RESUMEN
Long-term potentiation (LTP) is accompanied by increased spine density and dimensions triggered by signaling cascades involving activation of the neurotrophin brain-derived neurotrophic factor (BDNF) and cytoskeleton remodeling. Chemically-induced long-term potentiation (c-LTP) is a widely used cellular model of plasticity, whose effects on spines have been poorly investigated. We induced c-LTP by bath-application of the N-methyl-d-aspartate receptor (NMDAR) coagonist glycine or by the K(+) channel blocker tetraethylammonium (TEA) chloride in cultured hippocampal neurons and compared the changes in dendritic spines induced by the two models of c-LTP and determined if they depend on BDNF/TrkB signaling. We found that both TEA and glycine induced a significant increase in stubby spine density in primary and secondary apical dendrites, whereas a specific increase in mushroom spine density was observed upon TEA application only in primary dendrites. Both TEA and glycine increased BDNF levels and the blockade of tropomyosin-receptor-kinase receptors (TrkRs) by the nonselective tyrosine kinase inhibitor K-252a or the selective allosteric TrkB receptor (TrkBR) inhibitor ANA-12, abolished the c-LTP-induced increase in spine density. Surprisingly, a blockade of TrkBRs did not change basal spontaneous glutamatergic transmission but completely changed the synaptic plasticity induced by c-LTP, provoking a shift from a long-term increase to a long-term depression (LTD) in miniature excitatory postsynaptic current (mEPSC) frequency. In conclusion, these results suggest that BDNF/TrkB signaling is necessary for c-LTP-induced plasticity in hippocampal neurons and its blockade leads to a switch of c-LTP into chemical-LTD (c-LTD).
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Espinas Dendríticas/fisiología , Hipocampo/metabolismo , Potenciación a Largo Plazo/fisiología , Depresión Sináptica a Largo Plazo/fisiología , Receptor trkB/metabolismo , Transducción de Señal/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo/antagonistas & inhibidores , Células Cultivadas , Espinas Dendríticas/efectos de los fármacos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Glicina/farmacología , Glicinérgicos/farmacología , Hipocampo/citología , Potenciación a Largo Plazo/efectos de los fármacos , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Ratas Wistar , Receptor trkB/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/agonistas , Transducción de Señal/efectos de los fármacos , Tetraetilamonio/farmacologíaRESUMEN
Clinical studies show an evident antidepressive effect of physical exercise and animal research corroborate such evidence. However, the neurobiological mechanisms underlying the antidepressive effect of exercise are not completely understood. Notwithstanding, it is known that exercise increases brain-derived neurotrophic factor (BDNF) expression in the hippocampus similarly to antidepressant drugs. BDNF is synthesized as a precursor molecule that undergoes a proteolytic cleavage to generate either a mature or a truncated isoform. Precursor and mature BDNF are assumed to elicit opposing biological effects in neuroplasticity. In the present study we investigated the effect of voluntary physical activity on precursor and mature brain-derived neurotrophic factor levels and on proBDNF cleavage related genes, p11 and tissue plasminogen activator (tPA), as well as the antidepressive and cognitive effects of voluntary physical activity. Mice had access to mobile or locked running wheels for 28 days and were submitted to forced-swim, tail suspension and water maze tests. Their hippocampi were dissected and analyzed by Western blot and real time RT-PCR. Voluntary physical activity, but not locked wheel exposure, induced a robust increase in hippocampal mature BDNF protein levels, as well as in p11 and tPA mRNA expression; and also promoted antidepressive effects and improved learning, when compared with sedentary mice. On the other hand, there were no significant differences between any groups in the expression of precursor or truncated isoforms of BDNF. Our data suggest that the antidepressive effect of the physical exercise may depend, at least in part, on changes in BDNF post-translational processing.
Asunto(s)
Anexina A2/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Condicionamiento Físico Animal/fisiología , Proteínas S100/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Animales , Anexina A2/genética , Western Blotting , Depresión/metabolismo , Hipocampo/metabolismo , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Procesamiento Proteico-Postraduccional , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas S100/genética , Activador de Tejido Plasminógeno/genéticaRESUMEN
Adducins are a family of proteins found in cytoskeleton junctional complexes, which bind and regulate actin filaments and actin-spectrin complexes. In brain, adducin is expressed at high levels and is identified as a constituent of synaptic structures, such as dendritic spines and growth cones of neurons. Adducin-induced changes in dendritic spines are involved in activity-dependent synaptic plasticity processes associated with learning and memory, but the mechanisms underlying these functions remain to be elucidated. Here, beta-adducin knockout (KO) mice were used to obtain a deeper insight into the role of adducin in these processes. We showed that beta-adducin KO mice showed behavioral, motor coordination and learning deficits together with an altered expression and/or phosphorylation levels of alpha-adducin and gamma-adducin. We found that beta-adducin KO mice exhibited deficits in learning and motor performances associated with an impairment of long-term potentiation (LTP) and long-term depression (LTD) in the hippocampus. These effects were accompanied by a decrease in phosphorylation of adducin, a reduction in alpha-adducin expression levels and upregulation of gamma-adducin in hippocampus, cerebellum and neocortex of mutant mice. In addition, we found that the mRNA encoding beta-adducin is also located in dendrites, where it may participate in the fine modulation of LTP and LTD. These results strongly suggest coordinated expression and phosphorylation of adducin subunits as a key mechanism underlying synaptic plasticity, motor coordination performance and learning behaviors.
Asunto(s)
Conducta Animal/fisiología , Proteínas de Unión a Calmodulina/metabolismo , Destreza Motora/fisiología , Plasticidad Neuronal/fisiología , Sinapsis/fisiología , Animales , Proteínas de Unión a Calmodulina/deficiencia , Proteínas de Unión a Calmodulina/genética , Dendritas/fisiología , Discapacidades para el Aprendizaje/etiología , Potenciación a Largo Plazo , Depresión Sináptica a Largo Plazo , Trastornos Mentales/etiología , Ratones , Ratones Noqueados , Trastornos de la Destreza Motora/etiología , Fosforilación , ARN Mensajero/metabolismoRESUMEN
Alternatively spliced brain-derived neurotrophic factor (BDNF) transcripts are targeted to distinct cellular compartments in neurons but the mechanisms underlying this sorting are unknown. Although only some BDNF isoforms are targeted to dendrites, we have found that the coding region common to all BDNF transcripts contains a constitutively active dendritic targeting signal and that this signal is suppressed in transcripts containing exons 1 or 4, which are restricted to the cell soma and proximal dendrites. This dendritic targeting signal is mediated by translin, an RNA-binding protein implicated in RNA trafficking, and is disrupted by the G196A mutation associated with memory deficits and psychiatric disorders. Molecular modeling and mutational studies indicate that the G196A mutation blocks dendritic targeting of BDNF mRNA by disrupting its interaction with translin. These findings implicate abnormal dendritic trafficking of BDNF mRNA in the pathophysiology of neuropsychiatric disorders linked to the G196A mutation.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Proteínas de Unión al ADN/metabolismo , Dendritas/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo , Animales , Western Blotting , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Compartimento Celular , Línea Celular Tumoral , Células Cultivadas , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Hibridación in Situ , Ratones , Modelos Moleculares , Mutación Missense , Neuronas/citología , Neuronas/metabolismo , Unión Proteica , Interferencia de ARN , Transporte de ARN , ARN Mensajero/química , ARN Mensajero/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Brain-derived neurotrophic factor (BDNF) may exert contrasting effects depending on its different subcellular sites of action (soma, dendrites, axons). These contrasting effects may explain contradictory findings, for example that BDNF may favour or oppose epileptogenesis. We determined the distribution of five BDNF splice variants in the soma and dendrites of rat hippocampal principal neurons, after application of stimuli that prompt BDNF mRNA accumulation in dendrites (epileptogenic seizures). Under basal conditions, no BDNF mRNA splice variant was detectable in dendrites, while specific splice variants were found in dendrites in response to epileptogenic seizures. Three hours after pilocarpine administration, exon VI and exon II splice variants were found in dendrites, while exons I and IV transcripts displayed a strictly somatic localization. Three hours after kainate administration, only exon VI was found in dendrites. These data suggest that the regulated expression of different splice variants may provide a spatial code to ensure the delivery of BDNF to precise destinations in the cell soma or along the dendrites.
Asunto(s)
Empalme Alternativo/fisiología , Factor Neurotrófico Derivado del Encéfalo/genética , Hipocampo/citología , Neuronas/citología , Neuronas/metabolismo , ARN Mensajero/metabolismo , Análisis de Varianza , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Dendritas , Epilepsia/inducido químicamente , Epilepsia/patología , Exones/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Hibridación in Situ , Ácido Kaínico , Masculino , Pilocarpina , Ratas , Ratas Sprague-DawleyRESUMEN
The Landau-Kleffner syndrome, the continuous spikes-waves during slow sleep syndrome and the benign epilepsy of childhood with rolandic spikes are rare childhood epilepsies with unknown etiology. Improvement in patients treated with immunoglobulin suggests an involvement of the immune system. We provide immunohistochemical evidence of autoantibodies against rat brain auditory cortex, brainstem and cerebellum, in children suffering with one or more of these syndromes. Only 1/14 patient was celiac.
Asunto(s)
Autoanticuerpos/biosíntesis , Química Encefálica/inmunología , Enfermedad Celíaca , Síndrome de Landau-Kleffner/inmunología , Adolescente , Animales , Autoanticuerpos/análisis , Tronco Encefálico/inmunología , Enfermedad Celíaca/inmunología , Cerebelo/inmunología , Corteza Cerebral/inmunología , Niño , Preescolar , Epilepsia/inmunología , Humanos , Células de Purkinje/inmunología , RatasRESUMEN
Maturation of the visual cortex is a visual experience-dependent process. It has been shown that visual input triggers changes in N-methyl-D-aspartate receptor (NMDAR) subunit expression in the visual cortex. However, no data are available on the layer distribution of these molecular changes. Here we describe the laminar distribution of the cells expressing the NMDAR subunits NR2A and NR2B in the rat primary visual cortex at postnatal day (P) 21 and 37 using anti-NR2A and anti-NR2B antibodies and a stereological method to count labelled neurons. The percentage of neurons expressing the NR2A subunit in the layers II-VI remained unchanged between P21 and P37 with a slight decrease in layer V. Dark-rearing from P21 to P37 induced a pronounced decrease of the staining intensity and a significant decrease in the percentage of NR2A-expressing neurons. The changes in NR2A expression caused by dark rearing occur at similar levels in layers II-VI. The percentage of NR2B-positive cells in the different cortical layers remains unchanged from P21 to P37. The NR2B pattern was not significantly affected by dark-rearing. Thusly, the expression of NR2A depends upon visual experience after P21.
Asunto(s)
Adaptación a la Oscuridad/fisiología , Regulación hacia Abajo/fisiología , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Privación Sensorial/fisiología , Corteza Visual/crecimiento & desarrollo , Corteza Visual/metabolismo , Envejecimiento/metabolismo , Animales , Recuento de Células , Diferenciación Celular/fisiología , Inmunohistoquímica , Neuronas/citología , Ratas , Ratas Wistar , Transmisión Sináptica/fisiología , Corteza Visual/citología , Percepción Visual/fisiologíaRESUMEN
PURPOSE: To analyze whether the subcellular localization of the messenger RNAs (mRNAs) coding for the neurotrophin brain-derived neurotrophic factor (BDNF), its receptor TrkB, and the alpha and beta subunits of calcium-calmodulin-dependent kinase II (CaMKII) are modified after pilocarpine and kindled seizures. METHODS: Epilepsy models: pilocarpine and kindling. Analysis of mRNA levels in the dendrites: high-resolution, nonradioactive in situ hybridization. RESULTS: Nonstimulated rats: BDNF, TrkB, and CaMKII-beta mRNAs localized in the soma and in the proximal dendrites of hippocampal pyramidal cells, and in the soma only of dentate gyrus (DG) granule cells; CaMKII-alpha mRNA localized throughout the dendritic length in neurons of all hippocampal subfields. Pilocarpine seizures: increased staining levels of CaMKII-alpha mRNA throughout the whole dendritic length in all hippocampal subfields; induction of CaMKII-beta, BDNF, and TrkB mRNAs dendritic targeting in CA1, CA3, and DG neurons. Class 2 kindled seizures: increase in dendritic staining intensity for CaMKII-alpha in CA1, CA3, and DG neurons; induction of dendritic localization of CaMKII-beta, BDNF, and TrkB mRNAs in CA3 neurons. Fully kindled seizures: no change in the subcellular distribution of BDNF, TrkB and CaMKII-beta mRNAs; reduction of CaMKII-alpha mRNA dendritic staining, as compared with unstimulated kindled animals. CONCLUSIONS: Data provide evidence that BDNF, TrkB, and CaMKII-alpha and -beta mRNAs are accumulated in the dendrites of specific hippocampal neurons during pilocarpine seizures and kindling development. The dendritic targeting of these genes may be causally involved in epileptogenesis and thus may represent a new therapeutic target for some forms of partial epilepsy.
Asunto(s)
Dendritas/fisiología , Epilepsia del Lóbulo Temporal/genética , Epilepsia del Lóbulo Temporal/metabolismo , Marcación de Gen , Plasticidad Neuronal/genética , ARN Mensajero/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Convulsivantes , Modelos Animales de Enfermedad , Excitación Neurológica , Masculino , Pilocarpina , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor trkB/genética , Receptor trkB/metabolismo , Valores de Referencia , Convulsiones/inducido químicamente , Convulsiones/etiología , Fracciones Subcelulares/metabolismoRESUMEN
To ascertain whether signaling due to peripheral inflammation affects motoneuron vulnerability, we examined in adult rats the reaction to axonal injury of facial motoneurons primed by muscle inflammation. In this double-hit paradigm, preconditioning was achieved by injections into the facial muscles of the T cell mitogen phytohemagglutinin, which was found in a previous study ( 11 ) to elicit a retrograde response in motoneurons. Facial nerve transection was used as test lesion. Intramuscular injections of saline prior to axotomy were used as control for lectin pretreatment. In rats pretreated with phytohemagglutinin injection, upregulation of the expression of the antiapoptotic bcl-2 gene, examined with in situ hybridization, was significantly higher in facial motoneurons at 2 days postaxotomy compared with saline-injected control cases. After repeated phytohemagglutinin injections followed by nerve transection, induction in facial motoneurons of nitric oxide synthase, revealed by histochemistry and immunohistochemistry, as well as activation of the surrounding microglia, was enhanced at 14 days postaxotomy with respect to the saline-treated control cases. At the same time point, no significant intergroup difference was detected in the intensity of astrocytic activation. At 1 month postaxotomy, stereological cell counts revealed that motoneuron loss was significantly greater in the cases pretreated with phytohemagglutinin than in the saline-treated cases. The data point out that the response of the facial motor nucleus to axonal damage is altered by previous exposure to peripheral inflammation and that such preconditioning stimulus enhances motoneuron vulnerability to nerve injury.
Asunto(s)
Antígenos CD , Antígenos de Neoplasias , Antígenos de Superficie , Proteínas Aviares , Proteínas Sanguíneas , Tronco Encefálico/fisiopatología , Músculos Faciales/inervación , Inflamación/fisiopatología , Neuronas Motoras , Animales , Axotomía , Basigina , Tronco Encefálico/patología , Recuento de Células , Músculos Faciales/inmunología , Proteína Ácida Fibrilar de la Glía/biosíntesis , Inmunohistoquímica , Hibridación in Situ , Inflamación/inducido químicamente , Inflamación/patología , Masculino , Glicoproteínas de Membrana/biosíntesis , Mitógenos , Neuronas Motoras/patología , Neuronas Motoras/fisiología , NADPH Deshidrogenasa/metabolismo , Neuroglía/metabolismo , Neuroglía/patología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Fitohemaglutininas , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Linfocitos T/inmunologíaRESUMEN
OBJECTIVE: To characterize humoral response to cerebellum in patients with gluten ataxia. BACKGROUND: Gluten ataxia is a common neurologic manifestation of gluten sensitivity. METHODS: The authors assessed the reactivity of sera from patients with gluten ataxia (13), newly diagnosed patients with celiac disease without neurologic dysfunction (24), patients with other causes of cerebellar degeneration (11), and healthy control subjects (17) using indirect immunocytochemistry on human cerebellar and rat CNS tissue. Cross-reactivity of a commercial IgG antigliadin antibody with human cerebellar tissue also was studied. RESULTS: Sera from 12 of 13 patients with gluten ataxia stained Purkinje cells strongly. Less intense staining was seen in some but not all sera from patients with newly diagnosed celiac disease without neurologic dysfunction. At high dilutions (1:800) staining was seen only with sera from patients with gluten ataxia but not in control subjects. Sera from patients with gluten ataxia also stained some brainstem and cortical neurons in rat CNS tissue. Commercial anti-gliadin antibody stained human Purkinje cells in a similar manner. Adsorption of the antigliadin antibodies using crude gliadin abolished the staining in patients with celiac disease without neurologic dysfunction, but not in those with gluten ataxia. CONCLUSIONS: Patients with gluten ataxia have antibodies against Purkinje cells. Antigliadin antibodies cross-react with epitopes on Purkinje cells.
Asunto(s)
Ataxia/inmunología , Glútenes/efectos adversos , Anciano , Animales , Formación de Anticuerpos , Calbindinas , Cerebelo/metabolismo , Cerebelo/patología , Femenino , Gliadina/química , Gliadina/genética , Glútenes/inmunología , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Células de Purkinje/metabolismo , Células de Purkinje/patología , Ratas , Ratas Sprague-Dawley , Proteína G de Unión al Calcio S100RESUMEN
To investigate whether motoneurons react to signals deriving from target inflammation, we studied the facial motor nucleus after injections of phytohaemagglutinin in the snout of adult rats. This plant lectin is a tool widely used to induce proliferation and activation of T lymphocytes, and we observed marked lymphocyte infiltration in the injected facial muscles. Retrograde labelling of motoneurons was not detected after peripheral injections of fluorochrome-conjugated phytohaemagglutinin. Nitric oxide synthase, revealed by NADPH-diaphorase histochemistry, OX-42-immunoreactive microglia, and expression of the cell death repressor gene bcl-2, investigated with nonradioactive in situ hybridization and immunohistochemistry, were evaluated in the facial nucleus. Daily phytohaemagglutinin injections for 4 days, mimicking repeated muscle exposure to inflammatory stimuli, resulted after 2-day survival in NADPH-diaphorase induction in motoneurons and marked activation of the surrounding microglia. Quantitative image analysis of NADPH-diaphorase staining, and OX-42 immunoreactivity and microglial cell counts indicated highly significant increases with respect to saline-injected control cases. The occurrence of a neuroprotective retrograde response was evaluated monitoring bcl-2 expression. Following single phytohaemagglutinin administration, bcl-2 mRNA was significantly upregulated at 6 h in facial motoneurons and returned to basal levels at 24 h. Bcl-2 immunoreactivity was markedly upregulated at 24 h and was still significantly higher than in controls at 7 days, when concomitant NADPH-diaphorase induction in motoneurons and microglia activation was also observed. No degenerative features were observed in motoneurons after phytohaemagglutinin injections at the examined time-points. The data point out that local muscle inflammation retrogradely elicits gene activation in motoneurons and their microenvironment.
Asunto(s)
Antígenos CD , Antígenos de Neoplasias , Antígenos de Superficie , Proteínas Aviares , Proteínas Sanguíneas , Nervio Facial/inmunología , Neuronas Motoras/enzimología , Músculo Esquelético/inmunología , Músculo Esquelético/inervación , Miositis/fisiopatología , Animales , Basigina , Nervio Facial/citología , Expresión Génica/inmunología , Linfocitos/inmunología , Masculino , Glicoproteínas de Membrana/análisis , Microglía/química , Microglía/fisiología , Neuronas Motoras/química , Miositis/inducido químicamente , Miositis/inmunología , NADPH Deshidrogenasa/análisis , Óxido Nítrico Sintasa/metabolismo , Fitohemaglutininas , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/análisis , Ratas , Ratas WistarRESUMEN
Celiac disease (CD) is an intestinal malabsorption characterized by intolerance to cereal proteins accompanied by immunological responses to dietary gliadins and tissue transglutaminase, an autoantigen located in the endomysium. Tissue transglutaminase belongs to the family of enzymes that catalyze protein cross-linking reactions and is constitutively expressed in many tissues as well as being activated during apoptosis. The role of gliadins in eliciting the immune response in CD and how transglutaminase is linked to the primary reaction are still unclear. In this work, we report the production and analysis of six phage Ab libraries from the peripheral and intestinal lymphocytes of three CD patients. We were able to isolate Abs to transglutaminase from all intestinal lymphocytes libraries but not from those obtained from peripheral lymphocytes. This is in contrast to Abs against gliadin, which could be obtained from all libraries, indicating that the humoral response against transglutaminase occurs at the local level, whereas that against gliadin occurs both peripherally and centrally. Abs from all three patients recognized the same transglutaminase epitopes with a bias toward the use of the V(H)5 Ab variable region family. The possible role of these anti-transglutaminase Abs in the onset of CD and associated autoimmune pathologies is discussed.
Asunto(s)
Autoanticuerpos/biosíntesis , Autoanticuerpos/genética , Enfermedad Celíaca/enzimología , Enfermedad Celíaca/inmunología , Proteínas de Unión al GTP/inmunología , Transglutaminasas/inmunología , Adulto , Secuencia de Aminoácidos , Afinidad de Anticuerpos , Autoanticuerpos/metabolismo , Autoantígenos/inmunología , Autoantígenos/metabolismo , Unión Competitiva/inmunología , Células Clonales , Mapeo Epitopo , Sangre Fetal/inmunología , Técnica del Anticuerpo Fluorescente Directa , Proteínas de Unión al GTP/metabolismo , Biblioteca de Genes , Reordenamiento Génico de Cadena Pesada de Linfocito B , Reordenamiento Génico de Cadena Ligera de Linfocito B , Vectores Genéticos/inmunología , Humanos , Sueros Inmunes/metabolismo , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/metabolismo , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/metabolismo , Inovirus/genética , Inovirus/inmunología , Datos de Secuencia Molecular , Fibras Musculares Esqueléticas/inmunología , Mutación , Proteína Glutamina Gamma Glutamiltransferasa 2 , Solubilidad , Transglutaminasas/metabolismoRESUMEN
Brain-derived neurotrophic factor (BDNF) messenger RNA (mRNA) expression in the rat visual cortex of young and postnatal day 90 (P90) animals is developmentally regulated and influenced by visual experience. In the present paper we compared the expression of BDNF mRNA to the actual changes of BDNF protein occurring during postnatal development and verified whether BDNF protein distribution is controlled by visual activity. To achieve this aim we analysed BDNF mRNA and/or BDNF protein cellular distribution in the rat visual cortex at different postnatal ages by using immunohistochemistry and highly sensitive in situ hybridization. We found that before eye opening (P13), in all cortical layers a large number of visual cortical neurons contain BDNF mRNA with no detectable amount of BDNF protein. At later ages (P23 and P90), the number of BDNF-immunostained cells increases; most neurons are double labelled for BDNF mRNA and protein, and a small group of neurons is labelled only for BDNF protein. The cellular increase of BDNF immunolabelling is blocked in animals deprived of visual experience from birth (dark rearing), with a large population of neurons containing BDNF mRNA but not BDNF protein. This is similar to what is observed before eye opening. Exposure of dark-reared rats to a brief period (2 h) of light restores a good match between BDNF mRNA and BDNF protein cellular expression. We propose that visual experience controls the neuronal content of BDNF mRNA and BDNF protein in developing visual cortex.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso/biosíntesis , ARN Mensajero/biosíntesis , Corteza Visual/metabolismo , Animales , Western Blotting , Factor Neurotrófico Derivado del Encéfalo/genética , Recuento de Células , Colchicina/administración & dosificación , Colchicina/farmacología , Oscuridad , Células HeLa/efectos de los fármacos , Humanos , Técnicas para Inmunoenzimas , Hibridación in Situ , Inyecciones Intraventriculares , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/genética , Plasticidad Neuronal , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Parvalbúminas/análisis , Estimulación Luminosa , Ratas , Ratas Wistar , Privación Sensorial , Método Simple Ciego , Corteza Visual/crecimiento & desarrolloRESUMEN
TrkB mRNA was shown to be localized in the somatodendritic compartment in vitro but no data are currently available on the subcellular distribution of the neurotrophin receptors mRNAs in vivo. Here we describe the subcellular distribution of TrkA, TrkB, TrkC and p75 mRNAs in the adult rat basal forebrain. We find that TrkA, TrkC and p75 mRNAs are restricted to the cell soma but in addition, p75 mRNA labelling extends in average for 8 microm within the proximal dendrites of 34% of the labelled neurons. TrkB mRNA has a somatodendritic distribution in 95% of the labelled neurons reaching variable distances in different neurons (23-84.5 microm) and forebrain regions (mean: 40, 51 and 55 microm for diagonal band, septum and ventral pallidum).
Asunto(s)
Compartimento Celular/fisiología , Dendritas/metabolismo , Prosencéfalo/metabolismo , Receptor de Factor de Crecimiento Nervioso/genética , Receptor trkA/genética , Receptor trkB/genética , Receptor trkC/genética , Animales , Dendritas/ultraestructura , Globo Pálido/citología , Globo Pálido/metabolismo , Hibridación in Situ , Masculino , Prosencéfalo/citología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Núcleos Septales/citología , Núcleos Septales/metabolismoRESUMEN
This study aims to understand the mechanisms of dendritic targeting of brain-derived neurotrophic factor (BDNF) and tyrosine kinase B (TrkB) mRNAs. We show that brief depolarizations are sufficient to induce accumulation of BDNF and TrkB mRNAs in dendrites of hippocampal neurons. Endogenous BDNF, secreted during the KCl stimulation, contributes significantly to the dendritic accumulation of BDNF-TrkB mRNAs. In the absence of depolarization, 1 min pulses of exogenous BDNF are sufficient to induce dendritic accumulation of BDNF-TrkB mRNAs. After binding to TrkB, BDNF exerts this action by activating a PI-3 kinase-dependent pathway. The accumulation of dendritic mRNA by BDNF is not mediated by BDNF-induced neurotransmitter release. Because most hippocampal neurons coexpress BDNF and TrkB receptors, these results show that the subcellular distribution of BDNF-TrkB mRNAs is under the control of an autocrine-paracrine BDNF-TrkB-dependent loop.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/fisiología , Dendritas/metabolismo , Hipocampo/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Receptor trkB/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/farmacología , Células Cultivadas , Dendritas/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Cloruro de Potasio/farmacología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Receptor trkB/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
In this study, we report a comparative analysis of the distribution of brain-derived neurotrophic factor messenger RNA in the binocular primary visual cortex of rats analysed at the end of the critical period for monocular deprivation (postnatal day 35) and during adulthood (postnatal day 90). High-resolution non-isotopic in situ hybridization coupled with Nissl staining allowed to determine the relative number of neurons expressing brain-derived neurotrophic factor messenger RNA. In postnatal day 90 rats, the relative number of neurons positive for brain-derived neurotrophic factor messenger RNA significantly decreases in layer II/III with respect to postnatal day 35 animals, being constant in all the other cortical layers. Moreover, we demonstrate that dark rearing for 22 days, starting from postnatal day 90, determines: (i) a decrease of the overall level of brain-derived neurotrophic factor messenger RNA with a consequent reduction of labelling intensity in all cells throughout cortical layers II-VI; (ii) an increase of cell numbers expressing brain-derived neurotrophic factor messenger RNA in layers IV and V; and (iii) a decreased intensity of staining for brain-derived neurotrophic factor messenger RNA in dendrites after dark rearing. A re-exposure to light for 2 h after the period of darkness almost restores the number of brain-derived neurotrophic factor RNA-positive neurons. We conclude that the maturation of brain-derived neurotrophic factor messenger RNA in neurons of layer II/III goes beyond postnatal days 35-40, which can be considered the end of the critical period [Fagiolini M. et al. (1994) Vis. Res., 34, 709-720]. Moreover, we show that the cellular expression of brain-derived neurotrophic factor messenger RNA is regulated by light in adult rats as well as during development.
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Factor Neurotrófico Derivado del Encéfalo/genética , Regulación del Desarrollo de la Expresión Génica , ARN Mensajero/biosíntesis , Corteza Visual/metabolismo , Animales , Northern Blotting , Oscuridad , Dendritas/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Hibridación in Situ , Luz , Neuronas/metabolismo , ARN Mensajero/genética , Ratas , Privación Sensorial/fisiología , Visión Monocular/genética , Corteza Visual/crecimiento & desarrolloRESUMEN
In the visual cortex, brain-derived neurotrophic factor expression is modulated through glutamate receptors, including the N-methyl-D-aspartate glutamate receptor. It has been proposed that the N-methyl-D-aspartate glutamate receptor subunit composition itself might be regulated by brain-derived neurotrophic factor. Here, we investigated the co-expression of the neurotrophin-4/brain-derived neurotrophic factor receptor TrkB with the N-methyl-D-aspartate glutamate receptor subunits NR1-C1, NR2A and NR2B, on postnatal days 10 and 22 and in the adult rat primary visual cortex. At both postnatal days 10 and 22, TrkB is co-expressed in all cortical layers with the studied N-methyl-D-aspartate glutamate receptor subunits. In the adult, in layers IV-V, co-expression is restricted to a subpopulation of neurons, while in layers II-III, VI nearly all neurons co-express TrkB with NR1-C1, NR2A and NR2B. We conclude that in layers IV-V, the co-expression of TrkB with subunits NR2B and NR2A is developmentally regulated.
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Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Corteza Visual/metabolismo , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/metabolismo , Inmunohistoquímica , Hibridación in Situ , Isoformas de Proteínas/metabolismo , Ratas , Receptor de Factor Neurotrófico Ciliar , Distribución Tisular/fisiologíaRESUMEN
The function and the regulation of the expression of the extracellular matrix molecule tenascin-C during embryonic development are still unclear. In the present study, the expression of tenascin-C was analyzed in the trunk of zebrafish at the end of the first embryonic day. An antiserum raised against a zebrafish tenascin-C (TN-C) fusion protein reacted with 220 (doublet), 200, and 160 KD peptides. In situ hybridization showed that in the zebrafish wild-type embryo, tn-c mRNA was expressed by somites, neural crest cells, roof plate, notochord, hypochord, and tail fin bud. Thus, the expression of tn-c mRNA is an excellent marker for the differentiation of most zebrafish trunk structures. Immunolabelling with the anti-TN-C antibody was detected in the migratory pathway of neural crest cells and in the intersomitic furrows. In situ hybridization analysis of the zebrafish cyclops mutants, lacking the midline floor plate cells, showed normal expression of tn-c mRNA in all trunk structures. Analysis of the floating-head mutant, lacking the notochord, showed that tn-c mRNA expression in neural crest cells, roof plate, and tail fin bud is normal, but it is defective in the somites. By showing that the notochord, but not the floor plate, cells are required for normal tn-c expression in the trunk, this work provides new information on the role played by the embryonic axial structures in the regulation of the expression of tn-c during the development of zebrafish and allows new conclusions about somite patterning in the cyclops and floating-head zebrafish mutants.
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Mutación/fisiología , Tenascina/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Animales , Embrión no Mamífero/metabolismo , Embrión no Mamífero/fisiología , Técnica del Anticuerpo Fluorescente , Sueros Inmunes , Inmunohistoquímica/métodos , Hibridación in Situ , ARN Mensajero/metabolismo , Tenascina/genética , Distribución Tisular/fisiologíaRESUMEN
In this study, we describe the distribution of brain-derived neurotrophic factor messenger RNA in the binocular primary visual cortex of the rat during postnatal development, starting at postnatal day (P) 13. High-resolution non-isotopic in situ hybridization combined with Nissl staining were used to quantify the number of cells expressing brain-derived neurotrophic factor messenger RNA. At P13, most of the cells express brain-derived neurotrophic factor messenger RNA. After eye opening (P14-P15), the relative number of brain-derived neurotrophic factor messenger RNA-positive cells decreases by a factor of two in layer IV, i.e. that receiving the visual input, and in layer V. To verify the hypothesis that light could trigger this decrease, pups were kept in complete darkness from birth. At P22, pups reared in the dark were killed and the visual cortex processed for in situ hybridization and northern blotting. The results obtained in dark-reared animals prove that light deprivation can: (i) decrease the general levels of brain-derived neurotrophic factor messenger RNA, and (ii) increase the relative number of brain-derived neurotrophic factor messenger RNA-positive cells in layers IV and V with respect to control rats. Exposure to light for five days after the period of darkness restored the number of brain-derived neurotrophic factor messenger RNA-positive cells. We conclude that the expression of brain-derived neurotrophic factor messenger RNA in the rat primary visual cortex is regulated during development and that this process is under the control of visual input.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Privación Sensorial/fisiología , Corteza Visual/crecimiento & desarrollo , Corteza Visual/fisiología , Animales , Recuento de Células , Período Crítico Psicológico , Oscuridad , Expresión Génica/fisiología , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Células Piramidales/química , Células Piramidales/fisiología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Visión Monocular/fisiología , Corteza Visual/citologíaRESUMEN
In order to reassess the role of nerve growth factor (NGF) on rat basal forebrain cholinergic neurons (BFCNs) survival and/or phenotype maturation during the early postnatal life, we immunoneutralized NGF in vivo. Hybridoma cells producing the neutralizing anti-NGF monoclonal antibody alphaD11 were implanted in the lateral ventricle of the rat at different postnatal ages (P2, P8 and P15) and the effects on the number and the soma size of cholinacetyltransferase (ChAT) positive neurons were analysed 1, 2 or 3 weeks after the injection. A marked decrease in the number and in the soma size of BFCNs was observed implanting hybridoma cells at P2 and performing the analysis 1 week later. These effects are reversed 3 weeks after the implant of hybridoma cells at P2. At this time point, the levels of alphaD11 antibodies in the brain parenchyma are still in a vast molar excess over endogenous NGF. No effects on BFCNs were observed implanting alphaD11 cells at P15 while LGN neurons showed marked shrinkage. Our results demonstrate that the reduction in the number of ChAT-positive neurons during the first two postnatal weeks of anti-NGF treatment is not due to cell death. We conclude that NGF is not a survival factor for BFCNs, and that the influence of NGF on BFCNs cell maturation during the first 2 postnatal weeks is transient and reversible. Our results on tyrosine kinase (Trk) coexpression, suggest that NGF may cooperate with other factors in the cholinergic phenotype differentiation and maintenance after the second postnatal week.