RESUMEN
Extensive evidence suggests that BDNF regulates neural function and architecture after depolarization. Expression of BDNF is increased after depolarization, and the ability of BDNF to modulate synaptic function is well documented. To further investigate BDNF signaling after activity, we analyzed the effects of depolarization or BDNF treatment on receptor mRNA expression in cultured basal forebrain neurons. Levels of mRNA coding for the cognate BDNF receptor, trkB, as well as the common neurotrophin receptor, p75, were quantitated simultaneously using a sensitive solution hybridization technique. Depolarization or BDNF treatment increased p75 mRNA expression 94% and 195%, respectively. In contrast, trkB message decreased 23% after depolarization but was unchanged by BDNF treatment. Together, these changes resulted in significant increases in the p75/trkB ratio after depolarization or BDNF treatment that could alter BDNF binding or signal transduction.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/farmacología , Neuronas/fisiología , Receptor de Factor de Crecimiento Nervioso/genética , Receptor trkB/genética , Animales , Células Cultivadas , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Hipocampo/citología , Potenciales de la Membrana/efectos de los fármacos , Plasticidad Neuronal/fisiología , Neuronas/citología , Hibridación de Ácido Nucleico/métodos , Cloruro de Potasio/farmacología , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Sensibilidad y Especificidad , Estimulación QuímicaRESUMEN
Previous studies suggest that oligodendrocytes express trophic molecules, including neurotrophins. These molecules have been shown to influence nearby neurons. To determine whether neuronal signals may, in turn, affect oligodendrocyte-derived trophins, we examined regulation of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) mRNA expression in cultured oligodendrocytes of the basal forebrain. Neuronal signals had distinct effects on individual neurotrophins. KCl elicited increases in BDNF mRNA, but did not affect expression of NGF or NT-3. The cholinergic agonist, carbachol, increased expression of NGF, but did not affect expression of BDNF or NT-3. Glutamate elicited a decrease in BDNF, but did not affect expression of NGF or NT-3. This glutamate effect is not due to toxicity, since the number of total cells was unchanged, while the number of mature myelin basic protein positive (MBP+) cells increased. Our observations suggest that individual neuronal signals distinctly influence the trophic function of oligodendrocytes.
Asunto(s)
Núcleo Basal de Meynert/metabolismo , Comunicación Celular/fisiología , Factores de Crecimiento Nervioso/genética , Neuronas/metabolismo , Oligodendroglía/metabolismo , Transducción de Señal/fisiología , Animales , Animales Recién Nacidos , Núcleo Basal de Meynert/citología , Núcleo Basal de Meynert/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/genética , Carbacol/farmacología , Comunicación Celular/efectos de los fármacos , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Agonistas Colinérgicos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Ácido Glutámico/farmacología , Factor de Crecimiento Nervioso/genética , Neuronas/citología , Neuronas/efectos de los fármacos , Neurotrofina 3/genética , Oligodendroglía/citología , Oligodendroglía/efectos de los fármacos , Potasio/farmacología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
Recent studies suggest that the injured adult spinal cord responds to brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) with enhanced neuron survival and axon regeneration. Potential neurotrophin sources and cellular localization in spinal cord are largely undefined. We examined glial BDNF localization in normal cord and its temporospatial distribution after injury in vivo. We used dual immunolabeling for BDNF and glial fibrillary acidic protein (GFAP) in astrocytes, adenomatous polyposis coli tumor suppressor protein (APC) for oligodendrocytes or type III CDH receptor (OX42) for microglia/macrophages. In normal cord, small subsets of astrocytes and microglia/macrophages and most oligodendrocytes exhibited BDNF-immunoreactivity. Following injury, the number of BDNF-immunopositive astrocytes and microglia/macrophages increased dramatically at the injury site over time. Most oligodendrocytes contained BDNF 1 day and 1 week following injury, but APC-positive cells were largely absent at the injury site 6 weeks postinjury. Glial BDNF-immunolabeling was also examined 10 and 20 mm from the wound. Ten millimeters from the lesion, astrocyte and microglia/macrophage BDNF-immunolabeling resembled that at the injury at all times examined. Twenty millimeters from injury, BDNF localization in all three glial subtypes resembled controls, regardless of time postlesion. Our findings suggest that in normal adult cord, astrocytes, oligodendrocytes, and microglia/macrophages play roles in local trophin availability and in trophin-mediated injury and healing responses directly within and surrounding the wound site.
Asunto(s)
Astrocitos/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Microglía/metabolismo , Oligodendroglía/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Proteína de la Poliposis Adenomatosa del Colon , Animales , Especificidad de Anticuerpos , Astrocitos/citología , Recuento de Células , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Técnicas para Inmunoenzimas , Inmunohistoquímica , Macrófagos/citología , Macrófagos/metabolismo , Microglía/citología , Oligodendroglía/citología , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología , Médula Espinal/metabolismo , Médula Espinal/patología , Traumatismos de la Médula Espinal/patología , Factores de TiempoRESUMEN
Potential roles of trophins in the normal and injured spinal cord are largely undefined. However, a number of recent studies suggest that adult spinal cord expresses neurotrophin receptors and responds to the neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3), particularly after injury. The data indicate that trophins may enhance regrowth after damage and may represent a new therapeutic approach to injury. Neurotrophins are reportedly present in the spinal cord, but the cellular localization is unknown. This information is critical to begin delineating mechanisms of actions. To approach this problem, we examined whether spinal cord glia express BDNF and NT3 in vivo and have begun to define cellular distribution. Specific antibodies directed against the neurotrophins were utilized to visualize neurotrophin protein. Initial studies indicated that small cells in the white matter of adult rat spinal cord express BDNF and NT3. Large neurotrophin-positive neurons were also identified in the ventral cord. To identify the neurotrophin-positive cells, co-localization studies were performed utilizing neurotrophin polyclonal antisera together with monoclonal antibodies directed against the astrocyte marker, glial fibrillary acidic protein (GFAP). In the white matter of adult spinal cord, GFAP-positive and GFAP-negative cells expressed BDNF and NT3. Our study suggests that astrocyte and non-astrocyte cells provide trophic support to the adult spinal cord.
Asunto(s)
Astrocitos/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Factores de Crecimiento Nervioso/genética , Neuroglía/metabolismo , Neuronas/metabolismo , Médula Espinal/metabolismo , Animales , Biomarcadores , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteína Ácida Fibrilar de la Glía/análisis , Inmunohistoquímica , Factores de Crecimiento Nervioso/metabolismo , Neuronas/clasificación , Neuronas/citología , Neurotrofina 3 , Ratas , Ratas Sprague-Dawley , Transcripción GenéticaRESUMEN
Subcortical damage at birth often produces more severe deficits than similar lesions in an adult. In the present study, effects of unilateral electrolytic hippocampal ablations made on postnatal day 1 or in 3-month-old adult rats, were compared. Exploratory behavior and spatial navigation in the Morris water maze (MWM) were assessed 8 and 20 weeks after hippocampal damage. Rats with neonatal damage did not respond to novelty in the environment and did not learn to find the hidden platform in the MWM. Rats lesioned as adults did learn the water maze task, but slower than controls. We hypothesized that behavioral deficits observed in rats lesioned at birth, may be due, in part, to neurochemical dysfunction of the contralateral hippocampus. Specifically, cholinergic and GABAergic neurotransmission were assessed by measuring choline-acetyltransferase (ChAT) and GABAdecarboxylase (GAD) activity. In addition, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNA levels were assayed in the remaining (contralateral) hippocampus. Of these molecules, only BDNF gene expression was significantly reduced (by 30%) at 8 and 20 weeks after neonatal and adult unilateral ablation. The similar reduction in BDNF mRNA in both treatment groups does not correspond with the lesion's differential effect on memory function. However, the more severe learning impairment after neonatal lesion may reflect increased dependence on trophins during development.
Asunto(s)
Animales Recién Nacidos/fisiología , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Factor Neurotrófico Derivado del Encéfalo/genética , Expresión Génica/fisiología , Hipocampo/fisiología , Percepción Espacial/fisiología , Envejecimiento/metabolismo , Animales , Biomarcadores , Colina O-Acetiltransferasa/metabolismo , Conducta Exploratoria/efectos de los fármacos , Femenino , Glutamato Descarboxilasa/metabolismo , Hipocampo/anatomía & histología , Aprendizaje por Laberinto/efectos de los fármacos , Factores de Crecimiento Nervioso/biosíntesis , Embarazo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-DawleyRESUMEN
We have examined the role of the p75 neurotrophin receptor in survival-promoting effects of nerve growth factor (NGF) and neurotrophin-3 (NT-3) on cultured Purkinje cells. Previously, we showed that NGF promotes Purkinje cell survival in conjunction with (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), an agonist of metabotropic excitatory amino acid receptors, whereas NT-3 by itself increases cell number. We now present evidence that p75 plays different roles in Purkinje cell responses to the two neurotrophins. A metabotropic receptor of the mGluR1 subtype may interact with p75 function, so as to regulate Purkinje cell responsiveness to neurotrophins. When cerebellar cultures were grown for 6 days in the presence of ACPD and a mutant form of NGF that does not bind to p75, no increase in Purkinje cell number was observed. Moreover, the survival-promoting effect of wild-type NGF and ACPD could be inhibited by a neutralizing antiserum to p75 or by a pyrazoloquinazolinone inhibitor of neurotrophin binding to p75. In contrast, the response to NT-3 was potentiated by anti-p75 treatment and by the quinazolinone. These data indicate the mediation of p75 in the trophic response to NGF-ACPD and a negative modulatory role of p75 in the action of NT-3. To probe the role of ACPD in the p75-dependent response to NGF, metabotropic receptor subtype-specific ligands were tested. The pattern of agonist specificity implicated the mGluR1 subtype, a receptor that is expressed at high levels by Purkinje cells and linked to activation of protein kinase C (PKC). Down-regulation or blockade of PKC abolished the response to NGF-ACPD. Consistent with the opposite roles of p75 in effects of the two neurotrophins, blockade of mGluR1 or PKC potentiated the survival response elicited by NT-3. In sum, our data suggest that afferent excitatory transmitters activate specific metabotropic receptors to elicit a p75-mediated action of NGF. NT-3 acts on Purkinje cells by a different mechanism that is not absolutely p75-dependent and that is reduced by neurotrophin access to p75 and metabotropic receptor activity.
Asunto(s)
Factores de Crecimiento Nervioso/farmacología , Células de Purkinje/citología , Receptores de Glutamato Metabotrópico/fisiología , Receptores de Factor de Crecimiento Nervioso/fisiología , Animales , Supervivencia Celular/efectos de los fármacos , Cicloleucina/análogos & derivados , Cicloleucina/farmacología , Inhibidores Enzimáticos/farmacología , Glicina/análogos & derivados , Glicina/farmacología , Fármacos Neuroprotectores/farmacología , Neurotrofina 3 , Proteína Quinasa C/metabolismo , Células de Purkinje/efectos de los fármacos , Células de Purkinje/enzimología , Ratas , Ratas Sprague-Dawley , Receptor de Factor de Crecimiento Nervioso , Receptores de Glutamato Metabotrópico/agonistas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
It is well recognized that the neurotrophin family of factors as well as neurotransmitters play critical roles in the ontogeny of the brain. Moreover, a growing literature suggests that these environmental signals do not operate individually, but interact in critical ways to enhance maturation. This review focuses on three brain systems where this collaboration is particularly evident: the cerebellum, the basal forebrain-hippocampus and locus coeruleus-hippocampus. The material presented indicates that cross-talk between neurotransmitters and neurotrophins may be a mechanism common to the development of multiple neuronal groups throughout the central nervous system. Moreover, this cross-talk appears to involve the interaction of both neuronal and glial cell populations.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Factores de Crecimiento Nervioso/fisiología , Neuroglía/fisiología , Neuronas/fisiología , Neurotransmisores/fisiología , Animales , Aminoácidos Excitadores/fisiología , Transducción de SeñalRESUMEN
The hippocampus and septum play central roles in one of the most important spheres of brain function: learning and memory. Although their topographic connections have been known for two decades and topography may be critical for cognitive functions, the basis for hippocamposeptal topographic projection is unknown. We now report for the first time that Elf-1, a membrane-bound eph family ligand, is a candidate molecular tag for the genesis of the hippocamposeptal topographic projection. Elf-1 is expressed in an increasing gradient from dorsal to ventral septum. Furthermore, Elf-1 selectively allows growth of neurites from topographically appropriate lateral hippocampal neurons, while inhibiting neurite outgrowth by medial hippocampal neurons. Complementary to the expression of Elf-1, an eph family receptor, Bsk, is expressed in the hippocampus in a lateral to medial gradient, consistent with a function as a receptor for Elf-1. Further, Elf-1 specifically bound Bsk, eliciting tyrosine kinase activity. We conclude that the Elf-1/Bsk ligand-receptor pair exhibits traits of a chemoaffinity system for the organization of hippocamposeptal topographic projections.
Asunto(s)
Hipocampo/embriología , Proteínas/metabolismo , Tabique Pelúcido/embriología , Animales , Efrina-A2 , Expresión Génica , Hibridación in Situ , Ratones , Proteínas de Neoplasias , Neuritas/ultraestructura , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero/genética , Ratas , Transducción de Señal , Sinapsis/ultraestructura , Familia-src QuinasasRESUMEN
It is generally assumed that central nervous system injury sustained during development produces less severe behavioral deficits than damage in the adult, due to increased plasticity of the immature brain. However, developmental plasticity may also exacerbate deficits, presumably through formation of anomalous connections. Previous studies showed that after unilateral neonatal, but not adult, electrolytic hippocampal lesion spatial memory is severely impaired. To determine whether the memory deficit is correlated with anatomical changes in a major hippocampal afferent system, the septal input, the anterograde tracer Phaeseolus vulgaris leucoagglutinin was injected into the medial septum 2 months after unilateral neonatal hippocampal lesion. The density of septal fiber projections into the entorhinal cortex (EC) was found to be increased. Choline-acetyltransferase activity increased significantly in the EC 2 months postlesion, suggesting that septal cholinergic fibers are sprouting. Finally, nerve growth factor (NGF), which can mediate sprouting, was measured in the EC, NGF protein increased transiently 7 to 12 days postlesion in the ipsilateral EC, suggesting that increased trophic support is associated with growth of septal afferents into the EC. Thus, neonatal hippocampal lesion causes a reorganization of axonal connections associated with elevated NGF in the target region of the increased septal input. Moreover, since previous studies showed that the neonatal lesion is accompanied by a spatial memory deficit, this plasticity may compromise function of the remaining circuitry.
Asunto(s)
Animales Recién Nacidos/fisiología , Corteza Entorrinal/fisiopatología , Hipocampo/patología , Hipocampo/fisiopatología , Plasticidad Neuronal , Tabique Pelúcido/patología , Transmisión Sináptica , Animales , Encefalopatías/patología , Encefalopatías/fisiopatología , Catálisis , Corteza Entorrinal/patología , Factores de Crecimiento Nervioso/metabolismo , Regeneración Nerviosa , Ratas , Tabique Pelúcido/fisiopatologíaRESUMEN
Neurotrophins regulate neuronal survival and phenotypic differentiation. Recent evidence also suggests a role in the modulation of synaptic activity. Using neuronal cell cultures from embryonic hippocampus, we previously found that application of brain-derived neurotrophic factor rapidly enhanced synaptic transmission. We now report that application of neurotrophin-4, another ligand for the trkB neurotrophin receptor, was equally effective in enhancing synaptic currents. In contrast, nerve growth factor, neurotrophin-3, basic fibroblast growth factor and epidermal growth factor did not share this action. Our results suggest that activation of trkB receptors plays a selective role in the regulation of synaptic efficacy in the hippocampus.
Asunto(s)
Hipocampo/fisiología , Neuronas/fisiología , Receptores de Factor de Crecimiento Nervioso/fisiología , Transmisión Sináptica/fisiología , Animales , Células Cultivadas , Hipocampo/citología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Receptor de Factor Neurotrófico Ciliar , Factores de TiempoRESUMEN
To analyze cell-specific brain gene expression, we have developed a PCR-based subtractive hybridization cloning method utilizing trace starting material, allowing isolation of novel genes expressed under specific conditions. Our previous studies indicated that local substantia nigra (SN) type 1 astrocytes elaborate an array of trophic molecules which support the survival of SN dopaminergic neurons. Therefore, the current study focused on astrocyte gene expression utilizing a type 1 astrocyte-enriched cDNA library. We report initial characterization of a novel cDNA, designated AT1-46, that is preferentially expressed in the olfactory-limbic system of the adult rat brain. Although AT1-46 is expressed widely in the periphery, it is regulated both developmentally and in a cell-specific fashion in the brain. Structurally, AT1-46 is predicted to encode a highly alpha-helical molecule with several domains of potential coiled coil formation, and exhibits a 28% amino acid sequence identity with the intermediate filament-associated protein, trichohyalin.
Asunto(s)
ADN Complementario/biosíntesis , Sistema Límbico/metabolismo , Bulbo Olfatorio/metabolismo , Secuencia de Aminoácidos , Animales , Astrocitos/metabolismo , Secuencia de Bases , Northern Blotting , Células Cultivadas , Clonación Molecular , Biblioteca de Genes , Hibridación in Situ , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , ARN/biosíntesis , ARN/aislamiento & purificación , Ratas , Homología de Secuencia de AminoácidoRESUMEN
Glial cell line-derived neurotrophic factor (GDNF) promotes survival of midbrain dopaminergic neurons and motoneurons. Expression of GDNF mRNA in cerebellum raises the possibility that cells within this structure might also respond to GDNF. To examine potential trophic activities of GDNF, dissociated cultures of gestational day 18 rat cerebellum were grown for < or = 21 days in the presence of factor. GDNF increased Purkinje cell number without affecting the overall number of neurons or glial cells. A maximal response (50% above control) was elicited with GDNF at 1 pg/ml. Effects of GDNF on Purkinje cell differentiation were examined by scoring the morphologic maturation of cells in treated and control cultures. GDNF increased the proportion of Purkinje cells that displayed relatively mature morphologies, characterized by dendritic thickening and the development of spines and filopodial extensions. Morphologic maturation of the overall neuronal population was unaffected. In sum, our data indicate that GDNF is a potent survival and differentiation factor for Purkinje cells, the efferent neurons of cerebellar cortex. Together with its other actions, these findings raise the possibility that GDNF might be a critical trophic factor at multiple loci in neuronal circuits that control motor function.
Asunto(s)
Cerebelo/citología , Proteínas del Tejido Nervioso/farmacología , Células de Purkinje/citología , Animales , Calbindinas , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Embrión de Mamíferos , Factor Neurotrófico Derivado de la Línea Celular Glial , Proteína Ácida Fibrilar de la Glía/análisis , Humanos , Cinética , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/análisis , Fosfopiruvato Hidratasa/análisis , Células de Purkinje/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Proteína G de Unión al Calcio S100/análisisRESUMEN
Although neurotrophins are primarily associated with long-term effects on neuronal survival and differentiation, recent studies have shown that acute changes in synaptic transmission can also be produced. In the hippocampus, an area critically involved in learning and memory, we have found that brain-derived neurotrophic factor (BDNF) rapidly enhanced synaptic efficacy through a previously unreported mechanism--increased postsynaptic responsiveness via a phosphorylation-dependent pathway. Within minutes of BDNF application to cultured hippocampal neurons, spontaneous firing rate was dramatically increased, as were the frequency and amplitude of excitatory postsynaptic currents. The increased frequency of postsynaptic currents resulted from the change in presynaptic firing. However, the increased amplitude was postsynaptic in origin because it was selectively blocked by intracellular injection of the tyrosine kinase receptor (Ntrk2/TrkB) inhibitor K-252a and potentiated by injection of the phosphatase inhibitor okadaic acid. These results suggest a role for BDNF in the modulation of synaptic transmission in the hippocampus.
Asunto(s)
Hipocampo/fisiología , Proteínas del Tejido Nervioso/farmacología , Neuronas/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Sinapsis/fisiología , Transmisión Sináptica/efectos de los fármacos , Animales , Factor Neurotrófico Derivado del Encéfalo , Carbazoles/farmacología , Células Cultivadas , Embrión de Mamíferos , Éteres Cíclicos/farmacología , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Alcaloides Indólicos , Cinética , Factores de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Ácido Ocadaico , Técnicas de Placa-Clamp , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Sinapsis/efectos de los fármacos , Factores de TiempoRESUMEN
Extensive evidence indicates that the survival and development of neurons is dependent on neurotrophins. However, the factors potentially required for glial development and function, and the sites of synthesis, are not well defined. To investigate the potential role of neurotrophins in glial development in vivo, we studied the trk family of receptors and their cognate neurotrophins in the postnatal rat optic nerve using reverse transcription-polymerase chain reaction. Our results indicate that trk A, B and C messenger RNAs are expressed throughout development, and in adulthood. Both trk B and trk C expression decreased during development. However, trk C expression decreased most markedly, reaching barely detectable levels by day 90. These findings suggest that neurotrophins can affect both immature and mature glial function and that their actions may be regulated through the modulation of putative receptors. To determine whether alternatively spliced forms of trk C potentially mediate neurotrophin-3 actions, we assessed expression of the different trk C isoforms. We employed reverse transcription-polymerase chain reaction using primers that selectively amplify the extracellular or intracellular domains. Optic nerve expressed both the full-length receptor and one form containing an insertion in the tyrosine kinase domain. In addition, the expression of the insert splice variant was developmentally regulated. Our observations suggest that, in glia, actions of neurotrophin-3 are probably mediated through the full-length receptor and that selected alternatively spliced forms may also be involved in trk C receptor function. To determine whether glia at different stages of differentiation elaborate neurotrophins, we analysed expression of nerve growth factor, brain-derived neurotrophic factor, neurotrophins-3 and -4/5 in the optic nerve during development. Messenger RNAs for all the neurotrophins were detected at all postnatal ages, suggesting that progenitor cells, immature and mature glia are potential sources of neurotrophins. However, neurotrophin expression was not developmentally regulated. The invariant neurotrophin messenger RNA levels, and the changing expression of trk B and trk C during ontogeny, suggest that trophic regulation of glial development is primarily governed through modulation of receptor expression.
Asunto(s)
Factores de Crecimiento Nervioso/farmacología , Neuroglía/efectos de los fármacos , Nervio Óptico/efectos de los fármacos , Proteínas Tirosina Quinasas/farmacología , Animales , Secuencia de Bases , Southern Blotting , Expresión Génica , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-DawleyRESUMEN
A number of studies have begun to describe the effects of nerve growth factor (NGF) and the closely related brain-derived neurotrophic factor (BDNF) on the function of basal forebrain neurons. Little is known, however, about the effects of neurotrophins on membrane calcium conductances, which may play a role in growth factor signal transduction as well as regulation of neuronal excitability. Using the whole-cell patch-clamp technique, we investigated the effects of both NGF and BDNF on voltage-gated Ca(2+)-channel currents in cultured embryonic basal forebrain neurons. Exposure for 4-6 d to NGF significantly increased both the L-type and N-type components of the whole-cell current. Conversely, similar exposure to BDNF had no effect on Ca(2+)-channel currents. Consequently, one of the important effects of NGF may be to enhance calcium entry via voltage-dependent channels.
Asunto(s)
Canales de Calcio/fisiología , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/farmacología , Neuronas/fisiología , Prosencéfalo/fisiología , Potenciales de Acción , Animales , Factor Neurotrófico Derivado del Encéfalo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Células Cultivadas , Medio de Cultivo Libre de Suero , Dihidropiridinas/antagonistas & inhibidores , Embrión de Mamíferos , Cinética , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , omega-Conotoxina GVIARESUMEN
The survival and development of cerebellar neurons are under the control of interacting epigenetic signals. In the present study, we have examined interactive effects of nerve growth factor (NGF) and acetylcholine on in vitro cerebellar Purkinje cell survival. In initial experiments, dissociated rat cerebellar cultures were grown for 6-7 days in the presence of NGF and the stable cholinergic agonist carbachol. Simultaneous exposure to carbachol and NGF selectively increased Purkinje cell number, whereas neither agent was effective when tested alone. The increase in survival was blocked by the muscarinic antagonists atropine (0.1 microM) and pirenzepine (10 nM), but not by methoctramine (25 nM). Nicotine had no effect on survival when tested alone or in combination with NGF. The cerebellar cultures exhibited cholinergic neuronal traits: high-affinity choline uptake, and choline acetyltransferase and acetylcholinesterase activities. To determine whether transmitter produced in vitro triggers Purkinje responsiveness to NGF, cells were exposed to physostigmine, an acetylcholinesterase inhibitor. Physostigmine alone induced an atropine-sensitive increase in cell survival that was enhanced in the presence of NGF. These data suggest that the early expression of cholinergic traits plays a role in Purkinje development. Activation of muscarinic receptors triggers enhanced Purkinje survival in the presence of NGF.
Asunto(s)
Acetilcolina/fisiología , Supervivencia Celular/fisiología , Cerebelo/embriología , Células de Purkinje/fisiología , Receptores Muscarínicos/fisiología , Acetilcolina/farmacología , Acetilcolinesterasa/metabolismo , Animales , Atropina/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colina O-Acetiltransferasa/metabolismo , Interacciones Farmacológicas , Factores de Crecimiento Nervioso/farmacología , Fisostigmina/farmacología , Pirenzepina/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Nerve growth factor (NGF) has been shown to promote the survival of cultured cerebellar Purkinje cells, when tested in conjunction with metabotropic receptor activation. In the present study, we examined the effects of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF). Following in vitro exposure to NT-3 for 6 days, survival of the calbindin positive Purkinje cells was increased, relative to vehicle-treated controls. The total number of neurons was not affected. These observations suggest a specific action of NT-3 on the Purkinje cell population. Moreover, autoradiographic analysis revealed high affinity [125I]NT-3 binding sites, consistent with a direct action of this neurotrophin. Simultaneous treatment with a metabotropic receptor agonist did not alter the NT-3-elicited increase in cell number. This suggests that NT-3 regulates Purkinje cell survival by a mechanism distinct from the NGF response. When tested alone, or in the presence of metabotropic agonist, BDNF did not affect Purkinje cell number.
Asunto(s)
Factores de Crecimiento Nervioso/farmacología , Células de Purkinje/efectos de los fármacos , Animales , Autorradiografía , Sitios de Unión , Recuento de Células/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Factores de Crecimiento Nervioso/metabolismo , Neurotrofina 3 , Células de Purkinje/fisiología , Ratas , Distribución TisularRESUMEN
The recent discovery of glial cell line-derived neurotrophic factor (GDNF) identified a novel trophin that selectively increases survival of substantia nigra dopaminergic neurons, which degenerate in Parkinson's disease. Our previous studies indicated that GDNF RNA can be amplified from cultured rat nigral type 1 astrocytes and from rat striatum in vivo, implying local as well as target trophic support. The current study establishes the regional pattern of GDNF RNA expression in adult human brain. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the highest expression of GDNF mRNA in the human caudate, with low levels in the putamen and no detectable message in the nigra, suggesting that GDNF is a target-derived factor in humans. We also report the isolation of two additional GDNF-related cDNAs, termed astrocyte-derived trophic factors (ATF), which apparently result from differential RNA processing. Sequence analysis of rat ATF-1 revealed a 78-bp deletion corresponding to a loss of 26 amino acids within the prepro region of the predicted GDNF protein. The RNA processing events responsible for ATF-1 formation in rat brain are conserved in humans; we report the isolation of a full-length human ATF-1 homologue. We identified a second alternative transcript, human ATF-2; the transcript encodes a protein which differs in its first 18 amino acids from the predicted mature GDNF and ATF-1 proteins and shares the terminal 115 residues with the other two forms. To begin assessing the biologic significance of multiple transcript expression we characterized the actions of COS-expressed GDNF and ATF-1 cDNAs.(ABSTRACT TRUNCATED AT 250 WORDS)
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Astrocitos/metabolismo , Proteínas de Unión al ADN , Proteínas del Tejido Nervioso/genética , ARN Mensajero/metabolismo , Factor de Transcripción Activador 1 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factor Neurotrófico Derivado de la Línea Celular Glial , Humanos , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Ratas , Ratas Sprague-Dawley , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Differential regulation of individual neurotrophins by impulse activity potentially allows transformation of instantaneous signalling into diverse, long-lasting neural alterations. To define the temporal profiles of trophin gene expression we examined nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNAs in dissociated cell cultures of rat hippocampus using an improved solution hybridization technique. Traditional methods lack the precision and sensitivity to detect small changes during brief intervals and the facility to process large sample numbers simultaneously. This improved method has now allowed us to better define the dynamics of depolarization-induced changes in expression of individual trophin genes. Using elevated K+ as a depolarizing stimulus, NGF mRNA increased 40% after 48 h. In contrast, BDNF message rose almost 4-fold within 3 h and attained a maximal 6-fold increase within 6 h. Similar increases in BDNF mRNA levels were exhibited following treatment of cultures with glutamate, an excitatory neurotransmitter. To document the sensitivity of BDNF mRNA to depolarizing conditions, we examined expression after K+ withdrawal. BDNF message began decreasing within one hour post-depolarization, and returned to basal levels after 6 h. Observations indicate that BDNF and NGF mRNAs are induced differentially in response to impulse activity; BDNF message is acutely responsive to ongoing changes, whereas NGF mRNA responds more slowly and sluggishly. The physiological implications of this differential regulation are discussed.
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Expresión Génica , Hipocampo/fisiología , Factores de Crecimiento Nervioso/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Neuronas/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo , Células Cultivadas , Embrión de Mamíferos , Ácido Glutámico/farmacología , Cinética , Potenciales de la Membrana/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Cloruro de Potasio/farmacología , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Our studies have been directed to the identification of local, naturally-occurring molecules that support substantia nigra (SN) dopaminergic (DA) neuron survival. We have previously demonstrated that local Type I astrocytes selectively increase the dopaminergic population [30,31]. However, the mechanism of action remains to be defined. To determine whether survival is elicited through diffusible agents, Type I astrocyte conditioned medium (CM) was tested on SN dissociates. After 7 days of exposure to CM, DA neuronal integrity was monitored immunocytochemically with antibody to tyrosine hydroxylase (TH), the DA biosynthetic enzyme, or by TH catalytic assay. CM increased TH+ cell number greater than 2-fold, suggesting that a soluble factor(s) promoted neuron survival. Neurons cultured in serum free medium (SFM) are known to contain few, but detectable numbers of glia [34]. To examine whether CM affected neurons directly, or indirectly through glia, glial populations were stained with antibody against the glial marker, glial fibrillary acidic protein (GFAP). We employed several approaches to define the potential role of glia. Initially, CM was compared to basic fibroblast growth factor (bFGF), a glial mitogen that reportedly enhances nigral DA neuron survival. bFGF enhanced TH activity in our system, as well, but the effect was blocked by the mitotic inhibitor 5-fluorodeoxyuridine (FDUR), which kills dividing glia. In parallel studies CM increased enzyme activity and TH cell number in cultures exhibiting GFAP+ cells. To define the role of these glial cells in the CM effect, we completely eliminated astrocytes in CM-treated cultures employing alpha-aminoadipic acid (AA; 10-30 microM), a specific gliotoxin. At a concentration of AA that eliminated detectable GFAP+ cells, CM continued to elicit a significant increase in TH cell number. These data suggest that, in contrast to effects of bFGF, the DA neurotrophic activity in CM acts directly on nigral neurons to enhance survival.