Spinal motoneuron synaptic plasticity after axotomy in the absence of inducible nitric oxide synthase

<p>Abstract</p> <p>Background</p> <p>Astrocytes play a major role in preserving and restoring structural and physiological integrity following injury to the nervous system. After peripheral axotomy, reactive gliosis propagates within adjacent spinal segments, influenced by the local synthesis of nitric oxide (NO). The present work investigated the importance of inducible nitric oxide synthase (iNOS) activity in acute and late glial responses after injury and in major histocompatibility complex class I (MHC I) expression and synaptic plasticity of inputs to lesioned alpha motoneurons.</p> <p>Methods</p> <p><it>In vivo </it>analyses were carried out using C57BL/6J-iNOS knockout (iNOS^-/-) and C57BL/6J mice. Glial response after axotomy, glial MHC I expression, and the effects of axotomy on synaptic contacts were measured using immunohistochemistry and transmission electron microscopy. For this purpose, 2-month-old animals were sacrificed and fixed one or two weeks after unilateral sciatic nerve transection, and spinal cord sections were incubated with antibodies against classical MHC I, GFAP (glial fibrillary acidic protein - an astroglial marker), Iba-1 (an ionized calcium binding adaptor protein and a microglial marker) or synaptophysin (a presynaptic terminal marker). Western blotting analysis of MHC I and nNOS expression one week after lesion were also performed. The data were analyzed using a two-tailed Student's <it>t </it>test for parametric data or a two-tailed Mann-Whitney <it>U </it>test for nonparametric data.</p> <p>Results</p> <p>A statistical difference was shown with respect to astrogliosis between strains at the different time points studied. Also, MHC I expression by iNOS^-/-microglial cells did not increase at one or two weeks after unilateral axotomy. There was a difference in synaptophysin expression reflecting synaptic elimination, in which iNOS^-/-mice displayed a decreased number of the inputs to alpha motoneurons, in comparison to that of C57BL/6J.</p> <p>Conclusion</p> <p>The findings herein indicate that iNOS isoform activity influences MHC I expression by microglial cells one and two weeks after axotomy. This finding was associated with differences in astrogliosis, number of presynaptic terminals and synaptic covering of alpha motoneurons after lesioning in the mutant mice.</p

A Voltage-Sensitive Dye-Based Assay for the Identification of Differentiated Neurons Derived from Embryonic Neural Stem Cell Cultures

BACKGROUND: Pluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC) and embryonic neural stem cell (NSC) cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca(2+)-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate. METHODOLOGY/PRINCIPAL FINDINGS: In this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD). We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters. CONCLUSIONS/SIGNIFICANCE: Our method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture

Expression of class I major histocompatibility complex (MHC I) in the central nervous system: role in synaptic plasticity and regeneration

It has been recently demonstrated that the major histocompatibility complex of class I (MHC I) expressed in the central nervous system (CNS) does not only function as a molecule of the immune system, but also plays a role in the synaptic plasticity. The expression of MHC I influences the intensity and selectivity of elimination of synapses apposed to neurons that were subjected to lesion, besides influencing the reactivity of neighboring glial cells. MHC I expression and the degree of synaptic rearrangement and glial response after injury correlate with differences in the regenerative potential and functional recovery of isogenic mice strains. In this way, the new aspects regarding MHC I functions in the CNS may guide further studies aiming at searching the involvement of MCH I in neurologic disorders, as well as the development of new therapeutic strategies.Foi demonstrado recentemente que o complexo de histocompatibilidade principal de classe I (MHC I), expresso no sistema nervoso central (SNC), não funciona somente como molécula com papel imunológico, mas também como parte de um mecanismo envolvido na plasticidade sináptica. A expressão de MHC I interfere na intensidade e seletividade da retração de sinapses em contato com neurônios que sofreram lesão e também influencia a reatividade das células gliais próximas a esses neurônios. A intensidade do rearranjo sináptico e resposta glial após lesão, ligadas à expressão de MHC I no SNC, repercute em diferenças na capacidade regenerativa e recuperação funcional em linhagens de camundongos isogênicos. Dessa forma, os novos aspectos sobre a função do MHC I no SNC direcionam futuras pesquisas no sentido de buscar o envolvimento do MHC I em doenças neurológicas e também o desenvolvimento de novas estratégias terapêuticas.El complejo mayor de histocompatibilidad de clase I (MHC I), expresado en el sistema nervioso central (SNC), no sólo funciona como una molécula con función inmunológica, sino que es crucial para las respuestas del tejido nervioso en casos de lesiones. El MHC I está involucrado con los procesos de plasticidad sináptica y las células gliales en el microambiente de la médula espinal después de realizada axotomía periférica. La expresión de MHC I interfiere con la intensidad y la forma en que se producen la contracción y la eliminación de sinapsis con relación a las neuronas, cuyos axones se han comprometido, y también influye en la reactividad de las células gliales, cerca de estas neuronas. La intensidad de estos cambios, que responden a la expresión de MHC I en el SNC, implica diferencias en la capacidad de regeneración axonal de las células dañadas por axotomía, por lo que el nivel de expresión de las moléculas MHC I se relaciona con el proceso de regeneración de los axones y, en consecuencia, con la recuperación funcional. Por consiguiente, estos nuevos aspectos sobre la función del MHC I en el SNC orientan nuevas investigaciones con miras a entender el papel del MHC I en las enfermedades neurológicas y a desarrollar nuevas estrategias terapéuticas.19319

Astrocytic reaction and spinal cord synaptic changes in different isogenic mice strains after sciatic nerve transection

Orientador: Alexandre Leite Rodrigues de OliveiraDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Astrócitos são elementos fundamentais para o funcionamento normal do sistema nervoso central (SNC). Estas células exibem inúmeras funções, podendo modular a excitabilidade neural e a transmissão de impulsos nervosos através de seus processos lamelares finos que se localizam nas adjacências das sinapses. Todavia, astrócitos também podem desempenhar funções na integridade da fisiologia do sistema nervoso (SN) após uma lesão. A astrogliose após lesão nervosa é caracterizada pela hiperplasia e hipertrofia do corpo celular e dos processos astrocitários, os quais bloqueiam a regeneração axonal. No entanto, a reatividade astrocitária também possui aspectos positivos, como a produção de fatores neurotróficos. Apesar de a plasticidade sináptica após lesão seja um fenômeno conhecido, os mecanismos envolvidos em tal evento ainda permanecem desconhecidos. Nesse sentido, células gliais, especialmente astrócitos, podem exibir importantes papéis nos processos de mudança do SN, influenciando a retração dos terminais sinápticos tanto quanto promovendo um ambiente peri-sináptico propício, afetando o reestabelecimento dos botões que foram retraídos. Nesse trabalho, estudou-se a resposta astrocitária após a axotomia em camundongos das linhagens C57BL/6J, Balb/cJ e A/J, utilizando-se técnicas de imunofluorescência, microscopia eletrônica de transmissão e cultura celular. Nesse sentido, foram apresentadas evidências de que camundongos das linhagens isogênicas C57BL/6J, Balb/cJ e A/J exibem diferentes intensidades de reatividade astrocitária após lesão periférica in vivo, sendo estas diferenças significativas entre A/J (3,87 ± 0,07, média ± erro padrão, GFAP e 3,76 ± 0,16, ezrina) e C57BL/6J (2,35 ± 0,024, p<0,0001; GFAP e 2,69 ± 0,26, ezrina, p<0,001). Ainda, astrócitos derivados de córtices de camundongos cultivados in vitro exibiram diferença significativa na imunomarcação com anti-GFAP e anti-ezrina entre as linhagens A/J (22,60 ± 1,63, média ± erro padrão, GFAP e 18,47 ± 1,31, ezrina) e C57BL/6J (13.22 ± 1,80; p<0,001, GFAP, 13,95 ± 1,16, ezrina p<0,05). Adicionalmente, a astrogliose nas adjacências dos motoneurônios axotomizados foi menor em animais C57BL/6J quando comparado com A/J, sugerindo que a diferença da reatividade astrocitária possui influência direta na sinaptogênese in vitro. Ainda, a análise estrutural quantitativa indicou maior retração sináptica nas linhagens A/J e Balb/cJ em relação ao C57BL/6J, uma semana após a transecção do nervo isquiático. Nossos resultados demonstram que o aumento da astrogliose pode influenciar o grau de plasticidade sináptica na medula espinhal, que possivelmente influencia a regeneração axonal dos motoneurônios lesionadosAbstract: Astrocytes are of major importance for normal functioning of the central nervous system (CNS). These cells have been shown to play a large number of different functions in the brain. They can modulate the neural excitability and signal transmission by their thin lamellar process surrounding synapses. Moreover, astrocytes play a major role in preserving and restoring structural and physiological integrity following injury to the CNS. Astrogliosis is characterized by hyperplasia and hypertrophy of cell bodies and processes, which has been considered to block axonal regeneration, but reactiveness of glial cells at the lesion site has also positive aspects, such as the production of neurotrophic factors. Although synaptic plasticity is a widespread phenomenon, the underlying mechanisms leading to its occurrence are virtually unknown. In this sense, glial cells, especially astrocytes, may have a role in network changes of the nervous system, influencing the retraction of boutons as well as providing a proper perisynaptic environment, thereby affecting the replacement of inputs. In the present work, we have studied the astrocytic response after axotomy in C57BL/6J, Balb/cJ and A/J mice, using immunofluorescence, transmission electron microscopy and cell cultures techniques. In this sense, we present evidence that A/J, Balb/cJ and C57BL/6J isogenic mice display different astrocyte reactivity after a peripheral lesion in vivo (A/J; 3.87 ± 0.07- GFAP and 3.76 ± 0.16 ¿ezrin / C57BL/6J; 2.35 ± 0.024 - GFAP, p<0.0001 and 2.69 ± 0.26 - ezrin, p<0.001). Further, astrocytes from mice cortices were isolated and expanded in vitro, showing a significant difference in GFAP and ezrin labeling between AJ (22.60 ± 1.63 - GFAP and 18.47 ± 1.31 - ezrin) and C57BL/6J mice (13.22 ± 1.80 ¿ GFAP, p<0.001 and 13.95 ± 1.16 ¿ ezrin, p<0.05). Also, astrogliosis surrounding axotomized motoneurons C57BL/6J is lower than A/J mice and such a difference has a direct influence on in vitro synaptogenesis. Indeed, ultrastructural quantitative analysis showed more intense synaptic detachment in A/J and Balb/cJ strains after sciatic nerve transection than in C57BL/6J mice. Our findings demonstrate that an increased astrogliosis influences the degree of synaptic plasticity in the spinal cord, which may in turn contribute to the axonal regeneration of lesioned motoneuronsMestradoAnatomiaMestre em Biologia Celular e Estrutura

Astrocyte Reactivity Influences The Number Of Presynaptic Terminals Apposed To Spinal Motoneurons After Axotomy.

Although synaptic plasticity is a widespread phenomenon, the underlying mechanisms leading to its occurrence are virtually unknown. In this sense, glial cells, especially astrocytes, may have a role in network changes of the nervous system, influencing the retraction of boutons as well as providing a proper perisynaptic environment, thereby affecting the replacement of inputs. Interestingly, the glial reaction does vary between strains of rats and mice. In this sense, we present evidence that C57BL/6J and A/J isogenic mice present different astrocyte reactivity after a peripheral lesion in vivo as well as in vitro, by analyzing primary cell cultures. Such a difference in the glial reaction has a direct influence on in vivo number of pre-synaptic terminals and on in vitro synaptogenesis.109535-4

Expressão do complexo de histocompatilidade principal de classe I (MHC I) no sistema nervoso central: plasticidade sináptica e regeneração

Foi demonstrado recentemente que o complexo de histocompatibilidade principal de classe I (MHC I), expresso no sistema nervoso central (SNC), não funciona somente como molécula com papel imunológico, mas também como parte de um mecanismo envolvido na plasticidade sináptica. A expressão de MHC I interfere na intensidade e seletividade da retração de sinapses em contato com neurônios que sofreram lesão e também influencia a reatividade das células gliais próximas a esses neurônios. A intensidade do rearranjo sináptico e resposta glial após lesão, ligadas à expressão de MHC I no SNC, repercute em diferenças na capacidade regenerativa e recuperação funcional em linhagens de camundongos isogênicos. Dessa forma, os novos aspectos sobre a função do MHC I no SNC direcionam futuras pesquisas no sentido de buscar o envolvimento do MHC I em doenças neurológicas e também o desenvolvimento de novas estratégias terapêuticas