Funding was provided by the Joint Science and Technology Office &#8211; Chemical Biological Defense (JSTO-CBD) Defense Threat Reduction Agency (DTRA) under sponsor project number CCAR# CB3675 and National Institutes of Health (1 R21 AI101387-01 and 5 U01AI082051-05).

Placa 20000 diferenciado células ES de ratón (MES) / pocillo en placas de 96 una lisina recubiertos con poli-D así y mantener en la neurona motora medios diferenciación terminal durante 5-7 días. Administración 1. El compuesto y la intoxicación con BoNT / A Lleve a cabo todos los trabajos siguientes en un recinto BSL2 para mantener el cumplimiento con las pautas de los CDC / NIH. <ol><li> Preparar soluciones madre 10 mM de cada compuesto de la biblioteca en 100% de DMSO en placas de 96 pocillos de polipropileno. Utilice el stock de 10 mM para preparar una placa de dilución intermedia que es 10 veces mayor que la concentración de detección deseado. Preparar 100 uM placa intermedia mediante la supresión de stock 10 mM en placa de 96 pocillos y se diluye a 100 uM usando medios de cultivo. Dispensar 10 l de los compuestos sobre a 90 l de los medios de comunicación sobre las células 11. Prueba de los compuestos a 10 uM concentración final y asegurar el porcentaje final de DMSO no sea superior al 0,5%. </li><li> Realizar todos espárragos que utilizan células vivas y toxina activa bajo nivel de bioseguridad 2 condiciones. Reemplazar los viejos medios de comunicación en las placas de 96 pocillos con 80 l de los medios de comunicación fresca diferenciación terminal utilizando una pipeta manual de 12 canales. Llevar a cabo la aspiración y dispensar pasos por filas para evitar la exposición de las células sin medios de comunicación para el aire ambiente. </li><li> Pretratar las células con compuestos antes de la aplicación de la toxina mediante la adición de 10 l de 10x compuestos a partir de la placa de fuente utilizando una pipeta de 12 canales, seguido de mezclado por aspiración (dos veces). Para cada placa de 96 pocillos, tratar dos columnas de 6 pozos con 10x DMSO diluido en los medios de comunicación para servir como señal alta y los controles de señal bajos. La columna sin tratamiento BoNT exhibe el nivel más alto de la longitud completa SNAP-25 (control alto de la señal). Tratar la otra columna con BoNT solo (sin ningún compuesto) como el control de señal baja. Utilice baja de control para observar la eficacia de escisión de BoNT SNAP-25 y su detección con el anticuerpo BACS (Figura 2). </li><li> Se incuban las células con los compuestos durante 30 min a 37 ° C y 6% de CO 2 en la incubadora de cultivo celular. </li><li> Preparar neurotoxina botulínica A partir de un 1 mg / fuente comercial ml en tampón fosfato salino (PBS) a una final 10x madre de trabajo diluyendo con medios diferenciación terminal. </li><li> Iniciar la intoxicación mediante la adición de 10 l de 10x BoNT / A de stock en los pocillos designados para los controles de tratamiento y baja señal usando una pipeta multicanal (Figura 2). Tratar a los pocillos designados como alto control con medio solo. </li><li> Descontaminar todos los artículos de plástico y otros materiales desechables que entra en contacto con BoNT / A durante el estudio por inmersión en solución de hipoclorito de 0,825% en agua durante al menos 20 min. Lleve a cabo todos los procedimientos en los gabinetes de seguridad biológica y descontaminar las áreas de trabajo, tanto antes como después de los experimentos con 5% de detergente limpiador desinfectante como solución MicroChem. </li><li> Etiquetar las placas claramente para indicar el uso de la toxina y incubcomieron placas tratadas con 1 nM / L de BoNT / A durante 4 horas a 37 ° C y 6% de CO2 en un incubador de cultivo celular. Pantalla de señalización apropiado para informar a los colegas que la toxina está en uso en el laboratorio. </li><li> Detener BoNT / A escisión proteolítica por la fijación de metanol. Retire los medios de comunicación que contienen toxinas y compuestos de cada pocillo con una pipeta multicanal y desechar en una solución de lejía al 10%. Añadir metanol enfriado con hielo (100 l) directamente a cada pocillo. </li><li> Incubar las placas durante 15 min a temperatura ambiente (RT) para permitir que se produzca la fijación. </li><li> Después de la fijación, manejar de forma segura los reactivos desechados (considerado neutralizados) con protocolos de nivel de bioseguridad 1 y descartar en consecuencia. </li><li> Descartar metanol y el uso de 100 l de PBS para lavar las células y rehidratar ellos antes de la inmunotinción. Realizar dos lavados con PBS adicionales. </li><li> Después del lavado final, deje de PBS en los pozos, placas de sellado con película adhesiva de microplacas y se almacena a 4 ° C hasta su análisis. Placas se almacenan a 4 ° C for varios días. </li></ol> 2. La inmunotinción El procedimiento de inmunotinción es una operación de múltiples pasos de trabajo intensivo que incluye ciclos de dosificación de reactivos / aspirado repetitivas y extensa de lavado de placas que pueden conducir a la posible introducción de una variabilidad significativa intraplaca y Interplate. Un enfoque semi-automatizado se aplica para ahorrar el tiempo del personal de laboratorio, aumentar el rendimiento del ensayo, y minimizar la variabilidad inmunotinción. <ol><li> Utilice una estación de trabajo automatizada equipada con un cabezal de 96 pocillos capaz de transferir volúmenes de hasta 250 l e integrado con un brazo robótico de agarre para mover el material de laboratorio en diferentes lugares de la cubierta modular (ver Material y Equipo) para este protocolo. <ol><li> La cubierta modular está diseñado para acoger diferentes tipos de material de laboratorio y puede soportar hasta 11 artículos en un momento dado. El manejador de microplacas automatizado está equipado con un cargador de doble apilador de microplacas con el fin de sostener ymanipular hasta 50 placas por ciclo. </li><li> La estación de trabajo automatizada se encuentra en el interior de una cabina de bioseguridad de Clase II diseñado para equipos de automatización de laboratorio para asegurar la esterilidad y seguridad. Para la inmunotinción automatizada, la cubierta está dispuesto como se muestra en la Figura 3 para permitir el acceso del reactivo según sea necesario y sin intervención humana. </li></ol></li><li> Placas pre-carga que contienen células fijadas en la revista placa y la transferencia a la cubierta, según sea necesario para las operaciones de manejo de líquidos. Use la cabeza 96 de la pipeta provista de 250 consejos para aspirar mu l de PBS a partir de los pozos de hasta 10 l. Permeabilizar las membranas celulares para facilitar la unión del anticuerpo a dianas intracelulares con tampón de permeabilización (0,1% Triton X-100). Dispensar tampón de permeabilización (100 l) en cada pocillo antes de regresar las placas a la segunda cargador de almacenamiento del apilador de microplacas para una incubación de 15 min a temperatura ambiente (RT). </li><li> Retire permeabilización de amortiguación y PElavado de placas rforma con PBS (dos veces), como se describe anteriormente en el paso 1.13. </li><li> Después de la última etapa de lavado, eliminar el tampón PBS y dispensar 100 l de tampón de bloqueo que contenía 1% de suero de caballo y 0,1% de Tween 20 en PBS en todo microplacas antes de devolverlos a la revista placa durante 1 hora de incubación a temperatura ambiente. </li><li> Use dos anticuerpos primarios para la inmunotinción: un anticuerpo policlonal de conejo anti-ratón de anticuerpos BACS, para la detección de longitud completa SNAP-25 monoclonal de ratón y un anti-III tubulina de anticuerpos para la detección de las neuronas (ver Material y Equipo). Después de 60 min de incubación, retirar el tampón de bloqueo y dispensar 50 l de 4UG / ml de anticuerpo BACS y 0,5 mg / ml de III-tubulina en tampón de bloqueo en todas las placas. </li><li> Incubar durante 1 hora a RT a eliminar los anticuerpos primarios y lavar 3 veces con 100 l de PBS que contenía 0,05% de Tween (PBST). </li><li> Utilice una IgG anti-ratón marcado con Alexa Fluor 647 para visualizar el -III tubulina y anti-IgG de conejo marcado con Alexa Fluor 568 para visualizar el anticuerpo BACS. Preparar ambos anticuerpos como una dilución / ml 2 g en tampón de bloqueo y dispensar 50 l de la mezcla en cada pocillo. NOTA: Los anticuerpos secundarios seleccionados para la inmunotinción se etiquetan con diferentes fluoróforos para permitir la detección de la luz fluorescente emitida en longitudes de onda distintas que utilizan diferentes canales dentro del detector del Alto Imager contenido. </li><li> Incubar durante 1 hora a RT. Aspirar los anticuerpos secundarios y lavar 3 veces con 100 l de PBST. </li><li> Después del lavado final, añadir 100 l de PBS que contiene el colorante Hoechst (32 mM) para teñir el ADN para la detección de los núcleos. </li><li> Sellar las placas con una película impermeable ligera para proteger los fluoróforos de photobleaching. Las placas pueden almacenarse a 4 &#160; ° C durante semanas sin pérdida significativa de la señal. </li></ol> 3. Imaging NOTA: Lleve a cabo la adquisición de imágenes usando contenido de alta Imaging System (Ver Materiales y Equipo). <ol><li> Especificación de alto contenido Imager Definiciones: <ol><li> Seleccione el tipo de placa, diseño, número de campos, objetivo: Greiner u claro, formato de 96 pocillos, 16, 20X agua respectivamente. </li><li> Seleccione los canales: núcleo de excitación EX: 405 nm como el canal 4; citoplasma EX: 644 nm como el canal 2; antígeno específico EX canal anticuerpo: 561 nm como el canal 3 y Configurar potencia de excitación de cada láser, experimento Configuración como dos exposiciones, exposición 1 con una excitación a 644 nm, la exposición 2 con dos excitaciones a 405 nm y 561 nm. Seleccione hurgar en la basura como BIN2. </li><li> Utilice los pocillos de control de alta para la optimización de la exposición con la máxima intensidad como canal de SNAP-25 y bajo control de los pozos de intensidad mínima. </li><li> Ajuste la exposición, de modo que la intensidad de cada canal es aproximadamente la mitad del nivel de saturación (2.000-2.500 unidades relativas). </li><li> Identificar el plano Z óptima en el canal máscara celular utilizando delta script de corrección. Ver Figura 5 para resultados después de inmunotinción y de imagen. Exportar datos al servidor donde el software de análisis de imágenes está residiendo. </li></ol></li></ol> 4. Análisis de Imágenes (Figura 6) NOTA: Los siguientes pasos describen la aplicación de los algoritmos de software Columbus. <ol><li> Seleccione un algoritmo apropiado para segmentar los objetos primarios (núcleos). Si es necesario, ajustar el umbral de fondo y los parámetros de contraste. El software de Colón proporciona 4 métodos de detección de los núcleos. Seleccione el método que los núcleos de segmentos con precisión mediante la inspección visual de los núcleos segmentados (en F igura método 6a M se selecciona). </li><li> Seleccione el algoritmo de neuritas (CSIRO neuritas Análisis 2) al segmento de las neuritas Si es necesario, ajustar los parámetros de umbral y contraste de fondo para eliminar el fondo. Véase la figura 6b para los parámetros utilizados para detectar las neuritas. </li><li> Identificar las regiones neuritas (segmentos) neuritas based en los objetos secundarios (neuritas) utilizando una expansión pixel -3 del canal de tubulina β-III (Alexa Harina 640) como máscara (Figura 6c). </li><li> Calcular la intensidad de los canales de señal (SNAP-25, (Alexa Harina 568) para la región de las neuritas y el canal de la tubulina β-III (Figura 6d y 6e). </li><li> Seleccione los parámetros deseados para la exportación, tales como la longitud de las neuritas, las intensidades medias de SNAP-25, β-tubulina III, número de núcleos, número de segmento de neuritas, etc. (Figura 6f). </li><li> Placas de transferencia de apiladores de placas utilizando el robot y la carga en el Alto de imágenes de contenido para la adquisición de imágenes. </li></ol> 5. Análisis de Datos: Para evaluar la robustez del ensayo diseñado, calcular los siguientes parámetros del experimento basado en la placa. <ol><li> La señal (S) a fondo (B): S / B = intensidad de la señal de control de alta media) / intensidad media de un buen control de la señal </li> &lt;li&gt; Coeficiente de Variación (CV) de la señal y el fondo (para medir la uniformidad de la señal específica): CV = (desviación estándar (SD) / media de la muestra) * 100 (%), para determinar la variabilidad en el manejo de líquidos, reactivos, etc. </li><li> Señal a ruido: S = (intensidad de la señal significa - intensidad media de un buen control de la señal) / SD de control de baja <ol><li> Calcular el factor de la Z &#39;con la intoxicación de una mitad de una placa de 96 pocillos y un simulacro de la intoxicación de la otra mitad. Z &#39;= 1 - 3 * (SD de alta control de señal + SD de bajo control de la señal) / (media de alto control de la señal - media de un buen control de la señal). Para el análisis posterior de los datos de cribado, normalizar algunos de los parámetros primas primarias sobre una base placa para permitir la comparación entre las placas y entre los diferentes días de experimentos. </li></ol></li><li> Porcentaje de escisión, lo que refleja la reducción de la señal asociada con la longitud completa SNAP-25 detectado por el anticuerpo específico (BACS): Cleavage% = 100% * (mean intensidad de la señal de control de alto - intensidad de la muestra) la intensidad de la señal de control de alta / media. </li><li> Porcentaje de inhibición de la escisión:% de inhibición = 100% * (intensidad de la muestra - significa la intensidad de un buen control de la señal) / (intensidad de la señal de control de alta significar - intensidad de un buen control de la señal de media). </li></ol>

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Krishna P Kota is an employee of Perkin Elmer Inc. Waltham, MA, that produces instruments and software used in this manuscript. The content of this publication does not necessarily reflect the views or policies of the U.S. Department of Health and Human Services, the U.S. Department of Defense, the U.S. Department of the Army, or the institutions and companies affiliated with the authors.

La alta potencia de las neurotoxinas botulínicas y la relativa facilidad de su militarización ha dado lugar a su clasificación en la categoría A (la más alta prioridad) agentes biothreat por los Centros para el Control y la Prevención de Enfermedades. Desafortunadamente, no existen terapias aprobadas por la FDA para contrarrestar BoNT después de la intoxicación por toxina ha sido interiorizado por las neuronas motoras. Cualquier mecanismo druggable que promueve la recuperación neuronal de BoNT intoxicación pod...

La bacteria Clostridium botulinum produce la neurotoxina botulínica, una de las toxinas biológicas más potentes conocidos por el hombre 1. Hay 7 diferentes serotipos de BoNT (BoNT / AG). BoNT / A-Gall induce parálisis en la unión neuromuscular debido a la proteolisis complejo SNARE 2,3. SNARE proteolisis evita neurotransmisor-vesícula de la membrana de fusión y por lo tanto bloquea la exocitosis de neurotransmisores 4. El objetivo SNARE específica depende del serotipo particular, BoNT involucrado en el proceso de intoxicación. BoNT / A y BoNT / E escinden SNAP-25 mientras que escinde BoNT / C ambos SNAP-25 y sintaxina 5. El sinaptobrevina cleave restantes serotipos (también llamada asociada a vesículas de proteína de membrana (VAMP). BoNT / A fue elegido para el desarrollo del ensayo, ya que es responsable de una alta proporción de botulismo de origen natural y tiene la duración más larga de acción 6. Desarrollo de molécula pequeña la terapéutica contra la BoNT / A es una meta importante paranuestro programa de descubrimiento de fármacos y ha utilizado métodos por objetivos tradicionales para identificar inhibidores proteolíticas sitio activo hace 7, 8-10. Sin embargo, la creación de los inhibidores del sitio activo con actividad de amplio espectro contra múltiples serotipos y eficacia después de la exposición es probable que sea un reto. Por ello, hemos implementado un enfoque innovador, fenotípica descubrimiento de fármacos que utiliza BoNT SNAP-25 de la escisión como un criterio de valoración funcional para identificar pequeñas moléculas que pueden bloquear mediada por BoNT intoxicación de las neuronas motoras. SNAP-25 se requiere para la liberación de neurotransmisores, como la degradación de la SNAP-25 es predictivo de la parálisis y la letalidad in vivo. Por ejemplo, el cribado basado en células podría conducir al descubrimiento de nuevos moduladores de factores celulares responsables de la inactivación de la toxina o la inhibición de las vías de la toxina en el interior de las células diana. Un factor importante en el desarrollo del ensayo fenotípico es la selección de los modelos biológicos fisiológicamente relevantes. WE y otros han descrito madre embrionarias de ratón (ES) neuronas motoras derivadas de células que recapitular el carácter inmunológico de las neuronas motoras primarias, incluyendo la expresión de SNAP-25 11- 13. Es importante destacar que estos sistemas celulares son muy sensibles a BoNT / A intoxicación y demuestran la escisión dependiente de la dosis de la SNAP-25 en respuesta a concentraciones crecientes de toxina 11,12. Las neuronas motoras diferenciados también se producen en cantidades que son suficientes para el análisis basado en la placa de alto rendimiento y permitieron el diseño de una serie de ensayos celulares. El ensayo fenotípico es un método de inmunofluorescencia utilizando dos anticuerpos diferentes para cuantificar la escisión de longitud completa expresado de forma endógena SNAP-25 durante la BoNT / A intoxicación de la cultura de ratón de la neurona motora. Un terminal carboxilo de BoNT / A (BACS) de anticuerpos de escisión sensible que reconoce sólo de longitud completa SNAP-25 permite la evaluación de la BoNT / A proteolisis mediada de SNAP-25expresión en las neuronas del motor del ratón 10. Un diagrama esquemático del ensayo de HCI se representa en la Figura 1.

<table border="0" cellpadding="0" cellspacing="0" width="834">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Name of Reagent/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:280px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Botulinum neurotoxin A&#160;</td>
			<td style="width:180px;">Metabiologics</td>
			<td style="width:119px;">NA</td>
			<td style="width:280px;">No catalog number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microtitre plates</td>
			<td style="width:180px;">Greiner&#160;</td>
			<td style="width:119px;">655946</td>
			<td style="width:280px;">Poly-D-Lysine 96-well plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BACs antibody</td>
			<td style="width:180px;">Lampire Biological</td>
			<td style="width:119px;">NA</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microchem</td>
			<td style="width:180px;">National&#160;</td>
			<td style="width:119px;">0255</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Methanol</td>
			<td style="width:180px;">Thermo Scientific</td>
			<td style="width:119px;">A412-20</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Formaldehyde</td>
			<td style="width:180px;">Thermo Scientific</td>
			<td style="width:119px;">28908</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Horse serum</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">16050</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">PE JANUS MDT Mini Automated Workstation</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">AJMDT01</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Opera</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td>OP-QEHS-01</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Triton X 100</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:119px;">9002-93-1</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BIII tubulin antibody</td>
			<td style="width:180px;">R&#38;D Systems</td>
			<td style="width:119px;">BAM1195</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tween 20</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:119px;">P1379-1L</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342dye&#160;</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">3570</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Antimouse IgG</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">A21236</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Anti rabbit IgG</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">A10042</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Columbus Image analysis software</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">Ver 2.4</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Spotfire</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">Ver 5.5</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Clorox bleach</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:119px;">18-861-284</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PlateStack</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:280px;"></td>
		</tr>
	</tbody>
</table>

a high content imaging assay for identification of botulinum neurotoxin inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

Los datos de los controles alto y bajo crearon dos poblaciones distintas con la diferencia de dos medianas superiores a 3 desviaciones estándar (Figura 7A). El objetivo del proceso de selección es encontrar los compuestos dentro de la población de la muestra con los valores más cercanos a la población control positivo, suponiendo una distribución normal dentro de la población de la muestra (Figura 7B, (i)). Los puntos de datos que están más allá de 3 desviaciones estándar de ...

Watch this Scientific Journal Video about A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors at JoVE.com

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

Un alto contenido de imágenes de Ensayo para la Identificación de la neurotoxina botulínica Inhibidores

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

a-high-content-imaging-assay-for-identification-botulinum-neurotoxin

Research

JoVE Journal

Neuroscience

8.5K vistas.  Perkin Elmer Inc.. Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

Video: A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Un ensayo Cortar organotípicos de alta resolución de imágenes a intervalos de tiempo de la migración neuronal en el cerebro postnatal

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Investigación

Neurociencias

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

De alta resolución Magnética Funcional por imágenes de los métodos de resonancia cerebro medio humano

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Imágenes funcionales de la corteza auditiva en los gatos adultos el uso de alto campo fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay&#39;s use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT&#39;s proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.

The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices.&#160;Here, we present a protocol for the detection and quantification of BoNT from both solid and liquid matrices and demonstrate the assay with BOTOX, tomatoes, and milk.

El aislamiento y la cuantificación de la neurotoxina botulínica A partir de matrices complejas utilizando el BoTest Matrix Ensayos

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

Alta resolución<em&gt; En Vivo</em&gt; Protocolo Segmentación Manual for Human hipocampo subcampos Usando 3T Imágenes por Resonancia Magnética

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Parénquima no invasiva, vascular y metabólico de alta frecuencia de ultrasonido y fotoacústica Rata cerebral profunda Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

De alta resolución de imagen de resonancia magnética estructural del subcorteza Humano<I&gt; En Vivo</I&gt; Y Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Análisis de alto rendimiento del comportamiento Locomotor en el ensayo de la isla de Drosophila

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

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.

Desarrollo de ensayos para cuantificación de contenido alto de Sod1 mutante de la proteína total formación en células vivas

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

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.