RESUMEN
The calyx of Held, a large axo-somatic relay synapse containing hundreds of presynaptic active zones, is possibly the largest nerve terminal in the mammalian CNS. Studying its initial growth in-vitro might provide insights into the specification of synaptic connection size in the developing brain. However, attempts to maintain calyces of Held in organotypic cultures have not been fruitful in past studies. Here, we describe an organotypic slice culture method in which calyces of Held form in-vitro. We made coronal brainstem slices with an optimized slice angle using newborn mice in which calyces have not yet formed; the presynaptic bushy cells were genetically labeled using the Math5 promoter. After six to nine days of culturing, we readily observed large Math5-positive nerve terminals in the medial nucleus of the trapezoid body (MNTB), but not in the neighboring lateral superior olive nucleus (LSO). These calyx-like synapses expressed the Ca2+- sensor Synaptotagmin-2 (Syt-2) and the Ca2+ binding protein Parvalbumin (PV), two markers of developing calyces of Held in vivo. Application of the BMP inhibitor LDN-193189 significantly inhibited the growth of calyx synapses, demonstrating the feasibility of long-term pharmacological manipulation using this organotypic culture method. These experiments provide a method for organotypic culturing of calyces of Held, and show that the formation of calyx-like synapses onto MNTB neurons can be preserved in-vitro. Furthermore, our study adds pharmacological evidence for a role of BMP-signaling in the formation of large calyx of Held synapses.
Asunto(s)
Axones/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/análisis , Tronco Encefálico/fisiología , Proteínas del Tejido Nervioso/análisis , Sinapsis/fisiología , Animales , Vías Auditivas , Axones/efectos de los fármacos , Axones/ultraestructura , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Tronco Encefálico/citología , Tronco Encefálico/efectos de los fármacos , Tronco Encefálico/ultraestructura , Ratones , Proteínas del Tejido Nervioso/genética , Técnicas de Cultivo de Órganos/métodos , Parvalbúminas/análisis , Regiones Promotoras Genéticas , Pirazoles/farmacología , Pirimidinas/farmacología , Sinapsis/efectos de los fármacos , Sinapsis/ultraestructura , Sinaptotagmina II/análisisRESUMEN
The homeodomain transcription factor Nkx2.1 (NK2 homeobox 1) controls cell differentiation of telencephalic GABAergic interneurons and oligodendrocytes. Here we show that Nkx2.1 also regulates astrogliogenesis of the telencephalon from embryonic day (E) 14.5 to E16.5. Moreover we identify the different mechanisms by which Nkx2.1 controls the telencephalic astrogliogenesis. In Nkx2.1 knockout (Nkx2.1-/-) mice a drastic loss of astrocytes is observed that is not related to cell death. Further, in vivo analysis using BrdU incorporation reveals that Nkx2.1 affects the proliferation of the ventral neural stem cells that generate early astrocytes. Also, in vitro neurosphere assays showed reduced generation of astroglia upon loss of Nkx2.1, which could be due to decreased precursor proliferation and possibly defects in glial specification/differentiation. Chromatin immunoprecipitation analysis and in vitro co-transfection studies with an Nkx2.1-expressing plasmid indicate that Nkx2.1 binds to the promoter of glial fibrillary acidic protein (GFAP), primarily expressed in astrocytes, to regulate its expression. Hence, Nkx2.1 controls astroglial production spatiotemporally in embryos by regulating proliferation of the contributing Nkx2.1-positive precursors.
Asunto(s)
Astrocitos/metabolismo , Diferenciación Celular , Desarrollo Embrionario , Telencéfalo/metabolismo , Factor Nuclear Tiroideo 1/fisiología , Animales , Astrocitos/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/genética , Ratones , Ratones Noqueados , Telencéfalo/fisiología , Factor Nuclear Tiroideo 1/metabolismoRESUMEN
The NG2(+) glia, also known as polydendrocytes or oligodendrocyte precursor cells, represent a new entity among glial cell populations in the central nervous system. However, the complete repertoire of their roles is not yet identified. The embryonic NG2(+) glia originate from the Nkx2.1(+) progenitors of the ventral telencephalon. Our analysis unravels that, beginning from E12.5 until E16.5, the NG2(+) glia populate the entire dorsal telencephalon. Interestingly, their appearance temporally coincides with the establishment of blood vessel network in the embryonic brain. NG2(+) glia are closely apposed to developing cerebral vessels by being either positioned at the sprouting tip cells or tethered along the vessel walls. Absence of NG2(+) glia drastically affects the vascular development leading to severe reduction of ramifications and connections by E18.5. By revealing a novel and fundamental role for NG2(+) glia, our study brings new perspectives to mechanisms underlying proper vessels network formation in embryonic brains.
Asunto(s)
Neovascularización Fisiológica , Neuroglía/fisiología , Telencéfalo/embriología , Animales , Femenino , Masculino , RatonesRESUMEN
Guidepost cells present at and surrounding the midline provide guidance cues that orient the growing axons through commissures. Here we show that the transcription factor Nkx2.1 known to control the specification of GABAergic interneurons also regulates the differentiation of astroglia and polydendrocytes within the mouse anterior commissure (AC). Nkx2.1-positive glia were found to originate from three germinal regions of the ventral telencephalon. Nkx2.1-derived glia were observed in and around the AC region by E14.5. Thereafter, a selective cell ablation strategy showed a synergistic role of Nkx2.1-derived cells, both GABAergic interneurons and astroglia, towards the proper formation of the AC. Finally, our results reveal that the Nkx2.1-regulated cells mediate AC axon guidance through the expression of the repellent cue, Slit2. These results bring forth interesting insights about the spatial and temporal origin of midline telencephalic glia, and highlight the importance of neurons and astroglia towards the formation of midline commissures.
Asunto(s)
Comisura Anterior Cerebral/embriología , Astrocitos/metabolismo , Neuronas GABAérgicas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Interneuronas/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Comisura Anterior Cerebral/citología , Comisura Anterior Cerebral/metabolismo , Astrocitos/citología , Axones , Movimiento Celular , Electroporación , Embrión de Mamíferos , Neuronas GABAérgicas/citología , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Técnicas In Vitro , Interneuronas/citología , Ratones , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/citología , Neuronas/metabolismo , Telencéfalo/citología , Telencéfalo/embriología , Telencéfalo/metabolismo , Factor Nuclear Tiroideo 1RESUMEN
Neuronal migration disorders such as lissencephaly and subcortical band heterotopia are associated with epilepsy and intellectual disability. DCX, PAFAH1B1 and TUBA1A are mutated in these disorders; however, corresponding mouse mutants do not show heterotopic neurons in the neocortex. In contrast, spontaneously arisen HeCo mice display this phenotype, and our study revealed that misplaced apical progenitors contribute to heterotopia formation. While HeCo neurons migrated at the same speed as wild type, abnormally distributed dividing progenitors were found throughout the cortical wall from embryonic day 13. We identified Eml1, encoding a microtubule-associated protein, as the gene mutated in HeCo mice. Full-length transcripts were lacking as a result of a retrotransposon insertion in an intron. Eml1 knockdown mimicked the HeCo progenitor phenotype and reexpression rescued it. We further found EML1 to be mutated in ribbon-like heterotopia in humans. Our data link abnormal spindle orientations, ectopic progenitors and severe heterotopia in mouse and human.
Asunto(s)
Coristoma/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/fisiología , Mutación/fisiología , Células-Madre Neurales/fisiología , Secuencia de Aminoácidos , Animales , Bromodesoxiuridina , Ciclo Celular/fisiología , Movimiento Celular/fisiología , Corteza Cerebral/citología , Corteza Cerebral/embriología , Lisencefalias Clásicas y Heterotopias Subcorticales en Banda , Proteína Doblecortina , Electroporación , Humanos , Inmunohistoquímica , Malformaciones Arteriovenosas Intracraneales/patología , Intrones/genética , Ratones , Microscopía Confocal , Microtúbulos/fisiología , Mitosis/fisiología , Datos de Secuencia Molecular , Retroelementos/fisiología , Huso Acromático/fisiologíaRESUMEN
The corpus callosum (CC) represents the major forebrain commissure connecting the 2 cerebral hemispheres. Midline crossing of callosal axons is controlled by several glial and neuronal guideposts specifically located along the callosal path, but it remains unknown how these cells acquire their position. Here, we show that the Gli3 hypomorphic mouse mutant Polydactyly Nagoya (Pdn) displays agenesis of the CC and mislocation of the glial and neuronal guidepost cells. Using transplantation experiments, we demonstrate that agenesis of the CC is primarily caused by midline defects. These defects originate during telencephalic patterning and involve an up-regulation of Slit2 expression and altered Fgf and Wnt/ß-catenin signaling. Mutations in sprouty1/2 which mimic the changes in these signaling pathways cause a disorganization of midline guideposts and CC agenesis. Moreover, a partial recovery of midline abnormalities in Pdn/Pdn;Slit2(-/-) embryos mutants confirms the functional importance of correct Slit2 expression levels for callosal development. Hence, Gli3 controlled restriction of Fgf and Wnt/ß-catenin signaling and of Slit2 expression is crucial for positioning midline guideposts and callosal development.
Asunto(s)
Cuerpo Calloso/crecimiento & desarrollo , Factores de Transcripción de Tipo Kruppel/fisiología , Proteínas del Tejido Nervioso/fisiología , Telencéfalo/crecimiento & desarrollo , Agenesia del Cuerpo Calloso/genética , Agenesia del Cuerpo Calloso/fisiopatología , Animales , Encéfalo/crecimiento & desarrollo , Análisis por Conglomerados , Cuerpo Calloso/embriología , Femenino , Inmunohistoquímica , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/fisiología , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Mutación/fisiología , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Técnicas de Cultivo de Órganos , Polidactilia/genética , Embarazo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Telencéfalo/embriología , Regulación hacia Arriba/fisiología , Vía de Señalización Wnt/fisiología , Proteína Gli3 con Dedos de Zinc , beta Catenina/fisiologíaRESUMEN
Major outputs of the neocortex are conveyed by corticothalamic axons (CTAs), which form reciprocal connections with thalamocortical axons, and corticosubcerebral axons (CSAs) headed to more caudal parts of the nervous system. Previous findings establish that transcriptional programs define cortical neuron identity and suggest that CTAs and thalamic axons may guide each other, but the mechanisms governing CTA versus CSA pathfinding remain elusive. Here, we show that thalamocortical axons are required to guide pioneer CTAs away from a default CSA-like trajectory. This process relies on a hold in the progression of cortical axons, or waiting period, during which thalamic projections navigate toward cortical axons. At the molecular level, Sema3E/PlexinD1 signaling in pioneer cortical neurons mediates a "waiting signal" required to orchestrate the mandatory meeting with reciprocal thalamic axons. Our study reveals that temporal control of axonal progression contributes to spatial pathfinding of cortical projections and opens perspectives on brain wiring.
Asunto(s)
Corteza Cerebral/fisiología , Vías Nerviosas/fisiología , Tálamo/fisiología , Factores de Edad , Animales , Axones/fisiología , Tipificación del Cuerpo/genética , Calbindina 2 , Corteza Cerebral/citología , Contactina 2/metabolismo , Proteínas del Citoesqueleto , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica/genética , Glicoproteínas/genética , Proteínas de Homeodominio/genética , Péptidos y Proteínas de Señalización Intracelular , Complejo de Antígeno L1 de Leucocito/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Factores del Dominio POU/genética , Proteínas Represoras/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Semaforinas , Proteínas de Dominio T Box , Tálamo/citología , Factor Nuclear Tiroideo 1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína Wnt3A/genética , Proteínas tau/genéticaRESUMEN
The corpus callosum (CC) plays a crucial role in interhemispheric communication. It has been shown that CC formation relies on the guidepost cells located in the midline region that include glutamatergic and GABAergic neurons as well as glial cells. However, the origin of these guidepost GABAergic neurons and their precise function in callosal axon pathfinding remain to be investigated. Here, we show that two distinct GABAergic neuronal subpopulations converge toward the midline prior to the arrival of callosal axons. Using in vivo and ex vivo fate mapping we show that CC GABAergic neurons originate in the caudal and medial ganglionic eminences (CGE and MGE) but not in the lateral ganglionic eminence (LGE). Time lapse imaging on organotypic slices and in vivo analyses further revealed that CC GABAergic neurons contribute to the normal navigation of callosal axons. The use of Nkx2.1 knockout (KO) mice confirmed a role of these neurons in the maintenance of proper behavior of callosal axons while growing through the CC. Indeed, using in vitro transplantation assays, we demonstrated that both MGE- and CGE-derived GABAergic neurons exert an attractive activity on callosal axons. Furthermore, by combining a sensitive RT-PCR technique with in situ hybridization, we demonstrate that CC neurons express multiple short and long range guidance cues. This study strongly suggests that MGE- and CGE-derived interneurons may guide CC axons by multiple guidance mechanisms and signaling pathways.
Asunto(s)
Axones/fisiología , Movimiento Celular/fisiología , Cuerpo Calloso/embriología , Neuronas GABAérgicas/citología , Animales , Cuerpo Calloso/ultraestructura , Femenino , Neuronas GABAérgicas/ultraestructura , Técnicas In Vitro , Interneuronas/citología , Masculino , Ratones , Ratones Noqueados , Neurogénesis/fisiología , Proteínas Nucleares/genética , Telencéfalo/citología , Telencéfalo/embriología , Factor Nuclear Tiroideo 1 , Factores de Transcripción/genéticaRESUMEN
The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3-deficient mice show several hallmarks of ciliopathies including left-right asymmetry defects and hydrocephalus. Here we show that Rfx3-deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies.
Asunto(s)
Cuerpo Calloso , Proteínas de Unión al ADN , Factor 8 de Crecimiento de Fibroblastos , Factores de Transcripción de Tipo Kruppel , Proteínas del Tejido Nervioso , Neuronas , Factores de Transcripción , Animales , Axones/metabolismo , Axones/fisiología , Cuerpo Calloso/crecimiento & desarrollo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Factor 8 de Crecimiento de Fibroblastos/genética , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Mutantes , Morfogénesis/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuronas/fisiología , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Proteína Gli3 con Dedos de ZincRESUMEN
Subventricular zone (SVZ) progenitors are a hallmark of the developing neocortex. Recent studies described a novel type of SVZ progenitor that retains a basal process at mitosis, sustains expression of radial glial markers, and is capable of self-renewal. These progenitors, referred to here as basal radial glia (bRG), occur at high relative abundance in the SVZ of gyrencephalic primates (human) and nonprimates (ferret) but not lissencephalic rodents (mouse). Here, we analyzed the occurrence of bRG cells in the embryonic neocortex of the common marmoset Callithrix jacchus, a near-lissencephalic primate. bRG cells, expressing Pax6, Sox2 (but not Tbr2), glutamate aspartate transporter, and glial fibrillary acidic protein and retaining a basal process at mitosis, occur at similar relative abundance in the marmoset SVZ as in human and ferret. The proportion of progenitors in M-phase was lower in embryonic marmoset than developing ferret neocortex, raising the possibility of a longer cell cycle. Fitting the gyrification indices of 26 anthropoid species to an evolutionary model suggested that the marmoset evolved from a gyrencephalic ancestor. Our results suggest that a high relative abundance of bRG cells may be necessary, but is not sufficient, for gyrencephaly and that the marmoset's lissencephaly evolved secondarily by changing progenitor parameters other than progenitor type.
Asunto(s)
Callithrix/anatomía & histología , Callithrix/embriología , Ventrículos Laterales/citología , Ventrículos Laterales/embriología , Neocórtex/anatomía & histología , Neuroglía/fisiología , Aminoácidos , Animales , Animales Recién Nacidos , Recuento de Células , Embrión de Mamíferos , Transportador 1 de Aminoácidos Excitadores/metabolismo , Proteínas del Ojo/metabolismo , Hurones , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Antígeno Ki-67/metabolismo , Neocórtex/embriología , Neuroglía/metabolismo , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción SOXB1/metabolismo , Células Madre/fisiologíaRESUMEN
The corpus callosum (CC) is the main pathway responsible for interhemispheric communication. CC agenesis is associated with numerous human pathologies, suggesting that a range of developmental defects can result in abnormalities in this structure. Midline glial cells are known to play a role in CC development, but we here show that two transient populations of midline neurons also make major contributions to the formation of this commissure. We report that these two neuronal populations enter the CC midline prior to the arrival of callosal pioneer axons. Using a combination of mutant analysis and in vitro assays, we demonstrate that CC neurons are necessary for normal callosal axon navigation. They exert an attractive influence on callosal axons, in part via Semaphorin 3C and its receptor Neuropilin-1. By revealing a novel and essential role for these neuronal populations in the pathfinding of a major cerebral commissure, our study brings new perspectives to pathophysiological mechanisms altering CC formation.
Asunto(s)
Axones/metabolismo , Cuerpo Calloso/embriología , Neuronas/metabolismo , Semaforinas/metabolismo , Síndrome Acrocallosal/metabolismo , Síndrome Acrocallosal/patología , Animales , Axones/patología , Línea Celular , Movimiento Celular , Técnicas de Cocultivo , Cuerpo Calloso/citología , Cuerpo Calloso/metabolismo , Humanos , Ratones , Vías Nerviosas/citología , Vías Nerviosas/embriología , Neuronas/citología , Neuropilina-1/metabolismoRESUMEN
The Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins is required for filopodia formation in growth cones and plays a crucial role in guidance cue-induced remodeling of the actin cytoskeleton. In vivo studies with pharmacological inhibitors of actin polymerization have previously provided evidence for the view that filopodia are needed for growth cone navigation in the developing visual pathway. Here we have re-examined this issue using an alternative strategy to generate growth cones without filopodia in vivo by artificially targeting Xena/XVASP (Xenopus homologs of Ena/VASP) proteins to mitochondria in retinal ganglion cells (RGCs). We used the specific binding of the EVH1 domain of the Ena/VASP family of proteins with the ligand motif FP4 to sequester the protein at the mitochondria surface. RGCs with reduced function of Xena/XVASP proteins extended fewer axons out of the eye and possessed dynamic lamellipodial growth cones missing filopodia that advanced slowly in the optic tract. Surprisingly, despite lacking filopodia, the axons navigated along the optic pathway without obvious guidance errors, indicating that the Xena/XVASP family of proteins and filopodial protrusions are non-essential for pathfinding in retinal axons. However, depletion of Xena/XVASP proteins severely impaired the ability of growth cones to form branches within the optic tectum, suggesting that this protein family, and probably filopodia, plays a key role in establishing terminal arborizations.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Conos de Crecimiento/fisiología , Proteínas de Microfilamentos/metabolismo , Mitocondrias/metabolismo , Fosfoproteínas/metabolismo , Seudópodos/fisiología , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Xenopus/embriología , Animales , Células Cultivadas , Proteínas Fluorescentes Verdes , Conos de Crecimiento/metabolismo , Microscopía por VideoRESUMEN
Ena/VASP proteins play important roles in axon outgrowth and guidance. Ena/VASP activity regulates the assembly and geometry of actin networks within fibroblast lamellipodia. In growth cones, Ena/VASP proteins are concentrated at filopodia tips, yet their role in growth cone responses to guidance signals has not been established. We found that Ena/VASP proteins play a pivotal role in formation and elongation of filopodia along neurite shafts and growth cone. Netrin-1-induced filopodia formation was dependent upon Ena/VASP function and directly correlated with Ena/VASP phosphorylation at a regulatory PKA site. Accordingly, Ena/VASP function was required for filopodial formation from the growth cone in response to global PKA activation. We propose that Ena/VASP proteins control filopodial dynamics in neurons by remodeling the actin network in response to guidance cues.
Asunto(s)
Proteínas de Caenorhabditis elegans , Proteínas Portadoras/fisiología , Moléculas de Adhesión Celular/fisiología , Factores de Crecimiento Nervioso/fisiología , Neuronas/fisiología , Fosfoproteínas/fisiología , Seudópodos/fisiología , Citoesqueleto de Actina/metabolismo , Análisis de Varianza , Animales , Anticuerpos/farmacología , Proteínas Sanguíneas/metabolismo , Western Blotting/métodos , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Recuento de Células , Células Cultivadas , Corteza Cerebral/citología , Pollos , Colforsina/farmacología , Citocalasina D/metabolismo , Proteínas del Citoesqueleto/metabolismo , Dendritas/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes , Conos de Crecimiento/efectos de los fármacos , Conos de Crecimiento/metabolismo , Hipocampo/citología , Humanos , Inmunohistoquímica/métodos , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Proteínas de Microfilamentos , Microscopía Electrónica/métodos , Mitocondrias/metabolismo , Netrina-1 , Neuroglía/citología , Neuroglía/efectos de los fármacos , Neuroglía/fisiología , Neuronas/citología , Neuronas/efectos de los fármacos , Fosforilación , Pruebas de Precipitina/métodos , Estructura Terciaria de Proteína/genética , Seudópodos/efectos de los fármacos , Seudópodos/ultraestructura , Factores de Tiempo , Transfección/métodos , Tubulina (Proteína)/metabolismo , Proteínas Supresoras de TumorRESUMEN
Very little is known about the biophysical properties and the lipid or protein composition of membrane domains presumably present in endocytic and biosynthetic organelles. Here we analyzed the membrane composition of late endosomes by suborganellar fractionation in the absence of detergent. We found that the internal membranes of this multivesicular organelle can be separated from the limiting membrane and that each membrane population exhibited a defined composition. Our data also indicated that internal membranes may consist of at least two populations, containing primarily phosphatidylcholine or lysobisphosphatidic acid as major phospholipid, arguing for the existence of significant microheterogeneity within late endosomal membranes. We also found that lysobisphosphatidic acid exhibited unique pH-dependent fusogenic properties, and we speculated that this lipid is an ideal candidate to regulate the dynamic properties of this internal membrane mosaic.
Asunto(s)
Endosomas/ultraestructura , Ácidos Grasos no Esterificados/análisis , Membranas Intracelulares/ultraestructura , Lípidos de la Membrana/análisis , Fosfolípidos/análisis , Animales , Línea Celular , Cricetinae , Endosomas/química , Concentración de Iones de Hidrógeno , Membranas Intracelulares/química , Riñón , Lisofosfolípidos/análisis , Fusión de Membrana , MonoglicéridosRESUMEN
This article reviews the current knowledge of the early onset of the monoaminergic innervation in the developing cerebral cortex in humans and of changes in the distribution of tyrosine hydroxylase (TH) immunoreactivity in different neuronal populations of the developing telencephalon. The early genesis of the central monoaminergic neurons in mammals has led to postulations of a trophic role of monoamines in brain morphogenesis--especially in the cerebral cortex. The developmental effects of amines can be linked to the transient expression of different molecules linked to dopamine or serotonin neurotransmission. We present novel data on the immunocytochemistry of the vesicular monoamine transporter (VMAT2) and of the high-affinity serotonin transporter (SERT) in human fetuses. SERT is a marker of the serotoninergic axons and allows visualization of the serotonin afferents of the raphe in the human telencephalon. In addition, during a restricted time period corresponding to 12-14 postovulatory weeks, we found SERT-immunolabeled fibers in the rostral and caudal limbs of the internal capsule that do not correspond to serotoninergic fibers, but do coincide with the calbindin D28k-labeled thalamocortical fiber tracts. The present observations are correlated with findings in rodents, in which a transient expression of SERT is visible in the thalamocortical axons during early postnatal life. The function of this transporter has been shown to be important for the fine-tuning of cortical sensory maps during the critical period of development of these maps. Although the present observation does not allow ascertainment of which neurons transiently express SERT, it lends support to the notion that serotonin and serotonin uptake could have important developmental roles, during the formation of brain connections in humans, as they have in rodents.
Asunto(s)
Proteínas Portadoras/metabolismo , Corteza Cerebral/enzimología , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso/metabolismo , Neuropéptidos , Serotonina/metabolismo , Animales , Transporte Biológico , Biomarcadores , Corteza Cerebral/embriología , Desarrollo Embrionario y Fetal , Edad Gestacional , Humanos , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Tirosina 3-Monooxigenasa/metabolismo , Proteínas de Transporte Vesicular de Aminas Biógenas , Proteínas de Transporte Vesicular de MonoaminasRESUMEN
We report that lipids contribute to regulate the bidirectional motility of late endocytic compartments. Late endocytic vesicles loaded with cholesterol lose their dynamic properties, and become essentially immobile, including in cells from Niemann-Pick C patients. These vesicles then retain cytoplasmic dynein activity, but seem to be unable to acquire kinesin activity, eventually leading to paralysis. Our data suggest that this defect depends on the small GTPase Rab7, since the motility of vesicles loaded with cholesterol can be restored by the Rab7 inhibitory mutant N125I. Conversely, wild-type Rab7 overexpression mimics the effects of cholesterol on motility in control cells. Consistently, cholesterol accumulation increases the amounts of membrane-associated Rab7, and inhibits Rab7 membrane extraction by the guanine nucleotide dissociation inhibitor. Our observations thus indicate that cholesterol contributes to regulate the Rab7 cycle, and that Rab7 in turn controls the net movement of late endocytic elements. We conclude that motor functions can be regulated by the membrane lipid composition via the Rab7 cycle.
Asunto(s)
Colesterol/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Androstenos/farmacología , Animales , Anticolesterolemiantes/farmacología , Antígenos CD/genética , Antígenos CD/metabolismo , Compartimento Celular , Línea Celular , Cricetinae , Complejo Dinactina , Endocitosis , Endosomas/metabolismo , Proteínas Fluorescentes Verdes , Inhibidores de Disociación de Guanina Nucleótido/farmacología , Células HeLa , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Lisofosfolípidos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Monoglicéridos , Enfermedades de Niemann-Pick/metabolismo , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tetraspanina 30 , Proteínas de Unión a GTP rab7RESUMEN
The disruptive effect of excessive serotonin (5-HT) levels on the development of cortical sensory maps is mediated by 5-HT1B receptors, as shown in barrelless monoamine oxidase A knock-out mice, in which the additional inactivation of 5-HT1B receptors restores the barrels. However, it is unclear whether 5-HT1B receptors mediate their effect on barrel formation by a trophic action or an activity-dependent effect. To test for a possible effect of 5-HT1B receptors on activity, we studied the influence of 5-HT on the thalamocortical (TC) synaptic transmission in layer IV cortical neurons. In TC slices of postnatal day 5 (P5)-P9 neonate mice, we show that 5-HT reduces monosynaptic TC EPSCs evoked by low-frequency internal capsule stimulation and relieves the short-term depression of the EPSC evoked by high-frequency stimulation. We provide evidence that 5-HT decreases the presynaptic release of glutamate: 5-HT reduces similarly the AMPA-kainate and NMDA components and the paired pulse depression of TC EPSCs. We show also that 5-HT1B receptors mediate exclusively the effect of 5-HT: first, the effect of 5-HT on the TC EPSC is correlated with the transient expression of 5-HT1B receptor mRNAs in the ventrobasal thalamic nucleus during postnatal development; second, it is mimicked by a 5-HT1B agonist; third, 5-HT has no effect in 5-HT1B receptor knock-out mice. Our results show that in the developing barrel field of the neonatal mice, 5-HT1B receptors mediate an activity-dependent regulation of the TC EPSC that could favor the propagation of high-frequency TC activity.