RESUMEN
INTRODUCTION: In motor neurons, cholera toxin B (CTB) binds to the cell-surface ganglioside GM1 and is internalized and transported via structurally unique components of plasma membranes (lipid rafts). METHODS: Lipid raft uptake by axon terminals adjoining type-identified rat diaphragm muscle fibers was investigated using CTB and confocal imaging. RESULTS: Lipid raft uptake increased significantly at higher frequency stimulation (80 Hz), compared with lower frequency (20 Hz) and unstimulated (0 Hz) conditions. The fraction of axon terminal occupied by CTB was â¼45% at 0- or 20-Hz stimulation, and increased to â¼65% at 80 Hz. Total CTB fluorescence intensity also increased (â¼20%) after 80-Hz stimulation compared with 0 Hz. DISCUSSION: Evidence of increased lipid raft uptake at high stimulation frequencies supports an important role for lipid raft signaling at rat diaphragm muscle axon terminals, primarily for motor units physiologically activated at the higher frequencies. Muscle Nerve 59:611-611, 2019.
Asunto(s)
Toxina del Cólera/metabolismo , Diafragma/inervación , Microdominios de Membrana/metabolismo , Unión Neuromuscular/metabolismo , Nervio Frénico/metabolismo , Terminales Presinápticos/metabolismo , Animales , Estimulación Eléctrica , Microdominios de Membrana/ultraestructura , Microscopía Confocal , Neuronas Motoras/metabolismo , Unión Neuromuscular/ultraestructura , Nervio Frénico/citología , Nervio Frénico/ultraestructura , Terminales Presinápticos/ultraestructura , RatasRESUMEN
Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) are now recognized as multi-system disorders also involving various non-motor neuronal cell types. The precise extent and mechanistic basis of non-motor neuron damage in human ALS and ALS animal models remain however unclear. To address this, we here studied progressive motor neuronopathy (pmn) mice carrying a missense loss-of-function mutation in tubulin binding cofactor E (TBCE). These mice manifest a particularly aggressive form of motor axon dying back and display a microtubule loss, similar to that induced by human ALS-linked TUBA4A mutations. Using whole nerve confocal imaging of pmn × thy1.2-YFP16 fluorescent reporter mice and electron microscopy, we demonstrate axonal discontinuities, bead-like spheroids and ovoids in pmn suralis nerves indicating prominent sensory neuropathy. The axonal alterations qualitatively resemble those in phrenic motor nerves but do not culminate in the loss of myelinated fibers. We further show that the pmn mutation decreases the level of TBCE, impedes microtubule polymerization in dorsal root ganglion (DRG) neurons and causes progressive loss of microtubules in large and small caliber suralis axons. Live imaging of axonal transport using GFP-tagged tetanus toxin C-fragment (GFP-TTC) demonstrates defects in microtubule-based transport in pmn DRG neurons, providing a potential explanation for the axonal alterations in sensory nerves. This study unravels sensory neuropathy as a pathological feature of mouse pmn, and discusses the potential contribution of cytoskeletal defects to sensory neuropathy in human motor neuron disease.
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Transporte Axonal/genética , Microtúbulos/metabolismo , Enfermedad de la Neurona Motora/complicaciones , Enfermedades del Sistema Nervioso Periférico/etiología , Enfermedades del Sistema Nervioso Periférico/patología , Nervio Sural/patología , Animales , Axones/metabolismo , Axones/patología , Células Cultivadas , Modelos Animales de Enfermedad , Embrión de Mamíferos , Ganglios Espinales/citología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes Neurológicos , Ratones Transgénicos , Microtúbulos/genética , Microtúbulos/ultraestructura , Chaperonas Moleculares/genética , Enfermedad de la Neurona Motora/genética , Enfermedad de la Neurona Motora/patología , Mutación Missense/genética , Neuronas/metabolismo , Neuronas/patología , Neuronas/ultraestructura , Nervio Frénico/patología , Nervio Frénico/ultraestructura , Polimerizacion , Nervio Sural/metabolismo , Nervio Sural/ultraestructuraRESUMEN
Bites by Bothrops snakes normally induce local pain, haemorrhage, oedema and myonecrosis. Mammalian isolated nerve-muscle preparations exposed to Bothrops venoms and their phospholipase A2 toxins (PLA2 ) can exhibit a neurotoxic pattern as increase in frequency of miniature end-plate potentials (MEPPs) as well as in amplitude of end-plate potentials (EPPs); neuromuscular facilitation followed by complete and irreversible blockade without morphological evidence for muscle damage. In this work, we analysed the ultrastructural damage induced by Bothrops jararacussu and Bothrops bilineatus venoms and their PLA2 toxins (BthTX-I and Bbil-TX) in mouse isolated nerve-phrenic diaphragm preparations (PND). Under transmission electron microscopy (TEM), PND preparations previously exposed to B. jararacussu and B. bilineatus venoms and BthTX-I and Bbil-TX toxins showed hypercontracted and loosed myofilaments; unorganized sarcomeres; clusters of edematous sarcoplasmic reticulum and mitochondria; abnormal chromatin distribution or apoptotic-like nuclei. The principal affected organelles, mitochondria and sarcoplasmic reticulum, were those related to calcium buffering and, resulting in sarcomeres and myofilaments hypercontraction. Schwann cells were also damaged showing edematous axons and mitochondria as well as myelin sheath alteration. These ultrastructural changes caused by both of Bothrops venoms and toxins indicate that the neuromuscular blockade induced by them in vitro can also be associated with nerve and muscle degeneration.
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Venenos de Crotálidos/toxicidad , Diafragma/efectos de los fármacos , Fosfolipasas A2 Grupo II/toxicidad , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/ultraestructura , Nervio Frénico/efectos de los fármacos , Animales , Bothrops , Diafragma/ultraestructura , Masculino , Ratones , Nervio Frénico/ultraestructuraRESUMEN
Acute and chronic respiratory failure is one of the major and potentially life-threatening features in individuals with myotonic dystrophy type 1 (DM1). Despite several clinical demonstrations showing respiratory problems in DM1 patients, the mechanisms are still not completely understood. This study was designed to investigate whether the DMSXL transgenic mouse model for DM1 exhibits respiratory disorders and, if so, to identify the pathological changes underlying these respiratory problems. Using pressure plethysmography, we assessed the breathing function in control mice and DMSXL mice generated after large expansions of the CTG repeat in successive generations of DM1 transgenic mice. Statistical analysis of breathing function measurements revealed a significant decrease in the most relevant respiratory parameters in DMSXL mice, indicating impaired respiratory function. Histological and morphometric analysis showed pathological changes in diaphragmatic muscle of DMSXL mice, characterized by an increase in the percentage of type I muscle fibers, the presence of central nuclei, partial denervation of end-plates (EPs) and a significant reduction in their size, shape complexity and density of acetylcholine receptors, all of which reflect a possible breakdown in communication between the diaphragmatic muscles fibers and the nerve terminals. Diaphragm muscle abnormalities were accompanied by an accumulation of mutant DMPK RNA foci in muscle fiber nuclei. Moreover, in DMSXL mice, the unmyelinated phrenic afferents are significantly lower. Also in these mice, significant neuronopathy was not detected in either cervical phrenic motor neurons or brainstem respiratory neurons. Because EPs are involved in the transmission of action potentials and the unmyelinated phrenic afferents exert a modulating influence on the respiratory drive, the pathological alterations affecting these structures might underlie the respiratory impairment detected in DMSXL mice. Understanding mechanisms of respiratory deficiency should guide pharmaceutical and clinical research towards better therapy for the respiratory deficits associated with DM1.
Asunto(s)
Distrofia Miotónica/patología , Distrofia Miotónica/fisiopatología , Respiración , Animales , Axones/patología , Tronco Encefálico/patología , Tronco Encefálico/fisiopatología , Diafragma/patología , Diafragma/fisiopatología , Modelos Animales de Enfermedad , Humanos , Hibridación Fluorescente in Situ , Ratones , Ratones Transgénicos , Neuronas Motoras/patología , Músculos/inervación , Músculos/patología , Músculos/fisiopatología , Vaina de Mielina/metabolismo , Unión Neuromuscular/patología , Unión Neuromuscular/fisiopatología , Nervio Frénico/patología , Nervio Frénico/ultraestructura , Pruebas de Función RespiratoriaRESUMEN
Spinal muscular atrophy (SMA) is caused by mutation of the Survival Motor Neurons 1 (SMN1) gene and is characterized by degeneration of spinal motor neurons. The severity of SMA is primarily influenced by the copy number of the SMN2 gene. Additional modifier genes that lie outside the SMA locus exist and one gene that could modify SMA is the Zinc Finger Protein (ZPR1) gene. To test the significance of ZPR1 downregulation in SMA, we examined the effect of reduced ZPR1 expression in mice with mild and severe SMA. We report that the reduced ZPR1 expression causes increase in the loss of motor neurons, hypermyelination in phrenic nerves, increase in respiratory distress and disease severity and reduces the lifespan of SMA mice. The deficiency of SMN-containing sub-nuclear bodies correlates with the severity of SMA. ZPR1 is required for the accumulation of SMN in sub-nuclear bodies. Further, we report that ZPR1 overexpression increases levels of SMN and promotes accumulation of SMN in sub-nuclear bodies in SMA patient fibroblasts. ZPR1 stimulates neurite growth and rescues axonal growth defects in SMN-deficient spinal cord neurons from SMA mice. These data suggest that the severity of disease correlates negatively with ZPR1 levels and ZPR1 may be a protective modifier of SMA.
Asunto(s)
Proteínas Portadoras/metabolismo , Atrofia Muscular Espinal/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Immunoblotting , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patología , Vaina de Mielina/metabolismo , Vaina de Mielina/patología , Vaina de Mielina/ultraestructura , Nervio Frénico/metabolismo , Nervio Frénico/patología , Nervio Frénico/ultraestructura , Nervio Ciático/metabolismo , Nervio Ciático/patología , Nervio Ciático/ultraestructura , Índice de Severidad de la Enfermedad , Médula Espinal/metabolismo , Médula Espinal/patología , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/metabolismoRESUMEN
Synapse formation requires the organization of presynaptic active zones, the synaptic vesicle release sites, in precise apposition to postsynaptic neurotransmitter receptor clusters; however, the molecular mechanisms responsible for these processes remain unclear. Here, we show that P/Q-type and N-type voltage-dependent calcium channels (VDCCs) play essential roles as scaffolding proteins in the organization of presynaptic active zones. The neuromuscular junction of double knock-out mice for P/Q- and N-type VDCCs displayed a normal size but had significantly reduced numbers of active zones and docked vesicles and featured an attenuation of the active-zone proteins Bassoon, Piccolo, and CAST/Erc2. Consistent with this phenotype, direct interactions of the VDCC ß1b or ß4 subunits and the active zone-specific proteins Bassoon or CAST/Erc2 were confirmed by immunoprecipitation. A decrease in the number of active zones caused by a loss of presynaptic VDCCs resembled the pathological conditions observed in the autoimmune neuromuscular disorder Lambert-Eaton myasthenic syndrome. At the synaptic cleft of double knock-out mice, we also observed a decrease of the synaptic organizer laminin ß2 protein, an extracellular ligand of P/Q- and N-type VDCCs. However, the transcription level of laminin ß2 did not decrease in double knock-out mice, suggesting that the synaptic accumulation of laminin ß2 protein required its interaction with presynaptic VDCCs. These results suggest that presynaptic VDCCs link the target-derived synapse organizer laminin ß2 to active-zone proteins and function as scaffolding proteins to anchor active-zone proteins to the presynaptic membrane.
Asunto(s)
Canales de Calcio Tipo N/fisiología , Canales de Calcio Tipo P/fisiología , Canales de Calcio Tipo Q/fisiología , Proteínas del Citoesqueleto/metabolismo , Laminina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Unión Neuromuscular/metabolismo , Sinapsis/metabolismo , Animales , Canales de Calcio Tipo N/genética , Canales de Calcio Tipo P/genética , Canales de Calcio Tipo Q/genética , Recuento de Células , Electromiografía , Embrión de Mamíferos , Ratones , Ratones Noqueados , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Nervio Frénico/metabolismo , Nervio Frénico/ultraestructura , Subunidades de Proteína/metabolismo , Médula Espinal/citología , Médula Espinal/metabolismoRESUMEN
Damage-induced neuronal endopeptidase (DINE) is a metalloprotease belonging to the neprilysin family. Expression of DINE mRNA is observed predominantly in subsets of neurons in the CNS and peripheral nervous system during embryonic development, as well as after axonal injury. However, the physiological function of DINE and its substrate remain unknown. We generated DINE-deficient mice to examine the physiological role of DINE. Shortly after birth, these mice died of respiratory failure resulting from a dysfunction of the diaphragm, which showed severe atrophy. As DINE was abundantly expressed in motor neurons and there was atrophy of the diaphragm, we analyzed the interaction between motor nerves and skeletal muscles in the DINE-deficient mice. Although there were no obvious deficiencies in numbers of motor neurons in the spinal cord or in the nerve trajectories from the spinal cord to the skeletal muscle in DINE-deficient mice, detailed histochemical analysis demonstrated a significant decrease of nerve terminal arborization in the diaphragm from embryonic day 12.5. In accordance with the decrease of final branching, the diaphragms from DINE-deficient mice exhibited only a few neuromuscular junctions. Similar changes in nerve terminal morphology were also apparent in other skeletal muscles, including the latissimus dorsi and the intercostal muscles. These data suggest that DINE is a crucial molecule in distal axonal arborization into muscle to establish neuromuscular junctions.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Metaloendopeptidasas/metabolismo , Unión Neuromuscular , Neuronas/metabolismo , Terminales Presinápticos/fisiología , Aminoácidos/metabolismo , Animales , Animales Recién Nacidos , Bungarotoxinas/metabolismo , Colina O-Acetiltransferasa/metabolismo , Diafragma/patología , Diafragma/fisiopatología , Diafragma/ultraestructura , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica/genética , Metaloendopeptidasas/deficiencia , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión/métodos , Proteínas de Neurofilamentos/metabolismo , Unión Neuromuscular/citología , Unión Neuromuscular/embriología , Unión Neuromuscular/crecimiento & desarrollo , Neuronas/clasificación , Neuronas/ultraestructura , Nervio Frénico/patología , Nervio Frénico/fisiopatología , Nervio Frénico/ultraestructura , Terminales Presinápticos/ultraestructura , Trastornos Respiratorios/genética , Médula Espinal/citologíaRESUMEN
The structural determinants of myotoxicity of bothropstoxin-I (BthTX-I), a Lys49 phospholipase A(2) from Bothrops jararacussu venom, were studied by measuring the resting membrane potential in the mouse phrenic nerve-diaphragm preparation. This method proved to be around 100-fold more sensitive than the creatine kinase release assay, and was used to evaluate a total of 31 site-directed BthTX-I alanine scanning mutants. Mutants that reduced the resting membrane potential were located in a surface patch defined by residues in the C-terminal loop (residues 115-129), positions 37-39 in the membrane interfacial recognition surface (Y46 and K54), and residue K93. These results expand the known structural determinants of the biological activity as evaluated by previous creatine kinase release experiments. Furthermore, a strong correlation is observed between the structural determinants of sarcolemma depolarization and calcium-independent disruption of liposome membranes, suggesting that a common mechanism of action underlies the permeabilization of the biological and model membranes.
Asunto(s)
Bothrops , Fosfolipasas A2 Grupo II/metabolismo , Liposomas/metabolismo , Proteínas Mutantes/metabolismo , Nervio Frénico/metabolismo , Animales , Calcio/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/fisiología , Venenos de Crotálidos/toxicidad , Masculino , Potenciales de la Membrana , Ratones , Técnicas de Cultivo de Órganos , Nervio Frénico/efectos de los fármacos , Nervio Frénico/ultraestructura , Sarcolema/efectos de los fármacos , Sarcolema/fisiologíaRESUMEN
We have demonstrated that phrenic nerves' large myelinated fibers in streptozotocin (STZ)-induced diabetic rats show axonal atrophy, which is reversed by insulin treatment. However, studies on structural abnormalities of the small myelinated and the unmyelinated fibers in the STZ-model of neuropathy are limited. Also, structural changes in the endoneural vasculature are not clearly described in this model and require detailed study. We have undertaken morphometric studies of the phrenic nerve in insulin-treated and untreated STZ-diabetic rats and non-diabetic control animals over a 12-week period. The presence of neuropathy was assessed by means of transmission electron microscopy, and morphometry of the unmyelinated fibers was performed. The most striking finding was the morphological evidence of small myelinated fiber neuropathy due to the STZ injection, which was not protected or reversed by conventional insulin treatment. This neuropathy was clearly associated with severe damage of the endoneural vessels present on both STZ groups, besides the insulin treatment. The STZ-diabetes model is widely used to investigate experimental diabetic neuropathies, but few studies have performed a detailed assessment of either unmyelinated fibers or capillary morphology in this animal model. The present study adds useful information for further investigations on the ultrastructural basis of nerve function in diabetes.
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Diabetes Mellitus Experimental/patología , Neuropatías Diabéticas/patología , Fibras Nerviosas Amielínicas/patología , Nervio Frénico/patología , Animales , Glucemia/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Recuento de Células , Tamaño de la Célula/efectos de los fármacos , Diabetes Mellitus Experimental/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Masculino , Fibras Nerviosas Mielínicas/patología , Fibras Nerviosas Mielínicas/ultraestructura , Fibras Nerviosas Amielínicas/ultraestructura , Nervio Frénico/ultraestructura , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Despite numerous literature reports on the morphometry of the myelinated fibers of phrenic nerves in rats, a systematic study of the longitudinal and lateral symmetry of the unmyelinated fibers morphometry is not available. In this study, we have undertaken ultrastructural and morphometric studies of the phrenic nerve in adult rats, assessing two different levels (proximal and distal) from both right and left sides. Phrenic nerves of adult male Wistar rats were prepared for epoxy resin embedding and transmission electron microscopy. Morphometric analysis was performed with the aid of computer software, which took into consideration the unmyelinated fiber number, density, area, and diameter, as well as ratio between myelinated and unmyelinated fibers, and the percentage of the fascicular area occupied by the myelinated and unmyelinated fibers. Comparison of data from proximal and distal segments on the same side and from the same levels between sides was performed. Differences were considered significant when P < 0.05. The most important finding is that morphometric parameters of the phrenic nerve unmyelinated fibers in adult rats are both longitudinally and laterally symmetric. This study adds important morphometric information about the unmyelinated fibers of the phrenic nerves in adult rats for proximal and distal levels on both sides of the animal.
Asunto(s)
Axones/ultraestructura , Diafragma/inervación , Neuronas Motoras/ultraestructura , Nervio Frénico/ultraestructura , Animales , Axones/fisiología , Diafragma/fisiología , Lateralidad Funcional/fisiología , Masculino , Microscopía Electrónica de Transmisión , Neuronas Motoras/fisiología , Fibras Nerviosas Mielínicas/fisiología , Fibras Nerviosas Mielínicas/ultraestructura , Fibras Nerviosas Amielínicas/fisiología , Fibras Nerviosas Amielínicas/ultraestructura , Nervio Frénico/fisiología , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructura , Ratas , Ratas Wistar , Fenómenos Fisiológicos Respiratorios , Especificidad de la EspecieRESUMEN
Recent evidence has shown that the activation of receptor tyrosine kinases is not only dependent on binding of their ligands but in addition requires adhesion molecules as coreceptors. We have identified CD44v6 as a coreceptor for c-Met in several tumor and primary cells. The CD44v6 ectodomain is required for c-Met activation, whereas the cytoplasmic tail recruits ERM proteins and the cytoskeleton into a signalosome complex. Here we demonstrate that c-Met (and hepatocyte growth factor and Gab1) is haploinsufficient in a cd44-/- background, as the cd44-/-; met+/- (and cd44-/-; hgf+/- and cd44-/-; gab1+/-) mice die at birth. They have impaired synaptic transmission in the respiratory rhythm-generating network and alterations in the phrenic nerve. These results are the first genetic data showing that CD44 and c-Met collaborate in vivo and that they are involved in synaptogenesis and axon myelination in the central and peripheral nervous systems.
Asunto(s)
Haploidia , Receptores de Hialuranos/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/patología , Factor de Crecimiento de Hepatocito/metabolismo , Pulmón/anomalías , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/patología , Fibras Nerviosas/patología , Nervio Frénico/patología , Nervio Frénico/ultraestructura , Transmisión SinápticaRESUMEN
Netrin signaling is important to guide migrating neurons and axons in many systems. Experiments with vertebrate CNS explants suggested netrin is bifunctional, attracting some axons and repelling others. Netrin1-expressing cells attracted spinal commissural axons and repelled trochlear cranial nerve axons in these experiments. Subsequent genetic studies demonstrated that multiple axon types, including those of the spinal commissural neurons, are attracted to netrin in vivo; however, an in vivo role for netrin signaling in trochlear nerve repulsion has not been observed. Here, we demonstrate that mice with a null mutation in the netrin receptor Unc5c on the inbred C57BL/6J (B6) genetic background have ventral/ipsilateral trochlear nerve misprojections. These misprojections are attenuated on a hybrid B6 x SJL background. In addition, B6.Unc5c(-/-) mice die as neonates of apparent respiratory distress and have incomplete phrenic nerve innervation of the diaphragm muscle. Neither the trochlear nerve misprojections nor the phrenic nerve phenotype was observed in B6 embryos lacking the netrin receptors DCC or Neogenin1, or the ligand netrin1, indicating these signaling molecules are dispensable for guidance of these axons. Like the trochlear nerve, the phrenic nerve phenotype is modified in a B6 x SJL hybrid background. To identify these modifier loci, we performed genome scans of the hybrid Unc5c(-/-) mice and found a major SJL-derived suppressor locus on Chromosome 17. Our results provide the first evidence that genes involved in netrin signaling are necessary for proper mammalian spinal motor axon development and trochlear axon guidance. In addition, they demonstrate the importance of modifier genes in vertebrate axonal guidance.
Asunto(s)
Axones/metabolismo , Axones/ultraestructura , Neuronas Motoras/metabolismo , Nervio Frénico/metabolismo , Nervio Frénico/ultraestructura , Receptores de Factor de Crecimiento Nervioso/metabolismo , Nervio Troclear/metabolismo , Nervio Troclear/ultraestructura , Animales , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/ultraestructura , Receptores de Netrina , Fenotipo , Sitios de Carácter Cuantitativo/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Factor de Crecimiento Nervioso/genéticaRESUMEN
In three human foetuses aged 15, 17, and 23 weeks the number of axons surrounded by single Schwann cells was counted. These Schwann cell/axon complexes form the Schwann units. The largest Schwann units in the foetus aged 15 weeks contained 232 axons, in the foetus of 17 weeks the number was 140 and in the foetus of 23 weeks the largest units contained 65 axons.
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Axones/ultraestructura , Feto/anatomía & histología , Fibras Nerviosas Mielínicas/ultraestructura , Nervio Frénico/ultraestructura , Células de Schwann/ultraestructura , Femenino , Edad Gestacional , Humanos , Nervio Frénico/embriología , EmbarazoRESUMEN
After ipsilateral injections of biotinylated dextran amine (BDA) into the Kölliker-Fuse (KF) nucleus and cholera toxin B subunit (CTb) into the ventral horn in C4 to C5 segments of the spinal cord, an overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found in the rostral ventral respiratory group (rVRG) region ipsilateral to the injection sites. After ipsilateral injections of BDA into the KF and Fluoro-Gold (FG) into the ventral horn in C4 to C5 segments of the spinal cord, BDA-labeled axons were found to make asymmetrical synapses with the somata and dendrites of FG-labeled neurons within the neuropil of the rVRG region. Using retrograde tracing combined with immunohistochemistry for phosphate-activated glutaminase (PAG), we observed that as many as 72% of the rVRG neurons projecting to the PhN were immunoreactive for PAG and that approximately 62% and 75% of the KF neurons projecting respectively to the rVRG region and PhN contain PAG immunoreactivity. Using anterograde tracing combined with immunohistochemistry for vesicular glutamate transporter 2 (VGluT2), we further demonstrated that the KF axon terminals in the rVRG and PhN regions as well as the rVRG axon terminals in the PhN region contain VGluT2 immunoreactivity. The present results suggest that the glutamatergic pathways from the KF to the PhN directly and indirectly via the rVRG region may exist and underlie the inspiratory responses that are elicited by activation of the KF neurons.
Asunto(s)
Biotina/análogos & derivados , Ácido Glutámico/metabolismo , Proteínas de Transporte de Membrana , Vías Nerviosas/ultraestructura , Nervio Frénico/ultraestructura , Puente/citología , Centro Respiratorio/citología , Médula Espinal/ultraestructura , Proteínas de Transporte Vesicular , Animales , Transporte Axonal/fisiología , Proteínas Portadoras/metabolismo , Toxina del Cólera , Dextranos , Colorantes Fluorescentes , Glutaminasa/metabolismo , Inmunohistoquímica , Masculino , Bulbo Raquídeo/citología , Bulbo Raquídeo/fisiología , Microscopía Electrónica , Neuronas Motoras/fisiología , Neuronas Motoras/ultraestructura , Vías Nerviosas/fisiología , Nervio Frénico/fisiología , Puente/fisiología , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Ratas , Ratas Wistar , Centro Respiratorio/fisiología , Fenómenos Fisiológicos Respiratorios , Médula Espinal/fisiología , Estilbamidinas , Transmisión Sináptica/fisiología , Proteína 2 de Transporte Vesicular de GlutamatoRESUMEN
Activity-dependent and -independent signals collaborate to regulate synaptogenesis, but their relative contributions are unclear. Here, we describe the formation of neuromuscular synapses at which neurotransmission is completely and specifically blocked by mutation of the neurotransmitter-synthesizing enzyme choline acetyltransferase. Nerve terminals differentiate extensively in the absence of neurotransmitter, but neurotransmission plays multiple roles in synaptic differentiation. These include influences on the numbers of pre- and postsynaptic partners, the distribution of synapses in the target field, the number of synaptic sites per target cell, and the number of axons per synaptic site. Neurotransmission also regulates the formation or stability of transient acetylcholine receptor-rich processes (myopodia) that may initiate nerve-muscle contact. At subsequent stages, neurotransmission delays some steps in synaptic maturation but accelerates others. Thus, neurotransmission affects synaptogenesis from early stages and coordinates rather than drives synaptic maturation.
Asunto(s)
Acetilcolina/deficiencia , Diferenciación Celular/genética , Colina O-Acetiltransferasa/deficiencia , Unión Neuromuscular/anomalías , Terminales Presinápticos/metabolismo , Transmisión Sináptica/genética , Acetilcolina/biosíntesis , Animales , Colina O-Acetiltransferasa/genética , Diafragma/anomalías , Diafragma/inervación , Diafragma/ultraestructura , Feto , Eliminación de Gen , Inmunohistoquímica , Ratones , Ratones Noqueados , Microscopía Electrónica , Neuronas Motoras/metabolismo , Neuronas Motoras/ultraestructura , Mutación/genética , Unión Neuromuscular/metabolismo , Unión Neuromuscular/ultraestructura , Nervio Frénico/anomalías , Nervio Frénico/ultraestructura , Terminales Presinápticos/ultraestructuraRESUMEN
Overexpression of mutated superoxide dismutase (SOD1) in transgenic mice causes a progressive motor neuron degeneration in the spinal cord similar to that in human amyotrophic lateral sclerosis (ALS). Ultrastructural analysis of motor neurons at different stages of the disease in transgenic C57BL/6 mice carrying the G93A mutation of SOD1 showed, at about 2 weeks of age, much earlier than the initial symptoms of the disease, microvacuoles in the cytoplasm, with marked swelling of the mitochondria. Nuclei with an apoptotic morphology were never observed in these motor neurons. Swollen mitochondria were also seen in the distal part of motor axons of phrenic nerves and in the large axons of sciatic nerves before the onset of the disease, but no mitochondrial alterations were seen in skeletal muscles or in the small sciatic nerve axons. Moreover, we found no apparent changes in the histochemical reactivity of cytochrome oxidase in motor neurons of transgenic mice even at the advanced stage of the disease, suggesting that partial neuronal activity in these cells may be maintained despite the altered mitochondria. Immunoreactivity for human SOD1 was high around vacuoles in the motor neurons of transgenic mice but no cytoplasmic intracellular SOD1 aggregates were observed. Our data indicate that mitochondrial swelling may be an important factor triggering the cascade leading to progressive motor neuron death. Activation of the mitochondrial permeability transition pore may be involved in this process, through excitotoxicity or other neurotoxic stimuli.
Asunto(s)
Esclerosis Amiotrófica Lateral/enzimología , Apoptosis , Complejo IV de Transporte de Electrones/metabolismo , Mitocondrias/patología , Neuronas Motoras/enzimología , Vacuolas/patología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Inmunohistoquímica , Región Lumbosacra , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/enzimología , Neuronas Motoras/patología , Músculo Esquelético/ultraestructura , Nervio Frénico/patología , Nervio Frénico/ultraestructura , Médula Espinal/patología , Médula Espinal/ultraestructura , Succinato Deshidrogenasa/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Vacuolas/ultraestructuraRESUMEN
The development of phrenic motoneurons and descending bulbospinal projections to the cervical spinal cord have been examined in prenatal and early postnatal rats with the aid of the carbocyanine dyes DiI and DiA. Phrenic motoneurons could be identified by retrograde labelling as early as E13, while aggregation of phrenic motoneurons into a column and the formation of dendritic bundles became apparent from E16. The initial phrenic motoneuron dendritic bundles were oriented in the dorsolateral and ventromedial directions, while ventrolaterally directed bundles entering the marginal zone appeared by E16, and rostrocaudal bundles were clearly visible by E21. The column of phrenic motoneurons extended rostrocaudally from C2 to C6 at E13 and E14, but this became confined to the C3-5 segments by E21. Two-way tracing of connections between putative brainstem respiratory centres and cervical spinal cord with the carbocyanine dyes, DiI and DiA, indicated that brainstem bulbospinal neurons in the position of the adult ventral respiratory group (VRG) and medial parabrachial (MPB) nuclei appeared to project to the cervical cord white matter as early as E15 and may contribute axons to the grey matter of the cervical cord as early as E17 These findings are consistent with electrophysiological studies of respiratory function development in the fetal rat, which found relatively regular rhythmic phrenic discharge by E20 to 21. In summary, our findings indicate that the structural differentiation of phrenic motoneurons is well-advanced prior to birth and that the descending pathways involved in the control of respiratory function are in place several days before birth.
Asunto(s)
Tronco Encefálico/crecimiento & desarrollo , Nervio Frénico/crecimiento & desarrollo , Sistema Respiratorio/inervación , Animales , Transporte Axonal , Tronco Encefálico/embriología , Carbocianinas , Dendritas/ultraestructura , Femenino , Colorantes Fluorescentes , Neuronas Motoras/fisiología , Neuronas Motoras/ultraestructura , Vías Nerviosas/embriología , Vías Nerviosas/crecimiento & desarrollo , Vías Nerviosas/ultraestructura , Nervio Frénico/embriología , Nervio Frénico/ultraestructura , Embarazo , Compuestos de Piridinio , Ratas , Médula Espinal/embriología , Médula Espinal/crecimiento & desarrolloRESUMEN
In the adult rat, there is a general correspondence between the sizes of motoneurons, motor units, and muscle fibers that has particular functional importance in motor control. During early postnatal development, after the establishment of singular innervation, there is rapid growth of diaphragm muscle (Dia(m)) fibers. In the present study, the association between Dia(m) fiber growth and changes in phrenic motoneuron size (both somal and dendritic) was evaluated from postnatal day 21 (D21) to adulthood. Phrenic motoneurons were retrogradely labeled with fluorescent tetramethylrhodamine dextran (3,000 MW), and motoneuron somal volumes and surface areas were measured using three-dimensional confocal microscopy. In separate animals, phrenic motoneurons retrogradely labeled with choleratoxin B-fragment were visualized using immunocytochemistry, and dendritic arborization was analyzed by camera lucida. Between D21 and adulthood, Dia(m) fiber cross-sectional area increased by approximately 164% overall, with the growth of type II fibers being disproportionate to that of type I fibers. There was also substantial growth of phrenic motoneurons ( approximately 360% increase in total surface area), during this same period, that was primarily attributable to an expansion of dendritic surface area. Comparison of the distribution of phrenic motoneuron surface areas between D21 and adults suggests the establishment of a bimodal distribution that may have functional significance for motor unit recruitment in the adult rat.
Asunto(s)
Diafragma/crecimiento & desarrollo , Diafragma/inervación , Neuronas Motoras/fisiología , Neuronas Motoras/ultraestructura , Desarrollo de Músculos , Nervio Frénico/fisiología , Nervio Frénico/ultraestructura , Envejecimiento/fisiología , Animales , Dendritas/fisiología , Dendritas/ultraestructura , Procesamiento de Imagen Asistido por Computador , Masculino , Microscopía Confocal , Fibras Musculares Esqueléticas/fisiología , Fibras Musculares Esqueléticas/ultraestructura , Ratas , Ratas Sprague-Dawley , Espectrometría de Fluorescencia , Médula Espinal/citología , Médula Espinal/fisiologíaRESUMEN
Phoneutria nigriventer (Labidognatha, Ctenidae) is a spider found in the warm regions of South America. Bites by this species cause intense local pain, autonomic dysfunction and paralysis. PhTx2, a neurotoxic fraction of the venom of this species, interferes with the physiology of sodium channel function. The present study describes the morphological changes in mouse phrenic nerve and diaphragm muscle after 15, 30, 45 and 60 min of incubation with 1 microg of PhTx2/ml. Light and transmission electron microscopy showed that PhTx2 caused progressive myonecrosis which involved swelling of the sarcoplasmic reticulum, mitochondrial damage, disorganization of the sarcomeres, zones of hypercontracted myofibrils and rupture of the plasma membrane. The intramuscular fascicles of the phrenic nerve showed vacuolated myelinated axons and Schwann cells. The neuromuscular junctions had vesicle-depleted nerve terminals with swollen mitochondria. The axolema was frequently invaginated and sequestered portions of the axoplasm, or was sometimes interrupted at the site of the synaptic gutter. The post-synaptic junctional folds were shallow and disperse. These morphological alterations in the muscle and nerve fibres were similar to those caused by osmotic disturbances and agree with the ability of PhTx2 to increase the permeability of sodium channels. An increase in sodium influx would probably be accompanied by an influx of water and an elevation in the concentration of cytosolic calcium as a result of calcium release by the sarcoplasmic reticulum and/or mitochondria and the entry of extracellular calcium. The morphological effects caused by PhTx2 were comparable to those seen with Phoneutria nigriventer whole venom which is known to activate and to delay the inactivation of sodium channels. We conclude that PhTx2 is probably the main toxic fraction responsible for such morphological alterations.
Asunto(s)
Diafragma/efectos de los fármacos , Neuropéptidos/toxicidad , Neurotoxinas/toxicidad , Venenos de Araña/toxicidad , Animales , Diafragma/inervación , Diafragma/ultraestructura , Masculino , Ratones , Microscopía Electrónica , Unión Neuromuscular/efectos de los fármacos , Nervio Frénico/efectos de los fármacos , Nervio Frénico/ultraestructura , Factores de TiempoRESUMEN
The development of the right phrenic nerve and the distribution of phrenic nerve afferents to the spinal cord have been examined with the aid of electron microscopy and carbocyanine dye retrograde diffusion along the phrenic nerve, respectively. The formation of fascicles in the right phrenic nerve commenced at E15, while Schwann cells penetrated the nerve from E17 and myelination began at P0. The total number of axons in the right phrenic nerve decreased from E15 (943, 965 in two animals) to E19 (539, 582), remained steady until P0 (564, 594) before rising to almost adult values by P7 (689, 934). The postnatal rise in number of axons appears to be due to a large influx of unmyelinated axons. Carbocyanine dye tracing revealed that at E13, neurons in dorsal root ganglia C(2) to C(6) contributed peripheral processes to the phrenic nerve. Phrenic afferents arrived in the spinal cord by E13 and penetrated the dorsal horn at E14. Three terminal fields for phrenic afferents became apparent by E17. These were:(1) in the central parts of laminae I to V, (2) medially in laminae V to VII or adjacent area X near the central canal, (3) in laminae VIII and IX, around the differentiating phrenic motoneurons. Around the time of birth, some phrenic afferents in the second group were distributed across the midline and could be seen to approach the ventromedial dendritic bundle of phrenic motoneurons on the contralateral side, but these were no longer seen by P4. Just before birth (E21), afferents in the third group divided into two further subsets, supplying the dorsolateral and ventromedial groups of phrenic motoneuron dendritic bundles, respectively. Our findings strongly suggest that phrenic afferent differentiation is largely complete by birth.