Este trabalho foi financiado pelo Prêmio IMPACTO do Departamento de Radiação Oncológica da Universidade de Alabama-Birmingham Comprehensive Cancer Center, da Fundação da Criança de combate o câncer, ea Gabrielle Anjo (Fundação para ESY.).

1. Cultura de Células <ol><li> Cultura de células neuronais e tratá-los conforme necessário. </li><li> Células de colheita em tubos de 15 ml: meios de aspiração, lavar com solução salina tamponada com fosfato (PBS, cálcio e magnésio livre), adicionar tripsina, recolher em tubos de 15 ml e neutralizar a tripsina com meio contendo soro apropriadas. </li><li> Centrifugação a 1000 xg durante 5 min. </li><li> Aspirar mídia. </li><li> Ressuspender as células em PBS. </li><li> Centrifugação a 1000 xg durante 5 min. </li><li> Aspirar mídia e Ressuspender as células em PBS fresco. </li><li> Contar as células usando um hemocitômetro ou seu contador de células preferido. </li><li> Amostras de células deve ser preparado imediatamente antes de iniciar o ensaio. </li><li> Todas as amostras devem ser tratadas à luz escura ou amarelo para impedir o dano ao DNA da luz ultravioleta. </li></ol> 2. Ensaio Cometa 1. Preparação de slides <ol><li> Melt agarose a 1% (1 g / 100 ml em 1X Tris Base, ácido bórico, EDTA, TBE) em um forno de microondas durante 3 minutos até que todos os grânulos desaparecer. </li><li> Dip slides em a agarose fundido e limpe um lado limpar com um Kimwipe. Permitir que a agarose de ar seco a uma película transparente. Isto pode ser feito com antecedência e as lâminas podem ser armazenadas. </li></ol> 2. Ensaio do Cometa Neutral <ol><li> Solução frio de lise (2,5 M de NaCl, 100 mM de EDTA pH 10, 10 mM Tris Base, 1% de sódio sarcosinato de laurilo e Triton 1% X-100, pH 10) a 4 ° C durante pelo menos 1 hora antes da utilização. </li><li> 2.2.2. Derreta 1% baixo de derretimento ponto de de agarose a (1 g / 100ml em 1X Tris Base de Dados de, o ácido Boric, EDTA), em um forno de microondas para 3 min até que todos os grânulos de desaparecer. A agarose tem de ser arrefecida a 37 ° C num banho de água para evitar a indução artificial da cauda do cometa. Idealmente, a agarose precisa de ser arrefecida durante meia hora, antes da utilização. </li><li> 2.2.3. Diluiu-se a suspensão de células, de modo que há 100.000 células por ml. Combina-se o suspensão de células de com o baixo de derretimento ponto de de agarose a (at 37 ° C) menosuma proporção de 1:10 (v / v), de forma breve vórtice e, imediatamente pipetar 50 uL para a corrediça Comet. Usar o lado da ponta da pipeta para espalhar a suspensão de células de maneira uniforme sobre a área da amostra. Cada grupo de tratamento deve ser pelo menos em triplicado. </li><li> Lugar desliza para plana em geladeira por 30 min até que uma círculo aparece em a periferia de o slide. </li><li> Prechill a solução de de lise e slides de submergiria em esta solução de para 30 min no escuro e à 4 ° C (ou em um refrigerador). </li><li> Decantar ou aspirar a solução para lise e adicionar 1X tampão de electroforese de ponto morto (base Tris, ácido bórico, EDTA 1X TBE). Deixe lâminas neste tampão por meia hora na geladeira. </li><li> Adicionar tampão Eletroforese previamente arrefecido 1X Neutro (TBE) em câmara de eletroforese, slides Colocar na bandeja deslizante eletroforese. Alinhe os slides para que eles sejam equidistantes eletrodos. </li><li> Despeje 1X eletroforese neutro tampão até 0,2 centímetros acima slides. Tampão em excesso será entreFere com eletroforese. </li><li> Definir a tensão de alimentação de 1 V por cm (medida de eléctrodo para eléctrodo) e funcionou durante 30 minutos a 4 ° C (ou o quarto frio), no escuro. </li></ol> 3. Ensaio Cometa alcalino <ol><li> Solução frio de lise (2,5 M de NaCl, 100 mM de EDTA pH 10, 10 mM Tris Base, 1% de sódio sarcosinato de laurilo e Triton 1% X-100, pH 10) a 4 ° C durante pelo menos 1 hora antes da utilização. </li><li> Melt 1% baixo ponto de fusão de agarose (1 g / 100 ml em 1X Tris Base, ácido bórico, EDTA) em um forno de microondas durante 3 minutos até que todos os grânulos desaparecer. Em seguida, arrefeceu-se num 37 banho de água ° C durante pelo menos 30 min. </li><li> Combinar as células a 1 x 105 ml / com fundido baixo ponto de fusão de agarose (a 37 ° C) numa razão de 1:10 (v / v), de forma breve vórtice e, imediatamente pipetar 50 uL para a corrediça Comet. Usar o lado da ponta da pipeta para espalhar a suspensão de células de maneira uniforme sobre a área da amostra. Cada grupo de tratamento deve ser pelo menos em triplicado. </li><li> Lugar deslizoues plana em geladeira por 30 min até que uma círculo aparece em a periferia de o slide. </li><li> Imergir as lâminas em solução de lise previamente arrefecido e deixar a 4 ° C durante 1 hora a durante a noite no escuro. </li><li> Remover as lâminas a partir da solução de lise, drenar as lâminas e lavar uma vez com tampão de neutralização fria durante 5 minutos para remover o detergente residual e sais antes do passo de álcali desenrolamento. </li><li> Colocar as lâminas numa câmara de electroforese em gel cheia com tampão de electroforese previamente arrefecido feita recentemente (300 mM de NaOH, 1 mM EDTA, pH&gt; 13) não superior a 0,5 cm acima slides. Alinhe os slides para que eles sejam equidistantes eletrodos. </li><li> Deixe lâminas sentar no tampão alcalino durante 30 min no escuro para permitir desenrolamento do DNA e a expressão de álcali susceptível de dano. </li><li> Definir a tensão de alimentação de 1 V por cm (medida de eléctrodo para eléctrodo) e funcionou durante 30 minutos a 4 ° C (ou na sala de frio). </li></ol> 4. Fixação e StaCélulas Ining <ol><li> Escorra Tampão Eletroforese excesso </li><li> Imergir desliza em pré-refrigerada a água destilada durante 5 min à TA. </li><li> Imergir as lâminas em pré-arrefecida de etanol a 70% durante 5 min à TA. </li><li> As amostras secas durante a noite. Não exponha slides para luz brilhante. As amostras podem ser armazenadas durante meses à temperatura ambiente antes de marcar nesta fase. </li><li> Corar as lâminas por imersão em SYBR Green (1X diluído em PBS) durante 20 minutos no frigorífico. </li><li> Retirar as lâminas e permitir que-los o secar completamente,, no escuro. A agarose vai se tornar transparente quando está completamente seca. </li></ol> 4. Aquisição e Análise de Imagem <ol><li> Adquirir imagens usando microscópio de fluorescência definida para o filtro verde (Zeiss AxioVision) e analisar usando Comet software Ensaio (Instrumentos perceptivos). </li><li> Clique no botão &quot;cabeça cometa&quot; (do núcleo) e o software calcula os parâmetros, incluindo o momento da cauda médio e quantidade de DNA nonúcleo. </li><li> Analisar, pelo menos, 200 células por tratamento. </li><li> Exportar dados para o Microsoft Excel. </li><li> Calcular significar momento da cauda para cada grupo de tratamento e dados de plotagem como apropriado. </li></ol> 5. Resultados representativos Um exemplo de análise cometa ensaio em células neuronais é mostrado na Figura 2 e Figura 3. Neste caso, a irradiação das células neuronais induz danos no ADN. À medida que as células são submetidas ao campo eléctrico, o DNA migra a taxas diferentes, devido às diferenças no tamanho, que é subsequentemente analisados ​​utilizando o software Assay Comet. Quanto mais os danos no DNA, o mais distante do DNA migra para fora do núcleo. Este diminui a intensidade de fluorescente em o núcleo a qual é subsequentemente escolhido acima por o software e os resultados da em momento mais elevado cauda. A Tabela 1 retrata uma tabela de representante a partir de análise de cometa ensaio de e A Figura 4 mostra um repretativo gráfico comparando danos ao DNA em células neuronais após radiação medida pelo ensaio cometa. <img src="/files/ftp_upload/50049/50049fig1.jpg" alt="Figura 1"/> Figura 1. Gráfico Fluxo de de o ensaio de cometa. <a href="https://www.jove.com/files/ftp_upload/50049/50049fig1large.jpg" target="_blank">Clique aqui para ver figura maior</a> . <img src="/files/ftp_upload/50049/50049fig2.jpg" alt="Figura 2"/> Figura 2. Imagens representativas de células neuronais (A), sem e (B) com a cauda do cometa. <img src="/files/ftp_upload/50049/50049fig3.jpg" alt="Figura 3"/> Figura 3. Análise ensaio do cometa usando um software de Ensaio Cometa. (A) tiros Representante de tela de imagem adquirida usando Carl microscópio Zeiss fluorescente e analisados ​​utilizando CometSoftware de Ensaio. (B) Clicando sobre o núcleo ou a &quot;cabeça cometa&quot; (circulado em verde) gera um mapa fluorescente e gráfico (circulado em amarelo) e (C) tabela de dados (circulado em vermelho). <a href="https://www.jove.com/files/ftp_upload/50049/50049fig3large.jpg" target="_blank">Clique aqui para ver maior figura</a> . <img src="/files/ftp_upload/50049/50049fig4.jpg" alt="Figura 4"/> Figura 4. Gráfico representativas obtidas através da representação gráfica do momento da cauda médio obtido por análise de danos ao DNA em irradiados e não irradiados células neuronais utilizando o ensaio cometa neutro. Como esperado, a radiação 3Gy (x-ray) induz danos no ADN, como representado pela maior momento médio cauda nas células irradiadas neuronais. Mostrado é o momento da cauda média (+ / - erro padrão), ** P &lt;0,01. <img src="/files/ftp_upload/50049/50049table1.jpg" alt="Tabela 1"/> Tabela 1. </strong&gt; tabela Representante obtido pelo cometa análise ensaio em irradiados e não irradiados células neuronais. <a href="https://www.jove.com/files/ftp_upload/50049/50049table1large.jpg" target="_blank">Clique aqui para ver maior figura</a> .

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O teste do cometa tem a capacidade única de analisar células individuais. Isto é vantajoso na identificação de subpopulações de células que demonstram respostas diferenciais aos agentes citotóxicos. Algumas limitações práticas têm de ser tidas em conta. O número de células que podem ser avaliadas individualmente podem variar, dependendo do indivíduo. O tamanho da amostra tem de ser aumentada, se há uma variedade de lesões do ADN dentro de uma população. Suspensão de uma única célula viável é cr?...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Tubos de 1,5 ml </td><td> Santa Cruz Biotechnology, Inc </td><td> Sc-200271 </td><td></td></tr><tr><td> TBE 10X (Tris-base, o ácido bórico, EDTA) </td><td> Fisher Scientific </td><td> BP13331 </td><td></td></tr><tr><td> 15 ml de tubo </td><td> Fisher Scientific </td><td> 0553851 </td><td></td></tr><tr><td> Agarose </td><td> Sigma </td><td> A5093 </td><td></td></tr><tr><td> Folha de alumínio </td><td> Fisher Scientific </td><td> 01213101 </td><td></td></tr><tr><td> Proveta </td><td> Fisher Scientific </td><td> FB102300 </td><td></td></tr><tr><td> Centrifugador </td><td> Thermo Scientific </td><td> 75004261PR </td><td></td></tr><tr><td> Cometa software Ensaio </td><td> Comet Assay IV Sistema de Análise de Imagem </td><td></td><td></td></tr><tr><td> Cilindro </td><td> Fisher Scientific </td><td> 08555F </td><td></td></tr><tr><td> EDTA </td><td> GIBCO </td><td> 15575 </td><td></td></tr><tr><td> Câmara de eletroforese </td><td> Thermo Scientific </td><td> 09528101 </td><td></td></tr><tr><td> Etanol </td><td> Fisher Scientific </td><td> A407P4 </td><td></td></tr><tr><td> Microscópio fluorescente </td><td> Zeiss Axio Vision </td><td></td><td> Qualquer microscópio de fluorescência com filtro verde bastará </td></tr><tr><td> Hemocitômetro </td><td> Fisher Scientific </td><td> 0267152 </td><td></td></tr><tr><td> Baixo ponto de fusão agarose </td><td> Promega </td><td> V2111 </td><td></td></tr><tr><td> Microonda </td><td> Sears </td><td></td><td></td></tr><tr><td> Salina tamponada com fosfato, cálcio, magnésio, livre livre </td><td> HyClone </td><td> SH3025601 </td><td></td></tr><tr><td> Fonte de alimentação </td><td> BioRad </td><td> 1645050 </td><td></td></tr><tr><td> Geladeira </td><td> Sears </td><td></td><td></td></tr><tr><td> Governante </td><td> Staples </td><td></td><td></td></tr><tr><td> Deslizar </td><td> Fisher Scientific </td><td> 12550143 </td><td></td></tr><tr><td> Cloreto de sódio </td><td> Sigma </td><td> S7653 </td><td></td></tr><tr><td> De hidróxido de sódio </td><td> Sigma </td><td> S5881 </td><td></td></tr><tr><td> Lauril sarcosinato </td><td> Fisher Scientific </td><td> S529 </td><td></td></tr><tr><td> SYBR Green </td><td> Invitrogen </td><td> S7585 </td><td></td></tr><tr><td> Bandeja </td><td> Fisela Científico </td><td> 15242B </td><td></td></tr><tr><td> Tris Base </td><td> Sigma </td><td> T6066 </td><td></td></tr><tr><td> Triton-X 100 </td><td> Sigma </td><td> T8787 </td><td></td></tr><tr><td> Tripsina </td><td> HyClone </td><td> SH3023601 </td><td></td></tr><tr><td> Vórtice </td><td> Fisher Scientific </td><td> 02216108 </td><td></td></tr><tr><td> Banho de água </td><td> Fisher Scientific </td><td> 154622Q </td><td></td></tr></tbody></table>

assaying dna damage in hippocampal neurons using the comet assay

Um certo número de drogas como alvo as vias de reparação do ADN e induzem a morte de células através da criação de danos no DNA. Assim, os processos para medir directamente danos no ADN têm sido extensivamente estudados. Os métodos tradicionais são o tempo de consuming, de recursos,, caro, intensivo e requerem células de replicadoras. Em contraste, o ensaio cometa, uma única célula de ensaio de electroforese em gel, é mais rápido, não invasivo, medida, de baixo custo directo e sensível de danos no DNA e de reparação. Todas as formas de danos no DNA, bem como a reparação do ADN pode ser visualizado no nível de célula única usando esta técnica poderosa. O princípio subjacente ao doseamento é que cometa DNA intacto é altamente ordenada ao passo que o dano ao DNA interrompe essa organização. Os danificadas escoa de ADN na matriz de agarose e quando sujeito a um campo eléctrico, o DNA carregado negativamente migra em direcção ao cátodo, que está carregado positivamente. As grandes cadeias de DNA não danificadas não são capazes de migrar longe do núcleo. Danos no DNAcria fragmentos menores de DNA que viajam mais longe do que o DNA intacta. Comet Assay, um software de de análise de imagem, as medidas de e compara o global de intensidade de fluorescente de o DNA em o com um núcleo com DNA de que tenha migrado para fora de o núcleo. Sinal fluorescente do DNA migrado é proporcional aos danos de DNA. Tail DNA mais brilhante significa danos no DNA aumentada. Alguns dos parâmetros que são medidos são momento da cauda, ​​que é uma medida de tanto a quantidade de ADN e de distribuição de ADN no comprimento da cauda a cauda, ​​e percentagem de DNA na cauda. Este ensaio permite medir a reparação do ADN, bem desde resolução de danos no DNA significa reparação teve lugar. O limite de sensibilidade é de aproximadamente 50 quebras na cadeia por diplóide 1,2 células de mamíferos. As células tratadas com quaisquer agentes que danificam o ADN, tais como o etoposido, pode ser utilizado como um controlo positivo. Assim, o teste do cometa é um procedimento rápido e eficaz para avaliar os danos do DNA.

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Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

O ensaio cometa é uma forma eficiente de detectar quebras simples e de fita dupla, incluindo álcali-lábeis sites e DNA-DNA/DNA-protein ligações cruzadas no DNA em todas as células, incluindo neurônios do hipocampo. O método tira partido da migração diferencial de DNA num campo eléctrico devido a diferenças na quantidade de danos no DNA.

Ensaio danos no DNA em neurônios do hipocampo Usando o Ensaio Cometa

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

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Research

JoVE Journal

Neuroscience

27.1K visualizações.  University of Alabama-Birmingham. O ensaio cometa é uma forma eficiente de detectar quebras simples e de fita dupla, incluindo álcali-lábeis sites e DNA-DNA/DNA-protein ligações cruzadas no DNA em todas as células, incluindo neurônios do hipocampo. O método tira partido da migração diferencial de DNA num campo eléctrico devido a diferenças na quantidade de danos no DNA.

Vídeo: Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Assaying Toxicidade β-amilóide utilizando um transgênico C. elegans Modelo

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Neurociência

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Neuromodulação e Transportes mitocondrial: Imagens ao vivo em neurônios do hipocampo durante muito tempo

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Isolamento e Cultura de neurônios do hipocampo de ratos pré-natal

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Fosfato de cálcio transfecção de primário neurônios do hipocampo

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Imagiologia espinhas dendríticas dos neurônios do hipocampo de rato primária usando Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Fluorescência e imagem de bioluminescência subcelular Ca<sup&gt; 2+</sup&gt; Aged em neurônios do hipocampo

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

Stripe Ensaio para estudar a atividade atração ou repulsão de um substrato de proteína utilizando dissociado neurônios do hipocampo

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Avaliação da complexidade dendrítica do hipocampo em ratos idosos usando o método de Golgi-Cox

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

Medindo a endocitose da vesícula sináptica nos neurônios Hippocampal culturas

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

Regulação específica do circuito de assaying de células precursoras neurais hipocampais adultas

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...