RESUMO
In the developing nervous system, glial cells guide axons to their target areas, but it is unknown whether they help neurons to establish functional synaptic connections. The role of glial cells in synapse formation and function was studied in cultures of purified neurons from the rat central nervous system. In glia-free cultures, retinal ganglion cells formed synapses with normal ultrastructure but displayed little spontaneous synaptic activity and high failure rates in evoked synaptic transmission. In cocultures with neuroglia, the frequency and amplitude of spontaneous postsynaptic currents were potentiated by 70-fold and 5-fold, respectively, and fewer transmission failures occurred. Glial cells increased the action potential-independent quantal release by 12-fold without affecting neuronal survival. Thus, developing neurons in culture form inefficient synapses that require glial signals to become fully functional.
Assuntos
Neuroglia/fisiologia , Células Ganglionares da Retina/fisiologia , Sinapses/fisiologia , Transmissão Sináptica , Potenciais de Ação , Animais , Astrócitos/fisiologia , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Microglia/fisiologia , Oligodendroglia/fisiologia , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/ultraestrutura , Sinapses/ultraestruturaRESUMO
Although astrocytes constitute nearly half of the cells in our brain, their function is a long-standing neurobiological mystery. Here we show by quantal analyses, FM1-43 imaging, immunostaining, and electron microscopy that few synapses form in the absence of glial cells and that the few synapses that do form are functionally immature. Astrocytes increase the number of mature, functional synapses on central nervous system (CNS) neurons by sevenfold and are required for synaptic maintenance in vitro. We also show that most synapses are generated concurrently with the development of glia in vivo. These data demonstrate a previously unknown function for glia in inducing and stabilizing CNS synapses, show that CNS synapse number can be profoundly regulated by nonneuronal signals, and raise the possibility that glia may actively participate in synaptic plasticity.
Assuntos
Astrócitos/fisiologia , Proteínas de Ligação ao Cálcio , Células Ganglionares da Retina/fisiologia , Sinapses/fisiologia , Animais , Cálcio/metabolismo , Comunicação Celular , Células Cultivadas , Técnicas de Cocultura , Potenciais Pós-Sinápticos Excitadores , Corantes Fluorescentes/metabolismo , Ácido Glutâmico/farmacologia , Ionomicina/farmacologia , Glicoproteínas de Membrana/metabolismo , Microscopia Eletrônica , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal , Técnicas de Patch-Clamp , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/ultraestrutura , Colículos Superiores/embriologia , Colículos Superiores/crescimento & desenvolvimento , Colículos Superiores/ultraestrutura , Sinapses/ultraestrutura , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Sinaptofisina/metabolismo , SinaptotagminasRESUMO
During the development of the vertebrate nervous system, up to 50 percent or more of many types of neurons normally die soon after they form synaptic connections with their target cells. This massive cell death is thought to reflect the failure of these neurons to obtain adequate amounts of specific neurotrophic factors that are produced by the target cells and that are required for the neurons to survive. This neurotrophic strategy for the regulation of neuronal numbers may be only one example of a general mechanism that helps to regulate the numbers of many other vertebrate cell types, which also require signals from other cells to survive. These survival signals seem to act by suppressing an intrinsic cell suicide program, the protein components of which are apparently expressed constitutively in most cell types.
Assuntos
Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Neurônios/citologia , Oligodendroglia/citologia , Animais , HumanosRESUMO
Two functionally different forms of the voltage-dependent sodium channel were observed in glia and in neurons of the mammalian nervous system. Both forms had identical conductance and tetrodotoxin sensitivity and displayed steady-state inactivation, a strongly voltage-dependent rate of activation, and a faster but weakly voltage-sensitive rate of inactivation. However, the glial form had significantly slower kinetics and a more negative voltage dependence, suggesting that it was functionally specialized for glia. This form was found in most glial types studied, while the neuronal form was observed in retinal ganglion cells, cortical motor neurons, and O2A glial progenitor cells. Both forms occurred in type-2 astrocytes. The presence of the glial form correlated with the RAN-2 surface antigen.
Assuntos
Astrócitos/análise , Ativação do Canal Iônico , Neurônios/análise , Canais de Sódio/análise , Sódio/metabolismo , Potenciais de Ação , Animais , Células Cultivadas , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Neurônios Motores/análise , Nervo Óptico/análise , Ratos , Células Ganglionares da Retina/análise , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/metabolismo , Tetrodotoxina/farmacologiaRESUMO
The signaling mechanisms that control the survival of CNS neurons are poorly understood. Here we show that, in contrast to PNS neurons, the survival of purified postnatal rat retinal ganglion cells (RGCs) in vitro is not promoted by peptide trophic factors unless their intracellular cAMP is increased pharmacologically or they are depolarized by K+ or glutamate agonists. Long-term survival of most RGCs in culture can be promoted by a combination of trophic factors normally produced along the visual pathway, including BDNF, CNTF, IGF1, an oligodendrocyte-derived protein, and forskolin. These results suggest that neurotransmitter stimulation and electrical activity enhance the survival of developing RGCs and raise the question of whether the survival control mechanisms of PNS and CNS neurons are different.
Assuntos
Divisão Celular , Sobrevivência Celular , Células Ganglionares da Retina/fisiologia , Transdução de Sinais/fisiologia , Animais , Astrócitos/fisiologia , Fator Neurotrófico Derivado do Encéfalo , Contagem de Células , Células Cultivadas , Fator Neurotrófico Ciliar , Colforsina/farmacologia , AMP Cíclico/metabolismo , Eletrofisiologia , Ácido Glutâmico/farmacologia , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Proteínas do Tecido Nervoso/farmacologia , Oligodendroglia/fisiologia , Potássio/farmacologia , Ratos , Retina/embriologia , Células Ganglionares da Retina/citologiaRESUMO
Two different monoclonal antibodies to the Thy-1 antigen, T11D7 and 2G12, were used to purify and characterize retinal ganglion cells from postnatal rat retina. Although Thy-1 has been reported to be a specific marker for ganglion cells in retina, retinal cell suspensions contained several other types of Thy-1-positive cells as well. Nevertheless, a simple two-step "panning" procedure allowed isolation of ganglion cells to nearly 100% purity. We found that postnatal ganglion cells differed in antigenic, morphological, and intrinsic electrophysiological characteristics, and that these properties were correlated with one another. Minor variations of this panning protocol should allow rapid, high yield purification to homogeneity of many other neuronal and glial cell types.
Assuntos
Antígenos de Superfície/metabolismo , Separação Celular/métodos , Retina/fisiologia , Células Ganglionares da Retina/fisiologia , Potenciais de Ação , Animais , Anticorpos Monoclonais , Ratos , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/imunologia , Antígenos Thy-1RESUMO
We describe the electrophysiological properties of acutely isolated type-1 astrocytes using a new "tissue print" dissociation procedure. Because the enzymes used did not destroy or modify the ion channels, and the cells retained many processes, the properties may reflect those in vivo. The types of ion channels in type-1 astrocytes changed rapidly during the first 10 postnatal days, when they attained their adult phenotype. This change was dependent on the presence of neurons. In culture, most of these channel types were not expressed, but a phenotype more typical of that in vivo could be induced by co-culture with neurons. The electrophysiological properties of astrocytes make some existing hypotheses of astrocyte function less likely.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Canais Iônicos/metabolismo , Neuroglia/metabolismo , Envelhecimento/fisiologia , Animais , Astrócitos/fisiologia , Encéfalo/citologia , Cálcio/farmacologia , Células Cultivadas , Técnicas Citológicas , Denervação , Eletrofisiologia , Glutamatos/farmacologia , Ácido Glutâmico , Imuno-Histoquímica/métodos , Ativação do Canal Iônico , Neurônios/fisiologia , Potássio/fisiologiaRESUMO
Compared to neurons, the intracellular mechanisms that control glial differentiation are still poorly understood. We show here that oligodendrocyte lineage cells express the helix-loop-helix proteins Mash1 and Id2. Although Mash1 has been found to regulate neuronal development, we found that in the absence of Mash1 oligodendrocyte differentiation occurs normally. In contrast, we found that overexpression of Id2 powerfully inhibits oligodendrocyte differentiation, that Id2 normally translocates out of the nucleus at the onset of differentiation, and that absence of Id2 induces premature oligodendrocyte differentiation in vitro. These findings demonstrate that Id2 is a component of the intracellular mechanism that times oligodendrocyte differentiation and point to the existence of an as yet unidentified MyoD-like bHLH protein necessary for oligodendrocyte differentiation.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/fisiologia , Sequências Hélice-Alça-Hélice , Oligodendroglia/citologia , Proteínas Repressoras , Células-Tronco/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Divisão Celular , Células Cultivadas , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Expressão Gênica , Imuno-Histoquímica , Proteína 2 Inibidora de Diferenciação , Camundongos , Camundongos Knockout , Oligodendroglia/química , Oligodendroglia/metabolismo , Nervo Óptico/citologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Ratos , Células-Tronco/química , Células-Tronco/metabolismo , Fatores de Tempo , Fatores de Transcrição/análise , Fatores de Transcrição/deficiência , Fatores de Transcrição/fisiologia , Tri-Iodotironina/farmacologiaRESUMO
Whereas PNS neurons in culture are intrinsically responsive to peptide trophic factors, retinal ganglion cells (RGCs) are not unless they are depolarized, or their intracellular levels of cyclic AMP (cAMP) are elevated. We show here that depolarization increases cAMP in cultured RGCs sufficiently to enhance their responsiveness and that the trophic responsiveness of developing RGCs in intact retinas depends on physiological levels of activity and cAMP elevation. Responsiveness is lost after axotomy but is restored by cAMP elevation. The death of axotomized RGCs can be prevented if they are simultaneously stimulated by several trophic factors together with cAMP elevation. Thus, the death of RGCs after axotomy is not caused solely by the loss of retrograde trophic stimuli but also by a profound loss of trophic responsiveness.
Assuntos
Fatores de Crescimento Neural/fisiologia , Células Ganglionares da Retina/fisiologia , Inibidores de Adenilil Ciclases , Animais , Axotomia , Morte Celular , Núcleo Celular/enzimologia , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/metabolismo , Eletrofisiologia , Imunofluorescência , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
We describe electrophysiological properties of the O-2A glial progenitor cell in a new serum-free culture system. O-2A progenitors have many properties characteristic of neurons: they have glutamate-activated ion channels, express the neuronal form of the sodium channel, fire single regenerative potentials, and synthesize the neurotransmitter GABA by an alternative synthetic pathway. Nearly identical properties were observed in acutely isolated O-2A progenitors, indicating that this phenotype is not an artifact of culture. The O-2A did not express a simple subset of channel types found in its descendant cells, the type-2 astrocyte and oligodendrocyte, studied in the same culture system. During development, these electrophysiological properties may contribute to O-2A function in vivo.
Assuntos
Canais Iônicos/fisiologia , Neuroglia/citologia , Potenciais de Ação , Animais , Anticorpos Monoclonais , Astrócitos/citologia , Astrócitos/fisiologia , Diferenciação Celular , Células Cultivadas , Charibdotoxina , Meios de Cultura , Glutamatos/fisiologia , Técnicas In Vitro , Potenciais da Membrana , Neuroglia/fisiologia , Oligodendroglia/citologia , Oligodendroglia/fisiologia , Potássio/fisiologia , Canais de Potássio/fisiologia , Ratos , Venenos de Escorpião/farmacologia , Canais de Sódio/fisiologia , Ácido gama-Aminobutírico/fisiologiaRESUMO
Na(v)1.6 is the main sodium channel isoform at adult nodes of Ranvier. Here, we show that Na(v)1.2 and its beta2 subunit, but not Na(v)1.6 or beta1, are clustered in developing central nervous system nodes and that clustering of Na(v)1.2 and Na(v)1.6 is differentially controlled. Oligodendrocyte-conditioned medium is sufficient to induce clustering of Na(v)1.2 alpha and beta2 subunits along central nervous system axons in vitro. This clustering is regulated by electrical activity and requires an intact actin cytoskeleton and synthesis of a non-sodium channel protein. Neither soluble- or contact-mediated glial signals induce clustering of Na(v)1.6 or beta1 in a nonmyelinating culture system. These data reveal that the sequential clustering of Na(v)1.2 and Na(v)1.6 channels is differentially controlled and suggest that myelination induces Na(v)1.6 clustering.
Assuntos
Sistema Nervoso Central/crescimento & desenvolvimento , Nervo Óptico/crescimento & desenvolvimento , Nós Neurofibrosos/metabolismo , Canais de Sódio/metabolismo , Animais , Bioensaio/métodos , Diferenciação Celular/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/efeitos dos fármacos , Neurônios/citologia , Neurônios/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Nervo Óptico/citologia , Nervo Óptico/metabolismo , Isoformas de Proteínas/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Nós Neurofibrosos/ultraestrutura , RatosRESUMO
In this study, we show that oligodendrocyte differentiation is powerfully inhibited by activation of the Notch pathway. Oligodendrocytes and their precursors in the developing rat optic nerve express Notch1 receptors and, at the same time, retinal ganglion cells express Jagged1, a ligand of the Notch1 receptor, along their axons. Jagged1 expression is developmentally regulated, decreasing with a time course that parallels myelination in the optic nerve. These results suggest that the timing of oligodendrocyte differentiation and myelination is controlled by the Notch pathway and raise the question of whether localization of myelination is controlled by this pathway.
Assuntos
Proteínas de Membrana/fisiologia , Oligodendroglia/citologia , Receptores de Superfície Celular/fisiologia , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Proteínas de Ligação ao Cálcio , Diferenciação Celular/fisiologia , Células Cultivadas , Senescência Celular/fisiologia , Técnicas de Cocultura , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Proteína Jagged-1 , Ligantes , Proteínas de Membrana/genética , Nervo Óptico/citologia , Nervo Óptico/crescimento & desenvolvimento , Nervo Óptico/metabolismo , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Notch , Retina/citologia , Retina/crescimento & desenvolvimento , Retina/metabolismo , Proteínas Serrate-JaggedRESUMO
In the embryonic nervous system, developing axons can be guided to their targets by diffusible factors secreted by their intermediate and final cellular targets. To date only one family of chemoattractants for developing axons has been identified. Grafting and ablation experiments in fish, amphibians, and birds have suggested that spinal motor axons are guided to their targets in the limb in part by a succession of chemoattractants made by the sclerotome and by the limb mesenchyme, two intermediate targets that these axons encounter en route to their target muscles. Here we identify the limb mesenchyme-derived chemoattractant as hepatocyte growth factor/scatter factor (HGF/SF), a diffusible ligand for the c-Met receptor tyrosine kinase, and we also implicate HGF/SF at later stages as a muscle-derived survival factor for motoneurons. These results indicate that, in addition to functioning as a mitogen, a motogen, and a morphogen in nonneural systems, HGF/SF can function as a guidance and survival factor in the developing nervous system.
Assuntos
Axônios/fisiologia , Fatores Quimiotáticos/fisiologia , Fator de Crescimento de Hepatócito/fisiologia , Neurônios Motores/fisiologia , Fatores de Crescimento Neural/fisiologia , Medula Espinal/fisiologia , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Extremidades/embriologia , Extremidades/inervação , Vias Neurais/fisiologia , Ratos/embriologia , Medula Espinal/citologia , Medula Espinal/embriologiaRESUMO
Here, we describe a novel mechanism for the rapid regulation of surface levels of the neurotrophin receptor TrkB. Unlike nodose ganglion neurons, both retinal ganglion cells (RGCs) and spinal motor neurons (SMNs) in culture display only low levels of surface TrkB, though high levels are present intracellularly. Within minutes of depolarization or cAMP elevation, surface TrkB levels increase by nearly 4-fold, and this increase is not blocked by cycloheximide. These findings suggest that activity and cAMP elevation rapidly recruit TrkB to the plasma membrane by translocation from intracellular stores. We propose that a fundamental difference between peripheral nervous system (PNS) and central nervous system (CNS) neurons is the activity dependence of CNS neurons for responsiveness to their peptide trophic factors and that differences in membrane compartmentalization of the receptors underlie this difference.
Assuntos
AMP Cíclico/metabolismo , Neurônios Motores/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Células Ganglionares da Retina/metabolismo , Medula Espinal/metabolismo , Animais , Transporte Biológico/fisiologia , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Membrana Celular/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , Eletrofisiologia , Fatores de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Nervos Periféricos/citologia , Nervos Periféricos/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptor do Fator Neutrófico Ciliar , Medula Espinal/citologiaRESUMO
Recent studies suggest that interactions between neurons, glial cells and endothelial cells are critical in determining the structure of the retina and the optic nerve. Dysregulation of these interactions can lead to disruption of retinal architecture and impairment of vision.
Assuntos
Astrócitos/metabolismo , Fatores de Crescimento Endotelial/metabolismo , Gânglios/metabolismo , Linfocinas/metabolismo , Neurônios/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Retina/crescimento & desenvolvimento , Retina/metabolismo , Animais , Astrócitos/citologia , Axônios/fisiologia , Vasos Sanguíneos/crescimento & desenvolvimento , Células Epiteliais , Gânglios/citologia , Humanos , Recém-Nascido , Modelos Biológicos , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Nervo Óptico/citologia , Oxigênio , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Retina/citologia , Retinopatia da Prematuridade/etiologia , Retinopatia da Prematuridade/prevenção & controle , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Visão OcularRESUMO
Many studies have shown that myelin in the central nervous system strongly inhibits the regeneration of axons, so it comes as a surprise to discover that adult neurons transplanted into the brain rapidly extend their axons through myelinated pathways.
Assuntos
Axônios/fisiologia , Gânglios Espinais/fisiologia , Regeneração Nervosa/fisiologia , Animais , Astrócitos/fisiologia , Humanos , Modelos BiológicosRESUMO
BACKGROUND: We have shown previously that oligodendrocytes and their precursors require signals from other cells in order to survive in culture. In addition, we have shown that about 50% of the oligodendrocytes produced in the developing rat optic nerve normally die, apparently in a competition for the limiting amounts of survival factors. We have hypothesized that axons may control the levels of such oligodendrocyte survival factors and that the competition-dependent death of oligodendrocytes serves to match their numbers to the number of axons that they myelinate. Here we test one prediction of this hypothesis - that the survival of developing oligodendrocytes depends on axons. RESULTS: We show that oligodendrocyte death occurs selectively in transected nerves in which the axons degenerate. This cell death is prevented by the delivery of exogenous ciliary neurotrophic factor (CNTF) or insulin-like growth factor I (IGF-1), both of which have been shown to promote oligodendrocyte survival in vitro. We also show that purified neurons promote the survival of purified oligodendrocytes in vitro. CONCLUSION: These results strongly suggest that oligodendrocyte survival depends upon the presence of axons; they also support the hypothesis that a competition for axon-dependent survival signals normally helps adjust the number of oligodendrocytes to the number of axons that require myelination. The identities of these signals remain to be determined.
RESUMO
It now appears that most of the ion channels discovered in glia are similar or identical to their neuronal equivalents. Recent studies show that glial cells can sense and respond to neuronal signals and that neurons may influence both the development and maintenance of ion channel expression of certain glial cells. Although they lack excitability, glia are probably active participants in brain function.
Assuntos
Canais Iônicos/metabolismo , Neuroglia/metabolismo , Animais , Humanos , Neuroglia/ultraestruturaRESUMO
Although glial cells ensheath synapses throughout the nervous system, the functional consequences of this relationship are uncertain. Recent studies suggest that glial cells may promote the formation of synapses and help to maintain their function by providing nerve terminals with energy substrates and glutamate precursors.
Assuntos
Neuroglia/fisiologia , Sinapses/fisiologia , Animais , Encéfalo/fisiologia , Encéfalo/ultraestrutura , Ácido Glutâmico/metabolismo , Mamíferos , Modelos Neurológicos , Terminações Nervosas/fisiologia , Neurotransmissores/fisiologiaRESUMO
Here, we review progress in our understanding of neuronal and glial cell biology during the past ten years, with an emphasis on glial cell fate specification, apoptosis, the cytoskeleton, neuronal polarity, synaptic vesicle recycling and targeting, regulation of the cytoskeleton by extracellular signals, and neuron-glia interactions.