Este estudio fue apoyado por subvenciones del Vanderbilt-Ingram Cancer Center de Apoyo Grant (P30 CA068485), la Fundación cerebrales infantiles del tumor y de los Institutos Nacionales de Salud (NS042205).

1. Microdisección de portadores de tumores del cerebelo y la disociación del tejido tumoral <ol><li> Recuperación del tejido tumoral <ol><li> Los ratones enfermos teniendo meduloblastoma son a menudo runted, y mostrar hidrocefalia y típicos síntomas neurológicos, incluyendo parálisis posterior y el fracaso para recuperar la posición en caso de vuelco. Para recuperar el tejido tumoral, la eutanasia a los ratones por inhalación de dióxido de carbono. Es imporante no llevar a cabo la dislocación cervical, un procedimiento que genera una presión en el cráneo posterior y pueden poner en peligro la integridad del tejido tumoral. </li><li> La decapitación se realiza inmediatamente después de la muerte con un par de tijeras, quitar el pelo y el tejido muscular tanto como sea posible para una buena visualización del cráneo. Limpie la superficie del cráneo con Kimwipe empapado con etanol al 95%. </li><li> Use tijeras finas para cortar una abertura a lo largo de la línea media del cráneo, y eliminar el tejido del cráneo con unas pinzas finas, momento en el que todo el cerebro incluyendo el cerebelo con tumores está expuesto. </li><li> Mientras que el cerebelo de los adultos sanos pantalla bien definida hemisferios y el vermis, el cerebelo de ratones portadores de tumores a menudo son ampliadas, amorfa con una superficie lisa y los vasos sanguíneos visibles. El uso de técnicas estériles, recuperar el tumor cerebeloso con unas pinzas y colocar en el hielo frío tampón fosfato salino (PBS) sin Mg 2 + y Ca 2 +. </li></ol> Nota: todos los instrumentos son desinfectados en etanol al 95% y el vapor en autoclave antes de su uso. </li><li> La disociación del tejido tumoral <ol><li> Transferir el tejido tumoral de PBS al 50% Accutase (diluido en PBS), que es de aproximadamente 4 veces el volumen del tejido del tumor, el picadillo de tejido con tijeras finas durante 3 minutos a temperatura ambiente, seguido de incubación a 37 ° C durante 4 minutos , tras lo cual el tejido se somete a pipetear repetitivo con un Pipetman de 1 ml por un período adicional de 3 minutos. Este método se debe dar una mezcla de células individuales y pequeños agregados celulares. </li><li> Diluir la suspensión celular 3 veces con PBS y centrifugar durante 5 minutos a 1000xg para precipitar las células. (Los procedimientos anteriores se han descrito en &quot;El aislamiento, el enriquecimiento y el mantenimiento de las células madre meduloblastoma&quot;, Jove, en prensa) </li> Resuspender el botón celular en el medio recién preparado de células madre neurales que consta de medio Neurobasal con glutamina, penicilina-estreptomicina, N2, B27, EGF humano (25 ng / mL) y el FGF básico (25 ng / mL). Calcular la densidad de las células de la solución y hacer las diluciones con medio adicional de células madre neurales de tal manera que uno puede cargar 4 L de solución con un número adecuado de células tumorales disociada, por ejemplo, 5x10 5, en una estéril bisel jeringa con punta de 10μL. El número exacto de las células para ser inoculados deben ser determinados por el investigador de acuerdo a las necesidades concretas de experimentación. Mantenga la solución de células en un tubo de microcentrífuga 200μL mini (tubo de PCR) en el hielo. </ol></li></ol> 2. Los procedimientos anestésicos de los ratones receptores Para el procedimiento quirúrgico, un área desinfectadas dedicadas exclusivamente a la cirugía de roedores es necesario para la duración del procedimiento. Lo ideal es establecer un banco largo, con áreas separadas pero contiguas para la preparación de los animales, el campo de operación y de recuperación de animales. Guantes quirúrgicos, guantes de examen no, son necesarios para la cirugía. La técnica aséptica se mantiene gracias a la mano enguantada por encima de la cintura y se utiliza sólo para manipular objetos estériles de una superficie estéril seca. <ol><li> Una mezcla de ketamina (100 mg de ketamina por 1 kg de peso del ratón) y xilazina (10 mg xilazina por 1 kg de peso del ratón) se utiliza para la anestesia. Administrar los anestésicos por vía intraperitoneal. </li><li> Por lo general, tarda 3-5 minutos para que la anestesia surta efecto completo. Determinar si el ratón está completamente anestesiado por una pizca tep / cola. Coloque una pequeña cantidad de ungüento oftálmico en los ojos del ratón. Lleve el ratón anestesiado completamente al área de operación, un seguimiento constante para asegurarse de que el ratón está respirando normalmente. </li></ol> 3. Intracraneal injerto de las células tumorales <ol><li> Afeitado de la región dorsal posterior de la cabeza del ratón, utilizando una cortadora de cabello, para revelar el cráneo posterior por encima del mesencéfalo y el cerebelo. </li><li> Aplique una solución de Betadine en el cuero cabelludo expuesto con un hisopo de algodón, seguido de la limpieza con una gasa con alcohol. Repita estos dos pasos de esterilización dos veces. </li><li> Haga una incisión de medio centímetro en el cuero cabelludo posterior con un bisturí esterilizado. Perforar un agujero de trépano de 0,5 mm 2 mm hacia la derecha y 2 mm por detrás del lambda con un taladro dental estéril. </li><li> Coloque el ratón sobre un marco estereotáxico, enganchando sus incisivos en la bodega del marco. Cuidadosamente descender y la posición del bisel en la punta una jeringa de 10 l cargado con 4 l de solución de células tumorales en el agujero de trépano. Una vez que el bisel de la aguja de la jeringa es por debajo de la superficie del cráneo, descend por otro de 3 mm y luego ascender a 0,5 mm. Inyecte lentamente la cantidad deseada de las células tumorales, con una fuerza constante en un marco de tiempo de 30 segundos, en el cerebelo de los receptores. Deje la aguja en su lugar después de la finalización de la inyección durante dos minutos. A continuación, retire la aguja y la jeringa. Clip de la herida utilizando una autoclip. </li><li> Mantenga el ratón sobre una manta térmica después de la cirugía para ayudar a mantener su temperatura corporal. Los patrones de movilidad y las vías respiratorias se observó de forma continua. Una vez que el ratón se recupera de la anestesia, volver a meterla en una jaula de vivienda estéril. Diario de los alimentos y la ingesta de agua, comportamiento, preparación y tinción con rojo de los ojos. Inspeccione en busca de signos de infección en el sitio de la incisión. Retire la autoclips después de la cirugía en el día 7. Mantener a los ratones durante un promedio de 2.6 meses y el sacrificio de los que presentan síntomas típicos de meduloblastoma para estudios posteriores. </li></ol> 4. Resultados representante Meduloblastomas secundaria desarrollados en ratones receptores muy parecidos a los de los tumores primarios. El cerebelo con tumores secundarios eran a menudo ampliada, amorfo con los vasos sanguíneos aparentes ectópico cuando se ve como todo el montaje. Análisis inmunohistoquímico del tejido tumoral secundaria reveló que las células tumorales son altamente proliferativas, expresan su gran SmoM2-YFP y los marcadores de las neuronas del cerebelo gránulo precursores, como Math1 y Pax6. Cuando disociadas y cultivadas con métodos reportados (&quot;El aislamiento, el enriquecimiento y el mantenimiento de las células madre meduloblastoma&quot;, Jove, en prensa), las células de meduloblastoma secundaria tumor puede extenderse rápidamente, expresan varios marcadores de células madre, se clonigénicas y además puede iniciar la formación de tumores en trasplantadas en otros las personas inmunodeprimidas. <img src="/files/ftp_upload/2153/2153fig1.jpg" alt="Figura 1" /> Figura 1. Típico de todo el montaje y vistas en sección de un tejido meduloblastoma secundaria Un ejemplo típico del tejido meduloblastoma secundaria desarrollado 26 días después de la inyección de 5x10 5 células del tumor primario. Tinción hematoxilina revela la morfología típica de celulares de meduloblastoma, incluidos los de alta nuclear / citoplasma y el polimorfismo nuclear. Estas células tumorales secundarias son altamente proliferativas, como se indica por expresión de Ki67 y también con firmeza expresar membranosa SmoM2-YFP. <img src="/files/ftp_upload/2153/2153fig2.jpg" alt="Figura 2" /> Figura 2. Las células secundarias meduloblastoma pueden ser cultivadas y expandidas in vitro Utilizando el protocolo descrito para células primarias de ratón meduloblastoma (&quot;El aislamiento, el enriquecimiento y el mantenimiento de las células madre meduloblastoma&quot;, Jove, en prensa), estas células de meduloblastoma secundarios pueden ser cultivados y ampliados para el paso múltiple. La imagen muestra la colonia representante tumor mismo culto a los 4 días y 14 días. Las células tumorales cultivadas son bipolares, alargada, con escaso citoplasma y emitir a menudo de un denso núcleo de agregados celulares. Ellos no muestran inhibición del crecimiento de contacto. Las células pequeñas y redondas son los glóbulos rojos.

<ol>
	<li>Schuller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+granule+neuron+precursor+identity+is+a+critical+determinant+of+progenitor+cell+competence+to+form+Shh-induced+medulloblastoma.">Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Shh-induced medulloblastoma.</a> Cancer Cell. 14, 123-134 (2008).</li><li>Yang, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medulloblastoma+can+be+initiated+by+deletion+of+Patched+in+lineage-restricted+progenitors+or+stem+cells.">Medulloblastoma can be initiated by deletion of Patched in lineage-restricted progenitors or stem cells.</a> Cancer Cell. 14, 135-145 (2008).</li><li>Kessler, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-myc+alters+the+fate+of+preneoplastic+cells+in+a+mouse+model+of+medulloblastoma.">N-myc alters the fate of preneoplastic cells in a mouse model of medulloblastoma.</a> Genes Dev. 23, 157-170 (2009).</li><li>Flora, A., Klisch, T. J., Schuster, G., Zoghbi, H. Y., Y, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+Atoh1+disrupts+Sonic+Hedgehog+signaling+in+the+developing+cerebellum+and+prevents+medulloblastoma.">Deletion of Atoh1 disrupts Sonic Hedgehog signaling in the developing cerebellum and prevents medulloblastoma.</a> Science. 326, 1424-1427 (2009).</li><li>Salsano, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+neurogenic+basic+helix-loop-helix+transcription+factor+NEUROG1+identifies+a+subgroup+of+medulloblastomas+not+expressing+ATOH1.">Expression of the neurogenic basic helix-loop-helix transcription factor NEUROG1 identifies a subgroup of medulloblastomas not expressing ATOH1.</a> Neuro Oncol. 9, 298-307 (2007).</li></ol>

Varios factores críticos para asegurar el éxito de células de meduloblastoma aloinjerto que la formación de los rendimientos de un tumor secundario son los siguientes: En primer lugar, utilice sólo Accutase 50% para el tejido de la disociación y no más de digerir el tejido como tratamiento enzimático prolongada conduce a una reducción significativa de la viabilidad celular. También utilizamos sólo la Pipetman 1 mL tamaño como pequeños consejos también pueden generar daño físico a las células disociadas. Es bueno tener una mezcla de células individuales y pequeños agregados de aloinjerto. En segundo lugar, mezclar y equilibrar la solución de las células tumorales antes de cada carga de la jeringa Hamilton. No use más de 4 l de cada inyección, como un mayor volumen generaría demasiada presión intracraneal y la resistencia. Por último, es importante esperar 2 minutos para dejar que la solución inyectada se disipan en el tejido del cerebelo después de cada inyección. Los procedimientos descritos aquí permiten la determinación del tipo de células marcadas genéticamente (s) que pueden iniciar y propagar los tumores. Este protocolo también se aplica a situaciones en las células que se inyecta no se derivan directamente de los tejidos del tumor primario, sino de establecer líneas de células cultivadas. Se puede preparar el número adecuado de células por digestión enzimática o bien y / o pipeteo repetitivo, y empezar desde el paso 2 de este protocolo. Por lo tanto, también puede probar las funciones de los genes candidatos y el efecto inhibidor de los compuestos químicos con un estudio in vivo de tumores de crecimiento de lectura.

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Neurobasal medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21103049</td>
<td>&nbsp;</td>
<tr>
<td>hEGF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>PHG0311</td>
<td>25ng/ml</td>
</tr>
<tr>
<td>bFGF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>PHG0023</td>
<td>25ng/ml</td>
</tr>
<tr>
<td>N2</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17502048</td>
<td>1X</td>
</tr>
<tr>
<td>B27(-RA)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12587010</td>
<td>1X</td>
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<tr>
<td>Accutase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A1110501</td>
<td>50% </td>
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<td>Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030081</td>
<td>2mM</td>
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<td>Stereotaxic instrument</td>
<td>&nbsp;</td>
<td>Stoelting </td>
<td>51730</td>
<td>&nbsp;</td>
<tr>
<td>Foredom flexible shaft drill, hang-up style</td>
<td>&nbsp;</td>
<td>Stoelting </td>
<td>58650</td>
<td>&nbsp;</td>
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<td>Large probe holder</td>
<td>&nbsp;</td>
<td>Stoelting </td>
<td>51633</td>
<td>&nbsp;</td>
<tr>
<td>10 &mu;L syringe, 26 ga., bevel tip</td>
<td>&nbsp;</td>
<td>Stoelting </td>
<td>51105</td>
<td>&nbsp;</td>
<tr>
<td>Drill bit .75mm</td>
<td>&nbsp;</td>
<td>Stoelting</td>
<td>51455-3</td>
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intracranial orthotopic allografting of medulloblastoma cells in immunocompromised mice

El meduloblastoma es el tumor pediátrico más común del sistema nervioso. Una gran cantidad de estudios realizados en animales se ha centrado en cerebelosa gránulo precursores de neuronas (CGNPs) como la célula de origen para el meduloblastoma 1-4. Sin embargo, las diversas presentaciones clínicas de los subtipos de meduloblastoma en pacientes humanos (nodular, desmoplásico, clásica y de células grandes / anaplásico), y el hecho de que el meduloblastoma se encuentra en un subgrupo de pacientes humanos, sin expresión ectópica de CGNP marcador 5, sugieren que la origen celular y molecular de meduloblastoma son más complejos y lejos de ser descifrado. Por lo tanto, es esencial para determinar si hay un tumor meduloblastoma alternativa de células de origen sobre la base de que tipo de células específicas de modalidad terapéutica puede ser desarrollada. Con este fin, ortotópico intracraneal aloinjerto de marcadas genéticamente tipos de células del tumor seguida de análisis posteriores del desarrollo del tumor secundario en los receptores que permiten determinar el origen celular de células iniciadoras del tumor. A continuación se describe el protocolo que se ortotópico intracraneal aloinjerto de células de meduloblastoma derivadas del tejido del tumor primario, y este procedimiento también se puede utilizar para el trasplante de células de líneas celulares establecidas.

Watch this Scientific Journal Video about Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice at JoVE.com

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Este protocolo describe el aislamiento y la disociación de los tejidos del ratón meduloblastoma, y ​​la posterior aloinjerto de las células tumorales en ratones inmunodeprimidos receptores a fin de iniciar el meduloblastoma secundaria.

Aloinjerto intracraneal ortotópico de células de meduloblastoma en ratones inmunodeficientes

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron ...

intracranial-orthotopic-allografting-medulloblastoma-cells

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.1K vistas.  Vanderbilt University. Este protocolo describe el aislamiento y la disociación de los tejidos del ratón meduloblastoma, y ​​la posterior aloinjerto de las células tumorales en ratones inmunodeprimidos receptores a fin de iniciar el meduloblastoma secundaria.

Video: Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been generally adopted to study the nature of neural stem cells, including those derived from normal and tumorous tissues. However, appreciable amounts of differentiation and cell death are common in cultured neurospheres likely due to sub-optimal condition such as accessibility of all cells within sphere aggregates to culture medium.
Medulloblastoma, the most common pediatric CNS tumor, is characterized by its rapid progression and tendency to spread along the entire brain-spinal axis with dismal clinical outcome. Medulloblastoma is a neuroepithelial tumor of the cerebellum, accounting for 20% and 40% of intracranial and posterior fossa tumor in childhood, respectively1. It is now well established that Shh signaling stimulates proliferation of cerebellar granule neuron precursors (CGNPs) during cerebellar development 2-4. Numerous studies using mouse models, in which the Shh pathway is constitutively activated, have linked Shh signaling with medulloblastoma 5-9.
A recent report has shown that a subset of medulloblastoma cells derived from Patched1LacZ/+ mice are cancer stem cells, which are capable of initiating and propogating tumors 10. Here we describe an efficient method to isolate, enrich and maintain tumor stem cells derived from several mouse models of medulloblastoma, with constitutively activated Shh pathway due to a mutation in Smoothened (11, hereon referred as SmoM2), a GPCR that is critical for Shh pathway activation. In every isolated medulloblastoma tissue, we were able to establish numerous highly proliferative colonies. These cells robustly expressed several neural stem cell markers such as Nestin and Sox2, can undergo serial passages (greater than 20) and were clonogenic. While these cultured tumor stem cells were relatively small, often bipoar with high nuclear to cytoplasmic ratio when cultured under conditions favoring stem cell growth, they dramatically altered their morphology, extended multiple cellular processes, flattened and withdrew from the cell cycle upon switching to a cell culture medium supplemented with 10% fetal bovine serum. More importantly, these tumor stem cells differentiated into Tuj1+ or NeuN+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes, thus highlighting their multi-potency. Furthermore, these cells were capable of propagating secondary medulloblastomas when orthotopically transplanted into host mice.

This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.

Aislamiento, enriquecimiento y mantenimiento de las células madre meduloblastoma

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been ...

Investigación

Neurociencias

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Inyección intracraneal de adeno-asociado vectores virales

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

La transfección de células de ratón del ganglio de la retina por In vivo La electroporación

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Células vivas imágenes de células de los órganos sensoriales Precursor en Intacto Drosophila Las pupas

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Un método simple y barato para la Determinación de la sensibilidad al frío y Adaptación en ratones

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

Análisis de dolor en roedores y farmacoterapia intracraneal

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

La detección de 5-Hidroximetilcitosina en células madre neuronales y cerebros de ratones

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

Medición cuantitativa de proteínas sintetizadas intratecalmente en ratones

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein&#160;cofilin&#160;using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is&#160;described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep.

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

Registro electrocorticográfico de áreas de la corteza cerebral manipuladas con un virus adenoaso asociado dirigido a la cofilina en ratones

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.