Este trabajo fue apoyado por el Impact Award del Departamento de Oncología de Radiación de la Universidad de Alabama-Birmingham Comprehensive Cancer Center, la Fundación de Niños con Cáncer de lucha, y el Ángel de Gabrielle Foundation (para ESY.).

1. Cultivo celular <ol><li> Cultura células neuronales y tratarlos según sea necesario. </li><li> Células cosecha en tubos de 15 ml: medios de aspirado, enjuague con tampón fosfato salino (PBS, calcio y magnesio libre), añadir tripsina, recoger en tubos de 15 ml y neutralizar la tripsina con apropiados medios que contienen suero. </li><li> Girar a 1.000 xg durante 5 min. </li><li> Aspirar medios de comunicación. </li><li> Resuspender las células en PBS. </li><li> Girar a 1.000 xg durante 5 min. </li><li> Aspirar medio y las células se resuspenden en PBS fresco. </li><li> Contar las células utilizando un hemocitómetro o su contador celular preferido. </li><li> Las muestras de células debe prepararse inmediatamente antes de iniciar el ensayo. </li><li> Todas las muestras deben ser manejados en la oscuridad o bajo luz amarilla para evitar daños en el ADN de la luz ultravioleta. </li></ol> 2. Comet Assay 1. Preparación del portaobjetos <ol><li> Derretir 1% de agarosa (1 g / 100 ml en 1X Tris Base, ácido bórico, EDTA, TBE) en un microondas durante 3 min hasta que todos los gránulos desaparecer. </li><li> Dip diapositivas en la agarosa fundida y limpie un lado limpiar con un Kimwipe. Permitir que la agarosa se seca al aire a una película transparente. Esto se puede hacer por anticipado y diapositivas se pueden almacenar. </li></ol> 2. Ensayo Cometa Neutral <ol><li> Enfriar la solución de lisis (2,5 M NaCl, 100 mM EDTA pH 10, 10 mM Tris base, 1% de sodio lauril sarcosinato, y 1% de Triton X-100, pH 10) a 4 ° C durante al menos 1 h antes de su uso. </li><li> 2.2.2. Derretir 1% de agarosa de bajo punto de fusión (1 g / 100 ml en 1X Tris Base, ácido bórico, EDTA) en un microondas durante 3 min hasta que todos los gránulos desaparecer. La agarosa necesita ser enfriado a 37 ° C en un baño de agua para evitar la inducción artificial de cola del cometa. Idealmente, la agarosa necesita ser enfriado durante media hora antes de su uso. </li><li> 2.2.3. Diluir la suspensión de células de modo que hay 100.000 células por ml. Combinar la suspensión celular con el bajo punto de fusión de agarosa (a 37 ° C) enuna proporción de 1:10 (v / v), agitar brevemente, e inmediatamente pipetear 50 l sobre el portaobjetos Comet. Use el lado de la punta de la pipeta para difundir la suspensión de células uniformemente sobre el área de la muestra. Cada grupo de tratamiento debería ser al menos por triplicado. </li><li> Place desliza plana en el refrigerador durante 30 min hasta que aparece un círculo en la periferia de la corredera. </li><li> Prechill la solución de lisis y los portaobjetos en esta solución durante 30 min en la oscuridad a 4 ° C (o en un refrigerador). </li><li> Vierta o aspirar la solución de lisis y añadir tampón de electroforesis 1X neutro (Tris base, ácido bórico, EDTA, 1X TBE). Agregar diapositivas en este tampón durante media hora en el refrigerador. </li><li> Añadir preenfriada buffer 1X Electroforesis Neutral (TBE) en la cámara de electroforesis, coloque los portaobjetos en la bandeja de electroforesis de diapositivas. Alinear las diapositivas de manera que sean equidistantes de electrodos. </li><li> Vierta electroforesis 1X neutral almacenar hasta 0,2 pulgadas por encima de las diapositivas. El exceso de buffer de entreinterferir con la electroforesis. </li><li> Ajuste el voltaje de suministro de energía a 1 V por cm (medido electrodo a electrodo) y una duración de 30 min a 4 ° C (o el cuarto frío) en la oscuridad. </li></ol> 3. Ensayo Cometa Alcalino <ol><li> Enfriar la solución de lisis (2,5 M NaCl, 100 mM EDTA pH 10, 10 mM Tris base, 1% de sodio lauril sarcosinato, y 1% de Triton X-100, pH 10) a 4 ° C durante al menos 1 h antes de su uso. </li><li> Derretir 1% de agarosa de bajo punto de fusión (1 g / 100 ml en 1X Tris Base, ácido bórico, EDTA) en un microondas durante 3 min hasta que todos los gránulos desaparecer. Luego enfriar en un baño de agua a 37 ° C durante al menos 30 min. </li><li> Combinar las células a 1 x 105 / ml con fundido bajo punto de fusión de agarosa (a 37 ° C) en una proporción de 1:10 (v / v), agitar brevemente, e inmediatamente pipetear 50 l sobre el portaobjetos Comet. Use el lado de la punta de la pipeta para difundir la suspensión de células uniformemente sobre el área de la muestra. Cada grupo de tratamiento debería ser al menos por triplicado. </li><li> Lugar deslizóes plana en el refrigerador durante 30 min hasta que aparece un círculo en la periferia de la corredera. </li><li> Sumergir los portaobjetos en solución de lisis previamente enfriado y salen a 4 ° C durante 1 hora hasta toda la noche en la oscuridad. </li><li> Retirar los portaobjetos de la solución de lisis, drenar los portaobjetos y se lava una vez con tampón de neutralización en frío durante 5 min para eliminar el detergente residual y las sales antes de la etapa de desenrollado alcalino-. </li><li> Colocar los portaobjetos en una cámara de electroforesis en gel de llenado con tampón de electroforesis preenfriada recién hecho (300 mM NaOH, 1 mM EDTA,&gt; 13 pH) que no exceda de 0,5 cm por encima de diapositivas. Alinear las diapositivas de manera que sean equidistantes de electrodos. </li><li> Vamos diapositivas sentarse en el tampón alcalino durante 30 min en la oscuridad para permitir el desenrollado del ADN y la expresión de álcali responsable daño. </li><li> Ajuste el voltaje de suministro de energía a 1 V por cm (medido electrodo a electrodo) y una duración de 30 min a 4 ° C (o el cuarto frío). </li></ol> 4. Fijación y StaLas células nando <ol><li> Escurrir el exceso de tampón de electroforesis </li><li> Sumergir desliza en pre-refrigerada agua destilada durante 5 min a TA. </li><li> Sumergir los portaobjetos en pre-enfriada de etanol al 70% durante 5 min a RT. </li><li> Las muestras secas durante la noche. No exponga las transparencias a la luz brillante. Las muestras pueden almacenarse durante meses a temperatura ambiente antes de la puntuación en esta etapa. </li><li> Mancha de diapositivas por inmersión en SYBR Green (1X diluido en PBS) durante 20 minutos en el refrigerador. </li><li> Retirar los portaobjetos y dejar que se sequen completamente en la oscuridad. La agarosa se convertirá en transparente cuando está completamente seco. </li></ol> 4. Adquisición de imágenes y análisis <ol><li> Adquirir imágenes con microscopio de fluorescencia establece en el filtro verde (Zeiss AxioVision) y analizar el uso de software Comet Assay (instrumentos perceptivos). </li><li> Haga clic en la &quot;cabeza cometa&quot; (el núcleo) y el software calcula los parámetros que incluye el momento de la cola media y la cantidad de ADN en lanúcleo. </li><li> Analizar al menos 200 células por tratamiento. </li><li> Exportar datos a Microsoft Excel. </li><li> Calcular significa momento de la cola para cada grupo de tratamiento y los datos de trama según proceda. </li></ol> 5. Los resultados representativos Un ejemplo de análisis de cometa ensayo sobre las células neuronales se muestra en la Figura 2 y la Figura 3. En este caso, la irradiación de las células neuronales induce daño en el ADN. Como las células son sometidas a un campo eléctrico, el ADN migra a velocidades diferentes debido a diferencias en el tamaño que posteriormente se analizaron mediante el programa ensayo cometa. Cuanto más el daño en el ADN, más lejos el ADN migra fuera del núcleo. Esto disminuye la intensidad de fluorescencia en el núcleo, el cual es posteriormente recogido por el software y los resultados en momento de la cola superior. Tabla 1 representa una tabla representativa de análisis ensayo cometa y la Figura 4 muestra una representacióntativo gráfico comparando el daño del ADN en las células neuronales después de la radiación, medido por el ensayo de cometa. <img src="/files/ftp_upload/50049/50049fig1.jpg" alt="Figura 1"/> Figura 1. Diagrama de flujo del ensayo cometa. <a href="https://www.jove.com/files/ftp_upload/50049/50049fig1large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> . <img src="/files/ftp_upload/50049/50049fig2.jpg" alt="Figura 2"/> Figura 2. Imágenes representativas de células neuronales (A) sin y (B) con cola de cometa. <img src="/files/ftp_upload/50049/50049fig3.jpg" alt="Figura 3"/> Figura 3. Comet análisis ensayo utilizando software Comet Assay. (A) disparos representativos de pantalla de imagen adquirida con Carl Zeiss microscopio fluorescente y se analizaron usando CometEnsayo de software. (B) Al hacer clic en el núcleo o la &quot;cabeza del cometa&quot; (con un círculo verde) genera un mapa fluorescente y gráfica (marcada en amarillo) y un cuadro (C) datos (círculo rojo). <a href="https://www.jove.com/files/ftp_upload/50049/50049fig3large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> . <img src="/files/ftp_upload/50049/50049fig4.jpg" alt="Figura 4"/> Figura 4. Gráfico representativo obtenido por el trazado de la momento de la cola media obtenida mediante el análisis de daño del ADN en las células neuronales irradiados y no irradiados mediante ensayo cometa neutral. Como era de esperar, la radiación 3GY (rayos X) induce daño en el ADN como se ha representado por el momento media cola mayor en las células neuronales irradiados. Se muestra el momento de la cola media (+ / - error estándar), ** P &lt;0,01. <img src="/files/ftp_upload/50049/50049table1.jpg" alt="Tabla 1"/> Tabla 1. </strong&gt; cuadro representativo obtenido por el análisis de prueba del cometa en células neuronales irradiados y no irradiados. <a href="https://www.jove.com/files/ftp_upload/50049/50049table1large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> .

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No hay conflictos de interés declarado.

El ensayo de cometa tiene la capacidad única de análisis de células individuales. Esto es ventajoso en la identificación de las subpoblaciones de células que muestran una respuesta diferencial a los agentes citotóxicos. A pocas limitaciones prácticas tienen que ser tomadas en cuenta. El número de células que pueden ser evaluados individualmente puede variar dependiendo de la persona. El tamaño de la muestra debe ser mayor si existe una variación en el daño del ADN dentro de una población. Viable sola célul...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> El número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> 1,5 ml tubos </td><td> Santa Cruz Biotechnology, Inc </td><td> Sc-200271 </td><td></td></tr><tr><td> 10X TBE (Tris base, ácido bórico, EDTA) </td><td> Fisher Scientific </td><td> BP13331 </td><td></td></tr><tr><td> 15 ml tubo </td><td> Fisher Scientific </td><td> 0553851 </td><td></td></tr><tr><td> Agarosa </td><td> Sigma </td><td> A5093 </td><td></td></tr><tr><td> Lámina de aluminio </td><td> Fisher Scientific </td><td> 01213101 </td><td></td></tr><tr><td> Vaso </td><td> Fisher Scientific </td><td> FB102300 </td><td></td></tr><tr><td> Centrífugo </td><td> Thermo Scientific </td><td> 75004261PR </td><td></td></tr><tr><td> Ensayo Cometa software </td><td> Comet Assay IV Image Analysis System </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> Electroforesis cámara </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> Microscopio de fluorescencia </td><td> Zeiss Axio Vision </td><td></td><td> Cualquier microscopio de fluorescencia con filtro verde será suficiente </td></tr><tr><td> Hemocitómetro </td><td> Fisher Scientific </td><td> 0267152 </td><td></td></tr><tr><td> Bajo punto de fusión de agarosa </td><td> Promega </td><td> V2111 </td><td></td></tr><tr><td> Microonda </td><td> Sears </td><td></td><td></td></tr><tr><td> Tampón fosfato salino, calcio magnesio libre, libre </td><td> HyClone </td><td> SH3025601 </td><td></td></tr><tr><td> Fuente de alimentación </td><td> BioRad </td><td> 1645050 </td><td></td></tr><tr><td> Refrigerador </td><td> Sears </td><td></td><td></td></tr><tr><td> Regla </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> De cloruro de sodio </td><td> Sigma </td><td> S7653 </td><td></td></tr><tr><td> Hidróxido de sodio </td><td> Sigma </td><td> S5881 </td><td></td></tr><tr><td> Sodio 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> Fisla Ciencia </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> Baño de agua </td><td> Fisher Scientific </td><td> 154622Q </td><td></td></tr></tbody></table>

assaying dna damage in hippocampal neurons using the comet assay

Un número de medicamentos se dirigen las rutas de reparación de ADN e inducir la muerte celular mediante la creación de daños en el ADN. Así, los procesos para medir directamente el daño del ADN han sido ampliamente evaluados. Los métodos tradicionales requieren mucho tiempo, los recursos costosos, y requieren células en replicación. En contraste, el ensayo de cometa, un gel de una sola célula del ensayo de electroforesis, es más rápido, no invasivo, de bajo costo medida, directa y sensible de daño y reparación del ADN. Todas las formas de daño en el ADN, así como la reparación del ADN puede ser visualizado en el nivel de células individuales utilizando esta técnica de gran alcance. El principio que subyace a la prueba del cometa es que el ADN intacto es muy ordenado, mientras que el daño del ADN altera esta organización. Las filtraciones de ADN dañado en la matriz de agarosa y cuando se somete a un campo eléctrico, el ADN cargado negativamente migra hacia el cátodo, que está cargado positivamente. Las hebras grandes no dañadas de ADN no son capaces de migrar lejos del núcleo. Daño en el DNAcrea pequeños fragmentos de ADN, que pueden desplazarse más lejos que el ADN intacto. Comet Assay, un software de análisis de imagen, y compara las medidas de la intensidad global fluorescente del ADN en el núcleo con ADN que ha migrado fuera del núcleo. Señal fluorescente del ADN migrado es proporcional al daño del ADN. Brillante cola más larga de ADN significa un mayor daño del ADN. Algunos de los parámetros que se miden son momento de la cola, que es una medida tanto de la cantidad de ADN y la distribución de ADN en la longitud de la cola de la cola, y el porcentaje de ADN en la cola. Este ensayo permite medir la reparación del ADN, así ya que la resolución de daños en el ADN significa reparación ha tenido lugar. El límite de sensibilidad es de aproximadamente 50 roturas de cadena por célula diploide 1,2 mamíferos. Las células tratadas con agentes que dañan el ADN, tales como etopósido, se puede utilizar como un control positivo. Así, el ensayo cometa es un procedimiento rápido y eficaz para medir el daño del ADN.

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Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

El ensayo cometa es una manera eficaz de detectar saltos de simple y doble hebra, incluyendo álcali-lábiles y sitios DNA-DNA/DNA-protein enlaces cruzados en el ADN en todas las células incluyendo neuronas del hipocampo. El método se aprovecha de la migración diferencial de ADN en un campo eléctrico debido a las diferencias en la cantidad de daño en el DNA.

Ensayo de daño del ADN en las neuronas del hipocampo utilizando el ensayo 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 ...

assaying-dna-damage-in-hippocampal-neurons-using-the-comet-assay

Research

JoVE Journal

Neuroscience

27.1K vistas.  University of Alabama-Birmingham. El ensayo cometa es una manera eficaz de detectar saltos de simple y doble hebra, incluyendo álcali-lábiles y sitios DNA-DNA/DNA-protein enlaces cruzados en el ADN en todas las células incluyendo neuronas del hipocampo. El método se aprovecha de la migración diferencial de ADN en un campo eléctrico debido a las diferencias en la cantidad de daño en el DNA.

Video: 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.

Ensayo β-amiloide toxicidad con un transgénico 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 ...

Investigación

Neurociencias

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.

La neuromodulación y mitocondrial de transporte: Imágenes en vivo en las neuronas del hipocampo durante largos períodos de tiempo

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.

Aislamiento y cultivo de neuronas del hipocampo de ratones prenatales

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 Calcio La transfección de Primaria neuronas del 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.

Imaging las espinas dendríticas de las neuronas del hipocampo de rata primaria utilizando Estructurado Iluminación Microscopía

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.

Fluorescencia y bioluminiscencia de imágenes de subcelular Ca<sup&gt; 2 +</sup&gt; Envejecido en las neuronas del 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.

Raya de ensayo para estudiar la actividad de atracción o repulsión de un sustrato de proteína usando las neuronas del hipocampo disociadas

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.

Evaluación de la complejidad dendrítica del hipocampo en ratones envejecidos utilizando el 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.

Medición de endocitosis vesícula sináptica en neuronas Hippocampal cultivadas

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.

Regulación específica del circuito de ensayo de células precursoras neuronales hipocampales 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 ...