucleic acid amplification techniques has provided a powerful tool to tackle this challenging enterprise. Recently, these techniques have been successfully used to correlate the functional properties of various CNS proteins with their specific mRNA expression patterns in the brain.