Los autores desean agradecer a H. Olivares y D. Ruge para las discusiones y consejos valiosos. Esta investigación fue financiada en parte por un premio NSF SBIR (IIP-1.127.245 a BioSentinel Inc.) y un Departamento de Defensa contrato (W81XWH-07-2-0045 a BioSentinel Inc.).

FM Dunning, TM Piazza, F. Zeytin y WC Tucker son empleados o propietarios de BioSentinel Inc. BioSentinel actualmente fabrica y ha comercializado algunos de los reactivos que se presentan en este informe.

Este protocolo describe los procedimientos para la cuantificación de BoNT / A complejo holotoxina o Clostridium sobrenadante de cultivo en matrices complejas. El protocolo es el mismo, sin embargo, al probar otra serotipos de BoNT (por ejemplo BoNT / B, E y F) con sus respectivos ensayos Matrix 56,60, aunque la sensibilidad del ensayo variará según los serotipos y ensayos. Este protocolo no tiene en cuenta para cada tipo de muestra posible y algunas modificaciones puede ser necesaria depe...

Neurotoxinas botulínicas (BoNT) son las sustancias más letales conocidos, con dosis letales intravenosas humanos estiman en 3.1 ng / kg 1,2. Siete serotipos de BoNT estructuralmente similares, etiquetadas A a la G, de existir, consistiendo cada uno en un dominio de cadena pesada responsable de la unión celular, la absorción, y la translocación en el citosol y una cadena ligera que codifica una endopeptidasa de zinc 3-5. La exquisita toxicidad de BoNT resulta de, en parte, de su entrada obligatoria y específica en las neuronas motoras en la unión neuromuscular 6. Una vez dentro de la neurona, la endopeptidasa de la cadena ligera escinde específicamente uno o más de la N-etilmaleimida-sensible receptor soluble de proteína de fijación del factor (SNARE) proteínas necesarias para la fusión de vesículas, la inhibición de la liberación de neurotransmisores y que conduce a parálisis flácida 7-14. Comúnmente conocida como la enfermedad &quot;botulismo&quot; parálisis del diafragma y los músculos intercostales por BoNT en última instancia se traduce ense reciben insuficiencia respiratoria y la muerte a menos que el diagnóstico y tratamiento tempranos. Botulismo alimentario humano es más comúnmente asociado con BoNT serotipos A, B, E y F (BoNT / D, BoNT / B, etc) y por lo general resulta de la ingestión de alimentos contaminados 15,16, aunque, varios casos de botulismo por heridas fueron reportados entre los usuarios de drogas por vía intravenosa 17,18. En los Estados Unidos, el botulismo infantil como resultado de la ingestión de esporas de Clostridium por los niños menores de uno es la forma más común de botulismo 19-21. Sin embargo, se reportaron brotes de origen alimentario BoNT resultantes de conservas caseras impropio y el procesamiento de alimentos, tanto en Estados Unidos como en el extranjero. Entre 2000-2009, se reportaron al menos 338 casos de botulismo transmitido por alimentos en todo el mundo incluyendo a seis víctimas mortales 22. La capacidad de detectar rápidamente y con sensibilidad brotes de botulismo de origen alimentario es un indicador crítico que podría ayudar al diagnóstico precoz 23,24. Por otra parte, los métodos de detección que permiten rentable y las pruebas de rutina de alimentos dará lugar a una mejora de la seguridad alimentaria. Especificidad neuronal de BoNT y larga vida media biológica también lo convierte en un potente terapéutico. En los Estados Unidos, los medicamentos basados ​​en la NTBo están aprobados por la Administración de Alimentos y Medicamentos para el tratamiento de afecciones cosméticas y trastornos relacionados neuromusculares incluidas las líneas del entrecejo, distonía cervical, dolores de cabeza por migraña, la vejiga hiperactiva, y estrabismo. Numerosas aplicaciones &quot;off-label&quot; están documentados, incluyendo los tratamientos de dosis alta para la disfunción muscular severa 25-28. Cuantificación de toxinas precisa es fundamental para la dosificación correcta, ya que una dosis insuficiente puede dar lugar a un tratamiento ineficaz, mientras que una sobredosis pone a los pacientes en riesgo de efectos secundarios potencialmente dañinos. Desafortunadamente, no existe un protocolo ensayo de potencia normalizada se comparte entre los fabricantes, lo que resulta en desigualdades de definición de unidad entre productores de medicamentos a base de BoNT29-31 cts. La prueba estándar para la NTBo es el bioensayo en ratones en los que las muestras que contienen NTBo se inyectan por vía intraperitoneal en ratones, y el número de muertes registradas más de 1-7 días 16,32,33. El bioensayo en ratones es muy sensible a los límites de detección (LOD) de 5-10 pg de BoNT / A 34, sin embargo, las preocupaciones éticas sobre el uso de animales, el alto costo de la capacitación del personal y el mantenimiento de instalaciones de los animales, los tiempos de ensayo de largo, y la falta de protocolos estandarizados resultaron en llamadas a desarrollar, las pruebas estandarizadas BoNT libre de productos animales y métodos de cuantificación 35-39. Recientemente, varios métodos alternativos de cuantificación BoNT fueron desarrollados que ofrecen ratón o casi ratón sensibilidad bioensayo 40-49. Estos métodos suelen utilizar la fluorescencia, la espectrometría de masa, o métodos inmunológicos y ofrecen tiempos de ensayo considerablemente más corto que el bioensayo en ratones sin el uso de animales. Mass-espectrometría de enfoques combinados con techniq inmunológicaues, se mostró a detectar y cuantificar BoNT contenida en los alimentos y otras muestras complejas, sin embargo, los requisitos de capacitación de personal y equipo especializado límite de estos ensayos de 50-55. La mayoría de los otros ensayos alternativos no son fácilmente aplicables a la prueba de la muestra compleja o carecen del rendimiento necesarios para las pruebas de rutina BoNT. La naturaleza altamente variable de la viscosidad de la muestra de alimentos, pH, contenido de sal, y constituyentes de la matriz presenta un desafío especialmente difícil cuando se trata de desarrollar métodos de ensayo in vitro con sensibilidad para que coincida con la potencia extrema de la NTBo. Por otra parte, incluso los sistemas de amortiguación simples y relativamente benignas, como las que resultan de la resuspensión de los productos farmacéuticos a base de BoNT, contienen sal, albúmina, azúcar y estabilizantes (por ejemplo, excipientes) que impactan significativamente in vitro BoNT potencia 56. Se requiere la purificación de toxinas de las pruebas de actividad precisa de todos pero la más sencilla de las muestras 56-59. LaBoTest ensayos de Matrix fue diseñada para una rápida y de alto rendimiento, y la cuantificación consistente de BoNT a partir de muestras de alta complejidad utilizando equipos que se encuentran comúnmente en los laboratorios de investigación 56,60. Estos ensayos utilizan perlas paramagnéticas unidas covalentemente a los anticuerpos anti-BoNT específicos de serotipo de atar y secuestrar BoNT de una muestra y luego eliminar la interferencia de compuestos de matriz mediante lavado. Después del lavado, atado actividad proteolítica BoNT se cuantifica entonces en un tampón de reacción optimizado utilizando un reportero compatible con el serotipo BoNT está probando. Estos reporteros son proteínas fluorogénicos que consisten en una proteína fluorescente cian N-terminal (PPC) y un resto derivado de la proteína fluorescente amarilla C-terminal de resto (Venus) unidos por un sustrato de BoNT, residuos de SNAP25 141 a 206 o los residuos de sinaptobrevina 33-94 que constituye la BoTest A / E o B / D / reporteros F / G, respectivamente 45. Reportero escisión por BoNT se vigila por medio de Förster de transferencia de energía de resonancia (FRET). Cuando tque el reportero está intacta, la excitación de CFP se traduce en FRET a Venus, apagando emisión PPC y emocionante emisión Venus. La escisión del reportero por BoNT impide FRET, lo que lleva a un aumento en la emisión de PPC y disminución en la emisión de Venus. La actividad de BoNT se puede medir cuantitativamente utilizando la relación de las emisiones de la PPC y Venus. LOD por debajo de 3 pg son posibles a partir de una amplia gama de alimentos utilizando un formato de placa de 96 pocillos de alto rendimiento 56. Aumento de la sensibilidad se puede conseguir utilizando volúmenes de muestra más grandes ya que el ensayo permite la concentración de la toxina en la superficie de la perla. Los ensayos de Matrix BoTest para BoNTs A, B, E y F se desarrollaron y probaron con alimentos, productos farmacéuticos, y las muestras ambientales 56,60. Aquí se describe los procedimientos para la ejecución de estos ensayos para la detección de la NTBo en baja complejidad (por ejemplo, productos farmacéuticos, la NTBo en la memoria intermedia) y alta complejidad (por ejemplo, alimentos, medio ambiente) muestras. Métodos de procesamiento específicospara se abordan varios tipos de muestras en este protocolo y tipos de muestras que no se describen aquí por lo general se pueden adaptar usando una combinación de los métodos presentados. El protocolo fue desarrollado y probado con BoNT / A, pero se puede adaptar a otros serotipos de NTBo utilizando sus respectivos ensayos como se ha demostrado en otras partes 56,60.

<table border="0" cellpadding="0" cellspacing="0" width="648">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:192px;">BoTest Matrix A Botulinum Neurotoxin Detection Kit</td>
			<td style="width:107px;">BioSentinel</td>
			<td style="width:104px;">A1015</td>
			<td style="width:245px;">Detection kits for BoNT/B and F are also available.</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:192px;">Varioskan Flash fluorescence microplate reader</td>
			<td style="width:107px;">Thermo-Fisher Scientific</td>
			<td style="width:104px;">5250040</td>
			<td style="width:245px;">Most monochromator- or filter-based units with 434 nm excitation and 470 and 526 nm emission capability can be used.</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:192px;">96-well Magnetic Bead Separation Plate</td>
			<td style="width:107px;">V&#38;P Scientific&#160;</td>
			<td style="width:104px;">VP771H</td>
			<td style="width:245px;">Other magnetic plates may be used, but the plate should be designed to separate the beads to the side of the well.</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:192px;">Magnetic Bead-Compatible Plate Washer</td>
			<td style="width:107px;">BioTek&#160;</td>
			<td style="width:104px;">ELx405 VSRM</td>
			<td style="width:245px;">Optional, only required for automated plate washing.&#160; Other magnetic bead-compatible plate washers may also be used, but should be tested before use.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:192px;">Microcentrifuge</td>
			<td style="width:107px;">Various</td>
			<td style="width:104px;">N/A</td>
			<td style="width:245px;">Optional, only required for samples needing centrifugation.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:192px;">MixMate plate mixer</td>
			<td style="width:107px;">Eppendorf</td>
			<td style="width:104px;">22674200</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:192px;">Orbital Shaker</td>
			<td style="width:107px;">Various</td>
			<td style="width:104px;">N/A</td>
			<td style="width:245px;">Used at room temperature or at 25 &#176;C If temperature control is available</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:192px;">EDTA-free Protease Inhibitor Tablets</td>
			<td style="width:107px;">Roche</td>
			<td style="width:104px;">4693132001</td>
			<td style="width:245px;">Only required for food or environmental testing.&#160; Protease inhibitors must be EDTA-free.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:192px;">BoNT/A&#160;</td>
			<td style="width:107px;">Metabiologics</td>
			<td style="width:104px;">N/A</td>
			<td style="width:245px;">Optional, only required for standardization and quantification purposes&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:192px;">Black, Flat-bottomed 96-well Plates</td>
			<td style="width:107px;">NUNC</td>
			<td style="width:104px;">237105</td>
			<td style="width:245px;">Plates should not be treated</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:192px;">96-well Plate Sealing Tape</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:104px;">15036</td>
			<td style="width:245px;"></td>
		</tr>
	</tbody>
</table>

isolation and quantification of botulinum neurotoxin from complex matrices using the botest matrix assays

Se requiere la detección y cuantificación de la neurotoxina botulínica (NTBo) en matrices complejas precisa para la industria farmacéutica, medioambiental, y las pruebas de muestra de alimento. Se necesita prueba rápida BoNT de los productos alimenticios durante el forense del brote, el diagnóstico del paciente, y las pruebas de seguridad de los alimentos mientras que se requiere la prueba de potencia precisa para la fabricación de productos de fármacos basado en la NTBo y la seguridad del paciente. El bioensayo en ratón ampliamente utilizado para la prueba de BoNT es altamente sensible, pero carece de la precisión y el rendimiento necesario para la prueba rápida y rutinaria de BoNT. Además, el uso del bioensayo de los animales ha dado lugar a los llamados de las autoridades reguladoras de medicamentos y productos proponentes derechos de los animales en los EE.UU. y en el extranjero para sustituir el bioensayo en ratones para probar la NTBo. Varios ensayos in vitro de recambio en se han desarrollado que funciona bien con purificada de BoNT en tampones simples, pero la mayoría no han demostrado ser aplicable a prueba en matrices muy complejas. Aquí, un protocolo para la detección deNTBo en matrices complejas utilizando la matriz de ensayos BoTest se presenta. El ensayo consta de tres partes: La primera parte implica la preparación de las muestras para las pruebas, la segunda parte es un paso inmunoprecipitación usando anti-BoNT recubiertas de anticuerpos perlas paramagnéticas para purificar la NTBo de la matriz, y la tercera parte cuantifica proteolítica de la BoNT aislado actividad utilizando un reportero fluorogenic. El protocolo está escrito para las pruebas de alto rendimiento en placas de 96 pocillos utilizando matrices de ambos líquidos y sólidos y requiere alrededor de 2 h de preparación manual con tiempos de ensayo total de 4-26 horas dependiendo del tipo de muestra, la carga de la toxina, y la sensibilidad deseada. Los datos se presentan para las pruebas de BoNT / A con solución salina tamponada con fosfato, un producto farmacéutico, el sobrenadante del cultivo, 2% de leche, y tomates frescos e incluye la discusión de los parámetros críticos para el éxito del ensayo.

Un diagrama que resume los pasos en el protocolo descrito se muestra en la Figura 2. El ensayo requiere entre 4-26 horas en completarse, dependiendo del tipo de muestra y la sensibilidad del ensayo deseado, pero sólo ~ 2 horas de tiempo de práctica. El ensayo se realiza en placas de 96 pocillos y, dependiendo del tipo de prueba que se lleva a cabo, permite realizar pruebas por triplicado de hasta 20 muestras incluidas las normas por placa. La Figura 3 mues...

Watch this Scientific Journal Video about Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays at JoVE.com

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

La neurotoxina botulínica Matriz BoTest (BoNT) ensayos de detección de purificar y cuantificar rápidamente BoNT a partir de una variedad de matrices de muestras. Aquí, se presenta un protocolo para la detección y cuantificación de BoNT a partir de matrices tanto sólidos como líquidos y demostrar el ensayo con BOTOX ®, los tomates y la leche.

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 ...

isolation-quantification-botulinum-neurotoxin-from-complex-matrices

Research

JoVE Journal

Neuroscience

15.9K vistas.  BioSentinel Inc., Madison, WI.  La neurotoxina botulínica Matriz BoTest (BoNT) ensayos de detección de purificar y cuantificar rápidamente BoNT a partir de una variedad de matrices de muestras. Aquí, se presenta un protocolo para la detección y cuantificación de BoNT a partir de matrices tanto sólidos como líquidos y demostrar el ensayo con BOTOX ®, los tomates y la leche. 

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

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

El aislamiento y la expansión de las células troncales nerviosas de ratones adultos mediante el ensayo de neuroesfera

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Investigación

Neurociencias

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Disección y Cultura de ratón dopaminérgico estriatal en explantes y tridimensionales ensayos matriz de colágeno

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

La inmunohistoquímica wholemount de Topografía Revelando Complex Brain

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Aislamiento y cultivo de células endoteliales del cerebro anterior embrionarias

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

Producción y aislamiento de los axones de las neuronas sensoriales para el análisis bioquímico Usando porosos Filtros

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

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.

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 ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

La excitación externa de neuronas usando campos eléctricos y magnéticos en culturas de una y dos dimensiones

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

Enumeración de células madre neurales Usando ensayos clonales

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Aislamiento y cultivo de células madre adultas neural de la Zona subcallosal Ratón

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the cells themselves. Here, we present the method of Light-Induced Molecular Adsorption of Proteins (LIMAP) using the PRIMO system as a patterning technique to produce micro-patterned extracellular matrix (ECM) substrates using a single or combination of proteins. The method enables printing of ECM patterns in micron resolution with excellent reproducibility. We provide a step-by-step protocol and demonstrate how this can be applied to study the processes of neuronal pathfinding. LIMAP has significant advantages over existing micro-printing methods in terms of the ease of patterning more than one component and the ability to generate a pattern with any geometry or gradient. The protocol can easily be adapted to study the contribution of almost any chemical component towards cell fate and cell behavior. Finally, we discuss common issues that can arise and how these can be avoided.

Our overall aim is to understand how cells sense extracellular cues that lead to directed axonal growth. Here, we describe the methodology of Light-Induced Molecular Adsorption of Proteins, used to produce defined micro-patterns of extracellular matrix components in order to study specific events that govern axon outgrowth and pathfinding.

Adsorción molecular inducida por la luz de proteínas utilizando el sistema PRIMO para micro-patrones para estudiar las respuestas celulares a las proteínas de matriz extracelular

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the ...