Queremos agradecer a los miembros del Laboratorio de Marder. Agradecemos a nuestro asesor, el Dr. Marder Eva, por su apoyo y aliento. Esta investigación fue financiada por el Instituto Nacional de Trastornos Neurológicos y Apoplejía Becas NS y NS-17813-058110.

1. La disección bruto <ol><li> Coloque un cangrejo en hielo durante 30 minutos para calmar los sistemas sensoriales y motoras del animal. Lo rodean por todas partes con hielo. Se deben usar guantes al manipular el cangrejo. </li><li> Prepararse para la disección bruto por colocar la bandeja de disección y las herramientas que va a utilizar. Gubias, tijeras de corte de hueso, pequeñas tijeras, pinzas dentadas, una espátula con un borde afilado, un plato negro Sylgard recubiertos, y los pernos de insectos son necesarios. </li><li> Quitar el cangrejo del cubo de hielo por sus patas traseras para evitar pellizcos y colocarlo en la bandeja de disección. </li><li> Quitar las garras por primera vez por cada giro hacia el interior una. A continuación, retire cada pierna con el mismo movimiento de torsión. </li><li> Uso de la gubia, retire el maxilar superior que cubre la boca. También quite la maxillule suave. </li><li> Uso de la gubia de nuevo, empezar desde el extremo lateral posterior del caparazón y obtener un buen agarre en el borde de la misma. Medidas drásticas con la gubia y romper ese trozo de caparazón de. Asegúrese de eliminar suficiente del caparazón para permitir la espátula para entrar en la apertura. Continuar con la eliminación del borde del caparazón, hasta los ojos y repetir en el otro lado. </li><li> Con el lado afilado de la espátula, raspar suavemente el tejido interior suave contacto con el techo del caparazón dorsal. Mantenga la espátula en contacto con el caparazón para evitar dañar la STNS. Utilice la gubia de romper con las partes de la caparazón que han sido separados de los tejidos. También por separado algunos de los tejidos blandos de la caparazón ventral asegurarse de separar los músculos aductores de colocar el tejido de la caparazón. </li><li> Romper el caparazón dorsal en lo posterior como el corazón, donde hay dos pequeños huesecillos que sobresale del caparazón. Cortar por lo anterior como sea posible. Estabilizar la Cephalon (la &quot;cara&quot;) con un dedo, mientras que romper la parte más anterior del caparazón dorsal. </li><li> En la parte ventral del caparazón, sacar la mandíbula a la vez, el anclaje de la gubia en la mandíbula, torciendo hacia el exterior para desalojar a la mandíbula de la epistome adjunta, y tirar a la basura. Dos huesecillos de largo con músculos deben estar unidos a la mandíbula extraída si se hace correctamente. </li><li> Aparte el tejido de los lados de la Cephalon cerca de los ojos con la espátula. Con tijeras de disección pequeñas, atraviesan el tejido tan cerca de la cordillera de los huesecillos como capa por capa, es posible. </li><li> El resto de cangrejo en una pared de la olla de disección y mantenerla hasta asegurarlo en su lugar (las garras se pueden utilizar para esto). Cortar el labrum (&quot;el labio superior&quot;) de la epistome asegurándose de no dejar ningún tejido adherido. Utilizando tijeras de corte de hueso hacer dos cortes diagonales en el caparazón ventral para eliminar el Cephalon. </li><li> Tome el labrum con pinzas dentadas y cortar el labio (&quot;labio inferior&quot;) de la huesecillos en la parte ventral. A continuación, levante suavemente el labio lejos del caparazón ventral restantes. Use las tijeras pequeñas para separar la parte ventral del estómago del tejido que recubre el caparazón. Cuando las ampollas pilórica son visibles, cortes en el estómago fuera de los tejidos a ambos lados de la misma hasta que el estómago se extrae de los cangrejos. </li><li> Todavía con el estómago por el labio con unas pinzas, descansar al estómago contra la palma de la mano y se vierte solución salina en la boca. Esto expande el estómago y hace que sea más fácil de abrir. Corte de la abertura de la boca hacia abajo entre el derecho a través del píloro huesecillos entre las ampollas. Luego se corta en diagonal hacia abajo a través de las dos ramas cardial. Cortar las puntas de los tres dientes en el interior del estómago. </li><li> Coloque el estómago a un grande, negro, recubierto Sylgard plato con el interior del estómago hacia abajo. Precisar las cinco esquinas de la preparación con alfileres de insectos y llenar el recipiente con solución salina fría, sumergir toda la preparación (ver figura 1). </li></ol> 2. La disección fina <ol><li> , Micro-disección se necesitan instrumentos para la disección fina. En particular, pinzas y tijeras de primavera son necesarias, así como un soporte de pasador con una aguja de tungsteno. Un claro Sylgard recubiertos, placa de Petri y algunos pines de alambre fino también debería haber estado preparado. </li><li> A partir del el aumento más bajo, comience por la fijación abajo las aletas cerca del borde que se utiliza para la apertura de la boca. Reorganizar el resto de los pines para hacer la preparación tensa y pin cualquier área que sea necesaria. </li><li> Levante con cuidado la hipodermis vio que cubre la parte inferior de la preparación y el ajuste que a través de dejar un trozo de la hipodermis, con dos puntos blancos. </li><li> Pelar el amarillo, el tejido ralo, membranosa en la parte inferior de la preparación. El mvns están débilmente unidas a la parte inferior de este tejido. Separe cuidadosamente los mvns del tejido amarillo que se levanta. Siga el tejido amarillo y corte junto con el cerebro. </li><li> Seguir los procesos de espesor de salir del cerebro que se extienden laterally. Estos son los nervios commisural. Una vez que el extremo del nervio comisural se alcanza, quite la masa de esponjosa, blanco tejido que ha sido cortada de la parte del cerebro. </li><li> Donde los iones y los hijos de cumplir con los engranajes, cortar el músculo y el tejido para revelar la duración de los iones y los hijos. </li><li> Volver a la revisión de la hipodermis visto y lo cortó. Esto debería dejar una abertura en la arteria que rodea el STG. Corte a través de la apertura para separar los dos músculos que flanquean el STG. Levante los extremos de estos músculos y cortado a la derecha por debajo de ellos para separarlos de la arteria y para exponer la alns y agns. Separar los músculos que flanquean tan arriba como el cartílago que está sobre la STN. </li><li> Cortar el tejido que rodea el STG arterial hasta donde el mvns, dgn, y dvn cumplir. Trabajar por la DVN a la LVNs y cortar a través del tejido blanquecino, delicada que cubre el LVN. </li><li> Localice el PSN en los huesecillos en la parte inferior de la preparación. El PSN se puede utilizar para localizar el LVN y la unión dlvn. </li><li> A continuación vienen las dlvn abajo para localizar el PYN y PDN. El PYN a menudo envuelve a la ampolla de la apertura del píloro. El PDN bifurca de la dlvn y se encuentra entre el píloro ampolla y los músculos cardio pilórica válvula. Deje un poco de músculo unido a uno de estos nervios, ya que pueden confundirse fácilmente cuando la preparación se transfiere a la clara fuente Sylgard. </li><li> Una vez que todos los nervios se han descubierto, separar las conexiones entre el resto de STNS y los nervios del tejido no deseado y del estómago. Cuidadosamente mueva el STNS lejos del resto del estómago, cortando las conexiones perdidas. </li><li> La condición del plato Sylgard claro con la masa restante de tejido del estómago por el roce sobre la superficie de la Sylgard hasta el Sylgard ya no se siente demasiado seca o pegajosa. Sylgard es hidrofóbica y la STNS se adhieren fuertemente a que si el plato Sylgard recubiertos no está condicionada de esta manera. </li><li> Agregue un poco de solución salina fría para el plato. Coge los extremos superiores de los nervios comisurales con fórceps y llevar a los STNS a la clara fuente Sylgard. </li><li> Pin de la STNS abajo sobre el Sylgard. Cortar el cerebro de los nervios comisurales y el uso de los pines minuten para asegurar los cuatro extremos de los engranajes en primer lugar. Asegúrese de que la preparación es la orientación adecuada, al verificar que la red IVN está apuntando hacia arriba fuera de la Sylgard. La DGN debería salir un poco por debajo de la STG, cuando el STG es lado derecho hacia arriba. Pin el resto de las terminaciones nerviosas hacia abajo con las patas de alambre fino. </li><li> Limpie cualquier resto de músculo o tejido del STNS. </li><li> Desheath la STG utilizando un soporte de pasador y un poco enganchado, con aguja fina, el tungsteno. Haz un pequeño agujero en un rincón de la vaina de STG lejos de los cuerpos celulares y el uso de ese agujero para llegar entre las capas de la vaina. Con cuidado, cortar una solapa de la cubierta separados para exponer el neuropilo STG y cuerpos celulares. </li><li> Pin cualquier solapas de la cubierta hacia abajo para aumentar la accesibilidad a los cuerpos celulares y para estabilizar la STG de registros intracelulares. Re-pin el resto de la STNS asegurar que cada nervio se tensa y repartidas para el registro extracelular (ver figura 3). </li></ol> 3. Resultados <ol><li> Lo ideal sería que todos los nervios deben estar libres de rasguños y daños, en particular los que se registrarán a partir. Ninguno de los nervios deben ser enredado o retorcido. El STG debe estar intacto con todas las células dispuestas en forma de barbas de todo el neuropilo. El STNS intacta es de simetría bilateral y se ve como un homúnculo con la LVNs como las piernas, la mvns como los brazos y el extremo anterior de la cabeza. </li></ol> Abreviaturas: <table border="1"><tbody><tr><td> STNS </td><td> Sistema nervioso Stomatogastric </td></tr><tr><td> STG </td><td> Ganglio Stomatogastric </td></tr><tr><td> COG </td><td> Ganglio comisural </td></tr><tr><td> OG </td><td> Ganglio esofágico </td></tr><tr><td> mvn </td><td> Nervio ventricular mediana </td></tr><tr><td> ion </td><td> Los nervios del esófago inferior </td></tr><tr><td> hijo </td><td> Los nervios del esófago superior </td></tr><tr><td> agn </td><td> Nervio anterior del estómago </td></tr><tr><td> aln </td><td> Nervio lateral anterior </td></tr><tr><td> dgn </td><td> Nervio dorsal gástrico </td></tr><tr><td> dvn </td><td> Nervio dorsal del ventrículo </td></tr><tr><td> LVN </td><td> Nervio lateral del ventrículo </td></tr><tr><td> psn </td><td> Los nervios del estómago posterior </td></tr><tr><td> PYN </td><td> Nervio pilórica </td></tr><tr><td> PDN </td><td> Nervio dilatador pilórica </td></tr><tr><td> dlvn </td><td> Rama dorsal del nervio lateral del ventrículo </td></tr><tr><td> ivn </td><td> Nervio inferior del ventrículo </td></tr></tbody></table><img src="/files/ftp_upload/1207/figure_1_90ppi.jpg" alt= &quot;Figura 1&quot; /&gt; Figura 1: Diagrama que muestra la ubicación aproximada de la STNS en el estómago antes de la disección. <img src="/files/ftp_upload/1207/figure_2_90ppi.jpg" alt="Figura 2" /> Figura 2: Diagrama de los distintos músculos en la parte inferior del estómago y una superposición de las STNS en verde claro. Cada tipo de célula en el STG está en la lista con los músculos que se sabe que inervan. Este mapa es útil en la planificación de la disección de un nervio que lleva la señal de una célula (cortesía del laboratorio de Nadim). <img src="/files/ftp_upload/old/1207_2009_1_12_5/figure_3.jpg" alt="Figura 3" /> Figura 3: Diagrama esquemático de la STNS con electrodos intracelulares y extracelulares pozos (de Marder y Bucher, 2007).

<ol>
	<li>Harris-Warrick, R. M., Marder, E., Selverston, A. I., Moulins, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dynamic Biological Networks: The stomatogastric nervous system. , (1992).</li><li>Marder, E., Bucher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+circuit+dynamics+using+the+stomatogastric+nervous+system+of+lobsters+and+crabs.">Understanding circuit dynamics using the stomatogastric nervous system of lobsters and crabs.</a> Annu. Rev. Physiol. 69, 291-316 (2007).</li><li>Maynard, D. M., Dando, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+stomatogastric+neuromuscular+system+in+Callinectes+sapidus,+Homarus+Americanus+and+Panulirus+argus+(Decapoda+crustacean).">The structure of the stomatogastric neuromuscular system in Callinectes sapidus, Homarus Americanus and Panulirus argus (Decapoda crustacean).</a> Philos. Trans. R. Soc. Lond. B Biol. Sci. 268 (892), 161-220 (1974).</li><li>Selverston, A. I., Russell, D. F., Miller, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Stomatogastric+Nervous+System:+Structure+and+function+of+a+small+neural+network.">The Stomatogastric Nervous System: Structure and function of a small neural network.</a> Prog. Neurobiology. 7, 215-290 (1976).</li></ol>

La disección STNS es el primer paso en la realización de un experimento de electrofisiología o inmunohistoquímica, o para la obtención de células para el cultivo celular. Independientemente del experimento previsto, la disección bruto se mantendrá sin cambios. Sin embargo, la disección fina puede variar. Es importante planificar de antemano que las células y los nervios que va a grabar desde. La mayoría de las neuronas en el STG tienen procesos que inervan al menos un músculo en el estómago (ver figura 2) y...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Black Sylgard 170</td>
<td>Other</td>
<td>Dow Corning</td>
<td>170</td>
<td>Gross Dissection</td>
</tr>
<tr>
<td>Fine Scissors</td>
<td>Surgery</td>
<td>FST</td>
<td>14090-09</td>
<td>&nbsp;</td>
<tr>
<td>Insect Pins size 6</td>
<td>Surgery</td>
<td>FST</td>
<td>26000-65</td>
<td>&nbsp;</td>
<tr>
<td>Mayo Scissors</td>
<td>Surgery</td>
<td>FST</td>
<td>14110-17</td>
<td>&nbsp;</td>
<tr>
<td>Micro-Spatula</td>
<td>Surgery</td>
<td>Ward Sci.</td>
<td>15 V 4313</td>
<td>&nbsp;</td>
<tr>
<td>Rongeurs</td>
<td>Surgery</td>
<td>FST</td>
<td>16000-14</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Forceps</td>
<td>Surgery</td>
<td>FST</td>
<td>11021-12</td>
<td>&nbsp;</td>
<tr>
<td>Fine Tungsten Wire (0.001 in. diameter)</td>
<td>Surgery</td>
<td>California Fine Wire</td>
<td>&nbsp;</td>
<td>Fine Dissection</td>
</tr>
<tr>
<td>Forceps #5</td>
<td>Surgery</td>
<td>FST</td>
<td>91150-20</td>
<td>&nbsp;</td>
<tr>
<td>Minuten Pins</td>
<td>Surgery</td>
<td>FST</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>Moria Spring Scissors (7mm blades)</td>
<td>Surgery</td>
<td>FST</td>
<td>15370-52</td>
<td>&nbsp;</td>
<tr>
<td>Petri Dish (100x15mm)</td>
<td>Other</td>
<td>Fischer	</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>Pin Holder</td>
<td>Surgery</td>
<td>FST</td>
<td>26018-17</td>
<td>&nbsp;</td>
<tr>
<td>Super fine forceps #5SF</td>
<td>Surgery</td>
<td>FST</td>
<td>11252-00</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 182</td>
<td>Other</td>
<td>Dow Corning</td>
<td>182</td>
<td>&nbsp;</td>
<tr>
<td>Tungsten Needles</td>
<td>Surgery</td>
<td>FST</td>
<td>10130-05</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>Microscope</td>
<td>Wild</td>
<td>MC3</td>
<td>Misc.</td>
</tr>
<tr>
<td>Jonah Crab (C. borealis)</td>
<td>Animal</td>
<td>Commercial Lobster</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
						
		</div>

cancer borealis stomatogastric nervous system dissection

El ganglio stomatogastric (STG) es un excelente modelo para el estudio de las interacciones celulares y la red, ya que contiene un número relativamente pequeño de las células (aproximadamente 25 en C. borealis), que están bien caracterizados. Las células de la STG exhiben una amplia gama de productos y son responsables de las acciones motrices del estómago. El estómago contiene el molino gástrico, que descompone los alimentos con tres dientes internos, y el píloro, que filtra los alimentos antes de llegar al intestino. El STG produce dos salidas para controlar el ritmo del molino gástrico y el píloro conocido como generadores de patrones centrales (GPC). Cada celda de la STG se puede participar en uno o ambos de estos ritmos. Estas GPC permiten el estudio de la neuromodulación, la homeostasis, la variabilidad celular y de redes, desarrollo de redes, y la recuperación de la red. La disección del sistema nervioso stomatogastric (STNS) del cangrejo de Jonás (cáncer borealis) se realiza en dos partes: la disección fina y gruesa. En la disección bruto de todo el estómago se diseca del cangrejo. Durante la disección fina del STNS se extrae del estómago usando un microscopio de disección y la disección de micro-herramientas (ver figura 1). El STNS incluye la STG, el ganglio esofágico (OG), y los ganglios de la comisura (COG), así como los nervios que inervan los músculos del estómago. Aquí, se muestra cómo realizar una disección completa de los STNS en la preparación de un experimento de electrofisiología en las células de la STG se registrará a partir de la célula y los nervios periféricos se utilizan para las grabaciones extracelular. La técnica adecuada para encontrar los nervios deseada se muestra, así como nuestra técnica de desheathing el ganglio para revelar la somata y neuropilo.

Watch this Scientific Journal Video about Cancer Borealis Stomatogastric Nervous System Dissection at JoVE.com

Cancer Borealis Stomatogastric Nervous System Dissection

El sistema nervioso stomatogastric (STNS) del cangrejo de Jonás (<em&gt; C. boreal</em&gt;) Se puede utilizar para electrofisiología, inmunohistoquímica y estudios en cultivos celulares. La extracción STNS se realiza en dos partes: la disección fina y gruesa.

Cáncer de Borealis Stomatogastric disección del sistema nervioso

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of ...

cancer-borealis-stomatogastric-nervous-system-dissection

Research

JoVE Journal

Biology

Cancer Borealis Stomatogastric Nervous System Dissection

12.5K vistas.  Brandeis. El sistema nervioso stomatogastric (STNS) del cangrejo de Jonás ( C. boreal) Se puede utilizar para electrofisiología, inmunohistoquímica y estudios en cultivos celulares. La extracción STNS se realiza en dos partes: la disección fina y gruesa.

Video: Cancer Borealis Stomatogastric Nervous System Dissection

Dissection of Drosophila Ovaries

La disección de los ovarios Drosophila

Investigación

Biología

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

La disección del sistema nervioso central de larvas de Drosophila melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

La disección de embriones de ratón 6,5 dpc

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Isolation of Mononuclear Cells from the Central Nervous System of Rats with EAE

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the immune response to an infection of the CNS, such as poliomyelitis, Lyme neuroborreliosis, or neurosyphilis, it is often necessary to isolate the CNS-infiltrating immune cells.
In this video-protocol we demonstrate how to isolate mononuclear cells (MNCs) from the CNS of a rat with EAE. The first step of this procedure requires a cardiac perfusion of the rodent with a saline solution to ensure that no blood remains in the blood vessels irrigating the CNS. Any blood contamination will artificially increase the number of apparent CNS-infiltrating MNCs and may alter the apparent composition of the immune infiltrate. We then demonstrate how to remove the brain and spinal cord of the rat for subsequent dilaceration to prepare a single-cell suspension. This suspension is separated on a two-layer Percoll gradient to isolate the MNCs. After washing, these cells are then ready to undergo any required procedure. 
Mononuclear cells isolated using this procedure are viable and can be used for electrophysiology, flow cytometry (FACS), or biochemistry. If the technique is performed under sterile conditions (using sterile instruments in a tissue culture hood) the cells can also be grown in tissue culture medium. A given cell population can be further purified using either magnetic separation procedures or a FACS.

In this video we demonstrate how to isolate mononuclear cells from the central nervous system of rats with experimental autoimmune encephalomyelitis.

Aislamiento de células mononucleares de la del sistema nervioso central de ratas con EAE

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

Drosophila larval NMJ Disección

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

La disección de Saccharomyces cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Homarus Americanus Stomatogastric Nervous System Dissection

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the activity of interest and are accessible and amenable to experimental methods. The stomatogastric nervous system (STNS) of the American lobster (Homarus americanus; also know was the Atlantic or Maine lobster) has been established as a model system for studying rhythm generating networks and neuromodulation of networks. The STNS consists of 3 anterior ganglia (2 commissural ganglia and an oesophageal ganglion), containing modulatory neurons that project centrally to the stomatogastric ganglion (STG). The STG contains approximately 30 neurons that comprise two central pattern generating networks, the pyloric and gastric networks that underlie feeding behaviors in crustaceans1,2. While it is possible to study this system in vivo3, the STNS continues to produce its rhythmic activity when isolated in vitro. Physical isolation of the STNS in a dish allows for easy access to the somata in the ganglia for intracellular electrophysiological recordings and to the nerves of the STNS for extracellular recordings. Isolating the STNS is a two-part process. The first part, dissecting the stomach from the animal, is described in an accompanying video article4. In this video article, fine dissection techniques are used to isolate the STNS from the stomach. This procedure results in a nervous system preparation that is available for electrophysiological recordings.

Homarus Americanus Stomatogastric Nervous System Dissection

We describe the fine dissection of the stomatogastric nervous system from the stomach of the American lobster (Homarus americanus).

Homarus americanus Stomatogastric La disección del sistema nervioso

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the ...

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia.
Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer.
DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia.
We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.

Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.

PMS2 deficiente, ERCC1, Ku86, CCOI en los defectos del campo durante la progresión de cáncer de colon

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

La disección de Oenocytes de Adultos Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

La disección del riñón adultos de pez cebra

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...