RESUMO
Extravillus trophoblast (EVT) invasion plays a critical role in placental development. Integrins bind to extracellular matrix (ECM) proteins to mediate EVT cell adhesion, migration, and invasion. Changes in O-glycans on ß1-integrin have been found to regulate cancer cell behavior. We hypothesize that O-glycosyltransferases can regulate EVT invasion through modulating the glycosylation and function of ß1-integrin. Here, we found that the GALNT1 and GALNT2 mRNA were highly expressed in HTR8/SVneo and first trimester EVT cells. Immunohistochemstry and immunofluorescence staining showed that GALNT2 was expressed in subpopulations of EVT cells in deciduas, but not in syncytiotrophoblasts and cytotrophoblasts of placental villi. The percentage of GALNT2-positive EVT cells increased with gestational ages. Overexpression of GALNT2 in HTR8/SVneo cells significantly enhanced cell-collagen IV adhesion, but suppressed cell migration and invasion. Notably, we found that GALNT2 increased the expression of Tn antigen (GalNAc-Ser/Thr) on ß1-integrin as revealed by Vicia Villosa agglutinin (VVA) binding. Furthermore, GALNT2 suppressed the phosphorylation of focal adhesion kinase (FAK), a crucial downstream signaling molecule of ß1-integrin. Our findings suggest that GALNT2 is a critical initiating enzyme of O-glycosylation for regulating EVT invasion.
Assuntos
Movimento Celular , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , N-Acetilgalactosaminiltransferases/metabolismo , Placentação , Trofoblastos/metabolismo , Adesão Celular , Linhagem Celular , Células Cultivadas , Decídua/citologia , Decídua/metabolismo , Feminino , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Glicosilação , Humanos , Cadeias beta de Integrinas/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , N-Acetilgalactosaminiltransferases/biossíntese , N-Acetilgalactosaminiltransferases/genética , Fosforilação , Gravidez , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Trofoblastos/citologia , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Using ulnar nerve as donor and musculocutaneous nerve as recipient we recently demonstrated that end-to-end neurorrhaphy in young adult male Wistar rats resulted in good recovery following protracted survival. Here we explored whether anti-inflammatory drug- methylprednisolone, regeneration/myelination-enhancing agent- methylcobalamin and neurite growth-enhancing and angiogenic factor- pleiotrophin accelerated its recovery. Methylprednisolone suppressed the perineuronal microglial reaction and periaxonal ED-1 expression while pleiotrophin increased the blood vessel density and nerve fiber densities in the reconnected nerve as expected. Neither methylprednisolone nor methylcobalamin altered the expression of growth associated protein 43 in the neurons examined suggesting that they did not interfere with axonal regeneration attempt. Surprisingly methylcobalamin enhanced the recovery of compound muscle action potentials and motor end plate innervation and the performance on sticker removal grooming test and augmented the diameters and myelin thicknesses of regenerated axons dramatically while enhancing S-100 expression in Schwann cells; remarkable recovery was achieved 1 month following neurorrhaphy. Simultaneous methylcobalamin and pleiotrophin treatment resulted in quick and persistent supernumerary reinnervation but failed to enhance the recovery over that of the former alone. Methylprednisolone transiently suppressed the enumeration of regrowing axons. In conclusion, methylcobalamin may be preferred over methylprednisolone to facilitate the recovery of peripheral nerves following end-to-end neurorrhaphy. The long-term effect of this treatment however remains to be clarified.
Assuntos
Proteínas de Transporte/farmacologia , Citocinas/farmacologia , Membro Anterior/inervação , Metilprednisolona/farmacologia , Músculo Esquelético/inervação , Regeneração Nervosa/efeitos dos fármacos , Transferência de Nervo/métodos , Nervo Ulnar/efeitos dos fármacos , Vitamina B 12/análogos & derivados , Animais , Anti-Inflamatórios/farmacologia , Modelos Animais de Doenças , Masculino , Fatores de Crescimento Neural/farmacologia , Fatores de Crescimento Neural/uso terapêutico , Regeneração Nervosa/fisiologia , Ratos , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/fisiologia , Nervo Ulnar/fisiologia , Nervo Ulnar/transplante , Vitamina B 12/farmacologia , Vitamina B 12/uso terapêuticoRESUMO
Head trauma and acute disorders often instantly compress the cerebral cortex and lead to functional abnormalities. Here we used rat epidural bead implantation model and investigated the immediate changes following acute compression. The dendritic arbors of affected cortical pyramidal neurons were filled with intracellular dye and reconstructed 3-dimensionally for analysis. Compression was found to shorten the apical, but not basal, dendrites of underlying layer III and V cortical pyramidal neurons and reduced dendritic spines on the entire dendritic arbor immediately. Dendrogram analysis showed that in addition to distal, proximal apical dendrites also quickly reconfigured. We then focused on apical dendritic trunks and explored how proximal dendrites were rapidly altered. Compression instantly twisted the microtubules and deformed the membrane contour of dendritic trunks likely a result of the elastic nature of dendrites as immediate decompression restored it and stabilization of microtubules failed to block it. Subsequent adaptive remodeling restored plasmalemma and microtubules to normal appearance in 3 days likely via active mechanisms as taxol blocked the restoration of microtubules and in addition partly affected plasmalemmal reorganization which presumably engaged recycling of excess membrane. In short, the structural dynamics and the associated mechanisms that we revealed demonstrate how compression quickly altered the morphology of cortical output neurons and hence cortical functions consequently.
Assuntos
Córtex Cerebral/fisiopatologia , Plasticidade Neuronal , Neurônios/fisiologia , Tratos Piramidais/fisiopatologia , Animais , Córtex Cerebral/patologia , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/ultraestrutura , Espaço Epidural/patologia , Feminino , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Paclitaxel/farmacologia , Tratos Piramidais/patologia , Ratos , Ratos Wistar , Estresse Mecânico , Moduladores de Tubulina/farmacologiaRESUMO
Fatigue could be induced following forced exercise, sickness, heat stroke or sleep disturbance and impaired brain-related functions such as concentration, attention and memory. Here we investigated whether fatigue altered the dendrites of central neurons. Central fatigue was induced by housing rats in cage with 1.5-cm deep water for 1-5 days. Three days of sleep deprivation seriously compromised rats' performance in weight-loaded forced swimming and spatial learning tests, and 5 days of treatment worsened it further. Combinations of intracellular dye injection and three-dimensional analysis revealed that dendritic spines on retrograde tracer-identified corticospinal neurons and Cornu Ammonis (CA)1 and CA3 pyramidal neurons were significantly reduced while the shape or length of the dendritic arbors was not altered. Three days of rest restored the spine loss and the degraded spatial learning and weight-loaded forced swimming performances to control levels. In conclusion, although we could not rule out additional non-hypothalamic-pituitary-adrenal stress, the apparent fatigue induced following a few days of sleep deprivation could change brain structurally and functionally and the effects were reversible with a few days of rest.
Assuntos
Córtex Cerebral/fisiopatologia , Espinhas Dendríticas/fisiologia , Fadiga/complicações , Hipocampo/fisiopatologia , Transtornos da Memória/fisiopatologia , Resistência Física/fisiologia , Animais , Córtex Cerebral/citologia , Proteína 4 Homóloga a Disks-Large , Fadiga/fisiopatologia , Hipocampo/citologia , Imageamento Tridimensional , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Deficiências da Aprendizagem/etiologia , Deficiências da Aprendizagem/fisiopatologia , Masculino , Aprendizagem em Labirinto/fisiologia , Proteínas de Membrana/metabolismo , Transtornos da Memória/etiologia , Vias Neurais/citologia , Vias Neurais/fisiopatologia , Plasticidade Neuronal/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Células Piramidais/citologia , Células Piramidais/fisiologia , Ratos , Privação do Sono/fisiopatologia , Medula Espinal/citologia , Medula Espinal/fisiopatologia , Natação/fisiologiaRESUMO
We studied the cytoarchitecture and dendritic arbors of the output neurons of the sensorimotor cortex of aged rats and found that although individual cortical layer became thinner, the overall cytoarchitecture and neuron densities remained comparable to those of young adults. To find out whether aging affects cortical outputs we studied the soma-dendritic arbors of layers III and V pyramidal neurons, main output neurons of the cerebral cortex, using brain slice intracellular dye injection technique. With a fluorescence microscope, selected neurons were filled with fluorescence dye under visual guidance. Injected slices were resectioned into thinner sections for converting the injected dye into non-fading material immunohistochemically. The long apical dendritic trunk and branches could be routinely revealed. This allowed us to reconstruct and study the dendritic arbors of these neurons in isolation in 300-microm-thick dimension. Analysis shows that their cell bodies did not shrink, but the densities of spines on dendrites and the total dendritic length significantly reduced. Among spines, those with long thin stalks thought to be involved in memory acquisition appeared to be reduced. These could underlie the compromise of sensorimotor functions following aging.
Assuntos
Córtex Cerebral/citologia , Dendritos/ultraestrutura , Células Piramidais/ultraestrutura , Fatores Etários , Animais , Dendritos/metabolismo , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Feminino , Corantes Fluorescentes/administração & dosagem , Corantes Fluorescentes/metabolismo , Células Piramidais/metabolismo , Ratos , Ratos WistarRESUMO
To evaluate the quantity of penile veins for use as patch material for the treatment of penile curvature, we devised two formulas: from the calculus of applied civil engineering and a diagram from the goniometry of the cadavers' penises, respectively, and the techniques for their application. From March 1995 to July 2003, a total of 65 consecutive patients with penile curvature - 41 men with Peyronie's deformity and 24 with congenital penile deviations - underwent grafting with autologous deep dorsal veins with or without cavernosal veins as patch material. The patched veins required were calculated from the formula (pi)r(2)theta/45 degrees , which is derived from calculus. The tunical incision sector was meticulously performed in accordance with our diagram which is interpolated from seven male cadavers via goniometry and consequently the length of patched veins required was 2 (pi)r(theta)(')/theta. The corporotomy defect was fashioned with the detubularized veins after they were adequately prepared and spliced. In these patients, the average available area of the veins was 4.9 x 2.2 cm(2) (range, 4.5 x 1.8 to 5.6 x 2.4), which seemed adequate in all cases for patching, although 11 of them required two patches. Overall, 21 patients required a modified Nesbit's procedure on the contralateral/ipsilateral tunica to attain a satisfactory penile shape. Because of its anatomical vicinity and its availability as material, the deep dorsal vein associated with the cavernosal vein may play an indispensable role in grafting even with local anaesthesia on an outpatient basis.
Assuntos
Induração Peniana/cirurgia , Pênis/irrigação sanguínea , Pênis/cirurgia , Veias/transplante , Adulto , Idoso , Humanos , Masculino , Matemática , Pessoa de Meia-Idade , Ereção Peniana , Pênis/anormalidades , Pênis/patologia , Procedimentos Cirúrgicos Urológicos Masculinos/métodosRESUMO
To find out whether close axonal injury resulted in greater free radical risk to cord-projection central neurons than distant ones, we studied the expressions of nitric oxide synthase, calcineurin, and superoxide dismutase in rat rubrospinal neurons following brainstem, C2 and T10 axotomies using immunohistochemical methods. We found that nitric oxide synthase expression was upregulated more following brainstem than C2 lesion while T10 lesion triggered no detectable changes. This response peaked at 1 week and returned to control level by 8-week-post-injury. At the same time, calcineurin, which activated nitric oxide synthase, was increased 1 week following brainstem and C2 axotomies. These suggest that close, but not distant, axotomy enhanced NO production, which appeared to be cytotoxic since blocking NO synthesis with N-nitro- l-arginine methyl ester reduced brainstem axotomy-induced rubrospinal cell loss. On the other hand, the mitochondrial Mn-superoxide dismutase, which competes with NO to prevent the formation of the cytotoxic free radical peroxynitrite, was notably reduced after brainstem but almost unaltered following C2 axotomy. Meanwhile, the cytosolic Cu/Zn-superoxide dismutase was not altered following C2 but increased after brainstem axotomy. Ultrastructurally, in rubrospinal neurons more mitochondria became swollen following brainstem than C2 axotomy. Based on these, we proposed that besides the NO-overproduction-induced toxicity, superoxide-loading-induced mitochondrial damage also added to hampering the survival of the closely axotomized neurons.
Assuntos
Axônios/fisiologia , Radicais Livres/metabolismo , Neurônios/metabolismo , Núcleo Rubro/metabolismo , Traumatismos da Medula Espinal/metabolismo , Medula Espinal/metabolismo , Animais , Axônios/patologia , Axotomia/métodos , Calcineurina/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Técnicas Imunoenzimáticas , NG-Nitroarginina Metil Éster/farmacologia , Neurônios/ultraestrutura , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo I , Ratos , Ratos Wistar , Núcleo Rubro/efeitos dos fármacos , Núcleo Rubro/patologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologia , Superóxido Dismutase/metabolismo , Fatores de Tempo , Regulação para CimaRESUMO
In investigating the fate of the cord-projecting CNS neurons following spinal axonal injury, we have demonstrated that surviving rat rubrospinal neurons have altered electrical membrane properties so that their input/output relationship was increased. Further, we found that the synaptic inhibition they received from nearby reticular formation was also reduced following injury. Whether or not these property changes were functional was dependent on the output connections of injured neurons. In the current communication, we examined the supraspinal efferents of the injured neurons recognizing that normal neurons innervate not only spinal but also supraspinal targets. To this end we conducted anterograde tracing on the injured red nucleus 8 weeks following spinal lesion. Results showed that injured rubrospinal neurons still innervated the same supraspinal targets, targeted by normal neurons. We subsequently evaluated the relative intensity of the sustained supraspinal connectivity by examining, in detail, the cerebellar projection of rubrospinal neurons of similarly injured animals using retrograde tracing technique. Here our data revealed that the number, distribution and labeling intensity of rubrospinal neurons projecting to the cerebellum were unchanged following cord injury. In conclusion, although spinal cord injury deprive cord-projecting CNS neurons of their spinal targets, injured neurons survived with altered electrical membrane properties and intact supraspinal projections. The sustained supraspinal connections might allow injured cord-projecting CNS neurons to exert a different weight of influence on higher centers following spinal cord injury.
Assuntos
Axônios/patologia , Axônios/fisiologia , Vias Eferentes/patologia , Vias Eferentes/fisiopatologia , Núcleo Rubro/patologia , Núcleo Rubro/fisiopatologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Animais , Axotomia/efeitos adversos , Biotina/análogos & derivados , Núcleos Cerebelares/patologia , Núcleos Cerebelares/fisiopatologia , Dextranos , Feminino , Corantes Fluorescentes , Plasticidade Neuronal/fisiologia , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Membrane properties and inhibitory synaptic connections of normal and axotomized rat rubrospinal neurons were examined using a coronal slice preparation. Rubrospinal neurons were axotomized at the C2 vertebral level in vivo. Retrograde labelling in vivo and intracellular biocytin injection following recording were combined to identify recorded axotomized rubrospinal neurons. Their input resistances decreased three and four days and became higher than normal four and 10 weeks following lesioning which coincided with a sequential increase and decrease of their soma area. On the other hand, although their membrane time-constant was reduced three and four days following lesioning, it returned to normal value four and 10 weeks following axotomy. Other than these, their membrane current-voltage relationship including an inward rectification in the hyperpolarizing direction was not altered. Normal rubrospinal neurons generated very fast spikes which were not affected by axotomy. Both normal and axotomized cells generated trains of repetitive spikes with a fast spike frequency adaptation at the beginning upon suprathreshold current injection. However, the slope of the steady-state spike frequency and applied current relationship was increased four and 10 weeks following axotomy which also showed an increased steady-state spike frequency in response to high-amplitude current injection. Synaptically, the amplitude and duration of the monosynaptic inhibitory potential evoked from nearby reticular formation were reduced following axotomy. In addition, fewer rubrospinal neurons were found to receive this inhibition 10 weeks following axotomy. Thus, our results show that spinal axotomy induces a time-dependent modification of the membrane properties and spike generating behaviour of rubrospinal neurons which probably represents an initial decrease and a later increase of their excitability. This is accompanied by a persistent decrease of synaptic inhibition which is expected to affect structures that remained innervated by the undamaged axon collaterals of these spinally axotomized neurons.
Assuntos
Axônios/fisiologia , Potenciais da Membrana/fisiologia , Neurônios/fisiologia , Núcleo Rubro/fisiologia , Medula Espinal/fisiologia , Animais , Feminino , Ratos , Ratos WistarRESUMO
Spinal tractotomy-induced perineuronal astrocytic reaction of the rat rubrospinal system was studied using an antiserum to the astrocyte-specific glial fibrillary acidic protein as a marker. The effect of the proximity of axonal cut to cell bodies was also studied by comparing astrocytic reactions elicited by upper cervical and lower thoracic tractotomy. Fast blue was used as a retrograde tracer to identify axotomized neurons, which were found to concentrate in the caudal part of the contralateral red nucleus. The length of reactive astrocytic processes in the dorsomedial and ventrolateral parts of the nucleus was quantified separately since neurons in these two parts project to cervical and lumbar spinal cord, respectively. Those of the ipsilateral nucleus were also quantified. Sham operation caused a transient increase in reactive astrocytic processes one day after surgery. An early and a late increase of reactive astrocytic processes was found 2-5 days and 2-8 weeks following both thoracic and cervical tractotomy. Cervical axotomy of lumbar-cord-projecting rubral neurons caused an increase of reactive astrocytic processes similar in magnitude to that generated by thoracic axotomy. Following thoracic axotomy, the uninjured dorsomedial area of the contralateral nucleus also displayed an increase concomitant with that which occurred within the neighboring, injured ventrolateral nuclear area suggesting the action of diffusible factor(s). Surprisingly, cervical and thoracic tractotomy also elicited a similar increase of reactive astrocytic processes in the ipsilateral nuclei, independent of the number of ipsilaterally projected neurons present in each nucleus. This may be attributed to the retrograde influence from the denervated spinal target sites which were carried by fibers of the intact rubrospinal tract known to terminate bilaterally. In the lesioned nucleus, reactive astrocytic processes were often located close to axotomized cell bodies as early as 3 days following upper cervical and also, to a lesser extent, lower thoracic tractotomy. However, reactive astrocytic processes in the ipsilateral nucleus usually remained in the neuropil. These results suggest that axotomy induces two levels of retrograde astrocytic reactions within the soma area of intrinsic central neurons. Reactive astrocytic processes located proximally to axotomized cell bodies may have a different functional role from those distributed in the neuropil.
Assuntos
Astrócitos/citologia , Axônios/fisiologia , Neuroglia/citologia , Núcleo Rubro/citologia , Medula Espinal/citologia , Animais , Sobrevivência Celular/fisiologia , Feminino , Ratos , Ratos WistarRESUMO
The effect of axotomy at cervical and lumbar spinal levels upon the ability of rubrospinal neurons to retrogradely transport tracer was compared. Unilateral rubrospinal tractotomy was performed first at C5 and, after a few days, at C2 vertebral levels. Different retrograde tracers were applied at the lesioned sites right after tractotomy. Tracer applied at C5 labeled both cervical and lumbar-cord-projecting neurons. Tracer applied at C2 also labeled both groups of neurons if performed 2 days after that at C5; however, only cervical-cord-projecting neurons were labeled when it was performed 3 or 5 days after that at C5. In another set of experiments, a T10 tractotomy without tracer application was performed 2 or 5 days prior to the C5/C2, series of tract lesions. When preceded by a T10 lesion 2 days in advance, tracer applied at C5 labeled both cervical and lumbar-cord-projecting neurons. However, a T10 lesion 5 days in advance resulted in the labeling of only cervical-cord-projecting neurons by the tracer applied at C5. In either case, tracer applied at C2 consistently labeled only cervical-cord-projecting neurons, irrespective of the intervals-2, 3, or 5 days-allowed between C5 and C2 lesions. Most neurons labeled from C2 were also double-labeled by the tracer applied at C5. Thus, unlike lumbar-cord-projecting counterparts, cervical-cord-projecting rubrospinal neurons retain the ability to uptake and/or transport retrograde tracer several days following axotomy. This implies that cervical-cord-projecting rubrospinal neurons survive in a different functional state from their lumbar-cord-projecting counterparts following axonal injury.
Assuntos
Transporte Axonal/fisiologia , Núcleo Rubro/citologia , Traumatismos da Medula Espinal/veterinária , Medula Espinal/cirurgia , Animais , Feminino , Região Lombossacral/lesões , Região Lombossacral/inervação , Pescoço/inervação , Lesões do Pescoço , Ratos , Ratos Wistar , Núcleo Rubro/fisiologia , Medula Espinal/fisiologia , Traumatismos da Medula Espinal/cirurgiaRESUMO
The distribution of calbindin-immunoreactive neurons in the red nucleus and the subcellular distribution of the calbindin and parvalbumin in tracer-identified rubrospinal neurons of the rat were studied. Only a fraction of the retrogradely labelled rubrospinal neurons was found to contain calbindin. These neurons filled the caudal part of the red nucleus and also appeared sporadically along the ventromedial border of the middle segment of the red nucleus. In addition to the somata, calbindin was found in the dendritic arbors of tracer-identified rubrospinal neurons, revealed by injecting the fluorescent dye Lucifer Yellow into their cell bodies. The axons of rubrospinal neurons located in the caudal red nucleus were marked by performing anterograde tracing with fluorescent dextran tracer in freshly prepared brainstem slices. Parvalbumin was found to locate in swellings along these tracer-identified axons as well as at their cut ends. The results indicate that calbindin and parvalbumin are segregated to the somadendritic and axonal compartments of the rat rubrospinal neurons, respectively. This anatomical segregation suggests that they may have different functions in neurons.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Parvalbuminas/metabolismo , Núcleo Rubro/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Medula Espinal/metabolismo , Animais , Calbindinas , Feminino , Imuno-Histoquímica , Ratos , Ratos WistarRESUMO
Microglial reactivity in the red nucleus of rats was studied following upper cervical and lower thoracic rubrospinal tractotomy using the lectin binding method. Following axotomy, the contralateral nucleus containing the axotomized neurons was identified using the retrograde tracer Fast blue. It was subdivided into dorsomedial (DM) and ventrolateral (VL) portions known to project to the cervical and lumbar spinal cord, respectively. Lectin-labeled microglial cells and processes on the contralateral as well as in the ipsilateral nucleus were then quantified. An early and a late increase in microglial reactivity was observed in the nucleus at 2-5 days and 2-8 weeks following thoracic and cervical tractotomy with the latter producing a more pronounced reactivity. In rats subjected to thoracic axotomy, a similar microglial increase also occurred in the intact contralateral DM nuclear area suggesting the possible action of diffusable factor(s) that might have triggered the microglial activation from the axotomized VL nuclear area. The uninjured ipsilateral nucleus also exhibited a similar pattern of microglial reactivity irrespective of the number of ipsilaterally projecting neurons following both cervical and thoracic axotomy. This could have been elicited by the retrograde influence from the denervated targets carried by the intact rubrospinal fibers of the opposite side since many of them in fact terminate bilaterally (Antal, M. et al., J. Comp. Neurol., 325 (1992) 22-37). In all the axotomized or intact nucleus, microglial processes did not appear to surround neuronal cell bodies. The characteristic responses of microglial cells in the red nucleus may be related to the failure of rubrospinal neurons to regenerate following the severance of their axons.
Assuntos
Axônios/fisiologia , Microglia/fisiologia , Neurônios/fisiologia , Lectinas de Plantas , Núcleo Rubro/fisiologia , Medula Espinal/fisiologia , Amidinas , Animais , Feminino , Corantes Fluorescentes , Lateralidade Funcional/fisiologia , Histocitoquímica , Lectinas , Microscopia de Fluorescência , Vias Neurais/citologia , Vias Neurais/fisiologia , Ratos , Ratos Wistar , Núcleo Rubro/citologia , Medula Espinal/citologiaRESUMO
Synaptic connections between the neurons in the red nucleus (RN) and its extrinsic neurons were studied using rat brain-stem slices. Intracellular records were obtained from the RN neurons. Ipsilateral stimuli to areas in the dorsolateral mesencephalic reticular formation (DLMRF) or substantia nigra (SN) elicited monosynaptic hyperpolarizing postsynaptic potentials (PSPs) in about 95% of RN neurons recorded. The hyperpolarizing PSPs could be reversibly blocked by bicuculline, indicating that they were GABAA receptor-mediated-Cl(-)-inhibitory PSPs. The sites of most inhibitory synapses arising from DLMRF and SN are possibly located on the proximal half of the soma-dendritic membrane of RN neurons, according to the analysis of the IPSPs with Rall's model. In addition, tracing dyes were employed to examine the morphological pathways. After rhodamine B, a retrograde tracer, was applied to the RN in brain slices, the cell bodies of a number of neurons in DLMRF and SN were labeled. These labeled neurons were also immunopositive for glutamic acid decarboxylase (GAD) as revealed from double labeling with an anti-GAD antiserum. The anterograde tracer, tetramethylrhodamine dextran, was applied to the DLMRF or SN and taken up by many neurons in the areas. A portion of these cells extended their processes toward and terminated within the RN. Moreover, electron microscopic examination confirmed that the tetramethylrhodamine dextran-decorated synaptic terminals were present in the RN. The results indicate that the rubral neurons receive direct GABAA receptor-mediated inhibitory inputs from neurons in the DLMRF and SN, which may participate in modulation of rubral outputs.
Assuntos
Tronco Encefálico/fisiologia , Neurônios/fisiologia , Núcleo Rubro/fisiologia , Animais , Eletrofisiologia , Microscopia Eletrônica , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Wistar , Fatores de TempoRESUMO
The effect of distal axonal injury on the soma-dendritic morphology of intrinsic central neurons was examined using adult rat lumbar spinal cord-projecting rubrospinal neurons as a model. The soma-dendritic morphology was revealed using an improved Golgi-aldehyde method. Impregnated neurons were reconstructed in the two-dimensional plane for analysis. Four weeks after axotomy, neurons had reduced soma sizes and remained multipolar in shape. Some dendrites were found to end not far from their cell bodies. In addition, no long dendrite was identified following axotomy. Sholl's analysis [The Organization of the Cerebral Cortex. London, Methuen, [1956] revealed that axotomized neurons had fewer dendritic branches than control neurons. Total dendritic length was also reduced. Subsequent analyses showed that the average number of dendritic trunks was not altered however the mean number of terminal branches per dendritic trunk was reduced. The dendritic membrane of the normal neurons was usually smooth with occasional short protuberances on the proximal dendrites and spines on the distal dendrites, which did not change after axotomy. In control neurons, we identified an elaborate type of dendritic structure named dendritic appendage aggregates. These aggregates were located preferentially on terminal dendrites and were classified into three categories according to their complexity. The incidence of occurrence for these aggregates decreased following distal axotomy. These phenomena indicate that rat lumbar spinal cord-projecting rubrospinal neurons retract their distal dendrites in response to distal axotomy. The observed anatomic restructuring following axonal injury is likely to be accompanied by an alteration of afferents which normally synapse on distal dendrites.
Assuntos
Dendritos/ultraestrutura , Vias Neurais/lesões , Núcleo Rubro/citologia , Medula Espinal/citologia , Animais , Axônios , Tamanho Celular , Degeneração Neural , Vias Neurais/patologia , Ratos , Ratos WistarRESUMO
1. The electrophysiological properties of rat corticospinal neurons (CSNs) were studied 3, 9, and 12 mo after axotomy in the cervical spinal cord, with the use of a combination of the in vitro neocortical slice technique, intracellular recordings, and a double-labeling method that allowed identification of CSNs studied in vitro. 2. CSNs retained the rhodamine-labeled microspheres employed as a retrograde marker and were functionally active in the longest survival group (1 yr). 3. The somatic area of axotomized CSNs became progressively smaller, a reduction that amounted to 37% for all cells at 1 yr. There were no obvious differences between normal and axotomized cells in terms of apical dendritic widths, numbers of apical dendritic branches, or basal dendritic arbors. Intracortical axonal arborizations of axotomized neurons were in general similar to those of normal CSNs in that most axons ended in layers V and VI with only occasional collaterals reaching supragranular layers. 4. Axotomized CSNs were grouped according to their spike firing patterns during depolarizing current pulses so that their electrophysiological behavior could be compared with that of regular spiking and adapting groups of normal CSNs. No significant differences were found in resting membrane potential, or spike parameters between axotomized neurons in any survival group and normal controls. Neurons surviving 1 yr after axotomy had a higher input resistance (RN) than normal CSNs. There was a reduction in the percentage of CSNs that generated prominent spike depolarizing afterpotentials in the axotomized group. 5. The steady-state relationship between spike frequency and applied current (f-I slope) became steeper over time and was significantly greater 9 mo after axotomy in regular spiking (RS) and adapting neurons than in normal CSNs in the same groups. The increase in steady-state f-I slope was in part related to increases in the RN of axotomized neurons. 6. There was a significant decrease in the generation of slow afterhyperpolarizations following trains of spikes in axotomized versus normal RS neurons, first detected at 3 mo and also present in 9 mo and 1 yr survival groups. 7. Biphasic inhibitory postsynaptic potentials (IPSPs) were evoked in only 1 of 11 axotomized neurons in the 3-mo group, 2 of 12 cells examined at 9 mo, and 3 of 15 neurons 1 yr after axotomy. The proportions of neurons generating IPSPs were significantly smaller than in comparable groups of control CSNs. As a consequence, longer duration evoked excitatory postsynaptic potentials were generated by axotomized CSNs. 8. Results show that axotomized CSNs undergo alterations in intrinsic membrane properties and inhibitory synaptic electrogenesis that would tend to make them more responsive to excitatory inputs.
Assuntos
Axônios/fisiologia , Mapeamento Encefálico , Regeneração Nervosa/fisiologia , Tratos Piramidais/fisiologia , Transmissão Sináptica/fisiologia , Animais , Sobrevivência Celular/fisiologia , Córtex Cerebral/fisiologia , Técnicas de Cultura , Dendritos/fisiologia , Masculino , Potenciais da Membrana/fisiologia , Inibição Neural/fisiologia , Plasticidade Neuronal/fisiologia , Ratos , Ratos Sprague-Dawley , Medula Espinal/fisiologiaRESUMO
We performed experiments to determine whether axonal sprouting occurs in neurons of chronic neocortical epileptogenic lesions. Partially isolated somatosensory cortical islands with intact pial blood supply were prepared in mature rats. Neocortical slices from these lesions, studied 6-39 d later, generated spontaneous and/or evoked epileptiform field potentials (Prince and Tseng, 1993) during which neurons displayed prolonged polyphasic excitatory and inhibitory synaptic potentials/currents. Single electrophysiologically characterized layer V pyramidal neurons in control and epileptogenic slices were filled with biocytin using sharp and patch-electrode techniques, their axonal arbors reconstructed and compared quantitatively. Neurons in injured cortex had a 56% increase in total axonal length, a 64% increase in the number of axonal collaterals and more than a doubling (115% increase) of the number of axonal swellings. The presumed boutons were smaller and more closely spaced than those of control cells. In some neurons the main descending axon had hypertrophic segments from which branches arose. These highly significant changes were most marked in the perisomatic region of layer V. The axonal sprouting was associated with a decrease in somatic area but no significant change in dendritic arbors. Results suggest that a significant degree of axonal reorganization takes place in the chronically injured cortex where it might be an adaptive mechanism for recovery of function after injury, or might be maladaptive and play an important role in the generation of epileptiform events by increasing the numbers and density of synaptic contacts between neurons.
Assuntos
Axônios/fisiologia , Córtex Cerebral/lesões , Córtex Cerebral/patologia , Epilepsia/etiologia , Células Piramidais/fisiologia , Animais , Córtex Cerebral/fisiopatologia , Doença Crônica , Eletrofisiologia , Plasticidade Neuronal , RatosRESUMO
Studies on the effect of axotomy on adult intrinsic central projection neurons have generally assumed that the severed proximal axonal stumps were still capable of retrogradely transporting tracer at varying times after injury. Failure of transport was interpreted as neuronal death, which is at odds with current understanding that central projection neurons survived distal axotomy. We used lumbar spinal cord-projecting rubrospinal neurons of the rat as a model to evaluate the ability of injured neurons to transport tracer retrogradely at different times after distal axotomy. We examined only the caudal part of the red nucleus, since rubrospinal neurons are concentrated here. In control animals, tracer applied to the rubrospinal tract at the T10 vertebral level labeled ventrolateral rubral neurons, while C3 application marked all rubral neurons. From 3 days after a T10 axotomy and tracer application, most ventrolateral neurons were no longer labeled by another tracer application at the C3 vertebral level via an axonal cut. The phenomenon was not caused by tracer toxicity, since a T10 tractotomy without tracer application also prevented these axotomized neurons from being labeled when treated similarly. Thus, neuronal retrograde transport capability was seriously retarded 3 days after a distal axotomy. Loss of retrograde transport may merely suggest that a mechanism no longer in service has been switched off, or perhaps it may insulate injured neurons from the effect of lesion site-derived factors. Using this property, we were able to localize cervical spinal cord-projecting rubrospinal neurons in the caudal red nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Axônios/fisiologia , Neurônios/fisiologia , Núcleo Rubro/fisiologia , Degeneração Retrógrada/fisiologia , Medula Espinal/fisiologia , Animais , Transporte Biológico , Denervação , Feminino , Ratos , Ratos Wistar , Fatores de TempoRESUMO
The present study revealed the efferent projections from the external cuneate nucleus (ECN) to various medullary nuclei in the gerbil as demonstrated in fresh living brainstem slices by using in vitro anterogradely tracing with the dextran-tetramethyl-rhodamine-biotin. The tracer-labelled ECN axon terminals were observed (1) in most of the vital autonomic-related nuclei: the nucleus solitary tractus, nucleus ambiguus, rostroventrolateral reticular nucleus and C2 adrenergic area, (2) in the reticular formation: the medullary, parvocellular, intermediate, gigantocellular, dorsal paragigantocellular and lateral paragigantocellular reticular nuclei and medullary linear nucleus, and (3) in sensory nuclei: the cuneate nucleus, spinal trigeminal nuclei caudalis and interpolaris, paratrigeminal nucleus, medial and spinal vestibular nuclei, inferior olive and prepositus hypoglossal nucleus. These new findings are discussed in relation to possible roles of the ECN in cardiovascular, respiratory and sensorimotor controls.