Noi riconosciamo con gratitudine i membri del Laboratorio Marder. Ringraziamo il nostro consulente, il dottor Eve Marder, per il suo sostegno e incoraggiamento. Questa ricerca è stata finanziata dal National Institute of Neurological Disorder and Stroke Grants NS-NS-17.813 e 058.110.

1. Dissezione lordo <ol><li> Posizionare un granchio in ghiaccio per 30 minuti opachi i sistemi sensoriali e motorie dell&#39;animale. Lo circondano su tutti i lati con ghiaccio. I guanti dovrebbero essere indossati quando si maneggia il granchio. </li><li> Preparati per la dissezione lordo ponendo la pentola dissezione e gli strumenti si intende utilizzare. Rongeurs, osso taglio forbici, forbicine, pinzette dentate, una spatola con un bordo affusolato, un nero Sylgard rivestita piatto, e perni insetti sono necessari. </li><li> Rimuovere il granchio dal secchiello del ghiaccio per la sua zampa posteriore per evitare che pizzicotti e metterla nella padella dissezione. </li><li> Rimuovere gli artigli prima ruotando verso l&#39;interno di ogni uno. Quindi rimuovere ogni gamba con lo stesso movimento torsione. </li><li> Utilizzando il Rongeurs, rimuovere la mascella che copre la bocca. Rimuovere anche il maxillule morbido. </li><li> Utilizzando il Rongeurs ancora una volta, partire dalla fine laterale posteriore del carapace e ottenere un buon grip sul bordo di esso. Reprimere con la Rongeurs e rompere quel pezzo di carapace off. Assicurarsi di aver tolto abbastanza del carapace per consentire la spatola di entrare l&#39;apertura. Continuare a rimuovere il bordo del carapace fino agli occhi e ripetete dall&#39;altro lato. </li><li> Con la parte conica della spatola, raschiare delicatamente i tessuti molli interiore, lontani dal tetto del carapace dorsale. Tenere la spatola a contatto con il carapace per evitare di danneggiare il STNS. Utilizzare il Rongeurs di rompere le parti del carapace che sono stati separati dal tessuto. Anche separati alcuni dei tessuti molli dal carapace ventrale facendo attenzione a separare ogni muscoli adduttori attaccare il tessuto al carapace. </li><li> Rompere la corazza dorsale fino posteriore come il cuore dove ci sono due piccoli ossicini che sporgono dal carapace. Tagliare il più anteriore possibile. Stabilizzare la Cephalon (il &quot;volto&quot;) con un dito mentre rottura con la parte più anteriore del carapace dorsale. </li><li> Sul lato ventrale del carapace, tirare fuori le mandibole uno alla volta grazie all&#39;ancoraggio del Rongeurs su una mandibola, torsione verso l&#39;esterno per rimuovere la mandibola dal epistome allegato, e tirandolo via. Due ossicini lungo con i muscoli devono essere allegati alla mandibola estratto se fatto correttamente. </li><li> Separare via il tessuto dai lati della Cephalon vicino agli occhi con la spatola. Con le forbici dissezione piccolo, attraversano il tessuto più vicino al crinale di ossicino come strato dopo strato possibile. </li><li> Resto il granchio contro un muro della padella dissezione e prop in su fissandolo in posizione (gli artigli possono essere utilizzate per questo). Taglio del labbro (&quot;labbro superiore&quot;) dal epistome avendo cura di non lasciare qualsiasi tessuto allegato. Utilizzando ossa di taglio forbici fare due tagli diagonali nel carapace ventrale per rimuovere la Cephalon. </li><li> Prendere il labbro con una pinza dentata e tagliare il labium (&quot;labbro inferiore&quot;) dal ossicino sul lato ventrale. Poi sollevare delicatamente il labbro dal carapace ventrale rimanenti. Utilizzare le forbici piccolo per separare la parte ventrale dello stomaco dal tessuto di rivestimento del carapace. Quando le ampolle sono visibili piloro, tagliare lo stomaco lontano dal tessuto su entrambi i lati fino a quando lo stomaco è estratto dal granchio. </li><li> Ancora in mano allo stomaco con il labbro con una pinza, il resto lo stomaco contro il palmo della mano e versare salina in bocca. Questo si espande lo stomaco e rende più facile da tagliare aperto. Tagliato dall&#39;apertura alla foce giù tra il diritto piloro attraverso la ossicino tra le ampolle. Poi tagliare in diagonale verso il basso attraverso i due rami cardiale. Tagliare le punte delle tre denti all&#39;interno dello stomaco. </li><li> Posizionare lo stomaco su un grande, nero, Sylgard rivestite piatto con l&#39;interno dello stomaco in giù. Definire con precisione i cinque angoli della preparazione con perni di insetti e riempire il piatto con soluzione fisiologica fredda, immergendo l&#39;intera preparazione (vedi figura 1). </li></ol> 2. Dissezione multa <ol><li> , Micro-dissezione strumenti sono necessari per la dissezione fine. In particolare, pinze e forbici a molla sono necessarie così come un supporto pin con un ago di tungsteno. Un chiaro, Sylgard rivestita, capsula di Petri e di alcuni perni di filo sottile avrebbe dovuto essere preparato. </li><li> A partire l&#39;ingrandimento più basso, iniziare pinning giù i lembi vicino al labbro che ha usato essere l&#39;apertura della bocca. Riorganizzare il resto dei perni per rendere la preparazione teso e pin altre aree necessarie. </li><li> Sollevare con cautela il ipoderma notato che copre la parte inferiore della preparazione e tagliare di traverso lasciando una chiazza di ipoderma con due puntini bianchi. </li><li> Sbucciate il giallo, esile, il tessuto membranoso vicino al fondo della preparazione. Il mvns sono debolmente attaccati alla parte inferiore di questo tessuto. Separare con attenzione il mvns dal tessuto giallo, come si è tirato su. Segui il tessuto giallo e tagliate a fianco del cervello. </li><li> Seguire i processi di spessore uscire il cervello che si estendono laterally. Questi sono i nervi commisural. Una volta alla fine del nervo commissurali viene raggiunto, rimuovere la massa spugnosa, di colore biancastro tessuto che è stato tagliato via dal lato del cervello. </li><li> Dove gli ioni e figli incontrare gli ingranaggi, tagliare via il muscolo e del tessuto di rivelare la lunghezza degli ioni e dei figli. </li><li> Ritorna alla patch ipoderma macchiato e tagliarlo fuori. Questo dovrebbe lasciare un&#39;apertura nel arteria che circonda la STG. Tagliare attraverso l&#39;apertura di separare i due muscoli che fiancheggiano la STG. Sollevare l&#39;estremità di questi muscoli e tagliare proprio sotto di loro per separarli dalla arteria e per esporre il alns e AGN. Separare i muscoli ai lati fino a che la cartilagine che avvolge il stn. </li><li> Tagliare il tessuto arterioso che circonda la STG fino a dove il mvns, DGN, e DVN si incontrano. Lavoro lungo la DVN al lvns e tagliare il biancastro, delicato tessuto che copre il lvns. </li><li> Individuare il PSN negli ossicino vicino al fondo della preparazione. Il PSN può essere utilizzato per individuare il LVN e giunzione dlvn. </li><li> Avanti seguire le dlvn giù per individuare il pyn e PDN. Il pyn avvolge spesso sopra l&#39;ampolla pilorica per l&#39;apertura. La PDN forking del dlvn ed è tra i pilorica ampolla ed i muscoli della valvola pilorica cardio. Lascia qualche muscolo collegato a uno di questi nervi, perché possono essere facilmente confusi quando la preparazione viene trasferito il piatto chiaro Sylgard. </li><li> Una volta che tutti i nervi sono stati scoperti, recidere le connessioni sussistenti tra le STNS ei nervi dei tessuti e indesiderati dello stomaco. Muovere con cautela il STNS lontano dal resto dello stomaco, tagliando tutte le connessioni perse. </li><li> Condizione il piatto Sylgard chiaro con la massa restante del tessuto dello stomaco per lo sfregamento sulla superficie della Sylgard fino alla Sylgard non si sente più troppo appiccicoso o secco. Sylgard idrorepellente ed il STNS aderisce fortemente ad esso se la Sylgard rivestite piatto non è condizionato in questo modo. </li><li> Aggiungete un po &#39;salina fredda per il piatto. Afferrare le estremità superiori dei nervi commissurali con una pinza e portare il STNS al piatto chiaro Sylgard. </li><li> Perno del STNS verso il basso sulla Sylgard. Sever il cervello dai nervi commissurali e utilizzare i pin minuten per fissare le quattro estremità della Cogs primo down. Garantire che la preparazione sia dal lato giusto controllando che il IVN è rivolta verso l&#39;alto dalla Sylgard. Il dgn dovrebbe uscire un po &#39;sotto la STG quando la STG è da destra verso l&#39;alto. Pin il resto del nervo termina verso il basso con i perni di filo sottile. </li><li> Pulire via qualsiasi residuo muscolare o tessuto dal STNS. </li><li> Desheath la STG all&#39;utilizzo di un supporto e un pin leggermente aquilino, fine, ago di tungsteno. Fai un piccolo foro in un angolo della guaina STG lontano dal corpo cellulare e utilizzare quel buco per arrivare tra gli strati della guaina. Accuratamente tagliato un lembo di guaina separati per esporre il neuropilo STG e corpi cellulari. </li><li> Pin qualsiasi lembi della guaina giù per aumentare l&#39;accessibilità ai corpi cellulari e di stabilizzare la STG per le registrazioni intracellulari. Re-pin il resto del STNS garantire che ogni nervo è teso e ben distanziati per la registrazione extracellulare (vedi figura 3). </li></ol> 3. Risultati <ol><li> Idealmente, tutti i nervi devono essere privi di ammaccature e danni, in particolare quelli che saranno registrate da. Nessuno dei nervi dovrebbe essere aggrovigliati o torto. La STG dovrebbe essere intatto con tutte le celle disposte in una formazione barba intorno al neuropilo. Il STNS intatta è simmetria bilaterale e assomiglia a un omuncolo con il lvns come le gambe, il mvns come le braccia e la fine anteriore come la testa. </li></ol> Abbreviazioni: <table border="1"><tbody><tr><td> STNS </td><td> Stomatogastric sistema nervoso </td></tr><tr><td> STG </td><td> Ganglio Stomatogastric </td></tr><tr><td> CoG </td><td> Ganglio commissurali </td></tr><tr><td> OG </td><td> Ganglio esofageo </td></tr><tr><td> mvn </td><td> Ventricolare nervo mediano </td></tr><tr><td> ione </td><td> Esofageo inferiore del nervo </td></tr><tr><td> figlio </td><td> Superiore nervo esofageo </td></tr><tr><td> AGN </td><td> Anteriore del nervo gastrico </td></tr><tr><td> ALN </td><td> Anteriore del nervo laterale </td></tr><tr><td> dgn </td><td> Dorsale del nervo gastrico </td></tr><tr><td> DVN </td><td> Ventricolare nervo dorsale </td></tr><tr><td> LVN </td><td> Ventricolare nervo laterale </td></tr><tr><td> PSN </td><td> Stomaco nervo posteriore </td></tr><tr><td> pyn </td><td> Nervo pilorica </td></tr><tr><td> PDN </td><td> Dilatatore nervo pilorica </td></tr><tr><td> dlvn </td><td> Ramo dorsale del nervo del ventricolo laterale </td></tr><tr><td> IVN </td><td> Ventricolare nervo inferiore </td></tr></tbody></table><img src="/files/ftp_upload/1207/figure_1_90ppi.jpg" alt= &quot;Figura 1&quot; /&gt; Figura 1: Diagramma che indica la posizione approssimativa del STNS nello stomaco prima di dissezione. <img src="/files/ftp_upload/1207/figure_2_90ppi.jpg" alt="Figura 2" /> Figura 2: Schema dei vari muscoli nella parte inferiore dello stomaco e una sovrapposizione della STNS in verde chiaro. Ogni tipo di cellula del STG è elencato con i muscoli è nota la loro innervare. Questa mappa è utile quando si pianifica di sezionare un nervo che trasporta un segnale da una determinata cella (per gentile concessione del laboratorio Nadim). <img src="/files/ftp_upload/old/1207_2009_1_12_5/figure_3.jpg" alt="Figura 3" /> Figura 3: Rappresentazione schematica del STNS con elettrodi intracellulari e pozzi extracellulare (da Marder e 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 dissezione STNS è il primo passo per l&#39;esecuzione di un esperimento o di elettrofisiologia immunoistochimica, o per l&#39;ottenimento di cellule per la coltura cellulare. Indipendentemente l&#39;esperimento previsto, la dissezione lordo rimarrà invariato. Tuttavia, la dissezione multa può variare. E &#39;importante pianificare in anticipo del tempo che cellule e nervi che si registra da. La maggior parte dei neuroni nel STG sono processi che innervano almeno un muscolo nello stomaco (vedi figura 2) e sono clas...

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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>
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cancer borealis stomatogastric nervous system dissection

Il ganglio stomatogastric (STG) è un modello eccellente per studiare le interazioni cellulari e di rete, perché contiene un numero relativamente piccolo di cellule (circa 25 in C. borealis) che sono ben caratterizzati. Le cellule del STG mostra una vasta gamma di uscite e sono responsabili delle azioni motorie dello stomaco. Lo stomaco contiene il mulino gastrico che scompone il cibo con tre denti interni, e il piloro che filtra il cibo prima che raggiunga il midgut. La STG produce due uscite ritmica per controllare il mulino gastrica e piloro conosciuto come generatori di pattern centrali (CPG). Ogni cella della STG possono partecipare a uno o entrambi questi ritmi. Queste CPGs permettono per lo studio della neuromodulazione, omeostasi, la variabilità cellulare e di rete, sviluppo della rete, e il recupero della rete. La dissezione del sistema nervoso stomatogastric (STNS) dal granchio Giona (Cancer borealis) è fatto in due parti: la dissezione lordo e fine. Nella dissezione lordo stomaco intero sezionato dal granchio. Durante la dissezione bene il STNS viene estratto dallo stomaco utilizzando un microscopio dissezione e micro-dissezione strumenti (vedi figura 1). Il STNS comprende la STG, il ganglio esofageo (OG), ed i gangli commissurali (COG) così come i nervi che innervano i muscoli dello stomaco. Qui mostriamo come eseguire una dissezione completa del STNS in preparazione per un esperimento di elettrofisiologia in cui le cellule del STG sarebbe registrato da cellule ed i nervi periferici sarebbe stato utilizzato per le registrazioni extracellulari. La tecnica corretta per trovare i nervi desiderata è mostrata così come la nostra tecnica di desheathing ganglio di rivelare la somata e neuropilo.

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

Cancer Borealis Stomatogastric Nervous System Dissection

Il sistema nervoso stomatogastric (STNS) del granchio Giona (<em&gt; C. Borealis</em&gt;) Può essere utilizzato per elettrofisiologia, immunoistochimica, e studi su colture cellulari. L&#39;estrazione STNS avviene in due parti: la dissezione lordo e fine.

Cancro Borealis Stomatogastric dissezione del Sistema Nervoso

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 Views.  Brandeis. Il sistema nervoso stomatogastric (STNS) del granchio Giona ( C. Borealis) Può essere utilizzato per elettrofisiologia, immunoistochimica, e studi su colture cellulari. L'estrazione STNS avviene in due parti: la dissezione lordo e fine.

Video: Cancer Borealis Stomatogastric Nervous System Dissection

Dissection of Drosophila Ovaries

Dissezione di ovaie Drosophila

Ricerca

Biologia

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.

Dissezione del sistema nervoso centrale larvali 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 ...

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]).

Dissezione di 6,5 embrioni di topo 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.

Isolamento di cellule mononucleate dal Sistema Nervoso Centrale di ratti 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 larvali NMJ Dissection

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.

Dissezione di 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 dissezione del Sistema Nervoso

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 carente, ERCC1, Ku86, CcOI in Difetti del campo durante la progressione a tumore del 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.

Dissezione di Oenocytes da adulti 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.

Dissezione del rene Zebrafish adulti

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