We would like to acknowledge our advisor, Dr. Eve Marder. This research was supported by National Institute of Health grants NS17813 to Eve Marder and NS059255 to A-E T.

Prior to beginning this protocol the stomach of the lobster must be removed from the animal and prepared as described in the companion JoVE article (Bierman &amp; Tobin, 2009). The majority of the steps outlined below are to be performed under a dissection microscope.
Part I- Isolating the Commissural Ganglia
<ol>
<li>Orient the dish so that the anterior end (by the lip) is away from you and the posterior end (by the pylorus) is towards you. </li>
<li>Ensure that the stomach is pinned so that it lies as flat as possible on the dish.<ol>
<li>Pin down the esophagus on each side of the lip.</li>
<li>If necessary, rearrange the other pins placed during the gross dissection.</li>
</ol></li>
<li>Remove the hypodermis.<ol>
<li>Grab the hypodermis near the gm2/gm3 muscles, detach it from these muscles and gently pull in the anterior direction.</li>
<li>Separate the hypodermis from the underlying fat by snipping any connections. Use increased care around the central artery, which encloses a key part of the stomatogastric nervous system. Leave the hypodermis intact within about a 0.5 cm radius of the stomatogastric ganglion, which sits between the gm1b muscles.</li>
<li>Cut off separated hypodermis.</li>
</ol></li>
<li>Detach the brain from the underlying tissue.<ol>
<li>Identify the two large commissural nerves that project bilaterally from the anterior portion of the brain to the commissural ganglia in the anterior portion of the stomach.</li>
<li>Leaving these nerves intact, gently lift the brain with forceps and cut to separate the brain from the rest of the tissue. Be careful as you do this, as the stomatogastric nerve is underneath the brain and must be kept intact.</li>
</ol></li>
<li>Separate the commissural ganglia from surrounding the tissue.<ol>
<li>Follow one the commissural nerves to one of the commissural ganglia.</li>
<li>Identify the ion and son &#x2013; these nerves must remain intact. The commissural ganglion will have many nerves radiating out. Cut most of them (except the ion and son), but leave one extra nerve attached to hold the commissural ganglion in place while you finish the dissection.</li>
<li>Repeat with the other commissural ganglion.</li>
</ol></li>
<li>Clean the ion and son from lateral to medial. Leave the labial nerve intact to anchor the nervous system to the stomach. The esophageal ganglion sits were the ions intersect. You can clear below it by grabbing the inferior ventricular nerve and pulling up to cut underneath.</li>
</ol>
Part II- Isolating the Stomatogastric Ganglion (STG) and remaining nerves.
<ol start="7">
<li>Separate the STG from surrounding the tissue.<ol>
<li>The STG sits inside an artery that runs between the gm1b muscles and down to the gm2a, b muscles. Look for the cut end of the artery near the gm2a, b muscles.</li>
<li>Pull the cut end of the artery up and anteriorly, to expose the dvn. Continue pulling up until you reach the level of the remaining hypodermis. Once separated, the artery can be trimmed to the level where it touches the remaining hypodermis.</li>
<li>Cut off the muscle flap that was situated in the rostrum of the lobster.</li>
<li>Lift up the hypodermis above the artery, observing that the artery makes a tunnel inside the fat above the STG. Put one blade of your scissors inside the artery and cut the muscle above it. Check that you can see the dvn as you cut to be sure you are safely above the STG. Continue cutting anteriorly, following the artery. As you cut above the STG, you will be able to see the stn emanating anteriorly from the STG and diving down into the muscle. Continue cutting anteriorly to fully separate the muscle above the STG.</li>
<li>Clear any tissue away from the stn.</li>
</ol></li>
<li>Separate the agns from the surrounding tissue. (Even if you do not plan to record from the agn, you will need a portion of the intact nerve to pin down the ganglion for intracellular recordings.)<ol>
<li>To better see the agns, remove the remaining hypodermis and a thin layer of the underlying muscle.</li>
<li>Just posterior to the STG, identify a dark tunnel in the fat and muscle on either side of the dvn.</li>
<li>Stick one scissor blade inside the tunnel and cut the tissue above the nerve. Continue cutting laterally, following the nerve, to isolate it. (A couple millimeters is sufficient for pinning the ganglion, but expose at least 5 mm of the agn if you plan to record from it.)</li>
<li>Detach the agn from the muscle it innervates by cutting the connections.</li>
<li>Repeat on the other side to isolate the other agn.</li>
<li>Once both agns have been dissected, cut all the muscles and tissue connecting the STG to the stomach.</li>
</ol></li>
<li>Isolate the mvns. They are a bilateral pair of small nerves branching off the lvn and traversing laterally underneath the fat.<ol>
<li>Either use scissors or two forceps to cut through the fat on one side of the dvn, near where it branches into the two lvns. Here you are far from the mvns and won't risk cutting them.</li>
<li>Lift up the fat layer, and identify the small mvn underneath. These nerves run medial to laterally just posterior to the level of the gm1b muscles.</li>
<li>Remove the layer of fat above the mvn, leaving the nerve intact for now so it won't become tangled. You will cut its lateral end from off the stomach at the end of the dissection.</li>
<li>Repeat the process on the other side to isolate the other mvn.</li>
</ol></li>
<li>Isolate the lvns and posterior nerves.<ol>
<li>Starting where the lvns branch laterally off the dvn, follow an lvn by cutting the fat above it. Depending on the preparation, sometimes the entire fat layer above the nerve can be pulled off.</li>
<li>Continue revealing the lvn until the point where the psn branches off.</li>
</ol></li>
<li>Isolate the pyn.<ol>
<li>Locate the pyn, it is a nerve travelling posteriorly over the pylorus.</li>
<li>Remove the thin membrane covering this region.</li>
<li>Isolate the pyn by cutting a small section of the pylorus, attached to the pyn.</li>
<li>Gently lift the pyloric section and cut away the tissue on either side of the pyn until you reach the branch that attaches to the pdn.</li>
</ol></li>
<li>Isolate the pdn.<ol>
<li>Locate the pdn, it is a nerve travelling posteriorly to the pyloric dilator muscle.</li>
<li>Cut a small section of the pyloric dilator muscle, attached to the pdn.</li>
<li>Gently lift the pyloric dilator muscle section, cut away the tissue on either side of the pdn until you reach the branch that attaches to the pyn.</li>
</ol></li>
<li>Isolate the ventral lvn, that connects the pyn and pdn to the lvn.<ol>
<li>At the triangular branch point where the pyn and pdn connect to the ventral lvn, isolate any small section of nerve that branches off the ventral lvn, such as the vpln. This will be used as a handle to pull on the ventral lvn.</li>
<li>Lift on the handle and cut away the tissue on either side of the ventral lvn, working in the anterior direction until you reach the point where the psn branches off.</li>
</ol></li>
<li>Repeat steps 10 &#x2013; 13 to isolate the lvn and posterior nerves on the other side. </li>
<li>Detach the stomatogastric nervous system from the stomach.<ol>
<li>To fully detach the lvn from the stomach, pull on the psn and cut all tissue and nerves attached to the lvn and ventral lvn, except for the pdn and pyn. Repeat this process on the other side.</li>
<li>Cut the lateral end of each mvn. Lift the lateral end and cut any extraneous attachments between the nerve and the underlying stomach tissue.</li>
<li>At the anterior end, cut any extraneous attachments to the commissural ganglia, and cut the labial nerve on each side.</li>
<li>Visually scan the stomatogastric nervous system system to identify and then cut any additional attachments to the stomach tissue.</li>
<li>Grab onto the anterior end of both commissural nerves, above the commissural ganglia, and peel the stomatogastric nervous system off the stomach and move it to the side of the dish. As you do this, cut any remaining attachments to free the nervous system.</li>
</ol></li>
<li>Prepare the small Sylgard dish.<ol>
<li>Rub some stomach tissue thoroughly on the surface of the Sylgard in the small dish. This will decrease the hydrophobicity of the Sylgard, so that saline will distribute evenly across the dish.</li>
<li>Rinse and fill the Sylgard dish with chilled saline.</li>
</ol> </li>
<li>Transfer the nervous system to the prepared Sylgard dish by grabbing the anterior ends of both commissural nerves, lifting the nervous system out of the large dish, and laying it in the small dish. </li>
<li>Pin the STNS in the small dish using a combination of minuten pins and thinner pins cut from tungsten wire.<ol>
<li>Ensure that the preparation is dorsal side up by checking that the inferior ventricular nerve is pointing away from the Sylgard.</li>
<li>Remove the brain and pin the commissural nerves.</li>
<li>Gently pull the nervous system tight and flat by pulling each of the psns posteriorly and laterally, and pinning them down.</li>
<li>Remove the pyloric dilator muscle from the pdns, and pin the pdns.</li>
<li>Remove the pyloric segment from the pyns, and pin the pyns.</li>
<li>Pin down any remaining isolated nerves such as the labial nerves, the mvns and agns.</li>
</ol></li>
<li>Desheath the STG.<ol>
<li>Ensure the STG is flat against the Sylgard and held taut by the lateral agns, the anterior stn and the posterior dvn.</li>
<li>Arrange the lighting to enable visualization of the axons within the dvn.</li>
<li>Using a small pin or fine tungsten needle, make a small shallow hole in sheath surrounding the dvn, just posterior to the STG.</li>
<li>Pull the needle across the dvn, dorsal to the axons, to create a horizontal tear in the nerve sheath.</li>
<li>Grab the flap of nerve sheath posterior to the STG and pull anteriorly to remove the sheath from the dorsal side of the STG.</li>
</ol></li>
</ol> 
<table border="1">
<tbody>
<tr>
<td colspan="2">Abbreviations:</td>
</tr>
<tr>
<td>STNS</td>
<td>Stomatogastric nervous system</td>
</tr>
<tr>
<td>STG</td>
<td>Stomatogastric ganglion</td>
</tr>
<tr>
<td>CoG</td>
<td>Commissural ganglion</td>
</tr>
<tr>
<td>stn</td>
<td>stomatogastric nerve</td>
</tr>
<tr>
<td>mvn</td>
<td>medial ventricular nerve</td>
</tr>
<tr>
<td>ion</td>
<td>inferior oesophageal nerve</td>
</tr>
<tr>
<td>son</td>
<td>superior oesophageal nerve</td>
</tr>
<tr>
<td>agn</td>
<td>anterior gastric nerve</td>
</tr>
<tr>
<td>dvn</td>
<td>dorsal ventricular nerve</td>
</tr>
<tr>
<td>lvn</td>
<td>lateral ventricular nerve</td>
</tr>
<tr>
<td>psn</td>
<td>posterior stomach nerve</td>
</tr>
<tr>
<td>vpln</td>
<td>ventral posterior lateral nerve</td>
</tr>
<tr>
<td>pyn</td>
<td>pyloric nerve</td>
</tr>
<tr>
<td>pdn</td>
<td>pyloric dilator nerve</td>
</tr>
</tbody>
</table>

<ol>
	<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=Central+pattern+generators+and+the+control+of+rhythmic+movements.">Central pattern generators and the control of rhythmic movements.</a> Curr. Biol. 11, R986-R996 (2001).</li><li>Nusbaum, M. P., Beenhakker, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small-systems+approach+to+motor+pattern+generation.">A small-systems approach to motor pattern generation.</a> Nature. 417, 343-350 (2002).</li><li>Clemens, S., Massabuau, J. C., Legeay, A., Meyrand, P., Simmers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+modulation+of+interacting+central+pattern+generators+in+lobster+stomatogastric+ganglion:+influence+of+feeding+and+partial+pressure+of+oxygen.">In vivo modulation of interacting central pattern generators in lobster stomatogastric ganglion: influence of feeding and partial pressure of oxygen.</a> J. Neurosci. 18, 2788-2799 (1998).</li><li>Bierman, H. S., Tobin, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gross+dissection+of+the+stomach+of+the+lobster,+Homarus+americanus.">Gross dissection of the stomach of the lobster, Homarus americanus.</a> J Vis Exp. 27, (2009).</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, 161-220 (1974).</li><li>Herrick, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+history+of+the+American+lobster.">Natural history of the American lobster.</a> Bull. U.S. Bur. Fish. 29, 219-252 (1909).</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>Bucher, D., Taylor, A. L., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+pattern+generating+neurons+simultaneously+express+fast+and+slow+rhythmic+activities+in+the+stomatogastric+ganglion.">Central pattern generating neurons simultaneously express fast and slow rhythmic activities in the stomatogastric ganglion.</a> J. Neurophysiol. 95, 3617-3632 (2006).</li><li>Mizrahi, A., Dickinson, P. S., Kloppenburg, P., F&#233;nelon, V., Baro, D. J., Harris-Warrick, R. M., Meyrand, P., Simmers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+maintenance+of+channel+distribution+in+a+central+pattern+generator+neuron+by+neuromodulatory+inputs+revealed+by+decentralization+in+organ+culture.">Long-term maintenance of channel distribution in a central pattern generator neuron by neuromodulatory inputs revealed by decentralization in organ culture.</a> J. Neurosci. 21, 7331-7339 (2001).</li><li>Schulz, D. J., Goaillard, J. M., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+channel+expression+in+identified+single+and+electrically+coupled+neurons+in+different+animals.">Variable channel expression in identified single and electrically coupled neurons in different animals.</a> Nat. Neurosci. 9, 356-362 (2006).</li><li>Schulz, D. J., Goaillard, J. M., Marder, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+expression+profiling+of+identified+neurons+reveals+cell-specific+constraints+on+highly+variable+levels+of+gene+expression.">Quantitative expression profiling of identified neurons reveals cell-specific constraints on highly variable levels of gene expression.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 13187-13191 (2007).</li></ol>

The anatomy of lobster stomach and STNS has been well described previously5,6, and variations of this dissection procedure have been utilized in numerous studies. We recommend becoming familiar with the anatomy of the stomach5-7and STNS5,8 prior to beginning dissections. Such familiarity will decrease the risk of accidental damage to nerves and tissue.
To maintain the health of the preparation, it is critical to maintain the preparation at a cool temperature. This is easily achieved by exchanging the saline every 30 mins with 4&#x02DA;C saline kept in a refrigerator. When immersed in cold saline, the isolated STNS preparation is viable for many hours. Recordings in related Homarus gammarus have demonstrated stable rhythmic activity from the STG throughout 5 days in vitro9; similar robustness has been seen in H. americanus (pers. obs.).
Modulatory input to the STG from the commissural ganglia is required to preserve the ongoing motor patterns. Therefore, to maintain a normal rhythmic activity these ganglia and the ion, son, and stn nerves must be kept intact. The dissection we describe preserves several motor nerves (containing motor neuron axons): the agns (containing Gastric Mill (GM) axons), the mvns (containing Lateral Gastric (LG) and Inferior Cardiac (IC) axons), the pyns (containing Pyloric neuron (PY) axons), the pdns (containing Pyloric Dilator (PD) axons), and the lvns (containing Lateral Pyloric (LP), Medial Gastric (MG), LPG axons as well as those from GM, PD and PY). By dissecting additional (or different) nerves, other motor neuron axons may be recorded. Each individual type of motor neuron axon may be recorded individually by dissecting and recording from the nerve innervating the muscle to which that type of neuron projects.
This preparation may be used for a variety of studies focused on nervous system function. The activity of the neurons may be recorded either using intracellular, sharp electrode recordings from somata or axons, or by recording electrical impulses extracellularly from the nerves. Many ion channels in the STG neurons have been partially cloned and mRNA can be measured from manually pulled somata10,11.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>Fine Tungsten Wire (0.001 in. diameter)</td>
<td>Tool</td>
<td>California Fine Wire</td>
<td>&nbsp;</td>
<td>Cut into 1-2mm pieces to use as fine pins.</td>
</tr>
<tr>
<td>Forceps #5</td>
<td>Tool</td>
<td>FST</td>
<td>91150-20</td>
<td>&nbsp;</td>
<tr>
<td>Minuten Pins</td>
<td>Tool</td>
<td>FST</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>Moria Spring Scissors (7mm blades)</td>
<td>Tool</td>
<td>FST</td>
<td>15370-52</td>
<td>These are the top of the line. There are more economical spring scissors available from the same company.</td>
</tr>
<tr>
<td>Petri Dish (100x15mm)</td>
<td>Tool</td>
<td>Fischer</td>
<td>08-757-12</td>
<td>Fill 2-4mm with Sylgard 182.</td>
</tr>
<tr>
<td>Sylgard 182</td>
<td>Other</td>
<td>Dow Corning</td>
<td>182</td>
<td>&nbsp;</td>
<tr>
<td>Pin Holder</td>
<td>Tool</td>
<td>FST</td>
<td>26018-17</td>
<td>Optional. Desheathing can be done with scissors and fine forceps alone.</td>
</tr>
<tr>
<td>Super fine forceps #5SF</td>
<td>Tool</td>
<td>FST</td>
<td>11252-00</td>
<td>&nbsp;</td>
<tr>
<td>Tungsten Needles</td>
<td>Tool</td>
<td>FST</td>
<td>10130-05</td>
<td>Optional. Desheathing can be done with scissors or with pins cut from fine tungsten wire.</td>
</tr>
<tr>
<td>Dissection microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Homarus Americanus Stomatogastric Nervous System Dissection at JoVE.com

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 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 ...

homarus-americanus-stomatogastric-nervous-system-dissection

Research

JoVE Journal

Biology

11.1K 조회수.  Brandeis. 우리는 미국의 바닷가재의 뱃속에서 stomatogastric 신경계의 미세 절개를 설명 ( Homarus americanus).

비디오: Homarus Americanus Stomatogastric Nervous System Dissection

Dissection of Drosophila Ovaries

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]).

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.

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.

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.

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

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of cells (approximately 25 in C. borealis) which are well characterized. The cells in the STG exhibit a broad range of outputs and are responsible for the motor actions of the stomach. The stomach contains the gastric mill which breaks down food with three internal teeth, and the pylorus which filters the food before it reaches the midgut. The STG produces two rhythmic outputs to control the gastric mill and pylorus known as central pattern generators (CPGs). Each cell in the STG can participate in one or both of these rhythms. These CPGs allow for the study of neuromodulation, homeostasis, cellular and network variability, network development, and network recovery.
The dissection of the stomatogastric nervous system (STNS) from the Jonah crab (Cancer borealis) is done in two parts; the gross and fine dissection. In the gross dissection the entire stomach is dissected from the crab. During the fine dissection the STNS is extracted from the stomach using a dissection microscope and micro-dissection tools (see figure 1). The STNS includes the STG, the oesophageal ganglion (OG), and the commissural ganglia (CoG) as well as the nerves that innervate the stomach muscles. Here, we show how to perform a complete dissection of the STNS in preparation for an electrophysiology experiment where the cells in the STG would be recorded from intracellularly and the peripheral nerves would be used for extracellular recordings. The proper technique for finding the desired nerves is shown as well as our technique of desheathing the ganglion to reveal the somata and neuropil.

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric nervous system (STNS) of the Jonah crab (C. borealis) can be used for electrophysiology, immunohistochemistry, and cell culture studies. The STNS extraction is done in two parts: the gross and fine dissection.

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

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...