Agradecemos a todos os membros do laboratório AI por seus comentários e sugestões úteis. Gostaríamos de agradecer ao Prof. Mark Hipp (Instituto Max Planck de Bioquímica) por nos fornecer o vetor shuttle-GFP. Este trabalho foi apoiado por bolsas da Fundação de Ciência Israelense (ISF # 124/14), a Fundação de Ciência Binacional (BSF # 2013325), FP7 Marie Curie Carreira Integração Grant (CIG # 333794), eo Instituto Nacional de Psicobiologia em Israel NIPI # b133-14 / 15).

1. Preparação da Linha Celular <ol><li> Descongelar aproximadamente 1.000.000 células de medula espinhal de neuroblastoma de rato (células NSC-34) que foram armazenadas num recipiente de azoto líquido. Utilize uma incubadora de água a 37 ° C até as células serem completamente descongeladas. </li><li> Adicionar meio fresco e quente (meio DMEM contendo 10% de soro fetal bovino (FBS), 1% de L-glutamina e 1% de antibióticos pen-strep). </li><li> Centrifugar as células descongeladas (SL16R) a 500 xg durante 5 min, formando uma pastilha celular. Descarte o meio e adicione 1 mL de novo meio para a ressuspensão celular. </li><li> Colocar as células ressuspensas num balão de 15 mL e adicionar 5 mL adicionais de meio. Incubar as células durante a noite numa incubadora estéril com 5% de CO2 a 37 ° C. </li><li> Em paralelo, colocar 8 lamínulas não revestidas (tampa de vidro: 12 mm, 0,13-0,17 mm de espessura) em um prato de cultura de 60 mm e esterilizar usando 70% etanol e exposição a 30 minutos de luz UV até que esteja seco. Adicionar meio a cada prato e descartar para lavar o etanol residual.</li><li> Adicionar tampão de tripsina (2,5 g de tripsina e 0,2 g de EDTA em 1 L de Solução de Sal Equilibrada de Hanks) às células depois de atingirem aproximadamente 90% de confluência (cerca de 6 milhões de células por frasco) para permitir que as células aderentes se dissociem. NOTA: As células NSC-34 são sensíveis e requerem apenas 15 s de incubação com tampão de tripsina e 1 minuto adicional de incubação sem tripsina antes da dissociação utilizando o meio. </li><li> Plantar as células nas placas de cultura de 60 mm que já contêm as lamelas com 3 mL de meio, aproximadamente 350.000 por placa. Deixá-los na incubadora por 24 h para permitir a fixação de células para as lamelas. </li></ol> 2. Transfecção celular NOTA: Após aproximadamente 24 h, as células NSC-34 atingem 40% de confluência (aproximadamente 800.000 células) e estarão prontas para transfecção. <ol><li> Preparar meio DMEM puro (300 μL de meio DMEM para cada recipiente de cultura de 60 mm) em uma cuba de microcentrífuga estérilEs. </li><li> Adicionar 2 μg de cada plasmídeo necessário para transfecção a cada tubo de microcentrífuga contendo DMEM. NOTA: O plasmídeo shuttle-GFP é obrigatório em todas as transfecções celulares. </li><li> Adicionar o reagente de transfecção a todos os tubos de microcentrífuga que contêm os seus plasmídeos necessários. Aqui, adicionar 4 μL do reagente de transfecção comercial para cada 2 μg de ADN. </li><li> Vortex os tubos de microcentrífuga contendo DMEM, plasmídeos e o reagente de transfecção e incuba-os à temperatura ambiente durante 25 min. </li><li> Adicionar cada tubo de microcentrífuga a um prato de cultura e agitar suavemente o prato para formar uma solução homogénea. Coloque o prato de volta na incubadora por 48 h. NOTA: Uma vez que a vaivï¿½-GFP deve ser expressa em todas as cï¿½ulas, a eficiï¿½cia de transfecï¿½o de shuttle-GFP pode ser parcialmente examinada e testada previamente utilizando microscopia de fluorescï¿½cia apï¿½ transfecï¿½o durante a noite. Um exame completo de proteínas expressas não fluorescentes pode ser conduzido utilizando uma análise de imunobloté. </li></ol> 3. Microscopia <ol><li> Anexar as lamelas contendo células NSC-34 transfectadas para um suporte de lamela e lave suavemente com solução salina tamponada com fosfato (PBS) para remover as células mortas destacadas. </li><li> Identificar um adesivo celular adequado utilizando um filtro fluorescente verde com um microscópio invertido. NOTA: Um adesivo de célula adequado é definido como um campo de visão de microscópio mostrando um grande número de células (20-30 células num campo) e em que os núcleos estão claramente definidos nas células. Os núcleos celulares dentro do remendo de células são primeiro identificados por olho e são marcados manualmente pelo software de imagem. </li><li> Adicionar 200 μL de um tampão de leptomicina B (LMB) inibidor da exportina-1 contendo 10 ng / mL de LMB em PBS aos lamínulas por gotejamento do tampão nos lamínulas sob o microscópio. NOTA: Este ponto no tempo é definido como intervalo zero. </li><li> Quantificar a intensidade fluorescente dos núcleos marcados em intervalos de 3 s por cerca de 400-500 s. </li><li&gt; Teste entre 3 e 5 lamínulas para cada grupo de transfecção em cada experiência individual. </li></ol> 4. Análise NOTA: Uma vez que diferentes células expressam diferentes níveis de transferência de GFP fluorescente, é importante normalizar a intensidade fluorescente nuclear de cada célula para a sua própria intensidade fluorescente inicial. <ol><li> Normalize a intensidade fluorescente dos núcleos marcados dividindo o intervalo fluorescente de 3 de cada núcleo com a média dos seis intervalos iniciais desse núcleo. </li><li> Após a normalização de cada núcleo, produzir uma curva de intensidade fluorescente representando a mudança na fluorescência dos núcleos em função do tempo para cada remendo de célula. Para fazer isto, calcule a média de todas as pilhas em cada remendo da pilha (que consiste em aproximadamente 20-30 pilhas em cada remendo). NOTA: A curva de intensidade de fluorescência dos núcleos se assemelha a uma curva de saturação de Michaelis-Menten, que é melhor observadaMe períodos. Isto é devido a uma diminuição nas quantidades citosólicas de shuttle-GFP com o tempo devido ao transporte nuclear. Esta diminuição diminui estatisticamente a probabilidade de uma vaivém-GFP se transportar para o núcleo, atingindo assim a saturação na fluorescência nuclear. A inclinação derivada da taxa inicial linear da curva produz a taxa de entrada de Vmax no núcleo. </li><li> Analise as inclinações de Vmax de cada remendo de célula, derivadas de três a cinco experimentos independentes, usando o software gráfico para produzir uma inclinação que representa cada grupo experimental. Realize a quantificação usando análise estatística ANOVA unidirecional. </li></ol> 5. Validação de Expressão de Proteína <ol><li> Colocar recipientes de cultura de 60 mm contendo as células residuais (não utilizadas na experiência anterior) de cada grupo experimental em gelo. </li><li> Eliminar o meio e lavar as células duas vezes com PBS para diluir e lavar o meio restante. </li><li> Tratar as células com 200-400ML de tampão de lise contendo PBS, 0,5% de Triton e comprimido de cocktail de inibidor de protease (PI). Raspe usando um raspador de células. </li><li> Recolher as células em tampão de lise num tubo de microcentrífuga e homogeneizar com um homogeneizador a 4000 rpm durante 1 min sob gelo. </li><li> Centrifugar as células homogeneizadas a 2.500 xg durante 10 min. Recolher o sobrenadante, quantificar a concentração de proteína utilizando um ensaio de Bradford e armazenar até à utilização a -20 ° C. </li><li> Recolher entre 30 e 70 μg de proteína total (dependendo da proteína) e carregá-lo em um gel de SDS-PAGE para testar a expressão da proteína usando um ensaio de imunoblot. </li></ol>

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Este protocolo demonstra um ensaio altamente sensível e quantitativo novo para avaliar mudanças minuciosas no transporte nucleocytoplasmic. Utilizando este sistema, é possível observar e medir o transporte nucleocitoplasmático e sua disfunção em tempo real, não só grandes defeitos que resultam em mudanças dramáticas na distribuição de proteínas. Este ensaio não só tem uma vantagem de sensibilidade, mas também requer pouca preparação, é barato e é um ensaio muito fácil e de baixo engajamento. <p...

O complexo de poros nucleares (NPC) controla a importação e exportação de grandes moléculas dentro e fora do núcleo da célula. Em contraste com pequenas moléculas, que podem entrar no núcleo sem a regulação 1 , o transporte de moléculas maiores é fortemente controlado pelo NPC. Essas grandes moléculas, como proteínas e RNA, associam-se a fatores de transporte, incluindo importações e exportações, para serem importadas e exportadas do núcleo 2 . Para serem importados, as proteínas devem conter um pequeno motivo peptídico, geralmente chamado de sinal de localização nuclear (NLS), que é limitado por importinas 2 . Estas sequências de aminoácidos actuam como um marcador e são diversas na composição 3 , 4 . As proteínas podem ser exportadas do núcleo para o citoplasma devido à sua associação com aS, que ligam uma seqüência de sinal, geralmente chamada de sinal de exportação nuclear (NES) 2 . Ambos importins e exportins são capazes de transportar sua carga devido à regulação da pequena proteína nuclear relacionada Ras GTPase (Ran) 2 . Ran demonstrou-se que existem em diferentes estados conformacionais dependendo se está ligado a GTP ou GDP. Quando ligado a GTP, Ran pode vincular a importins ou exportins. Após a vinculação ao RanGTP, importins liberar sua carga, enquanto exportins deve vincular RanGTP para formar um complexo com sua carga de exportação. O estado de ligação de nucleótidos dominante de Ran depende da sua localização no núcleo (RanGTP) ou do citoplasma (RanGDP). Recentemente, vários estudos têm demonstrado uma ligação entre deficiências na via nucleocitoplasmática e tanto a esclerose lateral amiotrófica (ELA) quanto a demência frontotemporal (FTD) 5 , 6 </sup7 , 8 , 9 , 10 , 11 , 12 . A ELA é uma doença neurodegenerativa progressiva e fatal que afeta os neurônios motores superiores e inferiores (MNs) 13 e resulta em paralisia e, em última instância, na morte, em média de 2 a 5 anos. A maioria dos casos de ELA é classificada como esporádica (SALS), sem qualquer ligação genética aparente, mas 10% são herdados de forma dominante (ALS familiar, FALS). Recentemente, foram identificadas as expansï¿½s de repetiï¿½o de hexanucleï¿½idos (HREs) no gene 72 de leitura aberta do cromossoma 9 (C9orf72) 14 , 15 como uma causa genï¿½ica de ALS e FTD. Esses mutantes respondem por 30-40% dos casos FALS 16 , e diferentes estudos alegaramQue causam toxicidade por afetar o transporte nucleocitoplasmático 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 . Estes estudos mostram principalmente os resultados finais e manifestações de HREs sobre o transporte nucleocytoplasmic, mas eles não demonstram disrupção nucleocytoplasmic em tempo real. Como resultado, apenas a deficiência grave de transporte nucleocitoplasmático foi avaliada 11 , 17 , 18 . Este protocolo descreve um novo eSensível para avaliar e quantificar a disfunção nucleocitoplasmática de transporte em tempo real. A taxa de importação de uma proteína NLS-NES-GFP (shuttle-GFP) pode ser quantificada em tempo real utilizando microscopia de fluorescência. Isto é feito utilizando um inibidor de exportação, tal como descrito anteriormente 18 , permitindo assim que a vaivém-GFP apenas entre no núcleo. Usando a microscopia fluorescente e um software capaz de quantificar alterações fluorescentes em tempo real, é possível quantificar mudanças graduais na intensidade de fluorescência da nave-GFP, localizada no núcleo. Como resultado, pode ser feita uma curva de saturação tipo Michaelis-Menten do eixo de fluorescência ao tempo, representando a quantidade de GFP nuclear de transporte em qualquer momento dado. Usando a taxa linear inicial das curvas, é possível gerar uma inclinação, que representa a taxa de entrada no núcleo antes da saturação de fluorescência. Para validar o ensaio, oForam utilizadas repetições de dipeptídeos C9orf72, poli (GR) e poli (PR), que foram previamente relatados como perturbadores do transporte nucleocitoplasmático, 5 , 7 , 8 , 10 , 12 . Utilizando este ensaio, observou-se uma diminuição de 50% na taxa de importação nuclear em comparação com o controlo. Usando este sistema, podem ser examinadas pequenas alterações no transporte nucleocitoplasmático. Além disso, a capacidade de diferentes factores para resgatar um defeito de transporte nucleocitoplasmático pode ser determinada.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mouse Motor Neuron-Like hybrid Cell Line (NSC-34)</td>
			<td>tebu-bio</td>
			<td>CLU140-A</td>
		</tr>
		<tr>
			<td>DMEM 4.5g/L L-glucose</td>
			<td>Biological Industries</td>
			<td>01-055-1A</td>
		</tr>
		<tr>
			<td>FBS European Grade</td>
			<td>Biological Industries</td>
			<td>04-007-1A</td>
		</tr>
		<tr>
			<td>SL 16R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004030</td>
		</tr>
		<tr>
			<td>Thermo Forma Series ii Water Jacketed CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>3111</td>
		</tr>
		<tr>
			<td>Cover glass 12mm dia. thick 0.13-0.17mm</td>
			<td>Bar Naor LTD</td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://www.thermofisher.com/order/catalog/product/R0531">TurboFect Transfection Reagent</a></td>
			<td>Thermo Scientific</td>
			<td><a href="https://www.thermofisher.com/order/catalog/product/R0531">R0531</a></td>
		</tr>
		<tr>
			<td>Axiovert 100 inverted microscope</td>
			<td>Zeiss</td>
			<td></td>
		</tr>
		<tr>
			<td>IMAGING WORKBENCH 2&#160;</td>
			<td>Axon Instruments</td>
			<td></td>
		</tr>
		<tr>
			<td>Leptomycin B</td>
			<td>Sigma</td>
			<td>L2913</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biological Industries</td>
			<td>02-023-1A</td>
		</tr>
		<tr>
			<td>Homogenizer motor/control/chuck/90-degree clamp</td>
			<td>Glas-Col</td>
			<td>099C K5424CE</td>
		</tr>
		<tr>
			<td>MicroCL 17R Centrifuge, Refrigerated</td>
			<td>Thermo Scientific</td>
			<td>75002455</td>
		</tr>
	</tbody>
</table>

assay to measure nucleocytoplasmic transport in real time within motor neuron-like nsc-34 cells

O transporte nucleocitoplasmático refere-se à importação e exportação de grandes moléculas do núcleo celular. Recentemente, vários estudos têm mostrado uma ligação entre a esclerose lateral amiotrófica (ELA) e deficiências na via nucleocitoplasmática. ALS é uma doença neurodegenerativa que afeta os neurônios motores e resultando em paralisia e, finalmente, na morte, em média 2-5 anos. A maioria dos casos de ELA são esporádicos, sem qualquer ligação genética aparente, mas 10% são herdados de forma dominante. Recentemente, as expansï¿½s de repetiï¿½o de hexanucleï¿½idos (HREs) no gene 72 de leitura aberta do cromossoma 9 (C9orf72) foram identificadas como uma causa genï¿½ica de ALS e demï¿½cia frontotemporal (FTD). É importante notar que diferentes grupos propuseram recentemente que estes mutantes afectam o transporte nucleocitoplasmático. Esses estudos mostram principalmente o resultado final e manifestações causadas por HREs sobre o transporte nucleocitoplasmático, mas eles não demonstram disfunção de transporte nuclear emtempo real. Como resultado, apenas a deficiência grave de transporte nucleocitoplasmático pode ser determinada, principalmente devido à alta sobreexpressão ou inserção de proteína exógena. Este protocolo descreve um ensaio novo e muito sensível para avaliar e quantificar a disfunção nucleocitoplasmática de transporte em tempo real. A taxa de importação de uma proteína NLS-NES-GFP (shuttle-GFP) pode ser quantificada em tempo real utilizando microscopia de fluorescência. Isto é realizado utilizando um inibidor de exportação, permitindo assim que a GFP de transporte apenas entre no núcleo. Para validar o ensaio, utilizaram-se as repetições de dipeptídeos traduzidos por HRE de C9orf72, poli (GR) e poli (PR), que anteriormente foram mostrados como perturbadores do transporte nucleocitoplasmático. Utilizando o ensaio descrito, observou-se uma diminuição de 50% na taxa de importação nuclear em comparação com o controlo. Usando este sistema, podem ser examinadas pequenas alterações no transporte nucleocitoplasmático e a capacidade de diferentes fatores para resgatar (mesmo parcialmente) um trato nucleocitoplasmáticoPode ser determinado.

Utilizando o procedimento aqui apresentado, as células NSC-34 do tipo neurónio motor foram transfectadas com uma proteína NLS-NES-GFP (shuttle-GFP). Esta proteína, que tem NLS e NES sinal seqüências ( Figura 1 ), pode shuttle entre o núcleo eo citoplasma. A GFP genï¿½ica pode entrar ou sair do nï¿½leo na ausï¿½cia de qualquer marca de sinal de localizaï¿½o apenas por difusï¿½ e, por conseguinte, ï¿½ distribuï¿½a uniformemente entre o nï¿½le...

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Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Este protocolo descreve um método para medir sensivelmente a taxa de transporte nucleocitoplasmático dentro de células NSC-34 do tipo neurônio motor, quantificando a mudança em tempo real na importação nuclear de uma proteína NLS-NES-GFP.

Ensaio para Medir o Transporte Nucleocitoplasmático em Tempo Real dentro de Células NSC-34 do tipo Motor-Neuron

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

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Neuroscience

8.6K visualizações.  Ben-Gurion University of the Negev.  Este protocolo descreve um método para medir sensivelmente a taxa de transporte nucleocitoplasmático dentro de células NSC-34 do tipo neurônio motor, quantificando a mudança em tempo real na importação nuclear de uma proteína NLS-NES-GFP. 

Vídeo: Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Lapso de tempo de imagens ao vivo de relação clonal células progenitoras neurais no desenvolvimento Zebrafish Encéfalo anterior

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Neurociência

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

Motor Nerve transecção e Time-lapso de Comportamentos de células gliais em Zebrafish ao vivo

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

Using Informational Connectivity to Measure the Synchronous Emergence of fMRI Multi-voxel Information Across Time

It is now appreciated that condition-relevant information can be present within distributed patterns of functional magnetic resonance imaging (fMRI) brain activity, even for conditions with similar levels of univariate activation. Multi-voxel pattern (MVP) analysis has been used to decode this information with great success. FMRI investigators also often seek to understand how brain regions interact in interconnected networks, and use functional connectivity (FC) to identify regions that have correlated responses over time. Just as univariate analyses can be insensitive to information in MVPs, FC may not fully characterize the brain networks that process conditions with characteristic MVP signatures. The method described here, informational connectivity (IC), can identify regions with correlated changes in MVP-discriminability across time, revealing connectivity that is not accessible to FC. The method can be exploratory, using searchlights to identify seed-connected areas, or planned, between pre-selected regions-of-interest. The results can elucidate networks of regions that process MVP-related conditions, can breakdown MVPA searchlight maps into separate networks, or can be compared across tasks and patient groups.

Informational connectivity measures the correspondence between time courses of multi-voxel information across different brain regions. Multi-voxel pattern discriminability time series are extracted from regions and compared, revealing networks that are not identified in a typical functional connectivity approach.

Usando Informativa Conectividade medir o Emergence Synchronous de fMRI Multi-voxel Informações Across Time

It is now appreciated that condition-relevant information can be present within distributed patterns of functional magnetic resonance imaging (fMRI) brain ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD) and Huntington&#8217;s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 &#956;m/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

Imagens em tempo real de axonal Transporte de Quantum Dot marcado BDNF na Primária Neurônios

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

Eletrofisiologia em tempo real: Usando protocolos de circuito fechado para sondar Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

Itoforese em tempo real com tetrametilamônio para quantificar fração de volume e tortuosidade do espaço extracelular cerebral

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

Viver-pilha medição de odorante Receptor de ativação usando um ensaio de campo em tempo real

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Desenvolvimento de ensaio para quantificação de conteúdo elevado de Sod1 proteína mutante formação agregada em células vivas

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

Transporte Axonal de Organelas em Culturas de Neurônios Motores usando sistema de câmaras microfluidas

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...

Real-Time fMRI Brain Mapping in Animals

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a real-time fMRI platform to guide experimenters to monitor fMRI responses instantaneously during acquisition, which can be used to modify the physiology of animals to achieve desired hemodynamic responses in animal brains. The real-time fMRI set-up is based on a 14.1T preclinical MRI system, enabling the real-time mapping of dynamic fMRI responses in the primary forepaw somatosensory cortex (FP-S1) of anesthetized rats. Instead of a retrospective analysis to investigate confounding sources leading to the variability of fMRI signals, the real-time fMRI platform provides a more effective scheme to identify dynamic fMRI responses using customized macro-functions and a common neuroimage analysis software in the MRI system. Also, it provides immediate troubleshooting feasibility and a real-time biofeedback stimulation paradigm for brain functional studies in animals.

Animal brain functional mapping can benefit from the real-time functional magnetic resonance imaging (fMRI) experimental set-up. Using the latest software implemented in the animal MRI system, we established a real-time monitoring platform for small animal fMRI.

Mapeamento cerebral por fMRI em tempo real em animais

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a ...